An eye drop and a preparation method and application thereof

Abstract

The application relates to the field of ophthalmic drugs, and particularly discloses an eye drop, which comprises the following components in mass fraction: 0.003%-0.2% of chlorhexidine gluconate; and 0.025%-0.05% of chloramphenicol. Since acanthamoeba usually feeds on bacteria, the chlorhexidine gluconate is used as a main component to kill acanthamoeba in the application, and the antibiotic chloramphenicol is added, so that the keratitis caused by bacterial infection can be avoided, the ocular surface bacteria can be killed, the nutrition source of acanthamoeba can be reduced, and the treatment of acanthamoeba keratitis can be synergistically strengthened.

Description

Technical Field

[0001] This invention relates to the field of ophthalmic drugs, and more particularly to an eye drop, its preparation method, and its application. Background Technology

[0002] Acanthamoeba keratitis is an infectious corneal disease caused by the protozoan Acanthamoeba. Acanthamoeba is commonly found in contaminated soil or water sources and exists in two forms: trophozoites and cysts. The trophozoite, the active and infectious form of Acanthamoeba, is elongated oval in shape and, under suitable conditions, extends numerous spiny projections called spiny pseudopodia. Acanthamoeba typically feeds on bacteria and other microorganisms, reproducing by binary fission, with an average reproductive cycle of about 10 hours (6–24 hours). When environmental conditions are unsuitable, the trophozoite shrinks and secretes thick, double-walled cysts, forming cysts. Cysts are nearly round, with a smooth, polygonal inner wall and a often wrinkled outer wall. A spiny pore forms at the junction of the inner and outer walls, serving as the metabolic pathway for the cyst. Cysts are extremely resistant to the external environment, showing little sensitivity to common antibiotics, chlorides, and chemical disinfectants, and can survive for several years in natural environments. Cysts can float in the air. Cysts have been isolated from the nasopharynx of asymptomatic individuals. When environmental conditions are suitable, especially when nutrients are plentiful, the cysts will detach and transform into trophozoites within 3 days.

[0003] Because Acanthamoeba can transform into cysts, it is difficult to eradicate completely with conventional anti-infective drugs. If left untreated, Acanthamoeba keratitis can easily lead to blindness. However, there are currently no specific drugs in China effective against Acanthamoeba keratitis. Summary of the Invention

[0004] This invention provides an eye drop solution, its preparation method, and its application for treating Acanthamoeba keratitis. The objective of this invention is achieved through the following technical solution: An eye drop comprises the following components by mass fraction: 0.003%~0.2% chlorhexidine gluconate; 0.025%~0.05% chloramphenicol.

[0005] Optionally, the pH of the eye drops is 6.0~7.5 and the osmotic pressure is 280~320 mOsm / kg.

[0006] Optionally, the eye drops may also include a pH adjuster.

[0007] Optionally, the pH adjuster includes at least one of the following: phosphate, citrate, bicarbonate, borate, Tris, glycerol, mannitol, sorbitol, tromethamine, histidine, glycine, and 4-hydroxyethylpiperazine ethanesulfonic acid buffer.

[0008] Optionally, the eye drops may also include an osmotic pressure regulator.

[0009] Optionally, the osmotic pressure regulator includes at least one of sodium chloride and potassium chloride.

[0010] Optionally, the eye drops may also contain 0.05-0.15% sodium hyaluronate by mass.

[0011] The present invention also proposes a method for preparing the above-mentioned eye drops, comprising the following steps: Chlorhexidine gluconate and chloramphenicol were dissolved in water and mixed evenly to obtain the eye drops.

[0012] Optionally, the preparation method further includes adding a pH adjuster, sodium hyaluronate, and an osmotic pressure adjuster to the eye drops to adjust the pH of the eye drops to 6.0~7.5 and the osmotic pressure to 280~320 mOsm / kg.

[0013] The present invention also proposes the use of the above-mentioned eye drops in the preparation of medicaments for inhibiting Acanthamoeba keratosis and / or bacterial keratosis.

