CN115721656A - Pharmaceutical composition containing rebamipide or salt thereof, preparation method thereof and application thereof in preparing ophthalmic preparation - Google Patents

Abstract

The invention discloses a pharmaceutical composition containing rebamipide or a salt thereof, a preparation method thereof and application thereof in preparing an ophthalmic preparation. The pharmaceutical composition comprises: quinolinone compounds and pharmaceutically acceptable glycyrrhetate; wherein the quinolinone compound is rebamipide or pharmaceutically acceptable salt thereof, and the mass ratio of the quinolinone compound to the glycyrrhetate is 1. The pharmaceutical composition DG-RBM containing rebamipide provided by the invention is safe and non-irritant, has a better curative effect on preventing, treating and/or relieving chemical injury of eyes, can repair oxidative stress injury of corneal epithelial cells and corneal epithelial injury, and promote injury healing, and can inhibit corneal neovascularization, corneal inflammatory factor expression, HMGB1 expression, TLR4 expression and/or RAGE expression, so that a new solution is provided for prevention and treatment of related diseases.

Description

Pharmaceutical composition containing rebamipide or salt thereof, preparation method thereof and application thereof in preparing ophthalmic preparation

Technical Field

The invention belongs to the field of ophthalmic medicines, and particularly relates to a pharmaceutical composition containing rebamipide or a salt thereof, a preparation method thereof and application thereof in preparing an ophthalmic preparation.

Background

Chemical injury to the eyes, a common non-mechanical injury in ophthalmic emergencies, often occurs in chemical plants, construction sites, laboratories, etc. According to different chemical substances, alkali burn and acid burn can be divided, wherein the alkali burn is more serious than the acid burn.

After the eyes are chemically damaged, the eye tissues are damaged and cause inflammation, and the death of the eye tissues can be further caused if the inflammatory reaction cannot be inhibited in time, so that a vicious circle is generated. The High Mobility Group Protein B1 (HMGB 1) is an endogenous danger signal sent by necrotic cells in the body and is also a pro-angiogenic and proinflammatory molecule. It has been shown that HMGB1 is closely related to alkali burns and its complications, and HMGBl can promote the generation of corneal neovascularization by Toll-like receptors (TLRs), one of its downstream receptors.

At present, the treatment means for such eye diseases are very limited, and besides the drug therapy, severe patients often need corneal transplantation, with the risk of blindness.

Rebamipide (RBM), a white powder, odorless, bitter, soluble in methanol and ethanol but insoluble in water, is usually formulated as a suspension formulation, and can be used clinically for the treatment of dry eye conditions, such as: CN 111107838A, CN 110638748A and the like.

Dipotassium Glycyrrhizinate (DG) is white or white-like powder, has the effects of resisting inflammation, resisting allergy, preserving moisture and the like, is mainly used for relieving cough, eliminating phlegm, gastric ulcer, acute and chronic gastritis, eczema and skin itch in the pharmaceutical industry, and is used for treating cancers, preventing and treating AIDS and the like.

Up to now, no relevant literature reports have been retrieved for the use of rebamipide or its combination with dipotassium glycyrrhizinate for the prevention, treatment and/or alleviation of ocular chemical injury (or for reducing the expression levels of HMGB1, TLR4 and/or RAGE).

Disclosure of Invention

In view of the problems and/or disadvantages of the prior art, it is an object of the present invention to provide a pharmaceutical composition comprising rebamipide or a salt thereof. The pharmaceutical composition is safe, effective and non-irritating, and provides a new potential choice for treating and/or preventing eye diseases such as chemical injury of eyes, xerophthalmia, keratitis and the like.

The present invention provides a nanomicelle pharmaceutical composition comprising: quinolinone compounds and pharmaceutically acceptable glycyrrhetate; wherein the quinolinone compound is rebamipide or pharmaceutically acceptable salt thereof, and the mass ratio of the quinolinone compound to the glycyrrhetate is 1.

In a preferred embodiment of the invention, the mass ratio of the quinolinone compound to the glycyrrhetate is 1; preferably, the mass ratio of the quinolinone compound to the glycyrrhetate is 1. More preferably, the mass ratio of the quinolinone compound to the glycyrrhetate is 1.

In a preferred embodiment of the present invention, the pharmaceutical composition satisfies one or more of the following conditions i to iii:

i. the quinolinone compound is rebamipide;

ii. The glycyrrhetate is selected from one or more of sodium glycyrrhetate, disodium glycyrrhetate, potassium glycyrrhetate, dipotassium glycyrrhetate, ammonium glycyrrhetate and diammonium glycyrrhetate; preferably, the glycyrrhetate is dipotassium glycyrrhizinate or disodium glycyrrhizinate;

iii, the encapsulation rate of the quinolinone compound is more than 80 percent; preferably, the encapsulation rate of the quinolinone compound is more than or equal to 90% or more than or equal to 95%;

preferably, the pharmaceutical composition simultaneously satisfies conditions i to iii.

In a preferred embodiment of the present invention, the quinolinone compound is rebamipide.

In a preferred embodiment of the present invention, the pharmaceutical composition is prepared by a method comprising the steps of: dispersing or dissolving quinolinone compounds and glycyrrhetate in a solvent, uniformly mixing, and then carrying out rotary evaporation at 35-45 ℃ to remove the solvent to obtain the quinolinone compounds;

the solvent is preferably an alcohol solvent, more preferably methanol or ethanol.

The dosage of the solvent is 5-100 mL (for example, 10mL, 15mL, 20mL, 25mL, 30mL, 40mL, 50mL, etc.) per gram of quinolinone compound.

In a preferred embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

In a preferred embodiment of the present invention, the pharmaceutical composition is a solid formulation or a liquid formulation.

In a preferred embodiment of the present invention, the quinolinone compound in the pharmaceutical composition is in a therapeutically effective amount.

In a preferred embodiment of the present invention, the pharmaceutical composition is a liquid preparation, and the solvent is selected from pharmaceutically acceptable water, PBS buffer solution or sodium carboxymethylcellulose aqueous solution.

In a preferred embodiment of the invention, the pH of the liquid formulation is 6 to 8, more preferably 6.8 to 7.4.

In a preferred embodiment of the present invention, the liquid preparation is a nanomicelle eye drop.

In a preferred embodiment of the present invention, the encapsulation efficiency of the quinolinone compound is 80% or more.

In a more preferred embodiment of the present invention, the encapsulation efficiency of the quinolinone compound is 90% or more or 95% or more.

In a preferred embodiment of the present invention, when the concentration of the quinolinone compound in the liquid preparation is 5mg/mL, the liquid preparation satisfies one or more of the following conditions (1) to (3):

(1) the average diameter of the micelle of the liquid preparation is 1-80 nm;

(2) the polydispersity of the liquid preparation is less than or equal to 0.5;

(3) the Zeta potential of the liquid preparation is-20-0 mV;

in a preferred embodiment of the present invention, the solvent formulation satisfies the conditions (1) to (3) simultaneously.

For the condition (1), the average diameter of the micelles of the liquid preparation is preferably 5 to 25nm.

With the condition (2), the polydispersity coefficient of the liquid preparation is preferably 0.4 or less.

For the condition (3), the Zeta potential of the liquid preparation is preferably-10 to-5 mV.

