CN106512022B - Hydroxyl radical carthamin yellow carthamus A-red blood cell sticks chondroitin sulfate A (CSA) receptor protein polypeptide compound in the application for preparing anti-tumor drug - Google Patents
Hydroxyl radical carthamin yellow carthamus A-red blood cell sticks chondroitin sulfate A (CSA) receptor protein polypeptide compound in the application for preparing anti-tumor drug Download PDFInfo
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Abstract
The present invention provides hydroxyl radical carthamin yellow carthamus A-red blood cells to stick chondroitin sulfate A (CSA) receptor protein polypeptide compound (VAR2CSA-PEG-Hydroxy Saffloryellow A) in the application for preparing anti-tumor drug, and the structural formula that hydroxyl radical carthamin yellow carthamus A-red blood cell sticks chondroitin sulfate A (CSA) receptor protein polypeptide compound (VAR2CSA-PEG-Hydroxy Saffloryellow A) is as follows:
Description
Technical field
The present invention relates to synthetic drugs, and in particular to hydroxyl radical carthamin yellow carthamus A-red blood cell sticks chondroitin sulfate A (CSA) receptor egg
White polypeptide complex (VAR2CSA-PEG-Hydroxy Saffloryellow A) is in the application for preparing anti-tumor drug.
Background technique
The growth of tumour needs blood vessel to be provided nutrition and exclude metabolite, if the not generation of new vessels,
Tumour can only be in the small state of 1 ~ 2mm for a long time.Once new capillary vessel progress tumor tissues, tumour then increase rapidly
And it invades profit and transfer ability also greatly reinforces.Therefore, Tumor Angiongesis how is blocked to have become treating cancer in the world
Hot spot.
In the generating process of new blood vessel, endothelial cell activation and be proliferated be most basic and most important link.It is logical
Crossing inhibition vascular endothelial cell proliferation can inhibit new vessels to generate.Since tumour cell and vascular endothelial cell are mutually interdependent
Rely and mediated neonate tumour blood vessel, therefore, studies the process of Vitro Tumor angiogenesis, it is desirable that total with tumour cell in analogue body
Raw process, it may be assumed that vascular endothelial cell and tumour cell are grown under conditions of co-cultivation.
Studies have shown that the associated angiogenesis of at least 30 kinds growth factors and tumour, wherein it is raw most directly to promote blood vessel
It is vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) at the factor.The blood vessel of tumour
Endothelial cell goes back the receptor of great expression VEGF and bFGF, passes through vasoactive endothelium other than high VEGF expression and bFGF
These receptors of cell surface and induction of vascular is newborn.
Hydroxyl radical carthamin yellow carthamus A (Hydroxy Saffloryellow A, HSYA) can by inhibit tumour VEGF and
The expression of bFGF reaches antitumor action to inhibit tumor vascular growth；Red blood cell sticks chondroitin sulfate A (CSA) receptor egg
The adherency of white (VAR2CSA) and placental receptor (Chondroitin sulfate A, CSA), which mediates, to be combined, and CSA is vigorous in growth
Tumour cell in highly express, hydroxyl radical carthamin yellow carthamus A and red blood cell are sticked into chondroitin sulfate A (CSA) receptor protein
(VAR2CSA) be coupled after, can by hydroxyl radical carthamin yellow carthamus A target into CSA height express tumour cell in, to reach
To orientation antineoplastic action.
Present invention discover that hydroxyl radical carthamin yellow carthamus A (Hydroxy Saffloryellow A, HSYA) and red blood cell are sticked
Chondroitin sulfate A (CSA) receptor protein (VAR2CSA) coupling, forms hydroxyl radical carthamin yellow carthamus A-VAR2CSA polypeptide complex, Neng Gouyou
Effect inhibits the growth of kinds of tumor cells, preferably further researchs and develops new drug.
Summary of the invention
Technical problem to be solved by the present invention lies in researching and designing hydroxyl radical carthamin yellow carthamus A-, that red blood cell sticks sulfuric acid is soft
Ossein A receptor protein polypeptide compound (VAR2CSA-PEG-Hydroxy Saffloryellow A) is preparing anti-tumor drug
Application.
