CN105859833A - Polypeptide for inhibition of blood vessel neogenesis or growth and application thereof - Google Patents
Polypeptide for inhibition of blood vessel neogenesis or growth and application thereof Download PDFInfo
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Abstract
The invention belongs to the biological medicine field, and in particular, relates to a polypeptide for inhibition of blood vessel neogenesis or growth, a coding sequence, a vector containing the coding sequence, host cells, pharmaceutical compositions and a pharmaceutical use of the polypeptide. The polypeptide has the advantages of small molecular weight, good water solubility, high safety and good stability, and can be prepared by a solid phase synthesis method; and the purity is high, the yield is high and the cost is low.
Description
Technical field
The invention belongs to biomedicine field, in particular it relates to a kind of angiogenesis inhibiting or the polypeptide of growth.
Background technology
The formation of new vessels is an extremely complex process, and it includes: the expansion of existing blood vessels, the increasing of vasopermeability
Add, the degraded of blood vessel surrounding substrate, the activationa and proliferation of endothelial cell, migration and the formation of new capillary sample tube chamber.
Under normal physiological conditions, the generation of blood vessel is a process tightly regulated by multiple links, to repairing and maintaining body
Normal function has important effect.But, if inside tumor blood vessel fast-growth, then it is one of clinical common phenomenon.
Substantial amounts of animal model and human clinical trial show, the formation blocking intra-tumor new vessels can stop tumour effectively
Growth and the death of triggering tumor cell, thus reach the therapeutic action to tumour.Therefore, angiogenesis inhibiting is the most anti-
One of important directions of tumour new drug research.Big molecule anti-neovascularization medicaments such as Avastin has passed through the upper of U.S. FDA
City ratifies, and the most more medicine is in different clinical front and clinical investigation phase.
At eye, the blinding disease of about 2/3 is all relevant with pathologic neovascularization, such as: Herpetic Stromal Keratitis
CNV in the cornea rebirth blood vessel that causes, AMD and diabetic retinopathy or
Retinal neovascularization etc. in retinopathy of prematurity.The most clinically, eye pathologic neovascularization routine is transported
With laser photocoagulation, photodynamic therapy (Photodynamic therapy, PDT) and through pupil thermotherapy (Thermal
Transpupillary therapy, TTT) etc. treat.But, these treatment means not only easily cause destruction to local organization,
Its late result is the most of great satisfaction.Therefore, people continuously attempt to the exploitation treatment new green blood of eye pathologic in recent years
Pipe more effective way；For senile retinal vascular lesion, (Age-related macular degeneration is referred to as
AMD), the multiple disease relevant to angiogenesis such as diabetic retinopathy, arthritis, the treatment of angiogenesis inhibiting
New drug also has extensive and important effect.
When developing effective angiogenesis inhibitors, the particularity of ophthalmic remedy should be fully taken into account.First, eye exists
Multiple anatomical and functional barrier: Formulations for systemic administration usually cannot be in eye group due to blood aqueous barrier and blood-retina barrier
Knit the drug concentration that local reaches enough；Topical, such as intravitreal, the big molecule more than 76.5kDa is in theory
On be difficult to penetrate retina and act on retina and CNV；Being administered for ocular, medicine have to successively penetrate
Lipophilic corneal epithelial cell compact siro spinning technology and hydrophilic corneal stroma, the most only possess the most fat-soluble, low molecule
Amount or can get to by the medicine that combines of transporter in-house with ocular (such as: amino acid transporter, oligopeptides transporter etc.)
Anterior chamber plays a role.Second, the degree that medicine dissolves in hydrophilic tear, aqueous humor, vitreous humor and its validity are in just
Relevant.3rd, based on above-mentioned main cause, the bioavilability of ophthalmic remedy is the lowest；It is allowed to improve, administration need to be strengthened
Concentration.But being used for treating tumor neovasculature compound toxic and side effect more substantially, whole body and local all cannot high agent
Amount is administered.Additionally, the bigger exogenous protein of molecular weight is also sensitive foreign matter source, can be to part tissue of eye (such as: uvea)
Cause immunologic mjury.4th, although having had a series of comparatively safe endogenous angiogenesis inhibitors successively to be demonstrate,proved at present
Real, such as angiostatin (angiostatin), it is by plasminogen Kringle domain 1-4 (plasminogen Kringle 1-4)
Composition, can substantially suppress the growth of blood vessel dependent tumors, but owing to its molecular weight is relatively big and space conformation is complicated, therefore in system
Have during Bei that recombinant expressed purifying process is loaded down with trivial details and the deficiency such as endotoxin residual.Just because of the restriction of above-mentioned all conditions,
The medicine being currently used for treating ocular angiogenesis is extremely limited, such as recombinant anti human VEGF monoclonal antibody
Bevacizumab (Avastin), recombinant anti human VEGF monoclonal antibody fragment ranibizumab (Lucentis) etc., but they valencys
Lattice are high, and need repeatedly through intravitreal administration, even can cause blood vessel embolism equivalent risk.
As can be seen here, find the micromolecular inhibitor with specific biological activities and biocompatibility, and low price, subtract
Few side effect locally and systemically, is of great significance the clinical prevention tool of neovascular diseases.
Summary of the invention
Present invention aim at providing a kind of new angiogenesis inhibiting or the polypeptide of growth, this polypeptide belongs to micromolecular inhibitor,
Low price, it is possible to angiogenesis inhibiting or growth effectively, and reduce side effect locally and systemically.
In order to achieve the above object, the invention provides techniques below scheme:
In one aspect, the present invention provides the polypeptide of a kind of angiogenesis inhibiting or growth, described polypeptide to contain SEQ ID NO:1
Shown sequence.
