CN109512821A - One kind gram azoles is for Buddhist nun and 17-AAG composition polymer nano-micelle injection, preparation method and its application - Google Patents
One kind gram azoles is for Buddhist nun and 17-AAG composition polymer nano-micelle injection, preparation method and its application Download PDFInfo
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- 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
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- A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
- A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
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- A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
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- A61K9/00—Medicinal preparations characterised by special physical form
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Abstract
The present invention provides a kind of with synergy gram of azoles for Buddhist nun and 17-AAG composition polymer nano-micelle injection, preparation method and its application in preparation treatment drug resistance ALK positive non-small cell lung cancer drug.1 parts by weight of Buddhist nun are replaced comprising gram azoles in the preparation；0.5~3 parts by weight of 17-AAG；10~100 parts by weight of nano-micelle polymer.Described gram of azoles is uniform for Buddhist nun and 17-AAG composite Nano micellar particle size, and average grain diameter is 20~200nm.Said preparation can greatly increase gram azoles for the stability of the water solubility and preparation of Buddhist nun and 17-AAG, extend gram azoles and replaces the circulation time of Buddhist nun and 17-AAG in blood, the situation (such as treating the patient for thering is the patient of antibody or multiple dosing to generate anti-PEG antibody PEG) that polyethylene glycol (PEG) can be overcome not applicable as the polymer nano micelle class preparation of shell, (EPR effect) passive target can be acted on to tumor locus by enhancing infiltration and delay, and effectively gram azoles is inhibited to replace the drug resistant ALK positive non-small cell lung cancer cell of Buddhist nun, there is drug combination synergy.
Description
Technical field
The invention belongs to field of pharmaceutical preparations, are related to one kind gram azoles and inject for Buddhist nun and 17-AAG composition polymer nano-micelle
Agent, preparation method and its purposes in treatment drug resistance ALK positive non-small cell lung cancer drug.
Background of invention
Lung cancer is one of highest cancer of global incidence, and non-small cell lung cancer (non-small cell lung
Cancer, NSCLC) account for the 80%~85% of lung cancer sum.Preliminary epidemiological study shows in NSCLC patient, becomes
Type lymphom kinase (ALK) positive rate is about 3%~5%, it means that the U.S. about 6500~11000 ALK every year
Positive NSCLC patient, whole world estimation have 40000 patients to be diagnosed as ALK positive NSCLC every year.Gram azoles replaces Buddhist nun
It (Crizotinib) is the first small molecule tyrosine kinase inhibitors for ALK positive NSCLC exploitation, according to PHA-665752
It is designed with the eutectic structure of non-phosphorylating MET kinase domain, the phosphorylation of ALK and c-Met capable of inhibiting cell are to press down
(such as c-Met/HGFR mutant or ALK melt by c-Met/ hepatocyte growth factor receptor (HGFR) processed and ALK and its carcinogenic variant
Hop protein) activity, and then its signal path and downstream signal are blocked, to the amplification of c-Met/HGFR gene loci or ALK occurs
The tumour cell of gene loci transposition/inversion (such as EML4-ALK or NPM-ALK merge variant) shows potent selectivity
Growth inhibition and the activity for promoting apoptosis of tumor cells.In carried out clinical research, ALK is detected and selected according to biomarker
For positive NSCLC as study population, observing has significantly superior objective remission rate (ORR) and Progression free survival compared with the past chemotherapy
Time (PFS).2011 by FDA approval for the Locally Advanced of the ALK positive or the non-small cell lung cancer of transfer, FDA in 2016
Approval gram azoles is for Buddhist nun for treating advanced stage ROS1- positive Patients with Non-small-cell Lung.Current gram of azoles is EML4-ALK fusion for Buddhist nun
The optimal selection of gene masculine Patients with Advanced Lung Cancer first-line treatment.
Although the benefit of ALK positive lung cancer patient is obvious, this some patients often occurs replacing Buddhist nun to gram azoles in 1-2
Drug resistance, and central nervous system recurrence progress it is relatively conventional.Its resistance mechanism be it is diversified, it is big that two can be divided into
Class: the conversion (i.e. the activation of signal bypass) of ALK medicament-resistant mutation and other signal paths.Therefore targetedly exploitation can be fought
Gram azoles can meet the needs of clinical treatment ALK positive non-small cell lung cancer for the drug resistant new formulation of Buddhist nun significantly.Cell in vitro
Discovery heat shock protein 90 (HSP90) inhibitor is positive to ALK in system's research handling for Buddhist nun without gram azoles or replacing to a gram azoles
The drug resistant cell line of Buddhist nun is active.Its action principle is the degradation that HSP90 inhibitor can promote tumor signal pathway protein,
Such as ALK (being related to the proliferation and survival of tumour cell), replace Buddhist nun's drug resistance but secondary mutation patient do not occur providing one for gram azoles
Kind therapeutic strategy.A series of joint HSP90 inhibitor and selectivity ALK inhibitor are in clinical investigation phase
(NCT01712217 and NCT01579994).
