CN102716464A - Application of oligopeptide to preparation of medicine for treating angiogenesis ophthalmic diseases - Google Patents

Abstract

The invention relates to an application of oligopeptide FpAT or pharmacy acceptable salt with the amino acid sequences shown by Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg to the preparation of medicine for preventing and/or treating ophthalmic diseases relevant to angiogenesis. The medicine prepared from the oligopeptide FpAT has the advantages of good curative effect and high safety on ophthalmic diseases relevant to angiogenesis, particularly on diseases relevant to CNV (choroidal neovascularization). In addition, because the chemical ingredients are polypeptide, the preparation technology is mature, the medication cost is low, and the oligopeptide is very suitable for clinic use or popularization.

Description

The application of small peptide in preparation treatment blood vessel hyperplasia property ophthalmic diseases medicine

Technical field

The present invention relates to the biological medicine technology field, be specifically related to angiogenesis and suppress the application of small peptide in preparation prevention or treatment blood vessel hyperplasia property ophthalmic diseases medicine.

Background technology

VEGF (vascular endothelial growth factor; VEGF) be a kind of homodimer glycoprotein that links to each other through disulfide bond, molecular weight 34～45kD, sequence high conservative; The important sequence that structurally has homology with PDGF is met heat and is met sour stable.VEGF is a member of platelet derived growth factor family, is the most direct vascular endothelial cell mitogen.VEGF is a kind of important angiogenesis factors, and it has permeability, the propagation that promotes endotheliocyte that increases blood vessel, the important biological functions such as generation that promote blood vessel.VEGF all has expression under physiology and pathological conditions.Under the physiological condition, in a lot of normal structures expression is arranged all, but expression is generally lower.Vegf expression and periodic associated angiogenesis, placenta tissue, embryonal tissue, corpus luteum, the endometrium of propagation phase etc. are owing to the needs of angiogenesis, and vegf expression is in higher level., blood vigorous in some metabolism supplies abundant tissue, and like cardiac muscle, prostate, adrenal cortex etc., the expression of VEGF is higher than its hetero-organization.Under the pathological conditions, VEGF its mRNA level and protein level in tumor cell all have overexpression.

Research shows that VEGF mediates its activity through its receptor (VEGFR), through signal conduction performance biological effect in the cell.Two the specific receptor Flt of VEGF (Fms-like tyrosine kinase) and KDR (Kinase insert Domain containing Receptor) can cause endothelial cell migration, differentiation, finally form tubulose spline structure (blood vessel blank), are the important target spots that suppresses angiogenesis.Therefore, through with the combining of vegf receptor, can reduce or suppress the effect of vegf receptor mediation vegf expression, thereby reduce or suppress the hair growth promoting of new vessels.Based on through combine to suppress this mechanism of vegf expression with vegf receptor; The research staff has developed the inhibitor of many VEGF; Such as WO9845331A, WO2005000900A, WO2008077077A, WO2008022746A, CN1406948A etc., the inhibitor that these patent documentations disclose mostly is used to prevent and/or treat tumor and relevant disease.

Longevity becomes superfine people in CN101153054A, to disclose a kind of endogenic angiogenesis inhibition small peptide, and called after FpAT (Fibrinopeptide A truncated).This small peptide has only 15 aminoacid sequences, is specially: Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg, and disclose the effect that this peptide has the inhibition tumor growth.

CNV (CNV) claim new vessels under the retina again; It is propagation blood vessel from CC; Breach through the Bruch film is expanded; Form it in propagation between Bruch film and the retinal pigment epithelium or between neural retina and the retinal pigment epithelium or between retinal pigment epithelium and choroid and be more common in macula lutea portion, thereby seriously damage central vision.CNV tube wall permeability is higher, be prone to hemorrhage with ooze out, the cicatrix that then forms of a specified duration causes the damage of macula lutea portion, has a strong impact on central vision, even blinding.The most common retinopathy, be the age related macular degeneration (Aging Macular Degeneration, AMD), (Diabeticretinopathy DR) etc., can cause vision to descend suddenly to diabetic retinopathy; Treatment is difficulty relatively.Wherein age related macular degeneration (AMD) is the modal diseases causing blindness of crowd more than 50 years old, and prevalence is then to rise to 30% more than 1.6%, 75 years old in 50～65 years old crowd according to statistics.Diabetic renal papillary necrosis it is reported that the average prevalence in diabetics is up to 50%, and diabetes are Duoed 25 times than ND blind person.

At present, the treatment means of the ophthalmic diseases that treatment CNV is relevant has: laser therapy, through pupil thermotherapy, PDT (PDT), operative treatment and Drug therapy, wherein class of medications is comparatively ripe, prognosis is better.The medicine that uses clinically has monoclonal antibody medicines such as Avastin, Lucentis, Macugen.Lucentis is the drug of first choice of present AMD treatment, goes on the market in the U.S. in 2006.The preferred institute of Britain's national health and clinical treatment is also recommended the active drug of Lucentis as all AMDs of treatment (AMD); But the every pin price of Lucentis is up to 1500 dollars; The patient needs multiple injection every year; This has just increased patient's burden greatly, and the unable at all so high medical expense of bearing of the patient of most of developing countries.Therefore, developing the medicine that a kind of cheap, curative effect prevents and/or treats the relevant ophthalmic diseases of CNV preferably, is very necessary.

The pharmaceutical market that whole world opthalmological is particularly treated AMD still is in Rapid development stage at present, and annual external sales volume is more than 50 hundred million dollars, and annual rate of growth is 10%～15%.And at home, survey result of people such as Zou Haidong shows that crowd's AMD prevalence is 15.5% more than 50 years old, and according to the demographic data data of China, be 4,000,000,000 RMB the dimensions of market every year that can extrapolate China treatment AMD.

Research worker creatively is applied to prepare the medicine that prevents and/or treats the ophthalmic diseases of blood vessel hyperplasia property with the small peptide FpAT that CN101153054A discloses, and this kind medicine particularly to the relevant disease of CNV (CNV), has unexpected curative effect.