[0014] Compared with the prior art, the present invention has the following beneficial effects: This invention provides an eye drop comprising the following components by mass fraction: 0.025%~0.2% chlorhexidine gluconate; 0.025%~0.05% chloramphenicol. Since Acanthamoeba often feeds on bacteria, this invention, while using chlorhexidine gluconate as the main component to kill Acanthamoeba, also adds the antibiotic chloramphenicol. This not only avoids keratitis caused by bacterial infection but also kills bacteria on the ocular surface, reducing the nutrient source for Acanthamoeba and achieving the treatment of Acanthamoeba keratitis.

[0015] Both chlorhexidine gluconate and chloramphenicol are acidic to neutral chemicals (chlorhexidine gluconate pH 7, chloramphenicol pH 4.5-7.5). Adding appropriate buffer solutions and excipients like sodium hyaluronate to the eye drops stabilizes the pH at 6.0-7.5, maximizing the drug's effectiveness while minimizing irritation to the ocular surface. To maintain the same osmotic pressure between the eye drops and corneal cells, preventing dehydration or edema, an appropriate osmotic pressure regulator is added to stabilize the osmotic pressure at 280-320 mOsm / kg, thus creating an isotonic solution. Attached Figure Description

[0016] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings required for the specific embodiments or the prior art are briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 To detect the toxic effects of different combination drugs on corneal epithelial cells at different time points using the CCK-8 assay.

[0018] Figure 2 The results of toxicity tests on rabbit eye surfaces are presented for each of the embodiments and comparative examples. Detailed Implementation

[0019] Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is only for describing particular embodiments and is not intended to limit the present invention.

[0020] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] This invention provides an eye drop solution comprising the following components by mass fraction: 0.025%~0.2% chlorhexidine gluconate; 0.025%~0.05% chloramphenicol.

[0024] Chlorhexidine gluconate, also known as chlorhexidine gluconate, has the chemical formula C. 22 H 30 Cl2N 10 .2C6H 12O7 is an organic compound and a broad-spectrum bactericide, with the following structural formula:

[0025] Chloramphenicol, chemical formula C 11 H 12 Cl2N2O5 is an antibacterial antibiotic. ， Its aqueous solution remains effective even after boiling for 5 hours. The structural formula is as follows:

[0026] Since Acanthamoeba often feeds on bacteria, this invention uses chlorhexidine gluconate as the main ingredient to kill Acanthamoeba while adding the antibiotic chloramphenicol. This can not only avoid keratitis caused by bacterial infection, but also kill bacteria on the ocular surface, reduce the nutrient source of Acanthamoeba, and achieve the treatment of Acanthamoeba keratitis.

[0027] The eye drops also include a pH adjuster to adjust the pH value of the eye drops to 6.0-7.5. The pH adjuster includes at least one of the following: phosphate, citrate, bicarbonate, borate, Tris, glycerin, mannitol, sorbitol, tromethorphan, histidine, glycine, and 4-hydroxyethylpiperazine ethanesulfonic acid buffer; the excipient includes 0.1% sodium hyaluronate. Both chlorhexidine gluconate and chloramphenicol are acidic to neutral chemicals. Adding an appropriate buffer solution and the excipient sodium hyaluronate to the eye drops will stabilize the pH at 6.0-7.5, maximizing the drug's effectiveness while avoiding irritation to the ocular surface.

[0028] The eye drops also include an osmotic pressure regulator, which is used to adjust the osmotic pressure of the eye drops to 280~320 mOsm / kg. The osmotic pressure regulator includes at least one of sodium chloride and potassium chloride. To maintain the same osmotic pressure between the eye drops and the cells in the corneal tissue, and to avoid adverse reactions such as corneal dehydration or edema, an appropriate osmotic pressure regulator is added to the eye drops.

[0029] The eye drops also contain 0.1% sodium hyaluronate.

[0030] The present invention also proposes the use of the above-mentioned eye drops in the preparation of medicaments for inhibiting Acanthamoeba keratosis and / or bacterial keratosis.

[0031] Example 1 The method for preparing the eye drops in this embodiment includes: The drug was prepared using a 20% aqueous solution of chlorhexidine gluconate, chloramphenicol (powder), sodium hyaluronate, and deionized water. The mass fraction of chlorhexidine gluconate in the solution was 0.003%, the mass fraction of chloramphenicol was 0.025%, and the mass fraction of sodium hyaluronate was 0.1%. Sodium chloride was used as an isotonic agent to adjust the osmotic pressure to 320 mOsm / kg; the pH was adjusted to 7.0 using phosphate buffer. The prepared composition was then filtered through a 0.22 μm filter for sterilization to obtain the drug solution.

