CN106177959A - Anti-cancer composition and preparation thereof and application - Google Patents
Anti-cancer composition and preparation thereof and application Download PDFInfo
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- CN106177959A CN106177959A CN201610586187.7A CN201610586187A CN106177959A CN 106177959 A CN106177959 A CN 106177959A CN 201610586187 A CN201610586187 A CN 201610586187A CN 106177959 A CN106177959 A CN 106177959A
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
- A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
- A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
Abstract
The invention discloses a kind of anti-cancer composition and preparation thereof and application, belong to preparation and the application of cancer therapy drug.First the present invention discloses a kind of anti-cancer composition, including: the dexamethasone of effective dose and cancer therapy drug in treatment.Compared with being used alone cancer therapy drug, the present invention is used in combination dexamethasone and cancer therapy drug has more preferable tumor inhibitory effect.The present invention further discloses a kind of anticancer preparation, including: acceptable adjuvant on the described anti-cancer composition of effective dose and pharmaceutics in treatment.When present invention discover that anticancer preparation is prepared as nanometer formulation to be applied, targeting and slow releasing function can be passed through, release dexamethasone, in tumor microenvironment, improves tumor microenvironment Related Component, increases the cancer therapy drug accumulation at tumor locus further, significantly inhibit growth and the transfer of tumor, killing tumor cell, strengthen antitumous effect, in-vivo tumour suppression ratio reaches 97%, the toxic and side effects of cancer therapy drug can also be alleviated, reduce treatment cost and the risk of cancer.
Description
Technical field
The present invention relates to anti-cancer composition, particularly relate to that dexamethasone and one or more cancer therapy drugs are made anticancer group
Compound, the invention still further relates to the application in preparing anticancer preparation of the described anti-cancer composition, belongs to the preparation of cancer therapy drug and answers
Use field.
Background technology
Cancer (Cancer) is to affect one of healthy major chronic disease of Chinese residents, it has also become the first of Chinese residents is dead
Cause.Cancer in China Epidemiological study shows, nearly 30 years Cancer in Chinas constitute ratio by 20 century 70s in the cause of the death
10.13% rises to 22.32%, and mortality rate is risen to 135.88/10 ten thousand by 73.99/10 ten thousand.Further, along with aged tendency of population
Etc. the aggravation of factor, morbidity and the mortality rate of tumor are the most in rising trend.Predict according to IARC, if do not taked
Effective measures, the year two thousand twenty is counted in Cancer in China morbidity number and death will rise to 4,000,000 people and 3,000,000 people, and the year two thousand thirty will rise
To 5,000,000 people and 3,500,000 people.
At present, the method for the treatment of malignant tumor mainly includes excision, radiotherapy, chemotherapy and targeted therapy etc..Chemotherapy exists
The treatment initial stage can produce certain curative effect, but prolongation over time, often produce drug resistance, tumor recurrence or transfer,
Cause Endodontic failure.
Dexamethasone (DEX) is the adrenocortical hormone of a kind of synthetic, is again fluorine first dehydrohydro-cortisone, moral
Sha Meisong, fluorine methylprednisolone.Dexamethasone has antiinflammatory, antiallergic, antishock effect, is mainly used in anaphylaxis clinically
And autoimmune disease, such as skin diseases, anaphylaxis, asthma, rheumatism, chronic obstructive pulmonary disease, tuberculosis
And the treatment of other serious infectious disease.In the therapeutic process of malignant tumor, often it is used for subtracting by dexamethasone clinically
The untoward reaction that light chemotherapeutics brings, such as nausea and vomiting etc..
Therefore, develop a kind of anti-cancer composition by dexamethasone Yu Anticancer drug combination, reach to suppress tumor
Growth and anti metastasis, reduce cancer therapy drug toxic and side effects in oncotherapy, reduces treatment cost and the risk of cancer,
To there is wide development space and application prospect.
Summary of the invention
First technical problem to be solved by this invention is to provide a kind of anti-cancer composition, including dexamethasone and at least
A kind of cancer therapy drug；Compared with being used alone cancer therapy drug, this anti-cancer composition has more preferable tumor inhibitory effect and relatively low
Toxic and side effects；
Another technical problem to be solved by this invention is to provide a kind of anticancer system prepared by described anti-cancer composition
Agent, this anticancer preparation can destroy tumor microenvironment, the growth of suppression tumor and transfer, increase medicine tumor locus point
Cloth, not only has more preferable anti-tumor activity, and untoward reaction is significantly reduced.
For solving above-mentioned technical problem, the technical solution used in the present invention is:
First the present invention discloses a kind of anti-cancer composition, including: in treatment the dexamethasone of effective dose and at least one
Cancer therapy drug.
Wherein, described dexamethasone is selected from: pharmaceutically can dexamethasone, dexamethasone salt or dexamethasone ester in
Any one.Preferably, dexamethasone salt of the present invention is dexamethasone sodium phosphate；Described dexamethasone ester is dexamethasone
Any one in acetate or dexamethasone acetas.The multi-form of above-mentioned dexamethasone has different physical chemistry
Matter, can choose according to needs prepared by preparation.Cancer therapy drug of the present invention is selected from: cisplatin, carboplatin, amycin, Ramulus et folium taxi cuspidatae
Alcohol, docetaxel, daunorubicin, doxorubicin, epirubicin, pirarubicin, idarubicin, mitoxantrone, camptothecine, hydroxyl
Base camptothecine, topotecan, irinotecan, vinblastine, vincristine, vindesine, vinorelbine, vinflunine, Changchun are sweet
Ester, teniposide or Sony are for any one or more in Buddhist nun；It is preferably cisplatin, amycin, paclitaxel, docetaxel or length
Any one or more in spring new alkali；Most preferably docetaxel.
In treatment of the present invention, at least one cancer therapy drug of effective dose and the dexamethasone of the upper effective dose for the treatment of, permissible
Identical time or different time are administered.When identical time administration, each component in described compositions is with same or independent system
Agent form uses；When different time is administered, each component of described compositions can use identical or different separate dosage forms.
Cancer of the present invention includes: appointing in primary solid tumor, drug-resistant tumor, metastatic tumo(u)r, malignant tissue or cell
Anticipate one or more；It is further preferred that described cancer includes: nasopharyngeal carcinoma, oral cavity epidermoid carcinoma, carcinoma sarcomatodes, ovarian cancer, pancreas
Adenocarcinoma, carcinoma of prostate, colon cancer (includes colorectal carcinoma), rectal cancer, metastatic colorectal cancer, non-squamous non-small cell lung
Cancer, breast carcinoma, metastatic breast cancer, transitivity HER2 negative breast cancer, the brain cancer, Adult Human Brain glioma, child's brain colloid
Glucagonoma, child-resistance cerebral glioma, glioma, astrocytoma, medulloblastoma, child becomes neurocele thin
Born of the same parents' tumor, glioma, oligodendroglioma, meningioma, renal carcinoma (such as advanced renal cell carcinoma), bladder cancer, cervical cancer, esophagus
Cancer, gastric cancer, incidence cancer, hepatocarcinoma, pulmonary carcinoma (small cell lung cancer and nonsmall-cell lung cancer), squamous nonsmall-cell lung cancer, melanoma,
Myeloma, sarcoma (includes osteosarcoma), and skin carcinoma (includes squamous cell carcinoma), gastric cancer, carcinoma of testis, thyroid carcinoma, uterus carcinoma,
In rind gall, cancer of biliary duct, leiomyosarcoma, liposarcoma, fibrosarcoma, neuroendocrine carcinoma or salivary-gland carcinoma any one or
Multiple.
