阅读说明：本技术 Fdi化合物在眼科疾病中的用途 (Use of FDI compounds in ophthalmic diseases ) 是由 刘旭阳 谭俊凯 刘果 蓝春林 王云 蔡素萍 于 2021-03-31 设计创作，主要内容包括：本发明涉及药物活性成分叉头结构域抑制剂FDI的新用途,用于眼部疾病。(The invention relates to a novel application of a pharmaceutical active ingredient of a forkhead domain inhibitor FDI, which is used for treating eye diseases.)

1. Use of a compound of formula (I) and salts, isomers, hydrates thereof in the manufacture of a medicament for the treatment and/or prevention of ocular damaging diseases, the compound of formula (I) having the structure:

wherein R1, R2, R3 and R4 are respectively and independently selected from H, halogen or C1-2 alkyl.

2. The use according to claim 1, wherein R1, R2, R3, R4 are each independently selected from halogen; such as F, Cl, Br, I respectively and independently; preferably, R1, R2, R3, R4 are F.

3. Use according to claim 1, wherein the compound of formula (I) is the forkhead domain inhibitor FDI-6.

4. Use of a forkhead domain inhibitor FD1 or a salt, isomer or hydrate thereof in the preparation of a medicament for treating and/or preventing ocular injury diseases.

5. The use according to claim 4, wherein the FD1 is selected from the group consisting of FDI-2, FDI-4, FDI-7, FDI-10 or FDI-11 and isomers or salts thereof.

6. The use according to any one of claims 1 to 5, wherein the ocular damaging condition is selected from inflammatory injury, oxidative injury, physical injury (e.g. trauma, surgery, radiation injury) or immunological injury.

7. The use according to any one of claims 1 to 5, wherein the ocular damaging disorder is selected from ocular fibrosis, ocular inflammation or ocular neovascular disorders.

8. The use of any one of claims 1 to 5, wherein the ocular damaging condition is ocular fibrosis, ocular inflammation or ocular neovascular disease associated with ocular surgery selected from laser eye surgery, refractive surgery, corneal transplantation, keratotomy, keratomileusis, cataract surgery, glaucoma filtration surgery, trabeculectomy, tunnel formation, scleral reinforcement surgery, corneal surgery, vitreoretinal surgery, retinal detachment repair, retinal fixation surgery, pneumatic retinal fixation, eye muscle surgery, surgery involving the lacrimal apparatus, insertion of an implant into the eye and removal of the eye, removal of an eye content or other ophthalmic surgery.

Technical Field

The application relates to application of a forkhead domain inhibitor FDI in preparing a medicament for treating ocular injury diseases, in particular to application of the FDI in ocular fibrosis and ocular inflammatory and neovascular diseases.

Background

The visual organs are one of the important senses of the body and are also the most sensitive organs of the human body. The eye diseases cover a wide range of people, from young people susceptible to xerophthalmia and ametropia to old people susceptible to glaucoma and cataract, and the eye diseases seriously disturb people's lives along with the change of eye using modes, the popularization of overuse of eyes and the further aggravation of aging of the eyes. Among the treatments for many ophthalmic diseases, pharmacological therapy undoubtedly plays an important role.

Because the eye has a precise structure and is protected by anatomy, physiological barrier action and systemic circulation, the ophthalmic drug shows unique pharmacokinetic characteristics. In local or systemic administration this barrier must be overcome to reach the retina and vitreous. Due to the existence of blood-eye barrier, including blood-aqueous humor barrier and blood-retinal barrier, which are special anatomical structures, ophthalmic drug needs to consider the unique pharmacokinetic and pharmacodynamic characteristics of the drug in the local region of the eye, in addition to the strict control of the indications. The drug may be absorbed from blood vessels in the ocular surface structure, such as the limbal and conjunctival blood vessels, through the blood circulation into the eyeball, or permeate into the eyeball through the conjunctiva, fascia, and sclera. The main route of entry into the intraocular tissue from the surface of the eye is transcorneal transport, which is first distributed to, from, to, the tear film, into the cornea, and from the cornea into the eye. Wherein, the cells of the corneal epithelial cell layer and the endothelial cell layer are tightly connected, and the medicine can not enter through the extracellular space and can only be transported by the cell membrane. Thus, drugs that are available in other tissues or organs are generally not directly applied to the eye for their therapeutic effect. In addition, tears, cornea, conjunctiva, anterior chamber, vitreous, sclera, and other areas of the back of the eye interact with drugs differently. Meanwhile, there are many factors that affect the penetration of the drug through the cornea, such as drug concentration, viscosity, lipid solubility, surface activity, etc., and the inadequacy of pH and osmotic pressure can also cause eye irritation or reflex tear secretion. The drug is excreted via aqueous humor or directly, such as venous reflux, after being metabolized in the eyes, and the unique metabolic pathway of the drug has great influence on whether the drug can safely act on the eyes. And because corneal epithelial cells and endothelial cells both have lipid barriers, and tears and corneal stroma are water-soluble, the requirements for ophthalmic drug administration are quite special. It is very difficult to find a new safe and effective eye medicine.

Primary Open Angle Glaucoma (POAG) is a lifelong, life-threatening optic neuropathy that has elevated intraocular pressure as a major risk factor and requires lifelong treatment. At present, the treatment of glaucoma mainly reduces intraocular pressure. Therapeutic approaches for lowering intraocular pressure, such as drug therapy, laser therapy, glaucoma filtration surgery (e.g., trabeculectomy), etc., have proven to be effective interventions for treating glaucoma. Among them, trabeculectomy is the most common operation for treating severe glaucoma, and its operation principle is to create a new aqueous outflow channel from the anterior chamber to the subconjunctival space of the eye, thereby lowering the intraocular pressure. However, the long-term results of such procedures are not always satisfactory, as fibrosis and scarring of the subconjunctival tissue at the filtration site may lead to blockage of the artificial outflow tract, ultimately leading to surgical failure.

Subconjunctival fibrosis is a complex, multi-stage pathological process. In the initial stage after surgery, subconjunctival fibroblasts differentiate into myofibroblasts. The differentiated cells then proliferate and migrate and synthesize extracellular matrices such as collagen type i and fibronectin, a series of changes that may result in the formation of twisted and nonfunctional aggregates of scar tissue at the surgical site that eventually block the artificial outflow tract, resulting in surgical failure. Current antiproliferative drugs, such as 5-fluorouracil and mitomycin C, have been widely used clinically to prevent subconjunctival fibrosis following filteration. However, the non-specific effects of these drugs may lead to side effects such as corneal and intraocular tissue toxicity, ocular hypotension, leakage or infection associated with the bleb.

