WO2015139511A1 - Endostatin突变体、Endostatin突变体与聚乙二醇的交联物以及它们的应用 - Google Patents

Endostatin突变体、Endostatin突变体与聚乙二醇的交联物以及它们的应用 Download PDF

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WO2015139511A1
WO2015139511A1 PCT/CN2015/000167 CN2015000167W WO2015139511A1 WO 2015139511 A1 WO2015139511 A1 WO 2015139511A1 CN 2015000167 W CN2015000167 W CN 2015000167W WO 2015139511 A1 WO2015139511 A1 WO 2015139511A1
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amino acid
protein
solution
neovascularization
endostatin
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黎晓新
黄旅珍
白玉婧
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北京仁和天通生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to an endostatin mutant, a cross-linker of an Endostatin mutant and polyethylene glycol, and their use, in particular to their use in the treatment of ocular neovascular diseases.
  • Retinal and choroidal diseases are currently a class of diseases in which the clinical incidence of ophthalmology is increasing and difficult to treat, including retinopathy of prematurity, age-related macular degeneration, high myopia macular degeneration, proliferative diabetic retinopathy, retinal vein occlusion, and retinal veins.
  • the incidence of proliferative diabetic retinopathy is 3.6%, and the incidence of age-related macular degeneration in the age group over 70 years old can reach 25%. Based on this, it is estimated that the number of patients facing the risk of blindness in China due to intraocular neovascular diseases will exceed 30 million.
  • DME diabetic macular edema
  • Diabetes is now the leading cause of new blindness cases in the United States.
  • DME plagues at least 560,000 diabetic patients in the United States, with 75,000 new cases each year.
  • Neovascularization is an important manifestation of these diseases, and effective and safe anti-angiogenic therapy has become a pressing treatment for clinical ophthalmologists.
  • the treatment of retinal and choroidal neovascularization mainly includes photodynamic therapy, transpupillary thermotherapy, laser photocoagulation, radiation therapy, etc., but these treatments only play a role in the localization of new blood vessels, and the clinical efficacy is limited. Local injury during treatment can induce new blood vessels, and it can not prevent the re-formation of new blood vessels.
  • Lucentis a joint sale of Novartis and Roche for the treatment of fundus neovascular diseases, was launched in 2006 with 2008 sales of $1.768 billion, surpassing latanoprost to become the world's best-selling ophthalmic drug.
  • AMD age-related macular degeneration
  • This disease can cause damage to the macula (a part of the center of the eye's retina), which can lead to blindness and severe vision loss.
  • Lucentis is a fragment of VEGF antibody, which belongs to exogenous vascular inhibitors. It is often caused by side effects such as conjunctival hemorrhage and eye pain. Serious adverse drug reactions such as arterial thrombosis, stroke, myocardial infarction, and acute coronary syndrome.
  • endostatin an endogenous angiogenesis inhibitor isolated from matrix proteins and a fragment of 20 kDa at the C-terminal end of collagen XVIII. Endostatin can inhibit the migration and proliferation of endothelial cells, thereby effectively inhibiting fibrosarcoma T241, Melanoma B16/F10 and Hemangioendothel ioma EOMA in animal models. Growing.
  • the present invention claims a protein (Endostatin mutant) obtained by mutating Endostatin from the N-terminal amino acid residue 1 and the amino acid residue 3 to other amino acid residues; the Endostatin is as follows (a) or (b): (a) a protein consisting of amino acid residues from position 1 to position 184 of the sequence 1 in the sequence listing; (b) substituting and/or deleting and/or adding (a) one or several amino acid residues and Proteins derived therefrom associated with neovascularization.
  • the other amino acid residue is a carboxyl group-containing amino acid residue, an amino group-containing amino acid residue, an amide group-containing amino acid residue or a benzene ring-containing amino acid residue.
  • the Endostatin mutant may specifically be as follows (c) or (d): (c) a protein consisting of amino acid residues from position N to position 184 of the sequence 3 in the sequence listing; (d) from sequence 5 in the sequence listing A protein consisting of amino acid residues 2-184 at the N-terminus.
  • the present invention also protects a protein comprising the Endostatin mutant, specifically (e) or (f): (e) a protein consisting of the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing; (f) from the sequence listing A protein consisting of the amino acid sequence shown in SEQ ID NO: 5.
  • the invention also protects the crosslinks of the Endostatin mutants with polyethylene glycol.
  • the invention also protects crosslinks of proteins containing the Endostatin mutants with polyethylene glycol.
  • the cross-linked product is prepared as follows: the Endostatin mutant (or contains the same) The protein of the Endostatin mutant) is reacted with monomethoxypolyethylene glycol propionaldehyde to give a crosslinked product.
  • the preparation method of the cross-linking substance is specifically as follows: a solution of the Endostatin mutant (or a protein containing the Endostatin mutant) having a protein concentration of 2 mg/ml is taken, and monomethoxypolyethylene glycol propionaldehyde is added. The concentration was 10 g/L, NaHBCN was added thereto and the concentration was 20 mM, and the mixture was allowed to stand at room temperature for 4 hours to obtain a solution containing a crosslinked product.
  • the invention also protects the Endostatin mutant, a protein comprising the Endostatin mutant, or the use of the cross-linker in the manufacture of a medicament for inhibiting and/or blocking the production of neovascularization.
  • the present invention also contemplates a medicament for inhibiting and/or blocking the production of neovascularization, the active ingredient of which is the Endostatin mutant, a protein containing the Endostatin mutant, or the cross-linker.
  • the neovascularization may specifically be a retinal neovascularization and/or a choroidal neovascularization.
  • the invention also protects the Endostatin mutant, a protein comprising the Endostatin mutant, or the use of the cross-linker in the manufacture of a medicament for inhibiting endothelial cell migration.
  • the present invention also protects a drug for inhibiting migration of endothelial cells, wherein the active ingredient is the Endostatin mutant, a protein containing the Endostatin mutant, or the cross-linker.
  • the endothelial cell may specifically be a HMEC cell.
  • the parameters of the monomethoxypolyethylene glycol propionaldehyde described above are specifically as follows: a molecular weight of 20,000 and a degree of dispersion of ⁇ 1.05.
  • Figure 1 is a SDS-PAGE of the M-ES solution.
  • Figure 2 is a SDS-PAGE map in a stability experiment.
  • Figure 3 is an HPLC chromatogram in the stability experiment for peptidase D.
  • Figure 4 is a photograph in an animal model of oxygen-induced retinopathy.
  • Figure 5 is the inhibition rate in an animal model of oxygen-induced retinopathy.
  • Figure 6 is a photograph of an animal model of choroidal neovascularization in mice.
  • Figure 7 is the inhibition rate in a mouse model of choroidal neovascularization.
  • Figure 8 is a pharmacokinetic curve.
  • HMEC cells Xingzhi Biotechnology Co., Ltd., the product number is AA-CELL-91.
  • C57BL/6J mice Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., SCXK (Beijing) 2012-0001.