Summary of the invention

The present invention provides a kind of small peptide or its pharmaceutically acceptable salt with the aminoacid sequence shown in the formula I to prevent and/or treat the purposes in the relevant ophthalmic diseases medicine of blood vessel hyperplasia in preparation,

Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg I。

Become the called after FpAT of superfine people in CN101153054A according to the longevity.

The corresponding aminoacid of representative write a Chinese character in simplified form in above-mentioned letter, for example Asp representes be aspartic acid (Aspartic acid, D), that Ser representes is serine (Serine; S), Gly representes be glycine (Glycine, G), that Glu representes is glutamic acid (Glutamic acid; E), Phe representes be phenylalanine (Phenylalanine, F), that Leu representes is leucine (Leucine; L), Ala representes be alanine (Alanine, A), that Val representes is valine (Valine; What V), Arg represented is that (Arginine, R) etc., above-mentioned small peptide FpAT can form through the aminoacid dehydrating condensation of routine arginine.

Further, in the pharmaceutical applications of above-mentioned small peptide FpAT, the aminoacid sequence of said peptide is through replacing, lack and/or adding one or several aminoacid and have the effect that suppresses the relevant ophthalmic diseases medium vessels hair growth promoting of blood vessel hyperplasia.For those of ordinary skill in the art; Can carry out conventional change to the aminoacid sequence of above-mentioned small peptide and not change its function; Such as operations such as replacement, disappearance or interpolations through routine but do not change its function, especially carry out between the nonpolar amino acid or the replacement between the polar amino acid.

In addition; Term " pharmaceutically acceptable salt " be meant in rational medicine judgement scope, be applicable to mammal particularly people's tissue contact and do not have excessive toxicity, stimulation, anaphylaxis etc. and with rational benefit/risk than the salt that matches; Pharmaceutically acceptable salt specific to this small peptide and conventional derivant thereof; Can be its acetate, two caproates, alginate, citrate, aspartate, benzoate, benzene sulfonate, disulfate, butyrate, camphorate, camsilate, glycerophosphate, Hemisulphate, enanthate, caproate, fumarate, hydrochlorate, hydrobromate, hydroiodic acid hydrochlorate, 2-isethionate, lactate, maleate, mesylate, nicotinate, 2-naphthalene sulfonate, oxalates, 3-phenylpropionic acid salt, propionate, succinate, tartrate, phosphate, glutamate, Glu, bicarbonate, tosilate and hendecane hydrochlorate; More excellent ground can the preferred salt hydrochlorate, hydrobromate, sulfate, phosphate, oxalates, maleate, succinate and citrate; In addition, also can be its alkaline addition salts, for example lithium salts, potassium salt, sodium salt, calcium salt, magnesium salt, aluminum salt etc.

Further; In the above-mentioned small peptide FpAT or the pharmaceutical applications of its salt, the ophthalmic diseases that described blood vessel hyperplasia is relevant is CNV (Choroidal Ncowascularization, CNV) a relevant disease; Wherein, CNV is claimed new vessels under the retina again.

Further preferably; The pharmaceutical applications of described small peptide FpAT or its salt; It is characterized in that; The ophthalmic diseases that described blood vessel hyperplasia is relevant is AMD (Aging Macular Degeneration; AMD), diabetic retinopathy (diabeticretinopathy; DR), special property chorioretinitis, degenerative myopia, optic disc glass-film wart, optical fundus blood vessel batten stricture of vagina, yellow spotting retinal degeneration, heritability constitutional glass-film wart, the PE atrophy of adult type macular area or the exudative choroidopathy of centrality (Rieger type) sent out, " CNV property disease " (People's Health Publisher published in 2007 years) relevant portion that information such as the pathology of above-mentioned disease can be write referring to Wang Yusheng.

Further preferably, in the above-mentioned small peptide FpAT or the pharmaceutical applications of its salt, the ophthalmic diseases that described blood vessel hyperplasia is relevant is AMD or diabetic retinopathy.

The present invention also provides a kind of pharmaceutical composition, contains small peptide or its pharmaceutically acceptable salt and the pharmaceutic adjuvant of the aminoacid sequence shown in the formula I.

Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg I

The medicine that utilizes this small peptide FpAT to process has good effect, safe advantage to the relevant disease of CNV (CNV), and because its chemical constituent is a polypeptide, technology of preparing is ripe, 1% of the not enough Lucentis of drug cost.

Description of drawings

Fig. 1 embodiment 3 fluoroscopic visualization analytic statistics rectangular histograms

Fig. 2 embodiment 3 choroid shop sheet analytic statistics rectangular histogram

Fig. 3 embodiment 3HE dyeing assessment CNV thickness statistic histogram

Fig. 4 embodiment 4 fluoroscopic visualization figure and statistic histogram as a result

Fig. 5 embodiment 4 choroid shop sheet analytic statistics rectangular histogram

Fig. 6 embodiment 4HE dyeing assessment CNV thickness chart and statistic histogram

Fig. 7 embodiment 5 fluoroscopic visualization figure and statistic histogram as a result

Fig. 8 embodiment 6ELISA method detects the statistic histogram of the VEGF of ocular tissue

Fig. 9 embodiment 7Western Bloting method detects the expressed proteins histogram of the VEGF of ocular tissue

The specific embodiment

The source and/or the model that should be noted that laboratory animal, medicine and instrument that the following example is used are following:

The brown rat of the male Norway of healthy adult wild type (Norway brown rat, BN rat) is available from Beijing dimension tonneau China Experimental Animal Center, average body quality 180～200 grams;