In a preferred embodiment, described polypeptide is to increase SEQ ID before aforementioned SEQ ID NO:1 sequence in order
Any one or the multiple amino acid that start from C end of NO:2 (AVSLLRATG)；Or after aforementioned SEQ ID NO:1
Face increases any one or more amino acid started from N end of SEQ ID NO:3 (HAVPV) in order；Or the most all
Any one in SEQ ID NO:2 and SEQ ID NO:3 or multiple amino acid is increased by above-mentioned order.
In further preferred embodiment, the polypeptide of the present invention is:
SK001: its amino acid sequence is SEQ ID NO:1；SK002: its amino acid sequence is SEQ ID NO:5；SK003:
Its amino acid sequence is SEQ ID NO:7；SK004: its amino acid sequence is SEQ ID NO:9；SK005: its amino
Acid sequence is SEQ ID NO:11；SK006: its amino acid sequence is SEQ ID NO:13；SK007: its amino acid sequence
It is classified as SEQ ID NO:15；SK008: its amino acid sequence is SEQ ID NO:17；SK009: its amino acid sequence is
SEQ ID NO:19；SK010: its amino acid sequence is SEQ ID NO:21；SK011: its amino acid sequence is SEQ ID
NO:23；SK012: its amino acid sequence is SEQ ID NO:25；SK013: its amino acid sequence is SEQ ID NO:27；
SK014: its amino acid sequence is SEQ ID NO:29；SK015: its amino acid sequence is SEQ ID NO:31；SK016:
Its amino acid sequence is SEQ ID NO:33；SK017: its amino acid sequence is SEQ ID NO:35；Or SK018: its
Amino acid sequence is SEQ ID NO:37.
On the other hand, the present invention provides the nucleotide sequence of encoding such polypeptides.In a preferred embodiment, described core
Nucleotide sequence is:
Sk001: nucleotides sequence is classified as SEQ ID NO:4；Sk002: nucleotides sequence is classified as SEQ ID NO:6；Sk003: core
Nucleotide sequence is SEQ ID NO:8；Sk004: nucleotides sequence is classified as SEQ ID NO:10；Sk005: nucleotides sequence is classified as
SEQ ID NO:12；Sk006: nucleotides sequence is classified as SEQ ID NO:14；Sk007: nucleotides sequence is classified as SEQ ID NO:
16；Sk008: nucleotides sequence is classified as SEQ ID NO:18；Sk009: nucleotides sequence is classified as SEQ ID NO:20；Sk010:
Nucleotides sequence is classified as SEQ ID NO:22；Sk011: nucleotides sequence is classified as SEQ ID NO:24；Sk012: nucleotide sequence
For SEQ ID NO:16；Sk013: nucleotides sequence is classified as SEQ ID NO:28；Sk014: nucleotides sequence is classified as SEQ ID NO:
30；Sk015: nucleotides sequence is classified as SEQ ID NO:32；Sk016: nucleotides sequence is classified as SEQ ID NO:34；Sk017:
Nucleotides sequence is classified as SEQ ID NO:36；Or sk018: nucleotides sequence is classified as SEQ ID NO:38.Wherein, above-mentioned nucleosides
The polypeptide that acid sequence is identical with numeral numbering is corresponding.
On the other hand, the present invention provides a kind of expression vector expressing described polypeptide or host cell, comprises aforementioned polypeptides
Nucleotide sequence.Described expression vector can be recombinant eukaryon expression vector, preferred mammal fibrocyte expression vector；Also may be used
To be recombinant virus expression vector, preferably adeno-associated virus or adenovirus vector.Described expression vector can be thin the host converted
Born of the same parents replicate and expresses, it is preferable that described host cell is Chinese hamster ovary celI and subbreed thereof or 293 cells and subbreed thereof.
On the other hand, the present invention provides a kind of method preparing described polypeptide, and described method is chemical synthesis or restructuring table
Reaching method, described recombinant expressed method includes introducing in suitable host cell above-mentioned expression vector, carries out the expression of polypeptide.
On the other hand, the present invention provides a kind of pharmaceutical composition, comprises aforementioned polypeptides or its pharmaceutically acceptable salt and medicine
Acceptable carrier or excipient on；Preferably, the formulation of described pharmaceutical composition is injection, eyedrops, ophthalmically acceptable solidifying
Glue or spongaion.
On the other hand, the present invention provides a kind of polypeptide of the present invention or pharmaceutically acceptable salt in preparation treatment or prevention
Application in the medicine of the disease caused by angiogenesis or growth.Described disease is preferably disease of eye, tumour, ischemic
Heart disease, non-inflammation cardiomyopathy, coronary sclerosis, arteriosclerosis, arterial embolism, arterial thrombus, Berger's
Disease, chronic inflammation, IBD, ulcer, rheumatic arthritis, scleroderma, psoriasis, sterility or sarcoma shape are sick；
Described disease of eye is preferably AMD, BDR, CNV, macula lutea
Cystoid macular edema, diabetic macular edema, retinal vascular occlusion, cornea rebirth blood vessel, corneal graft new vessels
Property glaucoma, pteryium or chronic conjunctivitis.
Main advantages of the present invention, including:
A () polypeptide molecular weight of the present invention is little, can pass through ocular tissue's barrier；
B () good water solubility, can keep higher concentration in neutral tear, aqueous humor and vitreous humor；
C () security is high, little to biological tissue's toxic and side effect；And eye local application bioavilability is high, can reduce dosage,
Thus reduce systemic side effects；
D () can be prepared by the method for synthesis in solid state, purity is high, and yield is big, low cost；
The good stability of (e) polypeptide of the present invention.
Accompanying drawing illustrates:
Fig. 1 is the first mass spectrometric figure of the Purity of the high performance liquid chromatography of little peptide SK011 and mass spectral analysis；
Fig. 2 is the second order ms figure of the Purity of the high performance liquid chromatography of little peptide SK011 and mass spectral analysis.
