CN108785310A - Application of the Vande Thani in the antitumor drug for preparing targeting EphB4 - Google Patents
Application of the Vande Thani in the antitumor drug for preparing targeting EphB4 Download PDFInfo
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- CN108785310A CN108785310A CN201810690352.2A CN201810690352A CN108785310A CN 108785310 A CN108785310 A CN 108785310A CN 201810690352 A CN201810690352 A CN 201810690352A CN 108785310 A CN108785310 A CN 108785310A
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
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- A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P35/02—Antineoplastic agents specific for leukemia
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Abstract
The invention discloses application of the Vande Thani (dasatinib) in the antitumor drug for preparing targeting EphB4, belong to pharmaceutical technology field.The present invention is by the above application of experimental verification Vande Thani, and molecular docking method, SPR methods and MST are the experimental results showed that Vande Thani can interact with EphB4.EphB4 kinase assays show that Vande Thani can obviously inhibit EphB4 kinase activities.Significantly inhibit K562 Leukaemia, the proliferation of JLTRG, H9.Meanwhile Vande Thani significantly inhibits signaling molecule p-PI3K p85/p55, PI3K the p85 expression in the downstreams EphB4, lowers the phosphorylation level of MEK and ERK.Therefore, experimental result of the invention fully supports application of the Vande Thani in the tumour medicine of anti-leukemia medicine and targeting EphB4.
Description
Technical field
The invention belongs to pharmaceutical technology fields, and in particular to Vande Thani is in the antitumor drug for preparing targeting EphB4
Application.
Background technology
Leukaemia is a kind of common malignant tumour of hematological system, is one of domestic ten big malignant tumours occurred frequently, China is each
The incidence of regional leukaemia accounts for the 6th in various tumours.Leukaemia is the extremely Clonal disease of candidate stem cell, is had
Height heterogeneity.Clonal leukaemia cell because proliferation out of control, dysdifferentiation, apoptosis are obstructed etc. mechanism marrow and other make
Largely proliferation accumulation in haemal tissue, and other non-hematopoietic tissues and organ are infiltrated, while inhibiting normal hematopoiesis function.By onset
Emergency can be divided into leukemia.Leukaemia is often clinically divided into lymphocytic leukemia, myelocytic leukemia, mixing
Chronic myeloid leukemia etc..
Eph belongs to known maximum RTKs subtribes, including Eph receptors and its Ephrin ligands.EphB4 and EphrinB2 exists
Signal shows unique mode when transmitting, and can mutually be activated by other side, generates " forward signal " and " reverse signal ", EphB4/
Proliferation, transfer and the invasion etc. of ephrinB2 and its unique two-way signaling transduction regulation and control cancer.EphB4 swells in all substantially
It is expressed in tumor, but the drug for not targeting EphB4 at present appears on the market.
Vande Thani Vandetanib, molecular formula C22H24BrFN4O2, white, circle, concave-concave, film coating.Vande Thani category
The more tyrosine kinase inhibitors of small molecule belong to aniline quinazoline class compound, act not only on EGFR, VEGFR of tumour cell
With RET tyrosine kinase, other tyrosine kinase and serine/threonine kinase may also suppress.Vande Thani is first
The medullary thyroid carcinoma medicine of approval, suitable for treat cannot cut off, Locally Advanced or transfer have symptom or a progress
Medullary thyroid carcinoma.
It there is no Vande Thani anti-leukocythemia at present and target the application report of the tumour of EphB4.
Invention content
The purpose of the present invention is to provide application of the Vande Thani in the antitumor drug for preparing targeting EphB4.
The present invention is to be achieved through the following technical solutions：
The invention discloses application of the Vande Thani in the antitumor drug for preparing targeting EphB4.
Preferably, the drug is the drug for inhibiting EphB4 expression.
Preferably, the drug is the medicine for regulating and controlling its signal path by the expression by inhibiting tumour cell EphB4
Object.
It is further preferred that the drug is signaling molecule p-PI3K p85/p55, the PI3K for inhibiting the downstreams EphB4
P85 is expressed, and lowers the drug of the phosphorylation level of MEK and ERK.
Preferably, the tumour is leukaemia.