17-AAG (17- allylamine -17-AAG) is one obtained to geldanamycin structure of modification
The potent HSP90 inhibitor of kind, also known as tanespimycin (tanespimycin).17-AAG has typeⅡ pneumocyte strain proliferation
Significant inhibiting effect.17-AAG is able to suppress active tyrosine kinase in the drug resistant T790M cell strain of EGFR-TKI
(receptor tyrosine kinase, RTK) receptor phosphorylation lowers EGFR and other RTK receptors, inhibits mammal thunder
The transmitting of pa mycin target spot signal, leads to the degradation of cell survival alternative activation albumen, reduces ataxia telangiectasia
The activation of burst gene (Ataxia telangiectasia mutated, ATM), correspondingly hinders DNA in multiple levels and cuts
Except the activation of repair mechanism and key enzyme, and can promote thin to the lung cancer of Gefitinib, Tarceva generation drug resistance
Born of the same parents' strain restores sensitive.17-AAG can increase the apoptosis effect of anticancer drug, and EGF-R ELISA (EGFR) is caused to degrade, resistance
Hinder downstream signal to transmit, play chemotherapeutics synergy, for example taxol can be reinforced at lung cancer H3255 (L858R EGFR)
Antiproliferative effect in cell strain.
Gram azoles belongs to insoluble medicine in water, poorly water-soluble (about 10~20 μM) for Buddhist nun and 17-AAG, and is the term traditionally used to
The different surfaces activating agent or organic solvent of solubilising design to anticancer delivery system and prepare band there are toxicity problem outstanding
Carry out great challenge.In addition if successive or simple superposition uses two drugs, since two kinds of drugs have different medicines in vivo
Object pharmacokinetic profile is difficult to reach drug concentration and ratio with mutual synergy in tumor locus, and distich shares
Medicine reaches for optimum efficiency, challenges especially prominent.So presently relevant drug delivery system research is dedicated to new nontoxic pair
The delivery media of effect replaces present organic solubilized agent, improves the solubility of insoluble anti-tumor medicament, will need united
Two or more drugs are loaded into drug delivery system simultaneously, (are used in combination to reach the Pharmacokinetic Characteristics of improvement
Drug simultaneously reach tumor locus and according to drug concentration and proportion with synergy), it is intended to overcome multidrug resistance
And increase drug in the distribution and concentration of tumor locus, to reduce drug in the distribution of normal tissue, reduce toxicity.It is domestic
Outer researcher has done many researchs, such as macromolecule conjugate to new drug delivery system；Liposome nano granule；It is poly- with block
Object micella etc. is closed, to increase water solubility, but energy industrialized production and the kind for meeting safety evaluatio are rare at present.Wherein receive
Rice micellar preparation recent two decades become research hotspot.The solubility of drug can be improved in it, and Drug controlled release can be imitated by EPR
Answer (the i.e. high-permeability of solid tumor and retention effect < enhanced permeability and retention effect
> refers to that relative to normal tissue, the molecule or particle of certain sizes tend to the property for being gathered in tumor tissues.Just
Often the microvascular endothelial gap densification in tissue, structural integrity, macromolecular and lipid granule are not easy through vascular wall, and solid tumor
Blood vessels in tissue is abundant, vascular wall gap is wider, poor structural integrity, and lymphatic return missing causes macromolecular substance and rouge
Matter particle has selective high-permeability and anelasticity, and this phenomenon is referred to as the high-permeability of solid tumor mass and is detained effect
It answers, abbreviation EPR effect.) passive target to tumor locus, can also be modified in micellar surface with reach active targeting work
With.
Current nano-micelle preparations mainly use amphiphilic polymer, and drugloading rate is relatively high, and stability is good.It is amphipathic
Polymer is block copolymer, including hydrophilic block and lipophilic block, and the nano-micelle of formation is core-shell structure, and hydrophilic block is
The shell on surface can be scattered in water and physiological saline, and lipophilic block then forms the core of micella, for loading hydrophobic drug.
