CN102805729A - Vinflunine liposome preparation and preparation method of vinflunine liposome preparation - Google Patents
Vinflunine liposome preparation and preparation method of vinflunine liposome preparation Download PDFInfo
- Publication number
- CN102805729A CN102805729A CN201110149433XA CN201110149433A CN102805729A CN 102805729 A CN102805729 A CN 102805729A CN 201110149433X A CN201110149433X A CN 201110149433XA CN 201110149433 A CN201110149433 A CN 201110149433A CN 102805729 A CN102805729 A CN 102805729A
- Authority
- CN
- China
- Prior art keywords
- liposome
- vinflunine
- phospholipid
- ammonium
- dspe
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to a vinflunine liposome preparation and the preparation method thereof. The vinflunine liposome preparation mainly comprises vinflunine or salt, phospholipid, cholesterin and polyethylene glycol-distearoyl phosphatidyl ethanolamine of the vinflunine, wherein salt, phospholipid, cholesterin and polyethylene glycol-distearoyl phosphatidyl ethanolamine are pharmaceutically acceptable, and the vinflunine liposome preparation can be prepared into an injection or a lyophilized preparation. The preparation method is simple in technology, easy to operate and suitable for industrial production, and long-circulating vinflunine liposomes prepared by using the method are high in encapsulation rate and good in stability.
Description
Technical field
The invention belongs to field of pharmaceutical preparations, be specifically related to Liposomal formulation of a kind of antitumor drug vinflunine and preparation method thereof.
Background technology
Liquor epinephrinae bitartratis ophthalmicus vinflunine (vinflunine ditartrate) is a kind of novel semisynthetic vinca alkali by the exploitation of French Pierre Fabre company, through suppressing its antitumor action of cell proliferation performance of tubulin.Be used to treat bladder cancer, nonsmall-cell lung cancer, breast carcinoma and ovarian cancer etc.The difference of it and Vinorelbine monotartrate is on C-20 ' position, to replace two hydrogen atoms by two fluorine atoms, and (Crit Rev Oncol Hematol.2001,40:159-173), liquor epinephrinae bitartratis ophthalmicus vinflunine structural formula is as shown in the formula shown in the I.Find that in the external anticancer test of people and 13 kinds of tumor models of muroid vinflunine has better active anticancer and less neurovirulent characteristics than vinorelbine, referring to Eur J Cancer, 1999,35:512-520.Yet still there is certain dose-limiting toxicity reaction in this medicine, mainly comprises mucositis, constipation, neutrophilic granulocytopenia, referring to Br J Cancer, 2006,95:1161-1166.
CN101695474A (application number 200910169123.7; Applying date 2004.12.17) a kind of vinflunine medical composition that is used for parenteral administration is disclosed; According to this invention; The vinflunine medical composition, it adopts the sterile water solution form of stable hydrosoluble vinflunine salt between pH3 to 4, perhaps optionally add buffer.Said parenteral administration is intravenous administration particularly, is used to treat cancer.
Liposome is a kind of novel form of targeting drug delivery system; It is one of focus of pharmaceutics research always; It can with drug selectivity be transported to tumor locus; The performance therapeutical effect does not influence the function of normal cell, tissue or organ again simultaneously, thereby reaches the purpose that improves curative effect, reduces toxic and side effects.Liposome has the structure that is similar to cell membrane, constitutes the film forming inner surface of the hydrophilic head shape of lipoid of bilayer, and lipophilic afterbody then is in the centre of film.Just because this membranelike structure of liposome makes it can carry various hydrophilic, hydrophobic and amphipathic materials, their are wrapped into the liposome interior water, insert the surface that type lipid bilayer or absorption are connected liposome.
Liposome had both played the protective effect to medicine as pharmaceutical carrier, had improved the targeting property of medicine to the body specific part again, therefore had a lot of superior characteristic aspect the raising drug effect.Mainly show the following aspects: 1. liposome is nontoxic or toxic and side effects is little to body, and its liposome bilayer and biomembrane have bigger similarity and tissue intersolubility, thereby are easy to by tissue absorption, have improved the absorption rate of medicine; 2. wrapping kmedicine by liposome is a physical process, does not change drug molecular structure, can not destroy ingredient, and the medicine of parcel then can be avoided being destroyed by hydrolytic enzyme in the body; 3. the medicine behind the parcel can reduce its toxicity to the body specific part, reduces the drug use amount, makes medicine have slow release and controlled-release function; 4. different with viral vector, liposome can biodegradation in host, non-immunogenicity; 5. in gene therapy, be easy to gene compoundly, higher targeting property is arranged, can realize the treatment of specific cells; 6. material is cheap, is convenient to a large amount of preparations.
Very soon by reticuloendothelial system phagocytic, and main targeting was relatively poor to the targeting property at other positions in the organs of reticuloendothelial system than horn of plenty such as liver, spleen, bone marrow after conventional liposome got in the body.And it is subject to the effect of albumen, enzyme etc. in vivo and seepage takes place, and affects the treatment and toxicity is increased.The eighties in last century; Discoveries such as Canada scientist Allen; Similar with monosialoganglioside; The DSPE of the Pegylation that in liposome, adds (PEG-DSPE) can prolong the RT of liposome in blood, so this liposome is called hidden liposome.There is the longer time in the lipid physical ability that palmityl glucosiduronate or PEG quasi-grease derivative are contained in the surface of developments such as Klibanov in blood circulation, and increases the absorption of target site, so be called spatial stability liposome or long circulating liposomes (LCL).Therefore; LCL adds a certain proportion of glycolipid or on its phosphoric acid molecules, connects the protectiveness polymer in the constituent of liposome; Like the derivant-DSPE-Polyethylene Glycol (PEG-DSPE) of the distearyl of amphipathic PEG acid phospholipid amide, polyacrylamide, polyvinylpyrrolidone (PVP) etc.; Make some hydrophilic polysaccharide or polyhydroxy group be exposed to surface of liposome; Thereby reduce opsonic combination the in liposome and the blood plasma, it is stable to increase its blood, prolong half-life and the medicine time in blood.Method for preparing lipidosome commonly used has film dispersion method, injection method, transmembrane gradient active loading method, ultrasonic dispersing method, freeze-drying, reverse evaporation etc.
Summary of the invention
Main purpose of the present invention provides the vinflunine Liposomal formulation of a kind of envelop rate height, good stability; Said preparation can be escaped reticuloendothelial system (RES) phagocytosis in vivo; And can obviously reduce common vinflunine injection toxic and side effects; Be a kind of injectable formulation, this preparation has the effect of tumor passive target, compares with normal injection and can improve the oncotherapy effect.
The present invention also provides the method for preparing of vinflunine Liposomal formulation.Compare with prior preparation method, method for preparing provided by the present invention has entrapment efficiency height, liposome stability and reaches advantages such as technology is easy well, more helps realizing suitability for industrialized production.
The term explanation:
Liposome: liposome according to the invention has the general sense on the pharmaceutics, means drug encapsulation in the lipoids bilayer and the micro-bubbles utricule that forms; With phospholipid is main film material.
Multilamelar liposome: liposome is different according to the number of plies of the lipoids bilayer that is comprised, and is divided into unilamelar liposome and multilamelar liposome.The vesicle of plurality of layers of double molecular layer is called multilamelar liposome.
The DSPE of PEGization (PEG-DSPE): the DSPE of Pegylation is a kind of long circulation film material; MPEG
2000-DSPE: methoxy poly (ethylene glycol) 2000 DSPEs; But market is buied, and also can prepare voluntarily by prior art.