Detailed description of the invention
Active peptides
In the present invention, the polypeptide of the present invention refers to albumen or the polypeptide with neovascularization inhibiting activity.Described term includes tool
Polypeptide that have angiogenesis inhibiting or growth function, that comprise sequence SEQ ID NO:1 and variant form thereof.These variations
Form include (but being not limited to): 1-3 (usually 1-2, more preferably 1) amino acid whose disappearance, insertion and/or
Replace, and add or disappearance one or several amino acid at C end and/or N-terminal.Additionally, described term also includes list
Body and the polypeptide of the present invention of multimeric forms, linear and nonlinear polypeptide (such as cyclic peptide).As used herein, term " fragment ",
" derivative " and " analog " refers to be kept substantially the polypeptide of angiogenesis inhibiting function or activity.The polypeptide of the present invention
Fragment, derivative or the like can be that (i) has one or several conservative or non-conservative amino acid residue (preferably conservative amino
Acid residue) polypeptide that is replaced, or (ii) have the polypeptide of substituted radical in one or more amino acid residues, or (iii) this
Bright polypeptide merges, with another compound (such as extending the compound of polypeptide half-life, such as polyethylene glycol), the polypeptide formed,
Or (iv) additional amino acid sequence is blended in this peptide sequence and the polypeptide that formed is (with targeting sequencing, secretion sequence or 6His etc.
The then albumen that sequence label merges and formed).According to teaching herein, these fragments, derivative and analog belong to ability
Scope known to the those of skill in the art of territory.
The polypeptide analog of the present invention can be the difference on amino acid sequence with the difference of the polypeptide of the present invention, it is also possible to is not
Affect the difference on the modified forms of sequence, or have both at the same time.Analog also includes having being different from natural L-amino acids
The analog of residue (such as D-amino acid), and have non-naturally-occurring or synthesis amino acid (such as β, gamma-amino acid)
Analog.Should be understood that the polypeptide of the present invention is not limited to the above-mentioned representational polypeptide enumerated.Modify and (do not change
Primary structure) form includes: chemically derived the form such as acetylation or carboxylated of inner or in vitro polypeptide.Modify and also include
Glycosylation, as those or are processed further in step by glycosylated enzyme (such as mammal in the synthesis of polypeptide and processing
Glycosylase or deglycosylating enzyme) and the modification that completes.Modified forms also includes having phosphorylated amino acid residue (such as phosphorus
Acid tyrosine, phosphoserine, phosphothreonine) sequence.Also include being modified thus improve its anti-proteolytic
The polypeptide of solubility property or can be optimized.
The polypeptide of the present invention can also be to be used by the salt form that the acceptable acid of pharmacy or physiology or alkali are derivative.These salt bags
Include the salt that (but not limited to) is formed with following acid: hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, breast
Acid, pyruvic acid, acetic acid, butanedioic acid, oxalic acid, fumaric acid, maleic acid, oxaloacetic acid, methanesulfonic acid, ethyl sulfonic acid, benzene sulphur
Acid or isethionic acid.Other salt includes: the salt formed with alkali metal or alkaline-earth metal (such as sodium, potassium, calcium or magnesium), and
With ester, carbamate or the form of " pro-drug " of other routines.
Coded sequence
The polynucleotides of the present invention can be DNA form or rna form.DNA can be coding strand or noncoding strand.This
Nucleotides full length sequence or its fragment of invention generally can use the method for PCR TRAP, recombination method or Prof. Du Yucang to obtain.
At present, it is already possible to obtained the DNA of code book invention polypeptide (or its fragment, or derivatives thereof) completely by chemical synthesis
Sequence.Then this DNA sequence dna can be introduced various existing DNA moleculars (or such as carrier) as known in the art and carefully
In born of the same parents.
The present invention also relates to the carrier of the polynucleotides comprising the present invention, and by the carrier of the present invention or the volume of polypeptide of the present invention
The host cell that code sequence produces through genetic engineering.
Preparation method
Polypeptide of the present invention can be recombinant polypeptide or synthesis polypeptide, and correspondingly, polypeptide of the present invention can use conventional method Prof. Du Yucang,
Also can produce with recombination method.A kind of it is preferable that with liquid phase synthesis techniques or solid phase synthesis technique, such as Boc solid phase
Method, Fmoc solid phase method or two kinds of methods are used in combination.Synthesis in solid state can quickly obtain sample, can be according to the sequence of purpose peptide
Feature selects suitable resin carrier and synthesis system.Such as, in Fmoc system, preferred solid phase carrier has C in peptide as connected
The Wang resin of terminal amino acid, Wang resin structure be the arm between polystyrene, and amino acid be 4-alkoxyl benzylalcohol；
By 25% hexahydropyridine/dimethylformamide room temperature treatment 20 minutes, to remove Fmoc blocking group, and according to given ammonia
Base acid sequence is extended to N end one by one by C end.After having synthesized, with the trifluoroacetic acid containing 4% p-methyl phenol by synthesis
Proinsulin related peptide cuts down from resin and removes protection group, may filter that except the thick peptide of ether sedimentation isolated after resin.
After lyophilized for the solution of products therefrom, the peptide needed for purifying by gel filtration and reverse phase HPLC method.When using Boc
When system carries out synthesis in solid state, preferred resin is the PAM resin connecting and having C terminal amino acid in peptide, and PAM resin structure is
Arm between polystyrene, and amino acid is 4-methylol phenyl acetamide；In Boc synthesis system, deprotection, neutralize,
In the circulation of coupling, remove blocking group Boc and with diisopropylethylamine (DIEA) two with TFA/ dichloromethane (DCM)
Chloromethanes neutralizes.After peptide chain has been condensed, at 0 DEG C, process 1 with the hydrogen fluoride (HF) containing p-cresol (5-10%)
Hour, peptide chain is cut from resin, removes blocking group simultaneously.Extract with 50-80% acetic acid (containing a small amount of mercaptoethanol)
Peptide, isolated and purified, the most again through high pressure liquid-phase pure with molecular sieve Sephadex G10 or Tsk-40f further after solution is lyophilized
Change and obtain required peptide.Known various coupling agents and each amino acid residue of coupling method coupling in can using chemistry of peptides field,
Such as can use dicyclohexylcarbodiimide (DCC), hydroxyl benzotriazole (HOBt) or 1,1,3,3-tetra-urea hexafluorophosphoric acid ester
(HBTU) direct coupling is carried out.The small peptide obtained for synthesis, its purity and structure can use RP-HPLC and mass spectral analysis
Confirm.