U.S. Dolly (compound recipe holder bicalutamide eye drop)	The towering Co., Ltd. of Japan
		Pentobarbital sodium	Sigma-Aldrich company
Levofloxacin (levofloxacin eye drop)	The towering Co., Ltd. of Japan
		Tobrex eye ointment (tobramycin)	Alcon company
Ai Er caine (proxymetacaine hydrochloride eye drop)	Alcon company
		10% fluorescein sodium injection	Alcon company
OCT frozen section embedding medium	U.S. Sakura company
		Poly-D-lysine	Biotech company of China fir Golden Bridge in Beijing
FITC-Dextran	Sigma-Aldrich company
		Mouse anti rat CD31 monoclonal antibody	Cell Signaling Technology company
The Mus pVEGFR2 of the rabbit Chinese People's Anti-Japanese Military and Political College (phospho Y1214)	ABcom company
		Goat anti-mouse IgG (Texas is red)	Biotech company of China fir Golden Bridge in Beijing

[0033]

Goat anti-rabbit igg (FITC)	Biotech company of China fir Golden Bridge in Beijing
		DAPI	Biotech company of China fir Golden Bridge in Beijing
Triton X-100 (Triton-100)	Sigma Aldrich company
		The RCA of Texas Red labelling	Vector?Lab.
VEGF ELISA test kit	Shanghai Xi Tang biotech firm
		Mouse anti rat pVEGFR2 (phosphor Y1095)	ABcom company
532nm frequency multiplication semiconductor laser (Twin)	France BVI company
		Micro-surgical instruments	Suzhou six or six Visual Co.,Ltd
The BX51 optical microscope	Olympus company
		Fluoview 300 laser confocal microscopes	Olympus company
CM1900 constant temperature freezing microtome	Leica company
		Stereomicroscope	Zeiss company
The many burnt eye electrophysiology instruments of RETLPORT	France rowland company
		HRA II laser co-focusing fundus fluorescein angiography	Germany Heidberg company
SP-OCT optical coherence retina tomoscan	Germany Heidberg company
		The S-3400N scanning electron microscope	Hitachi company
Transmission electron microscope(TEM)	Hitachi company
		The HAMILTON1701RN microsyringe	Hamilton company
532nm frequency multiplication semiconductor laser (Twin)	France BVI company
		Micro-surgical instruments	Suzhou six or six Visual Co.,Ltd
The BX51 optical microscope	Olympus company
		Fluoview 300 laser confocal microscopes	Olympus company
CM1900 constant temperature freezing microtome	Leica company
		Stereomicroscope	Zeiss company
The many burnt eye electrophysiology instruments of RETLPORT	France rowland company
		HRA II laser co-focusing fundus fluorescein angiography	Germany Heidberg company

[0034]

SP-OCT optical coherence retina tomoscan	Germany Heidberg company
		The S-3400N scanning electron microscope	Hitachi company
Transmission electron microscope(TEM)	Hitachi company
		The HAMILTON1701RN microsyringe	Hamilton company

The preparation of embodiment 1 small peptide FpAT

The synthetic post of peptide that will load 4-(2 ', 4 ' 2-Dimethoxyphenyl-Fmoc-amino methyl) phenoxy group acetylamino ethylamide resin places the PioneerTM peptide synthesizer, under nitrogen, carries out the synthetic of peptide according to following order:

1) with resin solvation 5min in DMF;

2), remove the protection base Fmoc of (or on aminoacid a-amino of resin-bonded) on the resin grafting group with 20% the about 15min of piperidine carboxylic acid process resin among the DMF;

3) with the about 20min of DMF washing resin;

4) with α-carboxyl of first aminoacid Arg of HBTU and HOBT 0.2M solution and the 0.4M diisopropyl ethyl amine solution activation C-terminal among the DMSO-NMP in DMSO-NMP (N-Methyl pyrrolidone);

The activatory aminoacid that 5) will obtain in step 4 in DMF with the about 30min of resin (or aminoacid of resin-bonded) coupling of step 2;

6) with DMF washing resin 5min;

7) with following aminoacid repeating step 2-6:Fmoc-Val, Fmoc-Gly, Fmoc-Gly, Fmoc-Gly, Fmoc-Glu (γ-0tBu), Fmoc-Ala, Fomc-Leu, Fomc-Phe, Fomc-Asp (β-0tBu), Fmoc-Gly, Fmoc-Glu (γ-0tBu), Fmoc-Gly, Fomc-Ser (tBu), Fomc-Asp (β-0tBu);

8) with THF washing resin 5min;

9) with resin and the freshly prepd cutting reagent sulfo-methoxybenzene that mixes: water: dimercaptoethane: trifluoroacetic acid (2: 1: 1: 36, volume ratio) stirs 10-15min for 0 ℃, then at stirring at room 2hr;

10) filter and will filtrate centrifugal in cold diethyl ether; Pour out supernatant and repeat that cold diethyl ether is centrifugal to be precipitated up to peptide fully; With gained peptide bullion at preparation property C18 purification by silica gel column chromatography, with acetonitrile/(water, 0.1%TFA) gradient elution; Collection contains the individual eluting and the lyophilization of target peptide, obtains 464.3mg peptide FpAT.

Embodiment 2 used method of experiment or technology

1. the method for induced with laser CNV:

1% pentobarbital sodium (50mg/Kg) BN rats by intraperitoneal injection; Compound recipe holder bicalutamide eye drop mydriasis; Big rathole is placed contact lens (ocular three mirror contact lens); Hold before rat places slit lamp by the assistant, with 532 double-frequency lasers (parameter: spot diameter 100 μ m, power 125mv; Time of exposure: 0.1s); Coagulate 8 points apart from optic disc 1-1.5PD vascular space light, light coagulates back generation bubble and is masked as the Bruch film with small amount bleeding (or accompanying light sound) and broken up, and is judged as effective laser spot.

2. fluoroscopic visualization (fluorescent angiography; FA): behind the laser 5～7 days, conventional intraperitoneal injection of anesthesia, mydriasis; Lumbar injection 0.2ml fluorescein sodium injection (Alcon lab.Inc); 30sec---1min uses Heidelberg laser co-focusing fundus fluorescein angiography appearance (Heidberg HRA II, Heidberg lab.Genrmany) record, gets early, middle and late phase picture storage respectively.There is experience retina doctor to read sheet respectively by two.Criterion: with radiography laser spot in mid-term hyperfluorescence group, late period enlarged areas, obscurity boundary and is judged to be CNV and generates.