Anti-cancer composition of the present invention can be applied to prepare anticancer preparation.
The present invention further discloses a kind of anticancer preparation, including: the described anti-cancer composition of effective dose and medicine in treatment
The upper acceptable adjuvant of agent.Wherein, described adjuvant includes: solvent, surfactant, solubilizing agent, cosolvent, lubricant, fill out
Fill any one or more in agent, isoosmotic adjusting agent, stabilizer or proppant.
The dosage form of anticancer preparation of the present invention includes: injection, tablet, capsule, granule, suspensoid, Emulsion, molten
In liquor, sol, lyophilized injectable powder, mucilage, aerosol, microcapsule, microsphere, nanometer formulation, slow releasing preparation or controlled release preparation
Any one or more；It is preferably nanometer formulation.Wherein, described nanometer formulation includes: liposome, nanocapsule, nanosphere, fat
Any one or two kinds in any one or more in matter nanoparticle or micelle, preferably liposome or micelle.The present invention
The particle diameter of described nanometer formulation is 10 nanometer-300 nanometers.The conventional material preparing described nanometer formulation includes: lipid, phospholipid,
Cholesterol, high molecular polymer, bi-block copolymer, triblock polymer, albumen, polypeptide, nucleic acid, chitosan, surface activity
Agent, solubilizing agent, cosolvent, filler, isoosmotic adjusting agent, stabilizer or proppant etc..
The present invention further discloses a kind of docetaxel and be combined liposome with dexamethasone, including following component: many
Xi Tasai, dexamethasone and fat material.According to mass ratio meter, docetaxel: dexamethasone: fat material=10:1-10:100-400, excellent
Elect 10:1-10:200-250 as, more preferably 10:1-10:200.
The invention also discloses a kind of docetaxel and be combined micelle with dexamethasone, including following component: docetaxel,
Dexamethasone and fat material.According to mass ratio meter, docetaxel: dexamethasone: fat material=10:1-10:100-500, preferably 10:
1-10:100-300, more preferably 10:1-10:200.
Fat material of the present invention includes: extract any one or more in phospholipid or synthetic phospholipid.Wherein, described extraction
Phospholipid is selected from any one in soybean phospholipid or lecithin；Described synthetic phospholipid be selected from hydrogenated soybean lecithin, hydrolecithin,
DPPC, DSPC, DMPC, DPPE, DSPE, DMPE, DOPE, DPPG, DSPG, DMPG, or there is the phospholipid of long circulating action
DSPE-PEG2000, DSPE-PEG5000, DSPE-PEG1000, DPPE-PEG2000, DPPE-PEG5000, DPPE-PEG1000, DMPE-
PEG, or the difference in functionality group replacement phospholipid of phospholipid molecule, such as mPEG-DSPE-MAL, mPEG-DSPE-NHS, COOH-PEG-
DSPE or NH2Any one or more in-PEG-DSPE, cholesterol or cholesteryl ester.
As the preferred technical solution of the present invention, described docetaxel and dexamethasone are combined the fat material of liposome by phospholipid
Form with DSPE-PEG, or be made up of with DSPE-PEG phospholipid, cholesterol；More preferably by phospholipid with DSPE-PEG according to matter
Measure and form than 5:1；Or it is made up of according to mass ratio 4:1:1 with DSPE-PEG phospholipid, cholesterol；It is further preferred that it is described
Docetaxel and dexamethasone are combined the fat material of liposome by phospholipid and DSPE-PEG2000Form according to mass ratio 5:1；Or by
Phospholipid, cholesterol and DSPE-PEG2000Form according to mass ratio 4:1:1；Wherein said phospholipid includes but not limited to soybean phospholipid
Or lecithin.
Docetaxel of the present invention is DSPE-PEG, DPPE-PEG or DMPE-with the fat material of dexamethasone combination micelle
The mixture that any one or more in PEG forms according to arbitrary proportion；It is preferably DSPE-PEG2000, DSPE-PEG5000,
DPPE-PEG2000Or DPPE-PEG5000In the mixture that forms according to arbitrary proportion of any one or more.
The present invention further discloses the preparation method of a kind of described docetaxel and dexamethasone combination liposome, bag
Include: any one in membrane-sonic method, reverse phase evaporation, freeze-drying, injection method, extrusion molding or supercritical ultrasonics technology.
Wherein, the step of described membrane-sonic method includes: docetaxel, dexamethasone and fat material are dissolved in organic solvent by (1),
Obtain mixed solution；(2) remove organic solvent with Rotary Evaporators and obtain adipose membrane；(3) normal saline or phosphate-buffered are added
Liquid, vortex demoulding；(4) carry out supersound process with ultrasonic cell disintegration instrument, or further by particle diameter extruder regulation particle diameter after,
Filtration sterilization, to obtain final product.
Wherein, step (1) described organic solvent is the mixed solution of chloroform, methanol, so that fat material is completely dissolved and is advisable, and
And The book of Changes rotation steaming volatilization removes；According to volume basis, chloroform: methanol=1-20:1, preferably 9:1.Step (2) is described to be rotated
Evaporimeter removing organic solvent obtains the rotation steaming bath temperature of adipose membrane and is 20-70 DEG C, preferably 40-50 DEG C；Time is 20-
40min.The time of step (3) described vortex is 3-20min, preferably 5-6min.The ultrasonic power of step (4) described supersound process
For 3%-20%, preferably 20%；Ultrasonic time is 3-10min, preferably 5min；The described filter membrane being filtered into 220nm.
Docetaxel of the present invention can use and docetaxel and ground with the preparation method of dexamethasone combination micelle
The method that Sai meter Song combination liposome is identical, wherein uses membrane-sonic method to prepare docetaxel and is combined glue with dexamethasone
Shu Shi, step (1) described organic solvent can be acetonitrile.Other cancer therapy drugs, including paclitaxel, cisplatin, vincristine, Ah mould
Element etc. also is able to be prepared as joining with dexamethasone according to the preparation method of docetaxel with dexamethasone combination liposome or micelle
Liposome or micelle.
Cell in vitro poison of the present invention test result indicate that, dexamethasone is when low concentration without obvious cytotoxicity, and it is dense
Degree is when 10 more than μ g/ml, and to KB, the cell line such as KBv, PC-3,4T1,3T3, Hela all has certain inhibitory action.With individually
Use the cancer therapy drugs such as docetaxel, paclitaxel or amycin to compare, dexamethasone is used in combination and cancer therapy drug has more preferably
Tumor inhibitory effect, inhibition rate of tumor cell improve about 10-50%, illustrate to have between dexamethasone and cancer therapy drug association
Same potentiation.
The present invention is it has furthermore been found that the anti-cancer composition of dexamethasone and cancer therapy drug composition is applied with nanometer formulation form
Time, targeting and slow releasing function can be passed through, in release dexamethasone to tumor microenvironment, dexamethasone itself can be by anti-
Scorching effect and regulation tumor microenvironment effect, activating immune system, repair tumor microenvironment, weaken the microenvironment protection to tumor
Effect, increases cancer therapy drug in the accumulation of tumor locus, and Synergistic anti-cancer medicine reaches higher suppression tumor growth, antitumor turns
Move and kill the effect of tumor cell, strengthen antitumous effect, Mutiple Targets treatment can be realized for tumor and microenvironment thereof, reach
Preferably drug effect, and toxic and side effects alleviates.
