CN110339345B - Recombinant human truncated keratinocyte growth factor-1 eye drops and preparation method and application thereof - Google Patents
Recombinant human truncated keratinocyte growth factor-1 eye drops and preparation method and application thereof Download PDFInfo
- Publication number
- CN110339345B CN110339345B CN201910696014.4A CN201910696014A CN110339345B CN 110339345 B CN110339345 B CN 110339345B CN 201910696014 A CN201910696014 A CN 201910696014A CN 110339345 B CN110339345 B CN 110339345B
- Authority
- CN
- China
- Prior art keywords
- growth factor
- recombinant human
- keratinocyte growth
- human truncated
- eye drops
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
- A61K38/1825—Fibroblast growth factor [FGF]
-
- 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
- A61K47/02—Inorganic compounds
-
- 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
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
-
- 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
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/12—Carboxylic acids; Salts or anhydrides thereof
-
- 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
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
-
- 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
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0048—Eye, e.g. artificial tears
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
Abstract
The invention discloses recombinant human truncated keratinocyte growth factor-1 eye drops, which consist of recombinant human truncated keratinocyte growth factor-1 and auxiliary materials; the preparation method comprises the following steps: preparing a thickening agent stock solution, preparing a recombinant human truncated keratinocyte growth factor-1 mixed solution, mixing the two solutions, performing sterile filtration and fixing the volume. The eye drops can be used for promoting cornea repair without promoting blood vessel growth, and have the advantages of improving the stability and bioactivity of the recombinant human truncated keratinocyte growth factor-1 in the eye drops; in addition, the method of the invention is used for obtaining sterile recombinant human truncated keratinocyte growth factor-1 eye drops; the eye drops of the invention are used for treating corneal injury, and have good industrial application prospect.
Description
Technical Field
The invention relates to the field of eye drops, in particular to recombinant human truncated keratinocyte growth factor-1 eye drops and a preparation method and application thereof.
Background
The cornea is connective tissue without blood vessel growth at 6/1 of the forearm of the eye, and is easily subjected to external stimulation such as chemical stimulation, physical stimulation and the like due to exposure to various severe environments, so that the cornea is slowly repaired and has a long course of disease. Meanwhile, the integrity and transparency of the cornea after the ophthalmic surgery are also particularly important for the recovery of vision.
At present, the biological products clinically used for cornea repair include calf serum eye ointment, bei Fushu (recombinant bovine basic fibroblast growth factor eye drops), yibei (recombinant human epidermal growth factor eye drops) and the like, wherein epidermal growth factors are combined with EGF receptors adjacent to a wound basement membrane to enable basement membrane cells at the edge of a cornea to move to the surface layer to form a single cell layer, then mitotic proliferation is started to form double-layer cells to accelerate the repair of the cornea; the recombinant bovine basic cell growth factor belongs to FGF family, and combines with a fibroblast specific receptor on cornea to promote mitosis and proliferation of cells, increase collagen fibers and increase capillaries, thereby playing a role in promoting cornea repair [ the growth factor has a function of repairing corneal epithelial cell damage ]. The biological cornea repair medicines on the market at present have certain limitations, and can promote the growth of capillary vessels, influence the transparency of the cornea and cause the reduction of vision.
According to the literature Werner S.Keratinocyte Growth Factor, a unique plane in epithelial repair process, cell Growth Factor Rev,1998,9 (2): 153-165, keratinocyte Growth Factor is also two members of Fibroblast Growth Factor (FGF) family, namely KGF-1 (FGF-7) and KGF-2 (FGF-10), and promotes the proliferation and differentiation of epithelial cells of different tissues by combining with specific receptors only expressed in the epithelial cells, reduces the enzyme activity of toxic substances in the external environment of the cells, thereby promoting the renewal and proliferation of the epithelial cells and promoting the repair and healing of traumatic tissues. Different from other factors in the fibroblast growth factor family, KGF has an insignificant proliferation promoting effect in fibroblasts and vascular endothelial cells, and the characteristic is expected to become a breakthrough point for repairing corneal injury.
KGF-1 (FGF-7) has been developed for the treatment of canker sores caused by radiotherapy and chemotherapy and is a safe and reliable growth factor. GuLsun, erdag et al found that human diploid keratinocytes were modified by the gene encoding FGF-7, and that skin secretion levels were significantly increased and keratinocyte migration increased using the genetically modified FGF-7, resulting in thicker, highly proliferative epidermis. And the bacteriostatic properties of FGF-7 expressing skin were found to be about 500-fold improved when tested in bacterial infections, a more prominent indication of the role of FGF-7 in skin repair. Therefore, KGF-1 has a wide application prospect in wound repair of epithelial cells.