The present invention provides hydroxyl radical carthamin yellow carthamus A-red blood cells to stick chondroitin sulfate A (CSA) receptor protein polypeptide compound
(VAR2CSA-PEG-Hydroxy Saffloryellow A) is in the application for preparing anti-tumor drug.
Hydroxyl radical carthamin yellow carthamus A-red blood cell sticks chondroitin sulfate A (CSA) receptor protein polypeptide compound (VAR2CSA-PEG-
Hydroxy Saffloryellow A) structural formula it is as follows:
。
Hydroxyl radical carthamin yellow carthamus A-red blood cell of the present invention sticks chondroitin sulfate A (CSA) receptor protein polypeptide compound
(VAR2CSA-PEG-Hydroxy Saffloryellow A) is prepared by following method:
By hydroxyl radical carthamin yellow carthamus A (Hydroxy Saffloryellow A, HSYA),N,N'-succinimido carbonic acid
Ester (N, N '-Disuccinimidyl Carbonate, DSC) and amino polyethylene glycol (NPEG) are in 1:20:20 in molar ratio
It is stirred to react in acetone 6 hours or more, by gained hydroxyl radical carthamin yellow carthamus A-polyethylene glycol complex (HSYA-NPEG), decompression
It is dry, after removing acetone, is dissolved with purified water, extracted repeatedly by ethyl acetate, by acetic acid ethyl ester extract 200-300 mesh
The separation of thin layer silica gel, carries out gradient elution with methylene chloride-methanol, thin-layer chromatography control test, collection hydroxyl safflower yellow
Plain A- polyethylene glycol complex (HSYA-NPEG) is dissolved in dimethyl sulfoxide (Dimethyl sulfoxide, DMSO)；It will be red
Cell adhesion chondroitin sulfate A (CSA) receptor protein (VAR2CSA) 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride
(1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, EDC),NHydroxysuccinimidyl
Acid imide (N- Hydroxysuccinimide, NHS) activation, the ratio of 1:30 is by the poly- second of hydroxyl radical carthamin yellow carthamus A-in molar ratio
Glycol compound (HSYA-NPEG) and activated red blood cell stick chondroitin sulfate A (CSA) receptor protein (VAR2CSA) and mix, and 4 DEG C
Stirring 24 hours, takes out dialysis desalination, over-molecular sieve, and collection final product obtains hydroxyl radical carthamin yellow carthamus A-red blood cell and sticks sulfuric acid
Chondroitin A receptor protein polypeptide compound (VAR2CSA-PEG-Hydroxy Saffloryellow A).
Hydroxyl radical carthamin yellow carthamus A-red blood cell is sticked chondroitin sulfate A (CSA) receptor protein polypeptide compound by the present invention
(VAR2CSA-PEG-Hydroxy Saffloryellow A) carries out animal efficacy test, as the result is shown Sydroxy carthamin
A- red blood cell sticks chondroitin sulfate A (CSA) receptor protein polypeptide compound (VAR2CSA-PEG-Hydroxy Saffloryellow
A the growth that) can inhibit the malignant cell of the epithelial tissues such as oophoroma, lung cancer, uterine cancer and carcinoma of testis, turns cancer cell
Shifting has apparent inhibiting effect；Hydroxyl radical carthamin yellow carthamus A-red blood cell sticks chondroitin sulfate A (CSA) receptor protein polypeptide compound
(VAR2CSA-PEG-Hydroxy Saffloryellow A) will be slower than nest cancer, lung to the inhibitory effect of other tumour cells
It is compound to show that hydroxyl radical carthamin yellow carthamus A-red blood cell sticks chondroitin sulfate A (CSA) receptor protein polypeptide for cancer, uterine cancer and carcinoma of testis etc.
Object (VAR2CSA-PEG-Hydroxy Saffloryellow A) is easy to be enriched in the tumour cell of CSA receptor overexpression, right
The effect of other cells is much smaller.Therefore, hydroxyl radical carthamin yellow carthamus A-red blood cell sticks chondroitin sulfate A (CSA) receptor protein polypeptide
Compound (VAR2CSA-PEG-Hydroxy Saffloryellow A) can be used for preparing treatment oophoroma, lung cancer, uterine cancer and
The drug of the malignant tumours such as carcinoma of testis.
