CN108187055A - A kind of anti-cancer composition with synergistic function - Google Patents
A kind of anti-cancer composition with synergistic function Download PDFInfo
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- CN108187055A CN108187055A CN201810182606.XA CN201810182606A CN108187055A CN 108187055 A CN108187055 A CN 108187055A CN 201810182606 A CN201810182606 A CN 201810182606A CN 108187055 A CN108187055 A CN 108187055A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
- A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
- A61K31/404—Indoles, e.g. pindolol
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
Abstract
The present invention relates to a kind of anti-cancer composition with synergistic function, the composition specially including a effective amount of at least one VEGF/VEGFR inhibitor and 2 (structure of aryl piperazines containing N) substituted quinazoline analog derivatives.The composition of the present invention can significantly inhibit the growth of tumour cell, reduce the use of anticarcinogen, reduce Operative risk and cost, have broad application prospects.
Description
Technical field
The present invention relates to a kind of anti-cancer composition with synergistic function, preparation method and the usages.
Background technology
Cancer (Cancer), also known as malignant tumour are caused by the abnormal hyperplasia of cell.The cell of these paraplasms
The other parts of body can be invaded.The conventional method for the treatment of cancer includes operation, radiotherapy, chemotherapeutic immunity therapy, gene at present
Treatment etc..It is sufficiently complex due to influencing tumor development, and drug resistance and tumour are also easy to produce in therapeutic process
Recurrence so that the difficulty bigger of oncotherapy.
Only have sub-fraction tumour cell that there is oncogenicity, i.e. tumor stem cell (Cancer in entire tumor tissues
Stem cell, CSC), the generation of CSC and the self-renewal capacity of cancer cell, classic chemotherapy drug resistance and tumor recurrence are shifted etc.
It is closely bound up.Sutent (SN) is the small molecule TKI of multiple target point, and action target spot includes vascular endothelial growth factor
(Vascular Endothelial Growth Factor, VEGFR), platelet-endothelial source property growth factor (Platelet
Derived growth factor receptor, PDGFR) etc., but SN treatment breast cancer obtains poor clinical effectiveness, SN meetings
Induce the increase of anoxic initiation CSC quantity in knurl.
Important compound in dopamine (Dopamine, DA) catecholamine and phenyl ethylamine family, there is research table in recent years
It is bright, DA can by exciting d1 dopamine receptor (D1Dopamine receptor, D1DR) reduce tumor stem cell ratio from
And increase the drug effect of SN treatment tumours.2 (structure of aryl piperazines containing N-) substituted quinazoline analog derivative C2 and C17 and D1DR have
Stronger compatibility, but and have not seen such compound on tumor effect relevant report.
Invention content
The present inventor has found that 2 (structure of aryl piperazines containing N-) substituted quinazoline classes derive in tumor therapeutic procedure is studied
Object has stronger compatibility with D1DR, and the effect of SN inhibition tumours can be significantly increased with SN combinations.
The present inventor carried out compound and SN and has been combined inhibition to two kinds of pancreatic cancer cell in-vitro multiplications at research initial stage
Effect, index of cooperation is less than 1 after as a result showing the combination of two medicines, and two medical instruments is prompted to play the role of synergistic (see embodiment 1).And
And two medicine combination can significantly reduce the Colony forming ability of tumour cell (referring to embodiment 4).In vivo experiment, people source pancreas
Compound can significantly increase the antitumor action of SN with SN combinations in gland cancer xenograft tumor models, and without apparent drug poison
Property.
Therefore, it is an object of the present invention to provide a kind of anti-cancer composition with synergistic function, including effective
At least one anticancer drug of amount and a kind of a effective amount of non-anticancer drug, the anticancer drug are VEGF/VEGFR inhibitor；
The non-anticancer drug is 2 (structure of aryl piperazines containing N-) substituted quinazoline analog derivatives；2 (piperazines of aryl containing N-
Piperazine structure) substituted quinazoline analog derivative can make using vascular endothelial growth factor VEGF or its receptor as the anticancer drug of target spot
Therapeutic effect generates synergistic function.
According to an aspect of the present invention, the VEGFR inhibitor of the VEGF/VEGFR inhibitor, preferably small molecule
With the monoclonal antibody of VEGF, wherein, the VEGFR inhibitor of small molecule is preferably Sorafenib (Sorafenib, Bayer/
Onyx, 2005), Sutent (Sunitinib, pFizer, 2006), pazopanib (Pazopanib, GSK, 2009), A Xi
For Buddhist nun (Axitinib, pFizer, 2012) and its pharmaceutically one or more of acceptable salt, Buddhist nun of most preferably relaxing replace
Buddhist nun (Sunitinib, SN) and its pharmaceutically acceptable salt.The monoclonal antibody of VEGF is preferably bevacizumab
(Bevacizumab, Genentech, 2004), ranibizumab (Ranibizumab, Genentech, 2006).
