WO2024114360A1 - Utilisation de ns le traitement de maladies de la surface oculaire - Google Patents

Utilisation de ns le traitement de maladies de la surface oculaire Download PDF

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WO2024114360A1
WO2024114360A1 PCT/CN2023/131428 CN2023131428W WO2024114360A1 WO 2024114360 A1 WO2024114360 A1 WO 2024114360A1 CN 2023131428 W CN2023131428 W CN 2023131428W WO 2024114360 A1 WO2024114360 A1 WO 2024114360A1
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dihydroquercetin
group
ocular surface
cells
corneal
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PCT/CN2023/131428
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English (en)
Chinese (zh)
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张东蕾
袁振丽
安元龙
王敏楠
龚琪
何向东
何伟
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辽宁何氏医学院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants

Definitions

  • the invention relates to the technical field of pharmaceuticals, and in particular to application of dihydroquercetin in treating ocular surface diseases.
  • Oxidative damage is widely present in diseases, especially playing an important role in aging and inflammation-related diseases.
  • the free radicals produced by oxidation can directly act on tissue cell membranes and destroy them. Free radicals can also enter cells through channels on the cell membranes, causing damage to proteins and DNA in cells.
  • Oxygen free radicals participate in the metabolism of arachidonic acid, which is an important process in the inflammatory response.
  • the lipid peroxides produced are chemokines, which can aggravate the inflammatory response.
  • oxidation products can also induce the production of chemokines different from arachidonic acid, inactivate protease inhibitors, increase collagenase, etc., and thus destroy connective tissue.
  • Common eye diseases caused by oxidative damage include dry eye and corneal alkali burns.
  • Dry eye is a common disease in ophthalmology. It mainly refers to a disease caused by insufficient tears or excessive tear evaporation, which is caused by multiple factors such as tear stability and ocular surface inflammation.
  • researchers at home and abroad have conducted in-depth research on the pathogenesis of dry eye, and believe that the occurrence and development of the disease is related to the body's immune inflammatory response, cell apoptosis and sex hormone levels.
  • T cell-mediated immune inflammatory response is the most critical factor in the onset of dry eye.
  • the decrease in tear secretion or increase in evaporation leads to high osmotic pressure in tears, which is also a key factor in causing the vicious cycle of dry eye symptoms.
  • the high osmotic pressure of tears can cause morphological changes such as apoptosis of conjunctival epithelial cells and a decrease in the number of functional goblet cells, and trigger an inflammatory cascade reaction, further causing the death of corneal epithelial cells.
  • morphological changes such as apoptosis of conjunctival epithelial cells and a decrease in the number of functional goblet cells, and trigger an inflammatory cascade reaction, further causing the death of corneal epithelial cells.
  • the content of mucin and lipids in tears will be reduced, thereby aggravating the instability of the tear film and promoting this vicious cycle.
  • the disease causes varying degrees of dry eyes, foreign body sensation, photophobia and pain. In severe cases, corneal damage occurs, and the patient's visual function is threatened. Therefore, clinical treatment research on dry eye is imminent.
  • Corneal alkali burns are a common type of ocular trauma in clinical practice, with a very high blindness rate.
  • cell apoptosis and activation of inflammatory response are important factors causing corneal damage; in the late stage of alkali burns, corneal neovascularization will seriously affect corneal transparency and visual acuity.
  • choroidal neovascularization CNV of the fundus
  • the formation of CNV is a complex pathological process, which is regulated by multiple factors.
  • the growth factor network formed by many cytokines precisely regulates the formation of CNV.
  • Vascular endothelial growth factor (VEGF) is the strongest angiogenic factor discovered so far.
  • VEGF vascular endothelial growth factor
  • glucocorticoids include Cushing's syndrome, infection, gastrointestinal reaction, edema, glucose metabolism disorder, electrolyte imbalance, abnormal nervous system excitement, etc.
  • anti-VEGF drugs include conjunctival congestion, eye pain, foreign body sensation, corneal abrasion, corneal edema, increased intraocular pressure, floating black shadows, intraocular infection, retinal detachment or vitreous hemorrhage, etc.
  • the technical problem to be solved by the present invention is to provide a use of dihydroquercetin in treating ocular surface diseases.
