CN105663116A - Medicine composition for treating neuroblastoma - Google Patents
Medicine composition for treating neuroblastoma Download PDFInfo
- Publication number
- CN105663116A CN105663116A CN201610240306.3A CN201610240306A CN105663116A CN 105663116 A CN105663116 A CN 105663116A CN 201610240306 A CN201610240306 A CN 201610240306A CN 105663116 A CN105663116 A CN 105663116A
- Authority
- CN
- China
- Prior art keywords
- bez235
- nvp
- rubescensine
- cell
- neuroblastoma
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
-
- 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
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to a medicine composition for treating neuroblastoma. According to the scheme, oridonin and NVP-BEZ235 are applied jointly to treat neuroblastoma; meanwhile, the invention provides a mixture or the medicine composition containing the oridonin and the NVP-BEZ235.
Description
Technical field
The invention belongs to oncology, more particularly it relates to an for the pharmaceutical composition treating neuroblastoma.
Background technology
Neuroblastoma (Neuroblastoma, NB) and other most of tumors are distinctive in that it is apt to occur in child, and it is initiated by the embryonal tumors of neural crest, is made up of undifferentiated sympathetoblast. Its sickness rate was about 1/,100,000 below 15 years old in child, be modal malignant tumor in child within 1 years old clinically. Due to retrofit to the diagnostic method of neuroblastoma and treatment means in recent years, the overall survival of neuroblastoma patient improves a lot, but the survival rate of high-risk children and quality of life are still without significantly improving.
Owing to high-risk group of grade malignancy is high, early stage bone marrow and (or) Bone tumour occurs, and there is the problems such as MRD (MRD), the surgical result of neuroblastoma is extremely limited. At present clinically, CiE (cisplatin+etoposide) and CDV (cyclophosphamide+daunorubicin+vincristine) alternative chemotherapy scheme is the First-line chemotherapy scheme of current high-risk group of neuroblastoma, but complete incidence graph (CR) and very well partial rcsponse (VGPR) are still below 40%.
Further, classic chemotherapy scheme is to the therapeutic effect of neuroblastoma and the tolerance degree of patient already close to greatest extent, and exploring safer and effective Combination chemotherapy based on molecular targeted agents is also the task of top priority.
To sum up, this area is in the urgent need to seeking neuroblastoma therapeutic scheme safer, effective, to promote cure rate, improves the quality of life of clinical infant.
Summary of the invention
It is an object of the invention to provide a kind of medicine for treating neuroblastoma.
In a first aspect of the present invention, it is provided that the purposes of the mixture of NVP-BEZ235 and rubescensine A, for preparing the pharmaceutical composition for the treatment of neuroblastoma.
In a preference, in described NVP-BEZ235 and the mixture of rubescensine A, NVP-BEZ235 and rubescensine A are 1:0.1～1:100 according to weight ratio; It is preferably 1:0.2～1:80; It is more preferably 1:0.3～1:60.
In another aspect of this invention, it is provided that the mixture of a kind of NVP-BEZ235 and rubescensine A, in this mixture, NVP-BEZ235 and rubescensine A are 1:0.1～1:100 according to weight ratio; It is preferably 1:0.2～1:80; It is more preferably 1:0.3～1:60.
In another preference, in described mixture, NVP-BEZ235 and rubescensine A are 1:0.3875～1:77.5 according to weight ratio, 1:0.4～1:50,1:0.5～1:40,1:0.5～1:30,1:0.5～1:20,1:0.5～1:15,1:0.5～1:10 or 1:1～1:10 etc.
In another aspect of this invention, it is provided that a kind of pharmaceutical composition for treating neuroblastoma, described pharmaceutical composition includes: the mixture of NVP-BEZ235 and rubescensine A; And pharmaceutically acceptable carrier.
In a preference, in described NVP-BEZ235 and the mixture of rubescensine A, NVP-BEZ235 and rubescensine A are 1:0.1～1:100 according to weight ratio; It is preferably 1:0.2～1:80; It is more preferably 1:0.3～1:60.
In another preference, the dosage form of described pharmaceutical composition is: injection, infusion solution, tablet, capsule, pill; It is preferably injection.
In another aspect of this invention, it is provided that a kind of medicine box for treating neuroblastoma, in described medicine box, contain: NVP-BEZ235; And rubescensine A.
In another aspect of this invention, it is provided that a kind of medicine box for treating neuroblastoma, in described medicine box, containing described mixture.
In another aspect of this invention, it is provided that a kind of medicine box for treating neuroblastoma, in described medicine box, containing described pharmaceutical composition.
In a preference, possibly together with operation instructions in described medicine box, the method that treatment neuroblastoma is described.
The other side of the present invention, due to this disclosure, is apparent to those skilled in the art.
Accompanying drawing explanation
Fig. 1 .NVP-BEZ235 suppresses neuroblast propagation, but not inducing cell apoptosis.
(A, B) CCK-8 detects the variable concentrations NVP-BEZ235 impact on SHSY-5Y and SK-N-MC cell proliferation; (C, D) variable concentrations NVP-BEZ235 detects MCM2, cleavedCaspase-3, cleavedPARP protein level situation after processing SHSY-5Y (C) and SK-N-MC (D) cell. Using GAPDH as internal reference.
Fig. 2 .NVP-BEZ235 inducing neural blastoma G0/G1 phase blocks.
