CN102233132B - Application of VEGF acceptor fusion proteins in preparation of drugs for inhibiting growth of ocular surface neovascularization - Google Patents
Application of VEGF acceptor fusion proteins in preparation of drugs for inhibiting growth of ocular surface neovascularization Download PDFInfo
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Abstract
The invention relates to the application of VEGFR fusion proteins FP1, FP3, FP7 and FP8 in the preparation of drugs for treating ocular surface neovascularization and relevant diseases and a combined preparation of fusion proteins FP1, FP3, FP7, FP8 and an immunosuppressant, wherein the immunosuppressant is one or a combination selected from the group consisting of corticosteroid, rapamycin, dexamethasone and cyclosporine A; and amino acid sequences of FP1, FP3, FP7 and FP8 are respectively as shown in SEQ ID NO:1, 2, 3, 4.
Description
Technical field
The present invention relates to vascular endothelial cell receptor (Vascular Endothelial Growth Factor receptor, VEGFR) medical usage of fusion rotein, particularly the VEGFR fusion rotein is for the preparation of the application in the medicine for the treatment of the disease that is caused by eye table neovascularization growth.
Background technology
The Cornea and conjunctiva of eye table is easy to be subject to various stimulations, comprise that infected by microbes, physical shock, chemical constituent damage, corneal contact lens cause cornea friction breakage, cornea anoxia etc., the capital causes cornea or conjunctiva generation pathological changes, and these diseases often produce eye table new vessels.The disease relevant with eye table new vessels comprises cornea rebirth blood vessel, neovascular glaucoma, pterygium, conjunctivitis, antibacterial keratitis, fungal keratitis, viral keratitis, phlyctenular keratoconjunctivitis, corneal ulcer, scleritis, corneal contact lens complication etc.
The cornea rebirth blood vessel disease comprises that the eye table that cornea wound (comprising a thorn of table thoroughly wound, lacerated wound, scuffing, sting wound, chemical burn, physics burn etc.), corneal graft rejection, anterior chamber of eye and back segment operation cause damages, the corneal stroma ulcer scars forms, foreign body stimulates, the relevant anoxia of contact lens causes cornea rebirth blood vessel, the angiogenesis disease that the reasons such as limbal stem cell deficiency, malnutrition and avitaminosis, adverse effect and iatrogenic injury cause.
Cornea is one of important refractive media of eyeball, is transparent and avascular under normal condition, and still, cornea is under the pathological states such as infection, damage, anoxia and corneal transplantation, and blood capillary is grown in from limbus of corneae to cornea, forms the new vessels of cornea.Although local removing that be conducive to of cornea rebirth blood vessel infected and promotion injury repairing (Curr Opin Ophthalmol.12:242-249,2001), but the generation of cornea rebirth blood vessel can cause change (the Exp EyeRes.78:579-589 of the immunological characteristic environment of cornea tissue, 2004), finally cause the clinical problems such as patient's visual deterioration, corneal injury and the forfeiture of eye table function of shielding.Cornea rebirth blood vessel often causes the scarring of persistence inflammation and tissue, and cornea rebirth blood vessel is the principal character in many blinding keratopathy pathological changes, also is the complication of the main inpairment of vision of corneal infection, chemical damage and corneal transplantation postoperative.Simultaneously, new vessels also affects the success rate of corneal transplantation.
At present, the ocular disease that developed country's cornea rebirth blood vessel causes accounts for the first place of blind reason, and it loses blind rate and reaches 57.2%.China accounts for 10% of whole keratopathy by the cornea rebirth blood vessel due to the ocular injury, and therefore, the prevention of cornea rebirth blood vessel and treatment become present urgent problem.
Although laser therapy and surgical intervention provide potential Therapeutic Method for the cornea rebirth blood vessel patient, still stay some difficult problems after the treatment.For example, in most cases, through can not rebuilding without blood vessel state and pellucidity of the patient's of laser therapy and surgical intervention cornea.In addition, if the clinical hormonal medicaments such as drug main dexamethasone that are used for the treatment of at present cornea rebirth blood vessel, there is widening angle membrane damage area after the medication in such hormonal medicaments, the defective such as corneal healing speed is slow, and side reaction is more.Therefore, provide a safely and effectively medicine, be used for prevention and early treatment and intervene an eye table neovascular disease and eye and show an inflammation tool and be of great significance.
Cornea rebirth blood vessel be the pathological process of a complexity, existing result of study confirms, cornea rebirth blood vessel occurs and the main cause of development is because eye table organization medium vessels generates the balance run-off the straight between the factor and the anti-angiogenesis, but the cytokine of some eye surface diseases Secondary cases high expressed angiogenic growths, induce the eye table to the future development that is conducive to corneal neovascularization, wherein, VEGF is the most important short vascular endothelial cell growth factor (Science246:1306-9,1989) of finding at present.