[0032] Example 2 The method for preparing the eye drops in this embodiment includes: The drug was prepared using a 20% aqueous solution of chlorhexidine gluconate, chloramphenicol (powder), sodium hyaluronate, and deionized water. The mass fraction of chlorhexidine gluconate in the solution was 0.0625%, the mass fraction of chloramphenicol was 0.025%, and the mass fraction of sodium hyaluronate was 0.1%. Sodium chloride was used as an isotonic agent to adjust the osmotic pressure to 320 mOsm / kg; the pH was adjusted to 7.0 using phosphate buffer. The prepared composition was then filtered through a 0.22 μm filter for sterilization to obtain the drug solution.

[0033] Example 3 The method for preparing the eye drops in this embodiment includes: The drug was prepared using a 20% aqueous solution of chlorhexidine gluconate, chloramphenicol (powder), sodium hyaluronate, and deionized water. The mass fraction of chlorhexidine gluconate in the solution was 0.0125%, the mass fraction of chloramphenicol was 0.025%, and the mass fraction of sodium hyaluronate was 0.1%. Sodium chloride was used as an isotonic agent to adjust the osmotic pressure to 320 mOsm / kg; the pH was adjusted to 7.0 using phosphate buffer. The prepared composition was then filtered through a 0.22 μm filter for sterilization to obtain the drug solution.

[0034] Example 4 The method for preparing the eye drops in this embodiment includes: The drug was prepared using a 20% aqueous solution of chlorhexidine gluconate, chloramphenicol (powder), sodium hyaluronate, and deionized water. The mass fractions of chlorhexidine gluconate, chloramphenicol, and sodium hyaluronate in the solution were 0.025% and 0.025% respectively. Sodium chloride was used as an isotonic agent to adjust the osmotic pressure to 320 mOsm / kg. The pH was adjusted to 7.0 using phosphate buffer. The prepared composition was then filtered through a 0.22 μm filter for sterilization to obtain the drug solution.

[0035] Example 5 The method for preparing the eye drops in this embodiment includes: The drug was prepared using a 20% aqueous solution of chlorhexidine gluconate, chloramphenicol (powder), sodium hyaluronate, and deionized water. The mass fraction of chlorhexidine gluconate in the solution was 0.05%, the mass fraction of chloramphenicol was 0.025%, and the mass fraction of sodium hyaluronate was 0.1%. Sodium chloride was used as an isotonic agent to adjust the osmotic pressure to 320 mOsm / kg. The pH was adjusted to 7.0 using phosphate buffer. The prepared composition was then filtered through a 0.22 μm filter for sterilization to obtain the drug solution.

[0036] Example 6 The method for preparing the eye drops in this embodiment includes: The drug was prepared using a 20% aqueous solution of chlorhexidine gluconate, chloramphenicol (powder), sodium hyaluronate, and deionized water. The mass fractions of chlorhexidine gluconate, chloramphenicol, and sodium hyaluronate in the solution were 0.05% and 0.05% respectively. Sodium chloride was used as an isotonic agent to adjust the osmotic pressure to 320 mOsm / kg. The pH was adjusted to 7.0 using phosphate buffer. The prepared composition was then filtered through a 0.22 μm filter for sterilization to obtain the drug solution.

[0037] Example 7 The method for preparing the eye drops in this embodiment includes: The drug was prepared using a 20% aqueous solution of chlorhexidine gluconate, chloramphenicol (powder), sodium hyaluronate, and deionized water. The mass fraction of chlorhexidine gluconate in the solution was 0.1%, the mass fraction of chloramphenicol was 0.025%, and the mass fraction of sodium hyaluronate was 0.1%. Sodium chloride was used as an isotonic agent to adjust the osmotic pressure to 320 mOsm / kg; the pH was adjusted to 7.0 using phosphate buffer. The prepared composition was then filtered through a 0.22 μm filter for sterilization to obtain the drug solution.