Another kind of method is to produce polypeptide of the present invention with recombinant technique.By conventional recombinant DNA technology, the available present invention
Polynucleotides express or produce the SK series polypeptide of restructuring.In general there are following steps:
(1) by the polynucleotides (or variant) of code book invention polypeptide, or convert with the recombinant expression carrier containing these polynucleotides
Or suitable host cell of transduceing；(2) in suitable culture medium, host cell is cultivated；(3) separate from culture medium or cell,
Protein purification.
Recombinant polypeptide can intracellular or on cell membrane express or be secreted into extracellular.If it is required, its physics can be utilized
, chemistry separated and the albumen of purification of Recombinant by various separation methods with other characteristic.These methods are art technology
Known to personnel.The example of these methods includes, but are not limited to: conventional renaturation processes, processes (salt with protein precipitant
Analysis method), centrifugal, the broken bacterium of infiltration, super processs, ultracentrifugation, sieve chromatography (gel filtration), adsorption chromatography, ion friendship
Change chromatography, high performance liquid chroma-tography (HPLC) and other various LC technology and the combination of these methods.
Owing to polypeptide of the present invention is shorter, it can be considered to multiple polypeptide are cascaded, recombinant expressed after obtain polymer
The expression product of form, then forms required little peptide by the method such as being digested.
Pharmaceutical composition and application process
Present invention also offers a kind of pharmaceutical composition, polypeptide of the present invention that it contains (a) safe and effective amount or its pharmaceutically can connect
The salt being subject to；And (b) pharmaceutically acceptable carrier or excipient.In pharmaceutical composition, the amount of polypeptide of the present invention is usually 10
Microgram-100 milligrams/agent, preferably 100-1000 microgram/agent.For the purposes of the present invention, effective dosage is individual for giving
Body about 0.01 mg/kg is to 50 mg/kg, and preferably 0.05 mg/kg is many to the present invention of 10 mg/kg body weight
Peptide.Additionally, the polypeptide of the present invention can be alone, it is possible to be used together with other therapeutic agent and (be such as formulated in same drug regimen
In thing).
Described pharmaceutical composition also can contain pharmaceutically acceptable carrier.Term " pharmaceutically acceptable carrier " refer to for
The carrier of Therapeutic Administration.This term refers to so some medicament carriers: themselves do not induce generation to accepting said composition
Individual harmful antibody, and there is no undue toxicity after administration.These carriers are well known to those of ordinary skill in the art.
Remington ' s Pharmaceutical Sciences (Mack Pub.Co., N.J.1991) can find about pharmaceutically connecing
Discussing fully of the excipient being subject to.This kind of carrier includes (but being not limited to): salt solution, buffer solution, glucose, water, glycerine,
Ethanol, adjuvant and combinations thereof.Pharmaceutically acceptable carrier can contain liquid, such as water, salt solution, glycerine and ethanol.It addition,
These carriers there is likely to be complementary material, such as wetting agent or emulsifying agent, pH buffer substance etc..Generally, can be by
Therapeutic composition makes injectable agent, such as liquid solution or suspension；May also be fabricated which and be suitable for before the injection allocating solution or outstanding into
In liquid, the solid form of liquid-carrier.
Once make the composition of the present invention, can be administered by conventional route, including (but being not limited to):
Ocular, near the eyes, intraocular (especially in vitreous chamber), intramuscular, intravenous, subcutaneous, intracutaneous or topical.Wait to prevent
Or the object for the treatment of can be animal；Especially people.
When the pharmaceutical composition of the present invention is used for actual therapeutic, the medicine of various different dosage form can be used according to service condition
Compositions.It is preferred that can enumerate have eyedrops, injection (especially intravitreal agent), gel for eye use and
Spongaion.
These pharmaceutical compositions according to conventional methods by mixing, diluting or dissolve and prepare, and can add conjunction once in a while
Suitable medicated premix, as excipient, disintegrant, adhesive, lubricant, diluent, buffer, isotonic agent (isotonicities),
Preservative, wetting agent, emulsifying agent, dispersant, stabilizer and cosolvent, and this process for preparation can utilize used according to formulation
Often mode is carried out.Such as, the preparation of eyedrops can be performed such that by the polypeptide of the present invention or its pharmaceutically acceptable salt with
Base substance is dissolved in sterilized water (being dissolved with surfactant in sterilized water) together, and regulation osmotic pressure and acid-base value are to physiology
State, and can at random add suitable medicated premix such as preservative, stabilizer, buffer, isotonic agent, antioxidant
And tackifier, then make it be completely dissolved.
The pharmaceutical composition of the present invention can be administered with sustained release formulation.Such as, the polypeptide of the present invention or its salt can be impregnated in
Release polymer is in the pill of carrier or micro-capsule, then this pill or micro-capsule is implanted in tissue to be treated by operation.
Additionally, the polypeptide of the present invention or its salt are pre-coated with the intraocular lens of medicine also by insertion and are applied.As sustained release
The example of polymer, can enumerate has ethylene-vinylacetate copolymer, poly-hydroxyl-metacrylate
(polyhydrometaacrylate), polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymer, lactic acid-
Ethanol copolymers etc., preferably can enumerate is biodegradable polymer such as lactic acid polymer and lactic acid-ethanol copolymerization
Thing.