The pharmaceutically acceptable salt is to be synthesized by conventional chemical method from parent compound, the parent
Compound includes alkalinity or acid part.In general, the salt is such as by the free acid of these compounds or alkali form
It in organic solvent or reacts in water or in the mixture of water and organic solvent and makes with stoichiometric suitable alkali or acid
Standby.In general, non-aqueous media such as ether, ethyl acetate, ethyl alcohol, isopropanol or acetonitrile are preferred.The example packet of acid-addition salts
Containing inorganic acid addition salt such as hydrochloride, hydrobromate, hydriodate, sulfate, nitrate, phosphate and organic acid addition salt
Such as acetate, trifluoroacetate, maleate, fumarate, citrate, oxalates, succinate, tartrate, apple
Tartaric acid salt, mandelate, analgin and tosilate.The example of base addition salts includes inorganic salts such as sodium, potassium, calcium and ammonium
Salt and organically alkali metal salt such as ethylenediamine, ethanol amine, N, the amino of bis- alkylene ethanol amines of N-, triethanolamine and alkalinity
Hydrochlorate.
According to an aspect of the present invention, the cancer means comprising tumour, knurl is formed and any other malignant tissue
Or cell.Specifically include colon cancer or the carcinoma of the rectum, metastatic colorectal cancer, lung cancer, non-squamous non-small cell lung cancer, mammary gland
Cancer, metastatic breast cancer, metastatic HER2 negative breast cancers, the cancer of the brain, spongioblastoma of being grown up, children's spongioblastoma,
Child-resistance spongioblastoma, glioma, ependymoma, astrocytoma, medulloblastoma, children are into nerve channel
Cytoma, glioma, oligodendroglioma or meningioma, kidney such as advanced renal cell carcinoma, carcinoma of urinary bladder, cervical carcinoma, knot
Intestinal cancer (including colorectal cancer), the cancer of the esophagus, gastric cancer, incidence cancer, liver cancer, lung cancer (Small Cell Lung Cancer and non-small cell lung
Cancer), squamous non-small cell lung cancer, melanoma, myeloma, neuroblastoma, oophoroma, cancer of pancreas, prostate cancer, sarcoma
(including osteosarcoma), cutaneum carcinoma (including squamous cell carcinoma), gastric cancer, carcinoma of testis, thyroid cancer, uterine cancer, celiothelioma, bile duct
Cancer, leiomyosarcoma, embryonal-cell lipoma, nasopharyngeal carcinoma, neuroendocrine carcinoma, oophoroma, salivary-gland carcinoma or carcinoma sarcomatodes.
According to an aspect of the present invention, a effective amount of at least one anticancer drug and a effective amount of non-anticancer drug
It is applied in same time or different time.When same time is applied, each component in the composition is with same or independent
Dosage form uses.In different time in use, identical or different dosage form can be used in each component of the composition.
According to an aspect of the present invention, specification uses in the whole text term " joint " or " combination " or " administering drug combinations " or
" drug combination " refers to the therapeutic agent being included in identical or independent pharmaceutical preparation in same time or different time
It uses.If in different time using therapeutic agent, the use of therapeutic agent should be close enough in time, to make synergy
Effect occur.
It is a further object of the present invention to provide a kind of anticancer preparation, including the anticancer with synergistic function
Composition and customary adjuvant pharmaceutically.The customary adjuvant includes lubricant, filler, surfactant, solubilizer, hydrotropy
Agent etc..
According to an aspect of the present invention, each drug in the anticancer preparation can be separately or cooperatively tablet, capsule
Agent, suspension, solution, injection etc..
The present invention realizes following advantageous effect：
(1) replaced by one or more anticarcinogens (VEGF/VEGFR inhibitor) and 2 (structure of aryl piperazines containing N-)
Quinazoline derivative joint is used for the treatment of certain tumour, can significantly inhibit the growth of tumour cell in vitro, in vivo can be notable
Inhibit gross tumor volume, significant effect is better than anticarcinogen and is applied alone.
(2) drug combination of the invention can greatly reduce making for anticarcinogen under the basis for ensureing identical active anticancer
With reducing Operative risk and toxic side effect under larger dose caused by anticancer drug and (toxic side effect of anticarcinogen be usually applied alone
It is all bigger).