Docetaxel prepared by the present invention and dexamethasone combination liposome or micelle, the envelop rate of dexamethasone is more than
90%, the envelop rate of docetaxel is more than 95%.Extracorporeal releasing experiment shows, it is flat that dexamethasone reached release at about 8 hours
The platform phase, release is very fast；Docetaxel burst size after 36 hours reaches about 40%, is that a kind of procedural drug release is existing
As.Experiment made on the living proves, dexamethasone can increase the cancer therapy drug distribution at tumor locus, reduces the distribution at each internal organs.Separately
Outward, dexamethasone can reduce cytokine VEGF (VEGF) and the TNF-α (tumor necrosis factor) of tumor
Secretion level.
Cell in vitro poison test result indicate that, compared with being used alone docetaxel liposome or dexamethasone liposome,
Docetaxel has more preferable tumor inhibitory effect with dexamethasone combination liposome to KB, KBv, PC-3 or 4T1 cell.And
And, docetaxel and dexamethasone combination liposome the migration of tumor cell is played obvious inhibitory action (tumor cell
Transfer ability represents its ability that transfer occurs to a certain extent), it was demonstrated that docetaxel and dexamethasone combination liposome
Can effectively suppress neoplasm metastasis.Internal effect experiment result shows, docetaxel and dexamethasone combination liposome suppression KB
Preferably (tumor control rate reaches the effect of tumor, KBv drug-resistant tumor, PC-3 height metastatic tumour or the growth of 4T1 height metastatic tumour
83%-97%), not only it is significantly better than and is used alone docetaxel liposome (tumor control rate 66%-94%) or dexamethasone
Liposome (tumor control rate 62%-65%), and it is substantially better than listing preparation taxotere (tumor control rate 60%-90%).
Internal safety evaluatio test result indicate that, for KBv or PC-3 tumor, docetaxel is combined Liposomal delivery with dexamethasone
The nude mice of group loses weight less than listing preparation taxotere, and obvious bone marrow depression does not occurs, and safety is more preferable, and lists
Preparation taxotere has obvious bone marrow depression.
The present invention experiments prove that, dexamethasone can reduce the interstitial fluid pressure of tumor；Dexamethasone liposome, Duo Xi
He can significantly reduce interstitial fluid pressure with dexamethasone combination liposome at match at 4 hours, and docetaxel is combined lipid with dexamethasone
Body still can significantly reduce interstitial fluid pressure at 24 hours.The immunohistochemical analysis of KB tumor tissue in vitro shows, dexamethasone is administered
Group and docetaxel (include NF-κ B, TNF-α, PFGFR-β with dexamethasone combination liposome group to the many indexes of microenvironment
Deng) all there is certain reduction effect, apoptosis is also had certain increase, illustrates that dexamethasone suppression tumor growth is many factors
The result of comprehensive function；Docetaxel administration group also has certain regulation effect to the Multiple components of microenvironment, but it mainly leads to
Cross the apoptosis-induced effect realizing suppressing tumor growth.
To sum up, the nanometer formulation that dexamethasone and cancer therapy drug are made by the present invention can be in the treatment for different targets
Point, increases the preparation distribution at tumor locus, reduces the distribution at each internal organs, destroys tumor microenvironment, suppression cytokine
Secretion, suppresses tumor cell migration, demonstrates fabulous tumor inhibitory effect, and the therapeutic activity of cancer therapy drug is greatly enhanced, far
More than being used alone cancer therapy drug, and toxic and side effects declines.
Anti-cancer composition of the present invention or anticancer preparation can be administered by clinically-acceptable mode, including injection, oral, suction
Enter, implant or perfusion etc..
Technical solution of the present invention compared with prior art, has the advantages that
(1) present invention is by by cancer therapy drug and dexamethasone administering drug combinations, the external life significantly inhibiting tumor cell
Long, the internal increase significantly inhibiting gross tumor volume, its effect is better than alone cancer therapy drug.
(2) compared with some chemotherapeutic preparations of listing, anti-cancer composition of the present invention or anticancer preparation can reduce bad
Reaction, reduces the Operative risk caused by larger dose cancer therapy drug.
(3) anti-cancer composition of the present invention or anticancer preparation not only significantly inhibit effect to general tumor, and to drug resistance
Tumor and high metastatic tumo(u)r also have preferable inhibition, can range wider.
Accompanying drawing explanation
The docetaxel that Fig. 1 is dynamic light scattering (A) and transmission electron microscope (B) obtains is (real with dexamethasone combination liposome
Execute example 8 to prepare) particle diameter and form；
Fig. 2 is the cell in vitro poison experiment of different preparation group；(A) it is that each preparation group is to human mouth epidermoid carcinoma cell (KB)
Cell toxicant, (B) be each preparation group be that each preparation group is to people prostatitis to human mouth epidermoid carcinoma mdr cell (KBv) cell toxicant (C)
Adenocarcinoma cell (PC-3) cell toxicant, (D) is that each preparation group is to Mus source breast cancer cell (4T1) cell toxicant；
Fig. 3 is the different preparation group scratch experiments to PC-3 cell；
Fig. 4 is the release profiles in docetaxel and dexamethasone combination preparation；
Fig. 5 is that the impact of cytokine secretion is tested by dexamethasone；
Fig. 6 is internal (KB tumor) effect experiment of different preparation group, the respectively tumor growth curve of different dosing group；
Fig. 7 is internal (KBv drug-resistant tumor) effect experiment of different preparation group, the respectively tumor growth of different dosing group
Curve；
Fig. 8 is internal (PC-3 height metastatic tumour) effect experiment, the respectively tumor of different dosing group of different preparation group
Growth curve；
Fig. 9 is that the tumor of internal (4T1 height metastatic tumour) effect experiment, respectively different dosing group of different preparation group is raw
Long curve；
Figure 10 is internal (KBv drug-resistant tumor) safety evaluatio of different preparation group；(A) be different dosing group body weight become
Changing curve, (B) is the clean body weight change situation of different dosing group, and (C) is the situation of the blood phase neutrophilic granulocyte of different dosing group；
Figure 11 is internal (PC-3 height metastatic tumour) safety evaluatio of different preparation group；(A) it is the body of different dosing group
Weight change curve, (B) is the clean body weight change situation of different dosing group；
Figure 12 is the distribution of different preparation groups internal (KB tumor)；(A) being the living imaging result of animal, (B) is different
The isolated organ imaging results of administration group, (C) is isolated organ semi-quantitative analysis result；
Figure 13 is the different preparation group impacts on KBv drug-resistant tumor interstitial fluid pressure；
Figure 14 be different preparation group internal (KB tumor) tumor microenvironment in the immunohistochemical analysis of NF-κ B；
Figure 15 be different preparation group internal (KB tumor) tumor microenvironment in the immunohistochemical analysis of TNF-α；
Figure 16 be different preparation group internal (KB tumor) tumor microenvironment in the immunohistochemical analysis of PFGFR-β；
Figure 17 be different preparation group internal (KB tumor) tumor microenvironment in the immunohistochemical analysis of α-SMA；
Figure 18 be different preparation group internal (KB tumor) tumor microenvironment in the immunohistochemical analysis of CD-31；
Figure 19 be different preparation group internal (KB tumor) tumor microenvironment in the immunohistochemical analysis of college-1；
Figure 20 is internal (KB tumor) tumor death TUNEL immunohistochemical analysis of different preparation group；
Note: in Fig. 2-20, Control is matched group, Free DEX is dexamethasone, and DEX-Lip is dexamethasone lipid
Body, Taxotere is listing preparation taxotere, and DTX-Lip is docetaxel liposome, (DTX+DEX)-Lip be docetaxel with
Dexamethasone combination liposome.