In 2008, chinese patent with the publication number CN101537172B discloses an eye drop with corneal epithelial injury repairing effect and a preparation method thereof, wherein the eye drop is composed of recombinant human keratinocyte growth factor-2, an antioxidant, a chelating agent, a stabilizer, an osmotic pressure regulator, a surfactant and a proper buffer system in a certain proportion. The above patent shows that the eye drops can promote the repair of corneal damage without promoting the growth of blood vessels. NMPA has approved clinical application of the company in 2018, 5, 18 and published data indicates that the eye drops have not started clinical trials.
The source of the recombinant human truncated keratinocyte growth factor-1 is prepared by fermenting, expressing and purifying strains purchased by the company. The preparation is prepared by utilizing the specificity of the recombinant human truncated keratinocyte growth factor-1, the biological activity of promoting division differentiation and migration, and the high activity and high stability of the mutant, is applied to promoting corneal injury repair, and does not promote blood vessel growth. The recombinant human keratinocyte growth factor-2 eye drops in the patent and the recombinant human truncated keratinocyte growth factor-1 eye drops in the patent are protein eye drops, and the preparations focus on the stability and the biological activity of proteins.
In the united states patent No. US8304387, it is mentioned that recombinant human truncated keratinocyte growth factor-1 has unstable properties of being susceptible to forming insoluble aggregates and being sensitive to acid and base. Therefore, controlling the factors such as pH and solubility of the eye drops is the key to improve the stability and bioactivity of the recombinant human truncated keratinocyte growth factor-1 in the eye drops.
Disclosure of Invention
Aiming at the defects in the prior art, the first purpose of the invention is to provide the recombinant human truncated keratinocyte growth factor-1 eye drops which are used for promoting cornea repair and not promoting blood vessel growth and have the advantages of higher stability and bioactivity.
The second purpose of the invention is to provide a preparation method of the recombinant human truncated keratinocyte growth factor-1 eye drops, which is used for preparing and obtaining sterile recombinant human truncated keratinocyte growth factor-1 eye drops.
The third purpose of the invention is to provide the application of the recombinant human truncated keratinocyte growth factor-1 eye drops, the recombinant human truncated keratinocyte growth factor-1 eye drops are used for treating corneal injury without promoting blood vessel growth, and the eye drops have good industrial application prospect.
In order to achieve the first object, the invention provides the following technical scheme:
the eye drops comprise recombinant human truncated keratinocyte growth factor-1 and auxiliary materials, wherein the amino acid sequence of the recombinant human truncated keratinocyte growth factor-1 is SEQ ID NO:1, and the auxiliary materials comprise a thickening agent, an osmotic pressure regulator, a protein protective agent, a surfactant and a citrate buffer salt system.
By adopting the technical scheme, the recombinant human truncated keratinocyte growth factor-1 formed by deleting 23 amino acids from the N end of the human keratinocyte growth factor has the advantages of higher stability and unchanged in vivo and external biological functions, and the property that the human keratinocyte growth factor-1 is favorable for promoting the proliferation and migration of corneal epithelial cells without influencing the growth of vascular endothelial cells is utilized, so that the aims of promoting corneal repair and not promoting the growth of blood vessels are fulfilled;
because the recombinant human truncated keratinocyte growth factor-1 has the unstable properties of easily forming insoluble aggregation and being sensitive to acid and alkali, in the auxiliary materials, the pH value of the eye drops is buffered and adjusted through a citric acid buffer salt system, and a protein protective agent is used for protecting the recombinant human truncated keratinocyte growth factor-1 from the influence of the change of external factors such as temperature, shock and the like, so that the stability of the recombinant human truncated keratinocyte growth factor-1 is improved; the osmotic pressure regulator regulates the osmotic pressure of the eye drops and is assisted with a surfactant to promote the dissolution of the recombinant human truncated keratinocyte growth factor-1, so that the recombinant human truncated keratinocyte growth factor-1 in the eye drops is isotonic with tears, and the cornea repair effect of the eye drops is improved while the stimulation is reduced; the thickener reduces the surface tension of the liquid, weakens the mutual collision among proteins and improves the storage stability of the eye drops and the compliance of patients by changing the physical properties of the recombinant human truncated keratinocyte growth factor-1;
in conclusion, the recombinant human truncated keratinocyte growth factor-1 is used as a main effective component to promote cornea repair without promoting blood vessel growth, and an auxiliary material and the recombinant human truncated keratinocyte growth factor-1 generate a synergistic effect to finally achieve the purpose of improving the stability and the bioactivity of the recombinant human truncated keratinocyte growth factor-1 in eye drops.
Further, the concentration of the recombinant human truncated keratinocyte growth factor-1 is 0.01g/L-0.1g/L.