(3) it is thin can to significantly reduce Tumor Stem to 2 (structure of aryl piperazines containing N-) substituted quinazoline analog derivatives of the invention
The ratio of born of the same parents (closely related with drug resistance of tumor) illustrates that the present invention can be used as good solution to solve field of cancer treatment
Make us intractable tumor drug resistance sex chromosome mosaicism.
Description of the drawings
Fig. 1 is the mono- medicine of C2, C17 and SN respectively to the effect of two kinds of Cell Proliferation of Pancreatic Cancer Cell inhibiting effect of SW1990 and PANC-1
Fruit is schemed.A and B is respectively that C2 acts on SW1990 and PANC-1 cell inhibitory effects；C and D be respectively C17 to SW1990 and
PANC-1 cell inhibitory effects act on；E and F is respectively that SN acts on SW1990 and PANC-1 cell inhibitory effects.
Fig. 2 is design sketch of the C2 and SN combinations to human pancreatic cancer cell inhibited proliferation.A and B is respectively that two prescriptions are used
Combination with various concentration is to the inhibiting effect of SW1990 and PANC-1 cell Proliferations；C and D is respectively the combination of two medicine various concentrations
Index of cooperation in SW1990 and PANC-1 cells, index of cooperation, which represents the two less than 1, has synergistic effect.
Fig. 3 is design sketch of the C17 and SN combinations to human pancreatic cancer cell inhibited proliferation.A and B is respectively that two prescriptions are used
Combination with various concentration is to the inhibiting effect of SW1990 and PANC-1 cell Proliferations；C and D is respectively the combination of two medicine various concentrations
Index of cooperation in SW1990 and PANC-1 cells, index of cooperation, which represents the two less than 1, has synergistic effect.
Fig. 4 is design sketch of the C2 and C17 to human pancreatic cancer cell Colony forming ability inhibiting effect.A and B is respectively difference
Concentration C 2 is to the inhibiting effect of SW1990 and PANC-1 cell colony Forming abilities；C and D is respectively C17 pairs of various concentration
The inhibiting effect of SW1990 and PANC-1 cell colony Forming abilities.
Fig. 5 is combined for C2 and SN to be inhibited to make to the external Colony formings of human breast carcinoma cell lines MCF-7/Adr of adriamycin-resistant
Design sketch.
Fig. 6 is the design sketch that C2 and C17 inhibits that tumor stem cell acts in human pancreatic cancer cell PANC-1.
Fig. 7 is that C2 and SN is combined the design sketch acted on Tumor growth inhibition in internal cancer of pancreas Transplanted tumor model.A, it is female
Property nu/nu nude mices give blank control, gemcitabine GEM 15mg/kg, SN 10mg/kg, C2 100mg/kg, SN respectively
The growth curve of gross tumor volume during 10mg/kg+C2 100mg/kg；B, each group tumour are taken pictures result；C, each group mouse weight become
Change；D is administered the 28th day, puts to death animal, mouse is dissected, core dirty, liver, spleen, lungs, kidney are weighed and calculated each dirty
The amount of thinking highly of relative to mouse net weight percentage and organ index；E is administered the 28th day, and mouse orbit adopts whole blood determination blood routine
As a result.
Fig. 8 is that C2 and SN is combined the design sketch acted on Tumor growth inhibition in internal cancer of pancreas people source Transplanted tumor model.
A, NOD/SCID mouse give blank control, SN 10mg/kg, C2 100mg/kg, SN 10mg/kg+C2 100mg/kg respectively
When gross tumor volume growth curve；B, each group tumour are taken pictures result；C, the variation of each group mouse weight；D is administered the 28th day, puts to death
Animal dissects mouse, and core dirty, liver, spleen, lungs, kidney weigh and calculate each organ weights relative to mouse net weight
Percentage and organ index；E is administered the 28th day, and mouse orbit adopts whole blood determination blood routine result.
Specific embodiment
In order to better understand the present invention, current inventor provides following embodiments, however, these embodiments are only
Illustratively purpose and provide, and be not interpreted as limitation of the present invention because many of which variation be it is possible,
Without departing from the spirit and scope of the invention.Although the compositions and methods of the invention are described in particular embodiment,
Person of ordinary skill in the field is it is clear that the step of can make the composition or/and method or the sequence of step occurs
Variation, but without departing from idea of the invention and scope.More specifically, it is clear that it is in chemistry and biologically relevant
The similar drug of other pharmacology processes can replace drug as described herein, while reach the same or similar effect.The skill
The technical staff in art field obviously should be considered in scope of the invention all this similar replacements or modification
In concept.