Detailed description of the invention
Below in conjunction with specific embodiment further describe the present invention, advantages of the present invention and feature will be with describe and
Apparent.It should be understood that described embodiment is only exemplary, the scope of the present invention is not constituted any restriction.This area
Skilled artisans appreciated that, lower without departing from the spirit and scope of the present invention can to the details of technical solution of the present invention and
Form is modified or replaces, but these amendments or replacement each fall within protection scope of the present invention.
Cell in vitro poison experiment associated with embodiment 1 docetaxel and dexamethasone
Using complete medium to cultivate KB (purchased from country's nanometer centers), KBv (is purchased from country's nanometer centers), PC-3 (in
Academy of Medical Sciences Institute of Basic Medical Sciences of state), 4T1 (U.S. ATCC), 3T3 (Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences),
Hela (Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences), MDA-MB-231 (Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences),
B16-F10 cell (Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences), when cell grows to logarithmic (log) phase, with 96 orifice plate every hole inoculations
180 μ l, about 5000 cells；Incubator is cultivated and within 24 hours, is made cell attachment, every kind of cell give dexamethasone respectively, many west he
Match, and give dexamethasone and docetaxel simultaneously；Dexamethasone administration group final concentration of 0.1,1,5,10,20,50,
80、100μg/ml；The administration concentration of docetaxel is 5 μ g/ml；Give dexamethasone and the administration concentration of docetaxel group simultaneously
Consistent with individually dosed concentration；Incubator is cultivated 48 hours, discards culture medium.Add the cultivation completely containing 5%CCK-8 solution
Base 100 μ l；The each group of light absorption value at 450nm is detected by microplate reader after hatching 2h.
From cytotoxic result, dexamethasone when low concentration without obvious cytotoxicity, concentration 10 μ g/ml with
Time upper, above-mentioned cell line all there is certain inhibitory action；Compared with alone with docetaxel, give dexamethasone and Duo Xi simultaneously
He improves about 10-40% by match group cell inhibitory rate, has more preferable tumor inhibitory effect.
Cell in vitro poison experiment associated with embodiment 2 docetaxel and dexamethasone
Using complete medium to cultivate KB, KBv, PC-3,4T1 cell, when cell grows to logarithmic (log) phase, with the 96 every holes of orifice plate
Inoculate 180 μ l, about 5000 cells；Incubator is cultivated and within 24 hours, is made cell attachment, every kind of cell give dexamethasone, Duo Xi respectively
Docetaxel is given again after he match, dexamethasone process 1h；Dexamethasone administration group final concentration of 0.1,1,5,10,20,50,
80,100 μ g/ml, docetaxel administration concentration is 5 μ g/ml, and incubator is cultivated 48 hours, discards culture medium.Add containing 5%
The complete medium 100 μ l of CCK-8 solution；The each group of light absorption value at 450nm is detected by microplate reader after hatching 2h.
From cytotoxic result, above-mentioned cell line, when 10 more than μ g/ml, is all had certain by docetaxel concentration
Inhibitory action；Compared with alone with docetaxel, order gives dexamethasone and improves 12-with docetaxel group Carbazole alkaloid effect
About 45%, there is more preferable tumor inhibitory effect.
Cell in vitro poison experiment associated with embodiment 3 paclitaxel and dexamethasone
Specific experiment method, with embodiment 1, substitutes docetaxel with paclitaxel.
From cytotoxic result, above-mentioned cell line, when 10 more than μ g/ml, is all had certain by dexamethasone concentration
Inhibitory action；Compared with alone with paclitaxel, give dexamethasone simultaneously and paclitaxel group Carbazole alkaloid effect improves a 10-40% left side
The right side, has more preferable tumor inhibitory effect.
Cell in vitro poison experiment associated with embodiment 4 paclitaxel and dexamethasone
Specific experiment method, with embodiment 2, substitutes docetaxel with paclitaxel.
From cytotoxic result, above-mentioned cell line, when 10 more than μ g/ml, is all had certain by dexamethasone concentration
Inhibitory action；Compared with alone with paclitaxel, order gives dexamethasone and improves a 12-42% left side with paclitaxel group Carbazole alkaloid effect
The right side, has more preferable tumor inhibitory effect.
Cell in vitro poison experiment associated with embodiment 5 amycin and dexamethasone
Complete medium is used to cultivate MCF-7 (Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences), A549 (in country's nanometer
The heart), KB, HT-1080 (Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) cell, when cell grows to logarithmic (log) phase, use 96 orifice plates
180 μ l, about 5000 cells are inoculated in every hole；Incubator is cultivated and within 24 hours, is made cell attachment, every kind of cell give dexamethasone respectively,
Amycin, gives amycin and dexamethasone simultaneously；Dexamethasone administration group final concentration of 0.1,1,5,10,20,50,80,
100 μ g/ml, doxorubicin concentration is 1 μ g/ml, gives the administration concentration of amycin and Dexamethasone group with individually dosed simultaneously
Concentration is consistent, and incubator is cultivated 48 hours, discards culture medium.Use mtt assay detection cytotoxicity.
From cytotoxic result, above-mentioned cell line, when 8 more than μ g/ml, is all had certain by dexamethasone concentration
Inhibitory action；Compared with alone with amycin, administering drug combinations group Carbazole alkaloid effect improves about 15-45%, has the most swollen
Tumor inhibition.
Cell in vitro poison experiment associated with embodiment 6 cisplatin and dexamethasone
Use complete medium to cultivate MCF-7, A549, KB, HT-1080 cell, when cell grows to logarithmic (log) phase, use 96 holes
180 μ l, about 5000 cells are inoculated in the every hole of plate；Incubator is cultivated and is made cell attachment in 24 hours, gives dexamethasone respectively, cisplatin,
Give cisplatin and dexamethasone simultaneously；Final concentration of 0.1,1,5,10,20,50,80, the 100 μ g/ml of dexamethasone administration group,
Cisplatin administration dosage is 20 μ g/ml, gives cisplatin consistent with individually dosed concentration with the administration concentration of Dexamethasone group simultaneously,
Incubator is cultivated 48 hours, discards culture medium.Use mtt assay detection cytotoxicity.
From cytotoxic result, above-mentioned cell line, when 10 more than μ g/ml, is all had certain by dexamethasone concentration
Inhibitory action；Compared with alone with cisplatin, cisplatin and dexamethasone administering drug combinations group Carbazole alkaloid effect improve about 10-50%,
There is more preferable tumor inhibitory effect.
Cell in vitro poison experiment associated with embodiment 7 vincristine and dexamethasone
Use complete medium to cultivate MCF-7, A549, KB, HT-1080 cell, when cell grows to logarithmic (log) phase, use 96 holes
200 μ l, about 5000 cells are inoculated in the every hole of plate；Incubator is cultivated and is made cell attachment in 24 hours, gives dexamethasone respectively, and Changchun is new
Alkali, gives vincristine and dexamethasone simultaneously；Final concentration of 0.1,1,5,10,20,50,80, the 100 of dexamethasone administration group
μ g/ml, vincristine administration concentration is 4 μ g/ml, give simultaneously the administration concentration of vincristine and Dexamethasone group with individually to
The concentration of medicine is consistent, and incubator is cultivated 48 hours, discards culture medium.Use mtt assay detection cytotoxicity.
From cytotoxic result, above-mentioned cell line, when 10 more than μ g/ml, is all had certain by dexamethasone concentration
Inhibitory action；Compared with alone with vincristine, vincristine and dexamethasone administering drug combinations group Carbazole alkaloid effect improve 10-
About 35%, there is more preferable tumor inhibitory effect.