  • Monomethoxy polyethylene glycol propionaldehyde mo weight 20,000, dispersion ⁇ 1.05): Beijing Kaizheng Bioengineering Development Co., Ltd. NaHBCN: sigma, 156159.
  • Calcein-AM Calbiochem, 206700.
  • Peptidase D Badison Bio, BD00101. Cyclodextrin: sigma, C4767.
  • Glycine sigma, 410225.
  • Calcein-AM was dissolved in DMSO to give a stock solution at a concentration of 1 mg/ml, which was stored at -20 °C.
  • Peptidase D was taken and dissolved in a 15 mM HAc aqueous solution to obtain a peptidase D solution.
  • the inventors of the present invention have found that the W-ES protein is mutated from the first amino acid residue at the N-terminus and the amino acid residue at the third amino acid residue to other amino acid residues, and then cross-linked with polyethylene glycol, according to a large number of experiments and verifications. The stability and activity of the resulting conjugates were significantly increased.
  • the W-ES protein is shown in Sequence 1 of the Sequence Listing from amino acid residues 2-184 of the N-terminus, and the coding gene thereof is shown in Sequence 2 of the Sequence Listing from the 5' end of the 5th -552.
  • the M-ES protein is shown in Sequence 3 of the Sequence Listing from amino acid residues 2-184 of the N-terminus, and the coding gene thereof is shown in Sequence 4 of the Sequence Listing from the 5'-end 4-552.
  • the M-ES protein is a protein obtained by mutating the W-ES protein from the first amino acid residue at the N-terminus and the amino acid residue at the third amino acid residue from H to D.
  • the double-stranded DNA molecule shown in SEQ ID NO: 4 of the Sequence Listing was synthesized and inserted into the NdeI and EcoRI cleavage sites of the vector pET-30a(+) to obtain a recombinant plasmid.
  • the recombinant plasmid obtained in the step 1 was introduced into Escherichia coli DH5 ⁇ to obtain a recombinant strain.
  • the precipitate obtained in the step 3 was taken, and the solution was added in a ratio of 1 g: 10 mL (the solvent was water, containing 6 M guanidine hydrochloride, 50 mM Tris-HCl, 20 mM DTT, pH 9.0), allowed to stand at room temperature for 10 hours, and then centrifuged at 12,000 rpm for 20 minutes. Take the supernatant.
  • step 4 Take the supernatant obtained in step 4 and place it in a dialysis bag in a dialysate (solvent is water, containing 5 mM Tris-HCl, 2 mM GSSG, 0.2 mM GSH, 30 mM cyclodextrin, 10 mM glycine, pH 7.5)
  • a dialysate solvent is water, containing 5 mM Tris-HCl, 2 mM GSSG, 0.2 mM GSH, 30 mM cyclodextrin, 10 mM glycine, pH 7.5
  • the cells were dialyzed for 8 hours at 4 ° C, then centrifuged at 12,000 rpm for 20 min, and the supernatant was taken.
  • step 6 Take 100 ml of the supernatant obtained in step 5 and perform anion exchange chromatography.
  • a HiTrapQHP anion exchange chromatography column was used with a column length of 5 cm and an inner diameter of 1.6 cm.
  • the mobile phase was 50 ml of Tris-HCl buffer (pH 8.5, 50 mM), and all of the permeate was collected.
  • the penetrating solution obtained in the step 6 is concentrated by using an ultrafiltration concentrating tube (protein cut-off molecular weight: 3 KD) to obtain a concentrated solution having a protein concentration of 2 mg/ml, which is an M-ES solution.
  • an ultrafiltration concentrating tube protein cut-off molecular weight: 3 KD
  • the SDS-PAGE pattern of the M-ES solution is shown in lane 1 of Figure 1, the molecular weight sequence of Marker (bottom up): 14.4, 18.4, 25, 35, 45, 66.2, 116.0 KDa.
  • the target band was recovered and sequenced, and the sequencing results showed that the first 10 amino acid residues at the N-terminus were as shown in Sequence 3 of the Sequence Listing from the 1st to 10th amino acid residues at the N-terminus.
  • step 8 Take the M-ES solution obtained in step 7, add monomethoxy polyethylene glycol propionaldehyde to a concentration of 10 g/L, and add NaHBCN (a reducing agent to reduce the double bond formed by the modification reaction to a single After the bond was more stable) and allowed to have a concentration of 20 mM, it was allowed to stand at room temperature for 4 hours, and a fraction of 100 mM NaCl was collected by cation column chromatography to obtain a PEG-M-ES solution.
  • NaHBCN a reducing agent to reduce the double bond formed by the modification reaction to a single After the bond was more stable
  • the double-stranded DNA molecule shown in SEQ ID NO: 4 of the Sequence Listing is replaced with the double-stranded DNA molecule shown in SEQ ID NO: 2, and the other steps are the same.
  • a W-ES solution and a PEG-W-ES solution were obtained.
  • the M-ES solution prepared in Example 1 was diluted with Tris-HCl buffer (pH 7.4, 5 mM) to a protein concentration of 1 ⁇ M to obtain an M-ES test solution.
  • EDTA and ZnCl 2 were added to the M-ES test solution (the concentration of both EDTA and ZnCl 2 was 100 ⁇ M), allowed to stand at room temperature for 10 hours, and then fully dialyzed against Tris-HCl buffer (pH 7.4, 5 mM), and then The zinc ion content was measured by an atomic absorption spectrometer.
  • the W-ES solution prepared in Example 1 was diluted with Tris-HCl buffer (pH 7.4, 5 mM) to a protein concentration of 1 ⁇ M to obtain a W-ES test solution.
  • EDTA and ZnCl 2 were added to the W-ES test solution (the concentration of both EDTA and ZnCl 2 was 100 ⁇ M), allowed to stand at room temperature for 10 hours, and then thoroughly dialyzed against Tris-HCl buffer (pH 7.4, 5 mM), and then The concentration of zinc ions was measured by an atomic absorption spectrometer.
  • Zinc ion concentration ( ⁇ M) Zinc ion/protein (molar ratio) W-ES test solution 0.97 ⁇ 0.07 0.97 M-ES test solution 0.18 ⁇ 0.03 0.18
  • the PEG-M-ES solution prepared in Example 1 was prepared, and a PEG-M-ES test solution having a protein concentration of 1 mg/ml was prepared by using Tris-HCl buffer (pH 8.0, 10 mM) as a solvent, and the solution was removed after filtration. Store in a sterilized vial. The PEG-M-ES test solution after filtration sterilization was allowed to stand at 37 ° C, and samples were taken at 0, 7 and 15 days later for SDS-PAGE.
  • the PEG-W-ES solution prepared in Example 1 was prepared, and a PEG-W-ES sample solution having a protein concentration of 1 mg/ml was prepared by using Tris-HCl buffer solution (pH 8.0, 10 mM) as a solvent, and the cells were filtered and sterilized. Store in a sterilized vial.
  • the PEG-W-ES test solution after filtration sterilization was allowed to stand at 37 ° C, and samples were taken at 0, 7 and 15 days later for SDS-PAGE.