When the pharmaceutical composition of the present invention is used for actual therapeutic, as the polypeptide of the present invention of active component or its pharmaceutically
The dosage of acceptable salt, reasonably can add according to the body weight of each patient to be treated, age, sex, symptom degree
To determine.Such as, when Local eye drop, generally its concentration is about 0.1-10wt%, preferably 1-5wt%, and every day can 2-6
Secondary administration, each 1-2 drips.
VEGF and angiogenesis
VEGF (vascular endothelial growth factor, VEGF) is in new vessels forming process
One main mediated factor, VEGF gene at least can produce 7 kinds of VEGF variants, wherein through the shearing of transcriptional level
VEGF165 (VEGF-A, i.e. prototype VEGF) is one most common, topmost.Its main biological function is:
Selective long-range DEPT vascular endothelial cell mitosis, stimulates vascular endothelial cell proliferation and promotes vascularization；Strengthen blood vessel outstanding
It is the permeability of thin vessels, makes big molecule (mainly fibrinogen) extravasations such as plasma protein be deposited on EV base
In matter, the foundation for new capillary vessel net provides nutrition.
Therefore, scientific research personnel has carried out a lot of research for the suppression of VEGF.Bevacizumab (bevacizumab) is a kind of weight
Organizing anti-human VEGF monoclonal antibody, it is first anti-new green blood being applied to treat metastatic colorectal cancer patients by U.S. FDA approval
Pipe medicine.Intravitreal injection bevacizumab also can effectively suppress retina and CNV.Another kind of VEGF
Inhibitor Ranibizumab, is a kind of recombinant anti human VEGF monoclonal antibody fragment.It is first and is approved by the fda in the United States for
The anti-new vessels class medicine for the treatment of ocular neovascular macular degeneration.At present, people are still constantly looking for new more peace
The complete effective medicine treating ocular angiogenesis.
The present inventor, through extensively in-depth study, is prepared for molecule amount first and is less than the little of 5kD (such as the most about 1-2kD)
Molecular polypeptide.Specifically, the present inventor applies the method for bioinformatics, based on homology analysis and biological characteristics etc.
Analyze, devise several candidate sequence, use solid phase method to be synthesized, the polypeptide of the highly purified present invention of isolated and purified acquisition,
Cell proliferation of human umbilical vein model, chick chorioallantoic membrane vascular pattern and cornea of rats suture model through VEGF induction
Screening, it is thus achieved that a class novel, have prevention and treatment angiogenesis function micromolecule polypeptide.
Experimental model
Human umbilical vein endothelial cells (HUVEC) detects
Angiogenesis refers in primitive vessel clump or the mistake that forms new vessels on the basis of having there is blood vessel in the way of sprouting
Journey.In pathological conditions, in body, already present blood vessel is stimulated by angiogenic factors, expands, Ink vessel transfusing
Endothelial cell activation, breed, migrate, lumen spline structure is formed.Wherein, endothelial cell self duplication is the base of angiogenesis
Plinth, it provides necessary cell quantity for forming new vessels.Therefore, the present invention does using endothelial cell proliferation as medicine
Pre-target spot, Human umbilical vein endothelial cells (HUVEC) is detected by application CCK-8 method (purchased from Dojindo), to inquire into
Polypeptide SK004 is the effect of inhibition of endothelial cell proliferation during anti-angiogenic rebirth.
Cornea of rats suture model
Cornea of rats suture model is classical ocular angiogenesis intervention model, favorable repeatability, can quantitative analysis and becoming
This is relatively low, is commonly used to the active function detecting anti-neovascularization medicaments.
Industrial applicability
Containing peptide of the present invention or its pharmaceutically-acceptable salts as the pharmaceutical composition of active component, angiogenesis or growth are had
Significantly inhibitory activity.Confirming through animal experiment, polypeptide of the present invention is possible not only to suppress the angiogenesis of chick chorioallantoic membrane, also
Rat corneal neovascularization can be suppressed, and the proliferation function of human umbilical vein endothelial cell can be suppressed.Therefore the present invention
Polypeptide is expected to develop into medicine, for treating neovascular eye diseases and relevant neovascular diseases, such as tumor neogenetic blood
Pipe etc..
Sequence illustrates:
Following polypeptide is as comparison polypeptide, from Chinese patent CN 201010235580.4.
Polypeptide SK050, its amino acid sequence is SEQ ID NO:39, and nucleotides sequence is classified as SEQ ID NO:40；
Polypeptide SK051, its amino acid sequence is SEQ ID NO:41, and nucleotides sequence is classified as SEQ ID NO:42；
Polypeptide SK052, its amino acid sequence is SEQ ID NO:43, and nucleotides sequence is classified as SEQ ID NO:44；
Polypeptide SK053, its amino acid sequence is SEQ ID NO:45, and nucleotides sequence is classified as SEQ ID NO:46.
In should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and each technology hereinafter specifically described
Can be combined with each other between feature, thus constitute new or preferred technical scheme.As space is limited, tire out the most one by one at this
State.
Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments be merely to illustrate the present invention and
It is not used in restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally according to normal condition
Such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press,
1989) condition described in, or according to the condition proposed by manufacturer.