In a preferred embodiment of the present invention, the pharmaceutical composition is an ophthalmic formulation.

In a preferred embodiment of the present invention, the ophthalmic formulation satisfies at least one of the following uses a to f:

a. the ophthalmic preparation is used for treating and/or preventing chemical injury of eyes;

b. the ophthalmic preparation is an ophthalmic preparation for treating and/or preventing dry eye;

c. the ophthalmic preparation is used for treating and/or preventing oxidative stress damage of corneal epithelial cells;

d. the ophthalmic preparation is used for treating and/or preventing corneal epithelial injury;

e. the ophthalmic preparation is used for treating and/or preventing keratitis;

f. the ophthalmic preparation is an ophthalmic preparation for inhibiting corneal neovascularization, corneal inflammatory factor expression, HMGB1 expression, TLR4 expression or RAGE expression.

In a more preferred embodiment of the invention, the ophthalmic formulation is an ophthalmic formulation for the treatment and/or prevention of chemical injury to the eye.

In a more preferred embodiment of the present invention, the ophthalmic preparation is an ophthalmic preparation for the treatment and/or prevention of dry eye.

In a more preferred embodiment of the invention, the ophthalmic preparation is an ophthalmic preparation for the treatment and/or prevention of oxidative stress damage of corneal epithelial cells.

In a more preferred embodiment of the invention, the ophthalmic formulation is an ophthalmic formulation for the treatment and/or prevention of corneal epithelial damage.

In a more preferred embodiment of the present invention, the ophthalmic formulation is an ophthalmic formulation for the treatment and/or prevention of keratitis.

In a more preferred embodiment of the invention, the ophthalmic formulation is an ophthalmic formulation that inhibits corneal neovascularization, corneal inflammatory factor expression, HMGB1 expression, TLR4 expression, or RAGE expression.

In a further more preferred embodiment of the invention, the chemical injury to the eye is an alkaline burn injury to the eye.

In a further more preferred embodiment of the invention, the corneal inflammatory factor is VEGF, TGF-. Beta.1, NF-. Kappa.B, TNF-. Alpha.IL-6 or IL-1. Beta.

In a preferred embodiment of the present invention, the method comprises the steps of:

dispersing or dissolving quinolinone compounds and glycyrrhetate in a solvent, uniformly mixing, and then removing the solvent by rotary evaporation at 35-45 ℃ to obtain a solid product;

optionally including thereafter: dissolving or dispersing the solid product in the solvent of the liquid preparation, adjusting the pH value of the liquid preparation to 6-8, and filtering and sterilizing.

In a more preferred embodiment of the present invention, the solvent is an alcohol solvent, and is further more preferably methanol or ethanol;

the dosage of the solvent is 5-100 mL (for example, 10mL, 15mL, 20mL, 25mL, 30mL, 40mL, 50mL and the like) corresponding to each gram of quinolinone compound;

adjusting the pH value of the liquid preparation to 6.8-7.4;

sodium hydroxide and/or potassium hydroxide are used for adjusting the pH of the liquid formulation.

The application of the pharmaceutical composition in preparing an ophthalmic preparation.

The "encapsulation efficiency" in the present invention means the ratio of the amount of DG-encapsulated (or bound) RBM to the total amount of RBM (the sum of the amount of unencapsulated RBM and the amount of encapsulated RBM) in DG-RBM (solid).

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows:

test results show that the pharmaceutical composition DG-RBM containing rebamipide provided by the invention is safe and non-irritant, has a good curative effect on preventing, treating and/or relieving chemical injury to eyes (such as alkali burn to eyes), and can repair oxidative stress injury of corneal epithelial cells and corneal epithelial injury and promote injury healing; in addition, the composition can also inhibit corneal neovascularization, corneal inflammatory factor expression, HMGB1 expression, TLR4 expression and/or RAGE expression, and provides a new solution for preventing and treating related diseases.

Drawings

Fig. 1 is an appearance diagram of dipotassium glycyrrhizinate-rebamipide nano-micelle eye drops and a comparison thereof.

FIG. 2 shows the envelope ratios of different mass ratios of RBM to DG.

Fig. 3 shows the encapsulation efficiency of the dipotassium glycyrrhizinate-rebamipide nano-micelle eye drops stored for 12 weeks.

FIG. 4 is a diagram showing the observation of eye irritation of rabbits.

FIG. 5 is a histopathological observation image of rabbit eyes.

FIG. 6 shows the blood vessel congestion in chick embryo allantoic membrane-trypan blue staining experiment.

FIG. 7 shows the trypan blue uptake for different experimental groups.

FIG. 8 is a graph of corneal fluorescence intensity after administration of DG-RBM.

FIG. 9 shows the short-term cytotoxicity of DG-RBM eye drops.

FIG. 10 shows the long-term cytotoxicity of DG-RBM eye drops.

FIG. 11 shows the effect of DG-RBM eye drops on HCECs cell migration.

FIG. 12 shows the results of cell migration in different experimental groups.

FIG. 13 shows the results of cell viability assay after hydrogen peroxide induction.

FIG. 14 is a graph of the measurement of cellular ROS levels after hydrogen peroxide induction.

FIG. 15 is DG-RBM treatment versus H ₂ O ₂ Results of cell viability after induction of HCECs.

FIG. 16 shows DG-RBM vs. H ₂ O ₂ Effect of ROS levels after induction of HCECs.

FIG. 17 shows DG-RBM vs. H ₂ O ₂ Effect of SOD levels after induction of HCECs.

FIG. 18 shows DG-RBM vs. H ₂ O ₂ Effect of MDA levels after induction of HCECs.

FIG. 19 is a slit lamp observation of sodium fluorescein staining images of mouse corneas of different experimental groups.

FIG. 20 shows the corneal epithelium defect rate of mice in different experimental groups based on statistics of sodium fluorescein staining.

FIG. 21 is a slit lamp image of tiger red staining of the cornea of mice in different experimental groups.

FIG. 22 shows the corneal epithelium defect rate of mice in different experimental groups according to the tiger red staining statistics.

Fig. 23 is a slit lamp examination image of corneal neovessels after alkali burn.

FIG. 24 is an image of mouse corneal neovascularization after FITC-dextran infusion.

FIG. 25 shows the results of fluorescence quantification of neovascular area of corneal sections.

Fig. 26 is the result of histopathological examination of the cornea after alkali burn.

Figure 27 is the result of apoptosis of corneal epithelial cells after alkali burn.

FIG. 28 shows the expression of the inflammatory factor VEGF in the corneal tissue of different experimental groups.

FIG. 29 shows the expression of the inflammatory factor TGF-. Beta.1 in corneal tissue from different experimental groups.

FIG. 30 shows the expression of the inflammatory factor NF-. Kappa.B in corneal tissue of different experimental groups.

FIG. 31 is a graph showing the expression of the inflammatory factor TNF-. Alpha.in corneal tissue from different experimental groups.

FIG. 32 shows the expression of the inflammatory factor IL-6 in corneal tissue of different experimental groups.

FIG. 33 shows the expression of the inflammatory factor IL-1 β in corneal tissue from different experimental groups.

FIG. 34 shows the result of Western blotting in different experimental groups.

Fig. 35 is the HMGB1 levels in corneal tissue of different experimental groups.

Figure 36 is a graph of TLR4 levels in corneal tissue of different experimental groups.

