A pharmaceutical composition and method for treating and / or preventing corneal damage.

Abstract

This disclosure provides the use of 7,8-dihydroxyflavone in the treatment of corneal injuries. Specifically, this disclosure provides the use of 7,8-dihydroxyflavone in the preparation of medicaments for treating corneal injuries.

Description

A pharmaceutical composition and method for treating and / or preventing corneal damage

[0001] This disclosure claims priority to Chinese patent application CN202311793641.2 filed on December 25, 2023, and the entire contents of the aforementioned patent application are incorporated into this disclosure by reference. Technical Field

[0002] The present disclosure relates to the field of biomedicine technology. Specifically, the present disclosure relates to a method and a pharmaceutical composition for treating and / or preventing corneal damage. Background Art

[0003] The eyes are the "windows to the soul". Eye health involves people of all ages throughout their lifespan and is a major public health issue that affects people's well-being. The cornea is a transparent tissue covering the front of the eyeball. It contains a wealth of corneal nerves and is one of the most sensitive tissues in the body. The corneal nerves play an important role in the perception of temperature, chemical stimulation, mechanical stimulation, and pain. They nourish corneal epithelial cells and corneal stromal cells, maintain ocular surface health and corneal sensitivity, and play an important role in protecting the integrity of the corneal epithelium, promoting the proliferation and migration of corneal epithelial cells and corneal stromal cells, and promoting the repair of corneal damage. Corneal damage is a common disease in ophthalmology clinics, mainly including trauma (such as chemical corneal burns, mechanical corneal damage, ophthalmic surgical injuries, biological injuries, etc.), infectious damage, inflammatory damage, damage caused by abnormal tear film function (including damage to the corneal epithelium and corneal nerves caused by tear deficiency or abnormal tear dynamics, abnormal mucin secretion, and abnormal lipid secretion, which is also the main cause of dry eye disease), etc. Corneal nerve damage caused by eye trauma is also the main cause of neurotrophic keratitis. If not treated in time, it can lead to corneal epithelial defects, corneal opacity and corneal neovascularization, and then cause corneal tissue ulcers or corneal dissolution, and even corneal perforation, seriously affecting visual function and leaving patients with lifelong pain.

[0004] The causes of corneal damage, neurotrophic keratitis, and dry eye are complex and diverse. Existing methods for treating corneal damage mainly include: (1) administering drugs that promote corneal epithelial repair, such as artificial tears, which help stabilize the tear film and protect the corneal epithelium. Treatment drugs include sodium hyaluronate eye drops, calf blood deproteinized extract eye drops or gels, and 20% to 100% autologous serum treatment; (2) for those with ocular surface inflammatory reactions, low-concentration glucocorticoids or immunosuppressants are administered for anti-inflammatory treatment, such as 0.02% fluorometholone eye drops or 0.05% cyclosporine A eye drops. However, clinical treatment drugs are still very limited. Many patients' eye symptoms (such as red eyes, eye stinging, photophobia and tearing, foreign body sensation, and dryness) cannot be relieved, and their vision is severely impaired.

[0005] 7,8-Dihydroxyflavone (code: 7,8-DHF, CAS number: 38183-03-8) is a flavonoid derivative. So far, there has been no report on the use of 7,8-dihydroxyflavone in the treatment of corneal damage.

[0006] Brief Description

[0007] The present disclosure aims to solve at least one of the technical problems in the related art to a certain extent. To this end, the present disclosure provides a method for treating and / or preventing corneal damage, neurotrophic keratitis, or dry eye using 7,8-dihydroxyflavone or a pharmaceutically acceptable salt thereof.

[0008] In a first aspect of the present disclosure, provided is a use of 7,8-dihydroxyflavone or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating and / or preventing corneal damage, neurotrophic keratitis, or dry eye.

[0009] In a second aspect of the present disclosure, a pharmaceutical composition for preventing and / or treating corneal damage, neurotrophic keratitis, or dry eye is provided, wherein the pharmaceutical composition contains 7,8-dihydroxyflavone or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0010] In some specific embodiments, the pharmaceutical composition is an oral preparation, an injectable preparation or an ophthalmic preparation; wherein, the oral preparation may be selected from tablets, capsules, granules, powders, oral solutions or oral suspensions; the injectable preparation may be selected from injections, powder injections or concentrated injections; the ophthalmic preparation may be selected from plasters, lotions, eye drops, eye ointments, eye gels, intraocular injection solutions, fumigants or eye masks.