Technical scheme of the present invention is following:
The Liposomal formulation of vinflunine comprises the DSPE (PEG-DSPE) of medicine vinflunine, phospholipid, cholesterol and Pegylation, wherein,
The DSPE of Pegylation (PEG-DSPE) is 0.05～0.5: 1 with the mass ratio of phospholipid;
The mass ratio of C/PL is 0.1～0.5: 1;
In vinflunine, the mass ratio of medicine and phospholipid is 1: 3～20, and said drug encapsulation is in liposome interior of being made up of phospholipid bilayer and phospholipid bilayer.
Because medicine is encapsulated in liposome interior, can effectively reduce the sickness rate of side effect such as common clinically blood vessel irritation of vinflunine and phlebitis after therefore getting in the body; In addition, drug encapsulation can effectively be avoided the phagocytosis of reticuloendothelial system in liposome interior behind the entering human body, has prolonged the interior half-life of body of medicine, reaches macrocyclic effect; Because the vascular permeability of tumor locus is higher, therefore can make more medicine concentrate on tumor locus simultaneously, reach the passive target effect.
According to the present invention, described vinflunine exists with the form of bitartrate, and perhaps the form with pharmaceutically acceptable other soluble-salts exists.
According to the present invention, described phospholipid is selected from a kind of or combination in synthetic or semi-synthetic phospholipid, the natural phospholipid; Wherein, Said synthetic or semi-synthetic phospholipid is selected from distearyl acid phosphatidylcholine (DSPC), two Palmic acid phosphatidylcholines (DPPC), dimyristoyl phosphatidyl choline (DMPC) or hydrogenated soya phosphatide (HSPC), and said natural phospholipid is egg yolk lecithin, soybean phospholipid or sphingomyelins.
In order to increase the spatial stability of liposome, give liposome long cycle performance, prolong the interior half-life of body of liposome, the present invention adopts the DSPE (PEG-DSPE) of long recycled material Pegylation; The mass ratio of PEG-DSPE and phospholipid is preferably 0.1～0.5: 1.
Adopt cholesterol further to improve the stability of liposome in the above-mentioned vinflunine Liposomal formulation, the mass ratio of C/PL is preferably 0.2～0.3: 1.
According to the present invention, described vinflunine Liposomal formulation is injection formulation or lyophilized formulations.
In addition; According to different preparation technologies; Also contain a small amount of buffer salt in the above-described vinflunine Liposomal formulation or other can help medicine to be written into the organic salt or the inorganic salt of liposome interior, buffer salt commonly used comprises citrate buffer, phosphate buffer, carbonate buffer solution etc.; Organic salt or inorganic salt that described help medicine is written into liposome interior are selected from sodium carbonate, sodium phosphate, sodium ethylene diamine tetracetate, sucrose octasulfate ammonium, ammonium sulfate, ammonium acetate, ammonium chloride or ethylenediaminetetraacetic acid ammonium etc.
According to the present invention, also comprise the buffer of ammonium salt or pH3.0-6.0 in the preferred described vinflunine Liposomal formulation.Ammonium salt and buffer add when the preparation of liposome, but major part is removed in the preparation process, therefore have only very small amount to be saved in the final preparation.For example ammonium sulfate is present in blank liposome inside with the form of sulfate radical and ammonium root, and the ammonium root becomes that ammonia vapors away and sulfate radical combines with medicine behind medicine carrying; The buffer of pH3.0-6.0 pH after medicine carrying can raise and exceed this scope of pH3.0-6.0 for another example.The above ammonium salt comprises ammonium sulfate, ammonium acetate, ammonium chloride, ethylenediaminetetraacetic acid diammonium, sucrose eight ammonium sulfate etc.Its effect is through liposome interior and the outside ammonium gradient method that forms medicine to be written into liposome interior.The concentration range of said ammonium salt is 50-500mM.The buffer of the above pH3.0-6.0 comprises phosphate buffer, citrate buffer, carbonate buffer solution, acetate buffer etc., and its concentration range is 50-500mM.
In the preferred scheme, also contain one or more freeze drying protectants in the described vinflunine lipidosome freeze-dried preparation according to the present invention, freeze drying protectant is selected from sucrose, maltose, trehalose, lactose, galactose, glucose, mannitol.
According to the present invention, one of preferred scheme, described vinflunine lipidosome injection preparation contains in every 1000ml preparation: liquor epinephrinae bitartratis ophthalmicus vinflunine 1g-20g, phosphatidase 13 g-400g, PEG-DSPE 1.5g-200g, cholesterol 0.3g-200g.
Above-mentioned vinflunine lipidosome injection preparation also contains citrate buffer, phosphate buffer, carbonate buffer solution, sodium carbonate, sodium phosphate, sucrose octasulfate ammonium, sodium ethylene diamine tetracetate or the ethylenediaminetetraacetic acid ammonium of the small amount of residual that in the preparation process, adds inevitably.
According to the present invention, one of preferred scheme, described vinflunine lipidosome freeze-dried preparation; Contain in every 1000ml preparation before the lyophilizing: liquor epinephrinae bitartratis ophthalmicus vinflunine 1g-20g; Phospholipid, PEG-DSPE, cholesterol, freeze drying protectant, wherein; Phospholipid is 5-15 times of liquor epinephrinae bitartratis ophthalmicus vinflunine quality, and the mass ratio of PEG-DSPE and phospholipid is 0.1～0.4: 1; The mass ratio of C/PL is 0.2～0.3: 1; Freeze drying protectant is the 5-20% of liquid preparation quality before the lyophilizing.
Freeze drying protectant is selected from one of sucrose, maltose, trehalose, lactose, galactose, glucose or mannitol or combination; Described vinflunine lipidosome freeze-dried preparation also can contain citrate buffer, phosphate buffer, carbonate buffer solution, sodium carbonate, sodium phosphate, sucrose octasulfate ammonium, sodium ethylene diamine tetracetate or the ethylenediaminetetraacetic acid ammonium salt of the small amount of residual that in the preparation process, adds.
The method for preparing of vinflunine Liposomal formulation according to the invention comprises the steps:
The preparation of a, blank liposome
(1) takes by weighing phospholipid, PEG-DSPE and cholesterol by prescription and be dissolved in an amount of organic solvent, fully dissolving;
(2) preparation contains the aqueous solution of ammonium salt or the pH of buffer salt is the buffer of 3.0-6.0, and it is added in the solution that above-mentioned steps (1) makes, and under 40-70 ℃ of temperature conditions stirring reaction 10-60 minute, obtains multilamelar liposome;
(3) adopt high pressure homogenize, extrude, the method for ultrasonic or freeze thawing is reduced to 80-500nm with the mean diameter of the multilamelar liposome that step (2) obtains, and promptly obtains blank liposome;
The preparation of b, gradient liposome solutions
The blank liposome that step a is made by prior art is processed the system with the inside and outside ion gradient of liposome or the system of the inside and outside proton gradient of liposome; Make the pH value of the outer water of liposome be higher than the liposome interior pH value, thereby perhaps make the ammonium salt of the outer aqueous phase of liposome remove formation liposome inside and outside ammonium ion gradient;
C, medicine carrying
The gradient liposome solutions that step b is obtained places 30-60 ℃ of water-bath temperature to bathe, and gets recipe quantity medicine vinflunine solution and is added in the liposome solutions, and 30-60 ℃ was stirred 5-60 minute, and can obtain the vinflunine liposome solutions;
D, the outer water free drug of removal
Adopt in the vinflunine liposome solutions that dialysis, ultrafiltration or gel filtration chromatography method obtain step c non-encapsulated free drug to remove;
Promptly obtain vinflunine long circulating liposomes injection formulation after e, the filtration sterilization of steps d gained solution;
Continue following steps and prepare vinflunine long circulating liposomes lyophilized formulations:
Add freeze drying protectant in the vinflunine long circulating liposomes injection that f, step e obtain, carry out lyophilizing, get lyophilized formulations by prior art; Freeze drying protectant is selected from one of sucrose, maltose, trehalose, lactose, galactose, glucose or mannitol or combination; The amount of freeze drying protectant accounts for the 5-20% of the preceding liquid preparation quality of lyophilizing.