FIG. 37 is a graph of RAGE levels in corneal tissue of different experimental groups.

Detailed Description

While the present invention will be described more fully hereinafter with reference to the accompanying specific embodiments, it is to be understood by those skilled in the art that the following descriptions are provided for purposes of illustration only and are not intended to limit the scope of the present invention.

In the present invention, those who do not specify specific conditions are performed according to conventional conditions or conditions recommended by the manufacturer, and those who do not specify the reagents or instruments used are conventional products commercially available.

For example:

dipotassium glycyrrhizinate, available from Siemens Biotech, inc.

Rebamipide, MTT, available from shanghai aladine biochemistry science and technology, inc.

Coumarin 6, DPH, 3% hydrogen peroxide, TEMED, available from sigma aldrich (shanghai) trade ltd.

PBS buffer: purchased from wuhan seiver biotechnology limited.

Fetal bovine serum, DMEM/F12 culture medium, trypsin, immunohistochemical kit, DAB color development kit and prestained rainbow protein Marker (10-170 KD), purchased from Saimer Feishell science and technology (China) Co.

RIPA lysate, PMSF, tris base, 30% acrylamide, 1.5M Tri-Cl pH =8.8, 1.0M Tri-Cl pH =6.8, 1.0M Tri-Cl pH =7.4, tween 20 (Tween 20), ECL substrate solution, purchased from beijing solibao technologies ltd.

The goat anti-rabbit secondary antibody labeled with HRP was purchased from Kyowa gold bridge Biotechnology Co., ltd.

The kit comprises a Reactive Oxygen Species (ROS) detection kit, a lipid oxidation (MDA) detection kit and a total SOD activity detection kit (WST-8 method), and is purchased from Shanghai Biyuntian biotechnology limited company.

Chicken embryo: the egg weight is about 40g, provided by Guillain-Gorgo trade Co., ltd, jiangsu Zhao, and the egg is placed in an incubator, the temperature is kept at 37 ℃, the relative humidity is 40% -70%, and the egg is used after being cultured for ten days.

Male new zealand rabbits: purchased from Qingdao Kangda Biometrics, inc. (Qingdao, china).

C57BL/6J mice: 6-8 weeks, male mice, and experimental animal breeding Limited of Ji nan pun Yue.

In the present invention, all analyses were performed using the sps 11.5 software (spssinc., chicago), and P <0.05 indicated significance.

With respect to definitions of terms used in the present disclosure, the initial definitions provided for the terms herein apply to the terms throughout, unless otherwise indicated; for terms not specifically defined herein, the meanings that would be afforded to them by a person skilled in the art, in light of the disclosure and/or the context, should be given.

Example 1 preparation of dipotassium glycyrrhizinate-rebamipide nano-micelle eye drops:

preparation method (refer to: zuo R, zhang J, song X, et al. Encapsulation Halofuginone Hydrobromide in TPGS Polymeric Micelles engineering effective ingredient Triple-Negative Breast Cancer Cells [ J ]. Int J Nanomedicine,2021,16: 750mg Rebamipide (RBM) and varying mass ratios (1, 1. Subsequently, the eggplant-shaped flask was placed on a rotary evaporator and the methanol was evaporated at 40 ℃ under reduced pressure until a dry film (DG-RBM) was attached to the wall of the eggplant-shaped flask. Taking down the eggplant-shaped bottle, adding PBS buffer solution to dissolve the film (DG-RBM) on the bottle wall, adjusting the pH value to 6.8-7.4 by sodium hydroxide and fixing the volume (150 mL), and filtering by a sterile microporous membrane to obtain the dipotassium glycyrrhizinate-rebamipide (DG-RBM) nano micelle eye drop sterile preparation which is shown in figure 1: DG-RBM (the mass ratio of the RBM to the DG is 1; PBS buffer (as colorless clear transparent solution), DG (PBS as solvent, 75mg DG per ml, as light yellow solution), DG & RBM (PBS as solvent, 75mg DG and 5mg RBM per ml, as light yellow suspension solution), RBM suspension (PBS as solvent, 5mg RBM per ml, as white suspension solution) as controls.

1.1 measurement of Critical micelle concentration of dipotassium glycyrrhizinate

1, 6-diphenyl-1, 3, 5-hexatriene (DPH) is used as a fluorescent probe to respectively determine the Critical Micelle Concentration (CMC) of the dipotassium glycyrrhizinate in the artificial tears, the PBS buffer solution and the water. The results showed that CMC of dipotassium glycyrrhizinate in artificial tears, PBS buffer and water was 1.18. + -. 0.09, 1.07. + -. 0.06, 1.23. + -. 0.06mg/mL, respectively.

1.2 encapsulation efficiency

The prepared DG-RBM nanomicelles were eye-dropped through 0.22 μm filters and separated by filtration from unencapsulated RBMs as determined by high performance liquid chromatography (reference: ding P, chen Y, cao G, et al. Solutol ((R)) HS15+ pluronic F127 and Solutol ((R)) HS15+ pluronic L61 mixed microorganism systems for oral delivery of genistein [ J ]. Drug Des Deel Ther,2019, 13. The DG-RBM pre-and post-filtration solutions are diluted with an appropriate solvent (e.g., methanol) to break down the micelles. The RBM concentration was determined by high performance liquid chromatography. Encapsulation efficiency, which is the ratio of the RBM concentration detected after filtration to the RBM concentration detected before filtration.

High performance liquid chromatography:

hippocastin LC2010A high performance liquid chromatograph, UV-visible light detector, reversed phase chromatography column Agilent ZORBAX SB-C18 (250 mm. Times.4.60mm, 5 μm); the column temperature is 40 ℃; the mobile phase was methanol and aqueous acetic acid at pH =3 (55,v/v); the detection wavelength is 245nm; the flow rate is 1.0mL/min; the injection volume is 10. Mu.L. Retention time of rebamipide was 7.4min, limit of quantitation 50ng/mL.

The detection result is shown in figure 2, when the mass ratio of the RBM to the DG is 1, the RBM encapsulation rate of the DG-RBM is 86.85 +/-0.59%, and the phenomenon of drug precipitation is observed by naked eyes at the moment; when the mass ratio of RBM to DG is 1.

1.3 micelle size, polydispersity index and zeta potential

As a result of measurement using a nano particle size meter (Markov instruments Co., ltd., england), DG-RBM nanomicelle eye drops (RBM to DG mass ratio of 1:15, and RBM concentration of 5 mg/mL) were found to have an average diameter of 16.56. + -. 0.91nm, a polydispersity index of 0.377. + -. 0.12, and a zeta potential of- (7.27. + -. 0.591) mV.

1.4 investigation of storage stability

The prepared DG-RBM nano-micelle eye drops (the mass ratio of RBM to DG is 1:15, the RBM concentration is 5 mg/mL), subpackaged, sealed and protected from light, one part is stored at 4 ℃, and the other part is stored at 25 ℃. As shown in FIG. 3, the RBM encapsulation efficiency of DG-RBM was 92.02. + -. 1.83% when stored at 25 ℃ for 12 weeks, and 96.78. + -. 1.96% when stored at 4 ℃ for 12 weeks, which was still a clear pale yellow solution when observed with naked eyes.