By adopting the technical scheme, the recombinant human truncated keratinocyte growth factor-1 is used as a main effective component to promote cornea repair without promoting blood vessel growth, and the DNA generation of corneal epithelial cells is dose-dependent to the recombinant human truncated keratinocyte growth factor-1, so that the concentration of the recombinant human truncated keratinocyte growth factor-1 is controlled to be beneficial to achieving a better effect of promoting cornea repair.
Further, the concentration of the auxiliary materials is as follows:
by adopting the technical scheme, after the concentration of the recombinant human truncated keratinocyte growth factor-1 is selected to be better, the concentration of the auxiliary materials is correspondingly adjusted to select the more optimal concentration of the auxiliary materials, so that the stability and the biological activity of the recombinant human truncated keratinocyte growth factor-1 in the eye drops are further ensured.
Further, the citrate buffer salt system consists of citric acid and sodium citrate, and the pH value of the citrate buffer salt system is 6-7.
By adopting the technical scheme, because the pH of the tear of the patient with corneal injury is usually higher than that of a normal person, the sodium citrate and the citric acid are matched to form a citrate buffer salt system, so that the tear buffer salt system has a stronger pH buffer effect, is favorable for reducing the pH variation in the mixed environment of the tear and the eye drops, is favorable for reducing the acerbity of the eye drops and improves the compliance of the patient.
Further, the protein protectant is histidine.
By adopting the technical scheme, histidine can achieve the purpose of protecting the recombinant human truncated keratinocyte growth factor-1 by increasing soluble aggregation, preventing thermal damage or increasing the transformation temperature of protein.
Further, the thickener is polyvinyl alcohol.
By adopting the technical scheme, the pharmaceutical grade polyvinyl alcohol is a synthetic polymer thickener which has good biocompatibility and can be applied to eye drop products; the surface tension of the eye drops is reduced by adding polyvinyl alcohol, intermolecular collision of the recombinant human truncated keratinocyte growth factor-1 is weakened, the eye drops can be used as an artificial tear component to moisten the corneal surface while the stability and the bioactivity of the recombinant human truncated keratinocyte growth factor-1 are considered, and the compliance of patients is improved.
Further, the surfactant is tween 20.
By adopting the technical scheme, the solubility of the recombinant human truncated keratinocyte growth factor-1 is favorably improved by adding the Tween 20 into the eye drops.
Further, the osmotic pressure regulator is sodium chloride and mannitol, wherein the concentration of the sodium chloride is 2g/L-4g/L, and the concentration of the mannitol is 15g/L-40g/L.
By adopting the technical scheme, the mannitol has the function of regulating intraocular pressure, the sodium chloride has the function of regulating the osmotic pressure of extracellular fluid, and the eye drops and tears are isotonic or hypotonic by regulating the type and concentration of an osmotic pressure regulator, so that the stimulation is reduced, and the cornea repair effect of the eye drops is improved.
In order to achieve the second object, the invention provides the following technical scheme:
a method for preparing recombinant human truncated keratinocyte growth factor-1 eye drops comprises the following steps:
s1, dispersing a thickening agent into water for injection, carrying out hot-pressing sterilization at 121 ℃, and cooling to room temperature to obtain a thickening agent stock solution;
s2, sequentially dissolving the recombinant human truncated keratinocyte growth factor-1, an osmotic pressure regulator, a protein protective agent, a surfactant and a citrate buffer salt system in water for injection, and performing sterile filtration by using a filter membrane of 0.22 mu m to obtain a recombinant human truncated keratinocyte growth factor-1 mixed solution;
and S3, under the aseptic condition, uniformly mixing the thickening agent stock solution obtained in the S1 and the mixed solution of the recombinant human truncated keratinocyte growth factor-1 obtained in the S2, and performing constant volume by using aseptic water for injection to obtain the recombinant human truncated keratinocyte growth factor-1 eye drops.
By adopting the technical scheme, the preparation process adopts a sterile three-step method for operation, and the preparation method is simple and quick, so that the recombinant truncated keratinocyte growth factor-1 eye drops with qualified sterile requirements can be obtained without adding a bacteriostatic agent, the stimulation of the eye drops to the eyes is favorably reduced, and the compliance of patients is improved.
In order to achieve the third object, the invention provides the following technical solutions:
the application of the recombinant human truncated keratinocyte growth factor-1 eye drops is applied to treatment of corneal injury, wherein the corneal injury comprises corneal acid-base burn, corneal scald, corneal ulcer and corneal surgery injury.