Organic solvent described in the above-mentioned steps a is selected from dehydrated alcohol, the tert-butyl alcohol, ether, dichloromethane, chloroform, methanol or ethyl acetate, wherein preferred dehydrated alcohol; The consumption of organic solvent gets final product with the DSPE and the cholesterol dissolving that can make phospholipid, PEGization, and the addition of preferred organic solvent and the mass ratio of phospholipid are 1: 5～20.
Ammonium salt described in the above-mentioned steps a is selected from sucrose eight ammonium sulfate, ammonium sulfate, ammonium acetate, ammonium chloride or ethylenediaminetetraacetic acid ammonium; Preferably, the concentration of said ammonium salt is 50-500mM; Described buffer is selected from citrate buffer, phosphate buffer or carbonate buffer solution; Preferably, the concentration of said buffer salt is 50-500mM.
Prior art described in the above-mentioned steps a mainly contains pH gradient method or ammonium ion gradient method etc., and the present invention is preferably following:
I, pH gradient method: adopt alkaline solution sodium hydroxide or sodium radio-phosphate,P-32 solution that the pH value of water outside the blank liposome of step a preparation is transferred to and be higher than liposome internal pH 3.0-6.0, outer aqueous pH values is adjusted to 6.5-8.0; Utilize the different of the inside and outside pH value of liposome, medicine is written into liposome interior.
Ii, ammonium ion gradient method: the ammonium salt removal of the outer aqueous phase of the blank liposome of step a preparation is made it to form liposome inside and outside ammonium ion gradient through dialysis, ultrafiltration, gel filtration chromatography method.
The ammonium ion gradient method comprises ammonium sulphate gradient, sucrose octasulfate ammonium salt gradient method, EDTA ammonium salt gradient method etc.
The vinflunine Liposomal formulation that the present invention is prepared, the mass ratio of vinflunine medicine and phospholipid are 0.05 :-0.5: 1, be called for short medicine fat ratio, and mean diameter is 80-500nm, entrapment efficiency is greater than 95%.
Need not when preparing blank liposome among the said method step a organic solvent of dissolving film material is removed, the residual quantity in the final preparation is less than 20wt%, preferably less than 10wt%.
Preferred according to the present invention, the method for preparing of vinflunine Liposomal formulation, adopt one of following method:
1. the liposome membrane material is hydrogenated soya phosphatide, cholesterol and PEG-DSPE, and mass ratio 1-5: 1: 0.5-1 with an amount of dissolve with ethanol, adds ammonium sulfate 200-300mmolL
-1, 50-70 ℃ of water-bath aquation 30-60 minute, extruded or supersound process reduces particle diameter to 100-300nm high pressure homogenize, adopts 0.22 μ m filtering with microporous membrane degerming then, removes outer water ammonium sulfate, must the ammonium sulphate gradient liposome.Liquor epinephrinae bitartratis ophthalmicus vinflunine normal saline solution is added in this gradient liposome, and liquor epinephrinae bitartratis ophthalmicus vinflunine content is the 10%-50% of hydrogenated soya phosphatide content; 30-60 ℃ of water bath heat preservation medicine carrying 20-40 minute, 0.22 μ m microporous filter membrane aseptic filtration, and after removing outer water free drug, make liposome.Gel column method or dialysis are measured envelop rate.
2. the liposome membrane material is hydrogenated soya phosphatide, cholesterol and PEG-DSPE, and mass ratio 5-10: 1: 0.1-0.5 with an amount of dissolve with ethanol, adds citric acid-sodium citrate buffer 200-300mmolL
-1, pH3.0-6.0,50-70 ℃ of water-bath aquation 10-60 minute obtains the blank liposome first product; High pressure homogenize, extrude, ultrasonic or freeze thawing treatment reduces particle diameter to 50-300nm, adopts 0.22 μ m microporous filter membrane degerming then, blank liposome.Get blank liposome suspension 10.0mL, add 200-300mmolL
-1Sodium radio-phosphate,P-32 solution mixes, and adds the liquor epinephrinae bitartratis ophthalmicus vinflunine subsequently, and liquor epinephrinae bitartratis ophthalmicus vinflunine content is the 20%-40% of hydrogenated soya phosphatide content; 40-60 ℃ is incubated 5-60 minute, 0.22 μ m microporous filter membrane aseptic filtration, and make the vinflunine liposome after removing outer water free drug.Gel column method or ultrafiltration are measured envelop rate.
3. the liposome membrane material is hydrogenated soya phosphatide, cholesterol and PEG-DSPE, and mass ratio 10-20: 1: 0.01-0.1 with an amount of dissolve with ethanol, adds ethylenediaminetetraacetic acid ammonium 100-300mmolL
-1, pH4-6.0,40-70 ℃ of water-bath aquation 20-60 minute; High pressure homogenize, freeze thawing, extrude or supersound process reduces particle diameter to 100-200nm; Adopt 0.22 μ m filtering with microporous membrane degerming then, outer water ethylenediaminetetraacetic acid ammonium (EDTA-ammonium salt) is removed in dialysis, gets EDTA-ammonium gradient liposome.The liquor epinephrinae bitartratis ophthalmicus vinflunine is added in this gradient liposome, and liquor epinephrinae bitartratis ophthalmicus vinflunine content is the 10%-40% of hydrogenated soya phosphatide content; 30-60 ℃ of water bath heat preservation medicine carrying 5-40 minute, 0.22 μ m microporous filter membrane aseptic filtration, and make the vinflunine liposome after removing outer water free drug.
4. the liposome membrane material is hydrogenated soya phosphatide, cholesterol and PEG-DSPE; Mass ratio 5-15: 1: 0.5-1 with an amount of dissolve with ethanol, adds the solution that contains the sucrose octasulfate ammonium salt; 50-70 ℃ of water-bath aquation 10-60 minute; High pressure homogenize, extrude, ultrasonic, freeze thawing etc. handles and reduces particle diameter to 80～200nm, outer water sucrose octasulfate ammonium salt is removed in ultrafiltration, must the gradient liposome.The liquor epinephrinae bitartratis ophthalmicus vinflunine is added in this gradient liposome, and liquor epinephrinae bitartratis ophthalmicus vinflunine content is the 20%-30% of hydrogenated soya phosphatide content; 30-60 ℃ of water bath heat preservation medicine carrying 5-60 minute, 0.22 μ m filtering with microporous membrane degerming, and make the vinflunine liposome after removing outer water free drug.
Because the liquor epinephrinae bitartratis ophthalmicus vinflunine is a kind of water solublity antitumor drug, so medicine is encapsulated in the internal water phase space that is surrounded by bilayer lipid membrane after being prepared into Liposomal formulation.Water soluble drug adopts its envelop rate of liposome and the drug loading of method preparation of passive medicine carrying all lower, so the present invention adopted the method for the active medicine carrying of comparatively using always at present, comprises pH gradient method and ammonium gradient method etc.The present invention uses this scheme, and the Liposomal formulation drug loading of preparation is high, and envelop rate can reach more than 98%, and the drug leakage rate is low.And liposome can be placed for a long time and the downgrade phenomenon can not take place.
Film dispersion method is generally adopted in the preparation of prior art empty liposome; Fabricating technology generally is that film materials such as phospholipid, cholesterol are dissolved in an amount of organic solvent; Fully all remove organic solvent through revolving processing steps such as steaming, drying the dissolving back, is prepared into exsiccant thin film and carries out aquation afterwards again.The present invention improves this technical process, and the film material is dissolved in an amount of organic solvent, fully can directly carry out aquation after the dissolving, therefore needn't process exsiccant thin film, thereby reduce the complexity of technology, helps suitability for industrialized production.