[0038] Example 8 The method for preparing the eye drops in this embodiment includes: The drug was prepared using a 20% aqueous solution of chlorhexidine gluconate, chloramphenicol (powder), sodium hyaluronate, and deionized water. The mass fraction of chlorhexidine gluconate in the solution was 0.2%, the mass fraction of chloramphenicol was 0.025%, and the mass fraction of sodium hyaluronate was 0.1%. Sodium chloride was used as an isotonic agent to adjust the osmotic pressure to 320 mOsm / kg; the pH was adjusted to 7.0 using phosphate buffer. The prepared composition was then filtered through a 0.22 μm filter for sterilization to obtain the drug solution.

[0039] Comparative Example 1 The preparation method of the eye drops in this comparative example includes: The drug was prepared using sodium hyaluronate and deionized water, with a sodium hyaluronate mass fraction of 0.1% in the solution. Sodium chloride was used as an isotonic agent to adjust the osmotic pressure to 320 mOsm / kg; the pH was adjusted to 7.0 using phosphate buffer. The prepared composition was then filtered through a 0.22 μm filter for sterilization to obtain the drug solution.

[0040] Comparative Example 2 The preparation method of the eye drops in this comparative example includes: The drug solution was prepared using chloramphenicol (powder), sodium hyaluronate, and deionized water. The mass fraction of chloramphenicol in the solution was 0.025%, and the mass fraction of sodium hyaluronate was 0.1%. Sodium chloride was used as an isotonic agent to adjust the osmotic pressure to 320 mOsm / kg. The pH was adjusted to 7.0 using phosphate buffer. The prepared composition was then filtered through a 0.22 μm filter for sterilization to obtain the drug solution.

[0041] Comparative Example 3 The preparation method of the eye drops in this comparative example includes: The drug was prepared using a 20% aqueous solution of chlorhexidine gluconate, sodium hyaluronate, and deionized water. The mass fraction of chlorhexidine gluconate in the solution was 0.03%, and the mass fraction of sodium hyaluronate was 0.1%. Sodium chloride was used as an isotonic agent to adjust the osmotic pressure to 320 mOsm / kg. The pH was adjusted to 7.0 using phosphate buffer. The prepared composition was then filtered through a 0.22 μm filter for sterilization to obtain the drug solution.

[0042] Comparative Example 4 The preparation method of the eye drops in this comparative example includes: The drug was prepared using a 20% chlorhexidine gluconate aqueous solution, chloramphenicol (powder), sodium hyaluronate, and deionized water. The mass fraction of chlorhexidine gluconate in the solution was 0.05%, the mass fraction of chloramphenicol was 0.025%, and the mass fraction of sodium hyaluronate was 0.1%. Sodium chloride was used as an isotonic agent to adjust the osmotic pressure to 320 mOsm / kg; the pH was adjusted to 9.0 using phosphate buffer. The prepared composition was then filtered through a 0.22 μm filter for sterilization to obtain the drug solution.

[0043] Comparative Example 5 The preparation method of the eye drops in this comparative example includes: The drug was prepared using a 20% aqueous solution of chlorhexidine gluconate, chloramphenicol (powder), sodium hyaluronate, and deionized water. The mass fraction of chlorhexidine gluconate in the solution was 0.05%, the mass fraction of chloramphenicol was 0.025%, and the mass fraction of sodium hyaluronate was 0.1%. Sodium chloride was used as an isotonic agent to adjust the osmotic pressure to 320 mOsm / kg; the pH was adjusted to 5.0 using phosphate buffer. The prepared composition was then filtered through a 0.22 μm filter for sterilization to obtain the drug solution.

[0044] Comparative Example 6 The preparation method of the eye drops in this comparative example includes: The drug was prepared using a 20% aqueous solution of chlorhexidine gluconate, chloramphenicol (powder), sodium hyaluronate, and deionized water. The mass fraction of chlorhexidine gluconate in the solution was 1%, the mass fraction of chloramphenicol was 0.025%, and the mass fraction of sodium hyaluronate was 0.1%. Sodium chloride was used as an isotonic agent to adjust the osmotic pressure to 320 mOsm / kg. The pH was adjusted to 7.0 using phosphate buffer. The prepared composition was then filtered through a 0.22 μm filter for sterilization to obtain the drug solution.