Embodiment 8 docetaxel is combined the preparation of liposome with dexamethasone
50mg soybean phospholipid, 10mgDSPE-PEG2000, 3mg docetaxel, 3mg dexamethasone joins in round-bottomed flask,
Dissolve by the chloroform/methanol (volume ratio 9:1) of 20ml；Using Rotary Evaporators to remove organic solvent film forming, bath temperature is 50
Degree, the time is 40min；Add the phosphate buffer of 3ml in round-bottomed flask, five minutes demouldings of 3000 turns/min vortex；Turn
Moving on in the centrifuge tube of 5ml, use the ultrasonic 5min of ultrasonic cell disintegration instrument, ultrasonic power is 20%；Cross the microporous filter membrane of 220nm
Degerming obtain required preparation.
Preparation (Fig. 1) particle diameter prepared by said method is 73nm, and polydispersity coefficient is 0.185, the envelop rate of dexamethasone
More than 90%, docetaxel envelop rate is more than 95%.
Embodiment 9 docetaxel is combined the preparation of liposome with dexamethasone
40mg soybean phospholipid, 10mg cholesterol, 10mgDSPE-PEG2000, 3mg docetaxel, 3mg dexamethasone joins
In round-bottomed flask, dissolve by the chloroform/methanol (9:1) of 20ml；Rotary Evaporators is used to remove organic solvent film forming, bath temperature
Being 50 degree, the time is 40min；Adding the phosphate buffer of 3ml in round-bottomed flask, 3000 turns/min vortex takes off for five minutes
Film；Transferring in the centrifuge tube of 5ml, use the ultrasonic 5min of ultrasonic cell disintegration instrument, ultrasonic power is 20%；Cross the micro-of 220nm
Hole filter membrane is degerming obtains required preparation.
Preparation particle diameter prepared by said method is 80nm, and polydispersity coefficient is 0.132, and the envelop rate of dexamethasone is more than
90%, docetaxel envelop rate is more than 95%.
Embodiment 10 docetaxel is combined the preparation of liposome with dexamethasone
50mg soybean phospholipid, 10mgDSPE-PEG2000, 3mg docetaxel, 0.45mg dexamethasone joins round-bottomed flask
In, dissolve by the chloroform/methanol (9:1) of 20ml；Using Rotary Evaporators to remove organic solvent film forming, bath temperature is 50 degree,
Time is 40min；Add the phosphate buffer of 3ml in round-bottomed flask, five minutes demouldings of 3000 turns/min vortex；Transfer to
In the centrifuge tube of 5ml, using the ultrasonic 5min of ultrasonic cell disintegration instrument, ultrasonic power is 20%；The microporous filter membrane crossing 220nm is degerming
Obtain required preparation.
Preparation particle diameter prepared by said method is 75nm, and polydispersity coefficient is 0.183, and the envelop rate of dexamethasone is more than
90%, docetaxel envelop rate is more than 95%.
Embodiment 11 docetaxel is combined the preparation of liposome with dexamethasone
50mg soybean phospholipid, 10mgDSPE-PEG2000, 3mg docetaxel, 1.5mg dexamethasone joins round-bottomed flask
In, dissolve by the chloroform/methanol (9:1) of 20ml；Using Rotary Evaporators to remove organic solvent film forming, bath temperature is 50 degree,
Time is 40min；Add the phosphate buffer of 3ml in round-bottomed flask, five minutes demouldings of 3000 turns/min vortex；Transfer to
In the centrifuge tube of 5ml, using the ultrasonic 5min of ultrasonic cell disintegration instrument, ultrasonic power is 20%；The microporous filter membrane crossing 220nm is degerming
Obtain required preparation.
Preparation particle diameter prepared by said method is 74nm, and polydispersity coefficient is 0.191, and the envelop rate of dexamethasone is more than
90%, docetaxel envelop rate is more than 95%.
Embodiment 12 paclitaxel is combined the preparation of liposome with dexamethasone
60mg soybean phospholipid, 10mg cholesterol, 5mgDSPE-PEG2000, 3mg paclitaxel, 3mg dexamethasone joins circle
In end flask, other experimental techniques are with embodiment 8.
Preparation particle diameter prepared by said method is 75nm, and polydispersity coefficient is 0.164, and the envelop rate of dexamethasone is more than
85%, paclitaxel envelop rate is more than 95%.
Embodiment 13 docetaxel is combined the preparation of micelle with dexamethasone
30mg DSPE-PEG2000, 1mg docetaxel, 1mg dexamethasone joins in round-bottomed flask, molten with 10ml acetonitrile
Solve；Rotary Evaporators is used to remove organic solvent film forming, in the phosphate buffer of addition 3ml to round-bottomed flask, 3000 turns/
Five minutes demouldings of min vortex；Transferring in the centrifuge tube of 5ml, use the ultrasonic 5min of ultrasonic cell disintegration instrument, ultrasonic power is
20%；Cross that the microporous filter membrane of 220nm is degerming obtains required preparation.
Preparation particle diameter prepared by said method is 30nm, and polydispersity coefficient is 0.124, and the envelop rate of dexamethasone is more than
90%, docetaxel envelop rate is more than 95%.
Embodiment 14 paclitaxel is combined the preparation of micelle with dexamethasone
Other are with embodiment 13.Substitute docetaxel with paclitaxel and prepare micelle.
Preparation particle diameter prepared by said method is 28nm, and polydispersity coefficient is 0.155, and the envelop rate of dexamethasone is more than
90%, paclitaxel envelop rate is more than 95%.
Embodiment 15 docetaxel and dexamethasone are combined the cell in vitro poison experiment of liposome
Using complete medium to cultivate KB, KBv, PC-3,4T1 cell, when cell grows to logarithmic (log) phase, with the 96 every holes of orifice plate
Inoculate 180 μ l, about 5000 cells；Incubator is cultivated and is made cell attachment in 24 hours, gives dexamethasone liposome respectively, many west he
Match liposome, docetaxel and dexamethasone combination liposome (above-mentioned each liposome is prepared according to the preparation method of embodiment 8,
The raw material wherein preparing dexamethasone liposome consists of: 50mg soybean phospholipid, 10mgDSPE-PEG2000, 3mg dexamethasone；
The raw material preparing docetaxel liposome consists of: 50mg soybean phospholipid, 10mgDSPE-PEG2000, 3mg docetaxel, under
With), the concentration of dexamethasone is 0.01-100 μ g/ml, and the concentration of docetaxel is 0.001-100 μ g/ml；It is little that incubator cultivates 48
Time, discard culture medium.Add the complete medium 100 μ l containing 5%CCK-8 solution；Each group is detected by microplate reader after hatching 2h
Light absorption value at 450nm.
From cytotoxic result (Fig. 2), dexamethasone is when low concentration, and cytotoxicity is the least；During high concentration, no
Same cell line is different to the response lag of dexamethasone；When concentration is at 10 more than μ g/ml, various kinds of cell system is all had necessarily
Inhibitory action, to KB, the cytotoxicity of PC-3,4T1 cell is more significantly；Compared with docetaxel liposome, combine to
Medicine group has more preferable tumor inhibitory effect.