  • the dihydroquercetin has a protective effect on H 2 O 2- induced oxidative stress damage of corneal epithelial cells (HCE).
  • the present invention provides the use of dihydroquercetin in preparing a medicine for preventing, alleviating and/or treating ocular surface diseases.
  • Dihydroquercetin also known as taxifolin, exists in many plants, and its content is higher in larch, especially Douglas fir. Dihydroquercetin was first extracted and separated from the leaves of the conifer Chamaecyparisobtusa by Japanese scholar Fukui. In recent years, dihydroquercetin has also been found in many fruits, such as grapes, oranges and grapefruits. Studies have shown that dihydroquercetin contains more phenolic hydroxyl groups, has multiple biological activities, and can inhibit or activate multiple enzymes, thereby producing different physiological effects. The structural formula of dihydroquercetin is as follows:
  • the present invention applies dihydroquercetin to the prevention, alleviation and/or treatment of ocular surface diseases for the first time.
  • the ocular surface disease is an ocular surface disease caused by corneal epithelial cell damage and/or apoptosis.
  • the corneal epithelial damage is oxidative damage.
  • the oxidative damage is damage caused by hydrogen peroxide.
  • the dihydroquercetin of the present invention has a protective effect on oxidative stress damage of corneal epithelial cells (HCE) induced by H 2 O 2 .
  • dihydroquercetin has a proliferation effect on HCE cells and can increase the relative cell viability of HCE cells after oxidative damage.
  • the present invention establishes a H2O2 - induced corneal epithelial cell (HCE) damage model, determines the H2O2 modeling concentration, then performs modeling at this concentration to cause oxidative damage to HCE cells, and finally acts on HCE cells with different concentrations of dihydroquercetin. It is found that a certain concentration of dihydroquercetin has a proliferation effect on HCE cells, while too high a concentration will produce certain toxicity to HCE cells.
  • HCE corneal epithelial cell
  • the modeling concentration is specifically 300 ⁇ M.
  • the concentration of dihydroquercetin having a proliferation effect on HCE cells is 25-200 ⁇ M, and the concentration of dihydroquercetin that can produce a certain degree of toxicity to HCE cells is 600 ⁇ M.
  • the ocular surface disease of the present invention is dry eye or a disease related to corneal neovascularization.
  • the present invention also provides a dihydroquercetin ophthalmic preparation, comprising dihydroquercetin and a solvent.
  • the mass concentration of the dihydroquercetin is 0.01% to 0.5%; more preferably, the mass concentration of the dihydroquercetin is 0.03% to 0.3%; further preferably, the mass concentration of the dihydroquercetin is 0.03% or 0.3%.
  • the preparation is in the form of eye drops, eye ointment, periocular and intraocular injection, eye gel or liposome.
  • the present invention provides the use of dihydroquercetin in the preparation of a drug for preventing, alleviating and/or treating ocular surface diseases.
  • the present invention applies dihydroquercetin to the prevention, alleviating and/or treating ocular surface diseases for the first time.
  • the dihydroquercetin has a good protective effect on diseases related to oxidative damage of corneal epithelial cells (HCE).
  • HCE corneal epithelial cells
  • the dihydroquercetin can promote the proliferation of HCE cells and improve cell viability, and can also increase the tear secretion and tear film stability of HCE cells to significantly improve ocular surface diseases.
  • Figure 1 is a graph showing the results of using the MTS method to detect different concentrations of dihydroquercetin (A), different concentrations of H 2 O 2 (B), and a combination of 300 ⁇ M hydrogen peroxide and different concentrations of dihydroquercetin (C) after acting on corneal epithelial cells for 24 hours, wherein P represents a significant level, ** represents P ⁇ 0.01, *** represents P ⁇ 0.001, ## represents P ⁇ 0.01, and ### represents P ⁇ 0.001;
  • FIG2 is a diagram showing the results of tear secretion testing of live mice in animal experiments
  • FIG3 is a diagram showing the tear film breakup time test results of live mice in animal experiments.