[0045] Comparative Example 7 The preparation method of the eye drops in this comparative example includes: The drug solution was prepared using ofloxacin (powder), sodium hyaluronate, and deionized water. The mass fraction of ofloxacin in the solution was 0.025%, and the mass fraction of sodium hyaluronate was 0.1%. Sodium chloride was used as an isotonic agent to adjust the osmotic pressure to 320 mOsm / kg. The pH was adjusted to 7.0 using phosphate buffer. The prepared composition was then filtered through a 0.22 μm filter for sterilization to obtain the drug solution.

[0046] Comparative Example 8 The preparation method of the eye drops in this comparative example includes: The drug was prepared using a 20% aqueous solution of chlorhexidine gluconate, ofloxacin (powder), sodium hyaluronate, and deionized water. The mass fraction of chlorhexidine gluconate in the solution was 0.03%, ofloxacin was 0.025%, and sodium hyaluronate was 0.1%. Sodium chloride was used as an isotonic agent to adjust the osmotic pressure to 320 mOsm / kg; the pH was adjusted to 7.0 using phosphate buffer. The prepared composition was then filtered through a 0.22 μm filter for sterilization to obtain the drug solution.

[0047] Experiment Example 1: Chlorhexidine Gluconate Eye Drops Anti-Acanthamoeba and Bacterial Experiment Chlorhexidine gluconate eye drops anti-Acanthamoeba test: Using 100 μL of the above drug composition, apply 1 × 10⁻⁶ doses respectively. 4After 48 hours, amoeba trophozoites and cysts were centrifuged at 1500 rpm for 5 minutes to remove the composition. The mixture was then washed with Ringer's solution and centrifuged at 1500 rpm for 5 minutes, repeated three times. A suspension of amoeba bodies in E. coli at a concentration of 0.5 McFarland units was then transferred to a nutrient-free culture medium and incubated at 30°C for 7 days. Evaluation method: Observe for the presence of amoeba growth in the culture medium.

[0048] Table 1. Inhibitory effects of different drug combinations on Acanthamoeba trophozoites and cysts

[0049] In the table, (+) indicates amoeba growth; (-) indicates no amoeba growth.

[0050] According to the results in Table 1, after 48 hours of drug treatment, amoebic trophozoites and cysts grew in comparative groups 1, 2, 3, 7, and 8, while no amoebic growth was observed in the other groups. The comparison shows that the combined use of chlorhexidine and chloramphenicol significantly reduces the dosage of chlorhexidine; an amoeba and its cysts can be eliminated at a chlorhexidine concentration of 0.003%. In contrast, the comparative groups, where chlorhexidine alone at 0.03%, still failed to eliminate the amoeba and its cysts. Therefore, the eye drops of this invention achieve good therapeutic effects using a lower concentration of chlorhexidine, thus improving the safety of the eye drops.

[0051] Furthermore, the chloramphenicol and chlorhexidine in this invention work well together, and when other antibiotics are used to replace chloramphenicol, they cannot achieve the effect of eliminating amoebae and their cysts.

[0052] Antibacterial efficacy test of chlorhexidine gluconate and its combination with 0.25% chloramphenicol: Stock solutions were prepared using 20% ​​chlorhexidine gluconate aqueous solution and chloramphenicol (powder) at concentrations of 0.00156%, 0.00313%, 0.00625%, 0.0125%, 0.025%, 0.05%, 0.1%, 0.2%, and 0.4% chlorhexidine gluconate, as well as stock solutions of chlorhexidine gluconate and chloramphenicol at concentrations 10 times higher than the corresponding concentrations.

[0053] Pseudomonas aeruginosa and Staphylococcus aureus were inoculated onto blood agar basal medium and incubated at 35°C for 18-24 hours. A single colony was used to prepare a 0.5 McFarland unit bacterial suspension, which was then diluted 1000-fold with sterile phosphate buffer to obtain 10... 6 CFU / mL bacterial suspension was prepared, with 180 μL of the suspension added to three wells of a 96-well plate. 20 μL of a 10-fold effective concentration of chlorhexidine gluconate was added to the bacterial suspension, and the plate was incubated at 35°C for 16 h. After incubation, 10 μL of the bacterial suspension was added to soybean tryptone agar and incubated at 35°C for 24 h.