Preferably, the drug is the drug for inhibiting K562 Leukaemia, JLTRG or H9 proliferation.
Preferably, the animal dosage of Vande Thani is 10~80mg/kg.
Compared with prior art, the present invention has technique effect beneficial below：
The invention discloses application of the Vande Thani (dasatinib) in the antitumor drug for preparing targeting EphB4.This
Invention is by the above application of experimental verification Vande Thani, and molecular docking method, SPR methods and MST are the experimental results showed that Vande Thani
It can interact with EphB4.EphB4 kinase assays show that Vande Thani can obviously inhibit EphB4 kinase activities.
Further, show that Vande Thani can significantly inhibit K562 Leukaemia by MTT experiment, JLTRG, H9's
Proliferation, K562 cyto-inhibitions especially high to EphB4 expression quantity become apparent.Low K562 cells are struck by siRNA interference
EphB4 after, Vande Thani obviously weakens the inhibiting effect of K562 cells；And after being overexpressed the EphB4 of K562 cells, all morals
The inhibiting effect of K562 cells is remarkably reinforced in his Buddhist nun.In the experiment of nude mouse of intravenous injection K562 cells, Vande Thani is through filling
After stomach is administered 14 days, carries out taking blood, measure the expression of EphB4.Vande Thani can obviously reduce the content of internal EphB4.
Further, Vande Thani significantly inhibits signaling molecule p-PI3K p85/p55, the PI3K p85 in the downstreams EphB4
The phosphorylation level of MEK and ERK is lowered in expression.
Therefore, experimental result of the invention fully supports Vande Thani (dasatinib) in anti-leukemia medicine and targeting
Application in the tumour medicine of EphB4.
Description of the drawings
Fig. 1：EphB4 contents in K562, JLTRG, H9 leukaemia cell.A：Cell protein histogram；B：The quantitative figure of A figures；
Fig. 2：To the growth inhibition effect of K562 and H9 cells after Vande Thani effect different time；
Fig. 3：Influence of the Vande Thani to EphB4 kinase activities；
Fig. 4：Vande Thani is analyzed with EphB4 protein-interactings SPR；A：Vande Thani is tied with EphB4 intracellular proteins SPR
Close curve；B：Vande Thani and EphB4 extracellular protein SPR binding curves；
Fig. 5：SiRNA interferes EphB4 experimental results；A：Protein expression figure after gene interference；B：The quantitative figure of A figures；C：Gene
MRNA expression figure after interference；D：Vande Thani acts on wild type and unites with inhibiting rate after EphB4 knockdown K562 cells
Meter figure；
Fig. 6：K562 and H9 transiently transfects EphB4 experimental results；A：Protein expression figure after transient transfection；B：The quantitative figure of A figures；
C：MRNA expression figure after transient transfection；D：After Vande Thani acts on wild type and EphB4 transfected K562 cells
Inhibiting rate statistical chart；E：Vande Thani acts on wild type and inhibiting rate statistical chart after EphB4 transfected H9 cells.
Specific implementation mode
With reference to specific embodiment, the present invention is described in further detail, it is described be explanation of the invention and
It is not to limit.
Embodiment 1
Western blot determination EphB4 expression：Western blot investigates the EphB4 tables of three kinds of different type leukaemia cells
Up to level.When cell is in exponential phase of growth, cell is subjected to cracking 30min with RIPA cell pyrolysis liquids, using freezing from
Scheming 12000g, 10min, 4 DEG C cell pyrolysis liquid is subjected to centrifugation.Simultaneously a certain amount of 5 × loading is added in Aspirate supernatant
Buffer solution, heating boil 5min and make albuminous degeneration, mark, be positioned over -20 DEG C it is spare.The protein sample handled well is being prepared
10%SDS-PAGE on carry out loading, 1 × electrophoretic buffer is added, opens power supply, 120mV voltage stabilizings is kept to carry out sample point
From.The sample of separator well is subjected to purposive cutting, and it is subjected to During migration using 120mA current stabilizations.After transferring film
Pvdf membrane is put into skimmed milk power confining liquid and carries out closing 2h.It is incubated EphB4 primary antibodies later, and it is allowed to be incubated overnight at 4 DEG C.