  • FIG4 is a diagram of the corneal morphology of mice in each experimental group after corneal fluorescein sodium staining
  • FIG5 is a graph showing the scoring results of corneal epithelial damage in mice examined by corneal sodium fluorescein staining
  • FIG6 is a morphological diagram of new blood vessels on the cornea of New Zealand rabbits in each experimental group detected by corneal slit lamp;
  • FIG. 7 is a graph showing the results of the detection of corneal neovascularization areas in New Zealand rabbits in each experimental group, where **** represents P ⁇ 0.0001.
  • dihydroquercetin provided by the present invention in treating ocular surface diseases is described in detail below in conjunction with examples.
  • Human corneal epithelial cells were purchased from the ATCC cell bank in the United States.
  • the cell culture medium used was high-glucose DMEM medium supplemented with 10% (V/V) fetal bovine serum, 100U/mL penicillin, and 100 ⁇ g/mL gentamicin.
  • the cells were cultured in an incubator containing 5% carbon dioxide at 37°C.
  • the cells were plated in a 96-well plate and the cells were used in the logarithmic growth phase.
  • the cells were plated at a density of 1.5 ⁇ 10 5 cells/mL, 100 ⁇ L per well, and 6 duplicate wells per group.
  • the cells were grouped into normal cell group, hydrogen peroxide model group, and dihydroquercetin intervention groups at different concentrations.
  • the detection method was the MTS kit method (represented by Promega), and the absorbance was detected by a microplate reader at 490 nm.
  • Survival rate survival rate of each group / survival rate of normal cell group ⁇ 100%
  • Human corneal epithelial cells were purchased from the ATCC cell bank in the United States.
  • the cell culture medium used high-glucose DMEM medium supplemented with 10% (V/V) fetal bovine serum, 100U/mL penicillin, and 100 ⁇ g/mL gentamicin.
  • the cells were cultured in an incubator containing 5% carbon dioxide at 37°C.
  • the cells were plated in a 96-well plate. Cells in the logarithmic growth phase were plated at a density of 1.5 ⁇ 10 5 /mL, 100 ⁇ L per well, and 6 wells per group. After 24 hours, the cells were treated with H 2 O 2 modeling or dihydroquercetin administration according to the purpose of the experiment.
  • Color development 20 ⁇ L of MTS solution was added to each well and incubated for 2 to 4 hours.
  • Colorimetry Select a wavelength of 490nm, measure the light absorption value of each well on an enzyme-linked immunosorbent monitor, record the results, and draw a cell growth curve with time as the horizontal axis and absorbance as the vertical axis.
  • HCE human corneal epithelial cell
  • HCE cells were subcultured, and the plate was dripped at 1.5 ⁇ 10 5 cell/mL, 100 ⁇ L/well. HCE cells were treated with different concentrations of H 2 O 2 (100 ⁇ M, 200 ⁇ M, 300 ⁇ m, 400 ⁇ M, 500 ⁇ M, 600 ⁇ M, 700 ⁇ M, 800 ⁇ M, 900 ⁇ M) for 24h, 6 replicates per group to ensure the stability and accuracy of the experimental data, and the degree of damage to cells by H 2 O 2 was observed. Color development: 20 ⁇ L of MTS solution was added to each well, and the incubation continued for 2-4h. Colorimetry: 490nm wavelength was selected, and the light absorption value of each well was measured on an enzyme-linked immunosorbent monitor, and the results were recorded.
  • H 2 O 2 100 ⁇ M, 200 ⁇ M, 300 ⁇ m, 400 ⁇ M, 500 ⁇ M, 600 ⁇ M, 700 ⁇ M, 800 ⁇ M, 900 ⁇ M
  • the cell growth curve was drawn with different concentrations of H 2 O 2 as the horizontal axis and the absorbance value as the vertical axis.
  • the H 2 O 2 concentration when the cell survival rate was about 50% detected by MTT method was used as the modeling concentration of the oxidative damage model.
  • Survival rate survival rate of each group / survival rate of normal cell group ⁇ 100%
  • Figure 1 is a graph showing the results of using MTT method to detect different concentrations of dihydroquercetin (A), different concentrations of H 2 O 2 (B), and 300 ⁇ M hydrogen peroxide and different concentrations of dihydroquercetin (C) acting on corneal epithelial cells for 24 hours, where P represents significant level, ** represents P ⁇ 0.01, *** represents P ⁇ 0.001, ## represents P ⁇ 0.01, and ### represents P ⁇ 0.001.