By adopting the technical scheme, the recombinant human truncated keratinocyte growth factor-1 is combined with the specific receptor only expressed in the epithelial cells to promote the proliferation and differentiation of the epithelial cells of different tissues and reduce the enzyme activity of toxic substances in the external environment of the cells, so that the renewal and proliferation of the epithelial cells are promoted, and the proliferation promoting effect of the recombinant human truncated keratinocyte growth factor-1 in fibroblasts and vascular endothelial cells is not obvious, so that the recombinant human truncated keratinocyte growth factor-1 eye drops are applied to corneal injury such as corneal burn, acid-base burn, corneal scald, corneal ulcer and corneal surgical injury, and have good industrial application prospect.
The invention has good industrial application prospect by applying the recombinant human truncated keratinocyte growth factor-1 in the field of pharmacy and using the recombinant human truncated keratinocyte growth factor-1 to manufacture the medicine.
In conclusion, the invention has the following beneficial effects:
1. the invention utilizes the characteristics that the recombinant human truncated keratinocyte growth factor-1 promotes cornea repair and does not promote blood vessel growth, and is assisted by auxiliary materials, so that the auxiliary materials and the recombinant human truncated keratinocyte growth factor-1 generate synergistic action, and finally, the aim of improving the stability and the bioactivity of the recombinant human truncated keratinocyte growth factor-1 in eye drops is fulfilled.
2. The method of the invention prepares the recombinant human truncated keratinocyte growth factor-1 eye drops by adopting a three-step method, and the operation is simple and rapid.
3. The invention treats corneal injury through the recombinant human truncated keratinocyte growth factor-1 eye drops, and has good industrial application prospect.
Drawings
FIG. 1 is a statistical graph of the rate of epithelial defect in rats according to example 8 of the present invention.
Figure 2 is a graph of staining of sodium corneal fluorescein from example 8 in which the invention was practiced.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples.
Preparation example of recombinant human truncated keratinocyte growth factor-1
A cDNA sequence designed based on the amino acid sequence (SEQ ID NO: 1) of recombinant human truncated keratinocyte growth factor-1 was inserted into a pUC-57 vector to construct a pUC-57-delta N23 KGF-insert nucleotide sequence.
Using pUC-57-delta N23KGF plasmid as template, double enzyme digestion with Nde I endonuclease and BamH I endonuclease, recovering target fragment, using T4DNA ligase to ligate with the recovered fragment of plasmid pET-3C (purchased from TaKaRa company of Dalian province) through Nde I and BamH I enzyme digestion, transforming Escherichia coli cloning host bacterium Top10, and screening recombinant plasmid pET-3C-delta N23KGF by enzyme digestion and PCR verification. After the cDNA sequence of delta N23KGF in the recombinant plasmid is proved to be correct by DNA sequencing, the recombinant plasmid is transformed into Escherichia coli expression host bacteria BL21 (DE 3) plysS (purchased from China), and finally pET-3C-delta N23KGF/BL21 (DE 3) pLysS recombinant engineering bacteria are obtained by expression screening.
Selecting pET-3C-delta N23KGF/BL21 (DE 3) pLysS recombinant engineering bacteria with optimal expression, inoculating the recombinant engineering bacteria into LB culture medium containing benzyl amine and chloramphenicol resistance, inoculating the recombinant engineering bacteria into YT culture solution when the recombinant engineering bacteria are cultured to a certain concentration, culturing at 30 ℃, controlling the dissolved oxygen to be more than 30%, and adjusting the pH value to be 7.0. Fermentation broth OD 600 When 10-14 hours are reached, IPTG with the final concentration of 0.2mmoL/L is added, and the fermentation is stopped after the culture and induction are continued for 4 hours. And (3) taking the centrifuged recombinant engineering bacteria, re-suspending the centrifuged recombinant engineering bacteria in a buffer solution with the pH of 9.0, crushing the recombinant engineering bacteria under high pressure, and collecting supernatant. Then, capturing the protein in the supernatant through strong cation exchange chromatography, diluting the protein to a certain concentration, supplementing cysteine with a proper concentration to help the formation of a disulfide bond, and finally purifying through Source30S column chromatography to obtain the recombinant human truncated keratinocyte growth factor-1.