In addition, the fibrotic diseases of the eye include scarring of the cornea, secondary cataract, proliferative vitreoretinopathy, and the like. Similar to the pathological process of subconjunctival fibrosis, ocular cells are also characterized by the transformation of myofibroblasts through epithelial interstitium. Due to complex etiology, the effect of the conventional medicines, operations or laser treatment methods used clinically at present is often unsatisfactory. There is therefore an urgent need for more effective and safer compounds to specifically inhibit postoperative subconjunctival fibrosis and other ocular fibrotic diseases. Ocular Neovascularization (NV) is a leading cause of blindness in many Ocular diseases, including Diabetic Retinopathy (DR), age-related macular degeneration (AMD), retinopathy of prematurity (ROP), central and branch retinal vein occlusion (CRVO and BRVO), infectious keratitis, trauma, and various inflammatory eye diseases, among others. Among the different tissues of the eye, the retina, choroid and cornea are the most common sites of ocular NV, which is also found in severe ischemic ocular diseases and neovascular glaucoma. The new blood vessels have increased blood vessel permeability due to incomplete pericyte coverage, altered vascular endothelial function, etc., and finally cause increased vascular leakage. Meanwhile, overgrowth of new blood vessels into the outer retina in the avascular zone causes structural disorder thereof, which together cause visual impairment of the body to different degrees.

The mechanism of neovascular eye disease has not yet been fully elucidated. More and more researches show that under various pathological conditions, such as hypoxia, ischemia, inflammation, infection, trauma and the like, the balance between angiogenesis stimulating factors and angiogenesis inhibiting factors is broken, the basement membrane of a normal blood vessel is degraded firstly, and then blood vessel endothelial cells migrate and penetrate out of the basement membrane and migrate to the damaged part, meanwhile, the proliferation capacity of the blood vessel endothelial cells is enhanced, the blood vessel budding is accelerated, and abnormal blood vessel cavity production is caused, so that the mechanism is a common mechanism of ocular neovascularization. The current clinical treatment modes of the neovascular eye disease comprise vitreous surgery, laser photocoagulation, photodynamic therapy, intravitreal injection of anti-VEGF drugs and the like, and particularly, the anti-VEGF treatment is the result of remarkably changing the treatment. However, these treatments have their own drawbacks and have limited therapeutic efficacy. Long-term treatment with anti-VEGF is reported to have retinal atrophy, Retinal Pigment Epithelium (RPE) tears, systemic adverse reactions, etc. Therefore, the deep research on the molecular mechanism of the ocular neovascular diseases and the development of new anti-neovascular drugs have extremely important clinical significance.

The forkhead box M1 transcription factor (forkhead box M1, FOXM1) is a member of the forkhead/winged-helix (forkhead/winged-helix) transcription factor family, and is mainly responsible for normal cell proliferation, survival and self-renewal, as well as tumor development, progression and drug resistance. Currently, many findings have demonstrated that aberrant overexpression of FOXM1 is a key feature in tumorigenesis and progression for many human cancers. WO2018026776A describes that FOXM1 inhibitors have neuroprotective effects for the treatment of disorders associated with androgen receptor activity or spinal bulbar muscular atrophy, etc., in a subject. Incorporated FOXM1 Expression by cissplatin inhibitors Paclitaxel-Related Apoptosis in cissplatin-Resistant Human Squamous Cell Carcinoma (OSCC) Cell Lines (Hyeong Sim Choi et al, 2020) detected the Expression of forkhead box protein M1(FOXM1) mRNA and protein on Oral Squamous Cell Carcinoma (OSC) Cell Lines by real-time qPCR and Western blot assays, and Cisplatin-induced over-Expression of FOXM1 showed the same trend only in cis-platin Resistant Cell Lines, indicating that it is associated with the inhibition of Paclitaxel-Related Apoptosis. Meanwhile, FOXM1 showed some increase in pulmonary fibrosis as described in Loss of FOXM1 in macrophages proteins pulmonary fibrosis by activating p38 MAPK signaling pathway PLoS Gene (Goda C et al, 2020).

The forkhead Domain inhibitor FDI (forkhead Domain inhibitor) belongs to the FOXM1 inhibitor, is a small molecule inhibitor, and related inhibitors relate to compounds disclosed mainly including FDI-2, FDI-4, FDI-6, FDI-7, FDI-10 or FDI-11 and salts thereof. For example, the forkhead domain inhibitor FDI-6 can selectively bind to the DNA binding domain of the FOXM1 protein, so that the FOXM1 is displaced from chromatin, and the transcription of the gene regulated by the FOXM1 is widely inhibited. The description of the Suppression of the overexpression of the FOXM1 transgenic program via novel small molecule inhibition (Michael v. gormally et al 2014) states that the screening of 54,211 small molecule libraries from a high throughput point of view identifies novel small molecule inhibitors of FOXM1 that block DNA binding, wherein FDI6 is characterized by deep binding and is capable of directly binding FOXM1 protein, displacing FOXM1 from the genomic target of MCF7 breast cancer cells and inducing concomitant transcriptional downregulation. Global transcriptional profile of MCF7, cells from RNAeq showed FDI6 specific down-regulated FOXM 1-activated genes. This small molecule mediated effect was selective for the gene controlled by FOXM1, and had no effect on genes regulated by the homologous Forkhead family factors. CN109432091A (2018) describes the use of FDI-6 in the preparation of a medicament for the treatment of colon cancer. CN108210494A (2018) describes that FDI-6 plays a corresponding anti-hepatic fibrosis role as FOXM1 inhibitor, and the diseases causing hepatic fibrosis mainly include but are not limited to: chronic viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver disease, toxin and drug, autoimmune liver disease, liver blood stasis, hereditary metabolic disease, etc. Recipical Regulation Between Forkhead Box M1/NF- κ B and Methionine Adenosyltransfer enzyme 1A drivers Cancer (Yuan Li et al, 2020) found that FOXM1 expression was induced in both hepatocellular carcinoma and cholangiocarcinoma, examined the effect of FDI-6 on Cancer in xenografts and syngeneic models in vitro and in Liver, found that FDI-6 inhibited hepatoma cell growth in vitro and in vivo, and had minimal effect in hepatoma cells that did not express MAT 1A. The effect of FOXM1 and compensatory signalling pathways on cancer cell viability was determined by analysis of the compensatory signalling pathway induced by FDI6 in ovarian cancer cells by analysis of the compensatory signalling pathway induced by FDI6, and by assessing the effectiveness of FOXM1 and compensatory signalling pathways in simultaneously inhibiting ovarian cancer cell survival.

As can be seen, the current research on FDI is mainly focused on the aspect of antitumor activity; the application of the compound in eye diseases such as eye injury diseases is not available; particularly, the application report in the field of ocular fibrosis or inflammatory diseases and neovascular diseases is not found.