  • the H 2 O 2 concentration is 300 ⁇ M
  • the cell survival rate is 49%, so 300 ⁇ M H 2 O 2 is determined as the modeling concentration of the oxidative damage model in subsequent experiments.
  • the experiment was divided into 8 groups: normal control group, H 2 O 2 model group, and different concentrations of dihydroquercetin (0.1-200 ⁇ M) + H 2 O 2 intervention groups.
  • HCE cells were modeled with 300 ⁇ M H 2 O 2 , and different concentrations of drugs within the safe range were applied to HCE cells after modeling.
  • the cell survival rate among the groups was detected by MTS method, and the protective effect of dihydroquercetin on the antioxidant damage of HCE cells was detected.
  • the cell survival rate of the H2O2 modeling group was lower than that of the control group (P ⁇ 0.001), and that of the 25-200 ⁇ M dihydroquercetin group was higher than that of the H2O2 modeling group (P ⁇ 0.01), indicating that H2O2 can induce oxidative stress damage in HCE cells, and dihydroquercetin has a protective effect on H2O2 - induced oxidative stress damage in HCE cells.
  • mice Thirty mice were selected from 50 healthy female C57/BL6 mice aged 8 weeks, and randomly divided into 6 groups with 5 mice in each group, which were recorded as blank control group (N), model group (M), positive drug group (CsA), low concentration group of Chinese herbal medicine compound (SL), medium concentration group of Chinese herbal medicine compound (SM), and high concentration group of Chinese herbal medicine compound (SH).
  • N blank control group
  • M model group
  • CsA positive drug group
  • SL low concentration group of Chinese herbal medicine compound
  • SM medium concentration group of Chinese herbal medicine compound
  • SH high concentration group of Chinese herbal medicine compound
  • mice were raised in the S-rated PF laboratory of the Animal Center of Shenyang He's Medical College. During the experiment, 5 mice were kept in each cage (length 40cm ⁇ width 20cm ⁇ height 20cm), for a total of 6 cages.
  • the bedding was corn cob bedding. This experiment used sterilized complete nutritious feed. (Purchased from Liaoning Changsheng Biological Co., Ltd.).
  • the drinking water was purified water, which was prepared by the HT-RO1000 water purification system of the Animal Center. The water quality is tested by a dedicated person every year. During the experiment, the mice were free to drink water. The mice were fasted the night before the dissection, but they were not forbidden to drink water.
  • the temperature and humidity of the laboratory are automatically controlled by the air conditioning unit at 20°C-25°C and 40%-70%, and the temperature and humidity changes are recorded daily.
  • the lighting system automatically controls the light cycle changes in the animal laboratory, and the light and dark are evenly distributed for 24 hours.
  • the noise in the animal laboratory is controlled to no more than 60 decibels.
  • This experiment was approved and supervised by the Institutional Animal Care Committee (IACUC) and strictly complies with the national animal welfare regulations "Guiding Opinions on Treating Animals Well".
  • IACUC Institutional Animal Care Committee
  • an intelligent dry environment control system combined with scopolamine injection was used to induce dry eye models in mice.
  • a two-step dehumidification method was used to control the humidity of the model environment.
  • an adjustable temperature industrial dehumidifier was used to reduce the humidity in the room and adjust the humidity to 40% ⁇ 5%.
  • an intelligent drying box (dehumidification range RH10%-80%) was used to further reduce the humidity of the mouse breeding environment to 15% ⁇ 3%.
  • a noiseless, adjustable speed (wind speed range 0-5m/s) fan was placed in the intelligent drying box. The fan was placed 20cm away from the mouse cage, and the fan was set at the same vertical height as the mouse.
  • mice in the model control group (M), positive drug group (CsA), low-concentration dihydroquercetin group (E1), high-concentration dihydroquercetin group (E2), and solvent control group were placed in the dry environment (humidity 15% ⁇ 3%; wind speed 2.1 ⁇ 0.2 m/s; temperature 21-23°C), and the mice in the blank control group were raised in a normal environment (humidity 60%-80%; temperature 21-23°C).