Embodiment one: the synthesis of little peptide SK011 and isolated and purified
Use commercially available SYMPHONY type 12 channel polypeptide synthesizer (Protein Technologies company of the U.S.), close
Becoming sequence is the SK011 polypeptide shown in SEQ ID NO:23.Concrete grammar is as follows: according to the software of Peptide synthesizer
(Version.201 version) calculates configuration reagent, by 2-Chlorotrityl Chloride Resin resin (Tianjin Southern folding Cheng Ke
Skill Co., Ltd) put in reaction tube, add DMF (15ml/g) (Dikma), vibrate 30min.Leach out molten by husky core
Agent, is separately added into the Fmoc-L-H-OH (Fmoc-leucine-histidine-OH) (little peptide SK011) of 3 times of molar excess
Amino acid (Suzhou heavenly steed Pharmaceutical Group fine chemicals Co., Ltd), adds the DIEA (traditional Chinese medicines of 10 times of molar excess
Solution on Chemical Reagents in Shanghai company of group), it is eventually adding DMF and dissolves, vibrate 30min.Remove DMF, add 20% piperidines (state
Solution on Chemical Reagents in Shanghai company of medicine group) DMF solution (15ml/g), 5min, removes DMF, then it is molten to add 20% piperidines DMF
Liquid (15ml/g), 15min.Take out piperidine solution, take tens grainy resins, wash three times with ethanol, addition ninhydrin, KCN,
Each one of phenol solution, 105 DEG C-110 DEG C heating 5min, deepen blue for positive reaction.Wash twice with DMF (10ml/g),
Methyl alcohol (10ml/g) is washed twice, and DMF (10ml/g) washes twice.Add protected amino acid (FOMC-Asp-OH) three times of excess,
Three times of excess of HBTU (Suzhou heavenly steed Pharmaceutical Group fine chemicals Co., Ltd), all dissolve with DMF less, add
Enter reaction tube, be added immediately ten times of excess of NMM, react 30min.Wash once with DMF (10ml/g), methyl alcohol (10ml/g)
Washing twice, DMF (10ml/g) washes twice.Repeat aforesaid operations step, be sequentially connected with little peptide SK011 sequence from right to left
In amino acid.After last amino acid connects, deprotection, wash twice with DMF (10ml/g), methyl alcohol (10ml/g)
Washing twice, DMF (10ml/g) washes twice, and DCM (10ml/g) washes twice, washes away resin with this, then drains 10min.From tree
On fat cut polypeptide (cutting liquid (10/g): TFA (J.T.Baker) 94.5%, water 2.5%, EDT (ALDRICH) 2.5%,
TIS (ALDRICH) 1%；Clipping time: 120min).By lysate with nitrogen (Shanghai is than Europe gas industry company) to the greatest extent
Amount dries up, and washes six times with ether (a chemical reagent Co., Ltd is tried in Shanghai), and then normal temperature volatilizes.Finally by after purification
Solution be lyophilized, both obtained high-purity (> 95%) little peptide SK011.
With HPLC (SHIMADZU high performance liquid chromatograph model: preparative, analytic type, software: Class-VP.Sevial
System, manufacturer: SHIMADZU) purified polypeptide, by thick peptide pure water or add a small amount of acetonitrile (Fisher) dissolve, according to
Following condition purifies little peptide SK011 respectively.
Chromatographic column: Agilent Eclipse plus C18RRHD 1.8 μm 2.1 × 150mm
Mobile phase A: 0.1% aqueous formic acid；Mobile phase B: 0.1% formic acid acetonitrile solution.Gradient such as table 1:
Table 1: condition of gradient elution
| Time (min) | Mobile phase A (%) | Mobile phase B (%) | Flow velocity (ml/min) |
| 0 | 90 | 10 | 0.2 |
| 10 | 90 | 10 | 0.2 |
| 25 | 40 | 60 | 0.2 |
| 26 | 90 | 10 | 0.2 |
| 30 | 90 | 10 | 0.2 |
Detection wavelength: 220nm column temperature: 30 DEG C
Embodiment two: the qualification of little peptide SK011 and preservation
Take a small amount of finished product little peptide SK011, do the Purity that HPLC analyzes, and the molecular weight identification of ESI-MS.Condition
As follows: electron spray ionisation, positive ion mode detects, and nitrogen is as sheath gas, auxiliary gas and purge gas.Spray voltage 4.7kV:
Sheath gas is 15arb, and auxiliary gas is 5arb, and purge gas is 0arb；Capillary temperature: 275 DEG C, capillary voltage is 40V,
Lens voltage is 120V.Full scan mass range: 300-2000amu.Second order ms data dependence type collection, sweeps entirely
In tracing spectrum+divalent molecular ion peak for second mass analysis, high-purity helium is used as collision gas, and collision energy is 35ev.
Result shows, little peptide SK011 molecular weight: 1523.83DA, and its structural information is correct, the amino acid of coincidence theory design
Sequence, without modifying (result is shown in Fig. 1-2) after site mutation and expression.
By the little peptide of white powder, pack, be placed in-20 DEG C long-term preservations.
Embodiment three: the derivative preparation of polypeptide SK001-018, qualification and Activity determination
Prepare polypeptide as shown in table 3 by the method for embodiment one, and carry out peptide identification by embodiment two.These polypeptide
Structural information is all correct, with the consensus amino acid sequence of design.And carry out Activity determination as follows.
1, Activity determination principle
VEGF has the activity promoting HUVEC cell proliferation, and polypeptide of the present invention has the effect of suppression VEGF, therefore originally
Secondary detection utilizes HUVEC cell to carry out BA to compare, and first inoculation HUVEC cell, utilizes 2%FBS-ECM
Diluting each sample, after dilution, each sample mixes with VEGF.After cell attachment, inhale and abandon supernatant in each loading hole
Liquid, adds sample, and does dilution comparison and VEGF comparison, and after cultivating 4 days, every hole adds 20 μ l CCK-8 colour developings
Cell is developed the color by liquid, then utilizes ELIASA to be measured the cell after colour developing, and the size measuring absorbance is direct
Reacting cells quantity, according to HUVEC viable count, infers the rush HUVEC cell of each sample suppression VEGF mediation
Proliferation activity.Wherein dilution control group is as negative control, and VEGF control group is as positive control.