[0054] Evaluation method: The lowest MIC concentration that effectively inhibits 90% of bacterial growth. 90 .

[0055] The results showed that for Staphylococcus aureus, the MIC of chlorhexidine gluconate alone and chlorhexidine gluconate combined with 0.25% chloramphenicol was [not specified]. 90 All <0.00156%. For Pseudomonas aeruginosa, the MIC of chlorhexidine gluconate alone... 90 The MIC of chlorhexidine gluconate combined with 0.25% chloramphenicol was 0.0125%. 90 The concentration was 0.00625%. Therefore, the combination of chlorhexidine gluconate and chloramphenicol can reduce the effective antibacterial concentration.

[0056] Since Acanthamoeba often feeds on bacteria, this invention uses chlorhexidine gluconate as the main ingredient to kill Acanthamoeba while adding the antibiotic chloramphenicol. This can not only avoid keratitis caused by bacterial infection, but also kill bacteria on the ocular surface, reduce the nutrient source of Acanthamoeba, and synergistically enhance the treatment of Acanthamoeba keratitis.

[0057] Experiment Example 2: CCK-8 Cytotoxicity Experiment with Eye Drops Eye drops were prepared using 20% ​​chlorhexidine gluconate aqueous solution and chloramphenicol (powder) according to the methods in Examples 1, 2, 3, 4, 5, 7, and 8, respectively. The prepared compositions were then filtered through a 0.22 μm filter for sterilization.

[0058] Immortalized human corneal epithelial cells were cultured to 80-90% confluence, digested with 0.25% trypsin, and centrifuged at 1200 rpm for 3 minutes to prepare 1×10⁻⁶ cells / day. 5 Cell suspensions at a concentration of [cells / mL] were seeded in 96-well plates, with 100 μL of the suspension seeded and incubated at 37°C for 24 h in a CO2 cell culture incubator. After washing twice with sterile phosphate buffer, different formulations were added and incubated for 12 h and 24 h, respectively. A control group was prepared with 5% glucose buffer, with four replicates per group. After incubation, the composition was removed, and the cells were washed twice with sterile phosphate buffer. 100 μL of cell culture medium and 10 μL of CCK-8 solution were added, and the cells were incubated at 37°C in a CO2 cell culture incubator for 1 h. The absorbance at 450 nm was measured using a microplate reader.

[0059] Evaluation method: Compare the absorbance at 450nm of different groups.

[0060] according to Figure 1 The results showed that different drug combinations had no toxic effect on corneal epithelial cells at 12 and 24 hours.

[0061] Experimental Example 3: Safety Test of Eye Drops for Rabbit Corneal Conjunctiva Eye drops from Examples 1, 2, 3, 4, 5, 7, 8, and Comparative Example 6 were used to conduct combined drug eye drop experiments on 24 New Zealand white rabbits (48 eyes) to observe the safety of the combined drugs on the rabbit conjunctiva and cornea. Three rabbits received each combination drug, applied to the right eye; simultaneously, 5% glucose injection was used as a control, applied to the left eye. The drops were applied once every hour, eight times a day, for one week. The conjunctiva and cornea were observed using a slit-lamp microscope before application and on days 1, 3, and 7 after application.

[0062] Evaluation method: Assess the degree of conjunctival hyperemia, secretions, and corneal epithelial integrity after instillation of medication.

[0063] according to Figure 2 The results showed that after frequent eye drops of different concentrations of combined drugs on New Zealand white rabbits, groups 1, 2, 3, 4, 5, 7, and 8 showed no obvious conjunctival hyperemia or discharge, and the corneal epithelium remained intact without significant damage. In contrast, control group 6 showed eyelid swelling, conjunctival hyperemia, increased discharge, and rough corneal epithelium one day after drug application, and corneal ulcers were visible seven days after application. Therefore, 0.20% chlorhexidine gluconate + 0.025% chloramphenicol and lower concentrations have good ocular surface safety, while 1% chlorhexidine gluconate + 0.025% chloramphenicol has strong corneal and conjunctival toxicity.

[0064] Example 5: Stability Test of Eye Drops Chlorhexidine gluconate determination method: Chromatographic conditions: C8 column (150 mm × 4.6, 5 µm), mobile phase: 1% triethylamine-methanol-glacial acetic acid (50:75:0.4), flow rate: 1 mL / min, detection wavelength: 259 nm, column temperature: 28 ℃, injection volume: 20 µL.