[0011] In a third aspect of the present disclosure, a method for preventing and / or treating corneal damage, neurotrophic keratitis, or dry eye is provided, comprising administering 7,8-dihydroxyflavone to a subject, or administering the pharmaceutical composition according to the second aspect.

[0012] In some embodiments, the neurotrophic keratitis or dry eye is caused by corneal injury.

[0013] In some embodiments, the corneal injury is corneal epithelial injury and / or corneal nerve injury.

[0014] In some embodiments, the corneal nerve damage is selected from the group consisting of corneal epithelial interlayer nerve terminal damage, and / or corneal subepithelial basal nerve plexus damage, and / or corneal stromal nerve trunk damage.

[0015] In some specific embodiments, the corneal injury can be a chemical burn of the cornea; a mechanical injury of the cornea; a foreign body injury to the cornea; a biological injury to the cornea; a thermal burn to the cornea; or a corneal injury caused by abnormal tear film function, corneal infection, corneal inflammation, ciliary nerve damage, trigeminal nerve ophthalmic branch damage, neurotrophic keratitis, dry eye or ophthalmic surgery.

[0016] In some embodiments, the effective amount of 7,8-dihydroxyflavone is in the range of 0.1-1000 mg / kg body weight, for example 1-100 mg / kg body weight, such as 50-100 mg / kg body weight. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is a statistical comparison of corneal fluorescence staining scores of mice in the 7,8-DHF treatment group, the model group, and the normal control group in Example 1.

[0018] Figure 2 is a comparison of tear secretion measurement results of mice in the 7,8-DHF treatment group, the model group, and the normal control group in Example 1.

[0019] FIG3 is a comparison of nerve staining signals after 7,8-DHF was used to treat corneal damage in mice caused by alkali burning in Example 2.

[0020] (A) and (D) are the staining signals of nerve endings in the corneal epithelium of mice in the corneal alkali burn model group one week after treatment.

[0021] (B) and (E) are the staining signals of the subepithelial basal nerve plexus in the mouse corneal alkali burn model group one week after treatment;

[0022] (C) and (F) are the corneal stromal nerve trunk staining signals in the mouse corneal alkali burn model group one week after treatment.

[0023] (G) and (J) are the staining signals of nerve endings in the corneal epithelium of mice after corneal alkali burning and 7,8-DHF treatment for 1 week;

[0024] (H) and (K) are the staining signals of the basal nerve plexus under the corneal epithelium of mice treated with 7,8-DHF for 1 week after corneal alkali burning;

[0025] (I) and (L) show the corneal stromal nerve trunk staining signals of mice treated with 7,8-DHF for 1 week after corneal alkali burning.

[0026] As shown in Figure 3, after corneal alkali burning, the damage to the nerve endings between the corneal epithelium, the basal nerve plexus under the corneal epithelium and the corneal stromal nerve trunks was significantly improved in mice that received 7,8-DHF treatment for one week compared with those who did not receive treatment. The corneal nerve staining signal density was significantly increased.

[0027] FIG4 is a comparison of corneal nerve staining signals after 7,8-DHF was used to treat corneal damage in mice caused by corneal epithelial scratches in Example 3.

[0028] (A) and (D) are the staining signals of nerve endings between corneal epithelial layers in the mouse corneal epithelial scratch model group one week after surgery;

[0029] (B) and (E) are the staining signals of the subepithelial basal nerve plexus of the cornea in the mouse corneal epithelium scratch model group one week after surgery;

[0030] (C) and (F) are the corneal stromal nerve trunk staining signals in the mouse corneal epithelial scratch model group one week after surgery.

[0031] (G) and (J) are the staining signals of nerve endings between the corneal epithelium of mice after corneal epithelial scratching and treatment with 7,8-DHF for 1 week;

[0032] (H) and (K) show the staining signals of the basal nerve plexus under the corneal epithelium of mice after corneal epithelial scratching and treatment with 7,8-DHF for 1 week;

[0033] (I) and (L) show the corneal stromal nerve trunk staining signals of mice after corneal epithelial scratching and 7,8-DHF treatment for 1 week.

