CN105663116B - A kind of pharmaceutical composition for being used to treat neuroblastoma - Google Patents
A kind of pharmaceutical composition for being used to treat neuroblastoma Download PDFInfo
- Publication number
- CN105663116B CN105663116B CN201610240306.3A CN201610240306A CN105663116B CN 105663116 B CN105663116 B CN 105663116B CN 201610240306 A CN201610240306 A CN 201610240306A CN 105663116 B CN105663116 B CN 105663116B
- Authority
- CN
- China
- Prior art keywords
- bez235
- nvp
- oridonin
- mixture
- 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.)
- Expired - Fee Related
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 present invention relates to a kind of pharmaceutical composition for being used to treat neuroblastoma.Present invention is disclosed one kind by Oridonin and NVP BEZ235 use in conjunction, treats the scheme of neuroblastoma；Meanwhile the present invention provides mixture or pharmaceutical composition containing Oridonin Yu NVP BEZ235.
Description
Technical field
The invention belongs to oncology, more particularly it relates to a kind of medicine for being used to treat neuroblastoma
Compositions.
Background technology
Difference lies in it to be apt to occur in children for neuroblastoma (Neuroblastoma, NB) and other most of tumours,
It is initiated by the embryonal tumors of neural crest, is made of undifferentiated sympathetoblast.Its incidence youngster below 15 years old
It is about 1/,100,000 in child, is clinically most common malignant tumour in children within 1 years old.Due in recent years to neuroblastoma
Diagnostic method and treatment means retrofit, the overall survival of neuroblastoma patient improves a lot, but high-risk
The survival rate of infant and quality of life are still without significantly improving.
Since high-risk group of grade malignancy is high, marrow and (or) Bone tumour occur for early stage, and there are minimal residual disease (MRD)
The problems such as, the surgical result of neuroblastoma is extremely limited.At present clinically, CiE (cis-platinum+etoposide) and CDV
(endoxan+daunorubicin+vincristine) alternative chemotherapy scheme is the First-line chemotherapy side of current high-risk group of neuroblastoma
Case, but complete incidence graph (CR) and very well part alleviate (VGPR) and are still below 40%.
Also, classic chemotherapy scheme is to the therapeutic effect of neuroblastoma and the tolerance degree of patient already close to maximum
Limit, exploration is safer and effectively the Combination chemotherapy based on molecular targeted agents is also the task of top priority.
To sum up, there is an urgent need in the art to seek safer, effective neuroblastoma therapeutic scheme, cured with being lifted
Rate, improves the quality of life of clinical infant.
The content of the invention
It is an object of the invention to provide a kind of medicine for being used to treat neuroblastoma.
In the first aspect of the present invention, there is provided the purposes of the mixture of NVP-BEZ235 and Oridonin, is used to prepare
Treat the pharmaceutical composition of neuroblastoma.
In a preference, in the mixture of the NVP-BEZ235 and Oridonin, NVP-BEZ235 and winter
It is 1 that careless A prime, which is insulted, according to weight ratio:0.1~1:100；Preferably 1:0.2~1:80；More preferably it is 1:0.3~1:60.
In another aspect of this invention, there is provided a kind of mixture of NVP-BEZ235 and Oridonin, in the mixture
NVP-BEZ235 is 1 according to weight ratio with Oridonin:0.1~1:100；Preferably 1:0.2~1:80；More preferably it is 1:
0.3~1:60.
In another preference, in the mixture, NVP-BEZ235 is 1 according to weight ratio with Oridonin:
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..
In another aspect of this invention, there is provided a kind of pharmaceutical composition for being used to treat neuroblastoma, the medicine
Compositions include：The mixture of NVP-BEZ235 and Oridonin；And pharmaceutically acceptable carrier.
In a preference, in the mixture of the NVP-BEZ235 and Oridonin, NVP-BEZ235 and winter
It is 1 that careless A prime, which is insulted, according to weight ratio:0.1~1:100；Preferably 1:0.2~1:80；More preferably it is 1:0.3~1:60.
In another preference, the formulation of the pharmaceutical composition is：Injection, infusion solution, tablet, capsule, ball
Agent；Preferably injection.
In another aspect of this invention, there is provided it is a kind of to be used to treating the medicine box of neuroblastoma, in the medicine box, contain
Have：NVP-BEZ235；And Oridonin.
In another aspect of this invention, there is provided it is a kind of to be used to treating the medicine box of neuroblastoma, in the medicine box, contain
There is the mixture.
In another aspect of this invention, there is provided it is a kind of to be used to treating the medicine box of neuroblastoma, in the medicine box, contain
There is the pharmaceutical composition.
In a preference, also contain in the medicine box：Operation instructions, illustrate the side for treating neuroblastoma
Method.
The other side of the present invention is apparent to those skilled in the art due to this disclosure
's.
Brief description of the drawings
Fig. 1 .NVP-BEZ235 suppress neuroblast propagation, but not inducing cell apoptosis.
(A, B) CCK-8 detects influences of the various concentrations NVP-BEZ235 to SHSY-5Y and SK-N-MC cell Proliferations；(C、
D) various concentrations NVP-BEZ235 detects MCM2, cleaved Caspase- after handling SHSY-5Y (C) and SK-N-MC (D) cell
3rd, cleaved PARP protein levels situation.Internal reference is used as using GAPDH.
Fig. 2 .NVP-BEZ235 inducing neural blastoma G0/G1 phases block.