Most common hydrophilic block is polyethylene glycol (PEG), and good water solubility, toxicity is low.However 25% patient's body has been deposited
In anti-PEG antibody (due to containing caused by a large amount of PEG in other daily chemical products such as shampoo and some food), and multiple
Anti- PEG antibody may be generated after administration, to limit the clinical application of PEG quasi polymer micella.It is close in amphoteric polymer
Rouge block is mostly that (such as polylactic acid-glycolic ethylhexyldithiophosphoric acid PLGA gathers widely used polymer in the drug and medical material of FDA approval
Lactic acid PLA etc.).
Summary of the invention
The purpose of the present invention is overcome the deficiencies of the prior art and provide one kind gram azoles to receive for Buddhist nun with 17-AAG composition polymer
Rice glue beam injection.
Amphiphilic polymer of the present invention uses poly- (2- methyl -2- oxazoline) or poly- (2- ethyl -2- oxazole
Quinoline) it is used as hydrophilic block to replace PEG, it uses PLA as lipophilic block, polymer nano micelle is used as gram azoles and replaces Buddhist nun and 17-
The common carrier of AAG provides a kind of polymer nano micelle injection that can be fought gram azoles and replace Buddhist nun's drug resistance for clinic, can be significantly
Increase gram azoles for the stability of the water solubility and preparation of Buddhist nun and 17-AAG, extends gram azoles for Buddhist nun and the circulation of 17-AAG in blood
Time eliminates the solubilizer toxic side effect in existing preparation, and by composite micelle feature, keeps two drugs with fixed ratio
Example by EPR effect passive target arrive tumor locus, generate synergistic antitumor effect, confrontation gram azoles for Buddhist nun treatment when there may be
Drug resistance.In addition, the problems such as antibody is generated after anti-PEG antibody and multiple dosing, can be overcome using such polymer micelle,
Clinical meaning is very great.
Technical scheme is as follows:
A kind of with synergy gram of azoles is for Buddhist nun and 17-AAG composition polymer nano-micelle injection, in the preparation
1 parts by weight of Buddhist nun, 0.5~3 parts by weight of 17-AAG, 10~100 parts by weight of nano-micelle polymer are replaced comprising gram azoles；Described receives
Rice glue beam polymer is poly- (2- ethyl -2- oxazoline)-polylactic acid, i.e. PEOZ-PLA or poly- (2- methyl -2- oxazoline) -
Polylactic acid, i.e. PMOZ-PLA.
It is poly- (2- ethyl -2- oxazoline) in poly- (2- ethyl -2- the oxazoline)-polylactic acid in i.e. PEOZ-PLA
The degree of polymerization of PEOZ is 20~60 units, average molecular weight 1700 to 5100Da, the degree of polymerization of polylactic acid PLA is 30~
115 units, average molecular weight is 850 to 3300Da.
Poly- (2- methyl -2- the oxazoline)-polylactic acid, i.e. in PMOZ-PLA, poly- (2- methyl -2- oxazoline) PMOZ's
The degree of polymerization is 20~60 units, and for average molecular weight 1700 to 5100Da, the degree of polymerization of the polylactic acid PLA is 30~115
A unit, average molecular weight is 850 to 3300Da.
Preferably,
1 parts by weight of Buddhist nun, 1 parts by weight of 17-AAG, 10 parts by weight of nano-micelle polymer are replaced comprising gram azoles in the preparation；
The nano-micelle polymer is poly- (2- methyl -2- oxazoline)-polylactic acid, i.e. PMOZ-PLA, wherein poly- (2- methyl -2- is disliked
Oxazoline) degree of polymerization of PMOZ is 50 units, for average molecular weight in 4400Da, the degree of polymerization of the polylactic acid PLA is 115 lists
Position, average molecular weight is in 3300Da.
Described gram of azoles is 20~200nm for the average grain diameter of Buddhist nun and 17-AAG composition polymer nano-micelle injection.
Described gram of azoles is 30~100nm for the average grain diameter of Buddhist nun and 17-AAG composition polymer nano-micelle injection.
Preferably,
Described gram of azoles is 35nm for the average grain diameter of Buddhist nun and 17-AAG composition polymer nano-micelle injection.