(A) the FCM analysis situation of cell cycle after variable concentrations NVP-BEZ235 process SHSY-5Y and SK-N-MC cell 24h;
(B) level of cyclin after variable concentrations NVP-BEZ235 process SHSY-5Y and SK-N-MC. Using GAPDH as internal reference.
After (C, D) variable concentrations NVP-BEZ235 processes SHSY-5Y (C) and SK-N-MC (D) cell, the level of cyclin cyclinD1 and cyclinE albumen. Using GAPDH as internal reference.
Fig. 3 .NVP-BEZ235 and rubescensine A (ORI) use in conjunction more significantly inhibit neuroblastoma propagation.
The situation of LC3-I/II, P62 protein expression after (A, B) variable concentrations NVP-BEZ235 process SHSY-5Y and SK-N-MC cell; Using GAPDH as internal reference;
(C, D) CCK-8 detects NVP-BEZ235 (500nM) and Oridonin (2 μMs) and processes the proliferative conditions of cell after SHSY-5Y, SK-N-MC cell 24h alone or in combination;
(E, F) NVP-BEZ235 (500nM) and Oridonin (2 μMs) process the situation of LC3-I/II, P62 protein expression after SHSY-5Y, SK-N-MC cell 24h alone or in combination.
Fig. 4 .NVP-BEZ235 and rubescensine A (ORI) have cooperative effect.
After (A, B) variable concentrations rubescensine A processes SHSY-5Y (A) and SK-N-MC (B) cell 24h, CCK8 detects cell proliferation level;
(C, D) variable concentrations NVP-BEZ235 (BEZ), rubescensine A (Ori) and compositions (Co) process SHSY-5Y (C) and the dose-effect curve of SK-N-MC (D) cell;
(E, F) variable concentrations NVP-BEZ235 and rubescensine A compositions Combination index (CombinationIndex, CI) scattergram;
Fig. 5 .NVP-BEZ235 and rubescensine A use in conjunction inducing neural blastoma apoptosis.
(A, B) AO/EB detects NVP-BEZ235 (500nM) and Oridonin (2 μMs) and processes apoptotic situation after SHSY-5Y, SK-N-MC cell 24h alone or in combination;
(C, D) NVP-BEZ235 (500nM) and Oridonin (2 μMs) process cell cleavedCaspase-3 and cleavedPARP protein expression situation after SHSY-5Y, SK-N-MC cell 24h alone or in combination.
Fig. 6 .NVP-BEZ235 and rubescensine A use in conjunction significantly inhibit the growth of transplanted tumor in nude mice.
(A) size of each group of group tumor;
(B) average quality of each process group tumor;
(C) the HE dyeing of each process group tumor, Ki-67 express and apoptotic situation.
Detailed description of the invention
The present inventor studies through for a long time and widely, it has unexpectedly been found that, by rubescensine A and NVP-BEZ235 use in conjunction, for treatment neuroblastoma, there is extremely excellent effect. The present invention is completed based on this.
Rubescensine A
Rubescensine A (Oridonin) CASNO:28957-04-2 is also called rubescensin, isodonin, is a kind of tetracyclic diterpene compound. The rubescensine A of purification is faint yellow acicular crystal, and molecular formula is C20H28O6. Chemical structural formula is as follows:
In the present invention, described " rubescensine A " can be the compound shown in formula (I) that respective pure form exists, or purity (is preferably greater than 90% more than 85%, such as 95%, 98%, 99%) the compound shown in formula (I), it is also possible to be containing 1-90wt%, the more preferably extract (extract as from plant) of the rubescensine A of 5-85wt%.
Described rubescensine A can extract from any plant containing rubescensine A. It is preferred that described plant is Rabdosia plant winter LINGCAO (Rabdosiarubescens). Extract rubescensine A method such as may is that employing ethanol merceration after reflux, extract; The method of purification such as may is that the ultrasonic decolouring of activated carbon, silica gel column chromatography.
When learning its chemical constitution, described rubescensine A obtains also by the mode of chemosynthesis.
Additionally, there has been commercial rubescensine A in prior art, therefore it is that those skilled in the art are easily obtained.
In the present invention, also including the pharmaceutically acceptable salt of rubescensine A, it also remains with the chemism of rubescensine A. Described " pharmaceutically acceptable salt " refers to the salt that rubescensine A generates with mineral acid or organic acid reaction.
In the present invention, also include the precursor of rubescensine A, described " precursor " refers to after taking by suitable method, the precursor of this compound carries out metabolism or chemical reaction in patient body and is transformed into a kind of compound of structural formula (I), or the salt that forms of a compound of chemical structural formula (I) or solution.
The plant extract of rubescensine A can be directly administered; Or can be undertaken purifying and processing by the plant extract of rubescensine A, after making the rubescensine A of respective pure form or the preparation of various forms of rubescensine A, be used for being administered.
In prior art, it is known that rubescensine A can pass through the paths such as PI3K and MAPK and suppress Leukemia Cell Proliferation inducing cell apoptosis. But, rubescensine A application effect in solid tumor is not very good, because the cytotoxicity of rubescensine A has serious dose dependent, the drug level required for its inducing entity apoptosis of tumor exceeds a lot than the concentration that neoplastic hematologic disorder needs, and toxic and side effects is serious.