(A) various concentrations NVP-BEZ235 handles the fluidic cell of cell cycle after SHSY-5Y and SK-N-MC cells 24h
Detection case；
(B) various concentrations NVP-BEZ235 handles the level of cyclin after SHSY-5Y and SK-N-MC.With GAPDH
As internal reference.
After (C, D) various concentrations NVP-BEZ235 processing SHSY-5Y (C) and SK-N-MC (D) cells, cyclin
The level of cyclinD1 and cyclinE albumen.Internal reference is used as using GAPDH.
Fig. 3 .NVP-BEZ235 more significantly inhibit neuroblastoma propagation with Oridonin (ORI) use in conjunction.
LC3-I/II, P62 protein expression after (A, B) various concentrations NVP-BEZ235 processing SHSY-5Y and SK-N-MC cells
Situation；Internal reference is used as using GAPDH；
(C, D) CCK-8 detection NVP-BEZ235 (500nM) with Oridonin (2 μM) handle alone or in combination SHSY-5Y,
The proliferative conditions of cell after SK-N-MC cells 24h；
It is thin that (E, F) NVP-BEZ235 (500nM) and Oridonin (2 μM) handles SHSY-5Y, SK-N-MC alone or in combination
The situation of LC3-I/II, P62 protein expression after born of the same parents 24h.
Fig. 4 .NVP-BEZ235 and Oridonin (ORI) have cooperative effect.
CCK8 detections are thin after (A, B) various concentrations Oridonin processing SHSY-5Y (A) and SK-N-MC (B) cells 24h
Born of the same parents breed horizontal；
(C, D) various concentrations NVP-BEZ235 (BEZ), Oridonin (Ori) and composition (Co) processing SHSY-5Y
(C) and SK-N-MC (D) cell dose-effect curve；
(E, F) various concentrations NVP-BEZ235 and Oridonin composition Combination index (Combination Index,
CI) distribution map；
Fig. 5 .NVP-BEZ235 and Oridonin use in conjunction inducing neural blastoma Apoptosis.
(A, B) AO/EB detection NVP-BEZ235 (500nM) with Oridonin (2 μM) handle alone or in combination SHSY-5Y,
The situation of Apoptosis after SK-N-MC cells 24h；
It is thin that (C, D) NVP-BEZ235 (500nM) and Oridonin (2 μM) handles SHSY-5Y, SK-N-MC alone or in combination
Cell cleaved Caspase-3 and cleaved PARP protein expression situations after born of the same parents 24h.
Fig. 6 .NVP-BEZ235 significantly inhibit the growth of transplanted tumor in nude mice with Oridonin use in conjunction.
(A) size of each group group tumour；
(B) average quality of each treatment group tumour；
(C) HE of each treatment group tumour is dyed, Ki-67 is expressed and the situation of Apoptosis.
Embodiment
The present inventor widely studies by long-term, it has unexpectedly been found that, Oridonin is combined with NVP-BEZ235
Using, for treatment neuroblastoma there is extremely excellent effect.The present invention is completed based on this.
Oridonin
Oridonin (Oridonin) CAS NO:28957-04-2 is also known as rubescensin, isodonin, is one kind four
Ring diterpene-kind compound.The Oridonin of purifying is in faint yellow acicular crystal, molecular formula C20H28O6.Chemical structural formula is such as
Under：
In the present invention, " Oridonin " can be the compound shown in formula existing for respective pure form (I), or
Purity is more than the compound shown in the formula (I) of 85% (be preferably greater than 90%, such as 95%, 98%, 99%) or contains
There are the extract (such as extract from plant) of 1-90wt%, the more preferably Oridonin of 5-85wt%.
The Oridonin can extract from any plant containing Oridonin.It is preferred that the plant
It is Rabdosia plant Rabdosia rubescens (Rabdosia rubescens).Extracting the method for Oridonin for example can be：Using
Refluxing extraction after ethanol cold soaking；The method of purifying for example can be：Activated carbon ultrasound decoloration, silica gel column chromatography.
In the case where learning its chemical constitution, the Oridonin can also be obtained by way of chemical synthesis.
In addition, had the Oridonin of commercialization in the prior art, therefore it is that those skilled in the art are easy to obtain
.
In the present invention, the pharmaceutically acceptable salt of Oridonin is further included, it also remains with Oridonin
Chemism." pharmaceutically acceptable salt " refers to the salt that Oridonin is generated with inorganic acid or organic acid reaction.
In the present invention, the precursor of Oridonin is further included, " precursor " refers to after being taken with appropriate method,
The precursor of the compound is transformed into a kind of compound of structure formula (I) being metabolized or chemically reacted in patient body, or changes
Learn salt or solution that a compound of structure formula (I) is formed.
The plant extracts of Oridonin can be directly administered；Or the plant extracts of Oridonin can be carried out
Purification and processing, after the Oridonin of respective pure form or the preparation of various forms of Oridonins is made, for being administered.
In the prior art, it is known that Oridonin can suppress Leukemia Cell Proliferation simultaneously by paths such as PI3K and MAPK
Inducing cell apoptosis.However, application effect of the Oridonin in solid tumor be not very good, because Oridonin
Cytotoxicity has serious dose dependent, its required drug concentration of inducing entity apoptosis of tumor compares neoplastic hematologic disorder
The concentration needed is higher by very much, and toxic side effect is serious.