Hydroxyl radical carthamin yellow carthamus A-red blood cell of the present invention sticks chondroitin sulfate A (CSA) receptor protein polypeptide compound
(VAR2CSA-PEG-Hydroxy Saffloryellow A) treats treatment oophoroma, lung cancer, uterine cancer and carcinoma of testis in preparation
Etc. the drugs of malignant tumours be that chondroitin sulfate A (CSA) receptor protein polypeptide compound sticked by hydroxyl radical carthamin yellow carthamus A-red blood cell
(VAR2CSA-PEG-Hydroxy Saffloryellow A) is as pharmaceutical composition made of active constituent and conventional pharmaceutical carrier
Object.Described pharmaceutical composition can be tablet, dispersible tablet, lozenge, oral disintegrating tablet, sustained release tablets, capsule, soft capsule, dripping pill,
Granula, injection, powder-injection or aerosol etc..There is biggish clinical value.
Detailed description of the invention
Fig. 1 red blood cell sticks chondroitin sulfate A (CSA) receptor protein (VAR2CSA) and tumour cell A549 specific binding is schemed
1:rContr, 2:rVAR2CSA group, 3:CSA group in figure
Fig. 2 red blood cell sticks chondroitin sulfate A (CSA) receptor protein (VAR2CSA) and tumour cell skov-3 is specifically bound
Figure
1:rContr, 2:rVAR2CSA group, 3:CSA group in figure
Fig. 3 red blood cell sticks chondroitin sulfate A (CSA) receptor protein (VAR2CSA) and tumour cell ishikawa specificity knot
Close figure
1:rContr, 2:rVAR2CSA group, 3:CSA group in figure
Fig. 4 red blood cell sticks chondroitin sulfate A (CSA) receptor protein (VAR2CSA) and tumour cell HUVEC specific binding is schemed
1:rContr, 2:rVAR2CSA group, 3:CSA group in figure
Others' venous endothelial cell proliferative conditions of Fig. 5 difference group；
1 in figure: normal group, 2: model group, 3: administration group；* P < 0.01, compared with normal；P < 0.05 *, with model
Group compares
Effect of Fig. 6 hydroxyl radical carthamin yellow carthamus A to abnormality proliferation HUVEC apoptosis
1 in figure: normal group, 2: model group, 3: administration group；* P < 0.01, compared with normal；P < 0.05 *, with model
Group compares
Fig. 7 hydroxyl radical carthamin yellow carthamus A-red blood cell sticks the external inhibition of chondroitin sulfate A (CSA) receptor protein polypeptide compound
Tumor experiment
1 in figure: control group, 2: it is compound that hydroxyl radical carthamin yellow carthamus A-red blood cell sticks chondroitin sulfate A (CSA) receptor protein polypeptide
Object high dose group, 3: hydroxyl radical carthamin yellow carthamus A-red blood cell stick chondroitin sulfate A (CSA) receptor protein polypeptide compound middle dose group,
4: hydroxyl radical carthamin yellow carthamus A-red blood cell stick chondroitin sulfate A (CSA) receptor protein polypeptide compound it is low, 5: cyclophosphamide group.