Disclosure of Invention

The present invention is proposed to overcome the current situation that the therapeutic effect of eye injury diseases in the prior art is unsatisfactory, and to find a new, safe and effective eye therapeutic agent. The invention effectively expands the application field of the pharmaceutical active ingredient of the forkhead domain inhibitor FDI and provides a new application of the FDI.

In one aspect, the present invention provides the use of an FDI compound of formula (I) and salts, isomers, hydrates thereof in the preparation of a medicament for the treatment and or prevention of ocular injury diseases, said FDI compound of formula (I) having the structure:

wherein R1, R2, R3, R4 are each independently selected from H, halogen or C1-2 alkyl;

further, wherein R1, R2, R3, R4 are each independently selected from halogen; further, each is independently selected from F, Cl, Br and I;

further, wherein R1, R2, R3, R4 are F.

Further, the invention provides a use of FDI compound of formula (II) and salt, isomer and hydrate thereof in preparing medicament for treating and/or preventing ocular injury diseases, wherein the compound of formula (II) has the following structure:

the invention provides an application of FDI with a structure shown in formula I or salts, isomers and hydrates thereof in preparing medicines for treating and/or preventing ocular injury diseases.

In a second aspect, the present invention provides the use of a compound of formula FDI-2, FDI-4, FDI-7, FDI-10 or FDI-11, or isomers or salts thereof, for the manufacture of a medicament for the treatment and or prevention of ocular damaging diseases, said compound having the structure:

further, the ocular damaging disease is selected from inflammatory injury, oxidative injury, physical injury (e.g., trauma, surgery, radiation injury) or immunological injury; further, selected from ocular fibrosis, ocular inflammation or ocular neovascular disease; further, the disease is selected from ocular fibrosis, ocular inflammation or ocular neovascular diseases related to ocular surgery.

The medicine can inhibit inflammatory reaction and neovascularization after corneal injury, and remarkably reduce the formation of fibrotic scar in an artificial outflow channel. The drug of the present invention itself did not cause any adverse reaction throughout the experimental monitoring. The medicine of the invention is innovatively applied in the field of ophthalmology, can improve the success rate of surgery and reduce scar formation, has the function of resisting ophthalmic inflammation and neovascular diseases, and has no obvious adverse reaction. Therefore, the compound can become a very potential drug in the treatment of ophthalmic related diseases.

Drawings

FIG. 1: western blot to detect the expression of FOXM1 protein after different concentrations of FDI-6(2.5, 5, 10, 20, 30, 40 mu M) treatment of primary rabbit conjunctival sac fibroblasts

FIG. 2: cell proliferation Capacity at different concentrations of FDI-6(2.5, 5, 10, 20, 40. mu.M), and corresponding cell number

FIG. 3: healing conditions of scratches at 0, 12 and 24 hours after FDI-6 treatment of primary rabbit conjunctival sac fibroblasts and quantitative analysis thereof

FIG. 4: gel contraction conditions at 0, 12 and 24 hours after FDI-6 treatment of primary rabbit conjunctival sac fibroblasts and quantitative analysis thereof

FIG. 5: mRNA expression analysis of alpha-SMA, COL1A1 and FN after FDI-6 treatment of primary rabbit conjunctival sac fibroblasts

FIG. 6: protein expression levels of FN, COL1A1, alpha-SMA, p-Smad2/3, and Total Smad3 following FDI-6 treatment of primary rabbit conjunctival sac fibroblasts

FIG. 7: variation in intraocular pressure (IOP) at various time points (POD indicates days after surgery)

FIG. 8: representative morphological images of blebs in groups of 7 and 14 days post-surgery and bleb scores thereof

FIG. 9: corneal sodium fluorescein staining and representative image of anterior segment of eye on day 14 post-surgery

FIG. 10: confocal biomicroscopy of laser images of conjunctival epithelium, superficial stroma, and deep stroma in follicular region on day 14 after surgery

FIG. 11: hematoxylin-eosin staining, Masson trichrome staining and alpha-SMA immunohistochemistry of surgical (bleb) tissue in each group on day 14 post-surgery

FIG. 12: sodium fluorescein staining of cornea and representative image of anterior segment of eye

FIG. 13: effect of FDI-6 on the transcriptional levels of the corneal FOXM1, CD31, VEGF, IL-6, alpha-SMA and MMP-9 genes

Detailed Description

The ocular injury diseases of the invention are selected from inflammatory injury, oxidative injury, physical injury (such as trauma, surgery, radiation injury), immune injury and the like; further, selected from pathological injuries caused by various primary or secondary eye diseases or injuries caused by eye surgery; including ocular fibrosis, ocular inflammation, ocular neovascular disease, and the like; selected from ocular fibrosis, ocular inflammation or ocular neovascular disease associated with ocular surgery; further, a therapeutic agent selected from the group consisting of glaucoma, retinal vasculitis, uveitis (e.g., posterior uveitis), keratoconjunctivitis sicca, conjunctivitis, retinitis secondary to glaucoma, episcleritis, scleritis, keratitis, dacryocystitis, optic neuritis, retrobulbar neuritis, Diabetic Retinopathy (DR), ocular rosacea, age-related macular degeneration, neovascular glaucoma, corneal neovascularization, retinal choroidal neovascularization, polypoidal choroidal vasculopathy, diabetic retinopathy, diabetic macular edema, ischemic proliferative retinopathy, retinitis pigmentosa, cone dystrophy, proliferative vitreoretinopathy, retinal artery occlusion, retinal vein occlusion, retinal detachment, retinal pigment epithelium detachment; ocular inflammation after ocular surgery, ocular inflammation caused by physical ocular trauma, cataract, ocular allergy, dry eye, blepharitis, meibomian gland dysfunction, retinal-specific disorders such as retinitis pigmentosa and age-related injury, and ocular diseases caused by microbial infection, such as trachoma, pinkeye, ocular herpes, etc.; and ocular diseases accompanied by these diseases.

Further, particularly in the case of ophthalmic surgery, preventive measures may include the prevention and treatment of post-operative fibrosis, as well as the minimization of post-operative inflammation and neovascular disease. Further, the ophthalmic surgery is selected from the group consisting of laser ophthalmic surgery, refractive surgery, corneal transplantation, keratotomy, keratomileusis, cataract surgery, glaucoma filtration surgery, trabeculectomy, tunnel plasty, Karmra Inlays (Karmra Inlays), sclera consolidation surgery, corneal surgery, vitreoretinal surgery, retinal detachment repair, retinal fixation surgery, pneumatic retinal fixation, eye muscle surgery, surgery involving the lacrimal apparatus, inserting an implant into an eye and eye removal, eye content removal or other ophthalmic surgery such as scleral incisions, epikeratophakia, ciliary dissection, ciliary excising, ciliary denervation, iridectomy, and the like.