The preparation method of the injection, includes the following steps:
1) gram azoles is dissolved in ethyl alcohol or acetonitrile for Buddhist nun and 17-AAG and nano-micelle polymer respectively, is made into stock solution,
Then it measures gram azoles according to prescription to mix for Buddhist nun, 17-AAG and nano-micelle polymer stock solution, oscillation mixes；
2) by obtained gram azoles for Buddhist nun, 17-AAG and nano-micelle mixed with polymers organic phase be injected into round-bottomed flask or its
In its sample bottle, in slightly heating process, organic solvent is slowly evaporated into completely dry under decompression or normal pressure, formed in bottom of bottle
Drug and nano-micelle polymer material mixing film layer；
3) 35~50 DEG C are warming up to, distilled water is added or water for injection, slight oscillatory to film layer are completely dissolved into clear
Clear solution；
4) clear solution placement is cooled to room temperature, is centrifuged, by gram azoles of the unloaded in micella for Buddhist nun and 17-AAG from
Heart precipitating removes, take supernatant be freeze-dried gram azoles is lyophilized for Buddhist nun and 17-AAG composition polymer nano-micelle injection
Powder.
Preferably,
In the step 3), it is warming up to 45 DEG C.
In the step 4), when freeze-drying, is added protective agent, the freeze drying protectant be selected from mannitol, sucrose,
Or mixtures thereof any one of lactose, trehalose, maltose and glucose, the dosage of the freeze drying protectant are 5-10g/
100ml grams of azoles is for Buddhist nun and 17-AAG composition polymer nano-micelle.Freeze drying protectant, which is added, can increase gram azoles for Buddhist nun and 17-
The stability of AAG composition polymer micella injection, extends its validity period.
Gram azoles is for Buddhist nun and 17-AAG composition polymer nano-micelle injection in preparation tumor
Using the tumour is ALK positive lung cancer in non-cellule type and drug resistance ALK positive non-small cell lung cancer drug.
The present invention has the following technical effect that
1) provided by the invention gram of azoles is simple for Buddhist nun and the preparation method of 17-AAG composition polymer nano-micelle, is easy to grasp
Make, it is reproducible, it can be achieved that industrial-scale production.
2) the present invention provides described in one kind grams of azoles for Buddhist nun and 17-AAG composition polymer nano-micelle injection, is treating
ALK positive lung cancer in non-cellule type, drug resistance ALK positive non-small cell lung cancer, the patient for having anti-PEG antibody and multiple dosing
The purposes in the patient of PEG antibody is generated afterwards, wherein preferably, the tumour is ALK positive lung cancer in non-cellule type, drug resistance
Property ALK positive non-small cell lung cancer etc..
Compared to existing gram azoles replaces Buddhist nun's preparation, and provided by the invention gram of azoles is for Buddhist nun and 17-AAG composition polymer nano-micelle
It has the advantage that
1. the polymer material good biocompatibility, highly-safe, the allergic reaction of solubilizer bring, body will not be generated
Liquid contracting such as stays at the toxic side effects.
2. the present invention contains gram azoles in polymer nano micelle for Buddhist nun and 17-AAG simultaneously, improve gram azoles replace Buddhist nun,
The water solubility and stability of 17-AAG, extends its validity period.
3. provided by the invention gram of azoles is smaller for Buddhist nun and 17-AAG composition polymer nano-micelle partial size, can effectively penetrate swollen
Tumor blood vessel is gathered in tumor locus by EPR effect, passive target effect is realized, to improve the therapeutic effect of drug.
4. provided by the invention gram of azoles for Buddhist nun and 17-AAG polymer nano micelle injection can greatly prolong gram azoles for Buddhist nun,
The circulation time of 17-AAG in blood, reduce its toxicity.
5. the amphipathic that provided by the invention gram of azoles is related to for Buddhist nun and 17-AAG composition polymer nano-micelle injection
Using poly- (2- methyl -2- oxazoline) or poly- (2- ethyl -2- oxazoline) in conjunction object is used as hydrophilic block to replace PEG,
Hydrophily and PEG are suitable, and have similar " stealth " effect with PEG, as the shell of micella, both can protect the kernel of micella
With contained drug, and can prevent by the phagocytosis of mononuclear macrophage.
6. provided by the invention gram of azoles can overcome polyethylene glycol for Buddhist nun and 17-AAG composition polymer nano-micelle injection
(PEG) as the polymer nano micelle class preparation of shell be not suitable for situation (such as treatment to PEG have antibody patient or
Multiple dosing generates the patient of anti-PEG antibody).