Examples
Example 1: a method for preparing recombinant human truncated keratinocyte growth factor-1 eye drops comprises the following steps:
s1, dispersing polyvinyl alcohol in 500mL of water for injection, carrying out hot-pressing sterilization at 121 ℃, and cooling to room temperature to obtain a thickening agent stock solution;
s2, dissolving the recombinant human truncated keratinocyte growth factor-1, a citrate buffer system, histidine, mannitol, sodium chloride, tween 20 and 2mg of sucrose in 400mL of water for injection, and performing sterile filtration by using a 0.22-micron filter membrane to obtain a recombinant human truncated keratinocyte growth factor-1 mixed solution;
s3, uniformly mixing the thickener stock solution obtained in the step S1 and the mixed solution of the recombinant human truncated keratinocyte growth factor-1 of the step S2 under an aseptic condition, setting the mixed solution to 1L by using aseptic water for injection, and filtering the mixed solution by using a 0.22 mu m sterilization-grade filter membrane to obtain the recombinant human truncated keratinocyte growth factor-1 eye drops;
s4, the recombinant human truncated keratinocyte growth factor-1 eye drops are filled in an aseptic mode to obtain finished product eye drops of 0.5mL to 1.0 mL.
Wherein the amino acid sequence of the recombinant human truncated keratinocyte growth factor-1 is SEQ ID NO. 1, the citrate buffer system consists of citric acid and sodium citrate, and the pH value is 6.5. The components and their amounts are shown in table 1.
Example 2: the difference from example 1 is that the components and their amounts are shown in table 1.
Example 3: the difference from example 1 is that the components and their amounts are shown in table 1.
Example 4: the difference from example 1 is that the components and their amounts are shown in table 1.
Example 5: the difference from example 1 is that the components and their amounts are shown in table 1.
Comparative example
Comparative example 1: the difference from example 1 is that the components and their amounts are shown in table 1.
Comparative example 2: the difference from example 1 is that the components and their amounts are shown in table 1.
TABLE 1
Because the recombinant human truncated keratinocyte growth factor-1 eye drops are protein eye drops, the preparation focuses on the stability and the biological activity of protein, and the preparation of the recombinant human truncated keratinocyte growth factor-1 eye drops should meet the requirements under the eye drops of 2015 editionAnd (4) specifying. Therefore, it is necessary to examine the appearance, pH, recombinant human truncated keratinocyte growth factor-1 content and cell activity (EC) of the above-mentioned eye drops 50 ) And the like.
Example 6: stability study of recombinant human truncated keratinocyte growth factor-1 eye drops
1) The content of the recombinant Human truncated keratinocyte growth factor-1 in the recombinant Human truncated keratinocyte growth factor-1 eye drops is measured by adopting a Human KGF/FGF-7 (Catalog Number: DY 251) kit to quantitatively measure the recombinant Human truncated keratinocyte growth factor-1.
The content determination method comprises the following steps: coating a mouse anti-FGF antibody on a 96-well plate; after proper cleaning, adding the recombinant human truncated keratinocyte growth factor-1 eye drops to be detected and a standard substance to react for 2 hours at room temperature; after proper washing, detecting an antibody by using a biotin-labeled goat anti-FGF antibody, and reacting for 2 hours at room temperature; and finally adding horseradish oxidase labeled streptavidin for reaction at room temperature for 20min, adding TMB color development solution for color development in dark for 20min, finally stopping the reaction with 2M sulfuric acid, and measuring at the wavelength of 450 nm.
2) The biological activity of the recombinant human truncated keratinocyte growth factor-1 eye drops is determined, and the biological activity of the recombinant human truncated keratinocyte growth factor-1 is determined in vitro by adopting recombinant BAF-3-FGFR2 III 3b cells transfected by FGFR2 III 3b receptors of the company.
The method for measuring the biological activity comprises the following steps: adding the recombinant human truncated keratinocyte growth factor eye drops and the standard substance with different dilution times into a 96-well plate according to the amount of 100 mu L/hole respectively and making multiple holes; centrifuging BAF-3-FGFR2 III 3b cell suspension in logarithmic growth phase at 1000rpm for 5min, washing with RPMI1640 for 3 times, and resuspending the cells to 5 × 10 with sample living culture solution 5 Adding the powder into the 96-well plate in turn, wherein each well is 100 mu L; then placing the 96-well plate at 37 ℃ 5% 2 Culturing the cells under conditions for 72 hours; adding CCK-8 reagent 10 μ L to each well after 72 hr, at 37 deg.C, 5% 2 The reaction was continued for 8 hours and the results were measured at 450 nm. The semi-Effective Concentration (EC) was calculated by fitting a four-parameter regression curve with SigmapLot software 50 )。
3) Stability test
The determination method comprises the following steps: the eye drops of examples 1 to 5 and comparative example 1 were filled in 3mL vials, stoppered, capped, and subjected to accelerated examination at 25. + -. 2 ℃ for appearance, sterility, pH, content, and cell viability tests at 0 day, 1 month, and 3 months, respectively. The test results are shown in table 2.