The medicine can be applied by the routes of ocular local administration, injection administration, oral administration or transdermal administration; further, topical administration includes eye drops, or other topical preparations suitable for direct ocular administration, such as lotions, gels, ointments, and the like; the injection administration comprises subconjunctival injection, intrascleral injection, periocular injection, intravitreal injection, ocular insert, surgical implant such as sustained release implant, local injection, intramuscular injection, intravenous injection, subcutaneous injection, and other injections or powder injections; oral administration includes tablets, capsules, pills, granules, powders, liquid preparations such as solutions, emulsions, suspensions and the like.

In the medicament of the present invention, the concentration of the active ingredient is 0.5 to 500. mu.M, further, the concentration of the active ingredient is 1. mu.M, 1.5. mu.M, 2. mu.M, 2.5. mu.M, 3. mu.M, 3.5. mu.M, 4. mu.M, 4.5. mu.M, 5. mu.M, 5.5. mu.M, 6. mu.M, 7. mu.M, 8. mu.M, 9. mu.M, 10. mu.M, 15. mu.M, 20. mu.M, 25. mu.M, 30. mu.M, 35. mu.M, 40. mu.M, 45. mu.M, 50. mu.M, 55. mu.M, 60. M, 65. mu.M, 70. mu.M, 75. mu.M, 80. M, 85. mu.M, 90. M, 100. M, 110. mu.M, 120. M, 130. M, 140. mu.M, 150. M, 160. mu.M, 170. mu.M, 180. M, 200. M, 220. mu.M, 250. M, 275. mu.M, 300. mu.M, 375, 500. mu.M, 475. mu.M; preferably, the concentration of the effective component is 1-400 μ M; preferably, the concentration of the effective component is 2-300 μ M; preferably, the concentration of the effective component is 5-250 μ M; preferably, the concentration of the effective component is 10-200 μ M; preferably, the concentration of the effective component is 20-150 μ M; preferably, the concentration of the effective component is 30-100 μ M; preferably, the concentration of the effective ingredient is 40-80. mu.M.

Salts of the compounds of the invention are in the form of pharmaceutically acceptable salts, such as acid addition salts or base addition salts; acid addition salts can be prepared from suitable inorganic acids or suitable organic acids. Examples of such inorganic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, sulfuric acid, and phosphoric acid. Examples of such organic acids are selected from the group consisting of aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic organic acids, such as formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, glucuronic acid, maleic acid, fumaric acid, pyruvic acid, aspartic acid, glutamic acid, benzoic acid, anthranilic acid, methanesulfonic acid, salicylic acid, 4-hydroxybenzoic acid, phenylacetic acid, mandelic acid, pamoic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, pantothenic acid, 2-hydroxyethanesulfonic acid, toluenesulfonic acid, sulfanilic acid, cyclamic acid, stearic acid, alginic acid, beta-hydroxybutyric acid, galactaric acid and galacturonic acid. Base addition salts include: metal salts such as salts made of aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc; or salts made from organic bases including primary, secondary and tertiary amines, substituted amines including cyclic amines such as caffeine, arginine, diethylamine, N-ethylpiperidine, histidine, glucosamine, isopropylamine, lysine, morpholine, N-ethylmorpholine, piperazine, piperidine, triethylamine and trimethylamine.

The medicine can be prepared into corresponding dosage forms with conventional auxiliary materials in the field.

For example, the eye drops can be prepared as needed by appropriately selecting: tonicity agents such as sodium chloride, glycerin, mannitol, or glucose; buffers such as sodium phosphate or sodium acetate; surfactants such as polyoxyethylene sorbitan monooleate, polyoxyl 40 stearate or polyoxyethylene hydrogenated castor oil; stabilizers such as sodium citrate or sodium edetate; preservatives, such as benzalkonium chloride or parabens; and the like. The pH of the eye drops is within a range acceptable for ophthalmic preparations, preferably in a range of 4 to 8, preferably in a range of 5 to 7, 5.5 to 6.5. As the pH adjuster, a general pH adjuster can be used.

For example, the injection may be appropriately selected from tonicity agents such as sodium chloride, glycerin, mannitol or glucose; buffers such as sodium phosphate or sodium acetate; surfactants such as polyoxyethylene sorbitan monooleate; thickeners, such as methylcellulose, and the like.

For example, eye ointments can be prepared using widely used bases such as white petrolatum or liquid paraffin; the intercalator can be prepared using a biodegradable polymer such as hydroxypropyl cellulose, hydroxypropylmethyl cellulose, carboxyvinyl polymer or polyacrylic acid, and appropriately selecting an excipient, a binder, a stabilizer, a pH adjuster, etc., when necessary; the intraocular implant can be prepared using a biodegradable polymer such as polylactic acid, polyglycolic acid, lactic acid-glycolic acid copolymer, or hydroxypropylcellulose, and appropriately selecting an excipient, a binder, a stabilizer, a pH adjuster, or the like as needed.

For example, in the case of tablets, capsules, granules, powders and the like, such a preparation can be prepared as appropriate by selecting as follows: excipients such as lactose, glucose, D-mannitol, anhydrous dibasic calcium phosphate, starch or sucrose; disintegrants such as carboxymethylcellulose, carboxymethylcellulose calcium, croscarmellose sodium, crospovidone, starch, partially gelatinized starch or low-substituted hydroxypropylcellulose; binders such as hydroxypropyl cellulose, ethyl cellulose, gum arabic, starch, partially gelatinized starch, polyvinylpyrrolidone or polyvinyl alcohol; lubricants, such as magnesium stearate, calcium stearate, talc, hydrous silicon dioxide, or hydrogenated oil; coating agents such as refined sucrose, hydroxypropylmethylcellulose, hydroxypropylcellulose, methylcellulose, ethylcellulose or polyvinylpyrrolidone; flavoring agents, such as citric acid, aspartame, ascorbic acid or menthol; and the like.

The medicament of the invention can be used for independent administration or combined administration with other active pharmaceutical ingredients. The other active ingredients are antiinflammatory (such as steroid or non-steroid antiinflammatory), antibiotic, antiviral agent, antifungal agent, anesthetic, and analgesic. Can be administered simultaneously, or sequentially; can be prepared into single preparation or combined preparation or kit. The kit comprises a container for holding the separate compositions, such as a separate bottle or a separate foil packet, however, the separate compositions may also be contained in a single, undivided container.