7. provided by the invention gram of azoles can be by preferred proportion for Buddhist nun and 17-AAG composition polymer nano-micelle injection
Gram azoles is loaded into polymer nano micelle simultaneously for Buddhist nun and 17-AAG, and the rate of release of composition is controllable.
8. provided by the invention gram of azoles passes through for Buddhist nun and 17-AAG composition polymer nano-micelle injection in tumor locus
There is gram azoles of synergy ratio to replace Buddhist nun and 17-AAG for controllable release, significantly improve gram azoles for the tissue point of Buddhist nun, 17-AAG
Cloth and Pharmacokinetic Characteristics reduce nonneoplastic tissue cytotoxicity.
9. provided by the invention gram of azoles reaches in tumour cell for Buddhist nun with 17-AAG composition polymer nano-micelle injection
To the concentration and ratio of synergy, the index of cooperation (CI) for inhibiting tumour preferably, replaces Buddhist nun's list medicine compared to 17-AAG or gram azoles
Antitumor curative effect is greatly improved.
10. provided by the invention gram of azoles is for Buddhist nun and 17-AAG composition polymer nano-micelle injection in drug resistance ALK sun
It is effective in cure in property non-small cell lung cancer cell.
11. provided by the invention gram of azoles uses for the preparation method of Buddhist nun and 17-AAG composition polymer nano-micelle injection
Preparation method is simple and easy to do, and material source is extensive, at low cost, and stable product quality is easy to implement industrialized production.
Detailed description of the invention
Fig. 1 is that gram azoles prepared in the embodiment of the present invention replaces Buddhist nun and 17-AAG composition polymer nano-micelle atomic force microscopy
Mirror (AFM) spectrogram.
Fig. 2 is that gram azoles prepared in the embodiment of the present invention is lyophilized for Buddhist nun and 17-AAG composition polymer nano-micelle injection
The grain size distribution of powder redisperse liquid.
Fig. 3 is that gram azoles prepared in the embodiment of the present invention is lyophilized for Buddhist nun and 17-AAG composition polymer nano-micelle injection
The outer releasing curve diagram of powder redisperse liquid.
Fig. 4 is that gram azoles prepared in the embodiment of the present invention replaces Buddhist nun with 17-AAG composition polymer nano-micelle injection resistance to
Inhibitory effect in pharmacological property ALK positive non-small cell lung cancer cell.
Fig. 5 is according to gram azoles prepared in the embodiment of the present invention for Buddhist nun and 17-AAG composition polymer nano-micelle injection
Synergy index (drug ratio preferred) in drug resistance ALK positive non-small cell lung cancer cell.
Specific embodiment
The poly- oxazoline PEOZ-OH or PMOZ-OH of hydroxyl by -2 oxazoline of monomer 2- ethyl (or 2- methyl -2- dislike
Oxazoline) it is made within reaction 24 hours with cation ring-opening polymerization method at 100 DEG C, initiator is 4- methyl tosylate (MeOTs).Slightly
Product is precipitated after crossing silica gel with ether, and gained PEOZ-OH or PMOZ-OH and Pfansteihl are in chlorobenzene solvent, 140 DEG C, octanoic acid Asia
Further it polymerize under tin catalytic condition 24 hours.Gained PEOZ-PLA (or PMOZ-PLA) passes through ether deposition and purification, molecular weight
With distribution using glue penetration chromatography (GPC).N,N-Dimethylformamide (DMF) is used as mobile phase, flow velocity 1.0m/l/min；GPC
Column uses 5 μm of Phenogel, and by polymethyl methacrylate standard items (being purchased from Polymer Laboratories).Institute
The composition for obtaining polymer detects confirmation using nuclear magnetic resonance (1H-NMR) in deuterated chloroform solvent.
Below with reference to embodiment, the present invention is further described, but the embodiment be not meant to be subject to the present invention it is any
Limitation.
Embodiment 1
In the present invention, gram azoles replaces the preparation of Buddhist nun and 17-AAG composition polymer nano-micelle, includes the following steps:
1) it weighs gram azoles and respectively takes 10mg that 1ml dehydrated alcohol ultrasonic dissolution is added respectively for Buddhist nun and 17-AAG；Weigh PMOZ-
10ml dehydrated alcohol ultrasonic dissolution is added in PLA polymer 100mg；Gram azoles is replaced into Buddhist nun, 17-AAG and PMOZ-PLA mixed with polymers.