Embodiment 16 docetaxel is combined the impact of liposome cell migration with dexamethasone
With complete culture medium culturing PC-3 cell, when cell length to logarithmic (log) phase with 12 orifice plate every hole inoculation 1ml, about 500,000
Individual cell, incubator is cultivated and is made cell attachment in 24 hours.Cell look densification time, discard complete medium, use the rifle head of 10 μ l
Cut.And give dexamethasone liposome, docetaxel liposome, taxotere (Taxotere), docetaxel and ground plug respectively
Rice pine combination liposome (above-mentioned each liposome is prepared according to the preparation method of embodiment 8).Wherein dexamethasone concentration is 2 μ g/
Ml, docetaxel concentration is about 0.5 μ g/ml, and the concentration of combination liposome group dexamethasone and docetaxel is respectively 0.5 μ g/
ml.Used fluorescence inverted microscope to take pictures respectively at 0 and 24 hour.
From scratch experiment result (Fig. 3), the cut of matched group heals substantially, and each administration group can press down to varying degrees
The migration of cell processed, independent Dexamethasone group is weaker than each docetaxel group.Tumor cell migration is the first step of neoplasm metastasis, swollen
The transfer ability of oncocyte represents its ability that transfer occurs to a certain extent.Dexamethasone can be bright with docetaxel combination
The healing of aobvious suppression cut, illustrates that the migration energy of tumor cell is played good inhibiting effect by it.
Embodiment 17 docetaxel is combined liposome extracorporeal releasing experiment with dexamethasone
The preparation of different preparations: Free DEX (dexamethasone) uses 10% methanol to dissolve, and (listing preparation is safe for Taxotere
Rope Supreme Being) prepare by listing preparation, DEX-Lip (dexamethasone liposome, prepares according to embodiment 8 method, and raw material consists of:
50mg soybean phospholipid, 10mgDSPE-PEG2000, 3mg dexamethasone), (docetaxel liposome, according to embodiment 8 for DTX-Lip
Prepared by method, raw material consists of: 50mg soybean phospholipid, 10mgDSPE-PEG2000, 3mg docetaxel), (DTX+DEX)-Lip
(docetaxel is combined liposome with dexamethasone) membrane-sonic method prepares (prepared by embodiment 8).
Release medium Free DEX and DEX-Lip release medium are the phosphate buffer of pH7.4, Taxotere, DTX-
The release medium of Lip and (DTX+DEX)-Lip is the pH7.4 phosphate buffer containing 0.5% tween 80.Free DEX and
DEX-Lip, respectively 0.5, takes a little in 1,2,4,6,8,12,24 hour, and Taxotere, DTX-Lip and (DTX+DEX)-Lip are respectively
1, within 2,4,6,8,12,24,36,48 hours, take a little, use high performance liquid chromatography detection dexamethasone and docetaxel not
Burst size with time point.
Being understood (Fig. 4) by release profiles, dexamethasone reached the release platform phase at about 8 hours, and release is very fast, many west he
Match burst size after 36 hours and reach about 40%, be a kind of procedural drug release phenomenon.
The impact on cytokine secretion of the embodiment 18 dexamethasone liposome
Cell is cultivated: using the KB being in exponential phase, Hela, KBv, PC-3 are inoculated in 25 bottles.Cell is cultivated adherent
After certain time, when cell is in eugonic growth stage, culture medium of inclining.Administration processes: every kind of cell is divided into comparison
Group, Free DEX (dexamethasone) group and DEX-Lip group (dexamethasone liposome, prepares according to embodiment 8, and raw material consists of:
50mg soybean phospholipid, 10mgDSPE-PEG2000, 3mg dexamethasone).Matched group is the fresh culture of 4ml, Free DEX group
It is that 100 μm ol/l, matched group and experimental group use 3 bottles of bottle cells respectively with the concentration of DEX-Lip group dexamethasone, co-cultures
24 hours.Discard culture medium, and wash three times with PBS.Sample collection: every bottle adds fresh culture 4 milliliters, co-cultures 12 hours
After, draw 1 milliliter from which, 3000 leave the heart 10 minutes, take supernatant.Sample detection: use VEGF (vascular endothelial growth factor
Son) and the ELISA kit of TNF-α (tumor necrosis factor), detect each group of VEGF and TNF-α protein excretion level.Additionally
Use BCA test kit, detect protein content.
The secretion level of different disposal group VEGF and TNF-α understands (Fig. 5), and dexamethasone can reduce VEGF and TNF-α
Secretion level.
Embodiment 19 docetaxel is combined the internal effect experiment of liposome with dexamethasone
Set up oxter KB tumor model, reach 100mm at gross tumor volume3Time, it is divided into 6 groups, gives physiology salt the most continuously
Water (Control), free dexamethasone, dexamethasone liposome, taxotere, docetaxel liposome, docetaxel and ground plug
Rice pine combination liposome (prepared according to embodiment 10 by each liposome；The raw material wherein preparing dexamethasone liposome consists of:
50mg soybean phospholipid, 10mgDSPE-PEG2000, 0.45mg dexamethasone；The raw material preparing docetaxel liposome consists of:
50mg soybean phospholipid, 10mgDSPE-PEG2000, 3mg docetaxel), within every three days, it is administered once, totally three (dexamethasone dosage
1.5mg/kg, docetaxel dosage 10mg/kg).Measure gross tumor volume upon administration every day, investigate the suppression situation to tumor,
And put to death mouse in the tenth day after being administered for the first time, solution cuts tumor and takes pictures.
Being understood (Fig. 6) by each group of tumor growth curve and Ex vivo Tumor size, compared with matched group, each administration group can not
With the growth of the suppression tumor of degree, preferably (tumor control rate is for docetaxel and dexamethasone combination liposome effect
97%), it is better than listing preparation taxotere (tumor control rate is 90%).
Embodiment 20 docetaxel is combined the internal effect experiment of liposome with dexamethasone
Set up oxter KBv tumor model, reach 100mm at gross tumor volume3Time, it is divided into 6 groups, gives physiology salt the most continuously
Water, free dexamethasone, dexamethasone liposome, taxotere, docetaxel liposome, docetaxel is combined fat with dexamethasone
Plastid (prepared according to embodiment 11, and the raw material wherein preparing docetaxel liposome consists of: 50mg Semen sojae atricolor phosphorus by each liposome
Fat, 10mgDSPE-PEG2000, 3mg docetaxel；The raw material preparing dexamethasone liposome consists of: 50mg soybean phospholipid,
10mgDSPE-PEG2000, 1.5mg dexamethasone, lower same), within every four days, it is administered once, totally three times (dexamethasone dosage 2.5mg/
Kg, docetaxel dosage 5mg/kg).Measure gross tumor volume upon administration every day, investigate the suppression situation to tumor, and first
Within after secondary administration the 12nd day, putting to death mouse, solution cuts tumor and takes pictures.
Being understood (Fig. 7) by each group of tumor growth curve and Ex vivo Tumor size, compared with matched group, each administration group can not
With the growth of the suppression tumor of degree, preferably (tumor control rate is for docetaxel and dexamethasone combination liposome effect
86%), it is better than listing preparation taxotere (tumor control rate is 60%).
Embodiment 21 docetaxel is combined the internal effect experiment of liposome with dexamethasone
Set up oxter PC-3 tumor model, reach 70mm at gross tumor volume3Time, it is divided into 5 groups, gives physiology salt the most continuously
Water, dexamethasone liposome, taxotere, docetaxel liposome, docetaxel is combined liposome (each liposome with dexamethasone
Prepare according to embodiment 11), within every three days, it is administered once, totally 5 times (dexamethasone dosage 4mg/kg, docetaxel dosage 8mg/
kg).Within the most every two days, measure gross tumor volume, investigate suppression situation to tumor, and after being administered for the first time the 15th day
Putting to death mouse, solution cuts tumor and takes pictures.