1.5 Security Studies

(1) In vivo rabbit eye irritation test

Eye irritation evaluation (see: 1, li X, fang J, xin M, et al. Rebaudiosides as a formulating nanovehicles for a nimodipine ocular delivery [ J ]. Drug Deliv Transl Res,2020 (1); 2, zhang F, li R, yan M, et al. Ultra-small-emulsions based on a polyvinyl pyrrolidone K-17PF.

Physiological saline group:

SDS (sodium dodecyl sulfate) group: normal saline is used as a solvent, and the concentration is 0.5%;

DG-RBM group: the DG-RBM nano-micelle eye drops have the mass ratio of RBM to DG of 1;

group DG: PBS is solvent, and each milliliter contains 75mg of DG;

RBM group: PBS was used as solvent, and 5mg of RBM per ml.

The Draize-Test scores were first performed after the above groups were dropped into the eye, and the results of observation are shown in FIG. 4. The results showed that 0.5% SDS had some irritation to rabbit eye tissue, the rabbit lacked compliance, had failed to blink autonomously, the eyeball was engorged with swelling, the iris was visible but with a darker color, the sclera and limbal engorgement were higher than normal, the conjunctiva had a marked edema with partial eyelid eversion and wetness, and the conjunctiva had large secretions. Other groups have no obvious ocular injury, and no clinical abnormal signs are seen in cornea, conjunctiva and iris.

(2) Histopathology

After the photographing and the scoring are finished, the rabbit is euthanized and then the eye is picked up by using toothless tweezers, in order to ensure that the eyeball is not pulled to cause the damage of eye tissues in the process, the picked eyeball is soaked in tissue fixing liquid for fixing, is dehydrated and then is embedded in a wax block, the section is subjected to hematoxylin-eosin (HE) staining and periodic acid snow's staining (PAS) of the cornea, and tissues such as the cornea, the conjunctiva, the angle of the atrium, the retina and the like are observed under a microscope and photographed.

The results are shown in FIG. 5,0.5% SDS groups showing a significant reduction in the number and density of rabbit ocular conjunctival goblet cells, and no significant abnormalities were found in other groups, further demonstrating the safety of DG-RBM for ocular use.

(3) Chick embryo allantoic membrane-trypan blue staining experiment (CAM-TBS)

Chick embryo allantoic membrane-Trypan blue staining (CAM-TBS) method for evaluating irritativeness of dipotassium glycyrrhizinate-rebamipide nanomicelle eyedrops (refer to Liu S, han X, liu H, et al., incorporation of ion exchange functionalized-monomorphonite into colloidal nanoparticles with low incidence of treatment process Drug bio-available for glaucomatous treatment [ J ]. Drug Deliv,2020,27 (1): 652-661.). Experimental chick embryos were randomly divided into six groups of 5:

physiological saline group:

SDS (sodium dodecyl sulfate) group: normal saline as solvent, with concentration 0.5%;

NaOH group: 0.1mol/L sodium hydroxide solution;

DG-RBM group: the DG-RBM nano-micelle eye drops have the mass ratio of RBM to DG of 1;

group DG: PBS is solvent, and each milliliter contains 75mg of DG;

RBM group: PBS was used as solvent, and 5mg of RBM per ml.

After the sample was contacted with the chick embryo allantoic membrane (CAM) for 5min, the blood vessel condition of the CAM is shown in FIG. 6, the blood vessels of the chick embryo allantoic membrane in 0.1M NaOH solution and 0.5% SDS solution (both positive control groups) had significant blood vessel rupture bleeding, and the blood vessels of the chick embryo allantoic membrane in the other groups and physiological saline (negative control) were visually observed to have no rupture and bleeding, indicating that DG-RBM, DG and RBM had no significant irritation to the chick embryo allantoic membrane.

As shown in FIG. 7, the absorption of trypan blue was about 1.91. Mu.g, and it was not substantially irritating to chick embryo allantoic membrane, and the normal saline group was a negative control group. 0.1M NaOH group as positive control, trypan blue absorption of 49.01. Mu.g, 0.5% SDS solution group trypan blue absorption of about 52.02. Mu.g, with strong irritativeness. The absorption amounts of Trypan blue by DG-RBM, DG and RBM were about 2.27. Mu.g, 1.22. Mu.g and 4.28. Mu.g, respectively.

(4) Investigation of corneal absorption in vivo mice

Respectively labeling DG-RBM and RBM with coumarin 6 (Cou-6) (the concentration of coumarin 6 is 5 μ g/mL), randomly dividing mice into two groups, and dripping coumarin 6-labeled DG-RBM nano micelle eye drops on the surface of one group; another group of coumarin 6 marked RBM suspension is dripped once every 10 minutes for three times; the mice were sacrificed 30, 60 and 90 minutes after the end of the last instillation, and the eyeballs were rinsed with physiological saline to remove the eye drop residues. The mouse cornea was cut out and spread on a flat glass slide, and observed with an upright fluorescence microscope with the exposure time set to 100 msec.

The results are shown in FIG. 8, corneal fluorescence intensity sequence for DG-RBM: 30 min > 60 min > 90 min, while little green fluorescence is observed by RBM, indicating that DG-RBM absorption and utilization by the cornea is far superior to RBM.

EXAMPLE 2DG-RBM Nanopalescent eyedrops for the treatment of H ₂ O ₂ Inducing oxidative stress injury of human corneal epithelial cells

2.1 evaluation of cytotoxicity

MTT method for detecting cytotoxicity: cells were seeded in 96-well plates at approximately 5X 10 cells per well ⁴ Individual cells were cultured in 96-well plates for 24h to allow the cells to adhere.

(1) Short time cytotoxicity

DG-RBM: the DG-RBM nano-micelle eye drops have the mass ratio of RBM to DG of 1;

DG: PBS is solvent, and each milliliter contains 75mg of DG;

RBM: PBS is solvent, and each milliliter contains 5mg of RBM;

the DG-RBM, DG and RBM are added into a 96-well plate respectively and cultured together with cells for 1h, the detection method is the same as the long-time cytotoxicity detection method, untreated cells are used as a blank control, the survival condition of the cells is examined, and the result is shown in figure 9.

The results show that DG-RBM, DG and RBM do not show obvious cytotoxicity, but the cell survival rate of the preservative of benzalkonium chloride (BAC) of 0.1mg/mL is only 40.91 +/-1.05%, and the obvious cytotoxicity is shown.

(2) Long term cytotoxicity

Diluting dipotassium glycyrrhizinate-rebamipide nano-micelles to final concentrations of 500, 250, 125, 62.5, 31.25, 15.63, 7.81 and 3.913 mu g/mL respectively by using a culture medium containing 2% fetal bovine serum; diluting dipotassium glycyrrhizinate solution according to the corresponding concentration of the prescription to make the final concentration of 7500, 3750, 1875, 937.5, 468.75, 234.38, 117.19 and 58.59 mu g/mL; the rebamipide suspension was diluted to the final concentrations of 500, 250, 125, 62.5, 31.25, 15.63, 7.81 and 3.91 μ g/mL according to the prescription.