NVP-BEZ235
NVP-BEZ235 (2- methyl -2- [4- [3- methyl -2- oxos -8- (quinoline -3- bases) -2,3- glyoxalidine simultaneously [4,
5-c] quinoline -1- bases] phenyl] propionitrile) it is a kind of dual ATP competitiveness PI3K and mTOR inhibitors.The chemistry of NVP-BEZ235
Structural formula is as follows：
The NVP-BEZ235 can be obtained by way of chemical synthesis.In addition, there is commercialization in the prior art
NVP-BEZ235, therefore it is that those skilled in the art are easily obtained.
In the present invention, the pharmaceutically acceptable salt of NVP-BEZ235 is further included, it also remains with NVP-BEZ235's
Chemism." pharmaceutically acceptable salt " refers to the salt that NVP-BEZ235 is generated with inorganic acid or organic acid reaction.
In the present invention, the precursor of NVP-BEZ235 is further included, " precursor " refers to after being taken with appropriate method,
The precursor of the compound is transformed into a kind of compound of structure formula (II) being metabolized or chemically reacted in patient body, or changes
Learn salt or solution that a compound of structure formula (II) is formed.
The combination purposes of Oridonin and NVP-BEZ235
NVP-BEZ235 can significantly inhibit the propagation of some tumour cells as PI3K/mTOR double inhibitors, but
The inhibitory effects on proliferation of NVP-BEZ235 relies primarily on its significant Cycle Arrest effect, effectively tumour cell can not be induced to wither
Die.Therefore, effect when NVP-BEZ235 is applied during preclinical animal studies is not very good.
To strengthen the anti-tumor effect of NVP-BEZ235, its potential clinical value is given full play to, the present inventor will
It plays the material of the anti-tumor effect strengthened with various native compound compatibilities to filter out to cooperate.By a large amount of
Screening operation, find native compound Oridonin and NVP-BEZ235 Combined Treatment neuroblastomas, not only can be with
SHSY-5Y, SK-N-MC neuroblast apoptosis are significantly induced in vitro, strengthen the antitumor activity of NVP-BEZ235, Er Qie
The growth of neuroblast Xenografts in nude mice, and inducing transplantation apoptosis of tumor can also be significantly inhibited in vivo, and are strengthened
Autophagy activity NVP-BEZ235 joint Oridonin co-induction neuroblastoma apoptosis in play a significant role.
Therefore, the present invention provides Oridonin and NVP-BEZ235 mixture or composition purposes, for making
The pharmaceutical composition of standby treatment neuroblastoma.
In a specific embodiment of the present invention, using neuroblast SHSY-5Y and SK-N-MC as experiment material, with nude mice
For animal model, the internal of NVP-BEZ235 and Oridonin use in conjunction is demonstrated on cellular level and animal level
Outer antitumor activity.
Composition or mixture
The present invention provides a kind of mixture, contain：NVP-BEZ235 and Oridonin are as active component.Preferably
Ground, in the mixture, NVP-BEZ235 is 1 according to weight ratio with Oridonin:0.1~1:100；Preferably 1:
0.2~1:80；More preferably it is 1:0.3~1:60.For example, it is 1 according to weight ratio: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..
The present invention provides a kind of pharmaceutical composition, contain：(a) a effective amount of Oridonin or its is pharmaceutically acceptable
Salt；(b) a effective amount of NVP-BEZ235 or its pharmaceutically acceptable salt；And (c) pharmaceutically acceptable carrier or tax
Shape agent.
In the present invention, term " containing " represents that various composition can be applied in the mixture or composition of the present invention together.
Therefore, term " mainly by ... form " and " consist of " are included in term " containing ".
In the present invention, " pharmaceutically acceptable " component apply to people and/or animal and without excessive bad side reaction (such as
Toxicity, stimulation and allergy) have rational benefit/risk than material.
In the present invention, " pharmaceutically acceptable carrier " is for the Oridonin of the present invention and NVP-BEZ235 to be passed
Give animal or pharmaceutically acceptable solvent, suspending agent or the excipient of people.Carrier can be liquid or solid.
Any conventional dosage form can be made by conventional method for the pharmaceutical composition or mixture of the present invention.Formulation
Can be diversified, as long as the formulation that active ingredient is effectively reached in mammal body can be made all to be possible.
For example it may be selected from：Injection, infusion solution, tablet, capsule, pill.Wherein Oridonin or NVP-BEZ235 may have
In the carrier or dilution of suitable solid or liquid.
The Oridonin of the present invention and the mixture or pharmaceutical composition of NVP-BEZ235 can also be stored in and be suitable for noting
In the disinfector penetrated or instiled.In general, in the pharmaceutical composition of the present invention, Oridonin and NVP-BEZ235 are as work
Property component can account for the 0.01-20% of pharmaceutical composition gross weight, remaining can be pharmaceutically acceptable carrier.
Oridonin used and the effective dose of NVP-BEZ235 can be with administration pattern and disease to be treated
The order of severity and change.However, the mixture that usually Oridonin and NVP-BEZ235 are formed is daily with about 0.01-50mg/
When the dosage of kg the weight of animals is given, gratifying effect can be obtained, is preferably given daily with 1-3 separated dosage,
Or it is administered with sustained release forms.For most of large mammal, daily accumulated dose is about 0.1-30mg, is preferably about
0.5-20mg.Suitable for dosage form for oral administration, comprising intimately mixed about with solid-state or liquid pharmaceutically acceptable carrier
The Oridonin and NVP-BEZ235 of 0.1-200mg.This dosage is adjusted to provide optimal treatment response.For example, by
An urgent demand for the treatment of situation, can be given once daily dosage separated several times, or dosage is reduced pari passu.