Chinese patent ZL200510073595.4 discloses fusion protein F P1-FP6 structure and preparation method and anticancer usage thereof, and is used for view order pathological changes, but does not relate to the control of cornea rebirth blood vessel.Chinese patent ZL200610066257.2 discloses structure of fusion protein F P1-FP8 and preparation method thereof, with and medicine in the ophthalmic diseases that preparation treatment is caused by neovascularization growth in application, do not relate to equally VEGFR fusion protein F P1, FP3, FP7 and FP8 and in the medicine of preparation treatment cornea rebirth blood vessel, use.
Neovascular glaucoma refers to that iris and girder surface have newborn fibrovascular membranes to cause the peripheral iris anterior synechiae, hinders aqueous humor and discharges the glaucoma that causes, its new vessels breaks easily, and hyphema occurs repeatedly, so weigh up again courageous and upright glaucoma.Such disease is that a kind of state of an illness is heavy, easily recurrence, comparatively obstinate secondary glaucoma, and general anti-glaucoma medicine and operative treatment often is difficult to gather effect at present, although adopt laser or cryosurgical treatment more, easily recurs, even causes losing one's sight.Patient's eye is congested, corneal edema, and violent ophthalmalgia, headache, in order to remove misery, some patients were has to extract eyeball.
The pterygial cause of disease is not bright so far, may be relevant with ultraviolet radiation, smoke contamination.In recent years, have research to think that this disease is relevant with new vessels, and anaphylaxis and elastic fibrosis also have certain relation.At present, larger on patient's impact although can operative treatment to such disease Therapeutic Method preferably not, be badly in need of preventing and treating safely and effectively medicine.
Conjunctivitis is ophthalmology common disease, frequently-occurring disease.Wherein, mainly use antibiotic or hormone for the control of acute conjunctivitis.Chronic conjunctivitis easily recurs, and is difficult to cure, and causes dry and astringent, the foreign body sensation of patient's eye, and long-term ill meeting causes visual deterioration, blood vessel hyperplasia, also can involve cornea, causes keratitis, corneal ulcer etc.Effective control of such disease also is badly in need of preventing and treating safely and effectively medicine.
The disclosed technology contents of Chinese patent ZL200510073595.4 and ZL200610066257.2 is all as the application's reference.
Summary of the invention
One object of the present invention is to provide a kind of VEGFR fusion protein F P1, FP3, FP7, FP8 for the preparation of the application in the medicine for the treatment of eye table new vessels associated conditions, wherein, the aminoacid sequence of FP1, FP3, FP7 and FP8 is respectively shown in SEQ ID NO:1,2,3,4.
In a preferred embodiment of the invention, described eye table new vessels associated conditions is selected from any or its complication of cornea rebirth blood vessel, conjunctiva new vessels, conjunctivitis, pterygium, neovascular glaucoma, keratitis, antibacterial keratitis, fungal keratitis, viral keratitis, corneal ulcer, phlyctenular keratoconjunctivitis, scleritis, corneal contact lens complication, is preferably any or its complication of cornea rebirth blood vessel, conjunctiva new vessels.
In one embodiment of the invention, described cornea rebirth blood vessel associated conditions is selected from that the damage of eye table, the corneal stroma ulcer scars that cornea wound, corneal graft rejection, anterior chamber of eye and back segment operation cause forms, foreign body stimulates, anoxia causes the cornea rebirth blood vessel hypertrophy, and any or its complication of the neovascular disease due to limbal stem cell deficiency, malnutrition, vitamin deficiency, adverse effect or the iatrogenic injury.
Any or its complication of in a preferred embodiment of the invention, described cornea wound thoroughly wound, lacerated wound, the scuffing of thorn that be selected from eye table, sting wound, chemical burn, physics burn.
In a preferred embodiment of the invention, described chemical burn is selected from acid burn or alkali burn.
In a preferred embodiment of the invention, described viral keratitis is selected from the viral keratitis that herpes simplex virus (HSV) infection causes.
In a preferred embodiment of the invention, described cornea rebirth blood vessel associated conditions is selected from any cornea rebirth blood vessel growth of bringing out or causing of herpes simplex infections, operation suture thread, corneal transplantation postoperative, acid burn or alkali burn.
VEGFR fusion protein F P1 of the present invention, FP3, FP7 and the FP8 a plurality of subclass by catching VEGF family are (such as VEGF-A, VEGF-B, VEGF-C, VEGF-D) and placental growth factor (PIGF), seal and suppress the activation of its receptor, thus performance sealing or the bioactive effect of inhibition vegf protein.