Example 1 Effect of FDI-6 on Primary Rabbit conjunctival sac fibroblasts

(1) Material

1) The concentrations of FDI-6 drug used in the experiment were: 2.5, 5, 10, 20 and 40 μ M.

2) Primary Rabbit conjunctival sac Fibroblasts (rabbittenon's Fibroblasts) and primary fibroblast culture medium thereof (Sainbur biotechnologies Co., Ltd., China).

3) Main biochemical reagents (cartridge) used for detection: cell counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Kumamoto, Japan), Neutral type I collegen from rat tail (A10483-01, Gibco, Thermo Fisher Scientific, former savant, MA, USA), RevertAId First Strand cDNA Synthesis Kit (Fermentas, Thermo Fisher Scientific, Pittsburgh, PA, USA), SYBR Green Real-Time PCR Master Mix (Toyobo, Osaka, Japan), TGF-. beta.1 (PeTechh, NJ, USA).

4) Primary antibody used for detection and its working concentration: FOXM1(ab207298, rabbitmonoclonal antibody,1:1,000; Abcam, cambridge, MA, USA), SMA (ab7817, mouse monoclonal antibody,1: 200; Abcam, cambridge, MA, USA), COL1A1(ab6308, mouse monoclonal antibody, 1-2. mu.g/mL; Abcam, cambridge, MA, USA), FN (ab6328, mouse monoclonal antibody, 1-5. mu.g/mL; Abcam, cambridge, MA, USA), Smad 399523, Rabbitmonoclonal antibody,1:1000, Cell Technology, Danvers, MA, USA), Phospho 399523, Rabbitmonoclonal antibody, USA (# 578, Lab 572, USA), Cell 5718, Cell 578, Cell 5718, Cell 577, USA, Cell 5718, Cell 577, Cell 578, Cell 52, Cell 5710, USA, Cell 5718, Cell 5710, Cell 52, Cell 32, Cell 32, Cell 577, Cell.

5) Main instruments used for detection: fluor ChemE (92-14860-00; protein simple, San Jose, Calif., USA), flame atomic absorbance photometry (Perkin Elmer, Boston, MA, USA), Leica DM4 microscope (DM 400B; Leica, Wetzlar, Germany), Rotor-Gene Q cycler (QIAGEN, Germantown, MD, USA).

(2) Experimental methods

1) Cell culture and treatment with FDI-6 working solution of various concentrations

Firstly, primary rabbit conjunctival sac fibroblast is cultured at 37 ℃ and 5% CO₂Culturing in an incubator. The matched cell culture medium is replaced once a day. And carrying out subsequent experiments when the cells are passaged to 3-6 generations.

② 5 groups of cell experiments are carried out, and the following methods are adopted for treatment: blank control, cells treated with media only; dimethyl sulfoxide group (DMSO, diluted to 0.04% concentration with medium); TGF-. beta.1 group, cells were pretreated with 0.04% DMSO for 1 hour, and then with 20ng/mL TGF-. beta.1 for 24 hours; FDI-6+ TGF-. beta.1 group, cells were pretreated with 10M FDI-6 for 1h, then with 20ng/mL TGF-. beta.1 for 24 h; in the FDI-6 group, cells were treated with FDI-6 at concentrations of 2.5, 5, 10, 20 and 40. mu.M (screening experiments), or FDI-6 at a concentration of 10. mu.M for 24 hours.

2) CCK-8 detection and cell counting

Primary rabbit conjunctival sac fibroblast is cultured at 4.5 x 10³Number per well cells were seeded in 96-well plates and treated with FDI-6 or 20ng/ml TGF-. beta.1 at concentrations of 2.5, 5, 10, 20 and 40. mu.M for 24h, respectively. The spent medium was discarded and added to each well with fresh medium containing CCK-8 reagent (10. mu.l/well) and incubated for 3 hours. Finally, absorbance values at 450nm wavelength were measured for each well using Fluor ChemE. At the same time as this is done,each group (per well) of adherent cells was counted.

3) Scratch test

Primary rabbit conjunctival sac fibroblasts were seeded into 12-well plates. When the cells grew to 80-90% confluence, the cell culture was scraped with a 200 μ l sterile pipette tip. Phosphate Buffered Saline (DPBS) was used to remove scraped cell debris, and then serum-free medium diluted FDI-6 (10. mu.M) or TGF-. beta.1 (20ng/ml) was added. Cell migration images were taken at 0, 12 and 24 hours, respectively, and ImageJ software was used to analyze the wound healing rate (original wound area-actual wound area)/original wound area x 100% for each group. After treatment, cells will grow into the wound at different rates. Healing (wound closure) is achieved when the cells on both sides of the scarred edge contact each other and heal the gap.

4) Gel shrinkage test

Neutral type I collagen from rat tail working solution was prepared according to the instructions and mixed with 2.5X 10⁵Primary rabbit conjunctival sac fibroblast cell suspensions of cells/ml were mixed on ice in a volume ratio of 9: 1. The 24-well plate was incubated with 1% Bovine Serum Albumin (BSA) in an incubator for 1 hour, then 0.5ml of the cell mixture was added per well, and further incubated for 1 hour to allow coagulation. The coagulated collagen gels were detached from the well walls using a 10. mu.l pipette tip and serum-free medium (0.5 ml/well) containing TGF-. beta.1, DMSO or FDI-6 (10. mu.M) was added to the surface of each gel, respectively. Fluor ChemE takes collagen gel contraction images. Gel size was measured with Image J software. The percentage of area contracted of each group is evaluated by the formula: (initial gel area-gel area at each time point)/initial gel area X100%.

5) Western blotting (Western blot)

Primary rabbit conjunctival sac fibroblasts were inoculated into 6-well plates. When the cells have grown to 80-90% confluence, they are treated separately with medium containing TGF-. beta.1, DMSO or FDI-6 (10. mu.M) for a period of time. Cells were collected and total protein was extracted using RIPA cell lysis buffer. The resulting protein samples of each group were loaded at 20-40. mu.g/well on a gradient polyacrylamide gel (4% -20%), electrophoretically separated, and transferred to PVDF membrane. PVDF membranes were blocked for 1 hour with 5% skim milk and then incubated overnight at 4 ℃ in the corresponding primary antibody dilutions, respectively. After washing with 1 × TBS-T for 30 minutes, the cells were incubated with the corresponding secondary antibody dilution for 1 hour at room temperature. Protein bands were visualized with Fluor ChemE and analyzed using ImageJ software.