  • the signal of the target band and the degradation zone was scanned by the Shanghai Tianneng Tanon-2500(R) instrument.
  • the degradation rate the signal of the degradation zone / (the signal of the degradation zone + the signal of the target zone) ⁇ 100%.
  • the M-ES solution prepared in Example 1 was taken, and a M-ES test solution having a concentration of 2 mg/ml was prepared using Tris-HCl buffer (pH 8.0, 10 mM) as a solvent.
  • 3 ml of M-ES test solution was mixed with 3 ml of 50 ⁇ g/ml peptidase D solution, and incubated at room temperature, and samples were taken at 10 min, 20 min, 30 min, 60 min, 90 min, 120 min, 150 min and 180 min, respectively.
  • the sample obtained by sampling 500 ⁇ l was added to 50 ⁇ l of glacial acetic acid to terminate the reaction, and then 50 ⁇ l was taken, and the enzyme digestion rate was examined by HPLC.
  • the W-ES solution prepared in Example 1 was used, and a W-ES test solution having a concentration of 2 mg/ml was prepared using Tris-HCl buffer (pH 8.0, 10 mM) as a solvent.
  • 3 ml of W-ES test solution was mixed with 3 ml of 50 ⁇ g/ml peptidase D solution, and incubated at room temperature, and samples were taken at 10 min, 20 min, 30 min, 60 min, 90 min, 120 min, 150 min and 180 min, respectively.
  • the sample obtained by sampling 500 ⁇ l was added to 50 ⁇ l of glacial acetic acid to terminate the reaction, and then 50 ⁇ l was taken, and the enzyme digestion rate was examined by HPLC.
  • the column was UniSil 5-120C18 (4.6 x 250 mm) from Namicrotechnology, item number QCS131109.
  • Solution A 0.1% TFA in water
  • Solution B 0.1% TFA in acetonitrile.
  • Liquid chromatography conditions the mobile phase was a mixture of solution A and solution B at a flow rate of 1.0 ml/min; the mobile phase was linearly increased from 15% solution B (85% solution A, volume ratio) to 75% solution in 25 minutes. B (25% solution A, volume ratio); detector wavelength was 280 nm; column temperature was 25 °C. The % in this paragraph represents the volume ratio.
  • the enzymatic cleavage rate is equal to the peak area of the peak representing the fragment after digestion and divided by the peak area of all peaks.
  • the M-ES solution prepared in Example 1, the PEG-M-ES solution or the W-ES solution was separately taken and adjusted to the desired concentration with physiological saline.
  • a sterile 6 mm diameter microporous membrane was placed at the minimum of the CAM vessels, and 15 ⁇ l of the solution to be tested was added to the center of the microfiltration membrane (the solution to be tested was: 5 ⁇ g/ml PEG-M-ES solution, 20 ⁇ g /ml of PEG-M-ES solution, 40 ⁇ g/ml PEG-M-ES solution, 5 ⁇ g/ml M-ES protein solution, 20 ⁇ g/ml M-ES protein solution, 40 ⁇ g/ml M-ES protein solution 20 ⁇ g/ml of W-ES protein solution and physiological saline), and then the window was closed with a sealant and culture was continued for 3 days.
  • the solution to be tested was: 5 ⁇ g/ml PEG-M-ES solution, 20 ⁇ g /ml of PEG-M-ES solution, 40 ⁇ g/ml PEG-M-ES solution, 5 ⁇ g/ml M-ES protein solution, 20 ⁇ g/ml M-ES protein solution, 40 ⁇
  • Vascular inhibition rate (number of blood vessels in the saline group - number of blood vessels in the experimental group) ⁇ Number of blood vessels in the saline group ⁇ 100%.
  • the M-ES solution prepared in Example 1, the PEG-M-ES solution or the W-ES solution was separately taken and adjusted to the desired concentration with physiological saline.
  • the solution to be tested was 1 ⁇ g/ml PEG-M-ES solution, 16 ⁇ g/ml PEG-M-ES solution, 160 ⁇ g/ml PEG-M-ES solution, 1 ⁇ g/ml M-ES protein solution, 16 ⁇ g/ M ml of M-ES protein solution, 160 ⁇ g/ml of M-ES protein solution, 16 ⁇ g/ml of W-ES protein solution and physiological saline, and the solvent used for each solution was physiological saline.
  • HMEC cells were taken and digested with 0.25% trypsin-EDTA at 37 ° C for 1 minute. Cells were taken and resuspended in DMEM high glucose medium supplemented with 100 ⁇ g/ml penicillin and 100 ⁇ g/ml streptomycin to obtain 8 ⁇ 10 5 cells. /ml of cell suspension.
  • a second 24-well plate was placed on which a 24-well Transwell chamber (8 ⁇ m pore size, Millipore) was placed, and 184 ⁇ l of the cell suspension obtained in the step 2 and 16 ⁇ l of the test solution were added to each chamber, and incubated at 37 ° C for 1 hour.
  • step 3 After completing step 3, take the Transwell chamber, place it on the 24-well plate that completed step 1, and incubate for 4 hours at 37 ° C in a 5% CO 2 incubator.
  • a solution of Calcein-AM at a concentration of 1 ⁇ g/ml was prepared using Triple Digest (Invitrogen) as a solvent.
  • step 7 After completing step 4, take the Transwell chamber, aspirate the liquid, place it in the 24-well plate that completes step 6, and incubate for 25 minutes.
  • Inhibition rate (fluorescence intensity of physiological saline group - fluorescence intensity of experimental group) ⁇ fluorescence intensity of physiological saline group ⁇ 100%.
  • the M-ES solution or the PEG-M-ES solution prepared in Example 1 was separately taken and adjusted to the desired concentration with physiological saline.
  • the solutions to be tested were 5 mg/ml PEG-M-ES solution and 5 mg/ml M-ES solution, respectively.
  • OIR model Olygen-induced retinopathy, animal model of oxygen-induced retinopathy
  • Fluorescein-dextran-FITC (Sigma, St. Louis, MO, formulation method: 50 mg Fluorescein-dextran-FITC dissolved in 1 ml of PBS buffer) having a molecular weight of 2 ⁇ 10 6 was injected into the left ventricle 20 mg via the heart.
  • mice eyeballs were taken out and placed in a 4% paraformaldehyde solution for 30 minutes at room temperature.
  • the retina was taken out, and retinal plating was performed under a dissecting microscope. Observation and photography were performed by a fluorescence microscope (Zeiss Axiophot, Thornwood, NY), without blood vessels.
  • the area of the perfusion area was analyzed and counted using ImageJ software to obtain the number of vascular endothelial cells.
  • the treatment of replacing the solution to be tested with an equal volume of physiological saline in the step (2) is used as a model control group (OIR); in the step (1), "75% ⁇ 2% oxygen is placed for 5 days" and the step (2) is not performed.
  • the treatment of replacing the solution to be tested with an equal volume of physiological saline was used as a normal control group (Normal), and the area of the non-vascular perfusion area was zero.