3. vitreous chamber injection:

1) all experimental group rats by intraperitoneal injection 1% pentobarbital sodium 1ml (50mg/Kg), compound recipe holder bicalutamide eye drop mydriasis, it is lax that anesthesia back rat is whole body, and blunt being placed under the stereomicroscope (Zeiss) of eyelashes corneal reflex focused;

2) drip topical anesthetic cream (Ai Er caine eye drop);

3) extract the corresponding dosage medicine with microsyringe;

4) left hand is held microforceps holding angle limbus of sclera, and the right hand is held microsyringe 1mm behind limbus of corneae, the oblique vitreous chamber that thrusts, and medicine is slowly injected in the sense back that falls through, and prevents that with left hand tweezers clamping puncture orifice medicine from refluxing when going out pin;

5) microscopically is observed and is had or not vitreous hemorrhage and lens lesion, the wounded occurs decreasing and excludes group.Injection back point Tobrex collyrium and eye ointment.

The eye electric physiological detection:

The experimental group animal is all injected the previous day in vitreous chamber, injection back 24h, 3D; 7D, the capable full visual field dark adaptation F-ERG of 14D (full-field dark-adapted flash ERG), record dark adaptation a, b ripple incubation period, amplitude and oscillatory potential (oscillatory potentials; OPS), record O ₂Wave amplitude.

Method:

1) laboratory animal dark adaptation 1hr.

2) conventional lumbar injection 1% pentobarbital sodium 1ml, compound recipe holder bicalutamide eye drop mydriasis,

3) place and look electric the special-purpose animal experimental table of physiology, drip the topical anesthesia of Ai Er caine, reference electrode is needled into the rat isthmus, and it is subcutaneous that the earth polar is needled into afterbody, the rearmounted miniature recording electrode of cornea cake artificial tears (carbomer).

4) write down ERG and Ops respectively.

5) data and picture copy uses the SPSS statistics.

5. perfusion is fixedly drawn materials:

Injection back this treated animal of 7d is drawn materials, and makes the transmission electron microscope BIAO and BEN.

1) conventional intraperitoneal injection of anesthesia is the same, treat that toe pain emission disappears after, be fixed in the dissection operating board; Cut off stomach wall and expose diaphram and cut along arcus costarum, with vascular forceps clamping presternum, rib is cut off in midclavicular line along both sides; Upwards dig and fix, catheter needle is thrust from apex, through left ventricle such as the about 2mm of ascending aorta to expose whole thoracic cavity; Fix with micro-vascular clamp, cut off the right auricle after, after open perfusion liquid-transport pipe-line injects the 200ml normal saline and is transparence to the right auricle trickle; Changing perfusion is 4% paraformaldehyde 100ml, visible rat body spasm, afterbody perk (indicating perfusion fixes successfully).

2) cut off immediately in outer canthus and tissue near the eyes, with micro-tweezers holding angle rust-coloured conjunctiva, after another hand-held curved tissue was cut and inserted ball, passivity was separated, and cuts off optic nerve, carefully wipes out tissue near the eyes, complete free eyeball is also extractd.Use 27G needle pierces cornea to be placed on back fixedly 2hr in 4% paraformaldehyde.

3) wipe out the outer attached conjunctiva muscular tissue of eyeball under the stereomicroscope; Cut off from the corneoscleral junction annular; Removal comprises cornea iris crystal and vitreous body; Only staying to comprise sclera, choroid, amphiblestroid eyecup, is the radial eyecup of cutting off in center with the optic disc, makes the tissue (sclera choroid retina complex) of 1 * 1mm.Drop in 3% glutaraldehyde solution rapidly and fix, place 4 ℃ of refrigerator 2hr.

6. transmission electron microscope preparation of specimen:

1) (Glutaraldehyde GA) fixes to such an extent that ocular tissue's BIAO and BEN is transferred to the 2h that (contains 0.18M sucrose) in the flushing liquor in the solution with glutaraldehyde.

2) be transferred to 1% osmic acid (Osmic acid) fixative 1hr.

3) after flushing liquor washes twice, spend the night under the room temperature.

4) dehydration of gradient acetone and block staining such as table 1 show:

Table 1 acetone dewatering process flow

Acetone concentration

?50％

?70％

Two dye liquors

80％

90％

100％

Dewatering time

?10min

?10min

Spend the night

10min

Spend the night

45min

Temperature

?4℃

?4℃

4℃

4℃

Room temperature

Room temperature

Dyeing liquor is: acetic acid uranium-lead citrate

5) add desiccant to 100% acetone and place exsiccator solution.

6) soak into and embedding, press table 2.

Table 2 embedding flow process

7) ultrathin section, through after repairing piece, microtome is made 1 μ m semithin section, places microscope slide, Toluidine blue staining, microscopically tissue visualization structure and location.

8) through diamant, carry net, supporting film is made back row ultrathin section.

9) under the JEM-EX2000 transmission electron microscope, observe, take the photograph sheet, the image storage.

7. Histological section's collection of specimens and making:

1. the 14th day, animal eyeball specimen sampling, eyeball placed the eyeball fixative, and room temperature is preserved.

2. washing, flowing water flushing 10h

3. dehydration: gradient alcohol dehydration, see table 5

Table 5 dewatering process flow

4. eyecup is made: take out during eyeball dehydrating and curing to 70-90% ethanol and make eyecup.

Stereomicroscope is removed the outer attached conjunctiva eye muscle tissue of eye down, cuts from the corneoscleral junction annular, removes leading portion tissues such as comprising cornea iris crystal, keeps ciliary processes intact.

5. it is transparent to immerse xylene 2min

6. twice waxdip

7. the piece of tissue through waxdip moves in the embedding frame that melts paraffin, repaiies piece after solidifying.