In the specific embodiment of the present invention, in the mixture for giving some NVP-BEZ235 and Oridonin, such as
In one embodiment, in the zoopery for mouse, it is proposed that NVP-BEZ235 is according to weight ratio with Oridonin
2:1 dosage regimen.The dosage being scaled from the dosage of mouse suitable for the mankind is that those skilled in the art are easy to
Make, such as can be calculated according to Meeh-Rubner formula：
Meeh-Rubner formula：A=k × (W2/3)/10,000。
A is body surface area in formula, with m2Calculate；W is weight, is calculated with g；K is constant, small with animal species without same
Mouse and rat 9.1, cavy 9.8, rabbit 10.1, cat 9.9, dog 11.2, monkey 11.8, people 10.6.
Using the formula, Oridonin 10mg/kg+NVP-BEZ235 20mg/kg in mouse；Convert as people (50kg)
Can be then Oridonin 0.83mg/kg+NVP-BEZ235 1.67mg/kg.
The Oridonin and NVP-BEZ235 and its mixture or pharmaceutical composition can by it is oral and intravenous,
The approach administration such as intramuscular or subcutaneous.Can also be administered orally.Being adapted to the medicament forms of injection includes：Aseptic aqueous solution divides
Dispersion liquid and aseptic powder (being used for extemporaneous preparation of sterile injection solution or dispersion liquid).In all situations, these forms must be nothing
Bacterium and must be fluid to be easy to syringe discharge fluid.It must be stable under conditions of manufacture and storage, and must be able to
Prevent the pollution effect of microorganism (such as bacterium and fungi).Carrier can be solvent or decentralized medium, wherein containing such as water, alcohol
(such as glycerine, propane diols and liquid polyethylene glycol), their appropriate mixture and vegetable oil.
When necessary, Oridonin and NVP-BEZ235 can also be with other active ingredients or administered in combination.
It is used to treating the medicine box of neuroblastoma present invention also offers a kind of, in the medicine box, contains：Hold
Device 1, and the VP-BEZ235 components being placed in container 1；And container 2 and the Oridonin component that is placed in container 2.
Alternatively, in the medicine box, the mixture containing the Oridonin and NVP-BEZ235, wherein NVP-
The ratio of BEZ235 and Oridonin is for example foregoing.
Alternatively, in the medicine box, containing including the mixture of NVP-BEZ235 and Oridonin, and pharmaceutically may be used
The pharmaceutical composition of the carrier of receiving.
In addition, can also also have the material of some adjuvant drugs in the medicine box, such as injection needle tubing etc..
In addition, can also contain operation instructions in the medicine box, the method for illustrating to treat neuroblastoma.
With reference to specific embodiment, the present invention is further explained.It is to be understood that these embodiments are merely to illustrate the present invention
Rather than limit the scope of the invention.The experimental method of actual conditions is not specified in the following example, usually according to conventional strip
Part such as J. Pehanorm Brookers etc. are write, Molecular Cloning:A Laboratory guide, the third edition, Science Press, the condition described in 2002, or
According to the condition proposed by manufacturer.
Material and method
1st, inhibitory action of the CCK-8 methods detection determinand for cell
It is inoculated in after pancreatin digestion SHSY-5Y, SK-N-MC cell (5 × 10 in 96 orifice plates3Cells/200 μ l/well),
The NVP-BEZ235 of various dose is added after 24h and (or) Oridonin continues to cultivate, and 2h adds 20 μ l's before terminating culture
CCK-8 measures the absorbance in each hole in each hole under 450nm wavelength.
2nd, flow cytomery
After SHSY-5Y, SK-N-MC cell are handled with NVP-BEZ235 respectively, pancreatin digests and prepares cell suspension, adds
Washed 2 times with PBS after the fixed 2h of -20 DEG C of 75% ethanol of precooling, add 500 μ l PI dye liquors (50 μ g/ml, RNase A 100 of PI
μ g/ml), flow cytometer detects after lucifuge 20min.
3rd, the double fluorescent staining detections of AO/EB
It is inoculated in after pancreatin digestion SHSY-5Y, SK-N-MC cell in 24 orifice plates, adds NVP-BEZ235 and (or) the winter insults
After careless A prime culture 24h, PBS is washed 2 times, adds 200 μ l 1:AO (100 μ g/ml), EB (100 μ g/ml) dye liquor of 1 mixing, are incubated
Educate and take pictures under fluorescence microscope after 3min.
4th, SDS-PAGE is detected
Precooling PBS is washed after cell 2 times plus SDS cell pyrolysis liquids extraction total protein of cell, 4 DEG C of 12,000rpm centrifugations are received
Clearly, after BCA methods survey protein concentration, sample-loading buffer is added, takes 40g to carry out SDS- polyacrylamide gels electricity after heat denatured
Swimming, is transferred to pvdf membrane, and 5% skim milk incubation at room temperature 60min adds 4 DEG C of overnight incubations of primary antibody, and TBST is washed 3 times, added
Secondary antibody the incubation at room temperature 1h, TBST of horseradish peroxidase-labeled wash film 3 times, add developer solution, gather image.