Compared with prior art, major advantage of the present invention comprises:
(1) preparation technology is simple: compare with existing Liposomal formulation preparation technology; The active loading method preparation technology that the present invention adopts is simple, operation is prone to row; Saved to revolve for a long time and steamed and dry run, reduced the probability of phospholipid oxidation, be particularly suitable for industrial amplification production.
(2) the present invention is single bottled preparation, and medicine wraps into behind the liposome more stable, is not easy to leak.Liposome mean diameter 80-500nm, envelop rate are more than 95%, and the liposome behind the medicine carrying can be stablized placement 36 months and the main all no significant change of quality index in 2-8 ℃; Medicine is present in liposome interior with the form of complex, all be not easy in vivo and in vitro to leak, so release is slow, and the body internal stability is better.
(3) preparation of the present invention can be a lyophilized formulations, is convenient to preserve.
(4) easy to use: as can be directly during clinical use liposome to be got final product with 5% glucose dilution.
(5) drug toxicity and zest reduce: because medicine is encapsulated in liposome interior, therefore can effectively reduce blood vessel irritation and the phlebitic sickness rate that occurs in the clinical use of injection; Change the tissue distribution and the pharmacokinetic property of medicine after processing Liposomal formulation, so reduced toxic and side effects such as haematics toxicity and gastrointestinal toxicity; In addition, drug encapsulation can effectively be avoided the phagocytosis of reticuloendothelial system in liposome interior behind the entering human body, prolonged the half-life of medicine, can improve therapeutic index; And smaller particle size can improve the release of medicine at tumor locus, has certain targeting property.
Description of drawings
The liquor epinephrinae bitartratis ophthalmicus vinflunine liposome release in vitro curve (n=3) of the different medicine fat of Fig. 1 ratio.
Fig. 2 liquor epinephrinae bitartratis ophthalmicus vinflunine liposome is to the inhibition effect of human colon carcinoma HT-29 transplanted tumor in nude mice, abscissa express time (unit be day), and vertical coordinate is represented relative tumour volume.Article four, curve is corresponding four groups respectively: group (medicine fat was than 1: 10 for I negative control group, II liquor epinephrinae bitartratis ophthalmicus vinflunine injection group (adopting the liquor epinephrinae bitartratis ophthalmicus vinflunine to add the preparation of injection water), III liquor epinephrinae bitartratis ophthalmicus vinflunine liposome-1; According to the preparation of embodiment 2 methods) and IV liquor epinephrinae bitartratis ophthalmicus vinflunine liposome-2 group (medicine fat is than 1: 5, according to the preparation of embodiment 3 methods).
The specific embodiment
Further specify the present invention below in conjunction with embodiment, but be not limited to practical range.Those skilled in the art can make various modifications or improvement according to basic thought of the present invention, but only otherwise break away from basic thought of the present invention, all within scope of the present invention.
" vinflunine or pharmaceutically acceptable soluble-salt " abbreviates " vinflunine " or " medicine " sometimes as in this description.
The calculating of envelop rate: envelop rate=[medicine/(the outside free medicine of medicine+liposome that liposome interior is sealed) that liposome interior is sealed] * 100%; The normal separation methods such as polydextran gel, supercentrifugation, dialysis that adopt are separated free medicine in the solution with liposome; Measure the computational envelope rate respectively.
Polydispersity index (PDI): reacted the distribution situation of liposome particle diameter, the more for a short time particle size distribution that shows of numerical value is concentrated more.Embodiment 1, passive medicine carrying legal system are equipped with vinflunine liposome (Comparative Examples)
The liposome membrane material of sealing vinflunine is: hydrogenated soya phosphatide, cholesterol and PEG-DSPE, mass ratio 1: 0.2: 0.1.
Film dispersion method
The liposome membrane material is dissolved in an amount of ethanol, and ethanol is removed in decompression, forms exsiccant thin film, adds liquor epinephrinae bitartratis ophthalmicus vinflunine normal saline solution (4mg/ml), and the hydrated phospholipid film makes liposome.Through microjet particle diameter is reduced to about about 200nm.
Reverse phase evaporation
An amount of dichloromethane-ether (1: 1V/V) dissolving film material, add liquor epinephrinae bitartratis ophthalmicus vinflunine normal saline solution (4mg/ml), carry out ultrasonic in short-term, until forming stable w/o type Emulsion; Removal of solvent under reduced pressure adds the normal saline aquation, makes liposome.Through microjet particle diameter is reduced to about 200nm.
Alcohol injection
The liposome membrane material is gone into aquation in the liquor epinephrinae bitartratis ophthalmicus vinflunine normal saline solution with an amount of dissolve with ethanol in the following marginal not that stirs of 60 ℃ of conditions, makes liposome.Through microjet particle diameter is reduced to about 200nm.
The liposome encapsulation of measuring method for preparing through the gel column method is respectively 23.8%, 47.6% and 36.2%; Wherein the liposome of alcohol injection preparation is placed 4 hours postprecipitations in room temperature.
Embodiment 2, ammonium sulphate gradient prepare the vinflunine liposome
The liposome membrane material is hydrogenated soya phosphatide, cholesterol and PEG-DSPE, and mass ratio 1: 0.2: 0.1 with an amount of dissolve with ethanol, adds ammonium sulfate 300mmolL
-1, 60 ℃ of water-bath aquations 40 minutes, high pressure homogenizer is handled and is reduced particle diameter to about 200nm, adopts 0.22 μ m filtering with microporous membrane degerming then, and outer water ammonium sulfate is removed in dialysis, gets the ammonium sulphate gradient liposome.Liquor epinephrinae bitartratis ophthalmicus vinflunine normal saline solution is added in this gradient liposome, and 60 ℃ of water bath heat preservation medicine carryings 10 minutes promptly get the vinflunine liposome after the aseptic filtration, and wherein the content of liquor epinephrinae bitartratis ophthalmicus vinflunine is 2mg/ml.After removing outer water free drug, it is 98% that the gel column method is measured envelop rate.
The liposome membrane material is hydrogenated soya phosphatide, cholesterol and PEG-DSPE, and mass ratio 1: 0.2: 0.1 with an amount of dissolve with ethanol, adds citrate buffer 300mmolL
-1, pH4.0,65 ℃ of water-bath aquations 20 minutes obtain the blank liposome first product; High pressure homogenizer is handled and is reduced particle diameter to 150～200nm, adopts 0.22 μ m microporous filter membrane degerming then, gets blank liposome.
Get blank liposome suspension 10.0mL, add 500mmolL
-1Sodium radio-phosphate,P-32 solution 6mL mixes, and adds 4mgmL subsequently
-1Liquor epinephrinae bitartratis ophthalmicus vinflunine normal saline solution 5mL, 60 ℃ are incubated 10 minutes, make liposome after the aseptic filtration.It is 95.4% that the gel column method is measured envelop rate.
Embodiment 4 EDTA ion gradient legal systems are equipped with the vinflunine liposome
The liposome membrane material is hydrogenated soya phosphatide, cholesterol and PEG-DSPE, and mass ratio 1: 0.2: 0.1 with an amount of dissolve with ethanol, adds EDTA-ammonium salt (300mmolL
-1, pH5.0), 60 ℃ of water-bath aquations 20 minutes, high pressure homogenizer is handled and is reduced particle diameter to 180～200nm, adopts 0.22 μ m filtering with microporous membrane degerming then, and outer water EDTA-ammonium salt is removed in dialysis, must EDTA-ammonium gradient liposome.Liquor epinephrinae bitartratis ophthalmicus vinflunine normal saline solution is added in this gradient liposome, and 60 ℃ of water bath heat preservation medicine carryings 10 minutes promptly get the vinflunine liposome, wherein the content 4mg/ml of liquor epinephrinae bitartratis ophthalmicus vinflunine.Remove free drug and outer water is replaced into the histidine sucrose buffer of pH7.0, it is 99.2% that the gel column method is measured envelop rate, and Ma Erwen particle size analyzer determination mean diameter is 125nm, and polydispersity index (PDI) is 0.05.