8. section: the wax stone refrigerator and cooled of fixing in advance but is loaded on microtome, and the section of cycle type microtome is utmost point portion to the back; To cut into slices holds up with brush pen, and 45 ℃ of water-baths launch, and microscope slide picks up; Serial section behind the CNV appears in the microscopically observation structure, and slivering floats on water-bath; After microscope slide picks up, exhibition sheet platform baking 30min, the numbering back moves into 60 ℃ of incubator baking 4hr.

9. dyeing

The a dewaxing

B dyeing

The c transparently sealing that dewaters

The natural gum mounting is by numbered sequence dress box.

Observe and analyze: Olympus BX51 optical microscope is observed serial section, by the sequence number registration, takes pictures.

Use photoshop7.0 that each CNV is measured, choose the maximum ga(u)ge record and include statistical value in, every eye is got the 5--6CNV data average, be these data.The SPSS11.5 statistical analysis has been regarded as significant difference with P＜0.05.

8. perfusion of carotid artery FIRC-Dextran and choroid are spread sheet:

1. perfusion of carotid artery

Get three rats for every group, conventional lumbar injection 1% pentobarbital sodium 1ml is fixed in dissecting table, fully exposes cervical region; Cut skin, subcutaneous fascia successively, shallow-layer muscle exposes trachea and both sides sternocleidomastoid, and passivity is separated sternocleidomastoid; Clamp hemostasis back cuts off, and separate with the microscissors passivity tight trachea both sides under the stereomicroscope, sees the carotid artery of beating under the fascia; Fully separate fascia, free two bilateral common carotid arteries that expose, a side is played slip-knot ligation (subsequent use) with silk thread; Opposite side presets silk ligature, punctures into carotid artery with No. 4.5 venous detaining needles, sees that blood back is the cerise arterial blood; Micro-vascular clamp clamping, (the rat artery tube wall is poor, and sharp keen nook closing member slides and very easily causes arteriorrhexis to assist to release sharp keen nook closing member by the assistant; As this side tremulous pulse of ligation rapidly that breaks, repeat from opposite side), slowly inject 10ml heparin sodium normal saline after; (Fluoresceinisothiocyanato-dextran, FITC-Dextran), the ligation bilateral carotid arteries presets suture to continue to push the glucosan of 2ml (50mg/ml) marked by fluorescein isothiocyanate.Extract eyeball after 10 minutes, visible vena orbitalis posterior flows out blackish green thickness blood prompting and pours into successfully, and conventional cervical vertebra dislocation is put to death.The eyeball BIAO and BEN is put fixedly 2h of 4% paraformaldehyde.

2. choroid is spread sheet

A. viviperfuse FITC-Dextran choroid preparation of specimen

Under stereomicroscope, wipe out outside the eyeball affiliated groups such as conjunctiva eye muscle through fixed eyeball BIAO and BEN, cut off, remove and comprise cornea iris crystal and vitreous body from the corneoscleral junction annular; Only stay to comprise sclera, choroid, amphiblestroid eyecup, separate retina from the careful passivity of ora serrata, connect to the optic disc place closely with micro-iris repositor; Cut off gently with the shears point; The complete retina that divests stays RPE-choroid-sclera complex, and high-visible 8 CNV focuses are distributed in around the canalis opticus.Do 4 otch of loosening from periphery to optic disc radiation appearance, note not damaging CNV.

The shop sheet concave surface of making is laid on microscope slide downwards, 50% glycerol mounting, 4 ℃ of preservations of numbering back lucifuge.

B. exsomatize and contaminate choroid shop sheet BIAO and BEN (contrasting two kinds of shop sheet methods)

A. get the same period and test rat outside the group, conventional perfusion is put to death and is got the eyeball BIAO and BEN, and fixedly 2h obtains RPE-choroid-sclera complex like preceding making under the stereomicroscope.

B.24 in the orifice plate, choroid is spread sheet place 0.2%Triton solution, lucifuge is hatched 24h.

C. BIAO and BEN moves in agglutinin (dilution factor: 1: the 1000)-HEPES solution of rhodamine labelling, and lucifuge is hatched 24h.

The d.TBS lucifuge is shaken and is washed 24h.

E. carefully BIAO and BEN is laid on the microscope slide with brush pen, 50% glycerol mounting, 4 ℃ keep in Dark Place.

3. observe and analyze: observe under Fluoview 300 laser confocal microscopes (Olympus) and take pictures, secondary light is the 488nm blue light, gathers 525-545nm wavelength green glow; Focus on the RPE plane, the up horizontal optical serial section of stepper motor (1 μ m) is looked the above hyperfluorescence group in RPE plane and is new vessels; Choosing each CNV maximum area tangent plane takes pictures; Simple PCI image analysis software (Cimage system, Olympus, JP).Calculate total green fluorescence area of each CNV and total fluorescence intensity.Adopt the SPSS11.5 statistical analysis, P＜0.05 is regarded as group difference and has statistical significance.

The pharmacodynamics evaluation of embodiment 3 log10 dose effect relations

1. fluoroscopic visualization analysis:

Vitreous chamber injection back 12-14d, capable in batches fluoroscopic visualization inspection, picture typing deposit is analyzed.

FA shows: blank group, PBS (NaCl:8.00g, KCl:0.20g, Na ₂HPO ₄: 1.145g, KH ₂PO ₄: 0.20g, water: 1000ml; Down together) group image laser district in mid-term all shows hyperfluorescence group, and obscurity boundary enlarges even presents fusion light group, and the later stage dyes by force.(design at random according to computer software, sequence is: Met-Asp-Asp-Asp-Asn-Gly-Ser-Gly-Cys) group presents hyperfluorescence group to irrelevant peptide equally, and obscurity boundary enlarges, but the retinal capillary layer owes clear.Lucentis (commercially available, available from Novartis Co.,Ltd) group presents irregular hypofluorescence ring or group, and central authorities become the dark space, and the later stage only has hypofluorescence to dye, and the border does not have expansion.