TABLE 2
As can be seen from Table 2, the eye drops obtained by the above preparation method during the examination period have appearance and sterility indexes meeting the specification of eye drops of 2015 edition. The pH of examples 1-5 varied within. + -. 0.1, with little change; whereas comparative examples 1-2 all varied by more than 0.1. The key quality attributes of the stability test are content and cell activity, and the examples 1-5 are superior to the comparative examples 1-2 when the test is carried out at 25 +/-2 ℃ for 1 month and 3 months. In conclusion, the eye drops prepared by adopting the recombinant human truncated keratinocyte growth factor-1, the citrate buffer system, histidine, mannitol, sodium chloride, tween 20 and polyvinyl alcohol meet the regulation under the eye drop item of 2015 edition, and have better stability and bioactivity.
Example 7: pharmacodynamic research on recombinant human truncated keratinocyte growth factor-1 eye drop alkali burn model
Healthy New Zealand white rabbits (2 kg-3kg body weight) were used in combination. 75 animals are randomly grouped, 25 animals in each group are soaked in NaOH solution by using filter paper discs, the filter paper discs are carefully pasted on the right cornea middle area of the right eye with the fixed eyeball position for a plurality of minutes, and after the filter paper discs are taken down, the cornea and the corneal pocket are immediately washed by using physiological saline to prepare the corneal alkali burn model. The negative control product is eye drops without recombinant human truncated keratinocyte growth factor-1, and the composition of auxiliary materials is the same as that in example 3; the sample of the present invention is the eye drop of example 3 as a test sample; the positive control drug is recombinant bovine basic fibroblast growth factor eye drop (Bei Fushu, zhuhihishen), and is administered 4 times per day at a dose of 1 drop each time for 14 days. And (3) standard photographing of fluorescein-dyed slit lamp microscope at 1 day, 2 days, 4 days, 6 days, 10 days and 14 days of treatment respectively, and measuring and analyzing corneal epithelial healing area and corneal epithelial healing rate of each group at different times by a computer image system. Meanwhile, the corneal neovascularization length of the animals in the invention group and the positive control group is observed and measured by a slit lamp microscope on 1 day, 6 days and 14 days of treatment, the neovascularization area is calculated by a Robert model, and the influence of different time of each group on neovascularization is analyzed.
The results show that: in the corneal alkali burn model of white rabbit in New Zealand, the area of corneal alkali burn before administration is 45-55mm2. The rabbit eyes which are completely healed can be seen after the group is administrated for 2 days, the number of the completely healed rabbit eyes is increased along with the increase of the administration days, and the complete healing rates of the rabbit eyes of a negative control group, the rabbit eyes of the group and the rabbit eyes of a positive control group are respectively 21%, 86% and 79% when the group is administrated for 10 days; the observation of the new blood vessels shows that the areas of the new blood vessels of the invention group and the positive control group are 6933 + -1022 and 15203 + -4100 at 14 days.
From the above, the recombinant human truncated keratinocyte growth factor-1 eye drops can remarkably promote healing of rabbit corneal alkali burn, and the treatment effect of the eye drops is not remarkably different from that of a positive control drug; in addition, the eye drops of the invention and the eye drops of the positive control group have significant difference in the aspect of promoting corneal neovascularization. The recombinant human truncated keratinocyte growth factor-1 eye drops can promote healing of alkali burn cornea and do not promote growth of new blood vessels.
Example 8: pharmacodynamic study of recombinant human truncated keratinocyte growth factor-1 eye drops operation injury model
After gas anesthesia, lidocaine hydrochloride is spotted on the surface of the right eye of a healthy SD rat without eye diseases, a cornea trephine with the diameter of 4mm is used for placing a central impression of the cornea, 40 mu L of 20% alcohol is injected into the ring, the collagen sponge is used for absorbing and removing the alcohol in the ring after the cornea is soaked for 30s, the cornea is immediately flushed by physiological saline, a central corneal epithelium with the same diameter as the trephine is scraped by a corneal epithelium knife, the corneal epithelium is immediately dyed by fluorescein after modeling, and the dyeing area is 93-100%, as shown in figure 2. After the molding is successful, the animals are randomly divided into 3 groups, each group comprises 35 rats, and the rats are respectively a negative control group: the auxiliary materials are the same as example 3, and do not contain the recombinant human truncated keratinocyte growth factor-1; the invention group: eye drops of example 3; positive control group: recombinant bovine basic fibroblast growth factor eye drops (Bei Fushu, zhahi sheng). The three groups are administered 3 times daily at a dose of 50 μ L each for 7 days. The corneal epithelial defect area was measured with 1% sodium fluorescein staining using ipp6.0 software, analyzed with origine 9.0 statistical software, corneal epithelial defect rate = corneal epithelial fluorescence staining area/total corneal area.