Adding the samples into a 96-well plate, culturing the samples together with cells for 24, 48 and 72 hours, sucking away the sample solution culture medium of each well after the incubation is finished, adding 200 mu l of sterile PBS into each well, washing for 3 times, and washing out sample residues. Adding MTT solution to continue incubation for 4h, sucking supernatant, adding 200 mu L of dimethyl sulfoxide into each hole, and detecting the absorbance value at 490nm by using a microplate reader. The cytotoxicity was examined simultaneously with the experimental groups using untreated cells as a blank, at concentrations of 5. Mu.g/mL and 10. Mu.g/mL benzalkonium chloride, and the results are shown in FIG. 10.

The results show that the cell survival rate of 500 mu g/mL DG-RBM incubated with the cells for 72 hours is 100.265 +/-0.01 percent, the cell survival rates of corresponding 500 mu g/mL rebamipide suspension and 7500 mu g/mL dipotassium glycyrrhizinate solution are 103.216 +/-1.09 percent and 100.976 +/-1.20 percent respectively, and no cytotoxic reaction is shown, but the cell survival rates of 10 mu g/mL and 5 mu g/mL benzalkonium chloride incubated with the cells for 72 hours are only about 4 percent, and the cytotoxicity is obvious.

2.2 Effect on cell migration of HCECs (Human Corneal Epithelial Cells)

The inoculation amount of the cells is that the cells are attached to the wall and just cover the bottom of the cell well plate on the next day. After the cells are exactly covered by one layer, the cells are scratched by using a 200 mu L pipette tip. The cells were gently washed with PBS, the dropped cell debris was washed off, and DG-RBM, DG, and RBM using a serum medium as a solvent were added. Placing into a cell incubator, observing with an inverted microscope at 0, 12 and 24h, and taking pictures and recording. The cell migration area was analyzed using ImageJ and the cell migration rate was calculated.

According to the cytotoxicity result, the dipotassium glycyrrhizinate-rebamipide nano-micelle eye drops are diluted by 10 times according to the prescription concentration, namely 500 mug/mL (DG-RBM), the corresponding rebamipide suspension is 500 mug/mL (DG), the dipotassium glycyrrhizinate solution is 7500 mug/mL (RBM), a cell migration experiment is carried out, the cell migration result is shown in a figure 11 through microscopic observation, and the cell migration rate is shown in a figure 12 (P is less than 0.05 compared with a control; P is less than 0.05 compared with DG-RBM, P is less than 0.05, and the cell migration rate is shown in a figure 12 through microscopic observation.

When the cell scratching experiment is carried out on the cells, the three samples remarkably promote the corneal epithelial cell scratching migration within 12 hours (compared with a control group, P is less than 0.05), and the cell migration rate of the dipotassium glycyrrhizinate-rebamipide nano-micelle of 500 mug/mL is 69.450 +/-1.25 percent and is remarkably greater than that of the other two groups of samples (P is less than 0.05). At 24 hours, the cells in the micellar group were almost completely fused, with a mobility of 82.977. + -. 0.96%, and the cells in the two remaining groups were further migrated, but the degree of fusion was lower than in the micellar group (P <0.05 compared to the DG-RBM group). This indicates that both rebamipide and dipotassium glycyrrhizinate have the effect of promoting cell migration, and dipotassium glycyrrhizinate-rebamipide has a synergistic effect, and the effect is superior to that of any single component.

2.3 to H ₂ O ₂ Effect of inducing HCECs oxidative stress

(1) Establishment of H ₂ O ₂ Model for inducing HCECs to generate oxidative stress

Establishing HCECs oxidative stress experimental model, inoculating cells on 96-well plate with serum-free culture medium, wherein the cell density is about 5 × 10 per well ³ Individual cells, post-adherent cell modeling. H ₂ O ₂ The concentration of (A) was set to 200, 300, 400, 500, 600, 700, 800, 900, 1000nM, prepared with a fresh medium blank without serum, and prepared immediately under dark conditions. The cells were incubated with hydrogen peroxide at different concentrations as described above in a 96-well plate for 2, 4 hours, and then washed with sterile PBS, and the cell viability was measured by the MTT method as described above, and the results are shown in fig. 13.

The results show that with H ₂ O ₂ The cell viability also decreased gradually with increasing concentrations from 200nM to 1mM, and the viability of the cells was less than 2 hours after 4 hours incubation.

Determination of Reactive Oxygen Species (ROS), see FIG. 14. Determination of 500nM H using the active oxygen detection kit ₂ O ₂ The ROS level generated after inducing the cells for 4H is obviously increased (P < 0.05) compared with the ROS level of the cells induced under the condition, and further, H is proved ₂ O ₂ The HCECs are induced to generate an oxidative stress model, and the establishment is successful.

(2) Determination of Reactive Oxygen Species (ROS)

Taking HCECs in logarithmic growth phase according to the proportion of 10 multiplied by 10 ⁴ Cell density per well cells were seeded in 6-well plates. A blank cell control group, a hydrogen peroxide model group and different administration groups are arranged. And selecting proper concentration for pretreatment according to the cytotoxicity experiment results in different administration groups. After the pretreatment is finished, washing away residual sample solution by serum-free medium, and adding hydrogen peroxide for molding. After molding, the supernatant was aspirated and the 6-well plate was rinsed 3 times with serum-free medium. The procedure was performed according to the ROS kit (Shanghai Bin Yuntian Biotechnology Co., ltd.). Collecting cells and detecting fluorescence intensity. The fluorescence intensity of the cell suspension without the loaded probe was subtracted from the measured fluorescence intensity and compared to the fluorescence intensity of the blank. Each group was provided with 3 parallel controls.

(3) Determination of superoxide dismutase (SOD) content

SOD activity was measured according to the instructions of the total SOD activity detection kit (WST-8 method). In data analysis, SOD enzyme activity units were calibrated with the protein concentration of the cells.

(4) Assay for Malondialdehyde (MDA)

Lipid oxidation levels in cells were determined according to the lipid oxidation (MDA) test kit instructions. The MDA content is calculated and calibrated by the protein concentration of the cells.

According to the results of the cytotoxicity and the cell migration, the concentration of the prescription of the dipotassium glycyrrhizinate-rebamipide nano-micelle eye drops is diluted by 10 times, namely 500 mug/mL is used as the administration treatment concentration, the corresponding rebamipide suspension is 500 mug/mL, and the dipotassium glycyrrhizinate solution is 7500 mug/mL. The above sample solution was incubated for 12 hours, and then the cell viability, ROS level, SOD and MDA contents were measured, and the results are shown in FIGS. 15 to 18 (P < 0.05; and H; respectively, in comparison with the control) ₂ O ₂ In contrast, P is less than 0.05; and $ 2: indicating P <0.05 compared to DG-RBM).

Predose followed by H ₂ O ₂ Induction of cell viability and H of cells, DG-RBM group ₂ O ₂ The group ratio was significantly improved (P < 0.05), as shown in FIG. 15. After the hydrogen peroxide induces the human corneal epithelial cells, the viability of the cells is obviously damaged, and the ROS level is obviously increased, which indicates that the cells generate oxidative stress. The detection result shows that compared with a normal cell control group, H ₂ O ₂ The ROS and MDA of the cells of the group are obviously increased (P is less than 0.05), and the SOD is obviously reduced (P is less than 0.05); and H ₂ O ₂ Compared with the group, the SOD of the DG-RBM group is obviously increased (P is less than 0.05), and the ROS and MDA are obviously reduced (P is less than 0.05), which shows that the dipotassium glycyrrhizinate-rebamipide nano micelle can effectively reduce the oxidative stress level and regulate and control the content of SOD and MDA generated by cells.