5th, internal anti-tumor effect detection
5-6 weeks athymic female nude mice 30 of selection is only tested, and collects 5 × 106A SHSY-5Y cells are in 100 μ l
Cell suspension inoculation nude mice is purchased in PBS, animal protection regulation is observed in all zooperies.Nude mice by subcutaneous is grown after 5-7 days
Size about 100mm3During tumour, 24 nude mices of the tumour without hemorrhagic necrosis are selected to be randomly divided into 4 groups：Control group, NVP-BEZ235
Group, Oridonin group and drug combination group (n=6).Control group 1%DMSO 100ml/day intraperitoneal administrations, continuous 4 weeks；
NVP-BEZ235 group 20mg/kg/day intraperitoneal administrations, continuous 4 weeks；Oridonin 10mg/kg/day intraperitoneal administrations, continuous 4
Week；Two kinds of drug concentrations of drug combination group are also carried out according to above-mentioned dosage.The transplantable tumor line of apsides is measured weekly, is administered the 4th week and is tied
Cervical dislocation puts to death nude mice after beam, takes out subcutaneous transplantation knurl and measures volume mass.
6th, Ki-67 immunohistochemistry and TUNEL experiments
Partial tumors tissue is taken, 10% neutral formalin solution is fixed, conventional to make paraffin section；
(a) conventional H E is dyed, observation tumor histology form；
(b) the conventional dewaxing of section is to water, 0.01M sodium citrate 750W microwave antigen retrievals 2 × 10min, and PBS is washed 3 times,
3% hydrogen peroxide methanol room temperature acts on 10min.PBS is washed 3 times, and 0.05%BSA room temperatures closing 1h, is added dropwise Ki-67 monoclonal antibodies
4 DEG C of overnight incubations, PBS are washed 3 times, add goat anti-rabbit igg-HRP to be incubated at room temperature 1h.PBS is washed 3 times, and DAB colour developings, haematoxylin is redyed often
Rule dehydration, transparent, mounting, observe tumor cell proliferation situation；
(c) the conventional dewaxing of section is to water, 37 DEG C of Proteinase K antigen retrieval 30min, and PBS is washed 3 times, and TUNEL reaction solutions are added dropwise
(the nucleotide cocktail buffer 1 that TdT is connected with fluorescein:9 mixing) 37 DEG C be incubated 1h.Before positive controls dropwise reaction liquid
37 DEG C of pretreatment 10min of 0.1%DNaseI, negative control group PBS replaces TdT liquid, other same.PBS is washed 3 times, 3% peroxide
Change hydrogen methanol room temperature effect 10min.PBS is washed 3 times, 37 DEG C of closing 30min of sheep blood serum.37 DEG C of POD transforming agents are added dropwise after drying to incubate
30min is educated, PBS is washed 3 times, and DAB colour developings, haematoxylin redyes conventional dehydration, transparent, mounting, observes apoptosis of tumor cells situation.
Embodiment 1, NVP-BEZ235 are to the effect and molecular mechanism of neuroblastoma cell
1st, proliferative effects of the CCK-8 methods detection NVP-BEZ235 to neuroblastoma
To inquire into effect and molecule machine of the PI3K/mTOR double inhibitors NVP-BEZ235 to neuroblastoma cell
System, the present inventor first by concentration is respectively 200,500, the NVP-BEZ235 of 1000nM handle Human Neuroblastoma Cell Line
SHSY-5Y and SK-N-MC, processing time are 12h or 24h, and NVP-BEZ235 is detected to neuroblastoma by CCK-8 methods
Proliferative effect.
It turns out that NVP-BEZ235 has the function that Human Neuroblastoma Cell Line significant Inhibit proliferaton, and
In obvious time and dose dependent, as shown in FIG. 1A and 1B.
2nd, inhibitory action of the Western blot detection NVP-BEZ235 to Human Neuroblastoma Cell Line
Further, the present inventor detects NVP-BEZ235 to Human Neuroblastoma Cell Line using Western blot
SHSY-5Y, SK-N-MC breed and the influence of apoptosis index.The apoptosis index of selection includes；MCM2、cleaved Caspase-
3rd, cleaved PARP albumen, observation cell their expression after NVP-BEZ235 handles different time.
As shown in Fig. 1 C, D, SHSY-5Y, SK-N-MC neuroblast proliferative index MCM2 after NVP-BEZ235 is handled
Expression significantly reduce, but compared with untreated cell, Apoptosis marker cleaved Caspase-3 and
Cleaved PARP are without significant change.
The above results illustrate that NVP-BEZ235 is inhibited to the propagation of Human Neuroblastoma Cell Line.
The influence of embodiment 2, NVP-BEZ235 cell cycles
1st, influences of the flow cytometric analysis NVP-BEZ235 to the cell cycle of neuroblastoma cell
Further to inquire into inhibitory effects on proliferation mechanism of the NVP-BEZ235 to Human Neuroblastoma Cell Line, using streaming
Cell art detects the influence of NVP-BEZ235 cell cycles.
As a result as shown in Figure 2 A and 2 B, Human Neuroblastoma Cell Line SHSY-5Y, SK-N-MC is through 500,1000nM
After NVP-BEZ235 effects 24h, S phases and G2/M phase cell numbers are significantly lowered, but do not occur sub-G1 groups.