NVP-BEZ235
NVP-BEZ235 (2-methyl-2-[4-[3-methyl-2-oxo-8-(quinoline-3-base)-2,3-glyoxalidine is [4,5-c] quinoline-1-base also] phenyl] propionitrile) it is a kind of dual ATP competitiveness PI3K and mTOR inhibitors.The chemical structural formula of NVP-BEZ235 is as follows:
Described NVP-BEZ235 can be obtained by the mode of chemosynthesis. Additionally, there has been commercial NVP-BEZ235 in prior art, therefore it is that those skilled in the art are easily obtained.
In the present invention, also including the pharmaceutically acceptable salt of NVP-BEZ235, it also remains with the chemism of NVP-BEZ235. Described " pharmaceutically acceptable salt " refers to the NVP-BEZ235 salt generated with mineral acid or organic acid reaction.
In the present invention, also include the precursor of NVP-BEZ235, described " precursor " refers to after taking by suitable method, the precursor of this compound carries out metabolism or chemical reaction in patient body and is transformed into a kind of compound of structural formula (II), or the salt that forms of a compound of chemical structural formula (II) or solution.
The coupling purposes of rubescensine A and NVP-BEZ235
NVP-BEZ235 is as PI3K/mTOR double inhibitor, it is possible to significantly inhibit the propagation of some tumor cells, but the inhibitory effects on proliferation of NVP-BEZ235 relies primarily on its significant Cycle Arrest effect, can not effective inducing apoptosis of tumour cell. Therefore, when NVP-BEZ235 applies, effect during preclinical animal studies is not very good.
For strengthening the Graft Versus Tumor of NVP-BEZ235, giving full play to its potential clinical value, it is played the material of the Graft Versus Tumor strengthened with various native compound compatibilities to filter out to cooperate by the present inventor. Through substantial amounts of screening operation, find native compound rubescensine A and NVP-BEZ235 Combined Treatment neuroblastoma, it is possible not only to significantly induce SHSY-5Y, SK-N-MC neuroblast apoptosis in vitro, strengthen the anti-tumor activity of NVP-BEZ235, and the growth of neuroblast Xenografts in nude mice can also be significantly inhibited in vivo, and inducing transplantation apoptosis of tumor, and play a significant role the autophagy strengthened active associating in the neuroblastoma apoptosis of rubescensine A co-induction at NVP-BEZ235.
Therefore, the invention provides the purposes of the rubescensine A mixture with NVP-BEZ235 or compositions, for preparing the pharmaceutical composition for the treatment of neuroblastoma.
In a particular embodiment of the present invention, with neuroblast SHSY-5Y and SK-N-MC for experiment material, with nude mice for animal model, cellular level and animal level demonstrate the inside and outside anti-tumor activity of NVP-BEZ235 and rubescensine A use in conjunction.
Compositions or mixture
The invention provides a kind of mixture, contain: NVP-BEZ235 and rubescensine A are as active component. It is preferred that in described mixture, NVP-BEZ235 and rubescensine A are 1:0.1～1:100 according to weight ratio; It is preferably 1:0.2～1:80; It is more preferably 1:0.3～1:60. Such as, it is 1:0.3875～1:77.5,1:0.4～1:50,1:0.5～1:40,1:0.5～1:30,1:0.5～1:20,1:0.5～1:15,1:0.5～1:10 or 1:1～1:10 etc. according to weight ratio.
The invention provides a kind of pharmaceutical composition, contain: the rubescensine A of (a) effective dose or its pharmaceutically acceptable salt; The NVP-BEZ235 of (b) effective dose or its pharmaceutically acceptable salt; And (c) pharmaceutically acceptable carrier or excipient.
In the present invention, term " contains " and represents that various composition can be applied in mixture or the compositions of the present invention together. Therefore, term " mainly by ... composition " and " by ... form " be included in term " containing ".
In the present invention, " pharmaceutically acceptable " composition applies to people and/or animal and namely has the material of rational benefit/risk ratio without excessive bad side reaction (such as toxicity, stimulation and allergy).
In the present invention, " pharmaceutically acceptable carrier " is for the rubescensine A of the present invention and NVP-BEZ235 send to the pharmaceutically acceptable solvent of animal or human, suspending agent or excipient. Carrier can be liquid or solid.
The pharmaceutical composition of the present invention or mixture can make the dosage form of any routine by conventional method. Dosage form can be diversified, as long as the dosage form that active component arrives in mammal body effectively can be made all to be possible. Such as it is selected from: injection, infusion solution, tablet, capsule, pill. Wherein rubescensine A or NVP-BEZ235 may reside in carrier or the diluent of suitable solid or liquid.
The rubescensine A of the present invention and the mixture of NVP-BEZ235 or pharmaceutical composition also can be stored in the disinfector being suitable for injection or instillation. Generally, in the pharmaceutical composition of the present invention, rubescensine A and NVP-BEZ235 can account for the 0.01-20% of pharmaceutical composition gross weight as active component, and all the other can be pharmaceutically acceptable carrier.