Being understood (Fig. 8) by each group of tumor growth curve and Ex vivo Tumor size, compared with matched group, each administration group can not
With the growth of the suppression tumor of degree, preferably (tumor control rate is for docetaxel and dexamethasone combination liposome effect
93%), it is better than listing preparation taxotere (tumor control rate is 73%).
Embodiment 22 docetaxel is combined the internal effect experiment of liposome with dexamethasone
Set up 4T1 breast cancer model in situ, reach 70mm at gross tumor volume3Time, it is divided into 4 groups, gives physiology the most continuously
Saline, dexamethasone liposome, docetaxel liposome, docetaxel and dexamethasone combination liposome (each liposome according to
Prepared by embodiment 11), within every three days, it is administered once, totally 4 times (dexamethasone dosage 4mg/kg, docetaxel dosage 8mg/kg).?
Within after administration every two days, measure gross tumor volume, investigate the suppression situation to tumor, and the 15th day after being administered for the first time puts to death old
Mus, solution cuts tumor and takes pictures.
Being understood (Fig. 9) by each group of tumor growth curve and Ex vivo Tumor size, compared with matched group, each administration group can not
With the growth of the suppression tumor of degree, preferably (tumor control rate is for docetaxel and dexamethasone combination liposome effect
83%) docetaxel liposome (tumor control rate is 66%), it is better than.
Embodiment 23 docetaxel is combined the internal safety evaluatio of liposome with dexamethasone
Set up oxter KBv tumor model, reach 100mm at gross tumor volume3Time, it is divided into 6 groups, gives physiology salt the most continuously
Water, free dexamethasone, dexamethasone liposome, taxotere, docetaxel liposome, docetaxel is combined fat with dexamethasone
Plastid (each liposome is prepared according to embodiment 11), is administered once for every four days, totally three times (dexamethasone dosage 2.5mg/kg, many
Xi Tasai dosage 5mg/kg).Weigh nude mice body weight upon administration every day, investigate nude mice body weight change, and after being administered for the first time
Within 12nd day, take blood and carry out neutrophilic granulocyte (GRN detection).
Being understood (Figure 10 A, B) by each group of nude mice body weight change and clean body weight change, docetaxel is combined fat with dexamethasone
Plastid loses weight less than listing taxotere, and from neutrophilic granulocyte result (Figure 10 C), docetaxel and dexamethasone
There is not obvious bone marrow depression (listing preparation taxotere has obvious bone marrow depression) in combination liposome.
Embodiment 24 docetaxel is combined the internal safety evaluatio of liposome with dexamethasone
Set up oxter PC-3 tumor model, reach 70mm at gross tumor volume3Time, it is divided into 5 groups, gives physiology salt the most continuously
Water (control), dexamethasone liposome, taxotere, docetaxel liposome, docetaxel is combined liposome with dexamethasone
(each liposome is prepared according to embodiment 11), within every three days, it is administered once, totally 5 times (dexamethasone dosage 4mg/kg, docetaxel agent
Amount 8mg/kg).The most every two days nude mice body weight, investigate nude mice body weight change.
Being understood (Figure 11) by each group of nude mice body weight change and clean body weight change, docetaxel is combined liposome with dexamethasone
Group loses weight less than listing preparation taxotere.
Impact-the experiment made on the living on drug distribution of embodiment 25 dexamethasone
Set up oxter tumor model (KB), reach 300-600mm at gross tumor volume3Time (about 12d), it is divided into two groups, respectively
Give the fluorescently-labeled liposome of DiR, fluorescently-labeled dexamethasone liposome (liposome is prepared according to embodiment 8) (DiR to
Pharmaceutical quantities is 200 μ g/kg, and dexamethasone is 3mg/kg).After being administered 2,6,12,24 hours, living imaging analysis and investigation preparation exists
Internal distribution situation.And respectively organize three nude mices of execution at each time point, take out the heart, liver, spleen, lung, kidney, tumor respectively,
Living imaging is taken pictures.
Living imaging result, isolated organ imaging results and semi-quantitative analysis result confirm (Figure 12), and dexamethasone can increase
Add the medicine distribution at tumor locus, reduce the distribution at each internal organs.
Embodiment 26 dexamethasone reduces the interstitial fluid pressure of tumor
Set up oxter tumor model (KBv), reach 100mm at gross tumor volume3(about 6d), is divided into 6 groups, gives the most continuously
Normal saline (control), Free DEX (dexamethasone), DEX-Lip (dexamethasone liposome), (how western 10mg is for taxotere
He matches addition 0.25ml Tween 80 and 13% ethanol of 0.75ml), DTX-Lip (docetaxel liposome), (DTX+DEX)-Lip
(docetaxel is combined liposome with dexamethasone) (each liposome is prepared according to embodiment 11)；(dexamethasone dosage 2.5mg/
Kg, docetaxel dosage 5mg/kg) within every 4 days, it is administered once, totally three times.After third time is administered, naked detection in 4 and 24 hours respectively
The interstitial fluid pressure (IFP) of Mus tumor locus.
Result confirms (Figure 13), and dexamethasone liposome, docetaxel can at 4 hours with dexamethasone combination liposome group
Significantly reducing interstitial fluid pressure, docetaxel still can significantly reduce interstitial fluid pressure with dexamethasone combination liposome group at 24 hours.
The immunohistochemical analysis of embodiment 27KB tumor tissue in vitro
Prepared by frozen section: take Ex vivo Tumor, is placed in 4% paraformaldehyde placement and fixes for 2 days, send dissection building to be cut into 6 μm
Frozen section, is put in-20 degree refrigerators and saves backup.
Fixing: to take (cold wind dries up 5min) after the placement of frozen section room temperature is dried for 15-30 minute, frozen section immerses and fills
The color jar of 4% paraformaldehyde fixative, room temperature (15～25 DEG C) fixes 20～30min；PBS washes 3 times, each 5min.
Penetrating: sample chips immerses penetrating liquid, room temperature promotees to ooze 3～5min, and PBS washes 3 times, each 5min；(penetrating liquid is prepared:
1 × the PBS of 99mL adds 1.0mL Triton X-100, mixing, instant joins).
Close: sample chips immerses in confining liquid 1, and room temperature closes 10min.PBS washes 3 times, each 5min.(confining liquid 1 is joined
System: 80mL methanol adds 10mL H2O and 10mLH2O2(30%), instant join).2%BSA 37 degree closes 60min or 4 and spends
At night, need not wash, confining liquid is blotted；
One anti-hatches: the 300 anti-working solutions of μ L mono-(1:50 dilution) of dropping dilution, sealed membrane covering, hatches 1～2 for 37 DEG C
Hour or 4 DEG C overnight, PBS washes 5min × 3.
Two anti-hatch: adding 200 μ L bis-anti-(diluting 200 times), 37 degree of calorstats hatch 60min, lucifuge.PBS wash 5min ×
3。
Dye core: blot with absorbent paper around sample, add 100 μ L Hochest33258 or DAPI, hatch for 37 DEG C
30min, lucifuge, PBS washes 5min × 3.
Mounting: preparation glycerol/PBS (6:4, v/v) mounting ,-20 DEG C keep in Dark Place.
The experiment of Tunel apoptosis and the immunohistochemical analysis of tumor microenvironment composition of KB tissue are done respectively, including NF-κ
B, TNF-α, PFGFR-β, α-SMA, CD-31, college-1 and TUNEL analyze.Dyeed feelings by confocal microscopy
Condition.