Example 3 DG-RBM nano-micelle eye drops can regulate and control HMGB1 (High Mobility Group B1, high Mobility Group protein B1) signal path to treat alkali burn of mouse cornea

3.1 modeling

The mouse product for constructing the corneal alkali burn is C57BL/6J, and the state of the eyes of the mouse is observed to be abnormal by a slit lamp.

Circular filter paper 2mm in diameter was immersed in a 1M NaOH solution. After the mice were deeply anesthetized, ocular surface anesthesia was performed by dripping 0.5% tetracaine on the cornea. The mouse's eyelashes and extra hair around the eyes were carefully cut off under a microscope, and the periphery of the eyes was gently wiped with a cotton swab dipped in physiological saline. Using sterile ophthalmic forceps, a piece of NaOH-soaked filter paper was placed in the center of the cornea for 60 seconds to produce an acute alkali burn of the mouse cornea. The right eye of each mouse was alkali-burned and the left eye was used as a control. The filter paper was removed and the eye was rinsed with normal saline to wash away the residual NaOH solution from the eye. The solutions of the following components were dropped onto the cornea. The administration was repeated 4 times per day for 14 days.

(1) A PBS group;

(2) group HA: 0.1% sodium hyaluronate;

(3) DG-RBM group: the DG-RBM nano-micelle eye drops have the mass ratio of RBM to DG of 1 to 15, and the RBM concentration of 500 mug/mL;

(4) DG & RBM group: PBS is solvent, containing 7.5mg DG and 0.5mg RBM per ml;

(5) group DG: PBS is used as a solvent, and the concentration is 7500 mu g/mL;

(6) RBM group: PBS is a dispersing solvent, 500. Mu.g/mL.

3.2 mouse anterior Ocular crack Lamp Observation

(1) Mouse corneal fluorescein sodium staining

And starting the slit lamp, and adjusting the light source to cobalt blue light. Randomly selecting each group of mice on 1,3,5, 7 and 14 days, applying a fluorescein sodium solution with the concentration of 1% to the eyes of the mice to cover the eyeballs, flushing the redundant fluorescein sodium solution after the eyes stay on the surface for 10s, exposing the eyeballs, observing in front of a slit lamp, and taking pictures to record the coloring condition of the fluorescein sodium on the corneal epithelium of the mice. The corneal epithelial staining area was measured by Image analysis software Image J, and the corneal epithelial repair area was calculated.

The results of fluorescein sodium staining at each time point after the alkali burn of cornea in six groups of mice are shown in FIG. 19. The results are very intuitive and show that when mice undergo alkali burn, the sodium hydroxide erodes the whole corneal epithelium, and all the corneal epithelia of all groups of mice are fluorescent green (0 day) after being stained by fluorescein sodium under cobalt blue light; after administration, the dipotassium glycyrrhizinate-rebamipide nano-micelle eye drops are effective for treating mouse corneal alkali burn, have the treatment effect of promoting epithelial injury healing, and are superior to DG & RBM groups, DG groups and RBM groups.

The corneal epithelial area loss rate was determined statistically based on fluorescein sodium staining and the results are shown in FIG. 20 (.: indicates P <0.05 compared to PBS #: indicates P <0.05 compared to sodium hyaluronate;: indicates P <0.05 compared to DG-RBM).

PBS as a negative control, corneal lesions healed slowly after treatment, 34.05 ± 2.55% of the cornea was not repaired on day 7 after burn injury, and 10.41 ± 0.98% of the corneal epithelium was not repaired until day 14. Sodium Hyaluronate (HA) is used as a positive control, corneal epithelium is repaired by about 80% (100% -defect rate) on the 7 th day, about 70% is repaired by DG & RBM group, and about 91.87% is repaired by dipotassium glycyrrhizinate-rebamipide nano-micelle eye drops (DG-RBM group). On day 14 of administration, corneal lesions in the DG-RBM group had been substantially completely repaired, while lesions of varying degrees were present in all five groups.

(2) Mouse corneal tiger red staining

Starting a slit lamp, adjusting a light source to natural light, randomly selecting each group of mice for 1,3,5, 7 and 14 days, dripping tiger red solution on the eyes of the mice to cover the eyeballs, flushing redundant tiger red solution after staying on the eye surfaces for 10 seconds, fully exposing the eyeballs, observing in front of the slit lamp, and taking a picture to record the red coloration condition of the cutworms on the cornea of the mice. The corneal epithelial staining area was measured by Image analysis software Image J, and the corneal epithelial repair area was calculated.

The tiger red can stain the cells which are dry and necrotic, has high sensitivity and can be observed under white light. The results of the tiger red staining observations at each time point are shown in FIG. 21. Similar to the staining result of the fluorescein sodium, the staining range of corneal epithelium is gradually reduced along with the prolonging of the administration time of different groups, the corneal epithelium injury is repaired to different degrees, and the dipotassium glycyrrhizinate-rebamipide nano-micelle eye drops (DG-RBM group) have the best effect.

The corneal epithelial area loss rate was determined by tiger red staining and is shown in FIG. 22 (.: P <0.05 compared with PBS; # P <0.05 compared with sodium hyaluronate; # P <0.05 compared with DG-RBM). Similar to the statistical results of the fluorescein sodium, the dipotassium glycyrrhizinate-rebamipide nano-micelle eye drops (DG-RBM group) have the best effect, and have a significant difference (P is less than 0.05) compared with other groups.

3.3 observation of corneal neovascularization in mice

The course of the neovascular changes was observed after 1,3,5, 7, and 14 days after the alkali burn, and on 14 days, the mice were subjected to FITC-dextran jugular vein angiography, and then the cornea was mounted and observed with an upright microscope, and the fluorescence area was analyzed using image J software.

A representative photograph is presented by 14 day observation with a neovascular slit lamp, see FIG. 23. After the eyeballs of the mice are subjected to alkali burn, new blood vessel buds begin to appear on the corneal limbus of the mice in the PBS group from day 1; on day 3, the mice in the PBS group were observed to have short, fine blood vessels growing rapidly from the limbus to the center of the cornea, long, calibre-thickened neovascularization had developed and formed a network of blood vessels on the cornea of the PBS group on days 5-7, and neovascularization had reached the central region of the cornea on day 14.

The growth of the new blood vessels of the dipotassium glycyrrhizinate-rebamipide nano-micelle eye drop group and the sodium hyaluronate group is lighter than that of the PBS group, on the 14 th day, the nano-micelle eye drop group has fine blood vessels growing from the corneal limbus to the corneal center, but the diameter of the blood vessels is small, the blood vessel density of the sodium hyaluronate group is slightly larger than that of the nano-micelle eye drop group, and the distance from the new blood vessels to the corneal center of the group is also closer than that of the nano-micelle eye drop group. The treatment of the remaining three groups did not significantly inhibit the growth of new blood vessels to the center of the cornea, and the blood vessels of the rebamipide suspension group were the ones closest to the center of the cornea except for the PBS group, and the lumen of the blood vessels was large.