Specific embodiment
The invention discloses a kind of hydroxyl radical carthamin yellow carthamus A-red blood cell, to stick chondroitin sulfate A (CSA) receptor protein polypeptide compound
Object (VAR2CSA-PEG-Hydroxy Saffloryellow A) is in the application for preparing anti-tumor drug, below with reference to specific reality
Applying example, the present invention will be further explained, but scope of protection of the present invention is not limited thereto, any to be familiar with this technology neck
The technical staff in domain in the technical scope disclosed by the present invention, is equal according to the technical scheme of the invention and its inventive conception
Replacement changes, and should be covered by the protection scope of the present invention.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
1 hydroxyl radical carthamin yellow carthamus A of embodiment-red blood cell sticks chondroitin sulfate A (CSA) receptor protein polypeptide compound
(VAR2CSA-PEG-Hydroxy Saffloryellow A) coupling
Take a clean round-bottomed flask, weigh respectively hydroxyl radical carthamin yellow carthamus A (Hydroxy Saffloryellow A,
HSYA) (purity >=95%, be purchased from middle inspection institute) 0.612g,N,N'-succinimidyl carbonate (N, N '-
Disuccinimidyl Carbonate, DSC) (purity >=95%, purchased from Sigma-Aldrich) 5.2g and the poly- second of amination
Glycol (NPEG) (high-grade pure is purchased from the U.S.) 6mL, is added 250mL acetone (chromatographically pure is purchased from Sigma-Aldrich), it stirs
It mixes reaction 6 hours or more；By gained hydroxyl radical carthamin yellow carthamus A-polyethylene glycol complex (HSYA-NPEG), acetone is removed under reduced pressure,
The dissolution of 100mL purified water is added, adds 100mL ethyl acetate and extracts repeatedly, extraction is three times；Acetic acid ethyl ester extract is collected,
It is dried under reduced pressure, concentration acetic acid ethyl ester extract to about 50mL；By the thin layer silica gel of acetic acid ethyl ester extract 200-300 mesh point
From carrying out gradient elution with methylene chloride-methanol, thin-layer chromatography control test collects hydroxyl radical carthamin yellow carthamus A-polyethylene glycol
Compound (HSYA-NPEG) component, is dried under reduced pressure；Solid crude product is mixed with 20 mL ethyl acetate/methanols (volume ratio 3:1)
Bonding solvent dissolution uses dry column-packing to make eluant, eluent with methanol and carries out pillar layer separation, obtains brown powder target product；It will
Gained object is dissolved in 20mL dimethyl sulfoxide (Dimethyl sulfoxide, DMSO), and (chromatographically pure is purchased from Sigma-
Aldrich in)；It accurately weighs 0.10g red blood cell and sticks chondroitin sulfate A (CSA) receptor protein (VAR2CSA) and be dissolved in 20mL MES
In buffer (pH6.0), 0.286g 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (1- (3- is added
Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, EDC) (chromatographically pure is purchased from
) and 0.365g Sigma-Aldrich NHOSu NHS (N- Hydroxysuccinimide, NHS) (chromatographically pure, purchase
Reaction 30min is stirred at room temperature from Sigma-Aldrich), the hydroxyl radical carthamin yellow carthamus A-polyethylene glycol for being dissolved in DMSO is compound
Object (HSYA-NPEG) and activated red blood cell stick chondroitin sulfate A (CSA) receptor protein (VAR2CSA) mixing, are stirred at room temperature
30min；Reaction terminates, and with 4 DEG C of dialysis desalinations of phosphate buffer, every 6h changes a not good liquor, collects final product and obtains hydroxyl safflower
Safflor yellow A-red blood cell sticks chondroitin sulfate A (CSA) receptor protein polypeptide compound (VAR2CSA-PEG-Hydroxy
Saffloryellow A).
2 red blood cell of embodiment sticks the specific binding of chondroitin sulfate A (CSA) receptor protein (VAR2CSA) and tumour
1. Preparatory work of experiment
Complete medium: the fetal calf serum (GIBCO) of incomplete culture medium+10%, spare after mixing, 4 DEG C of preservations.Wherein
Incomplete culture medium selects RPMI1640 culture medium (GIBCO)
The preparation of various concentration CSA: electronic balance weighs a small amount of tested material (1-3mg), is dissolved with a small amount of PBS, with corresponding
Culture medium is configured to the solution of 7 concentration gradients, and after 0.22 μm of first concentration filtering, subsequent concentration doubling dilution is prepared.
2. experimental group and experimental program
4 cells are shared, each cell is respectively divided into 3 groups, every group of 3 holes.Respectively rContr, rVAR2CSA group, CSA
Group.A549, skov-3, ishikawa, HUVEC logarithmic growth phase cell (being purchased from Sheng Ke institute, Chinese Academy of Sciences cell bank) is taken respectively,
It being inoculated in 96 orifice plates (purchased from Corning company), every 100 μ L of hole is separately added into rContr, rVAR2CSA and CSA afterwards for 24 hours,
The every hole of control group adds 100 μ L of culture medium, and control group and detection group are all provided with 3 multiple holes, separately set blank control group, cell is not added and only adds
The conduct zeroing group of culture medium.96 orifice plates are placed in 37 DEG C, 5%CO2Incubator culture, cell grow to 70%-80% density
When, it removes supernatant, is incubated for PBS, 4 DEG C of incubations 30min with the fetal calf serum containing 2%, V5-FITC antibody progress the is then added
Secondary incubation, flow cytometer detection is analyzed after incubation.