VEGFR fusion protein F P1 of the present invention, FP3, FP7 and FP8 form by the different VEGFR of people functional areas albumen and/or the fusion of human IgG FC Fragment Protein.Particularly, FP1 is comprised of the 2nd immunoglobulin-like district of VEGFR1 and the 3rd immunoglobulin-like district of VEGFR2; FP3 is comprised of the 2nd immunoglobulin-like district of VEGFR1, the 3rd and the 4th immunoglobulin-like district of VEGFR2 and the FC section albumen of human IgG immunoglobulin; FP7 is comprised of the 2nd immunoglobulin-like district of VEGFR1, the 3rd and the 4th immunoglobulin-like district of VEGFR2 and the FC section albumen of human IgG immunoglobulin; FP8 is comprised of the 2nd immunoglobulin-like district of VEGFR1, the 3rd immunoglobulin-like district of VEGFR2 and the FC section albumen of human IgG immunoglobulin.
Method provided by the invention also can be used for treating cornea Acuteinjury and subacute stage damage.Acuteinjury is in 1 week after hindering, and clinical manifestation is edema and the thrombosis of eye table organization, and degeneration and necrosis appear in the cornea of getting involved, conjunctiva and vascular endothelial cell.2 in thoughtful 6 months after subacute stage was generally and hinders, clinical manifestation is the various hyperplasias of eye table and a migration, repair damage and damaged, and corneal epithelial cell occurs and replaced by conjunctival epithelium, and cornea has a large amount of new vesselses (NV) to form, and has a large amount of leukocyte to invade under the damaged tissue.In various concrete application facet, the treatment of Acuteinjury should be carried out in rear 24 hours in damage, and the treatment of subacute stage damage should be shown the Local synthesis situation according to eye, selects golden hour.
Another object of the present invention is to provide the combination formulations of VEGFR fusion protein F P1, FP3, FP7 and FP8 and immunosuppressant, described immunosuppressant is selected from any or its combination of corticosteroid, rapamycin, dexamethasone, ciclosporin A, is preferably dexamethasone.
Combination formulations of the present invention is administration or successively administration sequentially, that is to say, the medical worker can use VEGFR fusion protein F P1, FP3, FP7 and FP8 preparation to the patient first when being patient's administration, use immunosuppressant of the present invention to the patient again; Perhaps use immunosuppressant of the present invention to the patient first, use VEGFR fusion protein F P1, FP3, FP7 and FP8 preparation to the patient again; Perhaps use VEGFR fusion protein F P1, FP3, FP7 and FP8 preparation and immunosuppressant of the present invention to the patient simultaneously.
Description of drawings
The impact of the rat corneal neovascularization that Fig. 1 fusion protein F P1, FP3, FP7 and FP8 bring out alkali burn
The inhibitory action of the mouse cornea angiogenesis that Fig. 2 fusion protein F P1, FP3, FP7 and FP8 bring out operation suture thread
The inhibitory action of the mouse cornea Lymphangiogenesis that Fig. 3 fusion protein F P1, FP3, FP7 and FP8 bring out operation suture thread
The inhibitory action of the rabbit corneal angiogenesis that Fig. 4 fusion protein F P3 brings out alkali burn
Fig. 5 fusion protein F P1, FP3 and immunosuppressant are to the inhibitory action of cornea of rats post-transplantation corneal vascularization
The specific embodiment
Further specify by the following examples the present invention, but should not be construed as the formation any limitation of the invention.
Therapeutical effect and the safety testing of the corneal vascularization that embodiment 1 fusion rotein brings out alkali burn
(the SD rat, female, body weight is the animal model of corneal vascularization of 250g ± 20g), the inhibitory action of the cornea of rats angiogenesis that research fusion protein F P1, FP3, FP7 and FP8 bring out alkali burn to adopt the alkali burn method to set up rat.
Get rat, under narcotism, take right eye as experimental eye, be the cornea of right eye central authorities that filter paper that 3mm has soaked into 1mol/L NaOH solution is affixed on rat with diameter, act on and remove filter paper after 1 minute, with a large amount of normal saline flushing conjunctival sacs, remove remaining NaOH, and dropping Chloramphenicol Eye Drop (manufacturer: Pharmaceutical Co Ltd, Changzhou Pharmaceutical Factory No.4, specification 8ml: 20mg) 20ul and 1% atropine eye ointment.