The FpAT log10 dose concerns three groups of demonstrations: 2 μ g/ml group radiography shows mid-term; Confirm the hot spot injection back 7d of CNV formation, show as hyperfluorescence group slightly, clear border; Do not have and enlarge; Part becomes stronger Eriocheir sinensis foot appearance fluorescence centre (diameter is less than 100 μ m), and periphery with hypofluorescence slightly around, integral body is the petal spline structure.Part laser spot ring formation appearance is hyperfluorescence slightly, and central dark space forms and turn out to be CNV before this hot spot art.Indivedual hot spots show as the hypofluorescence ring, and this hot spot is invalid hot spot before interfering, and this group retinal capillary layer structure is clear, and angioplerosis is good.20 μ g/ml group focus is rendered as than hypofluorescence group or ring-type fluorescence, does not have obvious garland spline structure, and clear border does not have expansion, and the later stage hypofluorescence and dyed.And utmost point portion retinal capillary layer structural fuzzy poor filling simultaneously, but trunk is full good.The early stage retina of 200 μ g/ml group radiography is full to postpone about 3-5 second, sees that the retina trunk is full mid-term to look into, and blood capillary pours into bad; Be greyish black background fluorescence; Focal zone is bigger unformed appearance hypofluorescence group or ring, the unclear but enlarged areas of obscurity boundary, and central authorities become the dark space.Later stage is still slight dyes, but retinal capillary and trunk perfusion do not have and improve.After the grade scale interpretation, the SPSS statistics makes log10 dose and concerns statistic histogram.

SPSS adds up demonstration; The blank group; There was no significant difference between PBS group and irrelevant peptide group, and each group of FpAt and Lucentis group all with between blank, PBS, irrelevant peptide group significant difference (P＜0.05) is arranged, 20 μ g/ml organize and Lucentis organize between there was no significant difference; Between three groups of FpAT significant difference is also arranged, wherein 200 μ g/ml group and all the other each significant difference (P＜0.001) is all arranged between organizing.

2. choroid is spread the sheet analysis result

Behind perfusion of carotid artery FITC-Dxtran, make choroid shop sheet, Laser Scanning Confocal Microscope observation shows, pours into successfully the CNV clear in structure.Having good measurement is worth.

Vitreous chamber injection back 7d, both 14d after the laser modeling, each is organized choroid shop sheet Laser Scanning Confocal Microscope and shows down: blank group and PBS group are typical garland spline structure; The big fluorescence intensity of area is high, and irrelevant peptide group presents atypical garland spline structure, and area is smaller; 2 μ g/ml group for bulk loose fluorescence; Area reduces than negative control group, and 20 μ g/ml are closely fluorogen, and area further reduces.But 200 μ g/ml group is rendered as loose hypofluorescence group, and area does not have and reduces, and conforms to FFA result.Lucentis forms irregular loose fluorogen, and area significantly dwindles than matched group.

Simple PCI image analysis software is adopted in the experiment of this group, and (Cimage system, Olympus JP) analyze picture, and this software can calculate the fluorescence overall strength and the gross area respectively, is convenient to statistics.After using this software labelling marker, generate X-Y scheme respectively and draw area (sqr total), the regeneration graphics draws area and fluorescence intensity figure, includes statistics in.

Statistics shows: between 2 μ g/ml group, 20 μ g/ml group and Lucentis group and all the other each groups significant difference (P＜0.05) is arranged relatively in twos, and there was no significant difference between 200 μ g/ml and PBS and irrelevant peptide group.Zero difference between 2 μ g/ml group and 20 μ g/ml group, and and between the Lucentis group significant difference (P＜0.05) is arranged.

3. paraffin section, HE dyeing assessment CNV thickness

HE dyeing is visible, and each organizes the clear demonstration of CNV, is fusiformis hyperplastic tissue; With pigment granule, the CNV top is with complete or incomplete successional pigment epithelium layer, and the below connects with tela chorioidea; It is thus clear that it is middle from disconnected RPE layer, visible vessels chamber spline structure in the hyperplastic tissue.Thus it is clear that external granular layer (outer nuclear layer, ONL) excalation, and with external plexiform layer (outer plexiform layer; OPL) be adhered to the CNV top, adhesion place forms speckle, in be rich in a large amount of pigment celles; Corresponding retinal area forms the funnel-like outward appearance; Particularly blank group, irrelevant peptide group and PBS stack features are remarkable, and each group of FpAT and Lucentis group ONL, OPL and CNV adhesion scope are less, and the corresponding regional retinal structure trend of CNV is normal.

The CNV form shows: the blank group, and the PBS group and the peptide group CNV focus that has nothing to do become typical fusiformis, and central thickness is big, and the pigment cell is few; Visible vessels chamber spline structure in the focus, and 2 μ g/ml group sees that the fusiformis structure reduces than matched group, form is irregular, and the pigment cell increases in the focus; 20 μ g/ml group CNV focus significantly reduces, and only is fine and close line appearance, and focal zone the pigment cell increase; 200 μ g/ml group CNV focus also significantly reduces, but there have part focus thickness to change to be not obvious, but focus is cavity appearance; Lack entity structure, inner no lumen of vessels spline structure, performance of Lucentis group and 20 μ g/ml categories are seemingly.

The CNV serial section is chosen maximum thickness, and variance analysis (ANOVA) is compared (Bonferroni method) analysis in twos and drawn: PBS organizes except nothing to do with peptide group and blank control group, with all the other each groups significant difference (P＜0.001) is arranged all; 2 μ g/ml group, 20 μ g/ml group, 200 μ g/ml group and PBS group, irrelevant peptide group all has significant difference (P＜0.001) between blank control group.

The pharmacodynamics evaluation of embodiment 4 multiple dose-effect relationships

1. fluoroscopic visualization:

Vitreous chamber injection back 12-14d, capable in batches fluoroscopic visualization inspection, picture typing deposit is analyzed.