FITC-dextran jugular vein injection was performed for angiography and the corneal plating results are shown in FIGS. 24 and 25 (.: indicates P <0.05 compared to PBS; # indicates P <0.05 compared to sodium hyaluronate; # indicates P <0.05 compared to DG-RBM). Healthy mice had no new blood vessels on the cornea (control group) except for the limbus. The PBS group and the rebamipide suspension group have more branches and high density. The nano micelle eye drop group has small blood vessel density and low fluorescence intensity. The vascular density of the dipotassium glycyrrhizinate group and the sodium hyaluronate group is similar. The results of the corneal slab were consistent with the observations on the slit lamp; therefore, in combination with the above results, it is considered that the nanomicelle eye drops have a significant inhibitory effect on neovascularization.

3.4 histopathological examination of corneal tissue and inflammatory cells

The medicine is administered according to groups after the model is made, mice are randomly selected in each group for cervical dislocation and death in 7 th and 14 th days, the picked eyeballs are soaked in tissue fixing liquid for fixation, the dehydrated eyeballs are embedded in wax blocks, the sections are subjected to H & E staining of the cornea, and the section is observed by a microscope and photographed and recorded.

Histological examination of the alkali-burned cornea better corroborates the results of clinical observations, see fig. 26.

For the PBS treated group, on day 7, the corneal stroma was destroyed, a large inflammatory cell infiltration occurred, and significant newly formed vascular cavities were observed in the stroma, with a large amount of inflammatory exudates in the anterior chamber; on day 14, the condition was more severe, the corneal stroma was severely damaged, and more inflammatory cells appeared.

In the sodium hyaluronate group, on the 7 th day, corneal epithelium becomes thin, a small amount of inflammatory cells appear, stroma arrangement is compact, and on the 14 th day, corneal stroma becomes edema and corneal epithelium thickens, namely sodium hyaluronate has a certain repairing effect.

The corneal tissue of the nano-micelle eye drop group is closest to a healthy mouse, the corneal epithelium has slight defect on the 7 th day, the corneal epithelium is completely repaired on the 14 th day, the neovascular lumen is thin, the corneal stroma is compact, only a small amount of inflammatory factors exist, the corneal endothelium is intact, and no exudate is observed in the anterior chamber.

After 7 days of treatment in the physical mixture group (DG & RBM), the corneal epithelium became thin, the corneal stroma was destroyed, newly formed blood vessels and inflammatory cell infiltration were observed in the corneal stroma; on day 14, the condition was relieved, but there were still a lot of inflammatory cells, which infiltrated the corneal stroma and the lumen of the new blood vessels was large.

In both the dipotassium glycyrrhizinate group and the rapeptamide suspension group, inflammatory exudates in the anterior chamber were clearly observed, but they were mainly adhered to the corneal endothelium.

3.5 mouse corneal apoptosis assay (TUNEL staining)

The preparation method comprises the following steps of (1) after the model is made, administering the medicine according to groups, randomly selecting mice in each group on 7 th and 14 th days, removing necks, killing the mice, then carrying out eye picking, soaking picked eyeballs in tissue fixing liquid for fixing, placing the fixed eyeballs into an embedding box, and carrying out TUNEL staining operation.

The alkali burn injury was very harmful to the cells of the corneal tissue, and the corneal epithelium of all treatment groups had a relatively severe apoptosis phenomenon, as shown in fig. 27. However, the dipotassium glycyrrhizinate-rebamipide nanomicelle eye drops group had relatively few apoptotic cells compared to the other groups, indicating that DG-RBM can promote epithelial repair, consistent with the previous conclusions.

3.6 ELISA for detecting inflammatory factors

14 days after dosing, each group of mice was sacrificed and the cornea was microscopically divided. The cornea was washed with 4 ℃ pre-cooled sterile PBS, frozen with liquid nitrogen, and crushed using ophthalmic scissors. PBS containing phenylmethylsulfonyl fluoride (PMSF) was added and the whole was broken with a sonicator and the whole was run on ice. After the cornea was broken, it was centrifuged at 12000rpm at 4 ℃ for 10 minutes, and the supernatant was transferred to a new centrifuge tube. The protein concentration of each sample was determined using the BCA protein kit. The subsequent operations were performed according to the kit instructions and the results were corrected by the sample protein concentration.

The results are shown in FIGS. 28 to 33 (P <0.05 in comparison with PBS, # P <0.05 in comparison with sodium hyaluronate, and # P <0.05 in comparison with DG-RBM). The expression of VEGF, TGF-. Beta.1, NF-. Kappa.B, TNF-. Alpha.IL-6 and IL-1. Beta. Was significantly increased in the cornea of mice in the PBS group compared to the normal healthy control group. Compared with other groups, the dipotassium glycyrrhizinate-rebamipide nano-micelle eye drops and the sodium hyaluronate show better effects on reducing the expression of the inflammatory factors.

3.7 Western Blotting (WB)

(1) Mouse cornea protein extraction and treatment

Six groups of mice on day 14 after alkali burn were decapitated and sacrificed, and the eyeballs were carefully removed with ophthalmologic forceps; carefully separating out mouse cornea under a microscope, flushing the cornea with precooled PBS, dipping filter paper to absorb water, and shearing the cornea with clean ophthalmic scissors; putting every two corneas into a centrifuge tube, adding 200 μ L of RIPA lysate containing PMSF, and crushing on ice by using an ultrasonic crusher; centrifuging the sample at 4 ℃, and transferring the supernatant into a new centrifuge tube; diluting the corneal protein sample by 10 times, adding 20 mu L of the diluted corneal protein sample into a 96-well plate respectively, and setting a PBS (phosphate buffer solution) hole as a blank control; then operating according to the BCA kit instruction and calculating the protein concentration of the sample to be detected; after the protein sample and the loading buffer solution are fully mixed, the mixture is heated for 5 minutes at 95 ℃ by a metal bath, and then the mixture is frozen and stored in a refrigerator at minus 80 ℃ after being cooled.

(2) Preparation of Polyacrylamide gels

The glass plates were thoroughly cleaned and tightly assembled prior to use. Adding the SDS-PAGE separating gel into the centrifuge tubes from top to bottom, mixing, and injecting the separating gel into the glass plates. Slowly injecting deionized water to flatten the glue surface, and standing at room temperature. After the separation gel is polymerized, preparing the concentrated gel according to the formula, adding various components in the order from top to bottom, and uniformly mixing. And pouring concentrated glue on the top of the separation glue, inserting a tooth comb, and polymerizing the concentrated glue. After the concentrated glue is polymerized, the glass plate can be taken down and the preservative film at 4 ℃ can be wrapped up for storage and used within a week.

(3) Electrophoresis

Taking out the prepared rubber plate, and slightly pulling out the comb in parallel. And (3) placing the glue on an electrophoresis tank, and adding an electrophoresis buffer solution, wherein the buffer solution needs to submerge the upper resistance wire and the lower resistance wire. And adding a sample to be detected by using a sample adding gun head according to a preset sequence, and pre-dyeing a Marker. The electrophoresis tank is placed in ice water, the voltage of 80V is used for concentrating gel electrophoresis for 20-30 minutes, the voltage of 120V is used for separating gel electrophoresis, bromophenol blue runs to the bottom edge for about 60-80 minutes, and the electrophoresis is finished.