Be equally divided into 7 group (6 every group) with rat next day, i.e. FP1 group, FP3 group, FP7 group, FP8 group, pharmaceutical carrier group (being PBS solution), Dexamethasone group and normal saline group (NS).Wherein, the administration concentration of fusion rotein is 20mg/ml in FP1, FP3, FP7 and the FP8 group, uses before use the PBS solution allocation, and wherein, PBS is by 0.24g potassium dihydrogen phosphate (KH2PO4), 1.44g sodium hydrogen phosphate (Na
2HPO
4), 8.0g sodium chloride (NaCl), 0.2g potassium chloride (KCl) adds water to 1000mL, adds NaOH and regulates pH7.4; The administration concentration of Dexamethasone group is 0.25mg/ml.Drip at each treated animal eye table and respectively organize solution 4 times every day, each 50 microlitres, and administration time is respectively 8:00,12:00,16:00,20:00, continuous application 14 days.Simultaneously, after modeling, carry out the photograph of cornea fluorescence staining in 15 days, observe the impact of the ulcer area that forms behind the medicine Corneal Alkali, estimate the safety of medicine.
After alkali burn the 15th day, with Animal Anesthesia, with the ink cornea rebirth blood vessel that dyes, examine under a microscope and images acquired, and press Robert formula A=C/12 * 3.1416[R
2-(R-L)
2] calculate cornea rebirth blood vessel growth area, wherein, A represents the new vessels area, C is the circumference hour number of new vessels accumulation cornea, to be new vessels stretch into the length of cornea from limbus of corneae to L, and R is animal subject cornea mean radius (this experiment is 2.6mm), the results are shown in Table 1.
The inhibitory action of cornea rebirth blood vessel behind the table 1 fusion rotein Corneal Alkali
| Group | Cornea rebirth blood vessel area (mm 2) |
| The FP1 treatment group | 7.4±1.5 |
| The FP3 treatment group | 3.0±0.7 |
| The FP7 treatment group | 4.2±1.1 |
| The FP8 treatment group | 4.1±1.3 |
| Dexamethasone group | 9.8±1.0 |
| The pharmaceutical carrier group | 10.2±1.6 |
| The normal saline group | 11.0±1.2 |
Table 1 result shows: compare with Dexamethasone group, VEGFR fusion protein F P1, FP3, FP7 and FP8 can obviously suppress the new life of cornea blood vessel, can be used for treatment eye table burn property disease, and, fusion protein F P3 is relatively stronger to the inhibition ability of eye table angiogenesis, and is more remarkable to the inhibitory action of rat corneal neovascularization length and area.And the swollen degree of fusion rotein treatment group horn film water is lighter, new vessels is more tiny and distribute sparse, multidigit is near limbus of corneae, blood vessel does not enter cornea central authorities burn district, and, rat corneal neovascularization length and the Area Ratio of FP3 group all are significantly less than other treatment group, pharmaceutical carrier group and normal saline group (p＜0.05), also significantly are better than Dexamethasone group.
The impact of corneal injury behind the table 2 fusion rotein Corneal Alkali
| Group | Medication is corneal injury area (mm after 14 days 2) |
| The FP1 treatment group | 2.5±0.4 |
| The FP3 treatment group | 2.0±0.9 |
| The FP7 treatment group | 2.4±1.3 |
| The FP8 treatment group | 2.7±1.2 |
| Dexamethasone group | 9.4±1.4 |
| The pharmaceutical carrier group | 2.3±1.2 |
| The normal saline group | 2.1±1.1 |
After 14 days, although cornea rebirth blood vessel has obtained inhibition, corneal injury increases the weight of the dexamethasone in treatment group at continuous use, damaged area ulcer.And the trend that the corneal injury of fusion rotein treatment group does not increase the weight of is suitable with the normal saline matched group, has good safety.
This shows that the fusion rotein group has suppressed the side effect that new vessels does not have the inhibition corneal healing that dexamethasone exists simultaneously, efficacy and saferry all is better than dexamethasone.
Adopt corneal scarification, the animal model of herpes simplex virus is infected in preparation mice (BalB/C mice, female, 6-8 age in week, body weight 20-25g) corneal injury.
Herpes simplex virus is uploaded culture at the vero cell, and is quantitative by conventional method.The mice right eye is experimental eye, under narcotism, (contains approximately 5 * 10 with being moistened with 2 μ l HSV-1 types virus liquid
5PFU herpes simplex 1 type virus) micro sample-adding pin, cut and massage conjunctiva gently on animal corneal.