  • mice in the other groups were subcutaneously injected with 0.5 mg/0.2 mL scopolamine solution 3 times a day (9:00 am; 12:00 noon; 3:00 pm), and the model establishment time lasted for 2 weeks.
  • the drug was administered through the eyes, and the dosage was 5 ⁇ L in the conjunctival sac of each eye twice a day (9:00 am; 3:00 pm).
  • Each drug group started drug administration the day before modeling and continued for 15 days.
  • the drug administration method was ocular administration, and all experimental mice were administered the day before modeling.
  • the blank control group and the model control group were not administered;
  • the positive drug group was administered with 0.05% cyclosporine A eye drops 5 ⁇ L/time, which was dripped into the conjunctival sac;
  • the low-concentration administration group was administered with 0.03% dihydroquercetin eye drops 5 ⁇ L/time, and the high-concentration administration group was administered with 0.3% dihydroquercetin eye drops 5 ⁇ L/time.
  • Each group of mice was administered twice/day (9:00 am and 3:00 pm).
  • mice were given an ophthalmic examination (including visual defects, eyeball abnormalities, and corneal damage) to ensure that there were no abnormalities in the experimental mice.
  • the body weight was measured once a week during the adaptation period and the experiment.
  • the experimental mice were observed before and after dosing every day to record their health status and whether they had abnormal physical signs.
  • the observation of the mouse condition included: appearance (eyes, ears, mouth, nose, vulva, fur, excrement, limb activity, and mental state), vomiting, activity, diet, gait, dying, death, etc.
  • mice were fixed under non-anesthesia, the lower eyelids of the mice were clamped with soft-tipped forceps, and then one end of the phenol red cotton thread was folded and placed in the lower conjunctival sac, and the lower eyelids of the mice were relaxed to close naturally.
  • the phenol red cotton thread was fixed, the phenol red cotton thread was taken out after 1 minute of timing, and the phenol red cotton thread was placed on a ruler paper, and the length of the red part was calculated and recorded.
  • the data were statistically analyzed using GraphPad Prism 8.0 software.
  • Figure 2 is a graph showing the results of tear secretion in live mice in animal experiments.
  • N is a blank control group
  • M is a model control group
  • R is a solvent control group
  • Y is a positive drug group (mass concentration is 0.05% cyclosporine A)
  • E1 is a low-concentration dihydroquercetin group
  • E2 is a high-concentration dihydroquercetin group.
  • P represents a significant difference
  • * represents P ⁇ 0.05
  • ** represents P ⁇ 0.01
  • *** represents P ⁇ 0.001.
  • the length of the phenol red cotton thread (mm) was counted. The longer the phenol red cotton thread, the more tear secretion, and vice versa.
  • the tear secretion of animals in each group was normal before modeling, and there was no significant difference between the groups (P>0.05).
  • the tear secretion of the positive drug group (CsA), the low concentration of dihydroquercetin group (E1), and the high concentration of dihydroquercetin group (E2) increased significantly relative to the model control group (M), and the differences were statistically significant (P ⁇ 0.001). There was no significant change in tear secretion between the solvent control group and the model control group.
  • the above results show that after dihydroquercetin treatment, the tear secretion of dry eye model mice has increased to a certain extent.
  • mice were fixed in a non-anesthetized state, and sodium fluorescein solution was dripped into the mouse conjunctival sac. After the mouse eyelids were passively closed several times, the eyelids were opened and the time when the first tear film rupture black spot was observed under the cobalt blue light of the slit lamp was recorded, which was the tear film rupture time. The data were statistically analyzed using GraphPad Prism 8.0 software.
  • Figure 3 is a graph showing the tear film breakup time test results of live mice in animal experiments.
  • N is the blank control group
  • M is the model control group
  • R is the solvent control group
  • Y is the positive drug group (mass concentration is 0.05% cyclosporine A)
  • E1 is the low-concentration dihydroquercetin group
  • E2 is the high-concentration dihydroquercetin group.
  • P represents significant difference, * represents P ⁇ 0.05, ** represents P ⁇ 0.01, and *** represents P ⁇ 0.001.