6) Real-time fluorescent quantitative PCR (qRT-PCR)

Primary rabbit conjunctival sac fibroblasts were inoculated into 6-well plates. When the cells have grown to 80-90% confluence, they are treated separately with medium containing TGF-. beta.1, DMSO or FDI-6 (10. mu.M) for a period of time. Total RNA was extracted from the cells using Trizol reagent (Invitrogen, CA, USA), and RNA content was measured. Reverse transcription reaction (RT-PCR) was performed using the RevertAid First Strand cDNA Synthesis kit. And (3) carrying out Real-Time fluorescent quantitative PCR in a Rotor-Gene Q cycler by adopting a SYBR Green Real-Time PCR Master Mix kit, and detecting the transcription level of each group of corresponding genes. By use of 2^-ΔΔCtThe relative expression level of the target gene is calculated.

(3) Results of the experiment

1) Western blotting detection of FOXM1 protein expression level

To examine the effect of FDI-6 on FOXM1 expression in primary rabbit conjunctival sac fibroblasts, we examined the expression levels of FOXM1 protein in different concentrations of FDI-6 treated cells. The results showed no significant difference in FOXM1 protein expression between the blank, DMSO, and 2.5 μ M FDI-6 groups (fig. 1; n ═ 3, p > 0.05). Compared with the DMSO group, the FDI-6 treated group showed significant dose-dependent down-regulation of FOXM1 expression in cells (FIG. 1; n: 3, p < 0.05). The results show that FDI-6 at these concentrations successfully induced a reduction in FOXM1 protein levels in primary rabbit conjunctival sac fibroblasts.

2) CCK-8 detection and cell counting

The CCK-8 method and cell count were used to determine the total metabolic activity or cell proliferative capacity of cells to exclude the effect of drug toxicity on the expression of FOXM1 and to optimize the FDI-6 concentration. As shown in fig. 2, FDI-6 significantly inhibited the cellular metabolic/proliferative activity of primary rabbit conjunctival sac fibroblasts. The degree of inhibition increases with increasing FDI-6 concentration (n-3, p < 0.05). After 24 hours of treatment with 5 μ M FDI-6, the cell number continued to increase (n-3, p < 0.05); there was still a slight but insignificant increase at 10 μ M (n-3, p < 0.05); however, in the higher concentration (20 and 40 μ M) group, the number of cells decreased significantly, indicating cell death (n-3, p < 0.05). The results show that FDI-6 can effectively inhibit the expression of FOXM1, while FDI-6 with the concentration of 10 mu M seems to be the optimal concentration for subsequent cell experimental study, and expected patient compliance is better, so that a longer-term treatment effect can be realized in the eyes.

3) Scratch test

The purpose of this experiment was to observe the effect of 10 μ M FDI-6 on the migratory capacity of primary rabbit conjunctival sac fibroblasts. To further investigate the anti-fibrotic effect of FDI-6 in cells, a TGF-. beta.1 (20ng/ml) treatment model was therefore established to mimic fibrotic lesions in vitro. As shown in fig. 3, the difference in migration area (%) between the blank control group and the DMSO group was not statistically significant (n ═ 3, p > 0.05). Compared with the DMSO group, the migration area of the 10 μ M FDI-6 group cells was reduced by 22.8% (n ═ 3, p < 0.05). The migration area of TGF- β 1 group cells increased by 11.5% (n-3, p < 0.05), while FDI-6 significantly reversed this increase (n-3, p < 0.05). The result shows that FDI-6 has strong inhibition effect on the migration and proliferation functions of primary rabbit conjunctival sac fibroblasts.

4) Gel shrinkage test

The ability of fibroblasts to shrink collagen gel is an important marker for their differentiation into myofibroblast phenotype. As shown in fig. 4, the average shrinkage area (%) of primary rabbit conjunctival sac fibroblasts was not significantly different between the DMSO group and the blank control group (n ═ 3, p > 0.05). Compared with the DMSO group, the average area of contraction of the FDI-6 group was significantly reduced by 11.6% (n-3, p < 0.05), indicating the ability to modulate myofibroblast activity. Furthermore, the average contractile area of the TGF- β 1 group increased by 7.7% (n-3, p < 0.05), while FDI-6 reversed this contractile capacity. The result shows that FDI-6 has strong inhibiting effect on the contractility of primary rabbit conjunctival sac fibroblasts.

5) Detection of expression of fibrosis-related gene by Western blotting and real-time fluorescent quantitative PCR

The effect of FDI-6 on the expression of fibrosis-associated genes, such as α -SMA, COL1A1 and FN, was observed. As shown in fig. 5 and 6, there was no significant difference in the expression of these genes between the DMSO group and the blank group (n-4 for real-time fluorescent quantitative PCR; n-3 for immunoblot experiments; p > 0.05). FDI-6 significantly reduced the expression of the above genes at the mRNA and protein levels compared to the DMSO group (n-4 for real-time fluorescent quantitative PCR; n-3 for immunoblot experiments; p < 0.05). Compared with the DMSO group, the TGF- β 1 group showed increased expression of the above genes in both mRNA and protein (n-4 for real-time fluorescence quantitative PCR; n-3 for immunoblot experiments; p < 0.05), but FDI-6 also reversed this increase (n-4 for real-time fluorescence quantitative PCR; n-3 for immunoblot experiments; p < 0.05). As shown in fig. 6, FDI-6 significantly reduced the phosphorylation level of Smad2/3 protein in cells (n-3; p < 0.05). The results show that FDI-6 regulates the expression of the fibrosis-related genes in primary rabbit conjunctival sac fibroblasts.

Example 2 Effect of FDI-6 against Rabbit eye fibrosis

(1) Material

1) The concentration of FDI-6 drug used in the experiment was 40. mu.M.

2) Adult New Zealand white rabbits (5 male and female, 10-12 weeks old, 1.5-2kg, institute of laboratory animals of Advance in medical sciences, Sichuan province).

3) The main reagent materials: BD Sharpe disposable sterile insulin syringes (BD, U-100, 0.5ml, 0.33mm/29 Gx 12.7mm), 0.3% of ofloxacin ophthalmic infection (Santen, Inc., Tokyo, Japan), 0.3% of ofloxacin eye drops (Minsheng Pharma, Hangzhou, China), 3% of pentabarbitsodium (30mg/kg, Sanofi, Paris, France), formaldehydehyde, acetic acid, and saline (FAS) injectable (Wuhan service, Wuhan, China).

4) Main instruments used for detection:rebound tonometer(Icare,Finland,Espoo, Finland)，slit lamp biomicroscope(S350；Shanghai Medi Works Precision Instruments, Hangzhou,China)，In vivo confocal microscopy(HRT II/RCM,Heidelberg Engineering GmbH,Heidelberg,Germany)，Leica DM4 microscope(DM400B；Leica, Wetzlar,Germany)。

(2) grouping method

10 rabbits were randomly assigned to each of the following groups

Remarking: the operation mode is trabeculectomy; the injection mode is subconjunctival injection.