The present invention has studied VEGFR inhibitor SN and 2 (structure of aryl piperazines containing N-) substituted quinazoline analog derivative connection
With the in-vitro multiplication to tumour cell, Colony forming and the antitumor drug effect to two kinds of pancreatic cancer xenograft knurl models.
Embodiment 1
The inhibiting effect to Cell Proliferation of Pancreatic Cancer Cell is applied alone in C2, C17 and SN
It takes the logarithm growth period cell, with 0.25% pancreatin -0.53mmol/L EDTA solution digestions, centrifugation, is suspended again simultaneously
It counts.SW1990 cells and PANC-1 cells are inoculated in 96 porocyte culture plates by 6000/hole.After culture for 24 hours, carry out
Administration.For SW1990 cells, the mono- medicine administration groups of C2 it is final concentration of：0.1、1、2.5、5、10、15、50、100μM；For
PANC-1 cells, the mono- medicine administration groups of C2 it is final concentration of：0.1、1、2、6、8、10、20、50μM.In two kinds of cell line, C17 is mono-
Medicine administration group it is final concentration of：1、5、10、15、20、25、30、50、100μM.In two kinds of cell line, the mono- medicine administration groups of SN
It is final concentration of：0.1、0.5、1、5、7、10、15、50、100μM.Every group sets 6 parallel holes.Continue to be incubated 48h.After incubation
Culture medium is discarded, 10% trichloroacetic acid (TCA, w/v) solution that 100 μ L are pre-chilled in advance, 4 DEG C of fixed 1h are added in per hole；Discard TCA
Solution, tap water rinse 5 times, naturally dry；100 μ l 0.4% Sulforhodamine B (SRB) dyestuff is added in per hole, is contaminated at room temperature
Color 30min；SRB dyestuffs are discarded, wash 5 times with 1% acetum, naturally dry；It is molten that 200 μ l 10mmol/L Tris are added in per hole
Liquid (pH10.5), shaking 10min dissolves the SRB combined with tumour cell alkaline amino acid residue on oscillator, microplate reader survey
Determine absorbance at 540nm wavelength.The absorbance of blank control group and drug-treated group is expressed as ODcontrol,540And ODsample,540。
Processing is not administered for one of 96 porocyte culture plates, and bed board is handled after for 24 hours with by above-mentioned steps.Its mean light absorbency is set as
OD0h, 540.Cell survival rate is calculated by formula 1.1：
As a result display is (referring to Fig. 1)：IC50s of the C2 in two cell lines of SW1990 and PANC-1 be respectively 4.32 μM and
6.40 μM (Figure 1A, B), IC50s of the C17 in two cell lines of SW1990 and PANC-1 are respectively 12.56 μM and 10.53 μM (figures
1C, D), IC50s of the SN in two cell lines of SW1990 and PANC-1 is respectively 2.41 μM and 8.26 μM (Fig. 1 E, F),
Embodiment 2
The inhibiting effect that C2 and SN combinations are proliferated human pancreatic cancer cell
It takes the logarithm growth period cell, with 0.25% pancreatin -0.53mmol/L EDTA solution digestions, centrifugation, is suspended again simultaneously
It counts.SW1990 cells and PANC-1 cells are inoculated in 96 porocyte culture plates by 6000/hole.After culture for 24 hours, carry out
Administration.For three of SW990 cells 96 parallel orifice plates, the concentration of SN is followed successively by 0,1,5 μm of ol/L, for PANC-1 cells
Four 96 parallel orifice plates, the concentration of SN is followed successively by 0,2,5,10 μm of ol/L, and in every 1 96 orifice plate, and the concentration of C2 is all provided with
0,1,2,4 μm of ol/L is set to, every group sets 6 parallel holes.Continue to be incubated 48h.Extinction of each hole under 540nm is measured with srb assay
Degree.The size of index of cooperation CI (combination index, CI) is calculated using the method for document report.It is counted according to formula 2.1
The theoretical absorbance of administering drug combinations group is calculated, the ratio for surveying absorbance and theoretical value is index of cooperation (formula 2.2).CI values are small
In, equal to and more than 1, respectively represent C2 and SN have collaboration, sum it up and antagonism.
As a result display is (referring to Fig. 2)：For SW1990 cells (Fig. 2A, C) and PANC-1 cells (Fig. 2 B, D), C2 significantly increases
Strong inhibiting effect of the SN to cancer cell, during 2,4 μm of ol/L of C2, index of cooperation (CI) illustrates two medicines less than 1 after two medicines are combined
Combination has synergistic function.