(4) Rotary film

PVDF membrane (0.45 μm) was soaked in methanol for 1 minute, and the filter paper, PVDF membrane and sponge were soaked in the membrane-transfer buffer. After electrophoresis, the gel was carefully removed and placed in the membrane transfer buffer. And opening the film rotating clamp, putting the filter paper, the sponge, the glue and the PVDF film in a specified sequence, and exhausting bubbles. The power was turned on, always at 4 ℃ for 90 minutes.

(5) Immune response and chemiluminescence

Adding 5% skimmed milk powder, sealing, and incubating at room temperature for 2 hr; primary antibodies (antibody dilution ratio HMGB1 (1) dilution, RAGE (1); sucking out primary anti-incubation liquid, and rinsing with TBST; adding the secondary antibody solution, and incubating for 60 minutes at room temperature; discarding the secondary antibody, not recovering, and rinsing with TBST; the PVDF membrane protein sample is placed on the preservative film with the side facing upwards, equal amounts of chemiluminescent agent and reinforcing agent are mixed, dripped on the PVDF membrane, and developed. The grey values of the protein bands in the pictures were analyzed with image J software.

The corneas of mice not subjected to alkali burn and corneas of mice in each group on day 14 after the model creation of alkali burn were collected, and after extraction of corneal proteins, HMGB1 and proteins downstream thereof, TLR4 (Toll-Like Receptors 4, toll-Like Receptor 4) and RAGE (Receptor of Advanced Glycation products) were detected, as shown in FIGS. 34 to 37 (P < 0.05; #: P < 0.05; and P < 0.05; compared with sodium hyaluronate, and DG-RBM, respectively).

The results show that the levels of HMGB1, TLR4 and RAGE were significantly elevated in the corneal tissue of the PBS group of mice compared to the cornea of healthy mice (control). DG-RBM (dipotassium glycyrrhizinate-rebamipide nano micelle eye drops) can effectively reduce the expression levels of HMGB1, TLR4 and RAGE, so that the expression levels of HMGB1, TLR4 and RAGE tend to normal levels. Therefore, DG-RBM is expected to be used as an inhibitor of a key medium HMGB1, and the chemical injury on the cornea of the eye can be effectively prevented and/or treated by regulating an HMGB1 signal channel.

The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof, and it is therefore intended that all such changes and/or modifications be considered within the scope of the appended claims.

Claims (10)

1. A pharmaceutical composition, comprising: quinolinone compounds and pharmaceutically acceptable glycyrrhetate; wherein the quinolinone compound is rebamipide or pharmaceutically acceptable salt thereof, and the mass ratio of the quinolinone compound to the glycyrrhetate is 1.

2. The pharmaceutical composition according to claim 1, wherein the mass ratio of quinolinone compound to glycyrrhetate is 1; preferably, the mass ratio of the quinolinone compound to the glycyrrhetate is 1; more preferably, the mass ratio of the quinolinone compound to the glycyrrhetate is 1.

3. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition satisfies one or more of the following conditions i to iii:

i. the quinolinone compound is rebamipide;

ii. The glycyrrhetate is selected from one or more of sodium glycyrrhetate, disodium glycyrrhetate, potassium glycyrrhetate, dipotassium glycyrrhetate, ammonium glycyrrhetate and diammonium glycyrrhetate; preferably, the glycyrrhetate is dipotassium glycyrrhizinate or disodium glycyrrhizinate;

iii, the encapsulation rate of the quinolinone compounds is more than 80%; preferably, the encapsulation rate of the quinolinone compound is more than or equal to 90% or more than or equal to 95%;

preferably, the pharmaceutical composition simultaneously satisfies conditions i to iii.

4. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is prepared by a process comprising the steps of: dispersing or dissolving quinolinone compounds and glycyrrhetate in a solvent, uniformly mixing, and then carrying out rotary evaporation at 35-45 ℃ to remove the solvent to obtain the quinolinone compounds;

the solvent is preferably an alcohol solvent, and more preferably methanol or ethanol;

preferably, the dosage of the solvent is 5-100 mL per gram of quinolinone compound.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the pharmaceutical composition further comprises pharmaceutically acceptable excipients; and/or, the pharmaceutical composition is a solid preparation or a liquid preparation; and/or the quinolinone compound in the pharmaceutical composition is in a therapeutically effective amount.

6. The pharmaceutical composition according to any one of claims 1 to 4, wherein the pharmaceutical composition is a liquid preparation prepared from a solvent selected from the group consisting of pharmaceutically acceptable water, PBS buffer, and sodium carboxymethylcellulose aqueous solution;

preferably, the first and second liquid crystal display panels are,

the pH value of the liquid preparation is 6-8, and more preferably 6.8-7.4; and/or the liquid preparation is nano-micelle eye drops.

7. The pharmaceutical composition according to claim 6, wherein when the quinolinone compound concentration in the liquid preparation is 5mg/mL, the liquid preparation satisfies one or more of the following conditions (1) to (3):

(1) the average micelle diameter of the liquid preparation is 1-80 nm; preferably 5 to 25nm;

(2) the polydispersity coefficient of the liquid preparation is less than or equal to 0.5; preferably ≤ 0.4;

(3) the Zeta potential of the liquid preparation is-20-0 mV; preferably-10 to-5 mV;

preferably, the solvent formulation satisfies the conditions (1) to (3) at the same time.

8. The pharmaceutical composition according to any one of claims 1 to 4, wherein the pharmaceutical composition is an ophthalmic formulation;

preferably, the ophthalmic preparation satisfies at least one of the following uses a to f:

a. the ophthalmic preparation is used for treating and/or preventing chemical injury of eyes;

b. the ophthalmic preparation is an ophthalmic preparation for treating and/or preventing dry eye;

c. the ophthalmic preparation is used for treating and/or preventing oxidative stress damage of corneal epithelial cells;

d. the ophthalmic preparation is used for treating and/or preventing corneal epithelial injury;

e. the ophthalmic preparation is used for treating and/or preventing keratitis;

f. the ophthalmic preparation is an ophthalmic preparation for inhibiting corneal neovascularization, corneal inflammatory factor expression, HMGB1 expression, TLR4 expression or RAGE expression;

more preferably, the amount of the organic solvent is,

the chemical injury of the eyes is alkali burn of the eyes; and/or, the cornea inflammatory factor is VEGF, TGF-beta 1, NF-kappa B, TNF-alpha, IL-6 or IL-1 beta.

9. A process for the preparation of a pharmaceutical composition according to any one of claims 1 to 8, comprising the steps of:

dispersing or dissolving quinolinone compounds and glycyrrhetate in a solvent, uniformly mixing, and then performing rotary evaporation at 35-45 ℃ to remove the solvent to obtain a solid product;

optionally thereafter: dissolving or dispersing the solid product in a solvent of the liquid preparation, adjusting the pH value of the liquid preparation to 6-8, and filtering and sterilizing the solution;

preferably, the first and second electrodes are formed of a metal,

the solvent is an alcohol solvent, and methanol or ethanol is more preferable;

the dosage of the solvent corresponding to each gram of quinolinone compound is 5-100 mL;

adjusting the pH value of the liquid preparation to 6.8-7.4;

sodium hydroxide and/or potassium hydroxide are used to adjust the pH of the liquid formulation.

10. Use of a pharmaceutical composition according to any one of claims 1 to 8 for the preparation of an ophthalmic formulation.

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