(3) Experimental methods

1) Trabeculectomy

Trabeculectomy was performed according to the above table grouping. Rabbits were anesthetized by 3% sodium pentobarbital intravenous injection. After the periocular skin is sterilized, the operating-side eyelid is opened with an eyelid retractor, the peripheral corneal stroma is penetrated with 8-0 suture, and the eyeball is pulled and rotated to one side to fully expose the operating area. A limbal-based conjunctival flap is established in the supranasal quadrant and the sclera is exposed. A square scleral flap of approximately 3 x 3mm was then made. After removal of approximately 0.5X 1 mm of tissue in the trabecular meshwork area, peripheral iridectomy was performed. The scleral flap was sutured with 10-0 nylon suture. The conjunctiva was sutured with 10-0 nylon thread. FDI-6 or physiological saline at 40. mu.M was injected into the subtopical subconjunctival area of the operated or non-operated eye by an insulin syringe with the needle facing the operation area. After the procedure, the operative field was coated with 0.3% of loxacin opthalmic element. Eye drops were applied to 0.3% of ofloxacin eye drops the following day, 4 times daily for 1 week.

2) Intraocular pressure measurement

The animals were conscious and measured for 14 days using a TonoVet rebound tonometer every two days after the operation. Rabbits were first trained to receive measurements in a resting state. The investigators were performed at the same time from 2 pm to 4 pm each day.

3) Clinical examination item

Rabbits were anesthetized by intravenous injection of 3% sodium pentobarbital at the ear margin, and then the following examination items were performed.

Secondly, observing the shape of the filtering bleb in the operation area and the condition of the anterior segment of the eye every day through a slit lamp after operation, and taking pictures on the 7 th and 14 th days. Krofeld scores were made for height and size of bleb on days 7 and 14, as follows:

1+, minimum height, thickening of conjunctiva, no filtering bleb;

2+, filtering bleb is seen;

3+, elevated filtering bleb covers 3 to 4 clock-wise areas of the eyeball;

4+, a significantly elevated filtering bleb covers an area of 5 clock directions beyond the eyeball.

0, indicating no observable blisters.

And thirdly, carrying out fluorescent staining on the cornea by using a sodium fluorescein eye strip, checking the cornea of each rabbit by using blue light of a slit lamp biomicroscope on the 14 th day, and analyzing whether the drug has adverse effect on the cornea.

And the laser confocal biomicroscope is used for checking the condition of the filtering bleb on the 14 th day after the operation and the connective tissue under the epithelium of the filtering bleb area.

All the clinical examination items are carried out by experienced ophthalmologists according to the recommended procedures of manufacturers, and no side effects related to the examinations are found.

4) Histological analysis

On day 14 post-surgery, animals were sacrificed by overdesistant anesthesia with 3% sodium pentobarbital and the bilateral eyeballs were immediately removed, and the surgical area and bleb tissues were marked with magenta. After labeling, the eyes were fixed in formaldehydee, acetic acid, and saline (FAS) fixive at 4 ℃ for 48 hours. Routine paraffin embedding was then performed, 4 μm thick tissue sections. After the sections are treated conventionally, hematoxylin-eosin staining is respectively carried out for histomorphology analysis, Masson trichrome staining is carried out for evaluating collagen expression, and alpha-SMA immunohistochemical staining is carried out for observing the degree of tissue muscle fibrosis. Collagen expression was quantified as the ratio (%) of the area of blue-stained tissue to the total area using the Collagen Volume Fraction (CVF) to quantify the Collagen expression in the conjunctival and scleral layers.

(4) Results of the experiment

1) Intraocular pressure monitoring

As shown in fig. 7, there was no significant difference in pre-operative intraocular pressure values for each group. There was also no significant difference in intraocular pressure between the non-operative groups (placebo, saline and FDI-6 groups) (n ═ 4; p > 0.05). Intraocular pressure was significantly reduced in both the operative group (operative + saline group and operative + FDI-6 group) after the operation, but was significantly lower in the operative + FDI-6 group than in the operative + saline group (n ═ 4; p < 0.05) starting at day 12.

The results suggest that FDI-6 stabilizes ocular tension that is reduced after trabeculectomy.

2) Clinical examination item

(1) As shown in fig. 8, the filtration bleb score was significantly better in the surgery + FDI-6 group than in the surgery + saline group at day 14 (n ═ 4; p < 0.05). Corresponding to bleb scoring results, blebs were small and flat in the surgery + saline group and vascularized in the operative area on days 7 and 14, while blebs were slightly elevated and vascularized in the operative area was less in the surgery + FDI-6 group.

(2) The results of corneal fluorescein sodium staining are shown in fig. 9, and no fluorescein sodium staining was seen in all rabbit eyes, indicating no adverse or toxic effects after FDI-6 treatment. In addition, no significant complications such as corneal or conjunctival epithelial defects, bleb leakage, ocular hypotension, cataracts, and endophthalmitis were observed in all groups. There were no other significant differences between the surgery + saline group and the surgery + FDI-6 group, except for the difference in scarring.

(3) The confocal laser biomicroscopy analysis result is shown in fig. 10, the conjunctival epithelium in the operation + saline group has obvious inflammatory cell infiltration, a plurality of bright white cell nuclei can be seen, and the conjunctival epithelium in the operation + FDI-6 group has no obvious inflammatory cell infiltration. In addition, the conjunctival epithelial subcutaneous connective tissue space was loose in the non-operative group. A large amount of dense fibrous tissue was visible in the superficial and deep interstitium of the conjunctiva of the surgery + saline group, while treatment with FDI-6 reduced the increase in fiber density after trabeculectomy, resulting in functional bleb after trabeculectomy.

The results indicate that FDI-6 treatment can significantly reduce subconjunctival fibrosis and further promote the formation of functional blebs.

3) Tissue morphology analysis

(1) Hematoxylin-eosin staining results showed that the thickness between conjunctiva and sclera increased (n-3; p < 0.05) in the operative group (operative + saline group and operative + FDI-6 group), while the average thickness was significantly thinner in the operative + FDI-6 group than in the operative + saline group, but the difference between groups was not statistically significant (n-3; p > 0.05).

(2) Masson trichrome staining results showed that the operative group accumulated a large amount of collagen subconjunctivally. The collagen volume fraction of the group + FDI-6 was significantly lower than that of the group + physiological saline (n-3; p < 0.05).

(3) The alpha-SMA protein immunohistochemical result shows that positive expression exists between conjunctival tissue and scleral tissue of the operation group, negative expression exists between non-operation group, and alpha-SMA expression of the operation + FDI-6 group is weaker than that of the operation + normal saline group, which indicates that myofibroblasts are fewer.