2nd, the protein level of the neuroblast cyclinD1 and cyclinE of Western blot NVP-BEZ235 processing
Further, in order to which clear and definite NVP-BEZ235 uses the G0/G1 phase retardations of neuroblast, the present inventor
The protein level of the neuroblast cyclinD1 and cyclinE of Western blot NVP-BEZ235 processing.
The results show that NVP-BEZ235 significantly lowers cyclin cyclinD1 and cyclinE.This prompting NVP-
BEZ235 is mainly blocked by the inducing cell G1 phases plays inhibited proliferation to neuroblastoma, such as Fig. 2 C and Fig. 2 D institutes
Show.
Embodiment 3, NVP-BEZ235 and Oridonin have cooperative effect
1st, proliferative effects of the CCK-8 methods detection NVP-BEZ235 to neuroblastoma
Using CCK8 detections cell after various concentrations Oridonin processing SHSY-5Y (A) and SK-N-MC (B) cells 24h
Propagation is horizontal.
As a result as it can be seen that Oridonin has the function that Human Neuroblastoma Cell Line significant Inhibit proliferaton, and
In obvious time and dose dependent, as shown in Figure 4 A and 4 B shown in FIG..
2nd, the cooperative effect of NVP-BEZ235 and Oridonin measures
Detect different formulations NVP-BEZ235 and Oridonin combination to neuroblastoma cell SH-SY-5Y,
The inhibitory action of the propagation of SK-N-MC.NVP-BEZ235 and Oridonin are respectively added to train according to the additive amount listed by table 1
It is thin by CCK-8 kit measurements when processing 24 is small in 96 orifice plates for supporting neuroblastoma cell SH-SY-5Y, SK-N-MC
Born of the same parents' proliferative conditions (value of OD490nm), Combination index (Combination Index, CI) value is calculated according to OD values.According to Chou
With the intermediate value effect principle of Talalay descriptions, CI values, which are less than 1 two kinds of medicines of display, has synergistic effect.The assay method of CI values
See reference document Chou TC, Talalay P.Quantitative analysis of dose-effect
relationships:the combined effects of multiple drugs or enzyme inhibitors.Adv
Enzyme Regul.1984；22:27-55.
Various concentrations NVP-BEZ235 (BEZ), Oridonin and compositions-treated SHSY-5Y and SK-N-MC cell
Dose-effect curve is as depicted in figs. 4 c and 4d.
Various concentrations NVP-BEZ235 and Oridonin composition CI exponential distributions figure such as Fig. 4 E and Fig. 4 F.
The results show that the concentration of NVP-BEZ235 is 200nM in composition, the concentration of Oridonin is in 0.1~20uM
In the range of, the two shows cooperative effect to neuroblastoma cell SH-SY-5Y and the SK-N-MC inhibitory action bred.
Table 1
| NVP-BEZ235(nM) | Oridonin (uM) | CI values | Effect | |
| Composition 1 | 200 | 0.1 | 0.41366 | Collaboration |
| Composition 2 | 200 | 0.5 | 0.39051 | Collaboration |
| Composition 3 | 200 | 1 | 0.38707 | Collaboration |
| Composition 4 | 200 | 2 | 0.37563 | Collaboration |
| Composition 5 | 200 | 4 | 0.37896 | Collaboration |
| Composition 6 | 200 | 8 | 0.3295 | Collaboration |
| Composition 7 | 200 | 10 | 0.27183 | Collaboration |
| Composition 8 | 200 | 20 | 0.20476 | Collaboration |
Table 2
Embodiment 4, NVP-BEZ235 and Oridonin are united and applied in suppression neuroblastoma cell
1st, the neuroblast of Western blot NVP-BEZ235 processing
The present inventor uses the albumen of the neuroblast P62 and LC3-II of Western blot NVP-BEZ235 processing
It is horizontal.
The results show that after various concentrations NVP-BEZ235 processing SHSY-5Y, SK-N-MC cells 12h or 24h, P62 albumen
Level is remarkably decreased, and LC3-II levels are significantly raised, as shown in Figure 3A and Figure 3B, illustrate, NVP-BEZ235 is to neural female
The influence of cell carcinoma cells may be related to autophagy.
2nd, the inhibition that NVP-BEZ235 and Oridonin are applied alone or in combination
The present inventor has further investigated 500nM NVP-BEZ235 and 2 μM of Oridonin (ORI) (molar ratios 1:4,
It is about 1 to switch to weight ratio:3.1, liquid storage is added directly into culture medium) individually and Combined Treatment SHSY-5Y, SK-N-MC is thin
After born of the same parents 12h or 24h, CCK-8 detects the proliferative conditions of neuroblastoma.
It turns out that NVP-BEZ235 and Oridonin Combined Treatment group cell be compared with the mono- medicine groups of NVP-BEZ235,
Cell inhibitory effect is more notable, as shown in figs. 3 c and 3d.
3rd, the neuroblastoma cell of Western blot NVP-BEZ235 processing
The present inventor uses the nerve that Western blot NVP-BEZ235 and Oridonin are handled alone or in combination
The protein level of mother cell P62 and LC3-II.
The results show that NVP-BEZ235 and Oridonin Combined Treatment group cell and NVP-BEZ235 or Oridonin
Single medicine group is compared, and P62 declines degree and LC3-II increase degrees are more notable, as shown in Fig. 3 E and Fig. 3 F.