The degree of polymerization of PMOZ is 50 units, and average molecular weight is in 4400Da.The degree of polymerization of polylactic acid PLA is 115 units, average mark
Son amount is in 3300Da.
2) 50ml round-bottomed flask is added in mixed liquor, depressurizes at 40 DEG C and slowly solvent volatilizees, and vacuumized to remove
Dissolvent residual, in round-bottomed flask bottom, (drug+PMOZ-PLA is poly- for formation drug and nano-micelle polymer material mixing film layer
Close object).
3) at 45 DEG C, distilled water 10ml is added, slight oscillatory to film layer is completely dissolved into clear solution；
4) clear solution placement is cooled to room temperature, 5000rpm is centrifuged 10 minutes, and a gram azoles of the unloaded in micella is replaced
Buddhist nun or 17-AAG centrifugation remove, take supernatant be freeze-dried gram azoles for Buddhist nun and 17-AAG composition polymer nanometre glue
Beam injection freeze-dried powder.
Gram azoles made from Example 1 carries out the survey of encapsulation rate for Buddhist nun and 17-AAG composition polymer nano-micelle freeze-dried powder
It is fixed.
Chromatographic condition: with octadecylsilane chemically bonded silica be filler [such as Inertsil ODS3 (and 5 μm, 150mm ×
4.6mm), YMC Pack Pro C18(5 μm, 150mm × 4.6mm) or other equivalent chromatographic columns]；With phosphoric acid-ammonium hydroxide-water
(1.5:1.0:1000) (adjusting pH value to 2.5 with phosphoric acid or ammonium hydroxide) is mobile phase A, using acetonitrile as Mobile phase B, by table 1
Carry out gradient elution；Flow velocity is 1.0ml/min；Detection wavelength is respectively 210 and 333nm；20 μ l of sample volume.
1 gradient elution program of table
Distilled water is added again for Buddhist nun and 17-AAG composition polymer nano-micelle freeze-dried powder in gram azoles made from Example 1
Gram azoles of about 1mg/ml is dispersed into for Buddhist nun's concentration, 20 μ 1 of dispersion liquid is taken, 180 μ, 1 acetonitrile is added, vibrates sufficiently dissolution, then from
The heart takes 20 μ 1 of supernatant in HPLC sample introduction.Separately take standard gram azoles for Buddhist nun or 17-AAG stock solution, being made into concentration is 10,20,50,
100 and 200 μ g/ml acetonitrile standard solution, take 20 μ l in HPLC sample introduction respectively, carry out curve to gained peak area under each concentration
The standard curve of recurrence.Buddhist nun or 17-AAG drug are replaced by gram azoles in standard curve and nano-micelle freeze-dried powder redisperse liquid
Concentration, calculate gram azoles for the encapsulation rate of Buddhist nun and 17-AAG composition polymer nano-micelle is about 88.1% and 91.1%.
Distilled water is added again for Buddhist nun and 17-AAG composition polymer nano-micelle freeze-dried powder in gram azoles made from Example 1
Gram azoles of about 0.1mg/ml is dispersed into for Buddhist nun's concentration, a drop is diluted into 500 times of gram azoles again for Buddhist nun and 17-AAG composite polymeric
Object nano-micelle freeze-dried powder redisperse drop is placed about 2 minutes in the mica negative terminal surface newly removed, removes extra dispersion
Liquid is washed with distilled water and dries (see Fig. 1) in an argon atmosphere.Nano-micelle is uniform ball to atomic force microscope as the result is shown
Shape structure, partial size about 20-30nm.By 0.1mg/ml grams of azoles for Buddhist nun and 17-AAG composition polymer nano-micelle freeze-dried powder redisperse
Liquid measures the partial size of micella in Malvern nanometer particle size instrument, and the average grain diameter of acquisition is in 35.1nm, dispersion degree PDI=
0.112 (see Fig. 2).
Precision pipettes gram azoles for preparing in embodiment 1 dividing again for Buddhist nun and 17-AAG composition polymer nano-micelle freeze-dried powder
Dispersion liquid 0.1ml is put into dialysis apparatus (molecular cut off 3500), is put into beaker, and dissolution medium is 1OOml PBS buffering
Liquid；It is 37 DEG C that temperature, which is arranged, and horizontal slow oscillation samples after 0.5,1,2,4,6,8,12 hour, is measured respectively with HPLC thoroughly
Do not discharge drug gram azoles in analysis apparatus for the concentration of Buddhist nun, 17-AAG, the accumulative release rate and time for calculating drug are made to release in vitro
Medicine curve, is shown in Fig. 3.The rate that two drugs are discharged from polymer micelle is suitable, and 75% or so medicine was about discharged at 24 hours
Object.