Rubescensine A used and the effective dose of NVP-BEZ235 can change with the order of severity of the pattern of administration and disease to be treated. But, when usual rubescensine A and NVP-BEZ235 are constituted mixture gives with the dosage of about 0.01-50mg/kg the weight of animals every day, gratifying effect can be obtained, it is preferred that every day gives with 1-3 dosage separately, or with sustained release forms administration. For major part large mammal, the accumulated dose of every day is about 0.1-30mg, it is preferred that be about 0.5-20mg. It is applicable to dosage form for oral administration, comprises the rubescensine A with the intimately mixed about 0.1-200mg of the pharmaceutically acceptable carrier of solid-state or liquid and NVP-BEZ235. This dosage of scalable is to provide optimal treatment response. Such as, by an urgent demand for the treatment of situation, several times dosage separately can be given every day, or dosage is reduced pari passu.
In specific embodiments of the invention, give in the mixture of some NVP-BEZ235 and rubescensine A, for instance in an embodiment, in the zoopery of mice, it is proposed that NVP-BEZ235 and rubescensine A are the dosage regimen of 2:1 according to weight ratio. Being scaled the dosage suitable in the mankind from the dosage of mice is that those skilled in the art are susceptible to, for instance can be calculated according to Meeh-Rubner formula:
Meeh-Rubner formula: A=k × (W2/3)/10,000。
In formula, A is body surface area, with m2Calculate; W is body weight, calculates with g; K is constant, follower kind and different, mice and rat 9.1, Cavia porcellus 9.8, rabbit 10.1, cat 9.9, Canis familiaris L. 11.2, monkey 11.8, people 10.6.
Utilize this formula, rubescensine A 10mg/kg+NVP-BEZ23520mg/kg in mice; Conversion can be then rubescensine A 0.83mg/kg+NVP-BEZ2351.67mg/kg for people's (50kg).
Described rubescensine A and NVP-BEZ235 and mixture or pharmaceutical composition thereof can pass through oral and intravenous, intramuscular or the administration such as subcutaneous. It can also be oral administration. The medicament forms being adapted to injection includes: aseptic aqueous solution or dispersion liquid and aseptic powder (for extemporaneous preparation of sterile injection solution or dispersion liquid).In all situations, these forms must be aseptic and must be that fluid discharges fluid to be prone to syringe. Must be stable under conditions of manufacture and storage, and must be able to prevent the pollution effect of microorganism (such as antibacterial and fungus). Carrier can be solvent or disperse medium, wherein containing, for example water, alcohol (such as glycerol, propylene glycol and liquid polyethylene glycol), their suitable mixture and vegetable oil.
Time necessary, rubescensine A and NVP-BEZ235 also can with other active component or administered in combination.
Present invention also offers a kind of for the medicine box for treating neuroblastoma, in described medicine box, contain: container 1, and be placed in the VP-BEZ235 component in container 1; And container 2 and be placed in the rubescensine A component in container 2.
Or, in described medicine box, containing the mixture of described rubescensine A and NVP-BEZ235, wherein NVP-BEZ235 is such as aforementioned with the ratio of rubescensine A.
Or, in described medicine box, containing the mixture including NVP-BEZ235 and rubescensine A, and the pharmaceutical composition of pharmaceutically acceptable carrier.
Additionally, described medicine box can also also have the material of some adjuvant drugs, for instance injection needle tubing etc.
Additionally, described medicine box also can contain operation instructions, the method that treatment neuroblastoma is described.
Below in conjunction with specific embodiment, the present invention is expanded on further. Should be understood that these embodiments are merely to illustrate the present invention rather than restriction the scope of the present invention. The experimental technique of unreceipted actual conditions in the following example, generally conventionally condition such as J. Pehanorm Brooker etc. are write, Molecular Cloning: A Laboratory guide, the third edition, Science Press, the condition described in 2002, or according to manufacturer it is proposed that condition.
Material and method
1, CCK-8 method detection determinand is for the inhibitory action of cell
It is inoculated in 96 orifice plates after trypsinization SHSY-5Y, SK-N-MC cell (5 × 103Cells/200 μ l/well), the NVP-BEZ235 and (or) the rubescensine A that add various dose after 24h continue to cultivate, and terminate cultivating front 2h and add the CCK-8 of 20 μ l in each hole, measure the absorbance in each hole under 450nm wavelength.
2, flow cytomery
After SHSY-5Y, SK-N-MC cell processes with NVP-BEZ235 respectively, trypsinization also prepares cell suspension, 2 times are washed with PBS after adding the fixing 2h of 75% ethanol-20 DEG C of pre-cooling, add 500 μ lPI dye liquor (PI50 μ g/ml, RNaseA100 μ g/ml), flow cytometer detection after lucifuge 20min.
3, the double; two fluorescence staining detection of AO/EB
It is inoculated in 24 orifice plates after trypsinization SHSY-5Y, SK-N-MC cell, after adding NVP-BEZ235 and (or) rubescensine A cultivation 24h, PBS washes 2 times, add the AO (100 μ g/ml) of 200 μ l1:1 mixing, EB (100 μ g/ml) dye liquor, take pictures under fluorescence microscope after hatching 3min.
4, SDS-PAGE detection
Pre-cooling PBS adds SDS cell pyrolysis liquid and extracts total protein of cell after washing cell 2 times, 4 DEG C 12,000rpm centrifugal receives supernatant, after BCA method surveys protein concentration, add sample-loading buffer, take 40g after heat denatured and carry out SDS-polyacrylamide gel electrophoresis, it is transferred to pvdf membrane, 5% skim milk incubated at room 60min, adds 4 DEG C of overnight incubation of primary antibodie, and TBST washs 3 times, add two anti-incubated at room 1h of horseradish peroxidase-labeled, TBST washes film 3 times, adds developer solution, gathers image.