Embodiment 3
The inhibiting effect that C17 and SN combinations are proliferated human pancreatic cancer cell
It takes the logarithm growth period cell, with 0.25% pancreatin -0.53mmol/L EDTA solution digestions, centrifugation, is suspended again simultaneously
It counts.SW1990 cells and PANC-1 cells are inoculated in 96 porocyte culture plates by 6000/hole.After culture for 24 hours, carry out
Administration.For four of two kinds of cells 96 parallel orifice plates, the concentration of C17 is followed successively by 0,1,4,10 μm of ol/L, every 1 96 orifice plate
In, the concentration of SN is disposed as 0,1,2,5 μm of ol/L, and every group sets 6 parallel holes.Continue to be incubated 48h.Each hole is measured with srb assay
Absorbance under 540nm.The big of index of cooperation CI (combination index, CI) is calculated using the method for document report
It is small.The theoretical absorbance of administering drug combinations group is calculated according to formula 3.1, the ratio for surveying absorbance and theoretical value is index of cooperation
(formula 2.2).CI values are less than, equal to and more than 1, represent that C17 and SN has collaboration, adduction and antagonism respectively.
As a result display is (referring to Fig. 3)：For SW1990 cells (Fig. 3 A, C) and PANC-1 cells (Fig. 3 B, D), C17 compared with
Inhibiting effect of the SN to cancer cell is significantly enhanced during high dose, index of cooperation (CI) after the combination of two medicines when C17 is 10 μm of ol/L
Less than 1, illustrate that the combination of two medicines has synergistic function.
Embodiment 4
The effect of C2 and C17 external Colony formings to human pancreatic cancer cell
The influence of C2 and C17 to human pancreatic cancer cell Colony forming ability in vitro has been probed into this implementation.
It takes the logarithm the SW1990 cells in growth period and PANC-1 cells, with 0.25% pancreatin -0.53mmol/L EDTA solution
Digestion, is suspended and counts at centrifugation again.SW1990 cells and PANC-1 cell suspensions are diluted to 2000 respectively with culture medium
A/mL and 1500/mL adds in 1mL culture mediums and 1mL cell suspensions per hole in 6 orifice plates, makes every hole cell be respectively
2000 and 1500.6 orifice plates after inoculation are placed in cell incubator.After culture for 24 hours, it is administered.For SW1990 cells,
The pastille culture medium of a concentration of 0.5 μm of ol/L and 2 μm of ol/L of C2 is prepared, prepares a concentration of 0.5 μm of ol/L and 0.75 μ of C17
The pastille culture medium of mol/L.For PANC-1 cells, the drug containing culture of a concentration of 0.5 μm of ol/L and 1 μm of ol/L of C2 is prepared
Base prepares the pastille culture medium of a concentration of 0.5 μm of ol/L and 1 μm of ol/L of C17.The culture medium of blank control group adds in isometric
DMSO, a concentration of the 0.5% of DMSO in all systems.
After 48h is administered, each hole culture medium is abandoned in suction, is rinsed once with 0.5mL PBS per hole, 2mL fresh cultureds are added in per hole
Base changes the liquid once for every 2 days.After inoculation 10 days, culture medium is abandoned in suction, and every hole addition 0.5mL PBS rinsings are primary, and suction is abandoned after PBS often
Hole adds in 1mL methanol, fixes 10min at room temperature.Methanol is abandoned in suction, and 10min is dyed with 0.5% crystal violet solutions of 1mL.Knot is abandoned in suction
Crystalviolet solution rinses 6 orifice plates to wash away unbonded dyestuff, naturally dry with tap water.Utilize Amersham Imager 600
6 orifice plates are taken pictures, and clone's Colony forming quantity is counted with Image Quant TL 7.0, each group is compared.
As a result display is (referring to Fig. 4 A, B, C, D)：C2 and C17 to human pancreatic cancer cell SW1990 and PANC-1 in vitro
Colony forming ability has a significant impact, and concentration is higher, and the effect for inhibiting soft agar clonogenic assay is stronger.
Embodiment 5
C2 and SN is combined the effect of the external Colony forming to human breast cancer cell
Influence of C2 and the SN combination to human breast cancer cell Colony forming ability in vitro has been probed into this implementation.