3, result
1, flow cytomery MFI value is analyzed
In this experiment, we test human cancer cell line A549, skov-3, the ishikawa and the mankind in patient source
The Percentage bound of the HUVEC and rVAR2CSA in normal cell source, each cell are respectively divided into 3 groups, every group of 3 multiple holes.RContr group
RContr is only added in (control group), and rVAR2CSA is only added in rVAR2CSA group, and rVAR2CSA and CSA is added simultaneously in CSA group.It adds
Taking-up when cell grows to 70%-80% density after drug carries out MFI with flow cytometer after V5-FITC antibody incubation is added
(average fluorescent strength) detection.
According to obtaining a result after MFI value analysis and assessment, rVAR2CSA and human cancer cell line show high-bonding-ratio, simultaneously
After inhibitor C SA is added, Percentage bound is substantially reduced, and illustrates that rVAR2CSA has the function of identifying human cancer cell.
Effect of 3 hydroxyl radical carthamin yellow carthamus A of embodiment to HUVEC
The HUVEC model of abnormality proliferation is established, HSYA(is added and is purchased from Nat'l Pharmaceutical & Biological Products Control Institute's standard items) after,
MTT detects cell proliferative conditions, Flow cytometry cell cycle and apoptosis rate.
Experimental group and experimental program:
Normal group: normal HUVEC
Model group: normal HUVEC+SGC7901 supernatant (50%)
Administration group: normal HUVEC+SGC7901 supernatant (50%)+HSYA (0.075g/L)
Normal HUVEC 80% is passed on after merging, by 1 × 105It is inoculated in 25cm2Culture bottle in, be added 2.5mL/ bottles
Culture solution, it is random to be grouped after the adherent 6h of cell, culture solution is sucked out, the 1640 culture medium that normal group is added 5mL/ bottles (is purchased from
GIBCO), 5ml/ bottles of 1640 culture medium containing 50% A549 tumor supernatant are added in model group, and administration group, which is added, contains 50% A549
The 250 μ L of 1640 culture medium 4.75mL and 0.075g/L HSYA of tumour supernatant is 5mL/ bottles total, continues after cultivating 20h, collects thin
Born of the same parents are used for FCM analysis.
HUVEC cell 80% passes on after merging, and cell concentration is adjusted to 0.5 × 10 with culture medium5A/ml, is inoculated in 96
Orifice plate is set in incubator after adherent 6 hours, and supernatant is sucked out, and 200 hole μ L/ of 1640 culture medium is added in normal group.37℃,5%CO2Point
5mg/mL MTT(Pei Yang not be added and be purchased from Sigma-Aldrich after 48 hours) 10 holes μ L/, continue to cultivate 4h, removes supernatant, then
100 hole μ L/ DMSO is added, microplate reader (purchased from match Mo Feishier company) surveys 560nm OD value.
Cell concentration is adjusted to 106A/mL is added ice ethyl alcohol (75%) and is resuspended, fixed, and the cold PBS(of 1mL is added and is purchased from
Hyclone, GE), gently concussion makes cell suspend.1000rpm, 4 DEG C centrifugation, 10 minutes, abandon supernatant, stay precipitating, the process into
Row is twice.Cell is resuspended in 200 μ L Binding Buffer.The dyeing of 10 μ L propidium iodides (PI) is added, mixes gently, keeps away
Light reacts 30 minutes for room temperature reaction 15 minutes or 4 DEG C, 300 μ L Binding Buffer is added, flow cytometer is (purchased from beauty
BD company, state) detection.
Experimental result:
1) MTT is detected
Administration group cell absorbance is significantly lower than model group.Compared with model group, administration group cell OD value is significantly lower than mould
Type group illustrates that the growth of administration group cell receives inhibition.