  • mice Nine mice were placed in each group and the results were averaged.
  • Inhibition rate 1 - (number of vascular endothelial cells in the administration group) / number of vascular endothelial cells in the OIR group.
  • the photo is shown in Figure 4.
  • the number of vascular endothelial cells in each group is shown in Figure 5.
  • the results showed that the inhibition rate of PEG-M-ES and M-ES on the avascular perfusion area of the retina was similar, reaching about 75%.
  • CNV mouse animal model choroidal neovascularization, choroidal neovascularization mouse animal model
  • mice were dilated with compound tropamide eye drops, and the ophthalmic oxybuproca eye drops were applied to the ocular surface.
  • the coverslips were placed in front of the eyes of the mice after dilation to flatten the cornea, and then the mice were placed in a laser machine for laser retinal photocoagulation, and the laser-induced CNV model was laser-parameter 532 nm. 150mW, 100ms, 50mm, when the laser induces a point blast on the retina, the laser is stopped immediately.
  • a vitreous cavity drug injection was performed, and a solution to be tested was injected into the vitreous cavity, and both eyes were injected, and the injection volume per eye was 2 ⁇ l.
  • mice After 14 days, the mice underwent fundus fluorescein angiography (intraperitoneal injection of fluorescein sodium contrast agent 0.03 ml) to measure the size of CNV and the area of leakage.
  • the measuring instrument was a Phoenix Micron IV small animal fundus imaging system with CNV size and The leakage area was analyzed and statistically analyzed using ImageJ software.
  • the treatment of replacing the solution to be tested with an equal volume of physiological saline in the step (4) is used as a model control group (the relative CNV area is defined as 1); the laser retinal photocoagulation is not performed in the step (3) and the step (4) is used.
  • the treatment of the equal volume of physiological saline in place of the solution to be tested was taken as a normal control group, and its relative CNV area was zero.
  • mice Ten suckling mice were set in each group, and the results were averaged.
  • Inhibition rate 1 - (relative leakage area of the drug-administered group / relative leakage area of the model control group).
  • the M-ES solution or the PEG-M-ES solution prepared in Example 1 was separately taken and adjusted to the desired concentration with physiological saline.
  • the solutions to be tested were 5 mg/ml PEG-M-ES solution and 5 mg/ml M-ES solution, respectively.
  • the time points for the materials are: 0h, 12h, 24h, 2 days, 6 days, 12 days, 45 days.
  • the Endostatin ELIA kit (R&D) was used to detect the plasma concentration and calculate the pharmacokinetic parameters.
  • the DNA molecules shown in SEQ ID NO: 6 were substituted for the DNA molecules shown in SEQ ID NO: 4, and Example 1 and Example 2 were sequentially carried out, and the results were in agreement with the results of SEQ ID NO:4.
  • Endostatin is an endogenous vascular inhibitor of the human body. Its systemic drug-based therapeutic drug Endo has been widely used in the market, and its safety is significantly better than that of the exogenous vascular inhibitor drug.