Laboratory animal is divided into 7 groups (8 every group), i.e. FP1 group, FP3 group, FP7 group, FP8 group, pharmaceutical carrier group, Dexamethasone group and normal saline group (NS).Wherein, the administration concentration of fusion rotein is 10mg/ml in FP1, FP3, FP7 and the FP8 group, before use with the configuration of PBS solution dilution; The pharmaceutical carrier group is PBS solution; The administration concentration of Dexamethasone group is 0.25mg/ml.Drip at each treated animal eye table and respectively organize solution 4 times every day, each 10 microlitres, and administration time is respectively 8:00,12:00,16:00,20:00, successive administration 14 days.
After treatment finishes, with Animal Anesthesia, with slit lamp observation eye table angiogenesis and degree of injury.The corneal injury of each treated animal damage index evaluation, concrete damage index is by following criteria, 0: normal cornea; 1: slight muddy; 2: moderate is muddy; 3: severe muddy (iris does not see); 4: the muddy companion of severe ulcer; 5: corneal rupture.Every treated animal is as shown in table 3 in the distribution situation of the size of animal of each impairment scale.Wherein, the little expression of damage index is less to the damage of eye table, and the size of animal that damage index is little is fewer to show that the damage of medicine corneal is less better with protection.
The result shows; after medication finishes; the rat of FP1 group, FP3 group, FP7 group, FP8 group damage index≤2 is respectively 50%, 75%, 62.5% and 50%; pharmaceutical carrier group and normal saline group are 25%; Dexamethasone group is 25%; show that FP3 treatment viral keratitis effect better and for corneal injury does not enlarge, and has preferably protective effect.
The animal statistics of table 3 corneal injury index
| Group | Damage index≤1 | Damage index=2 | Damage index=3 | Damage index=4 |
| The |
25％ | 25％ | 25％ | 25％ |
| The FP3 treatment group | 37.5％ | 37.5％ | 12.5％ | 12.5％ |
| The FP7 |
25％ | 37.5％ | 25％ | 12.5％ |
| The FP8 |
25％ | 25％ | 12.5％ | 37.5％ |
| Dexamethasone group | 12.5％ | 12.5％ | 50％ | 25％ |
| The pharmaceutical carrier group | 12.5％ | 12.5％ | 25％ | 50％ |
| The normal saline group | 12.5％ | 12.5％ | 25％ | 50％ |
Corneal vascularization degree angiogenesis index assessment, concrete angiogenesis index is divided the scope of mainly invading cornea based on new vessels, adopt 16 minutes systems, be about to cornea and be divided into 16 districts (4 quadrants of cornea and 4 districts in each quadrant), 1 of vascular invasion is distinguished to get 1 minute, and final angiogenesis index is the summation that the vascular invasion cornea is respectively distinguished quantity.
Observe respectively the 7th day and the 15th day of administration, calculate the angiogenesis index of each treated animal cornea rebirth blood vessel.Compare with the normal saline group with the pharmaceutical carrier group, the growth trend of FP1 group, FP3 group, FP7 group, FP8 treated animal corneal vascularization index obviously reduces (p＜0.05), and the ability to function of FP3 albumen is better than the most by force and obviously Dexamethasone group, the results are shown in Figure 1.
As seen, fusion protein F P1, FP3, FP7, FP8 can obviously suppress corneal vascularization and reduce the corneal injury degree, can be used for treating viral keratitis, and safety.
The inhibitory action of the mouse cornea angiogenesis that embodiment 3 fusion rotein bring out operation suture thread
Adopt the interior implant operation of mouse cornea substrate to sew up the animal model that collimation method is set up corneal vascularization.
Get male BalB/c mice in 6 ages in week, body weight 18-25g is under narcotism, take right eye as experimental eye, with 3 suturess (11-0 nylon wire) being planted in the corneal stroma (every line arises near the limbus of corneae, terminates in the cornea central area, and adjacent two wire clamp angles are 120 °).
After 3 days, animal is divided into following 7 groups (6 every group), i.e. FP1 group, FP3 group, FP7 group, FP8 group, pharmaceutical carrier group, Dexamethasone group and normal saline group (NS) at the plantation suture.Wherein, the administration concentration of fusion rotein is 10mg/ml in FP1, FP3, FP7 and the FP8 group, before use with the configuration of PBS solution dilution; The pharmaceutical carrier group is PBS solution; The administration concentration of Dexamethasone group is 0.25mg/ml.Drip at each treated animal eye table and respectively organize solution 4 times every day, each 50 microlitres, and administration time is respectively 8:00,12:00,16:00,20:00, continuous application 14 days.