Precision pipettes gram azoles for preparing in embodiment 1 dividing again for Buddhist nun and 17-AAG composition polymer nano-micelle freeze-dried powder
Dispersion liquid 0.1ml, in cell culture medium gradually 10 times be diluted to series of concentrations, be added in the cell of 96 orifice plate cultures of culture
(NCI-H3122 grams of azoles of Non-small cell lung carcinoma replaces Buddhist nun's drug-resistant cell strain, 4000 cells/wells), after culture 24 hours, by drug containing
Culture medium, which removes and new culture medium is added, to be continued after cultivating 72 hours, and MTT reagent (100 micro- g holes) are added at 37 DEG C and are further cultured for 3
Hour, culture medium containing MTT is removed, 100 microlitres of DMSO are added and read data with plate reading machine under 562 nano wave lengths
(SpectraMax M5, Molecular Devices plate reading machine).Cells survival rate by with the non-dosing cell number meter of control group
It obtains, and average half-inhibitory concentration (IC50) is obtained by survival rate and cell concentration curve；Same operation will be independent
Drug, which is respectively added in cell, measures IC50 (Fig. 4).Drug combination is substantially reducing at NCI-H3122 grams of azoles and replaces compared with single medicine
IC50 in Buddhist nun's mdr cell.
Synergy index (combination index, CI) analysis is based on Chou and Talalay method and to adopt
It is calculated with CompuSyn software.Briefly, the case where being combined for double medicines, in each inhibiting rate degree (fraction of
Affected, Fa) on synergy index can be to be calculated by following equations: CI=(D) 1/ (Dx) 1+ (D) 2/
(Dx) 2, wherein (D) 1 and (D) 2 are with reaching specific Fa degree concentration of each drug in compound needed for cell inhibitory rate；And
(Dx) 1 and (Dx) 2 is single use a drug with can generating Fa degree concentration needed for cell inhibitory rate.Then collaboration is imitated
Fruit index CI maps to inhibiting rate degree.The CI obtained between Fa=0.2 and Fa=0.8 under normal conditions is considered as effectively, and
CI when IC50 (i.e. Fa=0.5) is used to evaluate whether medicaments compound has strong synergy in different ratio.Such as
Fruit CI value is to have synergy, and being greater than 1 is neutralization effect, is considered as pharmaceutical composition equal to 1 and does not cooperate with effect less than 1
Fruit, only simple superposition (Fig. 5).Index of cooperation when Fig. 5 is shown in IC50 is 0.32, indicate gram azoles of embodiment 1 for Buddhist nun with
17-AAG composition polymer nano-micelle has very strong inhibition tumour cell (NCI-H3122 grams of azoles is for Buddhist nun's drug-resistant cell strain) growth
Synergy.
Claims (11)
1. a kind of with synergy gram of azoles is for Buddhist nun and 17-AAG composition polymer nano-micelle injection, it is characterised in that:
1 parts by weight of Buddhist nun, 0.5~3 parts by weight of 17-AAG, 10~100 weight of nano-micelle polymer are replaced comprising gram azoles in the preparation
Part；The nano-micelle polymer is poly- (2- ethyl -2- oxazoline)-polylactic acid, i.e. PEOZ-PLA or poly- (2- methyl -
2- oxazoline)-polylactic acid, i.e. PMOZ-PLA.
2. injection according to claim 1, it is characterised in that: in poly- (2- ethyl -2- the oxazoline)-polylactic acid
That is in PEOZ-PLA, the degree of polymerization of poly- (2- ethyl -2- oxazoline) PEOZ is 20~60 units, average molecular weight 1700 to
The degree of polymerization of 5100Da, polylactic acid PLA are 30~115 units, and average molecular weight is 850 to 3300Da.
3. injection according to claim 1, it is characterised in that: poly- (2- methyl -2- the oxazoline)-polylactic acid, i.e.,
In PMOZ-PLA, the degree of polymerization of poly- (2- methyl -2- oxazoline) PMOZ is 20~60 units, average molecular weight 1700 to
The degree of polymerization of 5100Da, the polylactic acid PLA are 30~115 units, and average molecular weight is 850 to 3300Da.