  • the corneal morphology of each group of experimental mice was observed before and 14 days after modeling, and the corneas of both eyes were stained with sodium fluorescein solution, and a slit lamp was used for observation and photography. After fixing the mice, 5 ⁇ L of 2% sodium fluorescein saline solution was dripped into the conjunctival sac of the mice. After 1 minute, the eyes were gently rinsed with 2 mL of saline, and the excess solution was absorbed with a cotton swab. The mouse cornea stained with sodium fluorescein was irradiated with cobalt blue light, and the slit lamp was used for observation, photography and recording. If there is green fluorescence residue on the surface of the cornea, it means that the corneal epithelium is damaged. The sodium fluorescein residue on the corneal surface was scored and recorded. The data were statistically analyzed using GraphPad Prism 8.0 software
  • Figure 4 is a corneal morphology of mice in each experimental group after corneal fluorescein sodium staining.
  • the corneas of mice in the blank control group were intact and smooth, without sodium fluorescein attachment, indicating that the corneal epithelium of mice in the blank control group was intact and undamaged.
  • the model group and each drug-treated group had varying degrees of sodium fluorescein attachment, indicating that the corneal epithelium of mice in the model control group and each drug-treated group had varying degrees of damage.
  • the positive drug group and the dihydroquercetin-treated group also had dense punctate fluorescent attachment, but compared with the model group, there was a significant reduction.
  • the corneal epithelial injury scores of the mice in each experimental group after corneal fluorescein sodium staining are shown in Figure 5, where N is the blank control group; M is the model control group; R is the solvent control group; Y is the positive drug group (mass concentration is 0.05% cyclosporine A); E1 is the low-concentration dihydroquercetin group; and E2 is the high-concentration dihydroquercetin group.
  • * represents P ⁇ 0.05, ** represents P ⁇ 0.01, and *** represents P ⁇ 0.001.
  • the corneal epithelium of mice in the model group and each drug-treated group was damaged to varying degrees.
  • the positive drug group and the dihydroquercetin-treated group also showed corneal epithelial cell damage, but compared with the model group, it was significantly reduced (P ⁇ 0.001). This indicates that the positive drug group and the dihydroquercetin-treated groups at various concentrations reduced the degree of damage to the corneal epithelium of mice and had a certain protective effect on the cornea.
  • N blank control group
  • M model control group
  • LUV positive drug group
  • E1 dihydroquercetin low concentration group
  • E2 dihydroquercetin high concentration group
  • R solvent control group
  • Feeding conditions The experimental animals were raised in the general rabbit laboratory of the Animal Center of Shenyang He's Medical College. During the experiment, one rabbit was kept in each cage, for a total of 6 cages. The animals were raised in stainless steel hanging cages, and they were allowed to eat and drink freely. No animals of other species were kept in the same room.
  • the temperature and humidity of the animal room are automatically controlled, the temperature is controlled at 20°C-25°C, and the humidity is controlled at 40%-70%.
  • Artificial lighting, the light cycle is automatically controlled, 12 hours light, 12 hours dark, and the noise is below 60dB.
  • Animal Welfare Animal use complies with the national animal welfare regulations "Guiding Opinions on Treating Animals Well” (2006, Ministry of Science and Technology). The animal use plan is approved by the Institutional Animal Care Committee (IACUC), and the experimental process is subject to its supervision. During the experiment, it may be necessary to humanely kill the animals to alleviate the animals' suffering or pain. The treatment of animals killed for humane reasons is the same as that of animals that died in the experiment. Surviving animals are euthanized at the end of the experiment. Animal carcasses are entrusted to professional institutions for disposal.
  • IACUC Institutional Animal Care Committee
  • the chemical injury method was used to establish a rabbit corneal neovascularization model.
  • 1% sodium pentobarbital was injected into the ear vein for general anesthesia, and proparacaine eye drops were used for ocular surface anesthesia.
  • a 6mm diameter filter paper was soaked in 1mol/L NaOH solution for 1 minute, and then the soaked filter paper was applied to the center of the cornea. After 30 seconds, the filter paper was removed, and then the ocular surface and conjunctival sac were rinsed with saline for 1 minute.
  • levofloxacin was added to the modeled eye to prevent postoperative infection.
