Use of calcium folinate in the preparation of a drug for promoting corneal repair

Abstract

The application provides application of calcium folinate in preparation of corneal repair promoting drugs and belongs to the technical field of ophthalmic drugs. The application uses calcium folinate as an active ingredient of a drug for treating corneal injury, has a good treatment effect on corneal epithelial injury and corneal nerve injury, and the calcium folinate is low in price and easy to store. The application provides a calcium folinate eye drop solution, which comprises calcium folinate and physiological saline; the mass percentage content of the calcium folinate in the calcium folinate eye drop solution is 0.1-1%. Compared with the drugs commonly used in the clinic for promoting repair after corneal injury, the calcium folinate eye drop solution provided by the application is low in price, can reduce the medical expenditure of patients to a certain extent, and reduces the medical burden of patients. Meanwhile, the composition of the calcium folinate is single, the calcium folinate is easy to store and not prone to deterioration, and the application of the calcium folinate to a certain extent avoids the risk that the existing corneal repair promoting eye drop solution is continuously used after being contaminated and deteriorated, which may cause or aggravate the infection risk.

Description

Technical Field

[0001] This invention relates to the field of ophthalmic drug technology, and in particular to the application of calcium leucovorin in the preparation of corneal repair drugs. Background Technology

[0002] The cornea is a transparent membrane located on the anterior wall of the eyeball, comprising about one-sixth of the fibrous membrane. It protrudes slightly forward and is easily affected by external trauma such as abrasions from dust, metal filings, grains, or fingernails; physical and chemical injuries such as acid and alkali damage; ultraviolet radiation; underlying eye diseases (such as entropion and trichiasis); and high intraocular pressure leading to corneal edema. The corneal epithelium, located on the outermost layer of the cornea, is richly innervated. Epithelial shedding results in severe irritation symptoms, such as eyelid redness and swelling, photophobia, tearing, and intense pain. Studies have shown that epithelial cell damage contributes to the pathogenesis of Pseudomonas aeruginosa keratosis. Furthermore, in diabetic patients, hyperglycemia delays corneal epithelial healing, leads to corneal nerve fiber degeneration, and increases the risk of corneal infection. Therefore, promoting corneal epithelial repair and preventing corneal infection are crucial after corneal epithelial defects occur.

[0003] Currently available medications for repairing corneal epithelial damage include recombinant bovine basic fibroblast growth factor eye drops and calf blood deproteinized basic extract ophthalmic gel. However, these medications have the following drawbacks: 1. They are relatively expensive, increasing the financial burden on patients. 2. Once opened, these medications must be used within one week, requiring strict storage conditions. 3. Improper storage and use can easily lead to environmental contamination and the growth of bacteria and other pathogens, potentially causing eye discomfort or even worsening infections with continued use. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide the application of calcium leucovorin in the preparation of corneal repair drugs. This invention uses calcium leucovorin as the active ingredient in a drug for treating corneal damage, exhibiting good therapeutic effects, and calcium leucovorin is inexpensive and easy to store.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides the application of calcium folinate in the preparation of drugs that promote corneal repair.

[0007] Preferably, the corneal repair-promoting drug is a corneal epithelial damage repair drug and / or a corneal nerve damage repair drug.

[0008] This invention provides a calcium folinate eye drop, the components of which include calcium folinate and physiological saline;

[0009] The calcium folinate content in the calcium folinate eye drops is 0.1% to 1% by mass.

[0010] Preferably, the ingredients also include one or more of acid-base regulators, osmotic pressure regulators, and thickeners.

[0011] Preferably, the acid-base regulator is one or more of sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, acetic acid, sodium acetate, boric acid, borates, borax, citric acid, sodium citrate, citric acid, and sodium citrate.

[0012] The osmotic pressure regulator is one or more of sodium chloride, potassium chloride, boric acid, borax, glucose, glycerol, propylene glycol, polyethylene glycol, and sodium hyaluronate.