After treatment finishes, put to death animal, get cornea tissue and carry out immunohistochemical staining.Show new vessels with FITC (Fluorescein isothiocyanate fluorescein isothiocyanate) labelling CD31 (PECAM-1), show Lymphangiogenesis with Cy3 (indocyanine green dyestuff) label L YVE-1 (lymphatic vessel endothelial hyaluronic acid receptor-1), under fluorescence microscope, cornea is divided into 8 districts, gather successively the fluorescent image (100 *) in each district, calculate new vessels and vasculolymphatic area with image analysis software, take normal saline group mouse cornea new vessels or micro-vessel area as 100%, analyze each treated animal cornea rebirth blood vessel or lymphatic vessel growing state, and represent with new vessels or micro-vessel area ratio.
The result shows: use the eye drop that contains fusion protein F P3 by the eye table, can obviously reduce cornea blood vessel and Lymphangiogenesis that the suture operation line is induced.And mouse cornea new vessels (p＜0.05) and the Lymphangiogenesis area ratio (p＜0.05) of FP3 group all are significantly less than matched group, see Fig. 2 and Fig. 3.
As seen, fusion protein F P1, FP3, FP7, FP8 can obviously suppress cornea blood vessel and vasculolymphatic new life, can be used for treating keratitis that traumatic eye table neovascular diseases, eye table inflammation, corneal contact lens cause etc.
Embodiment 4 subconjunctival injection fusion protein F P3 bring out the rabbit corneal angiogenesis to alkali burn inhibitory action
With embodiment 2, adopt the alkali burn method to set up the animal model of rabbit corneal angiogenesis.
Get 18 of new zealand white rabbits, male and female are not limit, body weight 2.0-2.5kg.Under narcotism, take right eye as experimental eye, be affixed on the cornea of right eye central authorities of rabbit with the filter paper (diameter is 5mm) that soaks into NaOH (concentration is 1mol/L) solution, act on and remove filter paper after 1 minute, with 10ml normal saline flushing conjunctival sac, remove remaining NaOH, and drip Chloramphenicol Eye Drop (manufacturer: Pharmaceutical Co Ltd, Changzhou Pharmaceutical Factory No.4, specification 8ml: 20mg) 20ul.
The modeling animal is divided into following 3 groups (6 every group) at random, i.e. FP3 first day administration group, the 1st day subconjunctival injection FP3 after the alkali burn; The 14th day administration group of FP3, the 14th day subconjunctival injection FP3 after the alkali burn; Pharmaceutical carrier PBS solution matched group, the 1st day injectable drug carrier after the alkali burn.The administration volume of FP3 treatment group is 0.05ml, and administration concentration is 10mg/ml, the same treatment group of administration volume of matched group.
After 21 days, with Animal Anesthesia, use the microscopic examination rabbit cornea, and the area of measured angular membranous part position new vessels, the area percentage rate that accounts for whole eye table with the angiogenesis area represents the angiogenesis degree, the results are shown in Figure 4.
As seen, the cornea rebirth blood vessel area percentage of FP3 first day administration treated animal is significantly less than matched group (p＜0.01) and the 14th day administration group (p＜0.05) of FP3, illustrate that FP3 can obviously suppress the corneal vascularization due to the alkali burn, and early application is better to injured blood vessel new life's inhibition.
The inhibitory action of the rat corneal neovascularization that embodiment 5 fusion rotein cause acid burn
Adopt the acid burn method to set up rat (SD rat, female, body weight 200-250g) cornea rebirth blood vessel model.
Internal diameter with same Zhi Hanyou defat cotton core is the sample loading gun head of 1.5mm, dip the mixing of 750g/L silver nitrate and 250g/L potassium nitrate and burn liquid, place it in the central 10s of cornea of rats (burning at random right eye or cornea of left eye), remove the sample loading gun head, use immediately normal saline flushing 1min.
Next day, (body weight is that 180g ± 20g) is divided into following 6 groups (6 every group), i.e. FP1 group, FP3 group, FP7 group, pharmaceutical carrier group, Dexamethasone group and normal saline group (NS) at random with the modeling rat.Wherein, the administration concentration of fusion rotein is 10mg/ml in FP1, FP3, the FP7 group, before use with the configuration of PBS solution dilution; The pharmaceutical carrier group is PBS solution; The administration concentration of Dexamethasone group is 0.25mg/ml.Drip at each treated animal eye table and respectively organize solution 4 times every day, each 20 microlitres, and administration time is respectively 8:00,12:00,16:00,20:00, successive administration 14 days.
15 days after operation, after ketamine (60mg/kg) and chlorpromazine (30mg/kg) intraperitoneal injection anesthesia, images acquired under slit lamp microscope is measured cornea rebirth blood vessel length and hour number.