  • the endostatin N-terminal Histidine mutant was constructed in the present invention, and the zinc ion binding of the Histidine N-terminal mutant was significantly decreased in the experiment, but the stability was not significantly changed and the activity was improved.
  • the N-terminus loses the site of action of peptidase D, which helps to improve the stability of N-terminal PEG or other substance modification.
  • the endostatin cleavage site was altered by the N-terminal mutation, and the PEG modification at the N-terminus of the mutated Endostatin was enhanced.
  • the half-life of the modified product after intraocular injection is 15 days, which can be administered once a month or every 2-3 months, which is more conducive to the treatment of ophthalmic ball injection.

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Abstract

提供了一种Endostatin突变体，所述突变体是将Endostatin自N末端第1位和第3位氨基酸残基突变为其它氨基酸残基得到的，所述Endostatin是如下(a)或(b)：(a)由序列表中序列1自N末端第2-184位氨基酸残基组成的蛋白质；(b)将(a)经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与新生血管形成相关的衍生蛋白。还提供了Endostatin突变体与聚乙二醇的交联物以及它们在抑制和/或阻断新生血管生成中的应用。

Description

Endostatin突变体、Endostatin突变体与聚乙二醇的交联物以及它们的应用 技术领域
本发明涉及一种Endostatin突变体、Endostatin突变体与聚乙二醇的交联物以及它们的应用，具体涉及它们在眼部新生血管疾病治疗中的应用。
背景技术
视网膜和脉络膜疾病是目前眼科临床发病率越来越高且难以治疗的一类疾病，包括早产儿视网膜病变、老年性黄斑病变、高度近视黄斑病变、增生性糖尿病视网膜病变、视网膜静脉阻塞以及视网膜静脉周围炎所导致的视网膜脉络膜的新生血管等。据文献报道，增生性糖尿病视网膜病变的发病率为3.6％，70岁以上年龄组老年黄斑变性发病率可达25％。据此预计，我国目前受到眼内新生血管性疾病困扰面临失明危险的患者人数将超过3000万。中国每年新增DME(diabetic macular edema,糖尿病性黄斑水肿)超过30万例。糖尿病现在是美国境内新增失明病例的主要原因，DME困扰着美国境内至少560000例糖尿病患者，每年新增病例达75000人次。新生血管形成是这些疾病的重要表现形式，有效而安全的抗新生血管治疗成为临床眼科医师迫切需要的治疗手段。
目前对视网膜、脉络膜新生血管(CNV)的治疗方法主要有光动力疗法、经瞳孔温热疗法、激光光凝、放射疗法等，但这些治疗方法仅在新生血管局部起作用，临床疗效有限，且治疗时的局部损伤又可诱发新生血管，更不能预防新生血管再形成。
诺华和罗氏联合销售的、用于治疗眼底新生血管疾病的Lucentis(兰尼单抗)于2006年上市，2008年销售额达到17.68亿美元，超越拉坦前列素成为全球最畅销的眼科用药。2012年1月，Lucentis获准在中国上市，用于治疗老年相关性黄斑变性(AMD，age-related macular degeneration)。这种疾病会造成黄斑部(眼睛视网膜中心的一部分)损伤，可能导致失明和严重的视力损失。Lucentis是一种VEGF抗体的片段，属于外源性血管抑制剂，常见结膜出血、眼睛疼痛等副作用，极少数的出现 动脉血栓、脑中风、心肌梗死及急性冠脉综合症等严重药物不良反应事件。
1971年，美国哈佛大学儿童医学院的Judah Folkman教授提出肿瘤血管阻断理论：肿瘤的生长和迁移依赖于新生血管生成，阻断新生血管的生成可能阻止肿瘤的生长和转移。1996年，Judah Folkman实验室的O'Reilly发现了血管内皮抑制素(endostatin)，Endostatin是在基质蛋白质中分离出来的内源性新生血管抑制因子，是胶原蛋白XVIII C末端分子量为20kDa的片段。血管内皮抑制素可以抑制内皮细胞的迁移和增殖，进而可有效抑制动物模型中成纤维细胞瘤(Fibrosarcoma)T241、黑色素瘤(Melanoma)B16/F10和恶性血管内皮细胞瘤(Hemangioendothel ioma)EOMA等的生长。
发明公开
本发明的目的是提供一种Endostatin突变体、Endostatin突变体与聚乙二醇的交联物以及它们的应用。
本发明要求保护将Endostatin自N末端第1位氨基酸残基和第3位氨基酸残基突变为其它氨基酸残基得到的蛋白质(Endostatin突变体)；所述Endostatin是如下(a)或(b)：(a)由序列表中序列1自N末端第2-184位氨基酸残基组成的蛋白质；(b)将(a)经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与新生血管形成相关的由其衍生的蛋白质。所述其它氨基酸残基为含羧基的氨基酸残基、含氨基的氨基酸残基、含酰胺基的氨基酸残基或含苯环的氨基酸残基。所述Endostatin突变体具体可为如下(c)或(d)：(c)由序列表中序列3自N末端第2-184位氨基酸残基组成的蛋白质；(d)由序列表中序列5自N末端第2-184位氨基酸残基组成的蛋白质。
本发明还保护含有所述Endostatin突变体的蛋白质，具体可为如下(e)或(f)：(e)由序列表中序列3所示的氨基酸序列组成的蛋白质；(f)由序列表中序列5所示的氨基酸序列组成的蛋白质。
本发明还保护所述Endostatin突变体与聚乙二醇的交联物。
本发明还保护含有所述Endostatin突变体的蛋白质与聚乙二醇的交联物。
所述交联物的制备方法如下：所述Endostatin突变体(或含有所述 Endostatin突变体的蛋白质)与单甲氧基聚乙二醇丙醛反应，得到交联物。
所述交联物的制备方法具体如下：取蛋白浓度为2mg/ml的所述Endostatin突变体(或含有所述Endostatin突变体的蛋白质)的溶液，加入单甲氧基聚乙二醇丙醛并使其浓度为10g/L，加入NaHBCN并使其浓度为20mM，室温放置4小时，得到含有交联物的溶液。
本发明还保护所述Endostatin突变体、含有所述Endostatin突变体的蛋白质、或所述交联物在制备抑制和/或阻断新生血管的生成的药物中的应用。
本发明还保护一种抑制和/或阻断新生血管的生成的药物，其活性成分为所述Endostatin突变体、含有所述Endostatin突变体的蛋白质、或所述交联物。
所述新生血管具体可为视网膜新生血管和/或脉络膜新生血管。
本发明还保护所述Endostatin突变体、含有所述Endostatin突变体的蛋白质、或所述交联物在制备抑制内皮细胞迁移的药物中的应用。
本发明还保护一种抑制内皮细胞迁移的药物，其活性成分为所述Endostatin突变体、含有所述Endostatin突变体的蛋白质、或所述交联物。
所述内皮细胞具体可为HMEC细胞。
以上任一所述的单甲氧基聚乙二醇丙醛的参数具体如下：分子量为2万，分散度＜1.05。
附图说明
图1为M-ES溶液的SDS-PAGE图谱。