Embodiment 5, sucrose octasulfate gradient method prepare the vinflunine liposome
The liposome membrane material is hydrogenated soya phosphatide, cholesterol and PEG-DSPE; Mass ratio 1: 0.2: 0.1; With an amount of dissolve with ethanol, add the solution contain the sucrose octasulfate ammonium salt, 60 ℃ of water-bath aquations 20 minutes; Microjet is handled and is reduced particle diameter to about 200nm, and outer water sucrose octasulfate ammonium salt is removed in the tangential flow ultrafilter ultrafiltration.Liquor epinephrinae bitartratis ophthalmicus vinflunine normal saline solution is added in this gradient liposome, 60 ℃ of water bath heat preservation medicine carryings 10 minutes, 0.22 μ m filtering with microporous membrane degerming promptly gets the vinflunine liposome, and wherein the content of liquor epinephrinae bitartratis ophthalmicus vinflunine is 3mg/ml.Remove free drug and outer water is replaced into the histidine sucrose buffer of pH7.0, it is 99.2% that the gel column method is measured envelop rate, and Ma Erwen particle size analyzer determination mean diameter is 110nm, and polydispersity index (PDI) is 0.06.
If no specified otherwise, it is 1: 10 in medicine fat weight ratio all that following factor is investigated, and 60 ℃ of insulations were carried out medicine carrying under the condition in 10 minutes.The film material is two Palmic acid phosphatidylcholines (DPPC), cholesterol, PEG-DSPE, and mass ratio is l: 0.3: 0.2.
1 outer water pH regulator agent is to the influence of envelop rate
Adopting different alkali liquor to regulate the outer water pH of liposome is 7.4, measures the envelop rate result and sees table l.
The outer water pH regulator agent of table l is to the influence of envelop rate
2 inside and outside water pH gradients (Δ pH) are to the influence of envelop rate
With Na
3PO
4Solution is regulated the outer water pH of liposome, sets up Δ pH and is respectively 0,1.0,2.0,2.4,3.0, and medicine carrying is measured envelop rate, and the result sees table 2.
The inside and outside water pH gradient (Δ pH) of table 2 is to the influence of envelop rate
| |
0 | 1.0 | 2.0 | 2.4 | 3.0 |
| Envelop rate % | 19.4 | 42.5 | 65.3 | 78.5 | 91.0 |
3 medicine carrying temperature are to the influence of envelop rate
Respectively at 30,40,50,60 and 70 ℃ of insulations 10 minutes, measure envelop rate, the result sees table 3.
Table 3 medicine carrying temperature is to the influence of envelop rate
| Temperature/℃ | 30 | 40 | 50 | 60 | 70 |
| Envelop rate % | 71.8 | 91.8 | 89.5 | 88.5 | 51.9 |
4 medicine carrying times are to the influence of envelop rate
Be incubated 5,10,20 and 30 minutes respectively in 60 ℃, measure envelop rate, the result sees table 4.
The table 4 medicine carrying time is to the influence of envelop rate
| Time/minute | 5 | 10 | 20 | 30 |
| Envelop rate % | 85.3 | 91.5 | 92.3 | 84.5 |
The influence of 5 medicine fat comparison envelop rate
Investigate medicine fat weight ratio and be respectively l: 5, l: 6, l: 8, l: 10 and l: 20,50 ℃ of insulations 10 minutes, measure envelop rate, the result sees table 5.
The influence of table 5 medicine fat comparison envelop rate
| Medicine fat compares w/w | l∶5 | l∶6 | l∶8 | 1∶10 | l∶20 |
| Envelop rate % | 85.6 | 88.2 | 88.3 | 94.5 | 97.2 |
Embodiment 7 amount of alcohol influence liposome encapsulation
With the liposome membrane material: dimyristoyl phosphatidyl choline DMPC, cholesterol, PEG-DSPE, mass ratio are l: 0.5: 0.3, with the ethanol of cumulative volume 5%, 10.0%, 20.0% after 65 ℃ of stirring in water bath dissolvings; Add the sucrose octasulfate ammonium salt solution; Aquation, the preparation blank liposome, and with high pressure homogenizer processing reduction particle diameter; Cross 0.22 μ m microporous filter membrane degerming, get blank liposome.Measure particle diameter, according to method medicine carrying among the embodiment 5, measure envelop rate, the result sees table 6.
Table 6 ethanol influences liposome encapsulation
| Amount of alcohol (v/v) | Particle diameter (nm) | |
| 0％ | 120.3 | 95.6 |
| 5.0％ | 117.9 | 93.4 |
| 10.0％ | 119.5 | 94.1 |
| 20.0％ | 251.3 | 96.8 |
The result shows, residual 5.0%～10.0% ethanol in the blank liposome, liposome particle diameter and envelop rate there are no significant difference.And when the ethanol residual quantity reached 20%, the liposome particle diameter obviously increased.
The preparation of embodiment 8 liquor epinephrinae bitartratis ophthalmicus vinflunine lipidosome injections
Present embodiment is intended to illustrate how to prepare aseptic liquor epinephrinae bitartratis ophthalmicus vinflunine liposome through formulation and technology provided by the present invention.
The preparation of blank liposome
Take by weighing hydrogenated soya phosphatide 1000 grams, mPEG2000-DSPE 100 grams, cholesterol 200 grams are dissolved in the 120ml dehydrated alcohol, 60 ℃ of fully dissolvings of heating.10 liters of the citrate buffer solutions of the 0.3M of preparation pH4.0 add in the above-mentioned film material, and in 70 ℃ of aquations 20 minutes.Afterwards, it is added in microjet instrument,, obtain the blank liposome about particle diameter 100nm with the pressures cycle of 18000psi 5 times.Respectively through the aseptic filtering with microporous membrane degerming of 0.8um, 0.45um and 0.22um and carry out packing.
The medicine carrying process
Prepare the sodium phosphate buffer of aseptic 0.5M and the liquor epinephrinae bitartratis ophthalmicus vinflunine aseptic aqueous solution (in vinflunine) of 20mg/ml respectively.Get above-mentioned blank liposome 10ml, add sodium phosphate buffer 4ml and transfer pH to 7.4, place 50 degree water-bath temperature to bathe.Get liquor epinephrinae bitartratis ophthalmicus vinflunine aqueous solution 7.2ml respectively, 3.6ml adds in the above-mentioned liposome; Can obtain (the vinflunine: hydrogenated soya phosphatide, w: w) vinflunine long circulating liposomes that medicine fat ratio is respectively 1: 5 and 1: 10 after hatching 20 minutes in 40 degrees centigrade.
Remove outer water free drug
Adopt tangential flow ultrafilter that the outer water of above-mentioned liquor epinephrinae bitartratis ophthalmicus vinflunine liposome is replaced into sucrose-histidine solution isosmotic solution, the pH scope is 7.4, displaced volume＞4 times liposome volume.
Measure envelop rate with method well known to those skilled in the art, like microtrabeculae centrifuging, ultrafiltration or dialysis.The vinflunine long circulating liposomes envelop rate that the present invention adopts cation exchange resin microtrabeculae centrifugal determination medicine fat ratio to be respectively 1: 5 and 1: 10 is 94.46% and 97.31%.Measuring drug loading with the HPLC method is 5.20mgmL-1 and 3.27mgmL-1.