2, laboratory apparatus and material (being shown in Table 2)
Table 2: test apparatus and material
| Title | Producer | Model |
| ELIASA | MD | VESRAmax |
| Carbon dioxide incubator | Thermo | Forma Series II 3111 |
| Biohazard Safety Equipment | ESCO | AC2-4E1 |
| Inverted microscope | OLYMPUS | IX51-12pn/30W |
| ECM | ScienCell | |
| ECGS | ScienCell |
| CCK-8 | Dojindo | |
| VEGF | R&D | |
| FBS | ScienCell |
3, experimental procedure
3.1 recovery cells
From liquid nitrogen, take out 1 HUVEC (ScienCell company) cell, 37 DEG C of fast melt, be centrifuged resuspended, use
ECGS-ECM 4ml suspension cell, carries out cell count, and adds ECGS-ECM culture medium (ScienCell company, goods
Numbers 7693) preparation 2.0 × 104The cell suspension of individual/ml.
3.2 inoculating cell
Obtained cell suspension 4ml, adds ECGS-ECM culture medium 16ml, adds in 96 orifice plates with 100 μ l/ holes after mixing, 37 DEG C,
5%CO2Incubator is cultivated 1 day.
3.3 Sample Dilution loadings
Each sample is diluted to 2.5mM and 1.25mM with ECGS-ECM culture medium respectively standby.
3.4 preparation VEGF working solutions
With 2%FBS-ECM, VEGF is diluted to 40ng/ml standby.
3.5 hatch
Taking each sample of 3.3 preparations, add equal-volume VEGF working solution, mixing, 37 DEG C of incubators hatch 3 hours.
3.6 sample-adding
Take well-grown 96 orifice plates, inhale and abandon the nutrient solution in 96 orifice plates, add samples with 100 μ l/ holes, 37 DEG C, 5%CO2
Incubator is cultivated 4 days
3.7 readings and analysis
After cultivation terminates, observation of cell, confirm that form is the most pollution-free, every hole adds 25 μ l CCK-8 working solutions, and 37 DEG C are anti-
Answer 4 hours, at 450nm, survey absorption value with ELIASA.
4, experimental result
All of testing result all represents with inhibiting rate (%), inhibiting rate can the response sample suppression situation to VEGF, calculate
Formula is as follows:
Inhibiting rate (%)=dilution control group result/sample result * 100
Activity determination the results are shown in Table 3.
Table 3: polypeptide and Activity determination result
During this measures, when sample concentration is 1.25mM, all samples result all 50%～80%, illustrates these polypeptide
Have certain inhibitory action under this concentration to VEGF, when concentration is 2.5mM, effect is more excellent.
Embodiment four: the mensuration of little peptide anti-cornea of rats pathologic neovascularization effect
Using cornea of rats suture model, concrete grammar is as follows: healthy SD rat, and 160-180g is male, with right eye as reality
Test eye.Preoperative 3 days all experiment eyespot 0.3% ofloxacin eye drops, every day 2 times.Checking experiment eye before experiment, gets rid of
Eye disease is also weighed oneself.By 3ml/kg body weight intraperitoneal injection 1% yellow Jackets, give general anesthesia, and give ocular
Anesthesia.First impression is gently made with the corneal trephine of diameter 3mm in cornea of rats central authorities, with the vertical impression side of 10-0 nylon wire
To and make corneal stroma with it for center line and sew up, every penetrates corneal stroma 1 pin, every pin span about 1mm, suture in temporo side
Outermost end is away from corneal limbus about 1mm, and art finishes conjunctival sac and is coated with ofloxacin eye ointment.Distinguish 4 eye drops (10 every day for postoperative each group
L/ time/eye of μ.Preparing eye drops by embodiment five, wherein the content of Avastin and little peptide is shown in shown in packet).
It is grouped as follows: blank group (suture+PBS), positive controls (suture+Avastin 10mg/ml), each polypeptide group
(suture+0.5mg/ml).Often 8 rats (n=8) of group.Cornea rebirth blood vessel (CNV) is observed after postoperative 7th day mydriasis
Situation, and measure new vessels length L, simultaneously record neovascularization growth hour number C with gauge.Calculate CNV area
S=0.4*3.1416*C*L.And take a picture.One-way ANOVA is used respectively to organize CNV area S (mean value ± SE),
Using SPSS16.0.1 to carry out statistical analysis, concrete outcome is shown in Table 4.
Table 4: polypeptide and Activity determination result
Result shows, polypeptide group of the present invention has substantially suppression cornea of rats pathologic neovascularization effect for the 7th day after surgery.
Embodiment five: the preparation of eyedrops
Utilizing routine techniques, mix following components, prepare 1% eye eye drops, its formula is as follows:
Little peptide 10mg
Hydroxypropyl methyl cellulose 0.03g
Sterilized water 10mL
Regulation osmotic pressure 300Osm
Acid-base value (pH) 6.8～7.1
The all documents mentioned in the present invention are incorporated as reference the most in this application, are individually recited just as each document
As with reference to like that.In addition, it is to be understood that after the above-mentioned instruction content having read the present invention, those skilled in the art are permissible
Making various changes or modifications the present invention, these equivalent form of values fall within the application claims limited range equally.
Claims (8)
1. angiogenesis inhibiting or a polypeptide for growth, described polypeptide contains sequence shown in SEQ ID NO:1.
Polypeptide the most according to claim 1, it is characterised in that described polypeptide is before SEQ ID NO:1 sequence
Face increases any one or the multiple amino acid that start from C end of SEQ ID NO:2 in order；Or after SEQ ID NO:1
Face increases any one or more amino acid started from N end of SEQ ID NO:3 in order；Or the most all press above-mentioned order
Increase any one in SEQ ID NO:2 and SEQ ID NO:3 or multiple amino acid.