  • the administration method was ocular administration, and all animals in the drug administration groups began to be administered the day before modeling. No drug was given to the blank control group and the model control group; the positive drug group was given levofloxacin eye drops 50 ⁇ L/time, which were dropped into the conjunctival sac; the low-concentration drug administration group was given 0.03% dihydroquercetin eye drops 50 ⁇ L/time, and the high-concentration drug administration group was given 0.3% dihydroquercetin eye drops 50 ⁇ L/time. Each group of New Zealand rabbits was given 2 times/day (9:00 am and 3:00 pm).
  • the ocular surface inflammation and neovascularization area of the animals in each experimental group were detected 14 days after administration. Photos were taken and the neovascularization area was calculated. The data were statistically analyzed using GraphPad Prism 8.0 software.
  • Figure 6 is a morphological diagram of corneal neovascularization detected by corneal slit lamp in New Zealand rabbits of each experimental group 14 days after administration.
  • the black circle area is the area of corneal neovascularization.
  • the cornea of the blank control group is transparent and has no neovascularization; a large number of dendritic neovascularization appeared in the cornea of the model group and the solvent control group, expanding along the corneal limbus toward the central area of the cornea, which was significantly different from the blank control group; only a small amount of neovascularization appeared on the cornea of the positive drug control group and the high and low concentrations of dihydroquercetin administration groups, which was significantly improved compared with the model group.
  • N is the blank control group
  • M is the model control group
  • R is the solvent control group
  • Y is the positive drug group (levofloxacin)
  • E1 is the low concentration of dihydroquercetin group
  • E2 is the high concentration of dihydroquercetin group.
  • ** represents P ⁇ 0.01
  • **** represents P ⁇ 0.0001.
  • the area of corneal neovascularization in the model group and solvent control group was significantly different from that in the blank control group (P ⁇ 0.0001); the area of corneal neovascularization in the positive drug control group and the high and low concentrations of dihydroquercetin groups was smaller, which was significantly improved compared with the model group (P ⁇ 0.01), indicating that dihydroquercetin can inhibit the appearance of neovascularization and reduce the degree of corneal alkali burns.
  • dihydroquercetin plays an outstanding role in the treatment of ocular surface diseases.
  • Dihydroquercetin at a concentration of 50-200 ⁇ M promotes the proliferation of HCE cells
  • dihydroquercetin at a concentration of 25-200 ⁇ M protects HCE cells from oxidative stress damage induced by H2O2 .
  • the tear secretion of dry eye mice increased to a certain extent, and the stability of the tear film of mice was improved.
  • dihydroquercetin can reduce the degree of damage to the corneal epithelium of mice, has a certain protective effect on the cornea, and can inhibit the appearance of new blood vessels and reduce the degree of corneal alkali burns.

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Abstract

L'invention se rapporte au domaine technique de la pharmacie et concerne l'utilisation de la dihydroquercétine dans le traitement de maladies de la surface oculaire. L'utilisation de la dihydroquercétine dans le traitement de maladies de la surface oculaire de la présente invention concerne plus particulièrement l'utilisation de la dihydroquercétine dans la préparation d'un médicament destiné à prévenir, atténuer et/ou traiter les maladies de la surface oculaire. La présente invention applique pour la première fois la dihydroquercétine à la prévention, à l'atténuation et/ou au traitement des maladies de la surface oculaire. La dihydroquercétine, utilisée comme antioxydant, présente un bon effet protecteur contre les maladies liées au stress oxydatif dans les cellules. En outre, dans une certaine plage de concentration, la dihydroquercétine peut favoriser la prolifération et améliorer la vitalité des cellules épithéliales de la cornée humaine, et peut également augmenter la quantité de sécrétion lacrymale et la stabilité du film lacrymal des cellules épithéliales de la cornée humaine, ce qui entraîne une atténuation notable des maladies de la surface oculaire.
PCT/CN2023/131428 2022-12-02 2023-11-14 Utilisation de ns le traitement de maladies de la surface oculaire WO2024114360A1 (fr)

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WO2021006856A1 (fr) * 2019-07-11 2021-01-14 Людмыла Грыгоривна АЛМАКАЕВА Agent médicamenteux sous forme de gouttes oculaires
CN115209892A (zh) * 2019-10-28 2022-10-18 科拉医疗股份有限公司 用于减轻或预防氧化应激损伤的制剂

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