图2为稳定性实验中的SDS-PAGE图谱。
图3为对肽酶D的稳定性实验中的HPLC图谱。
图4为氧诱导视网膜病变动物模型中的照片。
图5为氧诱导视网膜病变动物模型中的抑制率。
图6为脉络膜新生血管小鼠动物模型中的照片。
图7为脉络膜新生血管小鼠动物模型中的抑制率。
图8为药代曲线。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
HMEC细胞：行知生物科技有限公司，货号为AA-CELL-91。载体pET-30a(+)：北京艾比根生物。大肠杆菌DH5α：北京艾比根生物。C57BL/6J小鼠：北京维通利华实验动物技术有限公司，SCXK(京)2012-0001。单甲氧基聚乙二醇丙醛(分子量为2万，分散度＜1.05)：北京凯正生物工程发展有限责任公司。NaHBCN：sigma，156159。Calcein-AM：Calbiochem，206700。肽酶D：拜迪森生物，BD00101。环糊精：sigma，C4767。甘氨酸：sigma，410225。
将Calcein-AM溶于DMSO，得到浓度为1mg/ml储液,-20℃保存。取肽酶D，用15mM HAc水溶液溶解，得到肽酶D溶液。
本发明的发明人根据大量实验和验证，发现将W-ES蛋白自N末端第1位氨基酸残基和第3位氨基酸残基突变为其它氨基酸残基，然后再与聚乙二醇交联，得到的偶联物的稳定性和活性均显著增加。W-ES蛋白如序列表的序列1自N末端第2-184位氨基酸残基所示，其编码基因如序列表的序列2自5’末端第4-552所示。M-ES蛋白如序列表的序列3自N末端第2-184位氨基酸残基所示，其编码基因如序列表的序列4自5’末端第4-552所示。M-ES蛋白是将W-ES蛋白自N末端第1位氨基酸残基和第3位氨基酸残基突变均由H突变为D得到的蛋白质。
实施例1、M-ES蛋白和W-ES蛋白的制备
一、M-ES蛋白和PEG-M-ES的制备
1、合成序列表的序列4所示的双链DNA分子并插入载体pET-30a(+)的NdeI和EcoRI酶切位点，得到重组质粒。
2、将步骤1得到的重组质粒导入大肠杆菌DH5α，得到重组菌。
3、将步骤1得到的重组菌接种至LB液体培养基，37℃、230rpm振荡培养至OD600＝0.5-0.8，加入IPTG并使其浓度为0.5mM，然后37℃、230rpm 振荡培养4h，然后8000rpm离心5min并收集菌体，用含150mM NaCl的Tris-HCl缓冲液(pH 9.0、50mM)悬浮菌体(5ml溶液/g湿菌体)并进行超声破碎(功率200W，每超声3秒停3秒，总时间为20min)，然后12000rpm离心10min，取沉淀(包涵体)。
4、取步骤3得到的沉淀，按照1g:10mL的比例加入溶解液(溶剂为水，含6M盐酸胍、50mM Tris-HCl、20mM DTT，pH 9.0)，室温静置10h，然后12000rpm离心20min，取上清液。
5、取步骤4得到的上清液，装入透析袋中，在透析液(溶剂为水，含5mM Tris-HCl、2mM GSSG、0.2mM GSH，30mM环糊精,10mM甘氨酸,pH7.5)中4℃透析8小时，然后12000rpm离心20min，取上清液。
6、取100毫升步骤5得到的上清液，进行阴离子交换层析。
采用HiTrapQHP型阴离子交换层析柱，柱子长度为5cm，内径为1.6cm，采用的流动相为50毫升Tris-HCl缓冲液(pH8.5、50mM)，收集全部的穿透液。
7、取步骤6得到的穿透液，采用超滤浓缩管(蛋白截留分子量为3KD)进行浓缩，得到蛋白浓度为2mg/ml的浓缩液，即为M-ES溶液。
M-ES溶液的SDS-PAGE图谱见图1的泳道1，Marker的分子量顺序(自下至上)：14.4、18.4、25、35、45、66.2、116.0KDa。回收目标带并测序，测序结果表明，N端前10个氨基酸残基如序列表的序列3自N端第1至10个氨基酸残基所示。
8、取步骤7得到的M-ES溶液，加入单甲氧基聚乙二醇丙醛并使其浓度为10g/L，加入NaHBCN(一种还原剂，使修饰反应形成的双键还原为单键后更稳定)并使其浓度为20mM，室温放置4小时，采用阳离子柱层析,收集100mM NaCl洗脱组分，得到PEG-M-ES溶液。
二、W-ES蛋白和PEG-W-ES的制备
用序列表的序列2所示的双链DNA分子代替序列表的序列4所示的双链DNA分子，其它同步骤一。得到W-ES溶液和PEG-W-ES溶液。
实施例2、性能比较
一、锌离子结合实验
将实施例1制备的M-ES溶液用Tris-HCl缓冲液(pH7.4、5mM)稀释 至蛋白浓度为1μM，得到M-ES待测液。在M-ES待测液中加入EDTA和ZnCl2(使EDTA和ZnCl2的浓度均为100μM)，室温静置10小时，然后用Tris-HCl缓冲液(pH7.4、5mM)充分透析，然后用原子吸收光谱仪检测锌离子含量。
将实施例1制备的W-ES溶液用Tris-HCl缓冲液(pH7.4、5mM)稀释至蛋白浓度为1μM，得到W-ES待测液。在W-ES待测液中加入EDTA和ZnCl2(使EDTA和ZnCl2的浓度均为100μM)，室温静置10小时，然后用Tris-HCl缓冲液(pH7.4、5mM)充分透析，然后用原子吸收光谱仪检测锌离子浓度。
进行五次重复实验，结果取平均值。
结果见表1。结果表明，与W-ES蛋白相比，M-ES蛋白结合锌离子的能力大大降低。
表1锌离子含量检测结果
  锌离子浓度(μM) 锌离子/蛋白(摩尔比)
W-ES待测液 0.97±0.07 0.97
M-ES待测液 0.18±0.03 0.18
二、稳定性实验
取实施例1制备的PEG-M-ES溶液，用Tris-HCl缓冲液(pH8.0、10mM)作为溶剂制备蛋白浓度为1mg/ml的PEG-M-ES待测液，过滤除菌后分装于灭菌的西林瓶中。将过滤除菌后的PEG-M-ES待测液37℃静置，分别于0时刻、7天后和15天后取样，进行SDS-PAGE。
取实施例1制备的PEG-W-ES溶液，用Tris-HCl缓冲液(pH8.0、10mM)作为溶剂制备蛋白浓度为1mg/ml的PEG-W-ES待测液，过滤除菌后分装于灭菌的西林瓶中。将过滤除菌后的PEG-W-ES待测液37℃静置，分别于0时刻、7天后和15天后取样，进行SDS-PAGE。
采用上海天能公司Tanon-2500(R)仪器自带软件扫描目标带和降解带的信号，降解率＝降解带的信号/(降解带的信号+目标带的信号)×100％。
进行五次重复实验，结果取平均值。单次实验的SDS-PAGE图谱见图2。
结果见表2。结果表明，PEG-M-ES的PEG降解率显著低于PEG-W-ES的PEG降解率，即M-ES蛋白的N端更稳定。
表2降解率的结果(％)
  7天 15天
PEG-W-ES待测液 5.07±0.23 8.98±0.21
PEG-M-ES待测液 0.04±0.03 0.05±0.03
三、对肽酶D的稳定性实验
取实施例1制备的M-ES溶液，用Tris-HCl缓冲液(pH8.0、10mM)作为溶剂制备浓度为2mg/ml的M-ES待测液。将3ml M-ES待测液与3ml 50μg/ml的肽酶D溶液混匀，室温孵育，分别于10min、20min、30min、60min、90min、120min、150min和180min后取样。向500μl取样得到的样品加入50μl冰醋酸终止反应，然后取50μl，通过HPLC法检测酶切率。
取实施例1制备的W-ES溶液，用Tris-HCl缓冲液(pH8.0、10mM)作为溶剂制备浓度为2mg/ml的W-ES待测液。将3ml W-ES待测液与3ml 50μg/ml的肽酶D溶液混匀，室温孵育，分别于10min、20min、30min、60min、90min、120min、150min和180min后取样。向500μl取样得到的样品加入50μl冰醋酸终止反应，然后取50μl，通过HPLC法检测酶切率。