Polypeptide the most according to claim 1, it is characterised in that described polypeptide is:
SK001: its amino acid sequence is SEQ ID NO:1；SK002: its amino acid sequence is SEQ ID NO:5；SK003:
Its amino acid sequence is SEQ ID NO:7；SK004: its amino acid sequence is SEQ ID NO:9；SK005: its amino
Acid sequence is SEQ ID NO:11；SK006: its amino acid sequence is SEQ ID NO:13；SK007: its amino acid sequence
It is classified as SEQ ID NO:15；SK008: its amino acid sequence is SEQ ID NO:17；SK009: its amino acid sequence is
SEQ ID NO:19；SK010: its amino acid sequence is SEQ ID NO:21；SK011: its amino acid sequence is SEQ ID
NO:23；SK012: its amino acid sequence is SEQ ID NO:25；SK013: its amino acid sequence is SEQ ID NO:27；
SK014: its amino acid sequence is SEQ ID NO:29；SK015: its amino acid sequence is SEQ ID NO:31；SK016:
Its amino acid sequence is SEQ ID NO:33；SK017: its amino acid sequence is SEQ ID NO:35；Or SK018: its
Amino acid sequence is SEQ ID NO:37.
4. the nucleotide sequence of the polypeptide encoded as described in any one of claim 1-3；Preferably, described nucleotides sequence
It is classified as sk001: nucleotides sequence and is classified as SEQ ID NO:4；Sk002: nucleotides sequence is classified as SEQ ID NO:6；Sk003:
Nucleotides sequence is classified as SEQ ID NO:8；Sk004: nucleotides sequence is classified as SEQ ID NO:10；Sk005: nucleotides sequence is classified as
SEQ ID NO:12；Sk006: nucleotides sequence is classified as SEQ ID NO:14；Sk007: nucleotides sequence is classified as SEQ ID NO:
16；Sk008: nucleotides sequence is classified as SEQ ID NO:18；Sk009: nucleotides sequence is classified as SEQ ID NO:20；Sk010:
Nucleotides sequence is classified as SEQ ID NO:22；Sk011: nucleotides sequence is classified as SEQ ID NO:24；Sk012: nucleotide sequence
For SEQ ID NO:16；Sk013: nucleotides sequence is classified as SEQ ID NO:28；Sk014: nucleotides sequence is classified as SEQ ID NO:
30；Sk015: nucleotides sequence is classified as SEQ ID NO:32；Sk016: nucleotides sequence is classified as SEQ ID NO:34；Sk017:
Nucleotides sequence is classified as SEQ ID NO:36；Or sk018: nucleotides sequence is classified as SEQ ID NO:38.
5. express expression vector or the host cell of polypeptide as described in any one of claim 1-3, comprise right such as and want
Seek the nucleotide sequence described in 4；Preferably, described expression vector is carrier for expression of eukaryon or virus expression carrier, more preferably
Ground, described carrier for expression of eukaryon is mammalian cell expression vector, and described virus expression carrier is adeno-associated virus or adenopathy
Poisonous carrier；Preferably, described host cell is Chinese hamster ovary celI and subbreed thereof or 293 cells and subbreed thereof.
6. the method preparing polypeptide as described in any one of claim 1-3, described method is chemical synthesis or restructuring
Representation.
7. a pharmaceutical composition, comprise the polypeptide as described in any one of claim 1-3 or its pharmaceutically acceptable salt and
Pharmaceutically acceptable carrier or excipient；Preferably, the formulation of described pharmaceutical composition is injection, eyedrops, ophthalmically acceptable
Gel or spongaion.
8. polypeptide or its pharmaceutically acceptable salt as described in any one of claim 1-3 in preparation treatment or prevent by blood vessel
Application in the medicine of the disease that newborn or growth causes；Preferably, described disease is disease of eye, tumour, ischemic cardiac
Popular name for, non-inflammation cardiomyopathy, coronary sclerosis, arteriosclerosis, arterial embolism, arterial thrombus, Berger's
Disease, chronic inflammation, IBD, ulcer, rheumatic arthritis, scleroderma, psoriasis, sterility or sarcoma shape are sick；
It is highly preferred that described disease of eye be AMD, BDR, CNV,
CME, diabetic macular edema, retinal vascular occlusion, cornea rebirth blood vessel, corneal graft are newborn
Neovascular glaucoma, pteryium or chronic conjunctivitis.
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| CN109970847A (en) * | 2017-12-27 | 2019-07-05 | 上海市第一人民医院 | A new class of polypeptide for inhibiting new vessels and its application |
| CN110870916A (en) * | 2018-09-03 | 2020-03-10 | 上海市第一人民医院 | Application of FBXW7 or its up-regulating agent in preparing medicine for treating diabetes and preventing and treating individual tumor of diabetes |
| CN110870917A (en) * | 2018-09-03 | 2020-03-10 | 上海市第一人民医院 | Application of EWS or its up-regulator in preparing medicine for treating diabetes and preventing and treating diabetes individual tumorigenesis |
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| CN104341486A (en) * | 2013-07-29 | 2015-02-11 | 上海市第一人民医院 | Novel polypeptide capable of inhibiting new vessels and application thereof |
| CN104341489A (en) * | 2013-07-29 | 2015-02-11 | 上海市第一人民医院 | Novel polypeptide capable of inhibiting new vessels and application thereof |
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| CN109970847A (en) * | 2017-12-27 | 2019-07-05 | 上海市第一人民医院 | A new class of polypeptide for inhibiting new vessels and its application |
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| CN110870916A (en) * | 2018-09-03 | 2020-03-10 | 上海市第一人民医院 | Application of FBXW7 or its up-regulating agent in preparing medicine for treating diabetes and preventing and treating individual tumor of diabetes |
| CN110870917A (en) * | 2018-09-03 | 2020-03-10 | 上海市第一人民医院 | Application of EWS or its up-regulator in preparing medicine for treating diabetes and preventing and treating diabetes individual tumorigenesis |
| CN110870916B (en) * | 2018-09-03 | 2022-03-15 | 上海市第一人民医院 | Application of FBXW7 or its up-regulating agent in preparing medicine for treating diabetes and preventing and treating individual tumor of diabetes |
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| CN109776656A (en) * | 2019-01-11 | 2019-05-21 | 广州领晟医疗科技有限公司 | It is a kind of for the peptide T IN7N of angiogenesis inhibiting and its application |
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