5, anti-tumor in vivo effect detection
Select 5-6 week athymic female nude mice 30 only to test, collect 5 × 106Individual SHSY-5Y cell purchases cell suspension inoculation nude mice in 100 μ lPBS, and all zooperies are all in accordance with animal protection regulation. After 5-7 days, nude mice by subcutaneous grows size and is about 100mm3During tumor, 24 tumors are selected to be randomly divided into 4 groups without the nude mice of hemorrhagic necrosis: matched group, NVP-BEZ235 group, rubescensine A group and drug combination group (n=6). Matched group 1%DMSO100ml/day intraperitoneal administration, continuous 4 weeks; NVP-BEZ235 group 20mg/kg/day intraperitoneal administration, continuous 4 weeks; Rubescensine A 10mg/kg/day intraperitoneal administration, continuous 4 weeks; Two kinds of drug level of drug combination group also carry out according to above-mentioned dosage. Measure weekly the transplanted tumor line of apsides, be administered and within the 4th week, terminate rear cervical dislocation execution nude mice, take out subcutaneous transplantation tumor and also measure volume mass.
6, Ki-67 SABC and TUNEL test
Taking Partial tumors tissue, 10% neutral formalin solution is fixed, conventional making paraffin section;
A () conventional H E dyes, observe tumor histology's form;
B () section routine dewaxing is to water, 0.01M sodium citrate 750W microwave antigen retrieval 2 × 10min, PBS wash 3 times, 3% hydrogen peroxide methanol room temperature effect 10min. PBS washes 3 times, and 0.05%BSA room temperature closes 1h, drips 4 DEG C of overnight incubation of Ki-67 monoclonal antibody, and PBS washes 3 times, adds goat anti-rabbit igg-HRP incubated at room 1h. PBS washes 3 times, and DAB develops the color, and haematoxylin redyes conventional dehydration, transparent, mounting, observes tumor cell proliferation situation;
C () section routine dewaxing is to water, E.C. 3.4.21.64 37 DEG C of antigen retrieval 30min, PBS wash 3 times, and dropping TUNEL reactant liquor (the nucleotide cocktail buffer 1:9 mixing that TdT is connected with fluorescein) 37 DEG C hatches 1h. Before positive controls dropwise reaction liquid, 0.1%DNaseI37 DEG C of pretreatment 10min, negative control group PBS replace TdT liquid, and other are identical. PBS washes 3 times, 3% hydrogen peroxide methanol room temperature effect 10min. PBS washes 3 times, and sheep blood serum 37 DEG C closes 30min. Dripping POD transforming agent 37 DEG C after drying to hatch 30min, PBS and wash 3 times, DAB develops the color, and haematoxylin redyes conventional dehydration, transparent, mounting, observes apoptosis of tumor cells situation.
Embodiment 1, NVP-BEZ235 are to the effect of neuroblastoma cell and molecular mechanism
1, the CCK-8 method detection NVP-BEZ235 proliferative effect to neuroblastoma
For the discussion PI3K/mTOR double inhibitor NVP-BEZ235 effect to neuroblastoma cell and molecular mechanism, the present inventor first by concentration respectively 200,500, the NVP-BEZ235 of 1000nM process Human Neuroblastoma Cell Line SHSY-5Y and SK-N-MC, the process time is 12h or 24h, detects the NVP-BEZ235 proliferative effect to neuroblastoma by CCK-8 method.
It was found that Human Neuroblastoma Cell Line is had the effect of significant Inhibit proliferaton by NVP-BEZ235, and in obvious time and dose dependent, as shown in FIG. 1A and 1B.
2, the immunoblotting detection NVP-BEZ235 inhibitory action to Human Neuroblastoma Cell Line
Further, the present inventor adopts the immunoblotting detection NVP-BEZ235 impact on Human Neuroblastoma Cell Line SHSY-5Y, SK-N-MC propagation and apoptosis index. The apoptosis index selected includes; MCM2, cleavedCaspase-3, cleavedPARP albumen, observation of cell processes their expression after different time through NVP-BEZ235.
As shown in Fig. 1 C, D, after NVP-BEZ235 processes, the expression of SHSY-5Y, SK-N-MC neuroblast proliferative index MCM2 significantly reduces, but compared with untreated cell, apoptosis mark cleavedCaspase-3 and cleavedPARP is without significant change.
The above results illustrates, NVP-BEZ235 is inhibited to the propagation of Human Neuroblastoma Cell Line.
Embodiment 2, NVP-BEZ235 cell cycle impact
1, the flow cytometric analysis NVP-BEZ235 impact on the cell cycle of neuroblastoma cell
For inquiring into the NVP-BEZ235 inhibitory effects on proliferation mechanism to Human Neuroblastoma Cell Line further, adopt the impact of Flow cytometry NVP-BEZ235 cell cycle.
As shown in Figure 2 A and 2 B, Human Neuroblastoma Cell Line SHSY-5Y, SK-N-MC are through 500, after 1000nMNVP-BEZ235 effect 24h, and S phase and G2/M phase cell number are significantly lowered, but sub-G1 group do not occur for result.