The results show (see fig. 11) that FDI-6 can prevent subconjunctival fibrosis after trabeculectomy by reducing myofibroblast differentiation and extracellular matrix synthesis, including FN and COL1a 1.

Example 3 Effect of FDI-6 on ocular neovascularization in rats

1 Material

(1) The concentration of FDI-6 drug used in the experiment was 40. mu.M.

(2) Sprague-Dawley rats (SD rats), male, weighing 175-200 g (Beijing Wintorlington laboratory animal technology, Inc., China).

(3) Main biochemical reagents (cartridge) used for detection: sodium hydroxide (Sigma Aldrich, St. Louis, MO, USA), Chloral hydrate (Sigma Aldrich, St. Louis, MO, USA), RevertAId First Strand cDNA Synthesis Kit (Fermentas, Thermo Fisher Scientific, Pittsburgh, Pa., USA), SYBR Green Real-Time PCR Master Mix (Toyobo, Osaka, Japan).

(4) Main instruments used for detection: slit lamp biomicroscope (S350; Shanghai Medi Works Precision Instruments, Hangzhou, China), Rotor-Gene Q cycler (QIAGEN, Germantown, MD, USA).

2 grouping mode

Remarking: the alkali burn is a corneal alkali burn model; the injection mode is subconjunctival injection.

3 Experimental methods

(1) Construction of corneal alkali burn model

SD rats were anesthetized with 10% chloral hydrate intraperitoneally, then the right eye was anesthetized with mydriasis and ocular surface, 3.5X 3.5mm filter paper was immersed in 1N (mol/L) sodium hydroxide for about 10 seconds, dipped in excess liquid and placed in the center of the rat cornea for 45 seconds, immediately followed by rinsing the cornea with 20ml of 0.9% sodium chloride injection for 1min, then 20. mu.l of 40. mu.M FDI-6 or physiological saline was injected under the upper bulbar conjunctiva, and 20. mu.l of 40. mu.M FDI-6 or physiological saline was injected under the left eye only subconjunctival.

Rats were eyedropped with 40. mu.M FDI-6 or physiological saline using a 1ml syringe after surgery. The eye drop is administered 3 times daily, one drop at a time (each administration is maintained for 2min), and the duration is up to 7 days after operation.

(2) Clinical examination item

1) Rats were anesthetized by intraperitoneal injection of 10% chloral hydrate and then subjected to the following examination items.

2) The corneal morphology, permeability and angiogenesis conditions, as well as the anterior segment condition were observed by slit lamps on postoperative days 1, 4 and 7, and pictures were taken. The observation indexes of the cornea neovascularization are as follows:

length: i.e. the length of the new blood vessels extending from the limbus into the cornea.

Area II: using the formula S ═ C/12 x 3.1416 × [ R × ]²-(R-r)²]Analyzing the area of corneal neovascularization growth; wherein C is the cornea neovascularization clock number; r is the corneal radius, and is unified to be 3.5 mm; r is the corneal neovascularization length.

3) Fluorescence staining of the cornea was performed with sodium fluorescein strips, the cornea of each rat was examined on day 7 with a slit-lamp biomicroscope in blue light, and the degree of corneal injury, as well as the effect of the drug on the injury, was analyzed.

(3) Real-time fluorescent quantitative PCR (qRT-PCR)

On day 7 post-surgery, animals were sacrificed by overanesthesia with 10% chloral hydrate and immediately bilateral eyes were removed and corneal tissue was cut along the corneal scleral rim. Total RNA was extracted from corneal tissue using Trizol reagent, and RNA content was measured. Reverse transcription reaction (RT-PCR) was performed using the RevertAid First Strand cDNA Synthesis kit. And (3) carrying out Real-Time fluorescent quantitative PCR in a Rotor-Gene Q cycler by adopting a SYBR Green Real-Time PCR Master Mix kit, and detecting the transcription level of each group of corresponding genes. By use of 2^-ΔΔCtThe relative expression level of the target gene is calculated.

4 results of the experiment

1) Clinical examination item

As shown in FIG. 12, no significant abnormality was observed in the anterior ocular segment in the non-alkali-burned group (blank control group, normal saline group and FDI-6 group), and the cornea was transparent and no new blood vessel was grown. While the alkali burn group showed significant corneal edema, corneal neovascularization and conjunctival congestion, the alkali burn + FDI-6 group was significantly less than the alkali burn + normal saline group. Especially on day 7 corneal edema and the length and area of neovessels were significantly lower than in the surgery + saline group (p < 0.05).

The result of corneal fluorescein sodium staining shows that no fluorescein sodium staining is seen in the non-alkali burn group on the 7 th day after operation, while fluorescein staining is seen in the alkali burn group. However, the alkali burn + FDI-6 group was significantly less colored than the alkali burn + saline group. Indicating that FDI-6 ameliorated the formation of corneal epithelial ulcers.

The result shows that FDI-6 can obviously inhibit the growth of new blood vessels after the corneal alkali burn and relieve the formation of corneal edema and epithelial ulcer.

2) Real-time fluorescent quantitative PCR

The effect of FDI-6 on corneal inflammation and neovascular associated gene expression was observed, as in CD31, VEGF, IL-6, α -SMA and MMP-9.

As shown in FIG. 13, there was no significant difference in the expression of these genes (p > 0.05) between the non-alkali burn groups (blank control group, normal saline group and FDI-6 group), but there was significant upregulation (p < 0.05) in both the alkali burn groups (alkali burn + normal saline group and alkali burn + FDI-6 group). The mRNA level of the gene in the alkali burn and FDI-6 group is obviously lower than that in the alkali burn and normal saline group (p is less than 0.05), which indicates that FDI-6 obviously reverses the up-regulation of the gene expression after corneal alkali burn.

The results indicate that FDI-6 exerts anti-inflammatory and anti-neovascular effects after corneal injury, possibly by inhibiting vascular inducing and inflammatory factors.

In conclusion, in the corneal neovascularization model induced by alkali burn, the FDI-6 treatment can obviously inhibit the growth, inflammation and fibrosis of corneal neovascularization, which is shown in that the area, length, corneal transparency and the like of corneal neovascularization observed under a slit lamp are improved compared with those of an untreated group. Pathological results show that after FDI-6 treatment, the expressions of a blood vessel related marker CD31, a fibrosis related marker alpha-smooth muscle actin (alpha-SMA) and the like are obviously reduced compared with those of an untreated group.

The foregoing shows and describes the general principles and features of the present invention, rather than limitations thereof. It will be understood by those skilled in the art that various equivalent modifications or changes may be made by those skilled in the art without departing from the spirit and scope of the present invention, and still be covered by the present invention.