色谱柱为纳微科技的UniSil5-120C18(4.6×250mm),货号QCS131109。溶液A：0.1％TFA水溶液；溶液B：0.1％TFA乙腈溶液。液相色谱条件：流动相为溶液A和溶液B的混合液，流速为1.0ml/min；在25分钟内流动相由15％溶液B(85％溶液A，体积比)线性上升至75％溶液B(25％溶液A，体积比)；检测器波长为280nm；柱温为25℃。本段中的％均代表体积比。酶切率等于代表酶切后片段的峰的峰面积除以全部峰的峰面积。
某个样本的谱图见图3。
进行五次重复实验，结果取平均值。
结果见表3。结果表明，与W-ES蛋白相比，M-ES蛋白对肽酶D的稳定性更好。
表3酶切率检测结果(％)
  10min 20min 30min 60min 90min 120min 150min 180min
W-ES 18.79 30.47 41.60 59.52 70.26 78.35 84.17 88.65
M-ES 0.07 0.05 0 0 0.11 0.08 0.03 0
四、对鸡胚绒毛尿囊膜(CAM)血管生成的抑制实验
分别取实施例1制备的M-ES溶液、PEG-M-ES溶液或W-ES溶液，用生理盐水调整至所需浓度。
将鸡蛋种蛋放入孵化箱(37℃±0.5℃)，气室向上，每天转动2-3次。第7天，在透射灯下观察并确定CAM，用牙科钻在气室顶端开1-2mm 小孔。在胚头前1cm、两条卵黄静脉之间的卵壳投影部分，用铅笔勾画出1.5cm×2cm区域，用碘酒及酒精消毒后用砂轮在蛋壳表面划刻出1mm凹痕，滴少量生理盐水于凹痕内。将无菌的直径为6mm的微孔滤膜放在CAM的血管最少处，在微孔滤膜中央加入15μl待测溶液(待测溶液分别为：5μg/ml的PEG-M-ES溶液、20μg/ml的PEG-M-ES溶液、40μg/ml的PEG-M-ES溶液、5μg/ml的M-ES蛋白溶液、20μg/ml的M-ES蛋白溶液、40μg/ml的M-ES蛋白溶液、20μg/ml的W-ES蛋白溶液和生理盐水)，然后用封口胶封闭窗口，继续培养3天。滴入10％福尔马林固定20min，以滤膜为中心剪下约3.5cm×3.5cm的尿囊膜，平铺于平皿中展开，晾干，置于显微镜下观察，记录以滤膜为中心、直径5mm范围内的血管数目(Blood vessel number，BVN)。计算血管抑制率(Inhibition rate，IR)。
血管抑制率＝(生理盐水组的血管数-实验组的血管数)÷生理盐水组的血管数×100％。
进行五次重复实验，结果取平均值。
结果见表4。结果表明，与W-ES蛋白相比，M-ES蛋白的血管抑制率更高，PEG-M-ES的血管抑制率相对于M-ES蛋白进一步提高。
表4血管数和血管抑制率的结果
  浓度(μg/ml) 血管数 血管抑制率(％)
生理盐水   79.49±3.51 0
PEG-M-ES 5 69.20±2.37* 12.9
PEG-M-ES 20 40.83±6.35*# 48.6
PEG-M-ES 40 24.19±2.38* 69.6
M-ES 5 70.41±3.76* 11.4
M-ES 20 42.00±1.96*# 47.2
M-ES 40 25.57±5.11* 67.8
W-ES 20 58.72±4.92* 26.1
注：*与生理盐水组比较P＜0.01，#与W-ES组比较P＜0.01。
五、对HMEC迁移抑制实验
分别取实施例1制备的M-ES溶液、PEG-M-ES溶液或W-ES溶液，用生理盐水调整至所需浓度。待测溶液分别为1μg/ml的PEG-M-ES溶液、16μg/ml的PEG-M-ES溶液、160μg/ml的PEG-M-ES溶液、1μg/ml的M-ES蛋白溶液、16μg/ml的M-ES蛋白溶液、160μg/ml的M-ES蛋白溶液、16μg/ml的W-ES蛋白溶液和生理盐水，各个溶液采用的溶剂均为生理盐水。
1、取第一个24孔板，每孔加入80μl待测溶液和920μl含0.5％FBS、100μg/ml青霉素和100μg/ml链霉素的DMEM高糖培养液。
2、取HMEC细胞，用0.25％胰蛋白酶-EDTA 37℃消化1分钟，取细胞，用100μg/ml青霉素和100μg/ml链霉素的DMEM高糖培养液重悬，得到8×105个细胞/ml的细胞悬液。
3、取第二个24孔板，其上放置24孔Transwell小室(8μm孔径，Millipore)，每个小室加入184μl步骤2得到的细胞悬液和16μl待测溶液，37℃孵育1小时。
4、完成步骤3后，取Transwell小室，置于完成步骤1的24孔板上，在37℃、5％CO2培养箱中孵育4小时。
5、用Triple消化液(Invitrogen)作为溶剂，制备浓度为1μg/ml的Calcein-AM溶液。
6、取第三个24孔板，每孔加入950μl Calcein-AM溶液。
7、完成步骤4后，取Transwell小室，吸弃液体后放入完成步骤6的24孔板中，孵育25分钟。
8、取完成步骤7的24孔板，轻轻摇晃几次，并轻轻敲打顶部与边缘，再孵育10分钟，轻轻摇晃几次，取出Transwell小室，用移液枪吹打混匀24孔板的各孔中的细胞，每孔取出200μl细胞悬液至96孔板中，以酶标仪检测各孔的荧光强度(激发波长为485nm，发射波长为500-550nm之间的峰值)。
抑制率＝(生理盐水组的荧光强度-实验组的荧光强度)÷生理盐水组的荧光强度×100％。
进行五次重复实验，结果取平均值。
结果见表5。结果表明，经PEG修饰后的M-ES活性较修饰前没有降低，与W-ES相比活性明显升高。此结果与鸡胚绒毛尿囊膜试验结果类似。
表5荧光强度的结果
样品 浓度(μg/ml) 荧光值 抑制率(％)
生理盐水 0 487.3±170.0 0
PEG-M-ES 1 405.0±161.9* 16.9
PEG-M-ES 16 265.3±100.3*# 45.6
PEG-M-ES 160 94.0±24.4* 80.7
M-ES 1 415.4±127.5* 14.8
M-ES 16 282.9±110.7*# 41.9
M-ES 160 99.2±18.6* 79.6
W-ES 16 350.69±124.71* 28.0
注：*与生理盐水组比较P＜0.01，#与W-ES组比较P＜0.01。
六、对鼠视网膜新生血管模型治疗效果的比较
分别取实施例1制备的M-ES溶液或PEG-M-ES溶液，用生理盐水调整至所需浓度。待测溶液分别为5mg/ml的PEG-M-ES溶液、5mg/ml的M-ES溶液。
1、OIR模型(Oxygen-induced retinopathy,氧诱导视网膜病变动物模型)
(1)生后第7天的C57BL/J6乳鼠随同母鼠放置到连接测氧仪的动物实验舱中，75％±2％氧气放置5天。
(2)取出乳鼠(乳鼠出生12天)，立即用水合氯醛麻醉，眼表滴用麻药(奥布卡因滴眼液)及散瞳剂(复方托品酰胺滴眼液)，待瞳孔散大后玻璃体腔注射待测溶液，双眼注射，每只眼的注射量为1.5微升。
(3)取乳鼠(乳鼠出生17天)，进行荧光素葡聚糖灌注视网膜铺片检测。
经心脏将分子量为2×106的Fluorescein-dextran-FITC(Sigma,St.Louis,MO，配制方法：50mg Fluorescein-dextran-FITC溶于1ml PBS缓冲液中)注射入左心室20mg。
(4)取出小鼠眼球放置于4％多聚甲醛溶液中室温固定30分钟，取出视网膜，在解剖显微镜下进行视网膜铺片,荧光显微镜(Zeiss Axiophot,Thornwood,NY)进行观察并照相,无血管灌注区面积应用ImageJ软件进行分析统计，得到血管内皮细胞核数。
以步骤(2)中用等体积生理盐水代替待测溶液的处理作为模型对照组(OIR)；以步骤(1)中不进行“75％±2％氧气放置5天”且步骤(2)中用等体积生理盐水代替待测溶液的处理作为正常对照组(Normal),其无血管灌注区面积为0。
每组设置9只乳鼠，结果取平均值。
抑制率＝1-(给药组血管内皮细胞核数)/OIR组血管内皮细胞核数。
照片见图4。各组的血管内皮细胞核数见图5。结果表明：PEG-M-ES与M-ES对视网膜无血管灌注区的抑制率相似，均达到75％左右。