[0065] Chlorhexidine gluconate test solution: Accurately weigh two vials of eye drops (approximately equivalent to 2 mg of chlorhexidine gluconate) into a 125 mL separatory funnel. Extract three times with chloroform (20, 15, 15 mL). Combine the chloroform phases and extract three times with 2 mol / L acetic acid solution (20, 15, 15 mL). Combine the aqueous phases and dilute to 100 mL in a volumetric flask with 2 mol / L acetic acid solution. Reference solution: Accurately weigh 25 mg of chlorhexidine gluconate reference standard into a 100 mL volumetric flask. Add 75 mL of 2 mol / L acetic acid solution to dissolve. Shake well and add 2 mol / L acetic acid solution to the mark. Shake well again.

[0066] Chloramphenicol determination method: A chromatographic condition and system suitability test was conducted, using octadecylsilane-bonded silica gel as the stationary phase; a mobile phase of acetonitrile (75:25) containing 0.1% sodium heptanesulfonate solution (500 mL of 0.1% sodium heptanesulfonate solution mixed with 5 mL of dimethylformamide and 0.5 mL of glacial acetic acid); a detection wavelength of 272 nm; appropriate amounts of chloramphenicol reference standard and chloramphenicol diol reference standard were dissolved in methanol and diluted with the mobile phase to prepare a solution containing 50 μg of each per mL. 10 μL of each solution was injected into the liquid chromatograph, and the chromatogram was recorded. The theoretical plate number, calculated based on the chloramphenicol peak, should not be less than 2500, and the resolution between the diol peak and the chloramphenicol peak should be greater than 2.0.

[0067] Chloramphenicol test solution: Dissolve approximately 25 mg of the above chloroform phase concentrate in 5 mL of methanol, then quantitatively dilute with the mobile phase to prepare a solution containing 0.1 mg per mL. Shake well, accurately measure 10 μL, inject into the liquid chromatograph, and record the chromatogram. Reference solution: Accurately weigh an appropriate amount of chloramphenicol reference standard, prepare and determine using the same method. Calculate the concentration of C in the test solution based on peak area using the external standard method. 11 H 12 The content of Cl2N2O5.

[0068] Table 2. Stability of chloramphenicol and chlorhexidine gluconate

[0069] The results in Table 2 show that chloramphenicol and chlorhexidine gluconate solutions exhibit excellent stability. Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. An eye drop, characterized in that, Components include the following mass fractions: 0.003%~0.2% chlorhexidine gluconate; 0.025%~0.05% chloramphenicol.

2. The eye drops according to claim 1, characterized in that, The eye drops have a pH of 6.0 to 7.5 and an osmotic pressure of 280 to 320 mOsm / kg.

3. The eye drops according to claim 2, characterized in that, The eye drops also include a pH adjuster.

4. The eye drops according to claim 3, characterized in that, The pH adjuster includes at least one of the following: phosphate, citrate, bicarbonate, borate, Tris, glycerol, mannitol, sorbitol, tromethamine, histidine, glycine, and 4-hydroxyethylpiperazine ethanesulfonic acid buffer.

5. The eye drops according to claim 2, characterized in that, The eye drops also include an osmotic pressure regulator.

6. The eye drops according to claim 5, characterized in that, The osmotic pressure regulator includes at least one of sodium chloride and potassium chloride.

7. The eye drops according to claim 1, characterized in that, The eye drops also contain 0.05-0.15% sodium hyaluronate by mass.

8. The method for preparing eye drops according to claim 1, characterized in that, Includes the following steps: Chlorhexidine gluconate and chloramphenicol were dissolved in water and mixed evenly to obtain the eye drops.

9. The method for preparing eye drops according to claim 8, characterized in that, It also includes adding a pH adjuster, sodium hyaluronate, and an osmotic pressure adjuster to the eye drops to adjust the pH of the eye drops to 6.0~7.5 and the osmotic pressure to 280~320 mOsm / kg.

10. The use of the eye drops according to any one of claims 1 to 7 in the preparation of a medicament for inhibiting Acanthamoeba keratitis and / or bacterial keratitis.

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