2) flow cytometer detection
Administration group Apoptosis peak is significantly raised；Administration group apoptosis rate is apparently higher than model group.Compared with model group,
Ratio shared by the administration group cell S phase is substantially reduced, and the variation of other each groups is unobvious.Illustrate that administration group HSYA inhibits cell
Growth, and apoptosis may occur.
Embodiment 4: hydroxyl radical carthamin yellow carthamus A-red blood cell sticks chondroitin sulfate A (CSA) receptor protein polypeptide compound
The external inhibition tumor experiment of (VAR2CSA-PEG-Hydroxy Saffloryellow A)
The HSYA of low concentration is a kind of stronger angiogenesis inhibitors, after HSYA chains VAS2CSA, specific recognition
Tumor tissues, the effect of being finally reached Growth of Tumors Transplanted.This experiment studies hydroxyl radical carthamin yellow carthamus A-by animal model
Red blood cell sticks chondroitin sulfate A (CSA) receptor protein polypeptide compound (VAR2CSA-PEG-Hydroxy Saffloryellow A)
To the inhibitory effect of Transplanted Gastric Carcinoma.
Experimental group and experimental program:
A549 cell strain is dressed to sufficient amount, when being in logarithmic growth phase with adherent cell, with 0.25% pancreatin
Digestion, counts under inverted microscope, and observes cell viability greater than 95%, adjusts concentration, is configured to 1 × 107A/mL is slender
Born of the same parents' suspension, it is subcutaneous to be only inoculated in C57BL/6 mouse (being purchased from Guangdong Province's animal experimental center) left rib abdomen by 0.2mL/, replicates mouse
Transplanted Gastric Carcinoma model.Five groups, every group 6 are randomly divided into after being inoculated with 1 week.It is inoculated with next day, hydroxyl radical carthamin yellow carthamus A-is red
Cell adhesion chondroitin sulfate A (CSA) receptor protein polypeptide compound (VAR2CSA-PEG-Hydroxy Saffloryellow A) is big,
In, small dose group be injected intraperitoneally respectively respective concentration drug (1.4,0.7,0.35mg/kg/ only, 2 times/d)；Model group is given
Measure sterile saline (0.2mL/, 2 times/d)；The 2nd day beginning intraperitoneal injection of cyclophosphamide (25mg/ of cyclophosphamide inoculation group
Kg/, every 1 time on the 2nd).Mouse weighing in 22nd day, puts to death.
It is inoculated with the 22nd day, after whole mouse are put to death, routine disinfection completely strips the left rib abdomen subcutaneous tumor of mouse, uses electronics
Balance weighs knurl weight, calculates the inhibition rate of tumor growth of each group.
Experimental result:
Whole C57BL/6 mouse naked eyes are visible at the 7th day after concentration microspike under the stomach wall of left side, and size is about
0.25 × 0.22cm or so subcutaneous nodule has gradually obviously gone out tumor, tumor formation rate 100%；From tumor growth curve it can be seen that
Tumor volume growth it is most fast be physiological saline group, growing most slow is cyclophosphamide group, medication the 14th day, each group gross tumor volume
There is difference, physiological saline group and the obvious quickening of HSYA large dosage group mice-transplanted tumor system growth, HSYA small dose group in establishment
Mice-transplanted tumor volume growth is relatively slow, and the growth of CTX group is most slow.Each group mice-transplanted tumor volume increases when 16d
All obvious to accelerate, wherein HSYA each group tumor volume change is most obvious.