The preparation of embodiment 9, vinflunine liposome freeze-drying agent
The liposome membrane material is lecithin, cholesterol and PEG-DSPE, and mass ratio 1: 0.2: 0.1 with an amount of dissolve with ethanol, adds EDTA-ammonium salt (300mmolL
-1, pH5.0), 60 ℃ of water-bath aquations 20 minutes, high pressure homogenizer is handled and is reduced particle diameter, adopts 0.22 μ m filtering with microporous membrane degerming, and outer water EDTA-ammonium salt is removed in dialysis, must EDTA-ammonium gradient liposome.Liquor epinephrinae bitartratis ophthalmicus vinflunine normal saline solution (4mg/ml) is added in this gradient liposome, and 60 ℃ of water bath heat preservation medicine carryings 10 minutes promptly get the vinflunine liposome.Remove free drug and outer water is replaced into the buffer that contains histidine, maltose, mannitol composition (maltose is freeze drying protectant, and mannitol is that freeze drying protectant also is an osmotic pressure regulator simultaneously, and histidine is the pH regulator agent) of pH7.0; It is 99% that the gel column method is measured envelop rate, and Ma Erwen particle size analyzer determination mean diameter is 108nm, and polydispersity index (PDI) is 0.05.Add freeze dryer after the packing ,-50 ℃ of pre-freezes 4 hours ,-30 ℃ of dryings (10-50Pa) 48 hours, 0 ℃ kept 2 hours, and 25 ℃ of dryings were taken out after 24 hours.Measure envelop rate 98.5%, particle diameter 102nm, PDI are 0.07, moisture 3%.
Embodiment 10, extracorporeal releasing test
Employing Dynamic Membrane dialysis is carried out extracorporeal releasing test by the liquor epinephrinae bitartratis ophthalmicus vinflunine liposome of two kinds of different medicine fat ratios of embodiment 5 methods preparation.Release medium is that the grade of pH 7.5 is oozed glucose/histidine/NH
4The Cl buffer.At first with the release medium dilution, make the HSPC concentration in the sample be about 4mg/ml in sample.Respectively get the 2mL sample and put into the bag filter of molecular cut off 10000Da, the bag filter two ends are hitched with cord, place 250ml tool plug conical flask, add the 100ml release medium.Be placed in the constant temperature water bath vibration shaking table, rotating speed is 50rmin-1, and temperature is (37 ± 0.5) ℃.Each prescription is set three parallel appearance.At the point in time sampling of setting, replenish the equal-volume thermostatic medium simultaneously.The HPLC method is measured vinflunine content in the medium, and calculating cumulative discharges percentage rate, draws release profiles, and release profiles is seen Fig. 1.
Visible from Fig. 1, in 0～6h release amount of medicine in time prolongation and increase sharply, drug release is slow behind the 6h, reaches platform.Can find out also simultaneously that the in-vitro release rate of medicine fat comparison liposome has certain influence, the liquor epinephrinae bitartratis ophthalmicus vinflunine liposome release in vitro of medicine fat than 1: 5 is more slow.
The study on the stability of embodiment 11, vinflunine liposome
To place 2-8 ℃ of environment to place according to the liposome of embodiment 3 methods preparation.Detect main quality index respectively at sampling in 0,3,6,12,24,36 month.The result is as shown in table 7:
The study on the stability of table 7 vinflunine liposome in 2-8 ℃ of environment
| 0 month | March | June | December | 24 months | 36 months | |
| Particle diameter | ?108nm | 112nm | 120nm | 116nm | 105nm | 110nm |
| Envelop rate % | ?95.6 | 96.2 | 95.1 | 94.2 | 95.7 | 94.3 |
| Lysophosphatide mg/ml | ?0.1 | 0.08 | 0.09 | 0.11 | 0.12 | 0.10 |
| Total impurities | ?0.21 | 0.24 | 0.20 | 0.25 | 0.28 | 0.26 |
The pharmacodynamics test of embodiment 12, liquor epinephrinae bitartratis ophthalmicus vinflunine liposome
Get under the aseptic condition and be in well-grown HT-29 tumor tissues (F5), cut into the even fritter about 2.0mm, with one of every nude mice right side of trocar axillary fossa subcutaneous vaccination, preparation transplanted tumor model treats that tumor growth is to about 270mm
3The time (d0) press gross tumor volume size random packet; Every group of 4 animals; Being respectively negative control group, liquor epinephrinae bitartratis ophthalmicus vinflunine injection group (adopting the liquor epinephrinae bitartratis ophthalmicus vinflunine to add the preparation of injection water), liquor epinephrinae bitartratis ophthalmicus vinflunine liposome-1, group (medicine fat was than 1: 10; According to embodiment 2 methods preparations) with totally four groups of liquor epinephrinae bitartratis ophthalmicus vinflunine liposomees-2 group (medicine fat prepared according to embodiment 3 methods than 1: 5).Each treatment group all adopts q3d * 4 (being administered once successive administration 4 times in per three days), and iv (intravenous injection) gives 20mg/kg the dosage regimen of (in vinflunine), and negative control group equivalent means equal-volume gives 5% glucose injection.
Measure the tumor footpath weekly for 2～3 times in the administration same day (d0) beginning; Investigate and respectively organize gross tumor volume dynamic change and relative tumor proliferation rate; Process of the test is with relative inhibition rate of tumor growth (relative tumor inhibition; RTI) be the leading indicator that dynamic observes, and general state such as record body weight.
Through q3d * 4 administration phases and thereafter the withdrawal time of 20d observe and to show, liposome-1 group was respectively 54.5% and 50.2% with liposome-2 a group RTI when administration finished, two groups of all risings gradually of RTI after this, 6d RTI peaks after the drug withdrawal.Liposome-1 group and liposome-2 group RTV (relative tumour volume) have compared significant difference (P＜0.05) result of the test and have seen Fig. 2 with the injection group.
Aspect the body weight influence of nude mouse; Injection group 6d (d18) after the last administration reduce as far as possible (11.2%); Liposome-1 (d5) group before the 3rd administration reduces as far as possible (6.1%), after this recovers, and finishes to maintain about-4.7% to the observation period always.Liposome-2 6d (d18) group after the last administration reduces as far as possible (12.6%), after this returns to about-3% until the observation period finishes, and each group does not all have animal dead.
Result of the test shows that liquor epinephrinae bitartratis ophthalmicus vinflunine liposome-1 (medicine fat was than 1: 10) and drug-loaded liposome-2 (medicine fat was than 1: 5) all obviously are superior to injection to the curative effect of human colon carcinoma HT-29 transplanted tumor in nude mice.
The toxicology test of embodiment 13, liquor epinephrinae bitartratis ophthalmicus vinflunine liposome
According to NCI standard maximum tolerated dose (maximal tolerate dose, MTD) method is estimated, key step is: 1) a certain dosage in proportion factor 1 .8 increase, signs of toxicity appears in all animals after acute death or injection 1d occurring; 2) reducing scale factor is 1.15, until finding the dosage that does not occur signs of toxicity after acute death or injection 1d not occurring, as preliminary MTD; 3) checking MT reconnaissance D; Increase size of animal, give to observe 11d continuously behind the medicine of this dosage, observation index comprises to external world IR, feed, drainage, by hair, breathing, gait etc.; If compare before the signs of toxicity and the body weight that do not occur death, progress in the observation period and the administration and descend 15%; Can think that this dosage is MTD, otherwise continue to grope, until finding qualified MTD value in the dosage of the ratio of successively decreasing.
Liquor epinephrinae bitartratis ophthalmicus vinflunine injection, liquor epinephrinae bitartratis ophthalmicus vinflunine liposome-1, liquor epinephrinae bitartratis ophthalmicus vinflunine liposome-2 pair KM mice (are kunming mice; Being the maximum outbreeding group of mean people Mus of China's volume of production, use amount) MTD of single dose iv administration is respectively 54.7,51.0,45.2mg/kg; The liquor epinephrinae bitartratis ophthalmicus vinflunine liposome that shows different medicine fat ratios is compared with its injection; Obvious increase or improvement are not seen in the acute toxicity aspect, and the KM mice is more or less the same to the toleration of injection and two drug-loaded liposomes.