2、CNV小鼠动物模型(choroidal neovascularization,脉络膜新生血管小鼠动物模型)
(1)6-8周C57BL/6J小鼠(体重为20-25g)经氯胺酮腹腔麻醉。
(2)用复方托品酰胺滴眼液对小鼠双眼进行散瞳，同时眼表滴用麻药奥布卡因滴眼液。
(3)将盖玻片置于散瞳后的小鼠眼睛前用于压平角膜，随后将小鼠放置于激光机前行激光视网膜光凝，采取的激光诱导CNV模型的激光参数为532nm、150mW、100ms、50mm，当激光在视网膜上诱导出点状爆破后即刻停止激光。
(4)在激光诱导CNV的即刻，进行玻璃体腔药物注射，玻璃体腔注射待测溶液，双眼注射，每只眼的注射量为2微升。
(5)14天后对小鼠进行眼底荧光造影(麻醉后腹腔注射荧光素钠造影剂0.03ml)，测量CNV的大小及渗漏的面积，测量仪器为PhoenixMicron IV小动物眼底成像系统，CNV大小及渗漏面积应用ImageJ软件进行分析统计。
以步骤(4)中用等体积生理盐水代替待测溶液的处理作为模型对照组(其相对CNV面积定义为1)；以步骤(3)中不进行激光视网膜光凝且步骤(4)中用等体积生理盐水代替待测溶液的处理作为正常对照组,其相对CNV面积为0。
每组设置10只乳鼠，结果取平均值。
抑制率＝1-(给药组相对渗漏面积/模型对照组相对渗漏面积)。
照片见图6。相对渗漏面积结果见图7。结果表明：PEG-M-ES与M-ES对脉络膜新生血管的抑制率相似，均为51％左右。
七、M-ES-PEG和W-ES-PEG在家兔眼球内注射药代半衰期的比较
分别取实施例1制备的M-ES溶液或PEG-M-ES溶液，用生理盐水调整至所需浓度。待测溶液分别为5mg/ml的PEG-M-ES溶液、5mg/ml的M-ES溶液。
1、成年雌性新西兰兔(体重为2-2.5kg，中国人民解放军军事医学科学院实验动物中心)经速眠新腹腔麻醉。
2、用复方托品酰胺滴眼液对新西兰兔双眼进行散瞳，同时眼表滴用奥布卡因滴眼液。
3、用胰岛素注射器行玻璃体腔注射，双眼注射，每只眼注射50μl待测溶液，棉签按压5秒。
4、取材时间点为：0h，12h，24h，2天，6天，12天，45天。耳缘动脉取血、耳静脉注射空气处死后取前房水每眼约0.1ml、玻璃体0.4ml、以及视网膜分别装于1.5ml Eppendrof管，4℃保存。
5、采用Endostatin ELIA试剂盒(R&D公司)检测血药浓度，并计算药代参数。
结果见表6和图8。PEG-M-ES半衰期达到14.67天，且在第45天血药浓度仍为有效浓度。因此可实现每月甚至每45天给药一次，方便患者使用。
表6药代动力学参数的结果
房室参数 M-ES PEG-M-ES
T1/2(d) 0.94 14.67
Ke(1/d) 0.737 0.047
V1/F(L/kg) 0.009 0.015
CL/F(L/d/kg) 0.007 0.001
AUC(0-t)(μg/L*d) 145446.445 1362757.32
AUC(0-∞)(μg/L*d) 145446.449 1387366.32
Ka(1/d) 0.738 0.052
t1/2Ka(d) 0.939 13.412
Tlag(d) 0 39.019
用序列6所示DNA分子代替序列4所示DNA分子依次进行实施例1和实施例2，结果与序列4的结果一致。
工业应用
Endostatin是一种人体本身具有的内源性血管抑制剂，其全身给药型肿瘤治疗药物恩度已在市场上广泛应用，安全性明显优于外源性血管抑制剂药物贝阀单抗。本发明构建了Endostatin N末端Histidine突变体，试验中发现Histidine N末端突变体锌离子结合明显下降，但其稳定性没有明显变化，且活性有提高。另外，突变后，其N末端失去了肽酶D的作用位点，有助于提高N末端PEG或是其他物质修饰的稳定性。因N端突变造成了Endostatin酶切位点的改变，在突变的Endostatin N末端进行PEG修饰，其稳定性增强。修饰产物在眼部球内注射后的半衰期为15天，可实现每月或是每2-3月给药一次，更利于眼科球部注射的治疗。

Claims (12)

  1. 将Endostatin自N末端第1位氨基酸残基和第3位氨基酸残基突变为其它氨基酸残基得到的蛋白质；所述Endostatin是如下(a)或(b)：(a)由序列表中序列1自N末端第2-184位氨基酸残基组成的蛋白质；(b)将(a)经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与新生血管形成相关的由其衍生的蛋白质。
  2. 如权利要求1所述的蛋白质，其特征在于：所述其它氨基酸残基为含羧基的氨基酸残基、含氨基的氨基酸残基、含酰胺基的氨基酸残基或含苯环的氨基酸残基。
  3. 如权利要求1所述的蛋白质，其特征在于：所述“将Endostatin自N末端第1位氨基酸残基和第3位氨基酸残基突变为其它氨基酸残基得到的蛋白质”为如下(c)或(d)：(c)由序列表中序列3自N末端第2-184位氨基酸残基组成的蛋白质；(d)由序列表中序列5自N末端第2-184位氨基酸残基组成的蛋白质。
  4. 一种蛋白质，为如下(e)或(f)：(e)由序列表中序列3所示的氨基酸序列组成的蛋白质；(f)由序列表中序列5所示的氨基酸序列组成的蛋白质。
  5. 权利要求1至4中任一所述的蛋白质与聚乙二醇的交联物。
  6. 权利要求1至4中任一所述的蛋白质在制备抑制和/或阻断新生血管的生成的药物中的应用。
  7. 权利要求5所述的交联物在制备抑制和/或阻断新生血管的生成的药物中的应用。
  8. 如权利要求6或7所述的应用，其特征在于：所述新生血管为视网膜新生血管和/或脉络膜新生血管。
  9. 一种抑制和/或阻断新生血管生成的药物，其活性成分为权利要求1至4中任一所述的蛋白质或权利要求5所述的交联物。
  10. 如权利要求9所述的药物，其特征在于：所述新生血管为视网膜新生血管和/或脉络膜新生血管。
  11. 权利要求1至4中任一所述的蛋白质或权利要求5所述的交联物 在制备抑制内皮细胞迁移的药物中的应用。
  12. 一种抑制内皮细胞迁移的药物，其活性成分为权利要求1至4中任一所述的蛋白质或权利要求5所述的交联物。
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WO1999029855A1 (en) * 1997-12-08 1999-06-17 Beth Israel Deaconess Medical Center Mutants of endostatin, 'em 1' having anti-angiogenic activity and methods of use thereof
CN1670035A (zh) * 2004-03-16 2005-09-21 哈尔滨医科大学 改变结构、增强抗肿瘤活性的内皮抑素及其应用
CN101265298A (zh) * 2008-04-30 2008-09-17 中国药科大学 含有非天然氨基酸的内皮抑素突变体及其衍生物
WO2013034116A1 (zh) * 2011-09-09 2013-03-14 清华大学 对atp结合位点进行突变的血管内皮抑制素突变体

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CN1670035A (zh) * 2004-03-16 2005-09-21 哈尔滨医科大学 改变结构、增强抗肿瘤活性的内皮抑素及其应用
CN101265298A (zh) * 2008-04-30 2008-09-17 中国药科大学 含有非天然氨基酸的内皮抑素突变体及其衍生物
WO2013034116A1 (zh) * 2011-09-09 2013-03-14 清华大学 对atp结合位点进行突变的血管内皮抑制素突变体

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