From ImmunohistochemistryResults Results (Figure 14-20): the many indexes of microenvironment is all had by Dexamethasone group upon administration
Certain reduction effect, also has certain increase to apoptosis, illustrates that dexamethasone suppression tumor growth is that many factors is comprehensively made
Result.Each group of Multiple components to microenvironment of docetaxel also has certain regulation effect, but it is mainly withered by induction
Die and realize suppressing the effect of tumor growth.
Claims (10)
1. an anti-cancer composition, it is characterised in that including: the dexamethasone of effective dose and cancer therapy drug in treatment.
2. according to the anti-cancer composition described in claim 1, it is characterised in that described dexamethasone is selected from: pharmaceutically can
Any one in dexamethasone, dexamethasone salt or dexamethasone ester；
Wherein, described dexamethasone salt is dexamethasone sodium phosphate；Described dexamethasone ester is dexamethasone acetate or ground plug
Rice pine acetas in any one；
Described cancer therapy drug is selected from: cisplatin, carboplatin, amycin, paclitaxel, docetaxel, daunorubicin, doxorubicin, table are soft
Than star, pirarubicin, idarubicin, mitoxantrone, camptothecine, hydroxy camptothecin, topotecan, irinotecan, vinblastine,
Vincristine, vindesine, vinorelbine, vinflunine, vinglycinate, teniposide or Sony for any one in Buddhist nun or
Multiple；It is preferably any one or more in cisplatin, amycin, paclitaxel, docetaxel or vincristine；The most
Xi Tasai.
3. according to the anti-cancer composition described in claim 1, it is characterised in that described cancer includes: primary solid tumor, drug resistance are swollen
Any one or more in tumor, metastatic tumo(u)r, malignant tissue or cell；
Preferably, described cancer includes: nasopharyngeal carcinoma, oral cavity epidermoid carcinoma, carcinoma sarcomatodes, ovarian cancer, cancer of pancreas, carcinoma of prostate,
Colon cancer, rectal cancer, metastatic colorectal cancer, breast carcinoma, metastatic breast cancer, transitivity HER2 negative breast cancer, the brain cancer,
Adult Human Brain glioma, child's cerebral glioma, child-resistance cerebral glioma, glioma, astrocytoma, become
Medulloblastoma, child's medulloblastoma, glioma, oligodendroglioma, meningioma, renal carcinoma, bladder cancer,
Cervical cancer, the esophageal carcinoma, gastric cancer, incidence cancer, hepatocarcinoma, pulmonary carcinoma, melanoma, myeloma, sarcoma, skin carcinoma, gastric cancer, carcinoma of testis,
Thyroid carcinoma, uterus carcinoma, mesothelioma, cancer of biliary duct, leiomyosarcoma, liposarcoma, fibrosarcoma, neuroendocrine carcinoma or saliva
Any one or more in adenocarcinoma.
4. the application in preparing anticancer preparation of the anti-cancer composition described in claims 1 to 3 any one.
5. an anticancer preparation, it is characterised in that including: anticancer described in the claims 1 to 3 any one of effective dose in treatment
Acceptable adjuvant in compositions and pharmaceutics.
6. according to the anticancer preparation described in claim 5, it is characterised in that the dosage form of described anticancer preparation includes: injection, sheet
Agent, capsule, granule, suspensoid, Emulsion, solution, sol, lyophilized injectable powder, mucilage, aerosol, microcapsule, micro-
Any one or more in ball, nanometer formulation, slow releasing preparation or controlled release preparation；It is preferably nanometer formulation；
Wherein, described nanometer formulation includes: in liposome, nanocapsule, nanosphere, lipid nanoparticle or micelle any one or
Multiple, preferably any one in liposome or micelle or two kinds.
7. a docetaxel is combined liposome with dexamethasone, it is characterised in that include following component: docetaxel,
Sai meter Song and fat material；
According to mass ratio meter, docetaxel: dexamethasone: fat material=10:1-10:100-400；Preferably, docetaxel: ground plug
Meter Song: fat material=10:1-10:200-250；It is furthermore preferred that docetaxel: dexamethasone: fat material=10:1-10:200.
8. a docetaxel is combined micelle with dexamethasone, it is characterised in that include following component: docetaxel, fill in
Meter Song and fat material；
According to mass ratio meter, docetaxel: dexamethasone: fat material=10:1-10:100-500；Preferably, docetaxel: ground plug
Meter Song: fat material=10:1-10:100-300；It is furthermore preferred that docetaxel: dexamethasone: fat material=10:1-10:200.
9. according to the how west described in the docetaxel described in claim 7 and dexamethasone combination liposome or claim 8
He is combined micelle with dexamethasone at match, it is characterised in that described fat material includes: extract in phospholipid or synthetic phospholipid any one
Or it is multiple；
Wherein, any one in soybean phospholipid or lecithin of described extraction phospholipid；
Described synthetic phospholipid is selected from: hydrogenated soybean lecithin, hydrolecithin, DPPC, DSPC, DMPC, DPPE, DSPE, DMPE,
DOPE, DPPG, DSPG, DMPG, DSPE-PEG, DPPE-PEG, DMPE-PEG, mPEG-DSPE-MAL, mPEG-DSPE-NHS,
COOH-PEG-DSPE, NH2Any one or more in-PEG-DSPE, cholesterol or cholesteryl ester；
Preferably, described docetaxel is made up of with DSPE-PEG phospholipid with the fat material of dexamethasone combination liposome, or by
Phospholipid, cholesterol form with DSPE-PEG；More preferably it is made up of according to mass ratio 5:1 with DSPE-PEG phospholipid, or by phosphorus
Fat, cholesterol form according to mass ratio 4:1:1 with DSPE-PEG；
The fat material of described docetaxel and dexamethasone combination micelle is any in DSPE-PEG, DPPE-PEG or DMPE-PEG
One or more are according to the mixture of arbitrary proportion composition.
10. docetaxel described in a claim 7 and docetaxel described in dexamethasone combination liposome or claim 8
Preparation method with dexamethasone combination micelle, it is characterised in that including: membrane-sonic method, reverse phase evaporation, freezing are done
Any one in dry method, injection method, extrusion molding or supercritical ultrasonics technology；
Wherein, the step of described membrane-sonic method includes: docetaxel, dexamethasone and fat material are dissolved in organic molten by (1)
In agent, obtain mixed solution；(2) remove organic solvent with Rotary Evaporators and obtain adipose membrane；(3) normal saline or phosphate are added
Buffer, vortex demoulding；(4) carry out supersound process with ultrasonic cell disintegration instrument, or after being adjusted particle diameter further, cross and filter
Bacterium, to obtain final product；
Preferably, step (1) described organic solvent is the mixed solution of chloroform, methanol, or is acetonitrile；Wherein, described chloroform,
In the mixed solution of methanol, according to volume basis, chloroform: methanol=1-20:1, preferably 9:1；
Step (2) described Rotary Evaporators removing organic solvent obtains the rotation steaming bath temperature of adipose membrane and is 20-70 DEG C, is preferably
40-50℃；Time is 20-40min；
The time of step (3) described vortex is 3-20min, preferably 5-6min；
The ultrasonic power of step (4) described supersound process is 3%-20%, preferably 20%；Ultrasonic time is 3-10min, preferably
5min；The described filter membrane being filtered into 220nm.
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| CN114807032A (en) * | 2022-05-20 | 2022-07-29 | 南京大学 | Application of dexamethasone in preparation of anti-inflammatory drugs for inducing extracellular vesicles produced by neutrophils |
| CN115089593A (en) * | 2022-05-20 | 2022-09-23 | 广东南国药业有限公司 | Dexamethasone preparation and preparation method thereof |
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