Liquor epinephrinae bitartratis ophthalmicus vinflunine injection, liquor epinephrinae bitartratis ophthalmicus vinflunine liposome-1, liquor epinephrinae bitartratis ophthalmicus vinflunine liposome-2 are identical with embodiment 12.
Claims (10)
1. the Liposomal formulation of a vinflunine mainly comprises the DSPE (PEG-DSPE) of medicine vinflunine, phospholipid, cholesterol and Pegylation, wherein,
The DSPE of Pegylation and the mass ratio of phospholipid are 0.05～0.5: 1; The mass ratio of preferred PEG-DSPE and phospholipid is 0.1～0.5: 1;
The mass ratio of C/PL is 0.1～0.5: 1, and the mass ratio of preferred C/PL is 0.2～0.3: 1;
In vinflunine, the mass ratio of medicine and phospholipid is 1: 3～20, and said drug encapsulation is in liposome interior of being made up of phospholipid bilayer and phospholipid bilayer.
2. the Liposomal formulation of vinflunine as claimed in claim 1 is characterized in that said medicine vinflunine is the liquor epinephrinae bitartratis ophthalmicus vinflunine.
3. according to claim 1 or claim 2 the Liposomal formulation of vinflunine is characterized in that described phospholipid is selected from distearyl acid phosphatidylcholine, two Palmic acid phosphatidylcholines, dimyristoyl phosphatidyl choline, hydrogenated soya phosphatide, egg yolk lecithin, soybean phospholipid or sphingomyelins.
4. according to claim 1 or claim 2 the Liposomal formulation of vinflunine is characterized in that it being injection formulation or lyophilized formulations.
5. the Liposomal formulation of vinflunine as claimed in claim 4; It is characterized in that; Described injection formulation contains in every 1000ml preparation: liquor epinephrinae bitartratis ophthalmicus vinflunine 1g-20g, phosphatidase 13 g-400g; The DSPE 1.5g-200g of Pegylation, cholesterol 0.3g-200g.
6. the Liposomal formulation of vinflunine as claimed in claim 4; It is characterized in that; Described lyophilized formulations; Contain in every 1000ml preparation before the lyophilizing: liquor epinephrinae bitartratis ophthalmicus vinflunine 1g-20g, phospholipid are 5-15 times of liquor epinephrinae bitartratis ophthalmicus vinflunine quality, and the mass ratio of PEG-DSPE and phospholipid is 0.1～0.4: 1; The mass ratio of C/PL is 0.2～0.3: 1; Freeze drying protectant is the 5-20% of liquid preparation quality before the lyophilizing.
7. the method for preparing of the said vinflunine Liposomal formulation of claim 1～6 comprises the steps:
The preparation of a, blank liposome
(1) DSPE and the cholesterol that take by weighing phospholipid, Pegylation by prescription are dissolved in an amount of organic solvent, fully dissolving;
(2) preparation contains the aqueous solution of ammonium salt or the pH of buffer salt is the buffer of 3.0-6.0, and it is added in the solution that above-mentioned steps (1) makes, and under 40-70 ℃ of temperature conditions stirring reaction 10-60 minute, obtains multilamelar liposome;
(3) adopt high pressure homogenize, extrude, the method for ultrasonic or freeze thawing is reduced to 80-500nm with the mean diameter of the multilamelar liposome that step (2) obtains, and promptly obtains blank liposome;
The preparation of b, gradient liposome solutions
The blank liposome that step a is made by prior art is processed the system with the inside and outside ion gradient of liposome or the system of the inside and outside proton gradient of liposome; Make the pH value of the outer water of liposome be higher than the liposome interior pH value, thereby perhaps make the ammonium salt of the outer aqueous phase of liposome remove formation liposome inside and outside ammonium ion gradient;
C, medicine carrying
The gradient liposome solutions that step b is obtained places 30-60 ℃ of water-bath temperature to bathe, and gets recipe quantity medicine vinflunine solution and is added in the liposome solutions, and 30-60 ℃ was stirred 5-60 minute, and can obtain the vinflunine liposome solutions;
D, the outer water free drug of removal
Adopt in the vinflunine liposome solutions that dialysis, ultrafiltration or gel filtration chromatography method obtain step c non-encapsulated free drug to remove;
Promptly obtain vinflunine long circulating liposomes injection formulation after e, the filtration sterilization of steps d gained solution;
Continue following steps and prepare vinflunine long circulating liposomes lyophilized formulations:
Add freeze drying protectant in the vinflunine long circulating liposomes injection that f, step e obtain, carry out lyophilizing, get lyophilized formulations by prior art; Freeze drying protectant is selected from one of sucrose, maltose, trehalose, lactose, galactose, glucose or mannitol or combination; The amount of freeze drying protectant accounts for the 5-20% of the preceding liquid preparation quality of lyophilizing.
8. method for preparing as claimed in claim 7 is characterized in that the organic solvent described in the step a is selected from dehydrated alcohol, the tert-butyl alcohol, ether, dichloromethane, chloroform, methanol or ethyl acetate, wherein preferred dehydrated alcohol; The addition of further preferred organic solvent and the mass ratio of phospholipid are 1: 5～20.
9. method for preparing as claimed in claim 7 is characterized in that the ammonium salt described in the step a is selected from sucrose octasulfate ammonium, ammonium sulfate, ammonium acetate, ammonium chloride or ethylenediaminetetraacetic acid ammonium; Preferably, the concentration of said ammonium salt is 50-500mM; Described buffer is selected from citrate buffer, phosphate buffer or carbonate buffer solution; Preferably, the concentration of said buffer salt is 50-500mM.