[0013] Preferably, the thickener is one or more of propylene glycol, polyethylene glycol, sodium hyaluronate, methylcellulose, hydroxypropylcellulose, chondroitin sulfate, polyvinylpyrrolidone, polyvinyl alcohol, and chitosan.

[0014] Preferably, the pH value of the calcium folinate eye drops is 6-8.

[0015] Preferably, the osmotic pressure of the calcium folinate eye drops is 260–320 mOsm / L.

[0016] Preferably, the viscosity of the calcium folinate eye drops is 15-25 mps.

[0017] Preferably, the dosage of the calcium folinate eye drops is 0.1 to 15 mL.

[0018] This invention provides the application of calcium folinate in the preparation of drugs promoting corneal repair. Calcium folinate (CF or LV) is the calcium salt of a tetrahydrofolate calcium formyl derivative, and is the activated form of folic acid in vivo. Calcium folinate does not require reduction by dihydrofolate reductase and directly participates in the biological reaction that uses folic acid as a carrier for transferring "one-carbon groups" in vivo. It is commonly used as an antidote for methotrexate and aminopterin overdose, and its simultaneous use with fluorouracil enhances the latter's therapeutic effect. It can also be used to treat megaloblastic anemia caused by folic acid deficiency and to promote the differentiation, maturation, and release of bone marrow hematopoietic cells. Calcium folinate is a water-soluble vitamin that mainly participates in DNA synthesis and repair, and is crucial for maintaining normal cellular metabolism. This invention uses calcium folinate as the active ingredient in drugs for treating corneal damage, showing good therapeutic effects on corneal epithelial damage and corneal nerve damage. Moreover, calcium folinate is inexpensive, stable, and easy to store.

[0019] The results of the examples show that 0.1% leucovorin calcium eye drops can significantly accelerate the healing speed of corneal epithelial wounds; compared with the saline control group, the corneal sensitivity of mice in the experimental group that received 5 μL of 0.1% leucovorin calcium eye drops 5 times a day was significantly increased; 0.1% leucovorin calcium can significantly promote the regeneration of corneal peripheral nerves.

[0020] This invention provides a calcium folinate eye drop, comprising calcium folinate and physiological saline; the calcium folinate content in the eye drop is 0.1%–1% by mass. Compared with currently used clinical drugs for promoting corneal repair, the calcium folinate eye drop provided by this invention is inexpensive, which can reduce patients' medical expenses and alleviate their medical burden to a certain extent. At the same time, the calcium folinate component is simple, easy to store, and not easily deteriorated, thus avoiding the risk of infection that may result from continued use of existing corneal repair eye drops that have become contaminated and deteriorated. Attached Figure Description

[0021] Figure 1 Images showing corneal epithelial wound healing in the experimental and control groups;

[0022] Figure 2 A statistical chart of corneal epithelial wound healing;

[0023] Figure 3 A comparison chart of corneal sensitivity after corneal epithelial healing;

[0024] Figure 4 The corneal nerve repair in two groups of mice 4 days after corneal epithelial scraping.

[0025] Figure 5 This is a corneal density statistics graph. Detailed Implementation

[0026] This invention provides the application of calcium leucovorin in the preparation of corneal repair-promoting drugs. In this invention, the corneal damage drug is preferably a corneal epithelial damage repair drug and / or a corneal nerve damage repair drug.

[0027] This invention provides a calcium folinate eye drop, the components of which include calcium folinate and physiological saline;

[0028] The mass percentage of calcium folinate in the calcium folinate eye drops is 0.1-1%, preferably 0.2-0.8%, and more preferably 0.4-0.5%.

[0029] The folinic acid calcium eye drops provided by this invention preferably include one or more of the following: an acid-base regulator, an osmotic pressure regulator, and a thickener. In this invention, the pH value of the folinic acid calcium eye drops is preferably 6-8, more preferably 7; the osmotic pressure of the folinic acid calcium eye drops is preferably 260-320 mOsm / L, more preferably 280-300 mOsm / L; and the viscosity of the folinic acid calcium eye drops is preferably 15-25 mps, more preferably 18-20 mps.