[0034] As shown in Figure 4, the damage to the nerve endings between the corneal epithelium, the basal nerve plexus under the corneal epithelium and the corneal stromal nerve trunks was significantly improved, and the corneal nerve staining signal density was significantly increased in mice that received 7,8-DHF treatment for one week after corneal epithelial scratching compared with those that did not receive treatment.

[0035] Figure 5 is a statistical comparison of corneal nerve density in the 7,8-DHF treatment group and the model group in Example 2.

[0036] Figure 6 is a statistical comparison of corneal nerve density in the 7,8-DHF treatment group and the model group in Example 3.

[0037] Detailed description

[0038] The present disclosure relates to a pharmaceutical composition for preventing or treating corneal damage, neurotrophic keratitis or dry eye, using 7,8-dihydroxyflavone or a pharmaceutically acceptable salt thereof as an active ingredient.

[0039] In some embodiments, the corneal injury can be corneal epithelial injury and / or corneal nerve injury.

[0040] In some embodiments, the corneal injury may be corneal injury caused by chemical corneal burns or mechanical corneal injury or related diseases.

[0041] Corneal chemical burns may be corneal alkali burns, corneal acid burns, corneal thermal burns, etc., where the alkali mentioned in corneal alkali burns can be inorganic alkalis such as sodium hydroxide, potassium hydroxide, calcium hydroxide, or organic alkalis; corneal acid burns can be organic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, or inorganic acids.

[0042] Mechanical corneal injury can be caused by scraping the corneal epithelium, foreign body injury, or eye surgery. Eye surgery refers to any procedure involving the eyeball, typically cataract surgery, glaucoma surgery, retinal surgery, LASIK, LASEK, or corneal transplants. Corneal scraping can be caused by contact lens wear or blunt trauma to the cornea.

[0043] The corneal damage caused by the above-mentioned related diseases may be corneal damage caused by tear film dysfunction, corneal infection, corneal inflammation, ciliary nerve damage, trigeminal nerve ophthalmic branch damage, neurotrophic keratitis, dry eye and other ophthalmic diseases.

[0044] In some specific embodiments, neurotrophic keratitis may be caused by corneal damage. Neurotrophic keratitis is usually a type of disease based on corneal nerve abnormalities, with weakened nerve sensory and secretory functions, leading to damage to the corneal epithelium and destruction of the integrity of the tear film, epithelium, and stroma. Specifically, the pathogenesis of neurotrophic keratitis is mainly due to the loss of corneal sensitivity caused by corneal nerve damage. After corneal denervation, it can lead to a series of corneal problems such as reduced tear secretion, reduced mitosis of corneal epithelial cells, reduced glycogen in corneal epithelial cells, and loss of microvilli of surface epithelial cells, causing activation of corneal inflammatory reactions, leading to a series of pathological changes such as corneal epithelial defects, delayed healing, and corneal ulcers.

[0045] In some specific embodiments, dry eye may be caused by corneal damage. In some cases, after corneal damage, changes in corneal structure may disrupt the lubrication and adhesion function of tear mucin to the ocular epithelium, disrupt tear dynamics, cause a decrease in tear secretion, and lead to a decrease in tear film stability, thereby causing dry eye. Corneal nerve damage caused by various factors can cause decreased corneal surface perception, decreased blink frequency, and reduced tear secretion, thereby causing dry eye. In other cases, corneal damage leads to keratitis, which can ultimately lead to the occurrence of dry eye by causing reduced tear secretion, tear film instability, aggravated inflammatory response, promoted cell apoptosis, and interfere with the neurophysiological system.

[0046] In other specific embodiments, 7,8-dihydroxyflavone and pharmaceutically acceptable salts thereof also play a positive role in treating dry eye caused by tear film damage and / or keratitis.

[0047] Since the corneal nerves are distributed in the cornea, corneal epithelial damage usually also causes corneal nerve damage.

[0048] The 7,8-dihydroxyflavone and pharmaceutically acceptable salts thereof disclosed herein may include all possible solvates, hydrates, racemates or stereoisomers.

[0049] In some embodiments, pharmaceutically acceptable salts may be salts that retain the biological effects and properties of 7,8-dihydroxyflavone and generally have no biological or other disadvantages, and may be acid addition salts and base addition salts, wherein acid addition salts can be formed with inorganic acids and organic acids, and pharmaceutically acceptable base addition salts can be formed with inorganic or organic bases and may have inorganic or organic counterions.

[0050] When preparing the pharmaceutical composition, commonly used fillers, extenders, binders, wetting agents, disintegrants, surfactants, diluents and other pharmaceutically acceptable excipients are used for preparation.

[0051] As used herein, the term "pharmaceutically acceptable excipient" means a pharmaceutically acceptable material, mixture, or vehicle that contributes to the consistency of a dosage form or pharmaceutical composition. Each excipient must be compatible with the other ingredients of the pharmaceutical composition when mixed to avoid interactions that would significantly reduce the efficacy of the disclosed pharmaceutical active ingredients when administered to a patient and interactions that would result in a pharmaceutical composition that is not pharmaceutically acceptable. In addition, each excipient must be pharmaceutically acceptable, e.g., possess a sufficiently high degree of purity.

[0052] Suitable excipients will vary depending on the specific dosage form selected. In addition, excipients may be selected based on their specific function in the composition. For example, certain excipients may be selected to facilitate the production of a uniform dosage form, certain excipients may be selected to facilitate the production of a stable dosage form, certain excipients may be selected to facilitate the carrying or transport of the active ingredient disclosed herein from one organ or part of the body to another organ or part of the body when administered to a patient, and certain excipients may be selected to enhance patient compliance.

[0053] The active ingredients of the present disclosure can be administered systemically or topically to the eye (e.g., topically, intracamerally, or via implant). The active ingredients can be incorporated into topical ophthalmic formulations for administration to the eye. The active ingredients can be combined with an ophthalmically acceptable preservative, surfactant, viscosity enhancer, penetration enhancer, buffer, sodium chloride, and hydrate to form a sterile aqueous ophthalmic suspension or solution. Ophthalmic solution formulations can be prepared by dissolving the active ingredient in a physiologically acceptable isotonic aqueous buffer. Isotonicity agents are used to adjust the osmotic properties of the eye drops and can typically be selected from sodium chloride or potassium chloride. Buffers have the function of adjusting the acidity or alkalinity of the eye drops. Commonly used buffers for preparing eye drops include aminocaproic acid, sodium monohydrogen phosphate, and sodium dihydrogen phosphate. Stabilizers stabilize the eye drops; sodium edetate and / or sodium perborate can be used as stabilizers. pH adjusters adjust the pH of the eye drop composition; examples include hydrochloric acid and / or sodium hydroxide. In addition, the ophthalmic solution may also contain an ophthalmically acceptable surfactant to aid in the dissolution of the active ingredient. In addition, the ophthalmic solution can also include materials such as hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, methylcellulose, polyvinyl pyrrolidone, etc., for increasing viscosity to improve the retention of the preparation in the conjunctival sac. Gelling agents can also be used, which include, but are not limited to, gellan gum and xanthan gum. In order to prepare sterile ophthalmic ointment, active ingredient and preservative can be mixed in a suitable matrix, wherein the suitable matrix is ​​such as mineral oil, liquid lanolin or white vaseline. Sterile ophthalmic gel preparation can be prepared according to the disclosed formula for similar ophthalmic preparations by suspending the active ingredient in a hydrophilic matrix obtained by a combination of, for example, Carbopol-940, etc.; preservative and tension agent can be mixed therein.

[0054] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and lozenges. These solid preparations contain one or more compounds of the present invention and at least one excipient, such as starch or carbonate. They are prepared by mixing calcium, sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium styrene and talc are also used. Oral liquid preparations include suspensions, internal solutions, emulsions, or syrups. In addition to the commonly used simple diluents water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavorings, and preservatives may also be added.

[0055] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, suppositories and the like.

[0056] As used in the present disclosure, the term "treating" any disease or condition refers to all diseases or conditions that can be slowed down, interrupted, prevented, controlled or stopped, but does not necessarily mean that all symptoms of all diseases or conditions disappear, and it also includes preventive treatment of the symptoms, especially in patients who are prone to such diseases or disorders. In some embodiments, it refers to improving the disease or condition (i.e., slowing down or preventing or alleviating the development of the disease or at least one clinical symptom thereof). In other embodiments, "treating" refers to alleviating or improving at least one physical parameter, including physical parameters that may not be perceived by the patient. In other embodiments, "treating" refers to regulating the disease or condition physically (e.g., stabilizing perceptible symptoms) or physiologically (e.g., stabilizing parameters of the body) or both. In other embodiments, "treating" refers to preventing or delaying the onset, occurrence or worsening of a disease or condition.

[0057] As used herein, the terms "effective amount," "therapeutically effective amount," or "therapeutically effective dose" refer to an amount of a compound of the present disclosure that is capable of inducing a biological or medical response in an individual (e.g., reducing or inhibiting enzyme or protein activity, or improving symptoms, alleviating symptoms, slowing or delaying disease progression, or preventing disease, etc.).

[0058] The dosage of active ingredient in the disclosed pharmaceutical composition can be changed, but the amount of active ingredient must be the amount that can obtain suitable dosage form.Active ingredient can be administered to the patient (animal and human) of this treatment needing with the dosage that provides optimal drug effect.Selected dosage depends on the therapeutic effect of expectation, depends on route of administration and treatment duration.Dosage will vary with patient, and this depends on attribute and severity of disease, patient's weight, patient's specific diet, medicine used simultaneously and other factors that those skilled in the art will recognize.

[0059] In some embodiments, the effective amount of 7,8-dihydroxyflavone of the present disclosure may vary according to the patient's age, gender, and weight, and is generally within the range of 0.1-1000 mg / kg body weight, for example, 1-100 mg / kg body weight, 50-100 mg / kg body weight, and may be administered every other day or divided into 1 to 3 doses per day. However, since it may increase or decrease depending on the route of administration, severity of the disease, gender, weight, age, etc., the dosage is not limited to the scope of the present disclosure in any way.

[0060] In some embodiments, the eye drop composition of the present disclosure can be used 1 to 3 drops per time, 5 to 6 times per day, and the dosage can be increased or decreased as appropriate based on the symptoms. The dosage level for a specific patient can vary depending on the patient's weight, age, sex, health status, administration time, administration frequency, severity of the disease, etc.

[0061] The present disclosure also provides the following technical solutions:

[0062] In some embodiments, the present disclosure provides use of 7,8-dihydroxyflavone in the preparation of a medicament for preventing and / or treating corneal damage or corneal epithelial damage.

[0063] In some specific embodiments, the corneal injury or corneal epithelial injury is selected from corneal chemical burns, corneal mechanical injury, corneal foreign body injury, corneal biological injury, corneal thermal burns, corneal injury after ophthalmic surgery, neurotrophic keratitis, and dry eye.

[0064] In some embodiments, the corneal injury is selected from the group consisting of injury to nerve endings within the corneal epithelium, and / or injury to the basal nerve plexus beneath the corneal epithelium, and / or injury to the corneal stromal nerve trunk.

[0065] In other embodiments, the present disclosure provides a pharmaceutical composition comprising 7,8-dihydroxyflavone and pharmaceutically acceptable excipients.

[0066] In some embodiments, the composition is any one of an ophthalmic preparation, suspension, granules, capsules, powders, tablets, solutions or drops.

[0067] In some embodiments, the ophthalmic preparation is eye drops, eye ointments, eye gels, or injections.

[0068] The subject of the pharmaceutical active ingredient of the present disclosure or its pharmaceutical composition refers to an animal. Typically, the animal is a mammal. The subject also refers to, for example, a primate (e.g., human, male or female), cattle, sheep, goat, horse, dog, cat, rabbit, rat, mouse, fish, bird, etc. In certain embodiments, the subject is a primate. In other embodiments, the subject is a human.

[0069] For the purpose of promoting the understanding of the principle of the present disclosure, reference will now be made to the embodiments illustrated in the accompanying drawings and the embodiments will be described in detail. However, these descriptions are not intended to limit the scope of the present disclosure in any way. Example

[0070] Example 1: Testing the effects of 7,8-DHF on corneal epithelial damage, neurotrophic keratitis and dry eye in the mouse corneal BAC eye drop model.

[0071] Materials and methods

[0072] PBS is phosphate buffered saline.

[0073] BAC refers to benzalkonium chloride.

[0074] 0.1% BAC means that the BAC content is 0.1 g per 100 mL.

[0075] 0.1% fluorescein sodium means that the content of fluorescein sodium is 0.1g per 100mL.

[0076] Normal control group: 5 μL of normal saline was dripped into each eye of the mice twice a day for 7 consecutive days.

[0077] Model group: 5 μL of 0.1% BAC was dripped into each eye of the mice twice a day for 7 consecutive days.

[0078] 7,8-DHF treatment group: 5 μL of 0.1% BAC was instilled into each eye twice daily for 7 consecutive days. Starting on the third day of 0.1% BAC administration, 7,8-DHF was orally administered twice daily at a dose of 100 mg / kg body weight in a volume of 10 μL / g for 7 consecutive days.

[0079] Corneal fluorescence staining: The first day of administration of the model group was recorded as day 1. On the 5th and 10th days thereafter, corneal fluorescence staining was performed on the model group, treatment group, and normal control group, respectively. The staining method was as follows: After anesthetizing the mouse, 5 μL of 0.1% sodium fluorescein was dropped into each eye of the mouse. The mouse's eyes were closed for several seconds to allow the sodium fluorescein to be distributed as evenly as possible on the cornea, and then observed under a slit lamp. The cornea was divided into four areas, and the score of each area was recorded separately. The scores of each area were added together to obtain the final score. The more severe the corneal damage, the higher the score. The scoring criteria are as follows:

[0080] lack, 0 points;

[0081] Slightly spot-like coloration with less than 30 points, 1 point;

[0082] Dot coloring with more than 30 points, 2 points;

[0083] Severe diffuse staining but without obvious flakes, 3 points;

[0084] Obvious flake staining, 4 points.

[0085] The statistical results of corneal fluorescence staining scores are shown in Figure 1.

[0086] Conclusion: Compared with the model group, the corneal fluorescence staining score of the 7,8-DHF treatment group was significantly lower, and the corneal fluorescence staining score of the normal control group was the lowest, indicating that 7,8-DHF can significantly promote the repair of corneal epithelial damage in the BAC eye drop model of mice.

[0087] Tear testing method: The first day of drug administration in the model group was designated as day 1. Tear testing was performed on the model group, treatment group, and normal control group on the 5th and 10th days thereafter. The testing method was as follows: After anesthetizing the mice, the lower eyelid was gently pulled open. A phenol red cotton thread was placed in the inner and outer third of the eyelid using forceps. A timer was immediately started for 1 minute, and the length of the phenol red cotton thread that turned red was measured and recorded. The statistical results are shown in Figure 2.

[0088] Conclusion: On the second day after the start of 7,8-DHF treatment, tear volume in the treatment group increased significantly compared with the model group. After 7 days of 7,8-DHF treatment, tear volume in the treatment group was comparable to that in the normal control group. This suggests that 7,8-DHF promotes the recovery of tear film function in the BAC eye drop model in mice.

[0089] Tear deficiency or abnormal tear dynamics impairs tear film function, which can cause damage to the corneal epithelium and / or corneal nerves, disrupting the interaction between the corneal epithelium and corneal nerves. This can lead to abnormal corneal sensitivity, structural integrity, and corneal inflammation, leading to dry eye and neurotrophic keratitis. In a BAC eye drop model in mice, the 7,8-DHF treatment group showed significant improvement in corneal damage and restored tear film function compared to the model group, demonstrating the therapeutic efficacy of 7,8-DHF in treating dry eye and neurotrophic keratitis.

[0090] Example 2: Testing the effects of 7,8-DHF on corneal neuroprotection and corneal nerve growth promotion in a mouse corneal alkali burn model

[0091] Materials and methods

[0092] A. Preparation of solution for alkali burning: Weigh 0.4 g of sodium hydroxide powder and dissolve it in 10 mL of deionized water.

[0093] B. Mouse corneal alkali burn model: Prepare a 2 mm diameter filter paper. After anesthetizing the mouse, draw 1 μL of alkali solution onto the filter paper. After the solution completely soaks the filter paper, place the filter paper on the center of the cornea. After 8 seconds, remove the filter paper immediately and rinse the eyeball with 10 mL of PBS.

[0094] 7,8-DHF treatment: The drug was administered once a day after model establishment at a dose of 50 mg / kg. After 7 days of administration, the cornea was perfused through the heart and samples were collected. The entire cornea was taken to make a flat-mounted slide and imaged with a fluorescence microscope.

[0095] Transgenic mice were used in Example 2 of this disclosure. Green fluorescent protein is specifically expressed in the corneal nerves of these transgenic mice, allowing the distribution of corneal nerves to be recorded using green fluorescent signals. The anatomical characteristics of the corneal nerves in the corneal epithelium, subepithelial stroma, and corneal base are as follows: They originate from the ophthalmic branch of the trigeminal nerve, pass through the ciliary nerve, reach the cornea, travel through the stroma, and rotate 90 degrees to form a nerve plexus in the subepithelial stroma. These nerve plexus then gives off small vertical branches, forming dense nerve endings within the corneal epithelium. Therefore, the main distribution characteristics of the corneal nerves are: corneal stromal nerve trunks, subepithelial basal nerve plexuses, and corneal epithelial nerve endings.

[0096] One week after alkali-burning the central cornea of ​​mice using sodium hydroxide, the corneal nerves were very sparsely distributed at the level of corneal stromal nerve trunks, subepithelial basal nerve plexus, and interepithelial nerve endings (see Figure 3 (A) to (F)). After alkali-burning the cornea of ​​mice followed by one week of 7,8-DHF treatment, the density of corneal nerve distribution at the level of corneal stromal nerve trunks, subepithelial basal nerve plexus, and interepithelial nerve endings was significantly increased compared to the model group (see Figure 3 (G) to (L)).

[0097] Images were acquired using a fluorescence microscope and analyzed and statistically analyzed using Image J, the image analysis software. Intergroup comparisons were then performed. In mice treated with corneal alkali burn followed by one week of 7,8-DHF, corneal nerve density was significantly higher than in the model group. The statistical results are shown in Figure 5.

[0098] Conclusion: In a mouse model of corneal injury induced by alkali burns, 7,8-DHF significantly increased corneal nerve density and promoted corneal nerve growth. Corneal nerves nourish corneal epithelial cells, playing an important role in protecting the integrity of the corneal epithelium and promoting its proliferation and repair. This suggests that 7,8-DHF is significantly effective in treating corneal injury caused by chemical burns.

[0099] The mouse corneal alkali burn model simulates corneal epithelial and corneal nerve damage, which are the primary causes of dry eye, while corneal nerve damage is the primary cause of neurotrophic keratitis. Example 2 demonstrates that 7,8-DHF has unexpected effects in treating neurotrophic keratitis and dry eye caused by corneal epithelial and / or corneal nerve damage.

[0100] Example 3: The effects of 7,8-DHF on corneal neuroprotection and promotion of corneal nerve growth in a mouse corneal epithelial scratch model were tested.

[0101] Materials and methods

[0102] A. Mouse corneal epithelial scraping model: After the mouse is anesthetized, the corneal epithelium is scraped to the corneal limbus with an electric epithelial scraper to expose the corneal stroma.

[0103] B. 7,8-DHF treatment: After the mouse model was established, the drug was administered once a day at a dose of 50 mg / kg. After 7 days of administration, samples were collected by cardiac perfusion, and the entire cornea was taken to make flat-mount slides and imaged under a fluorescence microscope.

[0104] In Example 3 of this disclosure, transgenic mice were used. Green fluorescent protein is specifically expressed in the corneal nerves of these transgenic mice, allowing for the labeling of corneal nerve distribution using green fluorescent signals. The main distribution characteristics of corneal nerves are: corneal stromal nerve trunks, subepithelial basal nerve plexuses, and corneal epithelial nerve endings.

[0105] One week after corneal epithelial scratching in mice, corneal nerves were very sparsely distributed at the level of corneal stromal nerve trunks, subepithelial basal nerve plexus, and interepithelial nerve endings (see Figure 4 (A) to (F)). After corneal epithelial scratching and one week of 7,8-DHF treatment, the density of corneal nerve distribution at the level of corneal stromal nerve trunks, subepithelial basal nerve plexus, and interepithelial nerve endings was significantly increased compared with the model group (see Figure 4 (G) to (L)).

[0106] Images were acquired using a fluorescence microscope and analyzed and statistically analyzed using Image J, the image analysis software. Intergroup comparisons were then performed. In mice treated with 7,8-DHF for one week after corneal epithelial scraping, corneal nerve density was significantly higher than in the model group. The statistical results are shown in Figure 6.

[0107] Conclusion: In a mouse corneal epithelial scratch injury model, 7,8-DHF increased corneal nerve density and promoted corneal nerve growth. Corneal nerves nourish corneal epithelial cells, playing an important role in protecting corneal epithelial integrity and promoting corneal epithelial cell proliferation and repair. This suggests that 7,8-DHF has a significant effect in treating mechanically induced corneal injuries.

[0108] The mouse corneal epithelial scratch model simulates corneal epithelial and corneal nerve damage, which are the main causes of dry eye, while corneal nerve damage is the main cause of neurotrophic keratitis. Example 3 demonstrates that 7,8-DHF has unexpected effects in treating neurotrophic keratitis and dry eye caused by corneal epithelial and / or corneal nerve damage.

[0109] The mouse corneal chemical alkali burn model, corneal epithelial scratch model and BAC corneal injury model in the examples can representatively simulate clinical corneal injuries, including but not limited to corneal chemical burns, corneal mechanical injury, corneal foreign body injury, corneal biological injury, corneal thermal burns, corneal injury after ophthalmic surgery, etc., as well as neurotrophic keratitis and dry eye caused by corneal injury, or corneal injury in neurotrophic keratitis and dry eye, the above-mentioned corneal injury includes corneal epithelial injury and / or corneal nerve injury.

[0110] Although various embodiments of the present disclosure have been described in great detail herein, such embodiments are provided merely as non-limiting examples of the application described herein. Therefore, it will be understood by those skilled in the art that various changes and modifications may be made to the application without departing from the scope of the application. Indeed, the present application is not intended to be exhaustive or to limit the scope of the application.

[0111] Further, in the description of representative embodiments, the present application content has given the method and / or process of the present application in a specific order of steps. However, the method or process should not be limited to the specific order of steps described. Other order of steps are possible. Therefore, the specific order of steps applied for herein should not be interpreted as limiting the present application. In addition, the application content for the method and / or process should not be limited to performing their steps in the order described. Such an order can be varied and still be within the scope of the present application.

[0112] The foregoing is merely a list of specific embodiments of the present application, intended to enable those skilled in the art to understand and implement the present application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application is not limited to the embodiments described herein, but is intended to conform to the broadest scope consistent with the principles and novel features of the present application.

Claims

1. Use of 7,8-dihydroxyflavone or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing and / or treating corneal injury or neurotrophic keratitis or dry eye disease.

2. According to the use described in claim 1, wherein The neurotrophic keratitis or dry eye disease is caused by corneal injury.

3. The use according to claim 1 or 2, wherein, The corneal injury is corneal epithelial injury and / or corneal nerve injury.

4. According to the use described in claim 3, wherein The corneal nerve injury is selected from corneal epithelial interlaminar nerve ending injury, and / or subepithelial basal nerve plexus injury of the cornea, and / or corneal stromal nerve trunk injury.

5. Use according to any one of claims 1 to 4, wherein The corneal injury is selected from corneal chemical burns; corneal mechanical injuries; corneal foreign body injuries; corneal biological injuries; corneal thermal burns; and corneal injuries caused by abnormal tear film function, corneal infection, corneal inflammation, ciliary nerve injury, ophthalmic branch injury of the trigeminal nerve, neurotrophic keratitis, dry eye disease or ophthalmic surgery.

6. A pharmaceutical composition comprising 7,8-dihydroxyflavone or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient, the pharmaceutical composition being used for preventing and / or treating corneal injury or neurotrophic keratitis or dry eye disease.

7. The pharmaceutical composition according to claim 6, characterized in that, The composition is an oral preparation, an injection preparation or an ophthalmic preparation; wherein, Optionally, the oral preparation is selected from tablets, capsules, granules, powders, oral solutions or oral suspensions; Optionally, the injection preparation is selected from injection solutions, powder for injection or concentrated solutions for injection; Optionally, the ophthalmic preparation is selected from patches, lotions, eye drops, eye ointments, ophthalmic gels, intraocular injection solutions, fumigants or eye masks.

8. A method for preventing and / or treating corneal injury or neurotrophic keratitis or dry eye disease, comprising administering 7,8-dihydroxyflavone or a pharmaceutically acceptable salt thereof to a subject, or administering the pharmaceutical composition according to any one of claims 6 or 7.

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