FA shows: blank group, the same 2.3.1 of PBS group image in mid-term.Lucentis group presents irregular hypofluorescence ring or group, and central authorities become the dark space, and the later stage only has hypofluorescence to dye, and the border does not have expansion.And FpAT 20 μ g/ml radiography findings such as preceding, 40 μ g/ml organize 60 μ g/ml group and present more weak ring-type fluorescence, and the border is still clear, and area does not have expansion.It is similar that 80 μ g/ml group CNV hypofluorescence ring and Lecentis organize, but have retinal capillary to pour into bad performance, particularly 14d, and back utmost point portion capillary network is inhomogeneous, and end has the high fluorescence of point-like.

Statistics shows: zero difference between 40 μ g/ml and 60 μ g/ml group, zero difference between 80 μ g/ml group is organized with Lucentis, but two groups all and other there were significant differences (P＜0.05) between organizing.

2. choroid is spread the sheet analysis result

PBS group, irrelevant peptide group, blank group and FpAT20 μ g/ml test with logarithm, but 40 μ g/ml organize 60 μ g/ml group and 80 μ g/ml have characteristic to change, wherein the rarely seen irregular ring line-transect property fluorescence of 40 μ g/ml; Lack group's appearance or petal spline structure; And 60 μ g/ml group demonstrates more weak fluorescence centre around with the high fluorescence ring of bud filament edge appearance, only sketches the contours of the focus profile, therebetween fluorescence lack as; 80 μ g/ml become incomplete garland appearance profile, and center fluorescence almost completely lacks.The Lucentis group shows as the very little spot film appearance fluorescence of area and assembles.

Variance analysis (ANOVA) is compared (Dunnett ' s method) in twos and drawn: sql green:PBS group, 20ul/ml group, 40ul/ml group have significant difference (P＜0.05) with each group; Lucntis group, 80 μ g/ml group all have significant difference (P＜0.05) with all the other each groups except that organizing with 60 μ g/ml; PBS group, 20 μ g/ml group, Lucentis group have significant difference (P＜0.05) with each group during mean green; 80 μ g/ml group has significant difference (P＜0.05) with all the other each groups except organizing with 40 μ g/ml group, 60 μ g/ml.

3. paraffin section, HE dyeing assessment CNV thickness

Multiple dose-effect experiment paraffin section shows; Irrelevant peptide group of PBS group and Lucentis group with before identical, FpAT 20 μ g/ml are with reference to preceding step experiment, 40 μ g/ml and 60 μ g/ml focuses are the change of cavity appearance; And the ONL adhesion is less; The OPL seriality is good, and the structure trend is recovered, and the maintenance of ONL form is normal relatively.80 μ g/ml group CNV form imperfection, rarely seen RPE layer thickens pigment and piles up.

Variance analysis (ANOVA) is compared (Dunnett ' s method) analysis in twos and drawn: PBS organizes except nothing to do with peptide group and blank control group, with all the other each groups significant difference (P＜0.001) is arranged all; 20 μ g/ml group, 40 μ g/ml group, 60 μ g/ml group, 80 μ g/ml group, Lucentis group and PBS group, irrelevant peptide group all has significant difference (P＜0.001) between blank control group.20 μ g/ml groups and 60ml, 80ml group have significant difference (P＜0.001).

The pharmacodynamics evaluation of embodiment 5 time effects relation

Vitreous chamber injection back 3d, 14d, 21d, capable in batches fluoroscopic visualization inspection, picture typing deposit is analyzed.

Vitreous chamber injection back 3d (9d after the laser modeling); Negative control group and blank group fluorescence leakage slightly weaken; And the FpAt group significantly weakens (P＜0.05) with the Lucentis group; Respectively organize the seepage situation to 7d and all slightly strengthen, but still have significant difference between PBS group and Lucentis group and FpAT group, the Lucentis group is not remarkable with the FpAT group difference.It is consistent that 14d respectively organizes fluorescence intensity trend, and zero difference between group all has the later stage and dyes, but leakage area enlarges.

Embodiment 6 ELISA methods detect the expression of the VEGF of ocular tissue

Get the injection of 24 BN rats and multiple dose-effect experiment synchronized packets modeling vitreous chamber; The 7d in the injection back, all animal lumbar injection excess l% pentobarbital sodiums (1.5ml), the cervical vertebra dislocation causes death; Get eyeball rapidly; Put and make eyecup on the ice bag, place EP pipe rapidly, it is subsequent use to be stored in-80 ℃ of refrigerators.

Detection method:

A. sample preparation: the using-system dismembyator grinds to form the homogenate shape in frozen tissue piece (comprising sclera, choroid, retina and segment glass body) the EP pipe, centrifugal 10 minutes place to go depositions of 2000rpm, and-80 ℃ of preservations are subsequent use after the packing.

B. titer preparation: press the preparation of test kit description, establish gauge orifice 8 holes, first hole adds 90 μ l diluents+10 μ l standard substance, 1 gets 10 μ l+90 μ l diluents formation gradient from the hole successively.

C. application of sample: add sample centrifugal liquid 100 μ l successively by sequence number.

D.37 ℃ hatch 120min.

E. wash plate: cleaning mixture (the 10ml concentrated solution adds 190ml distilled water, 1: 20).

Discard stock solution, add diluent shaking table 3min, 4 times repeatedly.Drain.

F. the anti-working solution of every Kong Jiayi (goat anti rat VEGFR2 phospho Y 1214) 100 μ l are hatched 60min for 37 ℃;

G. discard stock solution, wash 4 times, drain as preceding shaking;

H. the enzyme-added mark working solution 100 μ l in every hole are hatched 60min for 37 ℃;

I. discard stock solution, wash 4 times, drain as preceding shaking;

J. every hole adds substrate working solution 100 μ l, 37 ℃ of lucifuge 10min

K. every hole adds stop buffer 50 μ l colour developing, and ELIASA 449nm measures light absorption value.

L. repeat drawing standard curve, conversion VEGF content, SPSS16.0 software statistics 3 times.P＜0.05 is for having statistical significance.