2, the protein level of neuroblast cyclinD1 and the cyclinE that Western blot NVP-BEZ235 processes
Further, for the clear and definite NVP-BEZ235 G0/G1 phase retardation to neuroblast, the present inventor adopts the protein level of Western blot NVP-BEZ235 neuroblast cyclinD1 and the cyclinE processed.
Result shows, NVP-BEZ235 significantly lowers cyclin cyclinD1 and cyclinE. This prompting NVP-BEZ235 blocks neuroblastoma performance inhibited proliferation mainly through the inducing cell G1 phase, as shown in Figure 2 C and 2 D shown in FIG..
Embodiment 3, NVP-BEZ235 and rubescensine A have cooperative effect
1, the CCK-8 method detection NVP-BEZ235 proliferative effect to neuroblastoma
After adopting variable concentrations rubescensine A to process SHSY-5Y (A) and SK-N-MC (B) cell 24h, CCK8 detects cell proliferation level.
Result is visible, and Human Neuroblastoma Cell Line is had the effect of significant Inhibit proliferaton by rubescensine A, and in obvious time and dose dependent, as shown in Figure 4 A and 4 B shown in FIG..
2, the cooperative effect of NVP-BEZ235 and rubescensine A measures
The NVP-BEZ235 of detection different formulations and the rubescensine A combination inhibitory action to the propagation of neuroblastoma cell SH-SY-5Y, SK-N-MC. NVP-BEZ235 and Oridonin is respectively added in 96 orifice plates of cultivation neuroblastoma cell SH-SY-5Y, SK-N-MC by the addition listed by table 1, process 24 hours, by CCK-8 kit measurement cell proliferative conditions (value of OD490nm), Combination index (CombinationIndex, CI) value is calculated according to OD value. According to Chou and the Talalay intermediate value effect principle described, CI value shows that less than 1 two kinds of medicines have synergism. The assay method of CI value sees reference document ChouTC, TalalayP.Quantitativeanalysisofdose-effectrelationships: thecombinedeffectsofmultipledrugsorenzymeinhibitors.AdvE nzymeRegul.1984; 22:27-55.
The dose-effect curve of variable concentrations NVP-BEZ235 (BEZ), rubescensine A and compositions-treated SHSY-5Y and SK-N-MC cell is as depicted in figs. 4 c and 4d.
Variable concentrations NVP-BEZ235 and rubescensine A compositions CI exponential figure such as Fig. 4 E and Fig. 4 F.
Result shows, in compositions, the concentration of NVP-BEZ235 is 200nM, and the concentration of rubescensine A is within the scope of 0.1～20uM, and the inhibitory action of neuroblastoma cell SH-SY-5Y and SK-N-MC propagation is all showed cooperative effect by the two.
Table 1
| NVP-BEZ235(nM) | Rubescensine A (uM) | CI value | Effect | |
| Compositions 1 | 200 | 0.1 | 0.41366 | Collaborative |
| Compositions 2 | 200 | 0.5 | 0.39051 | Collaborative |
| Compositions 3 | 200 | 1 | 0.38707 | Collaborative |
| Compositions 4 | 200 | 2 | 0.37563 | Collaborative |
| Compositions 5 | 200 | 4 | 0.37896 | Collaborative |
| Compositions 6 | 200 | 8 | 0.3295 | Collaborative |
| Compositions 7 | 200 | 10 | 0.27183 | Collaborative |
| Compositions 8 | 200 | 20 | 0.20476 | Collaborative |
Table 2
Embodiment 4, NVP-BEZ235 and rubescensine A are united and applied in suppression neuroblastoma cell
1, the neuroblast that Western blot NVP-BEZ235 processes
The present inventor adopts the protein level of Western blot NVP-BEZ235 neuroblast P62 and the LC3-II processed.
Result shows, after variable concentrations NVP-BEZ235 processes SHSY-5Y, SK-N-MC cell 12h or 24h, P62 protein level is all remarkably decreased, LC3-II level is all significantly raised, as shown in Figure 3 A and Figure 3 B, illustrating, the impact of neuroblastoma cell is likely to relevant to autophagy by NVP-BEZ235.
2, the inhibition that NVP-BEZ235 and rubescensine A are applied alone or in combination
The present inventor has investigated 500nMNVP-BEZ235 and 2 μM rubescensine A (ORI) further, and (mol ratio is 1:4, transfer weight ratio to and be about 1:3.1, liquid storage is added directly in culture medium) individually and after Combined Treatment SHSY-5Y, SK-N-MC cell 12h or 24h, CCK-8 detects the proliferative conditions of neuroblastoma.
It was found that NVP-BEZ235 with rubescensine A Combined Treatment group cell compared with the mono-medicine group of NVP-BEZ235, cell inhibitory effect is more notable, as shown in figs. 3 c and 3d.
3, the neuroblastoma cell that Western blot NVP-BEZ235 processes
The present inventor adopts the protein level of neuroblast P62 and the LC3-II that Western blot NVP-BEZ235 and rubescensine A process alone or in combination.
Result shows, NVP-BEZ235 is with rubescensine A Combined Treatment group cell compared with NVP-BEZ235 or rubescensine A list medicine group, and it is more notable that P62 decline degree and LC3-II increase degree, as shown in Fig. 3 E and Fig. 3 F.