The promotion cells apoptosis of embodiment 5, NVP-BEZ235 and Oridonin use in conjunction
1st, the double fluorescent staining detections of AO/EB
The present inventor is using the double fluorescent staining detection NVP-BEZ235 of AO/EB and Oridonin use in conjunction to neural female
The effect of cytoma apoptosis.
The results show that SHSY-5Y, SK-N-MC cell, after NVP-BEZ235 handles 24h, compared with control group, cell increases
Reduction is grown, but without obvious apoptosis；Compared with the control, cell Proliferation and apoptosis become Oridonin treatment group cell without obvious
Change；With Oridonin joint group compared with the control, SHSY-5Y, SK-N-MC cell number are substantially reduced and withered NVP-BEZ235
Dying cell substantially increases, as fig. 5 a and fig. 5b.
2nd, immune-blotting method NVP-BEZ235 and Oridonin handle cell after neuroblastoma alone or in combination
The expression of apoptosis-related protein
The present inventor handles nerve alone or in combination by immune-blotting method list medicine NVP-BEZ235 and Oridonin
Cleaved Caspase-3 and cleaved PARP protein levels after mother cell 24h.
The result shows that NVP-BEZ235 and Oridonin Combined Treatment group be compared to single medicine and control group tumour cell,
The expression of Apoptosis marker cleaved Caspase-3 and cleaved PARP dramatically increase (Fig. 5 C, D).
The above results show that NVP-BEZ235 and Oridonin Combined Treatment, realize more excellent suppression tumour
Effect.
The internal anti-tumor effect of embodiment 6, NVP-BEZ235 and Oridonin use in conjunction
1st, SHSY-5Y subcutaneous transplantations knurl nude mice model is tested
To further look at the internal anti-tumor effect of NVP-BEZ235 and Oridonin use in conjunction, the present inventor builds
SHSY-5Y subcutaneous transplantation knurl nude mice models are found, tumor cell inoculation nude mice is after 3 days, and (20mg/kg, is prepared with NVP-BEZ235
In DMSO, during use PBS be diluted to DMSO concentration for 1%) and (or) Oridonin (10mg/kg, is formulated in DMSO,
PBS is diluted to DMSO concentration as 1%) intraperitoneal injection daily during use, totally 28 days.
During terminal, NVP-BEZ235 and Oridonin list medicine treatment group can reduce nude mice model compared with control group
The volume and quality of knurl, but the reduction of Combined Treatment group is more notable, as shown in Figure 6 A and 6 B.
2nd, Ki-67 immunohistochemical experiments
Ki-67 immunohistochemistry shows, the single medicine treatment group of NVP-BEZ235 (20mg/kg), Oridonin (10mg/kg) with
Control group compares ki-67 positive rate no significant differences, and Combined Treatment group is positive compared to single medicine treatment group and control group Ki-67
Cell number significantly reduces (Ki-67 positive cells show that tumour cell enlivens more), as shown in Figure 6 C.
TUNEL experiments detect apoptosis of tumor cells situation, and TUNEL is positive in the results show control group, single medicine treatment group knurl body
Property cell proportion no significant difference, TUNEL positive cells significantly increase that (TUNEL positive cells increase in Combined Treatment group knurl body
Show the cytosis of apoptosis), as shown in Figure 6 C.
To sum up, the research of the present inventor shows, NVP-BEZ235 and Oridonin use in conjunction can be in cells and dynamic
Significant anti-tumor effect is produced to neuroblastoma in thing level, this is female thin for the invalid progressive stage nerve of current chemotherapy
Born of the same parents' knurl patient explores effective combined treatment and provides new direction.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To be made various changes or modifications to the present invention, such equivalent forms equally fall within the model that the application the appended claims are limited
Enclose.
Claims (16)
- The purposes of the mixture of 1.NVP-BEZ235 and Oridonin, is used to prepare the drug regimen for the treatment of neuroblastoma Thing；Wherein NVP-BEZ235 is 1 according to weight ratio with Oridonin:0.2~1:100.
- 2. purposes as claimed in claim 1, it is characterised in that the NVP-BEZ235 and the mixture of Oridonin In, NVP-BEZ235 is 1 according to weight ratio with Oridonin:0.2~1:80.
- 3. purposes as claimed in claim 2, it is characterised in that NVP-BEZ235 is 1 according to weight ratio with Oridonin: 0.3~1:60.
- 4. the mixture of a kind of NVP-BEZ235 and Oridonin, it is characterised in that in the mixture, NVP-BEZ235 and winter It is 1 that careless A prime, which is insulted, according to weight ratio:0.2~1:100.
- 5. mixture as claimed in claim 4, it is characterised in that in the mixture, NVP-BEZ235 is pressed with Oridonin It is 1 according to weight ratio:0.2~1:80.
- 6. mixture as claimed in claim 5, it is characterised in that in the mixture, NVP-BEZ235 is pressed with Oridonin It is 1 according to weight ratio:0.3~1:60.
- 7. a kind of pharmaceutical composition for being used to treat neuroblastoma, it is characterised in that the pharmaceutical composition includes： The mixture of NVP-BEZ235 and Oridonin, wherein NVP-BEZ235 are 1 according to weight ratio with Oridonin:0.2~ 1:100；And pharmaceutically acceptable carrier.
- 8. pharmaceutical composition as claimed in claim 7, it is characterised in that the NVP-BEZ235 and Oridonin it is mixed In compound, NVP-BEZ235 is 1 according to weight ratio with Oridonin:0.2~1:100.