As shown in FIG. 1, at each time point after molding, the cornea was not significantly abnormal, and appeared to be relatively transparent, and the rat lens was visible. The corneal epithelium is repaired rapidly, and the corneal defect rate of each group of animals after one week is respectively as follows: negative control group: 30.2 +/-2.28%; the invention group: 4.8 +/-2.45%; the positive control group is 7.23 +/-3.21%. The basic repair of the corneal epithelial defects of rats in the invention group and the positive control group is demonstrated, and the recombinant human truncated keratinocyte growth factor-1 eye drops have the function of promoting the repair of corneal injury.
The present embodiment is only for explaining the present invention, and it is not limited to the present invention, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present invention.
Sequence listing
<110> Chongqing Paijin Biotechnology Co., ltd
<120> recombinant human truncated keratinocyte growth factor-1 eye drops and application thereof
<141> 2019-07-04
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 140
<212> PRT
<213> Artificial Sequence
<400> 1
Ser Thr Ala Thr Met Gly Gly Gly Ala Ile Ala Val Ala Ala Leu Pro
1 5 10 15
Cys Ala Thr Gly Thr Thr Leu Ala Ile Ala Leu Ala Gly Leu Val Leu
20 25 30
Gly Thr Gly Gly Met Leu Ala Ala Thr Ala Ile Met Gly Ile Ala Thr
35 40 45
Val Ala Val Gly Ile Val Ala Ile Leu Gly Val Gly Ser Gly Pro Thr
50 55 60
Leu Ala Met Ala Leu Gly Gly Leu Leu Thr Ala Leu Leu Gly Cys Ala
65 70 75 80
Gly Ala Cys Ala Pro Leu Gly Leu Ile Leu Gly Ala His Thr Ala Thr
85 90 95
Thr Ala Ser Ala Leu Thr Thr His Ala Gly Gly Gly Met Pro Val Ala
100 105 110
Leu Ala Gly Leu Gly Ile Pro Val Ala Gly Leu Leu Thr Leu Leu Gly
115 120 125
Gly Leu Thr Ala His Pro Leu Pro Met Ala Ile Thr
130 135 140
Claims (7)
1. The eye drops are characterized by consisting of recombinant human truncated keratinocyte growth factor-1 and auxiliary materials, wherein the amino acid sequence of the recombinant human truncated keratinocyte growth factor-1 is SEQ ID NO. 1, and the auxiliary materials consist of a thickening agent, an osmotic pressure regulator, a protein protective agent, a surfactant, a citrate buffer salt system and water for injection;
the concentration of the recombinant human truncated keratinocyte growth factor-1 is 0.01g/L-0.1g/L;
the concentration of the auxiliary materials is as follows:
5-50 mmol/L citrate buffer salt system;
protein protective agent 0.5g/L-20g/L;
1g/L-15g/L of thickening agent;
0.05g/L-0.2g/L of surfactant;
17g/L-44g/L of osmotic pressure regulator;
the citrate buffer salt system is composed of citric acid and sodium citrate, and the pH value of the citrate buffer salt system is 6-7.
2. The recombinant human truncated keratinocyte growth factor-1 eye drop as claimed in claim 1, wherein the protein protectant is histidine.
3. The recombinant truncated human keratinocyte growth factor-1 eye drop as claimed in claim 1, wherein the thickening agent is polyvinyl alcohol.
4. The recombinant human truncated keratinocyte growth factor-1 eye drop as claimed in claim 1, wherein the surfactant is tween 20.
5. The recombinant human truncated keratinocyte growth factor-1 eye drop as claimed in claim 1, wherein the osmotic pressure regulator comprises sodium chloride and mannitol, wherein the concentration of sodium chloride is 2g/L-4g/L, and the concentration of mannitol is 15g/L-40g/L.
6. The process for preparing the eye drops of recombinant human truncated keratinocyte growth factor-1 according to any one of claims 1-5, comprising the following steps:
s1, dispersing a thickening agent into water for injection, carrying out hot-pressing sterilization at 121 ℃, and cooling to room temperature to obtain a thickening agent stock solution;
s2, sequentially dissolving the recombinant human truncated keratinocyte growth factor-1, an osmotic pressure regulator, a protein protective agent, a surfactant and a citrate buffer salt system in water for injection, and performing sterile filtration by using a filter membrane of 0.22 mu m to obtain a recombinant human truncated keratinocyte growth factor-1 mixed solution;
s3, under the aseptic condition, uniformly mixing the thickener stock solution obtained in the step S1 with the recombinant human truncated keratinocyte growth factor-1 mixed solution obtained in the step S2, and carrying out volume fixing by using aseptic water for injection to obtain the recombinant human truncated keratinocyte growth factor-1 eye drops;
the amino acid sequence of the recombinant human truncated keratinocyte growth factor-1 is SEQ ID NO. 1.