Claims (4)
1. hydroxyl radical carthamin yellow carthamus A-red blood cell sticks chondroitin sulfate A (CSA) receptor protein polypeptide compound (VAR2CSA-PEG-
Hydroxy Saffloryellow A) in the application for preparing anti-tumor drug, which is characterized in that the Sydroxy carthamin
A- red blood cell sticks chondroitin sulfate A (CSA) receptor protein polypeptide compound (VAR2CSA-PEG-Hydroxy Saffloryellow
A structural formula) is as follows:
Hydroxyl radical carthamin yellow carthamus A-the red blood cell sticks chondroitin sulfate A (CSA) receptor protein polypeptide compound (VAR2CSA-PEG-
Hydroxy Saffloryellow A) it is prepared by following method:
By hydroxyl radical carthamin yellow carthamus A (Hydroxy Saffloryellow A, HSYA), N, N'- succinimidyl carbonate
(N, N '-DisuccinimidylCarbonate, DSC) and amino polyethylene glycol (NPEG) are in 1:20:20 acetone in molar ratio
In be stirred to react 6 hours or more, gained hydroxyl radical carthamin yellow carthamus A-polyethylene glycol complex (HSYA-NPEG) is dried under reduced pressure,
After removing acetone, is dissolved with purified water, extracted repeatedly by ethyl acetate, acetic acid ethyl ester extract is thin with 200-300 purpose
Layer silica gel separation carries out gradient elution with methylene chloride-methanol, and thin-layer chromatography control test collects hydroxyl radical carthamin yellow carthamus A-
Polyethylene glycol complex (HSYA-NPEG) is dissolved in dimethyl sulfoxide (Dimethyl sulfoxide, DMSO), and red blood cell is sticked
Attached chondroitin sulfate A (CSA) receptor protein (VAR2CSA) uses 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (1- (3-
Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, EDC), n-hydroxysuccinimide
(N-Hydroxysuccinimide, NHS) activation, the ratio of 1:30 is multiple by hydroxyl radical carthamin yellow carthamus A-polyethylene glycol in molar ratio
It closes object (HSYA-NPEG) and activated red blood cell sticks chondroitin sulfate A (CSA) receptor protein (VAR2CSA) mixing, 4 DEG C of stirrings 24
Hour, dialysis desalination, over-molecular sieve are taken out, collection final product obtains hydroxyl radical carthamin yellow carthamus A-red blood cell and sticks chondroitin sulfate
A receptor protein polypeptide compound (VAR2CSA-PEG-Hydroxy Saffloryellow A).
2. application according to claim 1, it is characterised in that hydroxyl radical carthamin yellow carthamus A-red blood cell sticks chondroitin sulfate A (CSA)
Receptor protein polypeptide compound (VAR2CSA-PEG-Hydroxy Saffloryellow A) is used as active constituent and pharmaceutical carrier
Pharmaceutical composition is made.
3. application according to claim 2, it is characterised in that described pharmaceutical composition is tablet, capsule, dripping pill, particle
Agent, injection, powder-injection or aerosol.
4. application according to claim 2, it is characterised in that the single dose hydroxyl safflower yellow of described pharmaceutical composition
Plain A- red blood cell sticks chondroitin sulfate A (CSA) receptor protein polypeptide compound (VAR2CSA-PEG-Hydroxy Saffloryellow
It A) is 1mg-5mg.
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| US20210278406A1 (en) * | 2018-07-13 | 2021-09-09 | Varct Diagnostic Aps | Isolation of circulating cells of fetal origin using recombinant malaria protein var2csa |
| CN109387627B (en) * | 2018-10-16 | 2021-09-24 | 中国科学院深圳先进技术研究院 | A reagent method for cancer screening and early diagnosis based on placenta-like chondroitin sulfate A |
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| CN101744802A (en) * | 2008-11-28 | 2010-06-23 | 张前 | Novel application of hydroxysafflor yellow A to preparation of anti-tumour drugs |
| CN104136041A (en) * | 2012-02-09 | 2014-11-05 | Var2制药有限公司 | Targeting of chondroitin sulfate glycans |
| WO2015095952A1 (en) * | 2013-12-27 | 2015-07-02 | The Centre For Drug Research And Development | Var2csa-drug conjugates |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101744802A (en) * | 2008-11-28 | 2010-06-23 | 张前 | Novel application of hydroxysafflor yellow A to preparation of anti-tumour drugs |
| CN104136041A (en) * | 2012-02-09 | 2014-11-05 | Var2制药有限公司 | Targeting of chondroitin sulfate glycans |
| WO2015095952A1 (en) * | 2013-12-27 | 2015-07-02 | The Centre For Drug Research And Development | Var2csa-drug conjugates |
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