- 9. pharmaceutical composition as claimed in claim 8, it is characterised in that the NVP-BEZ235 and Oridonin In mixture, NVP-BEZ235 is 1 according to weight ratio with Oridonin:0.2~1:80.
- 10. pharmaceutical composition as claimed in claim 9, it is characterised in that the NVP-BEZ235 and Oridonin In mixture, NVP-BEZ235 is 1 according to weight ratio with Oridonin:0.3~1:60.
- 11. the pharmaceutical composition as described in claim 7~10 is any, it is characterised in that the formulation of the pharmaceutical composition It is：Injection, infusion solution, tablet, capsule, pill.
- 12. pharmaceutical composition as claimed in claim 11, it is characterised in that the formulation of the pharmaceutical composition is injection Agent.
- 13. a kind of medicine box for being used to treat neuroblastoma, it is characterised in that in the medicine box, contain：NVP- BEZ235；And Oridonin；The content of wherein NVP-BEZ235 and Oridonin is 1 according to weight ratio:0.2~1: 100。
- 14. a kind of medicine box for being used to treat neuroblastoma, it is characterised in that in the medicine box, contain：Claim 4 The mixture.
- 15. a kind of medicine box for being used to treat neuroblastoma, it is characterised in that in the medicine box, contain：Claim 7- 12 any pharmaceutical compositions.
- 16. the medicine box as described in claim 13-15 is any, it is characterised in that also contain in the medicine box：Operation instruction Book, the method for illustrating to treat neuroblastoma.
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 CN105663116A (en) | 2016-06-15 |
| CN105663116B true 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 |
|---|
| Sensitization to TRAIL-induced apoptosis in human neuroblastoma SK-N-AS cells by NF-jB inhibitors is dependent on reactive oxygen species (ROS);Tom Gatsinzi等;《J Neurooncol》;20110113;第104卷;第459-472页 * |
| Sequential Dosing in Chemosensitization: Targeting the PI3K/Akt/mTOR Pathway in Neuroblastoma;Mike-Andrew Westhoff等;《PLOS ONE》;20131231;第8卷(第12期);e83128第1-14页 * |
| Therapeutic Targeting of Autophagy in Disease: Biology and Pharmacology;Yan Cheng等;《Pharmacol Rev》;20131031;第65卷;第1162-1197页 * |
| 冬凌草甲素抗肿瘤活性及其机制;李翔 等;《细胞生物学杂志》;20091231;第31卷(第3期);第313-318页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105663116A (en) | 2016-06-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104436194A (en) | Anti-cancer composition with synergistic effect | |
| CN104906558B (en) | The pharmaceutical composition containing ulinastatin for the treatment of cervical cancer | |
| CN107550900A (en) | Application of the Oridonin in terms of protein kinase B inhibitor is prepared | |
| CN104758292B (en) | PD-0332991 is preparing the purposes of prevention drug-resistant tumor drug | |
| CN106265722A (en) | The application in prevention and control of cancer of the ozone carburetion | |
| CN106377496A (en) | Anti-cancer transdermic absorption preparation | |
| CN110123809A (en) | 5- methyl-dihydro benzofuran-application of the imidazole salt compound in pharmacy | |
| CN101125140B (en) | Application of dihydroartemisinin in reinforcing chemotherapy medicine antitumor curative effect | |
| CN101167787B (en) | Albizzia plant extraction composition for inhibiting angiogenesis and preparation and application thereof | |
| CN107569485A (en) | A kind of compound preparation for treating BRAF inhibitor drug-resistant type melanomas | |
| CN103393638A (en) | Application of formononetin in preparing medicine for treating breast cancer | |
| CN103251585A (en) | Effects and application of artemisinin and its derivative in inhibition of platelet-derived growth factor receptor A | |
| CN109602752B (en) | Triptolide is in induction cancer cell autophagy and hdac inhibitor is cooperateed with to treat the application in tumour | |
| CN108125974A (en) | Application of the tilianin and combinations thereof in anti-angiogenic medicaments are prepared | |
| CN105663116B (en) | A kind of pharmaceutical composition for being used to treat neuroblastoma | |
| CN105534966A (en) | Application of gamma-aminobutyric acid as active ingredient for enhancing chemotherapeutic sensitivity | |
| CN105251023A (en) | Application of AXL/c-Met inhibitor in preparation of drug for reversing renal cancer sunitinib drug resistance | |
| CN105434432B (en) | N-Hydroxyphthalimide class compound application in preparation of anti-tumor drugs | |
| CN105560224A (en) | Application of salinomycin to preparation of angiogenesis inhibiting medicine | |
| CN108853236A (en) | A kind of Chinese medicine compound prescription application in preparation of anti-tumor drugs | |
| CN110935027B (en) | Pharmaceutical composition of arsenic trioxide and FLT3 inhibitor and application thereof | |
| CN112870193B (en) | Application of melatonin in preparation of medicine for treating HER2 positive breast cancer resistant to targeted medicine | |
| CN102210725B (en) | Application of hypericum japonicum thunb general flavone in preparing medicament for treating hepatic fibrosis | |
| Cao et al. | Rosiglitazone enhances 5-fluorouracil-induced cell growth inhibition in hepatocellular carcinoma cell line Hep3B | |
| CN107670033A (en) | Antineoplastic pharmaceutical compositions and its application |
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 | ||
| 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 |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20180413 Termination date: 20210418 |