The promotion cells apoptosis of embodiment 5, NVP-BEZ235 and rubescensine A use in conjunction
1, the double; two fluorescence staining detection of AO/EB
The present inventor adopts the double; two fluorescence staining detection NVP-BEZ235 of AO/EB and the effect to neuroblastoma apoptosis of the rubescensine A use in conjunction.
Result shows, SHSY-5Y, SK-N-MC cell processes after 24h through NVP-BEZ235, and compared with matched group, cell proliferation reduces, but without obvious apoptosis; Compared with the control, cell proliferation and apoptosis have no significant change rubescensine A process group cell; NVP-BEZ235 combines group compared with the control with rubescensine A, and SHSY-5Y, SK-N-MC cell number substantially reduces and apoptotic cell substantially increases, as fig. 5 a and fig. 5b.
2, immune-blotting method NVP-BEZ235 and rubescensine A process the expression of cell death related protein after neuroblastoma alone or in combination
The present inventor processes cleavedCaspase-3 and cleavedPARP protein level after neuroblast 24h alone or in combination by immune-blotting method list medicine NVP-BEZ235 and rubescensine A.
It is shown that NVP-BEZ235 and rubescensine A Combined Treatment group are compared to single medicine and matched group tumor cell, the expression of apoptosis mark cleavedCaspase-3 and cleavedPARP dramatically increases (Fig. 5 C, D).
The above results shows, NVP-BEZ235 and rubescensine A Combined Treatment, it is achieved that the effect of more excellent suppression tumor.
The anti-tumor in vivo effect of embodiment 6, NVP-BEZ235 and rubescensine A use in conjunction
1, SHSY-5Y subcutaneous transplantation tumor nude mice model experiment
For further looking at the anti-tumor in vivo effect of NVP-BEZ235 and rubescensine A use in conjunction, the present inventor establishes SHSY-5Y subcutaneous transplantation tumor nude mice model, after tumor cell inoculation nude mice 3 days, with NVP-BEZ235 (20mg/kg, being formulated in DMSO, during use, PBS is diluted to DMSO concentration is 1%) and (or) rubescensine A (10mg/kg, is formulated in DMSO, during use, PBS is diluted to DMSO concentration is 1%) every day lumbar injection, totally 28 days.
During terminal, NVP-BEZ235 with rubescensine A list medicine process group, all can reduce volume and the quality of transplanted tumor in nude mice, but Combined Treatment group reduces more notable, as shown in Figure 6 A and 6 B compared with matched group.
2, Ki-67 immunohistochemical experiment
Ki-67 SABC shows, the single medicine process group ki-67 positive rate no significant difference compared with matched group of NVP-BEZ235 (20mg/kg), rubescensine A (10mg/kg), and Combined Treatment group significantly reduces (Ki-67 positive cell shows that tumor cell enlivens more) compared to single medicine process group and matched group Ki-67 positive cell number, as shown in Figure 6 C.
TUNEL experiment detection apoptosis of tumor cells situation, TUNEL positive cell ratio no significant difference in result display matched group, single medicine process group tumor body, in Combined Treatment group tumor body, TUNEL positive cell significantly increases (TUNEL positive cell increases the cytosis showing apoptosis), as shown in Figure 6 C.
To sum up, the research of the present inventor shows, neuroblastoma can be produced significant Graft Versus Tumor by NVP-BEZ235 and rubescensine A use in conjunction on cell and animal level, and the progressive stage neuroblastoma patient that this chemotherapy being current is invalid explores effective combined treatment and provides new direction.
The all documents mentioned in the present invention are incorporated as reference all in this application, are individually recited as reference such just as each section of document. In addition, it is to be understood that after the above-mentioned teachings having read the present invention, the present invention can be made various changes or modifications by those skilled in the art, these equivalent form of values fall within the application appended claims limited range equally.
Claims (10)
- The purposes of the mixture of 1.NVP-BEZ235 and rubescensine A, for preparing the pharmaceutical composition for the treatment of neuroblastoma.
- 2. purposes as claimed in claim 1, it is characterised in that in described NVP-BEZ235 and the mixture of rubescensine A, NVP-BEZ235 and rubescensine A are 1:0.1～1:100 according to weight ratio; It is preferably 1:0.2～1:80; It is more preferably 1:0.3～1:60.
- 3. the mixture of a NVP-BEZ235 and rubescensine A, it is characterised in that in this mixture, NVP-BEZ235 and rubescensine A are 1:0.1～1:100 according to weight ratio; It is preferably 1:0.2～1:80; It is more preferably 1:0.3～1:60.
- 4. the pharmaceutical composition being used for treating neuroblastoma, it is characterised in that described pharmaceutical composition includes: the mixture of NVP-BEZ235 and rubescensine A; And pharmaceutically acceptable carrier.
- 5. pharmaceutical composition as claimed in claim 4, it is characterised in that in described NVP-BEZ235 and the mixture of rubescensine A, NVP-BEZ235 and rubescensine A are 1:0.1～1:100 according to weight ratio; It is preferably 1:0.2～1:80; It is more preferably 1:0.3～1:60.
- 6. the pharmaceutical composition as described in claim 4 or 5, it is characterised in that the dosage form of described pharmaceutical composition is: injection, infusion solution, tablet, capsule, pill; It is preferably injection.
- 7. the medicine box being used for treating neuroblastoma, it is characterised in that in described medicine box, contain: NVP-BEZ235; And rubescensine A.