Cornea rebirth blood vessel growth area is pressed Robert formula A=C/12 * 3.1416[R
2-(R-L)
2] calculate, wherein A represent the new vessels area, and C is the circumference hour number of new vessels accumulation cornea, and to be new vessels stretch into the length of cornea from limbus of corneae to L, and R is animal subject cornea mean radius (this experiment is 3mm).
The result shows: 15 days after operation, and the swollen degree of fusion rotein treatment group horn film water is lighter, and new vessels is more tiny and distribute sparsely, and multidigit is near upper and lower limbus of corneae.And, to compare with matched group, cornea rebirth blood vessel length and the area of FP1 group, FP3 group, FP7 group rat significantly reduce (p＜0.05), the results are shown in Table 4.
As seen, fusion protein F P1, FP3, FP7 have obvious inhibitory action to the struvite cornea rebirth blood vessel that acid burn causes, can be used for treatment eye table inflammatory diseases, and FP3 are better to the inhibition of eye table inflammatory disease angiogenesis.
The inhibitory action of cornea rebirth blood vessel behind the table 4 fusion rotein corneal acid burn
| Group | Cornea rebirth blood vessel area (mm 2) |
| The FP1 treatment group | 6.7±1.5 |
| The FP3 treatment group | 3.0±0.5 |
| The FP7 treatment group | 4.3±0.6 |
| Dexamethasone group | 8.8±1.1 |
| The pharmaceutical carrier group | 10.9±1.5 |
| The normal saline group | 11.2±1.6. |
Adopt the standby rabbit conjunctivitis animal model of alcohol induced legal system.
Choose 42 of Japanese white big ear rabbits, male and female are not limit, body weight 1.8-2.2kg.Be divided at random 7 groups, 6 every group, i.e. FP1 group, FP3 group, FP7 group, FP8 group, pharmaceutical carrier PBS solution group, Dexamethasone group and normal saline group (NS).
Rabbit left eye lower eyelid is pulled into cup-shaped, splash into medical-grade absolute ethanol in the eye conjunctival sac with micro sample adding appliance, dripped 0.135ml on the 1st day, dripped 0.09ml on the 2nd day, dripped 0.045ml on the 3rd day, right eye is as own control.Observed in the 4th day, and conjunctival congestion, edema occurred, close the order low-light, secretions increases, ciliary congestion then is considered as the modeling success.
Give respectively to organize medicine after the modeling success, wherein, the administration concentration of fusion rotein is 10mg/ml in FP1, FP3, FP7 and the FP8 group, before use with the configuration of PBS solution dilution; The pharmaceutical carrier group is PBS solution; The administration concentration of Dexamethasone group is 0.25mg/ml.Respectively organize the solution dropping in each treated animal eye table dropping and respectively organize solution 4 times every day, each 50 microlitres, and administration time is respectively 8:00,12:00,16:00,20:00, continuous application 7 days.
After treatment finishes, observe animal conjunctiva and eye expression condition.The conjunctivitis order of severity of each treated animal is pressed following criteria: 1. conjunctival congestion: without congested (-); Mild hyperaemia (+); Obviously congested, be peony (++); Diffusivity is congested, is aubergine (+++); 2. blepharoedema: without edema (-); Mild edema (+); Obviously congested, part ectropion of lid (++); Obvious edema, the eyelid semi-closure (+++); 3. secretions: without unusual secretions (-); A small amount of secretions (+); Secretions makes eyelid or eyelashes are moist or adhesion (++); Secretions makes the moist or adhesion in whole eye district (+++), the results are shown in Table 5.
Table 5 fusion rotein is on the impact of rabbit conjunctivitis
The result shows that after medication finished, fusion rotein treatment group and Dexamethasone group all can significantly be improved the conjunctivitis symptom, wherein FP3 to conjunctivitis to improve effect best.
Embodiment 7 fusion rotein are to the inhibitory action of cornea of rats post-transplantation corneal vascularization
By making the inhibitory action of cornea of rats transplantation model research fusion rotein in corneal transplantation postoperative angiogenesis.
(body weight is that 180g ± 20g) is donor, and (body weight is 180g ± 20g) be receptor, Mus 2-3 in age month for 96 SD female rats with 48 Wistar female rats.The receptor right eye is accepted corneal transplantation, the rear execution of drawing materials of donor eyes.Donor provides cornea of both eyes, and receptor is all selected the right eye operation.Front 20 minutes of donor, receptor art are with 1% atropine eye drop and 0.5% tropicaide ophthalmic solution mydriasis, and 10% chloral hydrate is with the 3ml/kg intraperitoneal injection of anesthesia, and eye is local simultaneously anaesthetizes with topical anesthetic cream.Drilling through diameter in donor's cornea is that 3.5mm plants sheet, places culture dish and plant sheet with viscoelastic agent protection for subsequent use.In the receptor right eye, be the trepan brill receptor plant bed of 3.0mm with diameter, plant bed is planted between sheet and is made interrupted suture 8 pins with atraumatic suture.Toe-in exposes not embedding.Then, the injected into anterior chambers balance liquid is to form the anterior chamber.Art finishes mydriasis, and subconjunctival injection gentamycin 0.1ml is coated with erythromycin eye ointment in the conjunctival sac.Silk suture margo palpebrae 1 pin, and in postoperative dismounting in the 1st day.