Variance analysis (ANOVA) is compared (Dunnett ' s method) in twos and drawn: each group of FpAT and PBS group difference be (P＜0.001) significantly, and the blank group does not have expression, becomes dose dependent more by a small margin between four groups of FpAT.

Embodiment 7 Western Bloting methods detect the expression of the VEGF of ocular tissue

1. protein extraction

The eyeball sample collection together Embodiment 2Described in, get-80 ℃ of frozen choroid retinal tissues (respectively organizing 2 on eyeball) and add 200 μ l phosphorylated protein lysates respectively, cracking 30min on ice, 4 ℃ of centrifugal 30min of 12000g/min get supernatant.Behind the protein quantification, sample adds 8 * loading buffer, and boiling water boils 5min, the centrifugal 5min.-20 of 12000g/min ℃ protein quantification

The preparation of A standard substance: 50 μ l concentration are followed successively by the BSA solution of 0,25,125,250,750,1000,1500 μ g/ml

B is equipped with BCA protein quantification working solution A liquid; B liquid 50: 1

The C sample preparation: every group of single doubly dilution in 1: 20,

The D application of sample: 25 μ l standard substance and sample add 96 orifice plates, add BCA protein quantification working solution 200 μ l; Mixing

E?37℃?30min

F measures OD570, drawing standard curve, conversion protein concentration (table 6)

Table 6 Western blot sample protein content is measured

Sample	Light absorption value	Protein content	Last appearance buffering
				1	0.161	2.73	14.7μl
2	0.225	4.02	10
				3	0.177	3.06	13.1
4	0.202	3.56	11.2
				5	0.128	2.07	19.3
6	0.200	3.51	11.4
				7	0.198	3.48	11.5

[0179]?

8×2	0.140	2.29	17.5
				9×3	0.146	2.42	16.5
10	0.236	4.24	9.4

Applied sample amount 40 μ g.

2. SDS-PAGE preparing gel; Progressively prepare separation gel and concentrate glue by test kit.

3. protein electrophoresis

Last appearance: according to the protein quantification result, equal protein sample and the marker application of sample that will contain sample-loading buffer are to each swimming lane

Electrophoresis: constant voltage 120-150V, 1.5-2h stops at the bottom of bromophenol blue migrates to glue

4. albumen electrotransfer

A prepares transfer membrane

Nitrocellulose membrane: shift 30min in the liquid, before the use

Pvdf membrane: 100% methanol soaks 10min, shifts liquid 3min, before the use

The B electrotransfer

On minus plate, prepare 2 layers in sponge successively, 3 layers of filter paper, glue, pvdf membrane, nitrocellulose membrane, 3 layers of filter paper, 2 layers in sponge, minus plate.100V voltage 1.5-2h

5. Protein Detection

A TBS embathes transfer membrane 2 * 10min

B filter paper drains, and is dipped to the protein band colour developing the beginning of spring in the red dye liquor, according to Marker cutting destination protein

C 5% defatted milk powder spends the night for 4 ℃

D TBS embathes 2 * 10min

E filter paper drains, and adds 4 ℃ of the anti-working solutions rabbit polyclonal to VEGF receptor2 (phosphoY1054+Y1059) that spends the night

F TBS embathes 3 * 10min

G filter paper drains, and adds two anti-working solutions and spends the night for 4 ℃

H TBS embathes 3 * 10min

I filter paper drains rearmounted Luminescent cases, adds luminescent solution, X-ray sheet exposure imaging in the darkroom

J interpretation of result: with software Bandscan 5.0 gray value analyses.

7d pVEGFR2 expressed after Western Bloting observed the injection of CNV rat vitreous chamber, and the result shows that each group of FpAT and the expression of Lucentis group weaken.

Embodiment 8 FpAT injection and preparations thereof

Get PBS (NaCl:8.00g, KCl:0.20g, Na that 300mg small peptide FpAT is dissolved in 10mL ₂HPO ₄: 1.145g, KH ₂PO ₄: 0.20g, water: 1000ml) solution, abundant stirring and evenly mixing, packing promptly gets.

Should be noted that; The above is merely preferred embodiment of the present invention; Be not limited to scope of the present invention, all any modifications of within spirit of the present invention and principle, having done, the replacement that is equal to and improvement etc. all should be included within protection scope of the present invention.

Claims (6)

1. small peptide or its pharmaceutically acceptable salt with the aminoacid sequence shown in the formula I prevents and/or treats the purposes in the relevant ophthalmic diseases medicine of blood vessel hyperplasia in preparation,

Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg I。

2. the pharmaceutical applications of small peptide according to claim 1 or its salt; It is characterized in that the aminoacid sequence of said peptide is through replacing, lack and/or adding one or several aminoacid and have the effect that suppresses the relevant ophthalmic diseases medium vessels hair growth promoting of blood vessel hyperplasia.

3. the pharmaceutical applications of small peptide according to claim 1 and 2 or its salt is characterized in that, the ophthalmic diseases that described blood vessel hyperplasia is relevant is the relevant disease of CNV.

4. the pharmaceutical applications of small peptide according to claim 3 or its salt; It is characterized in that the ophthalmic diseases that described blood vessel hyperplasia is relevant is AMD, diabetic retinopathy, the special property sent out chorioretinitis, degenerative myopia, optic disc glass-film wart, optical fundus blood vessel batten stricture of vagina, yellow spotting retinal degeneration, heritability constitutional glass-film wart, the PE atrophy of adult type macular area or the exudative choroidopathy of centrality.

5. the pharmaceutical applications of small peptide according to claim 4 or its salt is characterized in that, the ophthalmic diseases that described blood vessel hyperplasia is relevant is AMD or diabetic retinopathy.

6. pharmaceutical composition contains small peptide or its pharmaceutically acceptable salt and the pharmaceutic adjuvant of the aminoacid sequence shown in the formula I,

Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg I。

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