10. method for preparing as claimed in claim 7 is characterized in that, adopts one of following method:
1. the liposome membrane material is the DSPE of hydrogenated soya phosphatide, cholesterol and Pegylation, and mass ratio 1-5: 1: 0.5-1 with an amount of dissolve with ethanol, adds ammonium sulfate 200-300mmolL
-1, 50-70 ℃ of water-bath aquation 30-60 minute, extruded or supersound process reduces particle diameter to 100-300nm high pressure homogenize, adopts 0.22 μ m filtering with microporous membrane degerming then, removes outer water ammonium sulfate, must the ammonium sulphate gradient liposome; Liquor epinephrinae bitartratis ophthalmicus vinflunine normal saline solution is added in this gradient liposome, and liquor epinephrinae bitartratis ophthalmicus vinflunine content is the 10%-50% of hydrogenated soya phosphatide content; 30-60 ℃ of water bath heat preservation medicine carrying 20-40 minute, aseptic filtration, and make the vinflunine liposome after removing outer water free drug;
2. the liposome membrane material is the DSPE of hydrogenated soya phosphatide, cholesterol and Pegylation, and mass ratio 5-10: 1: 0.1-0.5 with an amount of dissolve with ethanol, adds citric acid-sodium citrate buffer 200-300mmolL
-1, pH3.0-6.0,50-70 ℃ of water-bath aquation 10-60 minute obtains the blank liposome first product; High pressure homogenize, extrude, ultrasonic or freeze thawing treatment reduces particle diameter to 50-300nm, adopts 0.22 μ m microporous filter membrane degerming then, blank liposome; Get blank liposome suspension 10.0mL, add 200-300mmolL
-1Sodium radio-phosphate,P-32 solution mixes, and adds the liquor epinephrinae bitartratis ophthalmicus vinflunine subsequently, and liquor epinephrinae bitartratis ophthalmicus vinflunine content is the 20%-40% of hydrogenated soya phosphatide content; 40-60 ℃ is incubated 5-60 minute, aseptic filtration, and make the vinflunine liposome after removing outer water free drug;
3. the liposome membrane material is the DSPE of hydrogenated soya phosphatide, cholesterol and Pegylation, and mass ratio 10-20: 1: 0.01-0.1 with an amount of dissolve with ethanol, adds ethylenediaminetetraacetic acid ammonium 100-300mmolL
-1, pH4-6.0,40-70 ℃ of water-bath aquation 20-60 minute; High pressure homogenize, freeze thawing, extrude or supersound process reduces particle diameter to 100-200nm; Adopt 0.22 μ m filtering with microporous membrane degerming then, outer water ethylenediaminetetraacetic acid ammonium is removed in dialysis, gets ethylenediaminetetraacetic acid ammonium gradient liposome; The liquor epinephrinae bitartratis ophthalmicus vinflunine is added in this gradient liposome, and liquor epinephrinae bitartratis ophthalmicus vinflunine content is hydrogenated soybean
The 10%-40% of content of phospholipid; 30-60 ℃ of water bath heat preservation medicine carrying 5-40 minute, aseptic filtration, and make the vinflunine liposome after removing outer water free drug;
4. the liposome membrane material is the DSPE of hydrogenated soya phosphatide, cholesterol and Pegylation; Mass ratio 5-15: 1: 0.5-1 with an amount of dissolve with ethanol, adds the solution that contains the sucrose octasulfate ammonium salt; 50-70 ℃ of water-bath aquation 10-60 minute; High pressure homogenize, extrude, ultrasonic, freeze thawing etc. handles and reduces particle diameter to 80～200nm, outer water sucrose octasulfate ammonium salt is removed in ultrafiltration, must the gradient liposome; The liquor epinephrinae bitartratis ophthalmicus vinflunine is added in this gradient liposome, and liquor epinephrinae bitartratis ophthalmicus vinflunine content is the 20%-30% of hydrogenated soya phosphatide content; 30-60 ℃ of water bath heat preservation medicine carrying 5-60 minute, aseptic filtration, and make the vinflunine liposome after removing outer water free drug.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110149433XA CN102805729A (en) | 2011-06-03 | 2011-06-03 | Vinflunine liposome preparation and preparation method of vinflunine liposome preparation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110149433XA CN102805729A (en) | 2011-06-03 | 2011-06-03 | Vinflunine liposome preparation and preparation method of vinflunine liposome preparation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102805729A true CN102805729A (en) | 2012-12-05 |
Family
ID=47229715
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110149433XA Pending CN102805729A (en) | 2011-06-03 | 2011-06-03 | Vinflunine liposome preparation and preparation method of vinflunine liposome preparation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102805729A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105188674A (en) * | 2013-03-14 | 2015-12-23 | 生物休眠有限公司 | Liposome formulation and manufacture |
| CN107362142A (en) * | 2016-05-13 | 2017-11-21 | 山东新时代药业有限公司 | A kind of fulvestrant lipidosome injection and preparation method thereof |
| CN107753430A (en) * | 2017-10-23 | 2018-03-06 | 天津市肿瘤医院 | A kind of long circulating liposomes of CLT 003 and preparation method thereof |
| US9993427B2 (en) | 2013-03-14 | 2018-06-12 | Biorest Ltd. | Liposome formulation and manufacture |
| CN110981837A (en) * | 2019-12-03 | 2020-04-10 | 沈阳药科大学 | Paclitaxel weakly acidic derivative active drug-loaded liposome and preparation and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1980637A (en) * | 2004-05-03 | 2007-06-13 | 赫尔姆生物科学公司 | Liposomes useful for drug delivery |
| CN101933904A (en) * | 2009-07-01 | 2011-01-05 | 齐鲁制药有限公司 | Vinorelbine long circulation liposome preparation and preparation method thereof |
-
2011
- 2011-06-03 CN CN201110149433XA patent/CN102805729A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1980637A (en) * | 2004-05-03 | 2007-06-13 | 赫尔姆生物科学公司 | Liposomes useful for drug delivery |
| CN101933904A (en) * | 2009-07-01 | 2011-01-05 | 齐鲁制药有限公司 | Vinorelbine long circulation liposome preparation and preparation method thereof |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105188674A (en) * | 2013-03-14 | 2015-12-23 | 生物休眠有限公司 | Liposome formulation and manufacture |
| CN105188674B (en) * | 2013-03-14 | 2017-12-08 | 生物休眠有限公司 | Liposomal formulation and manufacture |
| US9993427B2 (en) | 2013-03-14 | 2018-06-12 | Biorest Ltd. | Liposome formulation and manufacture |
| US10265269B2 (en) | 2013-03-14 | 2019-04-23 | Biorest Ltd. | Liposome formulation and manufacture |
| US11633357B2 (en) | 2013-03-14 | 2023-04-25 | Zuli Holdings, Ltd. | Liposome formulation and manufacture |
| CN107362142A (en) * | 2016-05-13 | 2017-11-21 | 山东新时代药业有限公司 | A kind of fulvestrant lipidosome injection and preparation method thereof |
| CN107753430A (en) * | 2017-10-23 | 2018-03-06 | 天津市肿瘤医院 | A kind of long circulating liposomes of CLT 003 and preparation method thereof |
| CN110981837A (en) * | 2019-12-03 | 2020-04-10 | 沈阳药科大学 | Paclitaxel weakly acidic derivative active drug-loaded liposome and preparation and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11858958B2 (en) | Blank liposome with ginsenoside Rg3 or its analog as membrane materials and preparations and uses thereof | |
| EP2123260A1 (en) | Liposome formulation and process for preparation thereof | |
| TWI362931B (en) | Irinotecan formulation | |
| CN103479578B (en) | The Liposomal formulation of a kind of maleic acid Pixantrone and preparation technology thereof | |
| CN101933904B (en) | Vinorelbine long circulation liposome preparation and preparation method thereof | |
| JP2009507049A (en) | Nanomicelle formulation of vinca alkaloid anticancer drug encapsulated in polyethylene glycol derivative of phospholipid | |
| CN1840193B (en) | Nanometer capsule of anthracene nucleus anticancer antibiotic with polyethylene glycol-phospholipid | |
| CN112137958A (en) | Doxorubicin and immunologic adjuvant-containing combined drug liposome and preparation method thereof | |
| CN102805729A (en) | Vinflunine liposome preparation and preparation method of vinflunine liposome preparation | |
| CN102935066B (en) | Irinotecan liposome preparation and preparation method thereof | |
| CN111450061A (en) | Hybrid mesenchymal stem cell exosome drug delivery system and preparation method and application thereof | |
| CN106727325A (en) | A kind of Liposomal formulation of imatinib mesylate and preparation method thereof | |
| CN101322681B (en) | Method for preparing nano micelle formulation of anthracene nucleus antineoplastic antibiotic | |
| CN102085189B (en) | Docetaxel liposome sterile lyophilized preparation and preparation method thereof | |
| CN109562068A (en) | Micro- rouge body composition containing faintly acid agent | |
| CN101849915A (en) | Vinorelbine stealth liposome freeze-dried powder injection and preparation method thereof | |
| CN102670509B (en) | Liposomal formulation containing slightly solubility camptothecine and preparation method thereof | |
| CN114788811A (en) | Gemcitabine hydrochloride chitosan micelle and preparation method thereof | |
| CN105796495B (en) | Irinotecan hydrochloride liposome pharmaceutical composition and preparation method thereof | |
| CN101147728A (en) | 6-methocy bideoxy bideoxy guanosine long circulating liposome preparation and preparing method | |
| CN115364218A (en) | Application of pharmaceutical composition with specific drug-to-lipid ratio in antitumor | |
| CN105616354A (en) | Neogambogic acid liposome injection and preparation method thereof | |
| CN101874788A (en) | 7-ethyl-10-hydroxycamptothecine liposome freeze-dried powder injection and preparation method thereof | |
| CN110496103B (en) | Docetaxel palmitate liposome and preparation method thereof | |
| CN113827546A (en) | Injectable hydrogel containing adriamycin and immunologic adjuvant combined drug liposome and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20121205 |