Next day, modeling receptor rat is divided into 12 groups (8 every group) at random, and namely FP3 group, FP1 group, Dexamethasone group, ciclosporin A group, rapamycin group, dexamethasone add FP3 group, ciclosporin A and add FP3 group, rapamycin and add FP3 group, dexamethasone and add that FP1 group, ciclosporin A add the FP1 group, rapamycin adds FP1 group, pharmaceutical carrier PBS solution group.Wherein, the administration concentration of fusion rotein is 10mg/ml in FP1, the FP3 group, before use with the configuration of PBS solution dilution; The pharmaceutical carrier group is PBS solution; The administration concentration of Dexamethasone group is 0.25mg/ml; The administration concentration of ciclosporin A is 1mg/ml; The administration concentration 1mg/ml of rapamycin is the administration of eye table.Wherein, FP3 group, FP1 group, Dexamethasone group, ciclosporin A group, rapamycin group, administration every day of pharmaceutical carrier group 4 times, each 20ul, administration time is respectively 8:00,12:00,16:00,20:00, continuous application 14 days; Dexamethasone adds FP3 group, ciclosporin A and adds FP3 group, rapamycin and add FP3 group, dexamethasone and add FP1 group, ciclosporin A and add FP1 group, rapamycin and add the FP1 group and take order administration or administering mode successively, FP1 and FP3 protein medicine-feeding are every day each 1 time sooner or later, each 20ul, chemicals is administered once every day, each 20ul, continuous application 14 days.
After 15 days after operation was anaesthetized with ketamine (60mg/kg) and chlorpromazine (30mg/kg) intraperitoneal injection, images acquired under slit lamp microscope was measured cornea rebirth blood vessel length and hour number.
Cornea rebirth blood vessel growth area is pressed Robert formula A=C/12 * 3.1416[R
2-(R-L)
2] calculate, wherein A represent the new vessels area, and C is the circumference hour number of new vessels accumulation cornea, and to be new vessels stretch into the length of cornea from limbus of corneae to L, and R is animal subject cornea mean radius (this experiment is 3mm), the results are shown in Figure 5.
The result shows: 15 days after operation, fusion protein F P1, FP3 and immunosuppressant (rapamycin, dexamethasone, ciclosporin A) drug combination, the corneal angiogenesis suppresses to have synergism (p＜0.05), and, the drug combination corneal angiogenesis inhibitory action of FP3, FP3 and immunosuppressant is the strongest, the corneal edema degree is lighter, and new vessels is more tiny and distribute sparse.
Sequence table
＜110〉Kanghong Biotech Co., Ltd., Chengdu
＜120〉application of vegf receptor fusion rotein in the medicine of preparation treatment eye table angiogenesis disease
＜130〉nothing
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<170>PatentIn version 3.5
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Claims (1)
1.VEGFR fusion protein F P3 is for the preparation of the application in the medicine for the treatment of conjunctivitis, wherein the aminoacid sequence of FP3 is shown in SEQ ID NO:2.
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| EP3753548A1 (en) | 2006-06-16 | 2020-12-23 | Regeneron Pharmaceuticals, Inc. | Vegf antagonist formulations suitable for intravitreal administration |
| SG10201509193UA (en) | 2011-01-13 | 2015-12-30 | Regeneron Pharma | Use of a vegf antagonist to treat angiogenic eye disorders |
| CN107115294B (en) * | 2012-01-19 | 2019-10-18 | 北京康弘生物医药有限公司 | A kind of eye drops containing VEGF antagonist |
| CN104548067A (en) * | 2014-12-23 | 2015-04-29 | 浙江省人民医院 | Application of recombinant human endostatin in preparing drugs for treating ocular neovascular diseases |
| CN113304245B (en) * | 2021-05-31 | 2022-03-15 | 河南大学 | Application of naphthalimide-polyamine derivative and cyclosporine A in preparation of angiogenesis inhibiting drugs |
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| US11806398B2 (en) | 2005-03-25 | 2023-11-07 | Regeneron Pharmaceuticals, Inc. | Citrate buffered VEGF antagonist formulations |
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