Film is subjected to cleaning 3 times, each 10min with TBST, then is incubated secondary antibody, it is made to react 1h.Film is subjected to cleaning 3 with TBST again
It is secondary, each 10min.Luminescent solution is eventually adding to develop.As a result referring to Fig. 1, the results showed that tri- plants of differences of K562, JLTRG, H9
Leukaemia cell in EphB4 expressions it is different, K562>JLTRG>H9.
Cell proliferation experiment：Vande Thani is investigated to K562 using mtt assay, the growth inhibition effect of JLTRG, H9 are examined
It examines.By the cell of exponential phase of growth with every hole 2 × 104The concentration of a cell is suspended in complete medium and with 180 μ L of every hole
Volume be inoculated in 96 orifice plates.After waiting for growth for 24 hours, it is added in 96 orifice plates with 20 μ L volumes of every hole.Wait for that it reacts 48h
Afterwards, the MTT (5mg/mL) of 20 μ L is added in 96 orifice plates.So that it is reacted 4h in 37 DEG C, using centrifuge (1000r/min) from
Heart 5min reuses pipettor and blots liquid in 96 orifice plates only, and is added in 150 μ L DMSO to 96 orifice plates, makes on shaking table
It is mixed well.It is finally selected in enzyme-linked immunosorbent assay instrument at 490nm and is detected and reads.As a result show K562 to all morals
His sensibility of Buddhist nun's effect is most strong, and IC50 values are minimum, and H9 cell EphB4 expression quantity is minimum, IC50 value highests.Vande Thani point
Not Zuo Yong K562 Leukaemia and H9 for 24 hours, after 48h, 72h, as a result referring to Fig. 2, when MTT results are shown with drug effect
Between increase, the sensibility of K562 and H9 also gradually increases, and IC50 values reduce, i.e. cell Proliferation of the Vande Thani to K562 and H9
Inhibiting effect and presentation dosage and time dependence.
Embodiment 2
Kinase assay investigates influence of the Vande Thani to EphB4 kinase activities：Using homogeneous time-resolved fluorescence (HTRF)
Method detects the effect of the phosphorylation of Drug inhibition EphB4 kinases.2 μ L EphB4 kinases and 2 μ L are separately added into 384 orifice plates
Substrate, then the Vande Thani of 4 μ L various concentrations is added wherein, 2 μ L ATP are finally added in 384 orifice plates, use knockout plate
Machine makes reaction system be uniformly mixed, and 384 orifice plates are at this moment put into 37 DEG C of baking ovens and are incubated.30min to be reacted and then to body
The Eu that addition is prepared with reaction terminating liquid in system3+In-TK- antibody working solution and each 5 μ L to 384 orifice plates of Streptavidin-XL665
Make reaction terminating.After reacting at room temperature 1h, 384 orifice plates are put into multi-function microplate reader and are detected, the condition of detection is：Choosing
It is excitation wavelength with 615nm, 665nm is launch wavelength.Referring to Fig. 3, the results showed that Vande Thani can effectively inhibit EphB4
Kinase activity, and be in concentration dependent.
Embodiment 3
SPR experiment confirmation target spots：One NTA chip is installed in OpenSPRTM instruments, it is slow then to take out PBS with maximum flow rate
Fliud flushing.After reaching signal base line, from the IPA (isopropanol) of loading mouth injection 80%, control valve is gone into inject, runs 10s
It rotates back into load and bubble is exhausted, after reaching baseline, wash buffer sample ring is used in combination air to empty.Reach baseline in signal
Afterwards, buffer flow rate is adjusted to 20 μ L/min.The imidazoles liquid of 250 μ L is imported to injection port and is injected, to initialize inductor table
It is consistent to initialize the 2-3 curve after injection for face.Import the NiCl of the 40mM of 250 μ L2It to injection port and injects, passes through
Ni2+Ion charges to NTA, and surface is now ready for carrying out functionalization with his labels ligand.Preparation is dissolved in 250 μ L fortune
His label ligands EphB4-1 (50 μ g/mL) in row buffering liquid, injection loading hole, interact at least 5min.Observation base
5min, to ensure to stablize.Amount by comparing the signal measurement ligand binding before and after injection ligand judges whether ligand is combined into
Work(.It is recycled successively by above-mentioned steps until the whole analysis of all analysis samples terminates.In this way measure Vande Thani with
EphB4 protein-interactings.As shown in figure 4, Vande Thani can cause with EphB4 intracellular proteins and extracellular protein interaction
The variation of absorbing wavelength.
Embodiment 4
SiRNA is interfered：By height expression EphB4 leukaemia cell be inoculated in 6 orifice plates with density appropriate, per hole 2mL without
Antibiotic medium is carried out to when 30%~50% fusion using 2000 transfection reagents of LipofectamineTM when cell growth
Transfection, EphB4 siRNA oligomer transfectional cells, after transfecting 48h, the inspection of real-time PCR and western blotting methods
Survey the mRNA and protein expression level of EphB4.
It transiently transfects：Leukaemia cell is inoculated in density appropriate in 24 orifice plates, is 1mL per hole nutrient solution volume.
Culture for 24 hours, makes it grow fusion in culture plate up to 70%~90%.It will using TurboFectTM In Vitro transfection reagents
EphB4 plasmid transfections are in cell.After transfecting 48h, real-time PCR and western blotting methods detect IL-2R's
MRNA and protein expression level.Cell proliferation experiment measures Vande Thani and makees to the inhibition of the cell of instantaneous low expression and high expression
With.Vande Thani weakens the inhibiting effect of the K562 cells of silence EphB4 genes, and IC50 values increase (Fig. 5), to K562 and H9
EphB4 genes (Fig. 6) are transiently transfected in leukaemia cell, Vande Thani is thin to the K562 cells and H9 of high expression EphB4 albumen
The inhibited proliferation of born of the same parents enhances, and IC50 values reduce.Illustrate Vande Thani mainly by being played with EphB4 protein-interactings
To the inhibited proliferation of leukaemia cell.
Claims (7)
1. application of the Vande Thani in the antitumor drug for preparing targeting EphB4.
2. application as described in claim 1, which is characterized in that the drug is the drug for inhibiting EphB4 expression.
3. application as claimed in claim 2, which is characterized in that the drug is the table by inhibiting tumour cell EphB4
Up to the drug for regulating and controlling its signal path in turn.
4. application as claimed in claim 3, which is characterized in that the drug is the signaling molecule p- for inhibiting the downstreams EphB4
PI3K p85/p55, PI3K p85 are expressed, and lower the drug of the phosphorylation level of MEK and ERK.
5. application as described in claim 1, which is characterized in that the tumour is leukaemia.
6. application as claimed in claim 5, which is characterized in that the drug be inhibit K562 Leukaemia, JLTRG or
The drug of H9 proliferation.
7. the application as described in any one of claim 1~6, which is characterized in that the animal dosage of Vande Thani is 10
~80mg/kg.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810690352.2A CN108785310A (en) | 2018-06-28 | 2018-06-28 | Application of the Vande Thani in the antitumor drug for preparing targeting EphB4 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113398267A (en) * | 2020-03-17 | 2021-09-17 | 中国医学科学院药物研究所 | Application of EphB4 as target in screening of drugs or models for increasing insulin sensitivity |
-
2018
- 2018-06-28 CN CN201810690352.2A patent/CN108785310A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113398267A (en) * | 2020-03-17 | 2021-09-17 | 中国医学科学院药物研究所 | Application of EphB4 as target in screening of drugs or models for increasing insulin sensitivity |
| WO2021184918A1 (en) * | 2020-03-17 | 2021-09-23 | 中国医学科学院药物研究所 | Use of ephb4 as target in screening of drugs or models for increasing insulin sensitivity |
| CN115279411A (en) * | 2020-03-17 | 2022-11-01 | 中国医学科学院药物研究所 | Application of EphB4 as target in screening of drugs or models for increasing insulin sensitivity |
| CN113398267B (en) * | 2020-03-17 | 2023-05-05 | 中国医学科学院药物研究所 | Application of EphB4 as target in screening of insulin sensitivity increasing drugs or models |
| CN115279411B (en) * | 2020-03-17 | 2023-10-20 | 中国医学科学院药物研究所 | Application of EphB4 as target in screening of insulin sensitivity increasing drugs or models |
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