- 8. the medicine box being used for treating neuroblastoma, it is characterised in that in described medicine box, contain: the mixture described in claim 3.
- 9. the medicine box being used for treating neuroblastoma, it is characterised in that in described medicine box, contain: the arbitrary described pharmaceutical composition of claim 4-6.
- 10. the medicine box as described in as arbitrary in claim 7-9, it is characterised in that possibly together with operation instructions, the method that treatment neuroblastoma is described in described medicine box.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610240306.3A CN105663116B (en) | 2016-04-18 | 2016-04-18 | A kind of pharmaceutical composition for being used to treat neuroblastoma |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610240306.3A CN105663116B (en) | 2016-04-18 | 2016-04-18 | A kind of pharmaceutical composition for being used to treat neuroblastoma |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105663116A true CN105663116A (en) | 2016-06-15 |
| CN105663116B CN105663116B (en) | 2018-04-13 |
Family
ID=56309843
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610240306.3A Expired - Fee Related CN105663116B (en) | 2016-04-18 | 2016-04-18 | A kind of pharmaceutical composition for being used to treat neuroblastoma |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105663116B (en) |
-
2016
- 2016-04-18 CN CN201610240306.3A patent/CN105663116B/en not_active Expired - Fee Related
Non-Patent Citations (4)
| Title |
|---|
| MIKE-ANDREW WESTHOFF等: "Sequential Dosing in Chemosensitization: Targeting the PI3K/Akt/mTOR Pathway in Neuroblastoma", 《PLOS ONE》 * |
| TOM GATSINZI等: "Sensitization to TRAIL-induced apoptosis in human neuroblastoma SK-N-AS cells by NF-jB inhibitors is dependent on reactive oxygen species (ROS)", 《J NEUROONCOL》 * |
| YAN CHENG等: "Therapeutic Targeting of Autophagy in Disease: Biology and Pharmacology", 《PHARMACOL REV》 * |
| 李翔 等: "冬凌草甲素抗肿瘤活性及其机制", 《细胞生物学杂志》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105663116B (en) | 2018-04-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Bartkowski et al. | Experiments on the efficacy and toxicity of locoregional chemotherapy of liver tumors with 5-fluoro-2′-deoxyuridine (FUDR) and 5-fluorouracil (5-FU) in an animal model | |
| TWI768686B (en) | Combination drug composition for anti-double hit lymphoma and application thereof | |
| CN110123809A (en) | 5- methyl-dihydro benzofuran-application of the imidazole salt compound in pharmacy | |
| CN103393638A (en) | Application of formononetin in preparing medicine for treating breast cancer | |
| CN105726527A (en) | Use and composition of small molecule compound | |
| CN101125140B (en) | Application of dihydroartemisinin in reinforcing chemotherapy medicine antitumor curative effect | |
| CN109602752B (en) | Triptolide is in induction cancer cell autophagy and hdac inhibitor is cooperateed with to treat the application in tumour | |
| CN103251585A (en) | Effects and application of artemisinin and its derivative in inhibition of platelet-derived growth factor receptor A | |
| CN107375258A (en) | A kind of antineoplastic combination medicine and its purposes in cancer therapy drug is prepared | |
| CN109745333A (en) | A kind of pharmaceutical composition and its application for bladder cancer treatment | |
| CN107569485A (en) | Compound preparation for treating drug-resistant melanoma of BRAF inhibitor | |
| CN104138369A (en) | Anticancer drug | |
| CN112870193B (en) | Application of melatonin in preparation of medicine for treating HER2 positive breast cancer resistant to targeted medicine | |
| CN105663116A (en) | Medicine composition for treating neuroblastoma | |
| CN106955292B (en) | A kind of pharmaceutical composition and purposes for treating the cancer of the esophagus | |
| CN108295085A (en) | Application of protodioscin in preparation of drug-resistant osteosarcoma drug | |
| CN103599111B (en) | Combination drug for treating pancreatic cancer | |
| CN107496417A (en) | Epigallo-catechin gallate (EGCG) prepares the application of targeted drug | |
| CN106924225A (en) | Application of the magnolol in antineoplastic sensitizer is prepared | |
| CN116036287A (en) | Application of Gefitinib combined with EGCG and/or EGF in preparation of medicines for treating EGFR wild type tumors | |
| CN116421590B (en) | Application of chlorhexidine diacetate in preparing medicine for preventing or/and treating liver cancer | |
| CN105560224A (en) | Application of salinomycin to preparation of angiogenesis inhibiting medicine | |
| Chen et al. | Experimental evidence of shikonin as a novel intervention for anti-inflammatory effects | |
| CN109260197A (en) | The purposes of indirubin compounds and bortezomib in the drug of preparation treatment Huppert's disease | |
| CN115887455B (en) | Application of azelnidipine serving as calcium channel blocker in preparation of medicines for treating endometrial cancer |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20180308 Address after: 201999 Mohe County Road, Baoshan District, Baoshan District, Shanghai Applicant after: The Affiliated No. 9 People's Hospital of Shanghai Jiaotong University School of Medicine Address before: 201999 Mohe County Road, Baoshan District, Baoshan District, Shanghai Applicant before: The three people Hospital, Shanghai Jiaotong University School of Medicine |
|
| TA01 | Transfer of patent application right | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20180413 Termination date: 20210418 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |