Composition for treating eye diseases

Abstract

The invention relates to a composition for treating eye diseases, which comprises human keratinocyte factor-2 and a protective agent, and the protective agent comprises: 1) one or more of 0.1%-2% (w / v) mannitol and 0.01%-0.05% (w / v) sodium hyaluronate; 2) 0.1%-2% (w / v) glycerol; 3) 0.1%-2% (w / v) of one or more of trehalose or 0.5% (w / v) of human serum albumin; and 4) 0.1%-2% (w / v) of one or more of lysine hydrochloride. The compatibility of an hKGF-2 stock solution and auxiliary materials is explored to obtain a better concentration range of a protective agent, a surfactant and a pH regulator in the composition, and on the basis of the obtained better concentration range of each pharmaceutic auxiliary material, the melting (denaturation) temperature and aggregation temperature of hKGF-2 protein in a solution system are explored to determine a better prescription, so that the pharmaceutical composition is suitable for clinical application. The hKGF-2 protein activity in the obtained prescription can be kept stable for a long time. In addition, compared with eye drops provided in the prior art, the hKGF-2 eye drops provided by the invention have a better effect in the aspect of xerophthalmia treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to pharmaceutical preparations containing organic active ingredients, and more particularly to a composition for treating eye diseases. Background Technology

[0002] Dry eye is a general term for various diseases caused by abnormal tear film quality or dynamics, leading to decreased tear film stability and resulting ocular surface tissue lesions. A healthy ocular surface is covered by a tear film structure composed of mucin, aqueous tears, and lipids secreted by accessory glands of the eye. The tear film maintains a normal microenvironment on the ocular surface and plays a role in adequate lubrication and protection of the eyeball. When the balance of substances in the tear film is disrupted and cannot be replenished or repaired in time, dry eye syndrome will occur.

[0003] In recent years, the incidence of dry eye syndrome has gradually increased, and the symptoms may continue to worsen with age. Existing products for relieving dry eye syndrome include Bepanthen eye drops, sodium hyaluronate eye drops, and recombinant bovine basic fibroblast growth factor eye drops, but the effects of the above eye drops in the treatment of dry eye syndrome are limited, and combination therapy is usually required. For example, Wu Cheng, Zhang Li, and Li Wei. Clinical efficacy and safety of recombinant bovine basic fibroblast growth factor eye drops combined with sodium hyaluronate eye drops in the treatment of dry eye syndrome [J]. Clinical Rational Drug Use, 2023, 16(36): 34-37. This study disclosed the relevant content of recombinant bovine basic fibroblast growth factor eye drops combined with sodium hyaluronate eye drops in the treatment of dry eye syndrome. Although the combination therapy improved the efficacy to a certain extent, it could only relieve mild dry eye symptoms, and most dry eye patients are elderly, which increases the difficulty of using this method. In addition, there is also a method that combines conventional treatment with traditional Chinese medicine treatment, such as Zhang Jing and Liu Shaorui. Observation on the efficacy of comprehensive therapy in treating meibomian gland dysfunction dry eye syndrome [J]. Laboratory Medicine and Clinical Practice, 2024, 21(02): 156-160+165. This method has a complicated treatment process and can only provide relief, so it is not practical.

[0004] Keratinocyte growth factor (KGF) belongs to the fibroblast growth factor family. KGF-2, also known as fibroblast growth factor-10 (FGF-10), is a basic protein growth factor secreted by subcutaneous tissue cells in the human body. It is characterized by strong tissue specificity, stability, and safety. KGF-2 stimulates the proliferation of newly formed or aging epithelial cells. Therefore, in the skincare field, it can be used to repair or renew damaged cell tissue and metabolism, for example, by promoting skin cell growth and regulating pigment balance to achieve skin care and delay aging. Existing research also provides nude mouse experiments, which have confirmed the unique developmental promoting effect of KGF-2 (promoting hair follicle development and hair growth). Current treatments for dry eye are mostly topical medications, which can only treat mild dry eye and temporarily improve symptoms, without addressing the root cause. Therefore, there is an urgent need for a new drug with good efficacy that can target mild to moderate-to-severe dry eye.

[0005] Furthermore, on the one hand, there are differences in understanding among those skilled in the art; on the other hand, the applicant studied a large number of documents and patents when making this invention, but due to space limitations, not all details and contents were listed in detail. However, this does not mean that the present invention does not possess the features of these prior art. On the contrary, the present invention already possesses all the features of the prior art, and the applicant reserves the right to add relevant prior art to the background art. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a composition for treating eye conditions, comprising human keratinocyte cytokine-2 (hKGF-2) and a protective agent, wherein the protective agent includes:

[0007] 1) 0.1%–2% (w / v) mannitol or 0.01%–0.05% (w / v) sodium hyaluronate;

[0008] 2) 0.1%–2% (w / v) glycerin;

[0009] 3) 0.1%–2% (w / v) trehalose or 0.5% (w / v) human serum albumin;

[0010] 4) 0.1% to 2% (w / v) of one or more of lysine hydrochloride.

[0011] According to a preferred embodiment, the concentration range of hKGF-2 is 50–200 μg / ml.

[0012] According to a preferred embodiment, the mass concentration of mannitol can be in the range of 0.5% or 1% to 2%.

[0013] According to a preferred embodiment, the mass concentration of mannitol can be 0.5%.

[0014] According to a preferred embodiment, the mass concentration of mannitol can be 1.6%.

[0015] According to a preferred embodiment, the mass concentration of glycerol can be in the range of 0.5% to 2%.

[0016] According to a preferred embodiment, the mass concentration of glycerol can be 1%.

[0017] According to a preferred embodiment, the mass concentration of glycerol can be 1.5%.

[0018] According to a preferred embodiment, the mass concentration of glycerol can be 0.5%.

[0019] According to a preferred embodiment, the mass concentration of trehalose can be in the range of 0.5% to 2%.

[0020] According to a preferred embodiment, the mass concentration of trehalose can be 2%.

[0021] According to a preferred embodiment, the mass concentration of lysine hydrochloride can be in the range of 0.5% to 1%.

[0022] According to a preferred embodiment, the mass concentration of lysine hydrochloride can be 0.5%.

[0023] According to a preferred embodiment, the composition for treating eye conditions further includes a surfactant, wherein the surfactant comprises 0.01% to 0.5% (w / v) poloxamer 188.

[0024] According to a preferred embodiment, the mass concentration range of poloxamer 188 can be 0.01% to 0.1%.

[0025] According to a preferred embodiment, the mass concentration of poloxamer 188 can be 0.05%.

[0026] According to a preferred embodiment, the mass concentration of poloxamer 188 can be 0.01%.

[0027] According to a preferred embodiment, the composition for treating eye conditions further includes a pH adjuster, wherein the pH adjuster includes citric acid, sodium citrate, or a combination thereof.

[0028] According to a preferred embodiment, the composition for treating eye conditions further includes a solvent, wherein the solvent can be water for injection.

[0029] According to a preferred embodiment, the pH of the composition for treating eye conditions is 6.0 to 7.0.

[0030] According to a preferred embodiment, the pH of the composition for treating eye conditions is 6.0 to 6.6.

[0031] In another aspect, the present invention provides the use of the aforementioned composition for treating eye conditions in the preparation of a medicament for treating dry eye syndrome.

[0032] In another aspect, the present invention provides the use of the aforementioned composition for treating eye conditions in the preparation of a medicament for treating benzalkonium chloride (BAC)-induced dry eye syndrome.

[0033] In another aspect, the present invention provides a container comprising the aforementioned composition for treating eye conditions.

[0034] According to a preferred embodiment, the container is an eye drop bottle.

[0035] Another aspect of the present invention provides a kit comprising the aforementioned container and instructions or label for applying and using the aforementioned composition for treating eye conditions.

[0036] Another aspect of the present invention provides a method for preparing a composition for treating eye conditions, comprising: preparing solution A, wherein solution A comprises glycerol, mannitol, lysine hydrochloride, trehalose, poloxamer 188, citric acid and sodium citrate; preparing solution B, wherein solution B comprises a prescribed amount of human keratinocyte cytokine-2 protein stock solution; and mixing solution A and solution B to obtain the composition for treating eye conditions.

[0037] Another aspect of the present invention provides a method for preparing a composition for treating eye conditions, comprising:

[0038] Add water for injection to the weighed prescription amount of pharmaceutical excipients and mix well to obtain solution A;

[0039] Add the prescribed amount of human keratinocyte cytokine (hKGF-2) protein stock solution to the weighed water for injection, mix well to obtain solution B;

[0040] Mix solution A and solution B thoroughly; and

[0041] Sterilization, among which,

[0042] Pharmaceutical excipients include glycerin, mannitol, lysine hydrochloride, trehalose, poloxamer 188, citric acid, and sodium citrate.

[0043] According to a preferred embodiment, the mixing method is stirring, wherein the stirring speed is 60-90 r / min.

[0044] According to a preferred embodiment, the mixing method is stirring, wherein the stirring speed is 60-80 r / min.

[0045] According to a preferred embodiment, the mixing method is stirring, wherein the stirring speed is 80-90 r / min.

[0046] According to a preferred embodiment, the mixing time of liquid A is 10 to 30 minutes.

[0047] According to a preferred embodiment, the mixing time of liquid A and liquid B is 10 to 45 minutes.

[0048] The beneficial effects of this invention are as follows: This invention first provides a composition for treating eye diseases, comprising hKGF-2, a protective agent, a surfactant, a pH adjuster, and a solvent. This invention investigates the compatibility of hKGF-2 stock solution with excipients, obtaining optimal concentration ranges for the protective agent, surfactant, and pH adjuster in the composition. Based on the obtained optimal concentration ranges for each pharmaceutical excipient, the melting (denaturation) temperature and aggregation temperature of hKGF-2 protein in the solution system are further investigated, determining an optimal formulation. The hKGF-2 protein activity in the obtained formulation remains stable for a long period.

[0049] The optimal concentration ranges of the protective agent, surfactant, and pH adjuster in the composition were obtained through investigation. Based on the optimal concentration ranges of each pharmaceutical excipient, the melting (denaturation) temperature and aggregation temperature of hKGF-2 protein in the solution system were further investigated to determine the optimal formulation. The hKGF-2 protein activity in the obtained formulation can remain stable for a long time.

[0050] On the other hand, this invention utilizes hKGF-2 eye drops to conduct Schirmer I tear film test, tear film breakup time (BUT) test, corneal fluorescein staining test, and pathological tissue investigation on a benzalkonium chloride (BAC)-induced rabbit dry eye model. The results show that the low-concentration, medium-concentration, and high-concentration groups of hKGF-2 eye drops all have good therapeutic effects on corneal damage and conjunctival pathological damage. Moreover, compared with the commercially available Bepanthen eye drops and sodium hyaluronate eye drops, the hKGF-2 eye drops provided by this invention have a better relieving effect on dry eye symptoms. Attached Figure Description

[0051] Figure 1 This is a summary of the activity values ​​of the prescription at different time points;

[0052] Figure 2 This is a summary of the peak areas of the prescription at different time points;

[0053] Figure 3 For different levels of T for different excipients m Distribution results of (melting temperature);

[0054] Figure 4For different levels of T for different excipients onset Distribution results of (denaturation initiation temperature);

[0055] Figure 5 For different levels of T for different excipients agg 266 and T agg Distribution results of 473 (protein aggregation temperature);

[0056] Figure 6 This is the pathological result of the effect of human keratinocyte growth factor-2 (hKGF-2) on the cornea. Detailed Implementation

[0057] The following is a detailed description with reference to the accompanying drawings. The embodiments described below are merely examples of the present invention, and the scope of the invention is not limited thereto. In this invention, human keratinocyte growth factor-2 eye drops are also referred to as hKGF-2 eye drops. In this invention, w / v represents mass concentration, and the concentration levels in the following embodiments refer to mass concentration. Lysine hydrochloride can also be represented as L-lysine hydrochloride. Lysine hydrochloride and lysine used in hKGF-2 eye drops can achieve corresponding technical effects to varying degrees.

[0058] The prescription quantity is the mass of the excipient obtained by multiplying the preparation volume by the proportion of the excipient in the prescription.

[0059] In this invention, the low-concentration hKGF-2 group (low-dose hKGF-2 group) refers to the treatment of test animals with hKGF-2 eye drops at a concentration of 50 μg / ml; the medium-concentration hKGF-2 group (medium-dose hKGF-2 group) refers to the treatment of test animals with hKGF-2 eye drops at a concentration of 100 μg / ml; and the high-concentration hKGF-2 group (high-dose hKGF-2 group) refers to the treatment of test animals with hKGF-2 eye drops at a concentration of 200 μg / ml.

[0060] Clinical use and dosage of human keratinocyte growth factor-2 eye drops: Instill 1-2 drops into each eye 4-6 times daily. In this application, the hKGF-2 eye drops can be packaged in an eye drop bottle; specifically, the hKGF-2 eye drops can be a single-use eye drop product, free of preservatives.

[0061] Example 1

[0062] This embodiment provides a composition for treating eye conditions, specifically an eye drop for treating dry eye syndrome.

[0063] Compositions used to treat eye conditions include hKGF-2, a protective agent, a surfactant, a pH adjuster, and a solvent.

[0064] Table 1. Ingredients of hKGF-2 Eye Drops

[0065] Element Dosage proportion Adding too much effect hKGF-2 80μg trace amounts none Main drug Mannitol 2mg 0.5％ none Protectant glycerin 6mg 1.5％ none Protectant Lysine hydrochloride 0.4mg 0.1％ none Protectant Trehalose 8mg 2％ none Protectant Polosham 188 0.2mg 0.05％ none surfactants Citric acid 0.05388mg 0.1347‰ none pH adjuster Sodium citrate 0.962mg 2.405‰ none pH adjuster Water for Injection 382.3841mg 95.596％ none solvent

[0066] Example 2

[0067] This embodiment provides an investigational experiment on the compatibility of hKGF-2 stock solution with excipients.

[0068] Specifically, glycerol, trehalose, mannitol, lysine hydrochloride, poloxamer 188, human serum albumin, and sodium heparin were selected as protectants for human keratinocyte growth factor eye drops (hKGF-2 stock solution). A citric acid buffer system (10 mM pH 6.5) was prepared with a protein concentration of 0.2 mg / ml. Single-factor screening experiments were designed with different excipient concentration levels to investigate the compatibility of each excipient with the stock solution.

[0069] The biological activity of hKGF-2 protein was tested after being placed at 37±2℃ for 7 days. The original solution with the same protein concentration under the same conditions was used as a control. The change rate of hKGF-2 protein activity was analyzed, and the results are shown in Table 2.

[0070] Table 2. Screening results of each excipient concentration and activity test.

[0071]

[0072] Note: The percentage in parentheses is the activity change rate (%) = (Dn - D0) / D0 * 100%

[0073] The results showed that mannitol, glycerol, trehalose, lysine hydrochloride and poloxamer 188 had different degrees of protective effect on protein activity, indicating that the above excipients were compatible with hKGF-2 and could be used as excipients for hKGF-2 eye drops.

[0074] Example 3

[0075] This embodiment provides the process for selecting the pH and buffer system of hKGF-2 eye drops.

[0076] Tears are a clear, slightly milky-white, watery fluid secreted by the lacrimal glands and goblet cells of the conjunctiva. The main organic components of tears are proteins, including albumin, globulins, lysozyme, and immunoglobulins IgA, IgG, and IgE.

[0077] The normal pH of human tears is between 6.4 and 7.7. Excessive acidity (pH below 4.8) can coagulate proteins in the ocular mucosa, while excessive alkalinity (pH above 9) can cause the epithelial cells of the ocular mucosa to swell. Therefore, both excessive acidity and alkalinity can cause significant irritation to the eyes. Strong irritation can increase tear secretion, leading to drug loss and failing to achieve the therapeutic purpose, and may even damage the cornea. Generally, the human eye can tolerate eye drops with a pH of 4.8 to 9.0. Considering the pH range that the human eye can tolerate, hKGF-2 eye drops with a pH range of 6.0 to 7.0 and a buffer system of 10mM citric acid (pH 6.6) were selected.

[0078] Example 4

[0079] This embodiment provides the process for selecting surfactants for hKGF-2 eye drops.

[0080] Surfactants (anionic and nonionic) can increase protein stability as additives. On one hand, surfactants compete with proteins for the gas-liquid interface, thus reducing protein exposure; on the other hand, surfactants can reduce non-specific adsorption of proteins onto surfaces such as pipe walls, bottle walls, and stoppers; furthermore, surfactants can increase protein solubility, thus increasing protein stability after their addition. Anionic surfactants like sodium dodecyl sulfate can affect protein denaturation and the deamidation reaction of food proteins. Nonionic surfactants such as Tween and polyethers can prevent protein adsorption on surfaces, inhibiting aggregation and precipitation, and preventing denaturation. Protein drugs, such as α-2b interferon, G-CSF, and tissue plasminogen activator, all incorporate small amounts of nonionic surfactants, such as Tween 80, to inhibit protein aggregation. The mechanism may be that surfactants tend to align at the gas-liquid interface, causing proteins to leave the interface and inhibiting denaturation. Poloxamer 188, a polyoxyethylene-polyoxypropylene ether block copolymer, is a novel type of high-molecular-weight nonionic surfactant. Poloxamer 188 is readily soluble in water and ethanol, soluble in anhydrous ethanol, ethyl acetate, and chloroform, and practically insoluble in diethyl ether or petroleum ether. It exhibits some foaming properties, and a 2.5% aqueous solution has a pH between 5.0 and 7.5. The pH of the water for injection is between 6.0 and 7.0. The aqueous solution is relatively stable in air, but its pH decreases upon exposure to light. Poloxamer 188 is stable to acidic and alkaline solutions and metal ions.

[0081] In this embodiment, poloxamer 188 was selected as the surfactant for hKGF-2 eye drops.

[0082] Example 5

[0083] This embodiment provides the process for selecting the protective agent for hKGF-2 eye drops.

[0084] In this embodiment, amino acids and sugars are selected as protective agents to prevent the hKGF-2 protein from experiencing decreased activity, degradation, and polymerization during storage.

[0085] Amino acids are commonly used protein protectants, preventing protein aggregation, degradation, and oxidation in solution. In this embodiment, lysine hydrochloride is selected as the protectant for hKGF-2 eye drops.

[0086] Sugars and polyols are non-specific protein stabilizers, and are among the most common and widely used protective agents. Sucrose, trehalose, glycerol, mannitol, and sorbitol (concentrations 1%–10%) are the most frequently used. The stabilizing effect of sugars and polyols is closely related to their concentration; the degree of stability of different sugars and polyols depends on the type of protein. Reducing sugars interact with amino acids and should therefore be avoided. Polyols such as glycerol and sugars stabilize proteins by selectively solubilizing them to prevent denaturation. At low concentrations, these additives allow more water molecules to encapsulate the protein, repelling more hydrophobic additives and thus increasing protein stability. At higher concentrations, they do not increase protein stability, and more hydrophobic organic solvents begin to denature the protein. Among various sugars, sucrose and trehalose are used most frequently. Trehalose, as a very stable disaccharide, is widely used in the preservation of vaccines, antisera, transfer factors, and bacterial vaccines. Trehalose possesses advantages such as non-reducing properties, a higher glass transition temperature, and lower hygroscopicity, effectively preventing protein structures from being damaged under extremely harsh conditions such as high cold, high temperature, high osmotic pressure, and dehydration. Simultaneously, exogenous trehalose also provides excellent non-specific protection for biological macromolecules and organisms.

[0087] In this embodiment, mannitol, glycerin, and trehalose were selected as protective agents for hKGF-2 eye drops.

[0088] Example 6

[0089] This embodiment provides the process of exploring the dosage of excipients.

[0090] 1. In this embodiment, eight pharmaceutical excipients, including mannitol, glycerol, trehalose, methionine, lysine hydrochloride, EDTA-2Na, poloxamer 188, and heparin sodium, were selected for screening tests at different concentration levels to investigate the protective effect of each excipient on 0.2 mg / ml (200 μg / ml) hKGF-2. The specific results are shown in Table 3.

[0091] Table 3 Single-factor experimental design table

[0092]

[0093] Table 3 shows the concentration levels of mannitol (0.1%, 0.5%, 1%, 2%), glycerol (0.1%, 0.5%, 1%, 2%), trehalose (0.1%, 0.5%, 1%, 2%), methionine (0.1%, 0.5%, 1%, 2%), lysine hydrochloride (0.1%, 0.5%, 1%, 2%), EDTA-2Na (0.05%, 0.1%, 0.5%, 1%), poloxamer 188 (0.01%, 0.1%, 0.3%, 0.5%), and heparin sodium (1 μg / ml, 10 μg / ml, 100 μg / ml, 500 μg / ml).

[0094] Table 4. Analysis of the Results of the Single-Factor Screening Experiment

[0095]

[0096]

[0097]

[0098] Note: The percentage in parentheses is the activity change rate (%) = (Dn-D0) / D0*100%. D0 refers to day 0; D7 refers to day 7; D14 refers to day 14.

[0099] The results of comparing the activity change rate and electrophoretic purity of each experimental group (mannitol, glycerol, trehalose, methionine, lysine hydrochloride, EDTA-2Na, poloxamer 188, and heparin sodium) with the control group (stock solution) at the same time point showed that: methionine, EDTA-2Na, and heparin sodium had no significant protective effect on hKGF-2 protein; mannitol, trehalose, poloxamer 188, and lysine hydrochloride had certain protective effects on hKGF-2 protein activity; mannitol, glycerol, and trehalose had significant and varying degrees of protective effects on hKGF-2 protein purity, with the following concentration ranges: mannitol concentration range preferably 0.5% or 1%–2%, glycerol concentration range preferably 0.5%–2%, trehalose concentration range preferably 0.5%–2%, lysine hydrochloride concentration range preferably 0.5%–1%, and poloxamer 188 concentration range preferably 0.01%–0.1%.

[0100] 2. Based on the results of the single-factor screening experiment and the principle of orthogonal experiment, mannitol, glycerol, trehalose, lysine hydrochloride and poloxamer 188 were selected as five influencing factors. The protein activity value was used as the analytical value. A five-factor, four-level orthogonal experiment was designed. The buffer system was a citric acid system (10mM pH 6.5) and the protein concentration was 0.2mg / ml.

[0101] Table 5. Design table for different concentration levels of protective agents.

[0102]

[0103] In Table 5, the concentration levels of mannitol are 1%, 1.3%, 1.6%, and 2%; the concentration levels of glycerol are 0.5%, 1%, 1.5%, and 2%; the concentration levels of trehalose are 0.5%, 1%, 1.5%, and 2%; the concentration levels of lysine hydrochloride are 0.5%, 0.6%, 0.8%, and 1%; and the concentration levels of poloxamer 188 are 0.01%, 0.05%, 0.08%, and 0.1%.

[0104] Table 6 Orthogonal Experimental Design Table

[0105]

[0106]

[0107] Note: In Table 6, 1 corresponds to level 1 in Table 5; 2 corresponds to level 2 in Table 5; 3 corresponds to level 3 in Table 5; and 4 corresponds to level 4 in Table 5.

[0108] Based on the activity change rate and the results of range-variance analysis, three optimal formulations were obtained in this embodiment. Specifically, the optimal formulations are: 1.6% mannitol, 1.5% glycerol, 2% trehalose, 0.5% lysine hydrochloride, and 0.05% poloxamer 188; 1.6% mannitol, 1% glycerol, 2% trehalose, 0.5% lysine hydrochloride, and 0.01% poloxamer 188; and 1.6% mannitol, 0.5% glycerol, 2% trehalose, 0.5% lysine hydrochloride, and 0.01% poloxamer 188.

[0109] The three formulations obtained so far have high osmotic pressure, and 0.5% mannitol has a good protective effect on protein activity. Therefore, the three formulations are adjusted as follows: ① 0.5% mannitol, 1% glycerol, 2% trehalose, 0.5% lysine hydrochloride, 0.01% poloxamer 188; ② 0.5% mannitol, 0.5% glycerol, 2% trehalose, 0.5% lysine hydrochloride, 0.01% poloxamer 188; ③ 0.5% mannitol, 1.5% glycerol, 2% trehalose, 0.5% lysine hydrochloride, 0.05% poloxamer 188.

[0110] Example 7

[0111] This embodiment combines univariate and multivariate analysis to screen the optimal prescription for eye drops through a more comprehensive study. See Tables 7 and 8 for the specific group design.

[0112] 1. This embodiment uses the non-chain UNcle multifunctional protein stability analysis system and UNcle Analysis Software (Version 5.04) to study the stability of proteins in different groups. The fluorescence and SLS (Static Light Scattering) modules of Unclear were used to detect the melting (denaturation) temperature and aggregation temperature of hKGF-2 protein in solution to obtain the conformational and colloidal stability of the protein. The DLS (Dynamic Laser Scattering) module of Unclear was used to detect the hydrated particle size and polydispersity of the protein in solution at room temperature or a specified temperature to obtain the particle size and dispersion of the sample. The DLS detection module monitored the changes in the average hydration kinetic diameter and scattered light intensity of the protein under thermal stress in real time. The monitoring results can reflect the protein denaturation / aggregation status, and therefore can be used as an orthogonal validation method for fluorescence detection of protein denaturation. Specific results are shown in Table 9.

[0113] Specifically, the test samples were lightly mixed at room temperature and centrifuged. 9 μl of sample was then pipetted into each well, with two replicates for each sample. Two Uncle detection modules were used in this study: full-spectrum fluorescence and static light scattering (SLS), corresponding to the melting (denaturation) temperature (T). m ) and aggregation temperature (T) agg ).

[0114] The following are different groups for protein stability analysis. In this embodiment, the melting (denaturation) temperature and aggregation temperature of the protein in the solution system were measured. This embodiment, while validating the original formulation, introduced 0.5% human serum albumin to replace trehalose, which improved the formulation's protective effect on protein activity while reducing costs. Furthermore, this embodiment introduced 0.01%–0.05% sodium hyaluronate to replace mannitol. The buffer system was a citric acid system (10 mM pH 6.5), and the hKGF-2 protein concentration was 0.2 mg / ml.

[0115] 2. Data Acquisition and Processing Methods

[0116] Temperature program settings: initial temperature 25℃, incubation time 180s, heating rate 0.4℃ / min, final temperature 95℃, heating rate 0.6℃ / min, waiting time 65 seconds. The full-spectrum fluorescence acquisition range is 250-720nm. This experiment uses the BCM (Barycentric Mean) method for data analysis. The static light scattering SLS module is divided into SLS266 and SLS473, corresponding to scattered light wavelengths of 266nm and 473nm, respectively. SLS266 has high detection sensitivity and is suitable for low-concentration proteins, mainly reflecting the formation of small aggregates; SLS 473 has a wider detection range and is suitable for high-concentration proteins, mainly reflecting the formation of large aggregates. The sample in this embodiment is prone to aggregation, and the signal response values ​​in both wavelength bands are valuable for reference.

[0117] 3. Determine the optimal formulation combination based on the analysis results of protein stability, as detailed in Table 9.

[0118] Table 7. Single-factor analysis design table for excipients at different concentration levels.

[0119]

[0120]

[0121] Table 7 includes 22 groups. The concentration levels of sodium hyaluronate are 0%, 0.01%, and 0.05%; the concentration levels of glycerol are 0.1%, 0.5%, and 2%; the concentration levels of trehalose are 0.1%, 0.5%, and 2%; the concentration levels of lysine hydrochloride are 0.1%, 0.5%, and 2%; the concentration levels of poloxamer 188 are 0.05% and 0.1%; the concentration levels of sodium heparin are 10 μg / ml and 100 μg / ml; the concentration levels of human serum albumin are 0.01%, 0.1%, and 0.5%; and the concentration levels of mannitol are 0.1%, 0.5%, and 1%.

[0122] Table 8. Multifactor analysis design for different prescriptions

[0123] Group Mannitol glycerin Trehalose Lysine Polosham 188 Human serum albumin 23 0.5％ 1％ 0 0.5％ 0.01％ 0.5％ 24 0.5％ 0.5％ 0 0.5％ 0.01％ 0.5％ 25 0.5％ 1.5％ 0 0.5％ 0.05％ 0.5％ 26 0.5％ 1％ 2％ 0.5％ 0.01％ 0 27 0.5％ 0.5％ 2％ 0.5％ 0.01％ 0 28 0.5％ 1.5％ 2％ 0.5％ 0.05％ 0 29 0.05％ 1％ 0 0.5％ 0.01％ 0.5％ 30 0.05％ 1％ 2％ 0.5％ 0.01％ 0 31 0.01％ 1.5％ 0 0.5％ 0.05％ 0.5％ 32 0.01％ 1.5％ 2％ 0.5％ 0.05％ 0

[0124] Note: The groups in Table 8 are numbered sequentially according to Table 7, with numbers 23 to 32 corresponding to prescriptions 1 to 10, respectively.

[0125] Table 9 Summary of Experimental Results

[0126]

[0127]

[0128]

[0129] The results are shown in Table 9 and... Figures 3-5 As shown, where, Figure 3 For different levels of excipients T m The melting temperature distribution results are shown, with the horizontal axis representing different levels of excipients and formulations, and the vertical axis representing melting temperature. The higher the melting temperature of the protein, the more stable the protein. (Based on Table 9 and...) Figure 3 As shown, high concentrations of sodium heparin, sodium hyaluronate, glycerol, and mannitol help improve the thermal stability of hKGF-2 protein.

[0130] Figure 4 For different levels of T for different excipients onset The distribution results of (denaturation initiation temperature) are shown on the horizontal axis, which represents different levels of excipients and formulations, and on the vertical axis, which represents the denaturation initiation temperature. Figure 5 For different levels of T for different excipients agg 266 and T agg 473 (protein aggregation temperature) distribution results, the horizontal axis represents different levels of excipients and formulations, and the vertical axis represents protein aggregation temperature, where T agg 266 refers to T at 266nm. agg T agg 473 refers to the T at 473nm. agg The higher the protein's aggregation temperature, the more stable the protein.

[0131] according to Figure 4 and Figure 5 The results showed that high concentrations of heparin sodium, glycerol, trehalose, poloxamer 188, and low concentrations of human serum albumin helped improve the thermal stability of hKGF-2 protein.

[0132] The results showed that the solution systems of formulations 1–10 exhibited high stability. At T agg At 473, lysine hydrochloride can inhibit protein aggregation, but the effect decreases significantly with increasing concentration; 0.1% lysine hydrochloride is preferred. In addition, while validating the original formulation, 0.5% human serum albumin was introduced to replace trehalose, aiming to improve the formulation's protective effect on protein activity while reducing costs.

[0133] Example 8

[0134] This embodiment provides the process of exploring the formulation stability verification test, specifically the process of exploring the conformational stability and colloidal stability of hKGF-2 protein in solution system.

[0135] In this embodiment, the buffer system was configured as a citric acid system (10 mM pH 6.5), and the hKGF-2 protein concentration was 0.2 mg / ml. Based on the results of Example 7, Formulas 3, 6, and 9 (Table 10) were selected from Formulas 1 to 10 to further investigate the protective effects of different formulas on the protein. That is, this embodiment is an optimization study of the formulas provided in Example 7.

[0136] Uncle's fluorescence and SLS (Static Light Scattering) modules were used to detect the melting (denaturation) temperature and aggregation temperature of proteins in solution systems. This embodiment found that lysine hydrochloride can inhibit protein aggregation in Tagg473, with the effect decreasing significantly with increasing concentration; 0.1% lysine hydrochloride is preferred. Furthermore, while validating the original formulation, 0.5% human serum albumin was introduced to replace trehalose, aiming to improve the protective effect of the formulation on protein activity while reducing costs. In this embodiment, the concentration of lysine hydrochloride in the formulation was reduced to 0.1%.

[0137] For detailed test data, please refer to... Figures 1-2 And Table 11.

[0138] Table 10 Prescription Composition Table

[0139] Group Mannitol glycerin Trehalose Lysine hydrochloride Polosham 188 Human serum albumin Prescription 3 0.5％ 1.5％ 0 0.5％ 0.05％ 0.5％ Prescription 6 0.5％ 1.5％ 2％ 0.5％ 0.05％ 0 Group Sodium hyaluronate glycerin Trehalose Lysine hydrochloride Polosham 188 Human serum albumin Prescription 9 0.01％ 1.5％ 0 0.5％ 0.05％ 0.5％

[0140] Table 11 Summary of Peak Area and Change Rate of Each Prescription at Different Time Points

[0141]

[0142] Note: Prescription 3 is in Figure 1 and Figure 2 The Chinese version is represented as XZ20230504-01; prescription 6 is in Figure 1 and Figure 2 The Chinese version is represented as XZ20230504-02; Prescription 9 is... Figure 1 and Figure 2 The Chinese version is represented as XZ20230504-03.

[0143] Figure 1 The results showed that formulation 6 exhibited the lowest rate of activity change within the 0–4.5 month period; Table 11, Figure 2 The results showed that formulation 6 exhibited the highest biological activity and the smallest rate of activity change within 6.5 months. Formulation 6 also had the largest peak area and the smallest rate of peak area change, indicating that the hKGF-2 protein activity of this formulation remained stable over a long period. Overall, formulation 6 performed well and can be observed for up to 9 months.

[0144] Prescription 6 (0.5% mannitol, 1.5% glycerol, 2% trehalose, 0.5% lysine hydrochloride, 0.05% poloxamer 188) is feasible and will be used for subsequent prescription optimization research.

[0145] Example 9

[0146] This embodiment provides the process of exploring the rationality of the selection of excipients in the prescription.

[0147] The excipients of hKGF-2 eye drops are: mannitol, glycerin, trehalose, poloxamer 188, lysine hydrochloride, citric acid, and sodium citrate. The rationality analysis of the selection of excipients for hKGF-2 eye drops is shown in Table 12.

[0148] Table 12 Analysis of the rationality of eye drop excipient selection

[0149]

[0150]

[0151] Based on the results of the single-factor screening experiment (Table 4), the preferred concentration ranges for mannitol were determined to be 0.5% or 1%–2%, for glycerol 0.5%–2%, for trehalose 0.5%–2%, for lysine hydrochloride 0.5%–1%, and for poloxamer 188 0.01%–0.1%.

[0152] Three formulations with good protective effects were selected based on orthogonal experiments (Tables 5 and 6). Formulation optimization and stability tests (Tables 7–11) were then conducted. Figure 1 and Figure 2 The final prescription included 0.5% mannitol, 1.5% glycerol, 0.1% lysine hydrochloride, 2% trehalose, and 0.05% poloxamer 188.

[0153] According to a preferred embodiment, the final formulation is determined to be: 0.5% mannitol, 1.5% glycerol, 0.1% lysine hydrochloride, 2% trehalose, 0.05% poloxamer 188, 0.1347‰ citric acid, and 2.405‰ sodium citrate.

[0154] Example 10

[0155] This embodiment provides the process of investigating the solvent volume required for the dissolution of each excipient. When water is used as the solvent, the solubility of mannitol is 18.9 g / 100 g (20°C); the solubility of lysine hydrochloride is 65 g / 100 g (20°C); the solubility of trehalose is 68.9 g / 100 g (25°C); poloxamer 188 is readily soluble in water; glycerol is miscible with water in any proportion; the solubility of citric acid is 59 g / 100 g (20°C); and the solubility of sodium citrate is 71 g / 100 g (25°C).

[0156] 1. Materials and Methods

[0157] (1) Weigh out the prescribed amounts of glycerin, mannitol, lysine hydrochloride, trehalose, poloxamer 188, citric acid, sodium citrate and other pharmaceutical excipients, add 100 ml of water for injection at 15-25℃, control the stirring speed at 60-80 r / min, observe the dissolution every 5 minutes, add water for injection as needed until the excipients are completely dissolved to obtain solution A, and record the stirring time and the total volume of water for injection added.

[0158] (2) Measure the remaining amount of water for injection at 15-25°C, mix well, and add the prescribed amount of protein stock solution to obtain solution B. Record the volume of water for injection and examine the appearance of the solution.

[0159] Table 13 Results of Solution Appearance Inspection

[0160] Control Items Liquid A Liquid B Water for injection (ml) 100 Approximately 80 Stirring speed (r / min) 60～80 / Stirring time (min) 20 / Appearance colorless clear liquid colorless clear liquid

[0161] 2. Results and Analysis

[0162] Table 13 shows the preparation details of each solution. As shown in Table 13, measuring 100 ml of water for injection, stirring at a speed of 60–80 r / min for at least 20 min, will completely dissolve all excipients in solution A, resulting in a colorless and clear liquid. Adding 80 ml of water for injection to the protein stock solution will produce a colorless and clear liquid in solution B.

[0163] Example 11

[0164] This embodiment provides the process of investigating the stirring time of liquid A and liquid B.

[0165] Since both liquid A and liquid B are solutions, and the volume ratio of liquid A to liquid B is approximately 1:1, a low-speed stirring method was chosen to mix them. Based on the existing equipment parameters, a stirring speed of 60–80 r / min was selected to investigate the suitable stirring time after mixing liquid A and liquid B.

[0166] 1. Materials and Methods

[0167] Add solution B to solution A and stir at a speed of 60-80 r / min. Measure the appearance and pH of the solution every 5 minutes and take 4 samples from different locations to measure their osmotic pressure at 3 time points.

[0168] 2. Results and Analysis

[0169] After mixing solutions A and B and stirring at 60–80 r / min for 5 min, the CV value of the osmotic pressure measured at each location was 0.57%; after stirring for 10 min, the CV value was 0.50%; and after stirring for 15 min, the CV value was 0.43%. The CV values ​​of the osmotic pressure measured at each location were all less than 1%. After stirring for 5 min, the solution pH was 6.48; after stirring for 10 min, the solution pH was 6.51; and after stirring for 15 min, the solution pH was 6.52. The pH fluctuation was very small after 5 min, and the solution remained stable. The results in Tables 14 and 15 demonstrate that the solution was uniformly distributed after stirring for 5 min.

[0170] Table 14 Mixing of Liquid A and Liquid B

[0171] Stirring time 0min 5min 10min 15min pH 6.28 6.48 6.51 6.52

[0172] Table 15 Osmotic pressure values ​​of samples after mixing solutions A and B for different stirring periods.

[0173]

[0174] Example 12

[0175] This embodiment provides the process of investigating the filtration flow rate when hKGF-2 stock solution is dissolved in other excipients and sterilized by filtration.

[0176] 1. Materials and Methods

[0177] This embodiment uses a Milink filtration membrane, material and specifications: PES membrane, size [missing information]. The pore size is 0.22μm. Specifically, the semi-finished product is filtered and sterilized using two 0.22μm filters connected in series, with a filtration rate not exceeding 0.1L / min, and the filtration results are observed.

[0178] 2. Results and Analysis

[0179] The filtration rate can meet the process requirements when it is controlled below 0.1 L / min. In order to control the process production time and risks, the filtration rate for the pilot test is determined to be 0.05 to 0.1 L / min.

[0180] Example 13

[0181] This embodiment provides a process for investigating the stability of a small-scale process.

[0182] Based on the process parameters obtained from the pilot-scale process, three consecutive batches of pilot production were conducted. The results of the control items for each process remained basically consistent, proving that the pilot-scale process is stable. Specific results are shown in Table 16.

[0183] Table 16 Results of three consecutive batches of small-scale tests

[0184]

[0185] Production was carried out according to the formulation table in Table 17, based on the parameters of each process determined by the pilot-scale process.

[0186] 1. Solution preparation

[0187] Solution A: Weigh the prescribed amounts of pharmaceutical excipients such as glycerin, citric acid, and sodium citrate, add 60% water for injection at 15-25℃, stir at 60-80 r / min for 30 min to dissolve them.

[0188] Solution B: Measure 30% water for injection at 15-25℃ and add the prescribed amount of protein stock solution.

[0189] Add solution B to solution A, rinse the container of solution B with 10% water for injection at 15-25°C, and stir at a speed of 60-80 r / min for 10 min.

[0190] 2. Sterilization

[0191] The prepared solution was filtered through two 0.22μm filter membranes for sterilization, with the filtration rate controlled at 0.05–0.1 L / min, to obtain a semi-finished solution.

[0192] 3. Filling

[0193] The packaging material is a low-density polyethylene eye drop bottle, and filling starts at 1ml / bottle.

[0194] 4. Affix labels and store.

[0195] Each item is labeled, stored in the warehouse at 2–8℃.

[0196] Table 17 Material Configuration Table for Small-Scale Trial Batch

[0197]

[0198] Example 14

[0199] This embodiment involves a pilot-scale (10 kg) scale-up of the small-scale process formulation to investigate suitable parameters for the solution preparation, sterilization, semi-finished product preparation, and filling processes in the corresponding facilities.

[0200] 1. Stability of solution preparation process

[0201] (1) Materials and Methods

[0202] ① Weigh out the prescribed amounts of pharmaceutical excipients such as glycerin, mannitol, lysine hydrochloride, trehalose, poloxamer 188, citric acid, and sodium citrate, add 6 kg of water for injection at 20-25℃, and stir at a speed of 60-80 r / min for 30 min.

[0203] ② Measure 6 kg of water for injection at 20-25℃, add the prescribed amount of hKGF-2 protein stock solution, mix well to obtain solution B, and test the appearance of the solution.

[0204] (2) Results and Analysis

[0205] After scaling up the solution preparation process, the two solutions obtained were consistent with the small-scale test results, namely, both solution A and solution B were colorless and clear liquids, proving that the scale-up of this process is feasible. The specific results are shown in Table 18.

[0206] Table 18 Appearance of each solution

[0207]

[0208]

[0209] 2. Sterilization

[0210] Considering the need to increase the volume of filtrate, a capsule filter was selected as the filtration device, and the filter media was the same as the PES membrane. The filtration flow rate was set to no more than 1 L / min, referring to the filter membrane instruction manual.

[0211] (1) Materials and Methods

[0212] Filter manufacturer: Pall filter membrane; material and specifications: PES; size: 5 inches; pore size: 0.22μm.

[0213] The semi-finished product was filtered and sterilized using two 0.22μm filters connected in series at a filtration rate of no more than 1L / min, and the filtration results were observed.

[0214] (2) Results and Analysis

[0215] The filtration rate can meet the process requirements when it is controlled below 1L / min. In order to control the process production time and risks, the filtration rate of the pilot test is determined to be 0.5 to 1L / min.

[0216] 3. Semi-finished product preparation process

[0217] (1) Materials and Methods

[0218] ① Add solution B to solution A and stir at a speed of 60-80 r / min. Take samples from 4 different locations every 5 minutes to measure osmotic pressure and pH, for a total of 4 time points.

[0219] (3) Results and Analysis

[0220] After mixing solutions A and B, the mixture was clear and transparent, and remained so over time. The mixing speed was 80–90 r / min. After 5 minutes of stirring, the pH value was 6.41; after 10 minutes, it was 6.43; after 15 minutes, it was 6.42; and after 20 minutes, it was 6.44, indicating that the pH value stabilized after 5 minutes of stirring. The CV (volume shift) of the osmotic pressure measured at various locations was 0.86% after 5 minutes of stirring, 0.92% after 10 minutes, 0.41% after 15 minutes, and 0.31% after 20 minutes. Based on these results, the CV of the osmotic pressure measured at various locations was less than 1%, proving that the solution was uniformly distributed and remained stable after 5 minutes of stirring. See Tables 19 and 20 for specific results.

[0221] Table 19 pH values ​​of semi-finished products at different time points

[0222] Stirring time 0 o'clock 5min 10min 15min 20min Appearance Clear and transparent Clear and transparent Clear and transparent Clear and transparent Clear and transparent pH 6.16 6.41 6.43 6.42 6.44

[0223] Table 20 Osmotic pressure of semi-finished products at different time points

[0224]

[0225] 4. Filling process

[0226] (1) Materials and Methods

[0227] The discharge port of the mixing tank is connected to a hose, which is connected to a BT300S peristaltic pump. The peristaltic pump is set to the dispensing mode, and the running speed and running time are adjusted to adjust the filling amount to 10-10.5g. Ten samples are prepared for each combination of process parameters. The minimum, maximum and average values ​​are analyzed to ensure that the process parameters that meet the specified filling amount can be produced stably.

[0228] (2) Results and Analysis

[0229] The peristaltic pump is set to distribution mode, with an operating speed of 76.8 r / min and an operating time of 2 s. The single-piece filling amount can be controlled between 9.9 and 10.1 g, with an average filling amount of 10.06 g. It can stably produce filling amounts that meet the specified requirements. For details, please refer to Table 21.

[0230] Table 21 Parameter Settings and Filling Status

[0231]

[0232] 5. Pilot-scale process validation

[0233] Based on the process parameters obtained from the pilot-scale process exploration, three consecutive batches of pilot-scale production were carried out. The results of the control items for each process were basically consistent with the results of the small-scale process in Example 13, proving that this pilot-scale process is stable.

[0234] 6. Refer to the pilot batch material preparation table for each production process, as detailed in Table 22.

[0235] Table 22 Material Preparation Table for Pilot Batch

[0236]

[0237]

[0238] (1) Solution preparation

[0239] Solution A: Weigh the prescribed amounts of pharmaceutical excipients such as glycerin, citric acid, and sodium citrate, add 60% water for injection at 15-25℃, stir at 60-80 r / min for 30 min to dissolve them.

[0240] Solution B: Measure 30% water for injection at 15-25℃ and add the prescribed amount of hKGF-2 protein stock solution.

[0241] Add solution B to solution A, rinse the container of solution B with 10% water for injection at 15-25°C, and stir at a speed of 60-80 r / min for 10 min.

[0242] (2) Sterilization

[0243] The prepared solution was filtered through two 0.22μm filter membranes for sterilization, with the filtration rate controlled at 0.5–1 L / min, to obtain a semi-finished solution.

[0244] (3) Filling

[0245] Using a peristaltic pump, set the mode to dispensing mode, the running speed to 76.8 r / min, the running time to 2 s, and after adjusting the filling amount to 10-10.5 g, stable production of filling amounts conforming to the specifications can be achieved, with a filling speed of 15-20 tubes / min.

[0246] (4) Label and store

[0247] Each item is labeled, stored in the warehouse at 2–8℃.

[0248] 7. Changes in production process

[0249] The main change from small-scale pilot production to pilot-scale production involves changes in production equipment. The relevant process parameters are basically the same as those in the small-scale pilot production. Specific changes can be seen in Table 23.

[0250] Table 23 Summary of Production Process Changes

[0251]

[0252] Table 24 lists the main production equipment for hKGF-2 eye drops.

[0253]

[0254]

[0255] Example 15

[0256] This embodiment provides a batch prescription for hKGF-2 eye drops.

[0257] The hKGF-2 eye drop batch formula consists of human keratinocyte growth factor stock solution, mannitol, glycerin, lysine hydrochloride, trehalose, poloxamer 188, citric acid and sodium citrate. There is no over-dosing of ingredients. See Table 25 for details.

[0258] Table 25hKGF-2 Eye Drops Batch Prescription

[0259] Element Dosage Adding too much Implementation Standards hKGF-2 2g no The quality standard for the stock solution is shown in 3.2.S.4.1. Mannitol 50g no Chinese Pharmacopoeia (2020 Edition, Part II) glycerin 150g no Chinese Pharmacopoeia (2020 Edition, Volume IV) Lysine hydrochloride 10g no Chinese Pharmacopoeia (2020 Edition, Part II) Trehalose 200g no Chinese Pharmacopoeia (2020 Edition, Volume IV) Polosham 188 5g no Chinese Pharmacopoeia (2020 Edition, Volume IV) Citric acid 1.347g no Chinese Pharmacopoeia (2020 Edition, Volume IV) Sodium citrate 24.05g no Chinese Pharmacopoeia (2020 Edition, Volume IV) Water for Injection 9559.6003g no Chinese Pharmacopoeia (2020 Edition, Part II) Made into 10kg --- ---

[0260] According to the sterilization method (General Chapter 1421, Chinese Pharmacopoeia 2020 Edition), the stirring speed is 80-90 r / min, the mixing time of solution A and solution B is 45 min, the mixing time of the semi-finished product is 20 min, the flow rate for filtration sterilization is 0.2-0.3 L / min, the foaming point pressure is greater than or equal to 0.34 MPa, and the sterility test of the finished product should be qualified. The single-vial fill weight should not be less than 93% of the labeled fill weight, and the average fill weight should not be less than the labeled fill weight.

[0261] Example 16

[0262] This embodiment is an experimental study on the effect of hKGF-2 eye drops provided in the previous embodiment on a benzalkonium chloride (BAC)-induced rabbit dry eye model. This embodiment establishes a dry eye model to observe the related symptoms of dry eye in the test animals. In this embodiment, the 50 μg / ml low-concentration hKGF-2 group refers to the group treated with hKGF-2 eye drops at a concentration of 50 μg / ml, the 100 μg / ml medium-concentration hKGF-2 group refers to the group treated with hKGF-2 eye drops at a concentration of 100 μg / ml, and the 200 μg / ml high-concentration hKGF-2 group refers to the group treated with hKGF-2 eye drops at a concentration of 200 μg / ml.

[0263] 1. Management of laboratory animals

[0264] The test animals in this embodiment were New Zealand rabbits. The ambient temperature in the animal housing was controlled between 16 and 26°C, with a maximum daily temperature difference of no more than 4°C. The relative humidity was controlled between 40% and 70%, with an air exchange rate of ≥8 times / hour. The system operated with 12 hours of lighting / 12 hours of darkness, alternating between light and dark (adjustable according to experimental needs). The working illuminance was ≥200 Lx, and the animal illuminance was 100–200 Lx. During the formal experiment, the weight of the test animals ranged from 2.0 to 2.5 kg. In daily feeding, hay could be added as a supplementary food for the rabbits as needed (decreased appetite, soft stools, etc.), with an amount of approximately 30–50 g added each time, while simultaneously reducing the corresponding amount of other feed.

[0265] In this embodiment, the negative control was a blank matrix control; the positive controls were Bepanthen eye drops and sodium hyaluronate eye drops.

[0266] 2. Main reagents and preparation methods

[0267] 0.1% benzalkonium chloride solution: Weigh 0.01g of benzalkonium chloride, dissolve it in 10ml of sterile physiological saline in a sterile workbench, and aliquot and store in 1ml sterile centrifuge tubes.

[0268] 1% fluorescein sodium solution: Weigh 0.1g of fluorescein sodium, add it to sterile physiological saline, prepare in the dark, and aliquot and store in 10ml sterile centrifuge tubes in the dark.

[0269] 3. Main Instruments

[0270] Electronic balances: BS224S, BSA2202S, BL3100, GP2100, and TE601-L (Beijing Sartorius Instrument Systems Co., Ltd.); Fully automatic autoclave: CLG-40M (ALP Corporation, Japan); Clean bench: SW-CJ-2FD (Suzhou Purification Equipment Co., Ltd.); Dehydrator: ASP300S (Leica GmbH, Germany); Paraffin embedding machine: EG1160 (Leica GmbH, Germany); Rotary microtome: RM2235 (Leica GmbH, Germany); Slice spreader: HI1210 (Leica GmbH, Germany); Slice oven: HI1220 (Leica GmbH, Germany); Image acquisition heads: OLYMPUS DDP71 and DP73 (OLYMPUS Corporation, Japan); Pathological image analysis software: IPP6.0 (Media Cybernetics, USA); CellSens Standard (OLYMPUS Corporation, Japan); ST5020 / CV5030 fully automatic staining / mounting workstation (OLYMPUS Corporation, Japan).

[0271] 4. Experimental Design

[0272] This embodiment of the experiment was conducted according to Table 26. The experiment included a normal control group, a model control group, a low-concentration hKGF-2 group (50 μg / ml), a medium-concentration hKGF-2 group (100 μg / ml), a high-concentration hKGF-2 group (200 μg / ml), a bepanthen eye drop group, and a sodium hyaluronate eye drop group, for a total of 7 groups. After grouping, the test animals were subjected to benzalkonium chloride eye drops to establish the hKGF-2 model. Starting from day 16 of modeling, the animals were treated with the drug at a fixed time every day for 21 consecutive days.

[0273] 4.1 The specific steps for animal grouping and modeling are as follows:

[0274] After the healthy New Zealand rabbits completed quarantine, fundus examination was performed using a slit lamp. Animals with abnormal fundus findings were removed, and the rabbits were grouped according to the experimental design. 0.1% BAC eye drops were administered at 9:00 AM and 3:00 PM, creating a bilateral model. This was continued for 36 days, with a negative control group receiving physiological saline. From day 16, medication was administered continuously for 21 days, creating a bilateral model according to the dosage design, with medication administered 4 or 5 times daily (with an interval of at least 30 minutes between administration and model creation). A blank matrix control was provided for both the normal control group and the model group. Schirmer I tear film test, tear film breakup time (BUT) test, and corneal fluorescein staining test were performed on days 0, 7, 15, 19, 23, 26, 30, and 37 (the day after the end of medication). On day 38 (the day after the end of medication), conjunctival and corneal tissue were removed for histopathological examination.

[0275] Table 26 Animal grouping experiment procedure

[0276]

[0277] Note: Each drop of eye drops contains approximately 50 μl of medication.

[0278] 4.2 Detection Indicators

[0279] All the following examinations were performed by the same person, with the same examination time, location, lighting, humidity, and temperature each time. Rabbits in each group were anesthetized on days 0, 7, 15, 19, 23, 26, 30, and 37 to perform the Schirmer I test, tear film breakup time test, and corneal fluorescein staining test.

[0280] (1) The Schirmer I experiment was used to collect the tear secretion of rabbits. The specific steps were as follows: Schirmer I experiment was performed before administering eye drops every day. Data was collected at fixed times every day on days 0, 7, 15, 19, 23, 26, 30 and 37 after the establishment of the model. Rabbits were anesthetized by intraperitoneal injection of an appropriate amount of chloral hydrate. Filter paper was placed in the outer third of the conjunctival sac, with the rest hanging on the skin surface. The length of the tear stain was measured and recorded after 5 minutes in mm. The measured length is the tear secretion of the rabbit.

[0281] (2) Specific steps of the tear film breakup time measurement experiment: On days 0, 7, 15, 19, 23, 26, 30 and 37 after model establishment, rabbits were anesthetized by intraperitoneal injection of an appropriate amount of chloral hydrate. After fixing the rabbits, 1% sodium fluorescein was applied to the conjunctival sac of the lower eyelid with a glass rod. After the eyelids were closed, the fluorescein was evenly distributed on the corneal surface. The upper and lower eyelids were fixed to fully expose the cornea. The rabbits were observed under a slit lamp with cobalt blue light. The time of the first breakup point on the tear film was recorded from the last blink. Each rabbit was tested 3 times, and the average value was calculated. The average value was taken as the tear film breakup time of the rabbit on that day.

[0282] (3) Specific steps of the corneal fluorescein staining test: Apply 1% sodium fluorescein to the lower palpebral conjunctival sac with a glass rod. Observe the corneal fluorescence staining after 1 minute and take pictures under a slit microscope. Divide the cornea and conjunctiva into four quadrants: cornea, temporal conjunctiva, inferior bulbar conjunctiva, and superior bulbar conjunctiva. Each quadrant is divided into 4 grades according to the degree and area of ​​staining: 0 points for no staining; 1 point for scattered punctate staining; 2 points for dense punctate staining; 3 points for filamentous, patchy, or blocky staining. The total score is 0-12 points. A total score less than 3 is negative; 3-6 is weakly positive; 6-9 is positive; and 9-12 is strongly positive. The scores of each eye are added together and analyzed. All sodium fluorescein staining scores are scored by a single observer using a blind method.

[0283] (4) Pathological examination of lacrimal gland and cornea

[0284] On day 38, the rabbits were euthanized, and the conjunctiva and cornea were removed, fixed with formalin, and sent for HE staining and pathological observation under a light microscope.

[0285] 5. Statistical Analysis Methods

[0286] All data were entered into GraphPad Prism for statistical analysis. The results are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons between groups.

[0287] 6. Experimental Results

[0288] 6.1 Effect of hKGF-2 on basal tear secretion in rabbits (Schirmer I tear test), the results are shown in Table 27.

[0289] Table 27 Schirmer I results before and after treatment in each group (mm)(x±s)

[0290] Group 0d 7d 15d 19d 23d 26d 30d 37d normal control group 15.92±5.20 16.38±5.25 15.96±5.62 15.67±3.56 16.25±4.18 16.79±3.56 16.67±5.12 17.25±5.30 Model control group 15.71.±3.34 11.63±5.51 10.25±3.66** 9.50±3.38** 9.58±4.50** 9.00±5.10** 8.96±4.10** 9.13±3.93** low concentration of hKGF-2 group 15.46±3.79 11.50±2.59 9.83±2.99** 9.83±3.35 9.88±4.09 9.13±4.85 13.83±4.54 14.38±5.05 hKGF-2 medium concentration group 16.33±4.09 12.04±4.62 10.33±3.18** 10.75±2.97 10.50±4.72 11.63±3.43 14.46±4.35# 15.96±4.20# high concentration of hKGF-2 group 16.54±4.30 12.63±4.06 10.50±3.18** 11.08±2.46 11.21±4.37 12.17±3.11 15.08±3.07# 16.75±4.74## Bepanthen eye drops group 17.38±4.27 11.96±2.84 11.04±2.36* 11.79±2.47 10.75±3.97 12.46±4.14 14.63±5.52# 15.63±6.02# Sodium hyaluronate eye drops group 15.75±3.12 11.50±3.21 11.42±2.60* 11.04±2.48 11.67±4.46 12.04±3.26 13.33±3.51 15.83±5.69#

[0291] Note: ① In Table 27, 0d, 7d, 15d, 19d, 23d, 26d, 30d, and 37d represent day 0, day 7, day 15, day 19, day 23, day 26, day 30, and day 37 of modeling, respectively. The low concentration group of hKGF-2 refers to hKGF-2 with a concentration of 50 μg / ml; the medium concentration group of hKGF-2 refers to hKGF-2 with a concentration of 100 μg / ml; and the high concentration group of hKGF-2 refers to hKGF-2 with a concentration of 200 μg / ml.

[0292] ② Compared with the normal control group, "*" indicates P<0.05, and "**" indicates P<0.01. Compared with the model control group, "#" indicates P<0.05, and "##" indicates P<0.01. Compared with Bepanthen eye drops, "^" indicates P<0.05, and "^^" indicates P<0.01. Compared with sodium hyaluronate eye drops, "&" indicates P<0.05, and "&&" indicates P<0.01.

[0293] The results in Table 2 show:

[0294] (1) On days 15, 19, 23, 26, 30 and 37 after modeling, compared with the blank control group, the basal tear secretion of rabbits in the model control group was reduced, and the result was statistically significant (P<0.01).

[0295] (2) On day 30 (day 15 of treatment) and day 37 (day 21 of treatment), compared with the model control group, the basal tear secretion in the positive control Bepanthen eye drops group was significantly increased, and the results were statistically significant (P<0.05). On day 37 (day 21 of treatment), compared with the model control group, the basal tear secretion in the positive control sodium hyaluronate eye drops group was significantly increased, and the results were statistically significant (P<0.05).

[0296] (3) On day 30 of modeling (day 15 of treatment) and day 37 of modeling (day 21 of treatment), compared with the model control group, the basal tear secretion of the medium concentration group (100 μg / ml) and the high concentration group (200 μg / ml) of hKGF-2 increased significantly, and the statistical results were statistically significant (P<0.01 or P<0.05). The basal tear secretion of the low concentration group (50 μg / ml) of hKGF-2 increased.

[0297] (4) With the extension of treatment time, the basal tear secretion of the low-concentration hKGF-2 group, the medium-concentration hKGF-2 group and the high-concentration hKGF-2 group showed an increasing trend.

[0298] The effects of 6.2hKGF-2 on rabbit tear film stability (BUT) are shown in Table 28.

[0299] Table 28 Results of tear film breakup time (BUT) before and after treatment in each group (s) (x±s)

[0300]

[0301] Note: ① In Table 28, 0d, 7d, 15d, 19d, 23d, 26d, 30d, and 37d represent day 0, day 7, day 15, day 19, day 23, day 26, day 30, and day 37 of modeling, respectively. The low concentration group of hKGF-2 refers to hKGF-2 with a concentration of 50 μg / ml; the medium concentration group of hKGF-2 refers to hKGF-2 with a concentration of 100 μg / ml; and the high concentration group of hKGF-2 refers to hKGF-2 with a concentration of 200 μg / ml.

[0302] ② Compared with the normal control group, "*" indicates P<0.05, and "**" indicates P<0.01. Compared with the model control group, "#" indicates P<0.05, and "##" indicates P<0.01. Compared with Bepanthen eye drops, "^" indicates P<0.05, and "^^" indicates P<0.01. Compared with sodium hyaluronate eye drops, "&" indicates P<0.05, and "&&" indicates P<0.01.

[0303] Table 28 shows the results:

[0304] (1) On days 7, 15, 19, 23, 26, 30 and 37 after modeling, the BUT measurement values ​​of rabbits in the model control group were reduced compared with those in the blank control group, and the results were statistically significant (P<0.01).

[0305] (2) On day 26 (day 11 of treatment), day 30 (day 15 of treatment), and day 37 (after day 21 of treatment), the BUT measurement value of the positive control Bepanthen eye drops group was significantly increased compared with the model control group, and the results were statistically significant (P<0.01 or P<0.05). On day 23 (day 8 of treatment), day 26 (day 11 of treatment), day 30 (day 15 of treatment), and day 37 (after day 21 of treatment), the BUT measurement value of the positive control sodium hyaluronate eye drops group was significantly increased compared with the model control group, and the results were statistically significant (P<0.01 or P<0.05).

[0306] (3) On day 30 (day 15 of treatment) and day 37 (day 21 of treatment), the BUT value of the low-concentration hKGF-2 group was significantly increased compared with the model control group, and the results were statistically significant (P<0.01 or P<0.05). On day 23 (day 8 of treatment), day 26 (day 11 of treatment), day 30 (day 15 of treatment), and day 37 (day 21 of treatment), the BUT value of the medium-concentration hKGF-2 group was significantly increased compared with the model control group, and the results were statistically significant (P<0.01 or P<0.05). On day 26 (day 11 of treatment), day 30 (day 15 of treatment), and day 37 (day 21 after treatment), the BUT values ​​of the high-concentration hKGF-2 group were significantly increased compared with the model control group, and the results were statistically significant (P<0.01).

[0307] (4) With the extension of treatment time, the tear film breakup time of the low-concentration hKGF-2 group, the medium-concentration hKGF-2 group and the high-concentration hKGF-2 group showed an increasing trend.

[0308] 6.3 Effect of human keratinocyte growth factor-2 on corneal fluorescein staining score. The results are shown in Table 29.

[0309] Table 29. Corneal fluorescein staining scores before and after treatment in each group (x±s)

[0310] Group 0d 7d 15d 19d 23d 26d 30d 37d normal control group 0.04±0.14 0.13±0.23 0.08±0.29 0.13±0.31 0.21±0.33 0.08±0.19 0.13±0.23 0.13±0.31 Model control group 0.17±0.33 1.83±1.03** 6.25±1.59** 5.79±1.29** 4.13±1.64** 4.21±1.54** 4.92±1.08** 3.96±1.47** low concentration of hKGF-2 group 0.17±0.44 1.67±0.61** 5.75±1.25** 5.21±1.57 2.19±1.22# 2.79±1.03# 2.92±0.939## 2.50±0.83# hKGF-2 medium concentration group 0.04±0.14 1.92±0.73** 4.92±2.10** 5.46±1.25 2.38±1.03## 2.13±0.93## 2.71±1.27## 2.46±1.32# high concentration of hKGF-2 group 0.04±0.14 1.58±0.63** 5.83±1.75** 4.92±0.90 2.25±0.69## 1.75±0.84## 2.54±0.99## 2.13±1.03## Bepanthen eye drops group 0.17±0.33 1.63±0.71** 6.29±1.94** 5.17±1.17 3.13±1.09 2.17±0.86## 3.13±1.32## 2.54±1.42# Sodium hyaluronate eye drops group 0.08±0.29 1.63±0.93** 5.71±1.99** 4.71±0.75 2.41±0.73## 2.33±0.96## 2.88±1.77## 2.33±0.81##

[0311] Note: ① In Table 29, 0d, 7d, 15d, 19d, 23d, 26d, 30d, and 37d represent day 0, day 7, day 15, day 19, day 23, day 26, day 30, and day 37 of modeling, respectively. The low concentration group of hKGF-2 refers to hKGF-2 with a concentration of 50 μg / ml; the medium concentration group of hKGF-2 refers to hKGF-2 with a concentration of 100 μg / ml; and the high concentration group of hKGF-2 refers to hKGF-2 with a concentration of 200 μg / ml.

[0312] ② Compared with the normal control group, "*" indicates P<0.05, and "**" indicates P<0.01. Compared with the model control group, "#" indicates P<0.05, and "##" indicates P<0.01. Compared with Bepanthen eye drops, "^" indicates P<0.05, and "^^" indicates P<0.01. Compared with sodium hyaluronate eye drops, "&" indicates P<0.05, and "&&" indicates P<0.01.

[0313] Table 29 shows the results:

[0314] (1) On days 7, 15, 19, 23, 26, 30 and 37 after modeling, compared with the blank control group, the corneal fluorescein staining score of rabbits in the model control group was significantly increased, and the results were statistically significant (P<0.01).

[0315] (2) On day 26 (day 11 of treatment), day 30 (day 15 of treatment), and day 37 (after day 21 of treatment), compared with the model control group, the corneal fluorescein staining score of the positive control Bepanthen eye drops group was significantly reduced, and the results were statistically significant (P<0.01 or P<0.05). On day 23 (day 8 of treatment), day 26 (day 11 of treatment), day 30 (day 15 of treatment), and day 37 (after day 21 of treatment), compared with the model control group, the corneal fluorescein staining score of the positive control sodium hyaluronate eye drops group was significantly reduced, and the results were statistically significant (P<0.01).

[0316] (3) On day 23 (day 8 of drug treatment), day 26 (day 11 of drug treatment), day 30 (day 15 of drug treatment), and day 37 (day 21 of drug treatment), compared with the model control group, the corneal fluorescein staining scores of the low, medium and high concentration hKGF-2 groups were significantly reduced, and the results were statistically significant (P<0.01 or P<0.05).

[0317] The results of the Schirmer I test, tear film breakup time measurement test, and corneal fluorescein staining score test provided in this embodiment show that, compared with the blank control group and the model control group, the rabbits' baseline tear secretion and tear film breakup time in the Schirmer I test were significantly lower than those in the normal control group, and their corneal fluorescein staining scores were higher, indicating more severe pathological conditions. Compared with the model control group, the positive control groups (Bepanthen eye drops group and sodium hyaluronate eye drops group) all showed a reduction in dry eye symptoms in rabbits.

[0318] Compared with the model control group, with increasing administration time, on day 21 after treatment, the basal tear secretion and tear film breakup time in the Schirmer test of rabbits in the low-concentration, medium-concentration, and high-concentration hKGF-2 groups all significantly increased, while the corneal fluorescein staining score significantly decreased. The tear film breakup time and corneal fluorescein staining score were significantly increased in the low-concentration, medium-concentration, and high-concentration hKGF-2 groups, respectively, suggesting that low-concentration, medium-concentration, and high-concentration hKGF-2 can all improve the symptoms of dry eye in rabbits.

[0319] The effects of 6.4hKGF-2 on corneal pathology results are shown in Table 30.

[0320] Table 30 Number of cases and severity of corneal and conjunctival lesions

[0321]

[0322] Levels: 1 = Mild; 2 = Mild; 3 = Moderate; 4 = Severe; 5 = Serious.

[0323] Table 30 and Figure 6 The results showed that, compared with the blank control group, the model control group rabbits exhibited corneal epithelial atrophy, significantly thinner epithelial cell layer, disordered cell arrangement, loose collagen fibers in the stroma layer, and visible fibroblast and angiogenesis proliferation, accompanied by inflammatory cell infiltration. Figure 6 A); The number of conjunctival goblet cells in the model control group rabbits was reduced ( Figure 6 B).

[0324] After 21 days of treatment, compared with the model group, corneal epithelial atrophy and inflammatory cell infiltration were improved in the low-concentration, medium-concentration, and high-concentration hKGF-2 eye drops groups, as well as the Bepanthen eye drops group and the sodium hyaluronate eye drops group; the severity and number of conjunctival epithelial goblet cell reduction lesions were also reduced. Corneal damage repair was better in the low-concentration, medium-concentration, and high-concentration hKGF-2 eye drops groups than in the Bepanthen eye drops group and the sodium hyaluronate eye drops group.

[0325] Therefore, the low-concentration, medium-concentration, and high-concentration groups of hKGF-2 eye drops, the Bepanthen eye drops group, and the sodium hyaluronate eye drops group can improve corneal and conjunctival pathological damage, and the low-concentration, medium-concentration, and high-concentration groups of hKGF-2 eye drops have a better effect on improving the cornea than the Bepanthen eye drops group and the sodium hyaluronate eye drops group.

[0326] Compared with the blank control group, the pathological severity in the model control group was more severe. Compared with the model control group, with the increase of drug administration time, on day 21 after treatment, the tear film breakup time of the low-concentration, medium-concentration, and high-concentration hKGF-2 groups all increased significantly, the corneal fluorescein staining score decreased significantly, and the pathological severity of the cornea was alleviated, suggesting that low, medium, and high concentrations can all improve the symptoms of dry eye in rabbits.

[0327] Therefore, under the experimental conditions of this embodiment, low concentrations (low dose), medium concentrations (medium dose), and high concentrations (high dose) of hKGF-2 can alleviate the symptoms of benzalkonium chloride-induced dry eye in rabbits.

[0328] It should be noted that the specific embodiments described above are exemplary. Those skilled in the art can devise various solutions inspired by the disclosure of this invention, and these solutions all fall within the scope of this invention and its protection. Those skilled in the art should understand that this specification and its accompanying drawings are illustrative and do not constitute a limitation on the claims. The scope of protection of this invention is defined by the claims and their equivalents. This specification contains multiple inventive concepts; phrases such as "preferredly" and "according to a preferred embodiment" indicate that the corresponding paragraph discloses an independent concept. The applicant reserves the right to file divisional applications based on each inventive concept. Throughout the text, the feature introduced by "preferredly" is only an optional mode and should not be construed as mandatory. Therefore, the applicant reserves the right to abandon or delete relevant preferred features at any time.

Claims

1. A composition for treating an eye condition, comprising human keratinocyte-2 and a protective agent, characterized in that, The protective agent includes: 1) 0.1%–2% (w / v) mannitol or 0.01%–0.05% (w / v) sodium hyaluronate; 2) 0.1%–2% (w / v) glycerin; 3) 0.1%–2% (w / v) trehalose or 0.5% (w / v) human serum albumin; 4) 0.1% to 2% (w / v) of one or more of lysine hydrochloride.

2. The composition according to claim 1, characterized in that, The mass concentration of mannitol can be in the range of 0.5% or 1% to 2%, preferably, the mass concentration of mannitol can be 0.5% or 1.6%.

3. The composition according to claim 1 or 2, characterized in that, The mass concentration of glycerol can range from 0.5% to 2%, and preferably, the mass concentration of glycerol can be 1%, 1.5%, or 0.5%.

4. The composition according to any one of claims 1 to 3, characterized in that, The mass concentration of trehalose can range from 0.5% to 2%, and preferably, the mass concentration of trehalose can be 2%.

5. The composition according to any one of claims 1 to 4, characterized in that, The mass concentration of lysine hydrochloride can range from 0.5% to 1%, and preferably, the mass concentration of lysine hydrochloride can be 0.5%.

6. The composition according to any one of claims 1 to 5, characterized in that, The composition for treating eye conditions further includes a surfactant, wherein the surfactant comprises 0.01% to 0.5% (w / v) poloxamer 188, preferably, the mass concentration of said poloxamer 188 can be in the range of 0.01% to 0.1%, and particularly preferably, the mass concentration of said poloxamer 188 can be in the range of 0.05% or 0.01%.

7. The composition according to any one of claims 1 to 6, characterized in that, The composition for treating eye conditions also includes a pH adjuster, wherein the pH adjuster includes citric acid, sodium citrate, or a combination thereof.

8. The composition according to any one of claims 1 to 7, characterized in that, The composition for treating eye conditions also includes a solvent, wherein the solvent can be water for injection.

9. The composition according to any one of claims 1 to 8, characterized in that, The composition for treating eye conditions has a pH of 6.0 to 7.0, preferably, the pH of the composition for treating eye conditions is 6.0 to 6.

6.

10. Use of the composition for treating eye conditions according to any one of claims 1 to 9 in the preparation of a medicament for treating dry eye syndrome.

11. Use of the composition for treating eye conditions according to any one of claims 1 to 9 in the preparation of a medicament for treating benzalkonium chloride-induced dry eye syndrome.

12. A container, characterized in that, The container contains the aforementioned composition for treating eye conditions, preferably an eye drop bottle.

13. A kit, characterized in that, The kit includes the container as described in claim 12 and instructions or labels for applying and using the composition for treating eye conditions as described in any one of claims 1 to 9.

14. A method for preparing a composition for treating eye diseases, characterized in that, include: Prepare solution A, wherein solution A comprises glycerol, mannitol, lysine hydrochloride, trehalose, poloxamer 188, citric acid and sodium citrate; Prepare solution B, wherein solution B comprises a prescribed amount of human keratinocyte cytokine-2 protein stock solution; The composition for treating eye conditions is obtained by mixing solution A and solution B.

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