Take the logarithm the MCF-7/Adr cells in growth period, with 0.25% pancreatin -0.53mmol/L EDTA solution digestions, from
The heart is suspended and counts again.To MCF-7/Adr cell suspensions be diluted to 1000/mL respectively with culture medium, added in per hole
For 1mL culture mediums with 1mL cell suspensions in 6 orifice plates, it is 1000 to make every hole cell.6 orifice plates after inoculation are placed in cell training
Support case.After culture for 24 hours, it is administered.Administration group setting is as follows：2 μm of ol/L+SN of C2 2 μm of ol/L, SN 0.5 μm of ol/L, C2
0.5μmol/L.The culture medium of blank control group adds in isometric DMSO, a concentration of the 0.5% of DMSO in all systems.
After 48h is administered, each hole culture medium is abandoned in suction, is rinsed once with 0.5mL PBS per hole, 2mL fresh cultureds are added in per hole
Base changes the liquid once for every 2 days.After inoculation 10 days, culture medium is abandoned in suction, and every hole addition 0.5mL PBS rinsings are primary, and suction is abandoned after PBS often
Hole adds in 1mL methanol, fixes 10min at room temperature.Methanol is abandoned in suction, and 10min is dyed with 0.5% crystal violet solutions of 1mL.Knot is abandoned in suction
Crystalviolet solution rinses 6 orifice plates to wash away unbonded dyestuff, naturally dry with tap water.Utilize Amersham Imager600
6 orifice plates are taken pictures, and clone's Colony forming quantity is counted with Image Quant TL 7.0, each group is compared.
As a result display is (referring to Fig. 5)：Colony formings of the 0.5 μm of ol/L of SN to human breast cancer cell line Bcap-37/Adr in vitro
Ability has no significant effect, but 2 μm of ol/L of C2 and SN, 0.5 μm of ol/L combination can significantly reduce the Colony forming energy of tumour cell
Power.
Embodiment 6
Influences of the C2 and C17 to human pancreas cancer stem cell ratio
It takes the logarithm the PANC-1 cells in growth period, with 0.25% pancreatin -0.53mmol/L EDTA solution digestions, centrifugation, again
Newly it is suspended and counts.PANC-1 cell suspensions are diluted to 6 × 10 with culture medium5A/mL, every bottle add in 5mL culture mediums with
1mL cell suspensions are in 25cm2In Tissue Culture Flask, it is 6 × 10 to make every bottle of cell5It is a.Culture bottle after inoculation is placed in cell training
Support case.After culture for 24 hours, it is administered.The setting of C2 and C17 administration groups is as follows：DMSO, C2 2 μm of ol/L, C2 4 μm of ol/L, C17
4 μm of ol/L of 2 μm of ol/L, C17, a concentration of the 1% of DMSO in all systems.
After 48h is administered, it is single cell suspension by cell dissociation, centrifuges 7min under 300 × g, discard supernatant liquid, adds in
Cell is resuspended in PBS that 10mL is pre-chilled in 4 DEG C of refrigerators, and 7min is centrifuged under 300 × g, discards supernatant liquid, add in 1mL balance to
Cell is resuspended in the ALDH buffer solutions of room temperature.With trypan blue staining to viable count.Cell is diluted to 1 with ALDH buffer solutions
×106A/mL.Each sample takes 0.5mL cell suspensions to be denoted as the control tube of the sample respectively in 2 2mL centrifuge tubes respectively
And detection pipe.Add in DEAB (the lignocaine benzaldehydes, for the specific inhibitor of ALDH, for carrying on the back of 5 μ L 1.5mmol/L
The control of scape fluorescence) storing solution is in control tube.The ALDH substrates of 2.5 μ L activation are separately added into control tube and detection pipe,
It is uniformly mixed immediately.Each control tube and detection pipe are placed in 37 DEG C of water-baths and are incubated 45min.5min is centrifuged under 4 DEG C of 300 × g,
Discard supernatant liquid, cell is resuspended in the ALDH buffer solutions for adding in 0.5mL middle precoolings on ice.200 mesh cell sieves are crossed, in fluidic cell
Sample detection on instrument.
As a result display is (referring to Fig. 6)：C2 and C17 can reduce the ratio of tumor stem cell in PANC-1 cells, and concentration is got over
Height, the effect for inhibiting tumor stem cell ratio are stronger.
Embodiment 7
SN and C2 combinations are to the inhibiting effect of tumour growth in internal cancer of pancreas Transplanted tumor model
It takes the logarithm the SW1990 cells in growth period, is prepared by 0.25% pancreatin -0.53mmol/L EDTA solution digestions
Single cell suspension, 1,000rpm centrifugation 5min, discards supernatant, adds in a small amount of 1640 culture medium of serum-free and blow and beat cell again
Mixing simultaneously counts, and cell density serum-free 1640 is adjusted to 1.75 × 10 according to count results7A/mL.In aseptic condition
It is lower by diluted cell suspension inoculation on the right side of the 5 week old female nu/nu nude mices oxter, every injection 0.2mL (about containing 3.5 ×
106A cell), observe inoculation situation.With the major diameter (Dmax) of vernier caliper measurement tumour and minor axis (Dmin), tumour body is calculated
Product V (V=Dmax × Dmin2/2)。
After inoculation, tumour growth to 50~100mm3When mouse is randomly divided into 5 groups, every group 5.Every group of dosage regimen
For：(1) blank control group：Daily gavage 0,2ml 1,2- propylene glycol solutions；(2) GEM groups：Every tail vein injection 0.1mL on the three
Physiological saline containing GEM, GEM dosages are 15mg/kg；(3) SN groups：The 1,2-PD of daily gavage 0.2ml SN is molten
Liquid, dosage 10mg/kg；(4) C2 groups：The 1,2-PD solution of daily gavage 0.2ml C2, dosage are
100mg/kg；(5) administering drug combinations group：The 1,2-PD solution of daily gavage 0.1ml SN and the 1,2-PD of 0.1mL C2
Solution, the dosage of SN is 10mg/kg, and the dosage of C2 is 100mg/kg.Wherein, blank control group is compareed to be stealthy
Group, GEM are gemcitabine, are positive controls.
It is administered the 0th day and starts, every other day measure the tumor size and weight of animal, observe the survival condition of animal, be
It is no adverse reaction occur.After administration 28 days, mouse orbit extraction 20 μ L of whole blood are rapidly added in 2mL dilutions, and slight concussion is mixed
Upper machine measures blood routine after closing uniformly.Animal is put to death, mouse is dissected, tumour is taken out, takes pictures, core after being put according to group
Dirty, liver, spleen, lungs, kidney, are soaked in physiological saline, weigh after being blotted with filter paper, calculate the internal organs of animal internal organs
Index.Calculation formula is：
As a result (Fig. 7) is shown：C2 is applied alone to gross tumor volume there is no significant inhibiting effect, but C2 and SN combinations can be significantly
It reduces tumor size and drug effect is slightly better than positive controls GEM (Fig. 7 A, B), nude mouse weight is without significant changes (figure during administration
7C), main organs are without apparent damage (Fig. 7 D), the apparent hematotoxicity of business (Fig. 7 E).The above result shows that C2 can be notable
Increase the tumor inhibition effect of xenograft tumor that SN is inoculated with human pancreatic cancer cell, C2 and SN is combined good security.
Embodiment 8
SN and C2 combinations are to the inhibiting effect of tumour growth in internal cancer of pancreas people source Transplanted tumor model
By the obtained Pancreatic Adenocarcinoma of excision of performing the operation out of clinical patients body, take out a part be cut into about 2mm ×
The fritter of 2mm × 3mm is injected into the NOD/SCID right side of mice subcutaneous abdomens of about 6 week old with the medullo-puncture needle of sterilizing, and referred to as
A generation source xenograft tumor (Patient-derived xenograft, PDX) model mouse (F1)；To tumour growth to about
500mm3When, it puts to death mouse and takes out tumour rapidly, a part is placed in cryopreservation tube, the FBS containing 10%DMSO is added in, is placed in liquid
It is frozen in nitrogen；Another part tumour is cut into the fritter of 2mm × 2mm × 3mm, is seeded to NOD/SCID mouse skins according to the method described above
Under, referred to as second generation PDX model mouses (F2), and so on.This experiment PDX model mouses used are the third generation (F3).
After third generation inoculation, tumour growth to 80~100mm3When mouse is randomly divided into 7 groups, every group 5.Every group gives
Prescription case is respectively：(1) blank control group：Daily gavage 0,2ml 1,2- propylene glycol solutions；(2) SN groups：Daily gavage 0.2ml
The 1,2-PD solution of SN, dosage 10mg/kg；(3) C2 groups：The 1,2-PD of daily gavage 0.2ml C2 is molten
Liquid, dosage 100mg/kg；(4) administering drug combinations group：The 1,2-PD solution and 0.1mLC2 of daily gavage 0.1ml SN
1,2-PD solution, the dosage of SN is 10mg/kg, and the dosage of C2 is 100mg/kg.