4. injection according to claim 1, it is characterised in that: replace 1 parts by weight of Buddhist nun, 17- comprising gram azoles in the preparation
1 parts by weight of AAG, 10 parts by weight of nano-micelle polymer；The nano-micelle polymer is poly- for poly- (2- methyl -2- oxazoline) -
Lactic acid, i.e. PMOZ-PLA, wherein the degree of polymerization of poly- (2- methyl -2- oxazoline) PMOZ is 50 units, and average molecular weight exists
The degree of polymerization of 4400Da, the polylactic acid PLA are 115 units, and average molecular weight is in 3300Da.
5. injection according to claim 1, it is characterised in that: described gram of azoles is for Buddhist nun and 17-AAG composition polymer nanometer
The average grain diameter of micella injection is 20~200nm.
6. injection according to claim 5, it is characterised in that: described gram of azoles is for Buddhist nun and 17-AAG composition polymer nanometer
The average grain diameter of micella injection is 30~100nm.
7. injection according to claim 6, it is characterised in that: described gram of azoles is for Buddhist nun and 17-AAG composition polymer nanometer
The average grain diameter of micella injection is 35nm.
8. such as the preparation method of injection of any of claims 1-7, it is characterised in that: the preparation method includes
Following steps:
1) gram azoles is dissolved in ethyl alcohol or acetonitrile for Buddhist nun and 17-AAG and nano-micelle polymer respectively, is made into stock solution, then
Gram azoles is measured according to prescription to mix for Buddhist nun, 17-AAG and the mixing of nano-micelle polymer stock solution, oscillation；
2) obtained gram azoles is injected into round-bottomed flask or other samples for Buddhist nun, 17-AAG and nano-micelle mixed with polymers organic phase
In product bottle, in slightly heating process, decompression or normal pressure under organic solvent is slowly evaporated into it is completely dry, bottom of bottle formed drug
With nano-micelle polymer material mixing film layer；
3) 35~50 DEG C are warming up to, distilled water or water for injection is added, it is molten that slight oscillatory to film layer is completely dissolved into clarification
Liquid；
4) clear solution placement is cooled to room temperature, is centrifuged, gram azoles of the unloaded in micella is centrifuged for Buddhist nun and 17-AAG and is sunk
Form sediment and remove, take supernatant be freeze-dried gram azoles for Buddhist nun and 17-AAG composition polymer nano-micelle injection freeze-dried powder.
9. preparation method according to claim 7, it is characterised in that: in the step 3), be warming up to 45 DEG C.
10. preparation method according to claim 7, it is characterised in that: in the step 4), be added when freeze-drying and protect
Protect agent, the freeze drying protectant be selected from any one of mannitol, sucrose, lactose, trehalose, maltose and glucose or its
Mixture, the dosage of the freeze drying protectant are 5-10g/100ml grams of azoles for Buddhist nun and 17-AAG composition polymer nano-micelle.
11. as of any of claims 1-7 gram of azoles exists for Buddhist nun and 17-AAG composition polymer nano-micelle injection
Prepare the application in tumor, it is characterised in that: the tumour is ALK positive lung cancer in non-cellule type and drug resistance
ALK positive non-small cell lung cancer drug.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104116710A (en) * | 2013-04-23 | 2014-10-29 | 北京大学 | Tumor-targeting pH-sensitive polymeric micelle composition |
| CN104116709A (en) * | 2013-04-23 | 2014-10-29 | 北京大学 | Tumor-targeting pH-sensitive polymeric micelle composition resisting tumor drug resistance |
| CN106456795A (en) * | 2014-03-03 | 2017-02-22 | 辛塔医药品有限公司 | Targeted therapeutics |
| CN106983719A (en) * | 2017-03-08 | 2017-07-28 | 江苏富泽药业有限公司 | A kind of docetaxel polymer nano micelle injection, its preparation method and its application in tumor is prepared |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104116710A (en) * | 2013-04-23 | 2014-10-29 | 北京大学 | Tumor-targeting pH-sensitive polymeric micelle composition |
| CN104116709A (en) * | 2013-04-23 | 2014-10-29 | 北京大学 | Tumor-targeting pH-sensitive polymeric micelle composition resisting tumor drug resistance |
| CN106456795A (en) * | 2014-03-03 | 2017-02-22 | 辛塔医药品有限公司 | Targeted therapeutics |
| CN106983719A (en) * | 2017-03-08 | 2017-07-28 | 江苏富泽药业有限公司 | A kind of docetaxel polymer nano micelle injection, its preparation method and its application in tumor is prepared |
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