[0030] In this invention, the pH adjuster is preferably one or more selected from sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, acetic acid, sodium acetate, boric acid, borate, borax, citric acid, sodium citrate, citric acid, and sodium citrate. This invention does not have specific requirements on the amount of the pH adjuster added; it is sufficient to make the pH of the folinic acid calcium eye drops 6-8.

[0031] In this invention, the osmotic pressure regulator is preferably one or more of sodium chloride, potassium chloride, boric acid, borax, glucose, glycerol, propylene glycol, polyethylene glycol, and sodium hyaluronate. This invention does not have specific requirements for the amount of the osmotic pressure regulator used; it is sufficient to make the osmotic pressure of the calcium folinate eye drops 260–320 mOsm / L.

[0032] In this invention, the thickener is preferably one or more of propylene glycol, polyethylene glycol, sodium hyaluronate, methylcellulose, hydroxypropylcellulose, chondroitin sulfate, polyvinylpyrrolidone, polyvinyl alcohol, and chitosan. This invention does not have specific requirements on the amount of the thickener used; it is sufficient to make the viscosity of the calcium folinate eye drops 15–25 mps.

[0033] In this invention, the dosage of the calcium folinate eye drops is preferably 0.1 to 15 mL, more preferably 0.1 to 1 mL or 1 to 15 mL.

[0034] In this invention, the method for preparing the calcium folinate eye drops preferably includes the following steps:

[0035] Calcium folinate and physiological saline are mixed to obtain calcium folinate eye drops.

[0036] In this invention, the leucovorin calcium is preferably injectable leucovorin calcium. As a specific embodiment of this invention, the injectable leucovorin calcium has a specification of 0.1g / 10mL, and is preferably manufactured by Hengrui, Lianyungang, China.

[0037] In this invention, during the mixing process, it is preferable to add one or more of the following: acid-base regulator, osmotic pressure regulator, and thickener.

[0038] The present invention preferably sterilizes the prepared calcium folinate eye drops. The present invention does not have specific requirements for the sterilization method; any sterilization method well known to those skilled in the art can be used. Preferably, the sterilization is filtration sterilization, and the filtration sterilization is preferably performed using a filtration sterilization filter with a pore size of 0.1–0.5 μm.

[0039] The following examples illustrate the application of calcium folinate provided by the present invention in the preparation of corneal repair drugs, but these examples should not be construed as limiting the scope of protection of the present invention.

[0040] Example 1: Corneal Epithelial Wound Healing Model

[0041] Commercially available calcium leucovorin (0.1 g, 10 mL, Hengrui, Lianyungang, China) was diluted to 0.1% with physiological saline. Store at 4°C protected from light.

[0042] Mice were anesthetized by intraperitoneal injection of ketamine and xylazine, followed by topical anesthesia with 2% promecaine hydrochloride eye drops. The central 2.5 mm diameter inner corneal epithelium was scraped off using an AlgerBrush II corneal epithelial scraper, with only one eye used per animal at a time. Corneal epithelial defects were observed at 0, 12, 24, and 36 hours after staining with 0.25% sodium fluorescein and imaging under a slit lamp. ImageJ software was used to analyze the stained areas and calculate the percentage of residual epithelial defect area compared to the original defect area at 0 hours. One hour after establishing the corneal epithelial wound healing model, mice in the experimental group were given 5 μL of 0.1% leucovorin ocular instillation, while the control group received the same dose of physiological saline, five times daily. The corneal epithelial wound healing models of the experimental and control groups are shown in the figures below. Figure 1 As shown in the figure. The results indicate that the experimental group healed faster than the control group.

[0043] Corneal epithelial wound healing was statistically analyzed using fluorescein-stained corneal slit-lamp photography at 0h, 12h, and 24h after corneal epithelial scraping, as shown in the following figure. Figure 2 As shown, it can be seen that 0.1% leucovorin calcium eye drops can significantly accelerate the healing speed of corneal epithelial wounds.

[0044] Example 2: Measurement of corneal sensitivity after corneal epithelial healing

[0045] Four days after corneal epithelial scraping in both experimental and control groups, corneal sensitivity in unanesthetized mice was measured using a Cochet-Bonnet esthesiometer. The test began at the maximum length of the nylon filament (6 cm), decreasing by 0.5 cm each time until the corneal sensory threshold was found. The maximum nylon filament length that elicited a positive response was considered the corneal sensory threshold and was verified three times.

[0046] A comparison of corneal sensitivity after corneal epithelial healing is shown in the figure below. Figure 3 As shown in the figure, the corneal sensitivity of the experimental group mice was significantly higher than that of the control group.

[0047] Example 3: Evaluation of corneal nerve regeneration

[0048] Mice were euthanized by injecting 0.6% sodium pentobarbital after corneal sensitivity measurement. The eyeballs were dissected and fixed with 4% fresh paraformaldehyde for 30 minutes at room temperature, followed by washing six times (5 minutes each) in 0.1M phosphate-buffered saline (0.1M) buffer (pH 7.4). 5% donkey normal serum and 0.3% Triton X-100 were added to 0.1M PBS, and the mixture was incubated overnight at 4°C. The entire cornea was then incubated with Alexa Flour 594 mouse monoclonal antibody III-tubulin antibody (1:200; Biolegend Antibody Services, San Diego, CA) in buffer for 4 hours, followed by thorough washing six times (10 minutes each) in 0.1M PBS. A radial incision was made in the cornea, and the incision was flattened with a coverslip. The cornea was placed flat on a glass slide (Citotest Labware Manufacturing Co. Ltd.), 50 μL of 0.1 M phosphate buffer was added, a coverslip was placed on top, and the cornea was examined and photographed using an LSM880 Zeiss inverted microscope (Carl Zeiss Meditec, Jena, Germany). Subsequently, the nerve density of the entire cornea was statistically analyzed using ImageJ.

[0049] The corneal nerve repair in the two groups of mice 4 days after corneal epithelium was scraped off is shown in the figure. Figure 4 As shown in the figure, the corneal nerve density in the experimental group mice was higher than that in the control group. The corneal density statistical graph is shown below. Figure 5 As shown in the figure, the corneal nerve density of the experimental group mice was significantly higher than that of the control group.

[0050] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of calcium folinate in the preparation of a corneal repair promoting drug; The corneal repair promoting drug is a corneal epithelial injury repair promoting drug; the corneal epithelial injury is a physical injury; The corneal repair promoting drug is a calcium folinate eye drop, and the components include calcium folinate and physiological saline; The mass percentage content of the calcium folinate in the calcium folinate eye drop is 0.1-1%.

2. Use according to claim 1, characterized in that, The components of the calcium folinate eye drop further include one or several of an acid-base regulator, an osmotic pressure regulator and a thickening agent.

3. Use according to claim 2, characterized in that, The acid-base regulator is one or several of sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, acetic acid, sodium acetate, boric acid, boric acid salt, borax, citric acid, sodium citrate, citric acid and sodium citrate; The osmotic pressure regulator is one or several of sodium chloride, potassium chloride, boric acid, borax, glucose, glycerol, propylene glycol, polyethylene glycol and sodium hyaluronate.

4. Use according to claim 2, characterized in that, The thickening agent is one or several of propylene glycol, polyethylene glycol, sodium hyaluronate, methyl cellulose, hydroxypropyl cellulose, chondroitin sulfate, polyvinyl pyrrolidone, polyvinyl alcohol and chitosan.

5. Use according to claim 1 or 2, characterized in that, The pH value of the calcium folinate eye drop is 6-8.

6. Use according to claim 1 or 2, characterized in that, The osmotic pressure of the calcium folinate eye drop is 260-320 mOsm / L.

7. Use according to claim 1 or 2, characterized in that, The viscosity of the calcium folinate eye drop is 15-25 mps.

8. Use according to claim 1 or 2, characterized in that, The filling specification of the calcium folinate eye drop is 0.1-15 mL.

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