It is administered the 0th day and starts, every other day measure the tumor size and weight of animal, observe the survival condition of animal, be
It is no adverse reaction occur.After administration 28 days, mouse orbit extraction 20 μ L of whole blood are rapidly added in 2mL dilutions, and slight concussion is mixed
Upper machine measures blood routine after closing uniformly.Animal is put to death, mouse is dissected, takes out tumour, peace ancestral takes pictures after putting, dirty, liver of coring
Dirty, spleen, lungs, kidney, are soaked in physiological saline, weigh after being blotted with filter paper, calculate the organ index of animal internal organs.
As a result display is (referring to Fig. 8)：C2 is applied alone to gross tumor volume there is no significant inhibiting effect, but C2 and SN is combined energy
Tumor size (Fig. 8 A, B) is significantly reduced, nude mouse weight is without significant changes (Fig. 8 C) during administration, and main organs are without apparent damage
(Fig. 8 D) and without apparent hematotoxicity (Fig. 8 E).Human tissue is inoculated with the above result shows that C2 can dramatically increase SN
Xenograft tumor tumor inhibition effect, C2 and SN are combined good security.
It discusses：From in vitro results as can be seen that C2, C17 and SN combination can dramatically increase SN to growth of tumour cell
Inhibiting effect, C2, C17 can significantly reduce the Colony forming ability of tumour cell and the ratio of pancreas cancer stem cell.In body
In interior experiment, C2 can dramatically increase the antitumor drug effect of SN.In two kinds of cancer of pancreas Transplanted tumor models, C2 and SN combination can be with
Antitumor drug effect is dramatically increased, and without apparent hematotoxicity and system toxicity, before illustrating that the combination scheme has wide application
Scape.
Claims (10)
1. a kind of anti-cancer composition with synergistic function, which is characterized in that the combination includes a effective amount of at least one
VEGF/VEGFR inhibitor and 2 (structure of aryl piperazines containing N-) substituted quinazoline analog derivatives C2 or C17.
2. the anti-cancer composition according to claim 1 with synergistic function, which is characterized in that the VEGF/
VEGFR inhibitor is VEGFR micromolecular inhibitors or VEGF monoclonal antibodies.
3. the anti-cancer composition according to claim 2 with synergistic function, which is characterized in that the VEGFR
Micromolecular inhibitor is selected from Sutent, pazopanib, Axitinib, Sorafenib and its pharmaceutically in acceptable salt
It is one or more of.
4. the anti-cancer composition according to claim 2 with synergistic function, which is characterized in that the VEGF is mono-
Clonal antibody is selected from one or both of bevacizumab or ranibizumab.
5. the anti-cancer composition according to claim 2 with synergistic function, which is characterized in that the VEGF/
VEGFR inhibitor is Sutent or its pharmaceutically acceptable salt.
6. the anti-cancer composition according to claim 1 with synergistic function, which is characterized in that described 2 (contain
N- aryl piperazines structure) structure of substituted quinazoline analog derivative is：
C2：
C17：
7. the anti-cancer composition according to claim 1 with synergistic function, which is characterized in that the cancer is real
Body cancer, preferably breast cancer, carcinoma of urinary bladder, cervical carcinoma, colon cancer, the cancer of the esophagus, incidence cancer, liver cancer, lung cancer (Small Cell Lung Cancer and
Non-small cell lung cancer), non-squamous non-small cell lung cancer, melanoma, myeloma, neuroblastoma, oophoroma, cancer of pancreas is preceding
Row gland cancer, kidney, advanced renal cell carcinoma, sarcoma (including osteosarcoma), cutaneum carcinoma (including squamous cell carcinoma), gastric cancer, carcinoma of testis,
Thyroid cancer, uterine cancer, celiothelioma, cholangiocellular carcinoma, leiomyosarcoma, embryonal-cell lipoma, nasopharyngeal carcinoma, neuroendocrine carcinoma, ovum
Nest cancer, kidney, salivary-gland carcinoma, Small Cell Lung Cancer or carcinoma sarcomatodes.
8. the anti-cancer composition according to claim 7 with synergistic function, which is characterized in that the cancer is pancreas
Gland cancer and breast cancer.
9. a kind of pharmaceutical preparation with antitumaous effect, the customary adjuvant of anti-cancer composition and pharmacy including claim 1,
The dosage form of the pharmaceutical preparation is the regular dosage form of art of pharmacy, wherein different types of drug is individually packed or common packaging,
Different types of drug is with one dosage type low temperature or different dosage forms.
10. the anti-cancer composition with synergistic function described in any one of claim 1-10 is in anticancer drug is prepared
Application.
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