7. The use of an ophthalmic solution of recombinant human truncated keratinocyte growth factor-1 according to any one of claims 1-5, wherein the ophthalmic solution is used for preparing a medicament for treating corneal injury, the corneal injury comprises corneal acid-base burn, corneal scald, corneal ulcer, corneal surgery injury; the amino acid sequence of the recombinant human truncated keratinocyte growth factor-1 is SEQ ID NO 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910696014.4A CN110339345B (en) | 2019-07-30 | 2019-07-30 | Recombinant human truncated keratinocyte growth factor-1 eye drops and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910696014.4A CN110339345B (en) | 2019-07-30 | 2019-07-30 | Recombinant human truncated keratinocyte growth factor-1 eye drops and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110339345A CN110339345A (en) | 2019-10-18 |
| CN110339345B true CN110339345B (en) | 2022-11-29 |
Family
ID=68179095
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910696014.4A Active CN110339345B (en) | 2019-07-30 | 2019-07-30 | Recombinant human truncated keratinocyte growth factor-1 eye drops and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110339345B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR102440312B1 (en) * | 2020-08-28 | 2022-09-05 | 한국해양과학기술원 | Thermally stable fgf7 polypeptide and use of the same |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101537172A (en) * | 2008-08-14 | 2009-09-23 | 温州医学院 | Recombinant human keratinocyte growth factor-2 containing eye drops and method for preparing same |
| CN109402130A (en) * | 2018-11-23 | 2019-03-01 | 成都中医药大学附属医院 | A kind of recombinant human horny cell growth factor-2-1 and its preparation method and application |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101084008B (en) * | 2004-12-15 | 2013-04-10 | 比奥维特罗姆股份公开公司 | Therapeutic formulations of keratinocyte growth factor |
-
2019
- 2019-07-30 CN CN201910696014.4A patent/CN110339345B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101537172A (en) * | 2008-08-14 | 2009-09-23 | 温州医学院 | Recombinant human keratinocyte growth factor-2 containing eye drops and method for preparing same |
| CN109402130A (en) * | 2018-11-23 | 2019-03-01 | 成都中医药大学附属医院 | A kind of recombinant human horny cell growth factor-2-1 and its preparation method and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110339345A (en) | 2019-10-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| TWI388349B (en) | Ophthalmic devices and related methods and compositions | |
| CN1092990C (en) | Modulation of cell proliferation and wound healing | |
| Seib | Reverse-engineered silk hydrogels for cell and drug delivery | |
| US8974803B2 (en) | Injectable biomaterials | |
| CN107308494A (en) | A kind of injection collagen, preparation method and filler | |
| Zhou et al. | Hydrogels derived from acellular porcine corneal stroma enhance corneal wound healing | |
| Suzuki et al. | Optimization of silk fibroin membranes for retinal implantation | |
| CN110339345B (en) | Recombinant human truncated keratinocyte growth factor-1 eye drops and preparation method and application thereof | |
| MXPA06012175A (en) | Corneal neuritogenesis promoter containing pacap and its derivative. | |
| Wang et al. | ECM‐Like Adhesive Hydrogel for the Regeneration of Large Corneal Stromal Defects | |
| RU2646804C1 (en) | Ophthalmic agent for regeneration of cornea of the eye | |
| BURSTEIN | Growth factor effects on corneal wound healing | |
| CN102188695B (en) | Ophthalmic gel composition | |
| CN103656622B (en) | Recombination human body keratinized cell factor K GF-2 environment sensitive type eye transmission system and application thereof | |
| CN101537172B (en) | Recombinant human keratinocyte growth factor-2 containing eye drops and method for preparing same | |
| JP2020533014A (en) | Method for Producing Soluble Recombinant Human Basic Fibroblast Growth Factor (rh-bFGF) | |
| CN110604811B (en) | Artificial tear containing recombinant human lysozyme and recombinant human epidermal growth factor | |
| WO1993016717A1 (en) | Method and agents for promoting wound healing | |
| CN113827706B (en) | Application of GIP and its derivative peptide in preparing skin wound treating medicine | |
| CN111346217B (en) | New application of polypeptide TDL23 | |
| TWI732372B (en) | A bifunctional angiogenesis inhibitor and the use thereof | |
| CN110742860B (en) | Eye drops, preparation method and application thereof in medicine for treating corneal injury | |
| WO2019151450A1 (en) | Nerve cell culture material and therapeutic agent for nerve damage | |
| US8207119B2 (en) | Ophthalmological composition | |
| CN101721681A (en) | Application of human Keratiocyte growth factor 1 in preparation of medicament for treating foot ulcers and leg ulcers of diabetes |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |