CN1813676A - Anthracene ring antitumor medicinal liposome and its production process - Google Patents
Anthracene ring antitumor medicinal liposome and its production process Download PDFInfo
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Abstract
The present invention discloses an anthracyclic anti-tumor medicine liposome and its production process. It is composed of anthracyclic medicine, liposome material and antioxidant, the medicine/liposome ratio (w/w) is 0.05-0.2:1, in which the liposome material includes phospholipids and cholesterol, the mole ratio of both them is 40-60:30-50 mol%; the antioxidant is formed from alpha-tocopherol and desferal, their concentractions respectively are 0.010-0.030 mg/ml and 0.05-0.30 mg/ml. Its production process includes the following steps: (1). Preparing multiplayer liposome; (2), preparing small single-layer liposome; and (3), preparing anthracyclic medicine liposome.
Description
Technical field
The present invention relates to a kind of anthracene ring antitumor medicinal liposome and production technology thereof, specifically the liposome of amycin, epirubicin and daunorubicin and their ammonium sulphate gradient production technology belong to chemical pharmacy field.
Background technology
Anthracene ring antitumor medicinal such as amycin, epirubicin and daunorubicin are broad-spectrum anti-cancer drugs commonly used clinically.Be mainly used in breast carcinoma, gynecological tumor, gastrointestinal tumor, head and neck tumor, pulmonary carcinoma, hematological system tumor, malignant lymphoma etc. at present.Wherein representing medicine is amycin, is called doxorubicin (doxorubicin) again, and its antitumor action principle is that to suppress DNA synthetic and rely on mRNA synthetic of DNA and the mitochondrial functional structure of influence.Though amycin is of many uses as a line medication, the toxic and side effects that is caused by it is serious.Be mainly the cardiac toxicity of dose limitation, mainly show as arrhythmia, electrocardiographic abnormality, irreversible heart failure and cardiomyopathy appear in serious meeting; Dose limitation toxicity also has bone marrow depression, shows as serious granulocytopenia; Also have gastrointestinal reaction, alopecia in addition, spill blood vessel and cause local soft tissue inflammation necrosis etc.These toxic and side effects have greatly limited the clinical practice of amycin, make the application accumulated dose can not surpass 550mg/m
2The short whole body simultaneously distribution of metabolism time is relevant in vivo with amycin to produce these toxicity.Reduce the heart bone marrow toxicity for effectively using amycin, semi-synthetic epirubicin obtains clinician's favor, and the different 4 ' position hydroxyls of amino sugar part that are of its structure with amycin are become trans by cis.Epirubicin (epirubicin) also is a broad-spectrum anti-cancer drug, and the heart bone marrow toxicity that it causes alleviates than amycin is obvious.Daunorubicin (daunorubicin) claim rubidomycin again, is mainly used in leukemia and some entity tumor chemotherapy at present, and toxicity and amycin are similar.
Liposome is the phospholipid bilayer membrane structure, and main component is phospholipid and cholesterol, and is close with human normal cell's film, thereby to human body avirulence non-immunogenicity, is used to the parcel of some antitumor drug in recent years.Increase because tumor vascular endothelial cell destroys permeability, add the shortage lymphatic return, liposome is very easy to spill tumor vessel in mesenchyma stroma of tumors.After medicine is devoted to lesions position, medicine slowly releases from liposome, enter tumor cell with simple diffusion form, reach and reduce the purpose that toxicity improves curative effect, therefore with can yet be regarded as a kind of method of ideal reduction toxicity raising curative effect of liposome anthracene ring antitumor medicinal.
External at present existing liposomal doxorubicin and daunorubicin product come out.Wherein liposomal doxorubicin is a hidden liposome of new generation, and drugs approved by FDA is used for the treatment of the gentle adenocarcinoma of acquired immune deficiency syndrome (AIDS) Ka Boji sarcoma, ovarian cancer and transitivity, and commodity are called Doxil (U.S.)/Caelyx (Europe).In addition, liposome daunorubicin (commodity are called DaunoXome) also obtains FDA approval listing.
Summary of the invention
The main technical problem to be solved in the present invention provides the anthracene ring antitumor medicinal liposome that a kind of toxic and side effects is little, stability is high, the body-internal-circulation time is long.
Another technical problem that the present invention will solve provides the production technology of this anthracene ring antitumor medicinal liposome.
For achieving the above object, the present invention is by the following technical solutions:
A kind of anthracene ring antitumor medicinal liposome is made up of anthracene ring antitumor medicinal, liposome material and antioxidant, and medicine/fat ratio (w/w) is 0.05～0.2: 1; Wherein the liposome material comprises phospholipid and cholesterol, and the mol ratio of the two is 40～60: 30～50mol%; Antioxidant is made up of α-tocopherol and desferal, and the concentration of the two is respectively 0.010～0.030mg/ml and 0.05～0.30mg/ml.Anthracene nucleus medicament is wrapped in the liposome aqueous phase.
Also contain DSPE-PEG in the liposome material
2000(poly ethylene-ethylene glycol-DSPE), phospholipid, cholesterol and DSPE-PEG
2000Three's mol ratio is 40～60: 30～50: 4～20mol%.Described phospholipid is selected from sphingomyelin (sphingomyelin, SM), distearoyl phosphatidylcholine (distearoylphosphatidylcholine, DSPC) or hydrogenated phospholipid phatidylcholine (hydrogenated soybeanphosphatidylcholine, HSPC) a kind of in, the three all belongs to rigidity phospholipid.
Described anthracene ring antitumor medicinal is selected from a kind of in amycin, epirubicin and the daunorubicin, and what the present invention used is their hydrochlorate, and domestic manufacturer all can produce.
We use DSPC, HSPC and SM to form the liposome of phospholipid bilayer membrane structure respectively with cholesterol, and are wherein best with the liposome stability that DSPC and cholesterol are formed.DSPC (perhaps SM, HSPC) is a phospholipid of forming duplicature, is the ultimate constituent of liposome.The phospholipid that uses in the prescription of the Doxil/Caelyx of external listing is HSPC.Use DSPC to be among the present invention because the acyl chain length unanimity of DSPC and phase transition temperature (phase transitiontemperature be a height than HSPC Tc), so DSPC should be more stable than the liposome of HSPC phospholipid bilayer as the liposome of phospholipid bilayer; The effect of cholesterol is to reduce the stability that the flowability of film improves duplicature.The matching principle of their concentration is for obtaining the liposome of optimum stabilization, DSPC (or SM, HSPC) 40～60mol%, cholesterol 30～50mol%, being higher or lower than this concentration ratio liposome can not stablize, there is the bibliographical information cholesterol concentration can not be lower than 30mol%, otherwise just can not play any effect, but cholesterol concentration can not be higher than 50mol% again, can accelerate packaging medicine like that on the contrary and leak out from liposome.
It is to realize by α-tocopherol and the desferal that adds trace in prescription that the present invention improves medicine stability.One of their effect is hydrolysis and the oxidation that suppresses phospholipid and cholesterol, and another effect is the oxidation that inhibition is wrapped in the anthracene nucleus medicament of liposome aqueous phase.Stability test shows, the percolation ratio of liposome of share these two antioxidants under the present invention fills a prescription dosage is well below only with a kind of liposome of antioxidant wherein.The main characteristics that reasonable these two kinds of antioxidants of use are patents of the present invention are different from external like product simultaneously.Use in the prescription as the Doxil/Caelyx of external listing be histidine (histidine) to improve the stability of liposomal doxorubicin, we have then selected α-tocopherol and desferal to realize liposome stability.Evidence (seeing below explanation), the prepared Evacet of patent of the present invention is consistent with external product stability.
Generally, medicine fat can be high more than the envelop rate of low more medicine, and stability also increases thereupon, and the medicine fat ratio of the liposome medicament that therefore a lot of envelop rates are higher was at 0.05: 1 or lower.Yet medicine fat is lower than more, and the medicament contg that enters in the liposome is few more, and the adjuvant for preparing liposome simultaneously is very expensive, and the utmost point is unfavorable for the clinical practice of medicine.But because the liposome finite capacity if add the macromole PEG that is uniformly distributed in the phospholipid bilayer both sides, takies part liposome capacity again, so improve medicine fat than very difficult.The present invention adopts above-mentioned prescription, and messenger drug fat can reach 0.2: 1 than the highest, and envelop rate is not less than 90% simultaneously.Have never seen in foreign literature and the liposome product and to be higher than this ratio, the medicine fat of the liposome medicament that domestic many research institutions are prepared is than well below this ratio.
Thereby the prolong drug of trying one's best when reducing poisonous side effect of medicine circulation time increase curative effect of medication in vivo also is the target that the liposome medicament research and development are pursued, and the present invention is by adding hydrophilic macromole poly ethylene-ethylene glycol-DSPE (DSPE-PEG in prescription
2000) achieve this end.For anthracene ring antitumor medicinal, used DSPE-PEG
2000The preferred concentration scope be 4～10mol%: but be lower than also body-internal-circulation time of prolong drug of 4mol%, but effect is very not remarkable; And the concentration of PEG is when surpassing 15mol%, can make liposome form cystose micelle and do not present double-decker.DSPE-PEG
2000Can strengthen the liposome membrane surface hydrophilicity, reduce of the combination of various plasma proteins to liposome, make liposome can hide engulfing of reticuloendothelial system very effectively, stability and envelop rate are improved, and the rate that spills reduces, and the body circulation time of liposome is obviously prolonged, redistribution in the body, reduce the toxic reaction of the medicine that wraps, the concentration in focus also significantly improves simultaneously, thereby increases therapeutic index.Medicine still is wrapped in the liposome during in addition owing to injection, has eliminated and after medicine spills the stimulation of organizing has been caused the inflammation necrosis.
For the ease of enforcement, the present invention selects ammonium sulphate gradient as basic production technology, by its every reaction condition of assay optimization, finally finds out the process route that is suitable for the anthracene ring antitumor medicinal liposome large-scale production.
A kind of production technology of anthracene ring antitumor medicinal liposome injection may further comprise the steps:
(1) preparation multilamellar liposome: the amount according to aforementioned formula is prepared chloroform or the chloroform/methanol solution that contains liposome material and antioxidant, and the rotary evaporation film forming adds 125～400mM, and pH value is 5～7 ammonium sulfate aquations;
(2) preparation small unilamellar vesicle: the ammonium sulfate that contains multilamellar liposome that step (1) is obtained is squeezed into the small unilamellar vesicle liquid that particle diameter is 80～120 nanometers through extruder after carrying out five freeze thaws circulations; Use the outer ammonium sulfate of the equilibrated Sephadex G-50 column chromatography of 5～15% sucrose liquid (pH5～7) eluting liposome subsequently.
(3) preparation anthracene ring antitumor medicinal liposome: it is in 5～15% the sucrose liquid (pH5～7) that the hydrochlorate injection of injection stage anthracene ring antitumor medicinal is dissolved in concentration, under 50～70 ℃ of conditions, hydrochloric acid anthracene nucleus medicine sucrose liquid joined in the small unilamellar vesicle liquid and mix, in 50～70 ℃ of water-baths, place more than 10 minutes, hydrochloric acid anthracene nucleus medicine is entered in the small unilamellar vesicle, be cooled to room temperature, remove sucrose and free anthracene nucleus medicament obtains finished product.
The effect of using 125～400mM ammonium sulfate in the step (1) mainly is to set up the envelop rate that ammonium sulphate gradient improves anthracene nucleus medicament.But 125～400mM stabilized liposome osmotic pressure, preferred concentration are 150～250mM; PH value adopts 5～7, and optimal pH is 6.5.
Five freeze thaws circulation in the described step (2) be meant with multilamellar liposome with dry ice (20 ℃) freezing after thawing naturally at room temperature, and then freezing with dry ice, so circulate five times.Effect is further to improve envelop rate and stability.In practical operation, also can omit the freeze thaw circulation step, and directly enter pressing steps.Omit the prepared liposome physicochemical property of freeze thaw circulation step and use the prepared liposome difference with insignificance of freeze thaw circulation step.Preferred 100 nanometers of the particle diameter of small unilamellar vesicle.
The method of removing free anthracene nucleus medicament in the described step (3) is chromatography and centrifugal separation, belongs to general knowledge known in this field, so not superfluous stating.The chromatographic column method can be used Sephadex G-50 post, and with liposome anthracene nucleus medicament upper prop, the normal saline eluting gets final product; Centrifuging is that liposome anthracene nucleus medicine is inserted centrifuge, 100, and under the 000g condition centrifugal 5～10 minutes, remove supernatant, get final product with physiological saline solution pellet.
Use 5～15% sucrose solutions in described step (2) and (3), the best is selected 10% isotonic solution for use; Sucrose liquid pH5～7, the best is selected pH6.5 for use.
Advantage of the present invention is: prescription disclosed by the invention can make envelop rate greater than 90% liposome, and toxicity is low, and stability is high, adds DSPE-PEG
2000After, further having prolonged body-internal-circulation time of medicine, the slow release and the curative effect that help medicine improve.Ammonium sulphate gradient after use optimizing is easy and simple to handle, save time the liposome encapsulation height that makes, the response rate 90%, phospholipid, cholesterol and DSPE-PEG
2000The response rate be about 85%.The liposome percolation ratio is low, and particle diameter is (100 ± 10 nanometer) evenly, and good stability is placed for 4 ℃ and preserved 1 year particle diameter no change, and envelop rate is still more than 85%.This method is convenient to sterilization and is removed pyrogen simultaneously, can realize large-scale production.
The invention will be further described below in conjunction with the specific embodiment.
The specific embodiment
Table 1: liposome prescription
| Phospholipid (mol%) | Cholesterol (mol%) | DSPE-PEG 2000 (mol％) | α-tocopherol (mg/ml) | Desferal (mg/ml) | |
| | 52 | 43 | 5 | 0.019 | 0.13 |
| | 46 | 42 | 12 | 0.019 | 0.13 |
| | 52 | 43 | 5 | 0.015 | 0.20 |
| Embodiment 4 | 46 | 42 | 12 | 0.015 | 0.20 |
| Embodiment 5 | 52 | 43 | 5 | 0.023 | 0.11 |
| | 46 | 42 | 12 | 0.023 | 0.11 |
| | 45 | 45 | 10 | 0.010 | 0.05 |
| | 52 | 42 | 6 | 0.03 | 0.30 |
| | 60 | 36 | 4 | 0.015 | 0.01 |
| | 50 | 50 | - | 0.020 | 0.15 |
Above material source is as follows:
Amycin, epirubicin and daunorubicin are purchased in the Zhejiang Haizheng Pharmaceutical Co.
Sphingomyelin (SM, sphingomyelin), DSPC (distearoylphosphatidylcholine), HSPC (hydrogenated soybean phosphatidylcholine), cholesterol and PEG
2000-DSPE purchases Avantipolar Lipids (Alabaster, AL) company in the U.S..
α-tocopherol and desferal purchase the Sigma company in the U.S..
Embodiment 1:
1. preparation multilamellar liposome
With injection stage DSPC, cholesterol and DSPE-PEG
2000The three is dissolved in (v/v, 2: 1) in the chloroform/methanol liquid, adds antioxidant α-tocopherol and desferal.Aforesaid liquid is put into rotary evaporation bottle rotary evaporation film forming, again this film is immersed in the 250mM pH6.5 ammonium sulfate.
2. preparation small unilamellar vesicle
After five freeze thaw circulations, aforesaid liquid is put into extruder repeatedly push, obtain the small unilamellar vesicle of particle diameter 100 nanometers.The carbon-fiber film that extruding is used is respectively 0.14 and 0.08 nanometer.Use the outer ammonium sulfate of the equilibrated Sephadex G-50 of 10% sucrose liquid (pH6.5) column chromatography eluting liposome subsequently.
3. preparation Evacet
The injection stage doxorubicin hydrochloride inj is dissolved in the 10% sucrose liquid.Under 65 ℃ of conditions, be that 0.2: 1 amount will amycin sucrose liquid adds in the small unilamellar vesicle and mixes by medicine/fat ratio, in 65 ℃ of water-baths, placed 15 minutes.Be cooled to room temperature, through the equilibrated Sephadex G-50 of 0.9% sodium chloride solution column chromatography eluting sucrose.Remove free doxorubicin.
Microporous membrane degerming, pyrogen and other impurity by 0.22 nanometer promptly obtain the Evacet finished product.
This product liposomal doxorubicin good stability, in the test of cultivating in the test of 10 times of buffer and with calf serum, the amycin of 75-80% still is wrapped in the liposome after 24 hours.
1. with injection stage DSPC, cholesterol and DSPE-PEG
2000The three is dissolved in (v/v, 2: 1) in the chloroform/methanol liquid, adds antioxidant α-tocopherol and desferal.Aforesaid liquid is put into rotary evaporation bottle rotary evaporation film forming, again this film is immersed in the 150mM pH5.5 ammonium sulfate.
2. preparation small unilamellar vesicle
Aforesaid liquid is put into extruder repeatedly push, obtain the small unilamellar vesicle of particle diameter 100 nanometers.The carbon-fiber film that extruding is used is respectively 0.14 and 0.08 nanometer.Use the outer ammonium sulfate of the equilibrated Sephadex G-50 of 5% sucrose liquid (pH5.5) column chromatography eluting liposome subsequently.
3. preparation Evacet
The injection stage doxorubicin hydrochloride inj is dissolved in the 5% sucrose liquid.Under 65 ℃ of conditions, be that 0.2: 1 amount will amycin sucrose liquid adds in the small unilamellar vesicle and mixes by medicine/fat ratio, in 65 ℃ of water-baths, placed 30 minutes.Be cooled to room temperature, through the equilibrated Sephadex G-50 of 0.9% sodium chloride solution column chromatography eluting sucrose.Remove free doxorubicin.
Microporous membrane degerming, pyrogen and other impurity by 0.22 nanometer promptly obtain the Evacet finished product.
1. preparation multilamellar liposome
Injection stage HSPC, cholesterol and DSPE-PEG
2000The three is dissolved in (v/v, 2: 1) in the chloroform/methanol liquid, adds antioxidant α-tocopherol and desferal.Aforesaid liquid is put into rotary evaporation bottle rotary evaporation film forming, again this film is immersed in the 250mM pH6.5 ammonium sulfate.
2. preparation small unilamellar vesicle
After five freeze thaw circulations, aforesaid liquid is put into extruder repeatedly push, obtain the small unilamellar vesicle of particle diameter 100 nanometers.The carbon-fiber film that extruding is used is respectively 0.14 and 0.08 nanometer.Use the outer ammonium sulfate of the equilibrated Sephadex G-50 of 10% sucrose liquid (pH6.5) column chromatography eluting liposome subsequently.
3. preparation Evacet
The injection stage doxorubicin hydrochloride inj is dissolved in the 10% sucrose liquid.Under 65 ℃ of conditions, be that 0.1: 1 amount will amycin sucrose liquid adds in the small unilamellar vesicle and mixes by medicine/fat ratio, in 65 ℃ of water-baths, placed 15 minutes.Be cooled to room temperature, through the equilibrated Sephadex G-50 of 0.9% sodium chloride solution column chromatography eluting sucrose.Remove free doxorubicin.
Microporous membrane degerming, pyrogen and other impurity by 0.22 nanometer promptly obtain the Evacet finished product.
This product liposome vincristine good stability, in the test of cultivating in the test of 10 times of buffer and with calf serum, the amycin of 75-80% still is wrapped in the liposome and (sees below the test explanation) after 24 hours.
Embodiment 4
1. preparation multilamellar liposome
Injection stage HSPC, cholesterol and DSPE-PEG
2000The three is dissolved in (v/v, 2: 1) in the chloroform/methanol liquid, adds antioxidant α-tocopherol and desferal.Aforesaid liquid is put into rotary evaporation bottle rotary evaporation film forming, again this film is immersed in the 150mM pH5.5 ammonium sulfate.
2. preparation small unilamellar vesicle
Aforesaid liquid is put into extruder repeatedly push, obtain the small unilamellar vesicle of particle diameter 100 nanometers.The carbon-fiber film that extruding is used is respectively 0.14 and 0.08 nanometer.Use the outer ammonium sulfate of the equilibrated Sephadex G-50 of 10% sucrose liquid (pH5.5) column chromatography eluting liposome subsequently.
3. preparation Evacet
The injection stage doxorubicin hydrochloride inj is dissolved in the 10% sucrose liquid.Under 65 ℃ of conditions, be that 0.1: 1 amount will amycin sucrose liquid adds in the small unilamellar vesicle and mixes by medicine/fat ratio, in 65 ℃ of water-baths, placed 30 minutes.Be cooled to room temperature, through the equilibrated Sephadex G-50 of 0.9% sodium chloride solution column chromatography eluting sucrose.Remove free doxorubicin.
Microporous membrane degerming, pyrogen and other impurity by 0.22 nanometer promptly obtain the Evacet finished product.
Embodiment 5
1. preparation multilamellar liposome
With injection stage SM, cholesterol and DSPE-PEG
2000The three is dissolved in (v/v, 2: 1) in the chloroform/methanol liquid, adds antioxidant α-tocopherol and desferal.Aforesaid liquid is put into rotary evaporation bottle rotary evaporation film forming, again this film is immersed in the 250mM pH6.5 ammonium sulfate.
2. preparation small unilamellar vesicle
After five freeze thaw circulations, aforesaid liquid is put into extruder repeatedly push, obtain the small unilamellar vesicle of particle diameter 100 nanometers.The carbon-fiber film that extruding is used is respectively 0.14 and 0.08 nanometer.Use the outer ammonium sulfate of the equilibrated Sephadex G-50 of 10% sucrose liquid (pH6.5) column chromatography eluting liposome subsequently.
3. preparation Evacet
The injection stage doxorubicin hydrochloride inj is dissolved in the 10% sucrose liquid.Under 65 ℃ of conditions, be that 0.05: 1 amount will amycin sucrose liquid adds in the small unilamellar vesicle and mixes by medicine/fat ratio, in 65 ℃ of water-baths, placed 15 minutes.Be cooled to room temperature, through the equilibrated Sephadex G-50 of 0.9% sodium chloride solution column chromatography eluting sucrose.Remove free doxorubicin.
Microporous membrane degerming, pyrogen and other impurity by 0.22 nanometer promptly obtain the Evacet finished product.
This product liposomal doxorubicin good stability, in the test of cultivating in the test of 10 times of buffer and with calf serum, the amycin of 75-80% still is wrapped in the liposome after 24 hours.
1. preparation multilamellar liposome
With injection stage SM, cholesterol and DSPE-PEG
2000The three is dissolved in (v/v, 2: 1) in the chloroform/methanol liquid, adds antioxidant α-tocopherol and desferal.Aforesaid liquid is put into rotary evaporation bottle rotary evaporation film forming, again this film is immersed in the 150mM pH5.5 ammonium sulfate.
2. preparation small unilamellar vesicle
Aforesaid liquid is put into extruder repeatedly push, obtain the small unilamellar vesicle of particle diameter 100 nanometers.The carbon-fiber film that extruding is used is respectively 0.14 and 0.08 nanometer.Use the outer ammonium sulfate of the equilibrated Sephadex G-50 of 10% sucrose liquid (pH5.5) column chromatography eluting liposome subsequently.
3. preparation Evacet
The injection stage doxorubicin hydrochloride inj is dissolved in the 10% sucrose liquid.Under 65 ℃ of conditions, be that 0.05: 1 amount will amycin sucrose liquid adds in the small unilamellar vesicle and mixes by medicine/fat ratio, in 65 ℃ of water-baths, placed 30 minutes.Be cooled to room temperature, through the equilibrated Sephadex G-50 of 0.9% sodium chloride solution column chromatography eluting sucrose.Remove free doxorubicin.
Microporous membrane degerming, pyrogen and other impurity by 0.22 nanometer promptly obtain the Evacet finished product.
1. preparation multilamellar liposome
With injection DSPC, cholesterol and DSPE-PEG
2000The three is dissolved in (v/v, 2: 1) in the chloroform/methanol liquid, adds antioxidant α-tocopherol and desferal.Aforesaid liquid is put into rotary evaporation bottle rotary evaporation film forming, again this film is immersed in the 350mM pH6.5 ammonium sulfate.
2. preparation small unilamellar vesicle
After five freeze thaw circulations, aforesaid liquid is put into extruder repeatedly push, obtain the small unilamellar vesicle of particle diameter 100 nanometers.The carbon-fiber film that extruding is used is respectively 0.14 and 0.08 nanometer.Use the outer ammonium sulfate of the equilibrated Sephadex G-50 of 10% sucrose liquid (pH6.5) column chromatography eluting liposome subsequently.
3. preparation Evacet
The injection stage doxorubicin hydrochloride inj is dissolved in the 10% sucrose liquid.Under 65 ℃ of conditions, be that 0.2: 1 amount will amycin sucrose liquid adds in the small unilamellar vesicle and mixes by medicine/fat ratio, in 65 ℃ of water-baths, placed 60 minutes.Be cooled to room temperature, through the equilibrated Sephadex G-50 of 0.9% sodium chloride solution column chromatography eluting sucrose.Remove free doxorubicin.
Microporous membrane degerming, pyrogen and other impurity by 0.22 nanometer promptly obtain the Evacet finished product.
This product liposomal doxorubicin good stability, in the test of cultivating in the test of 10 times of buffer and with calf serum, the amycin of 75-80% still is wrapped in the liposome after 24 hours.
1. preparation multilamellar liposome
With injection DSPC, cholesterol and DSPE-PEG
2000The three is dissolved in (v/v, 2: 1) in the chloroform/methanol liquid, adds antioxidant α-tocopherol and desferal.Aforesaid liquid is put into rotary evaporation bottle rotary evaporation film forming, again this film is immersed in the 250mM pH6.5 ammonium sulfate.
2. preparation small unilamellar vesicle
After five freeze thaw circulations, aforesaid liquid is put into extruder repeatedly push, obtain the small unilamellar vesicle of particle diameter 100 nanometers.The carbon-fiber film that extruding is used is respectively 0.14 and 0.08 nanometer.Use the outer ammonium sulfate of the equilibrated Sephadex G-50 of 10% sucrose liquid (pH6.5) column chromatography eluting liposome subsequently.
3. prepare the epirubicin liposome
Injection stage Farmorubine Hydrochloride injection is dissolved in the 10% sucrose liquid.Under 65 ℃ of conditions, be that 0.2: 1 amount will epirubicin sucrose liquid adds in the small unilamellar vesicle and mixes by medicine/fat ratio, in 65 ℃ of water-baths, placed 30 minutes.Be cooled to room temperature, through the equilibrated Sephadex G-50 of 0.9% sodium chloride solution column chromatography eluting sucrose.Remove free epirubicin.
Microporous membrane degerming, pyrogen and other impurity by 0.22 nanometer promptly obtain epirubicin liposome finished product.
This product liposome epirubicin good stability, in the test of cultivating in the test of 10 times of buffer and with calf serum, the epirubicin of 75-80% still is wrapped in the liposome after 24 hours.
1. preparation multilamellar liposome
With injection HSPC, cholesterol and DSPE-PEG
2000The three is dissolved in (v/v, 2: 1) in the chloroform/methanol liquid, adds antioxidant α-tocopherol and desferal.Aforesaid liquid is put into rotary evaporation bottle rotary evaporation film forming, again this film is immersed in the 250mM pH6.5 ammonium sulfate.
2. preparation small unilamellar vesicle
After five freeze thaw circulations, aforesaid liquid is put into extruder repeatedly push, obtain the small unilamellar vesicle of particle diameter 100 nanometers.The carbon-fiber film that extruding is used is respectively 0.14 and 0.08 nanometer.Use the outer ammonium sulfate of the equilibrated Sephadex G-50 of 10% sucrose liquid (pH6.5) column chromatography eluting liposome subsequently.
3. prepare the epirubicin liposome
Injection stage Farmorubine Hydrochloride injection is dissolved in the 10% sucrose liquid.Under 65 ℃ of conditions, be that 0.2: 1 amount will epirubicin sucrose liquid adds in the small unilamellar vesicle and mixes by medicine/fat ratio, in 65 ℃ of water-baths, placed 15 minutes.Be cooled to room temperature, through the equilibrated Sephadex G-50 of 0.9% sodium chloride solution column chromatography eluting sucrose.Remove free epirubicin.
Microporous membrane degerming, pyrogen and other impurity by 0.22 nanometer promptly obtain epirubicin liposome finished product.
This product liposome epirubicin good stability, in the test of cultivating in the test of 10 times of buffer and with calf serum, the epirubicin of 75-80% still is wrapped in the liposome after 24 hours.
1. preparation multilamellar liposome
Injection DSPC, cholesterol are dissolved in (v/v, 2: 1) in the chloroform/methanol liquid, add antioxidant α-tocopherol and desferal.Aforesaid liquid is put into rotary evaporation bottle rotary evaporation film forming, again this film is immersed in the 250mM pH6.5 ammonium sulfate.
2. preparation small unilamellar vesicle
After five freeze thaw circulations, aforesaid liquid is put into extruder repeatedly push, obtain the small unilamellar vesicle of particle diameter 100 nanometers.The carbon-fiber film that extruding is used is respectively 0.14 and 0.08 nanometer.Use the outer ammonium sulfate of the equilibrated Sephadex G-50 of 10% sucrose liquid (pH6.5) column chromatography eluting liposome subsequently.
3. preparation daunorubicin liposome
Injection stage daunorubicin hydrochloride injection is dissolved in the 10% sucrose liquid.Under 65 ℃ of conditions, be that 0.2: 1 amount will daunorubicin sucrose liquid adds in the small unilamellar vesicle and mixes by medicine/fat ratio, in 65 ℃ of water-baths, placed 15 minutes.Be cooled to room temperature, through the equilibrated Sephadex G-50 of 0.9% sodium chloride solution column chromatography eluting sucrose.Remove free daunorubicin.
Microporous membrane degerming, pyrogen and other impurity by 0.22 nanometer promptly obtain the daunorubicin liposome finished product.
This product liposome daunorubicin good stability, in the test of cultivating in the test of 10 times of buffer and with calf serum, the daunorubicin of 75-80% still is wrapped in the liposome after 24 hours.
Embodiment 11, acute toxicity test
One. material:
Kunming mice is purchased the zooscopy institute in the Chinese Academy of Medical Sciences
Doxorubicin hydrochloride is purchased in the Zhejiang Haizheng Pharmaceutical Co.
The hydrochloric doxorubicin liposome injection, embodiment 3.
Two. method:
Relatively acute toxic reaction after doxorubicin hydrochloride and the disposable mouse tail vein administration of hydrochloric doxorubicin liposome injection and dead distribution.
With the mice random packet, 10 every group, totally 90.Wherein the doxorubicin hydrochloride inj group is 40,50 of hydrochloric doxorubicin liposome injection groups.Inject doxorubicin hydrochloride and hydrochloric doxorubicin liposome injection respectively, doxorubicin hydrochloride inj dosage is respectively 15,20,25,30 milligrams/kg body weight, and hydrochloric doxorubicin liposome injection dosage is respectively 20,30,40,50,60 milligrams/kg body weight.Observed 14 days, and observed mice body weight change, acute poisoning symptom and mortality every day and determine median lethal dose(LD 50) (LD50).The results are shown in Table 2.
Three. the result
The acute toxicity test of table 2. amycin and liposomal doxorubicin (LD50)
| Amycin | The Evacet injection | |
| LD50(mg/kg) | 23.179 | 43.572 |
Conclusion: Evacet effectively reduces the toxicity of load medicine, reduces by 1.88 times of toxicity in this test.
Embodiment 12: compare liposome anthracene nucleus medicine stability leak test hydrochloric doxorubicin liposome injection with external like product, embodiment 3.
Doxil/Caelyx is available from U.S. Schering Plough company (Schering-Plough).Caelyx is at the commodity of China pattern Lay by name.
The hydrochloric doxorubicin liposome and the Caelyx of isodose are inserted respectively in the bag filter, cultivate dialysis together with PBS buffer+10% calf serum of 1,000 times under 37 ℃ of temperature, at different time, sampling detects in dialysis solution respectively.
Conclusion: hydrochloric doxorubicin liposome is consistent with the seepage speed of Caelyx, illustrates that both stability is consistent.
Description of drawings:
Fig. 1 is that the embodiment of the invention 12 this product compare liposome anthracene nucleus medicine stability leak test figure with external like product
Claims (10)
1. an anthracene ring antitumor medicinal liposome is characterized in that being made up of anthracene ring antitumor medicinal, liposome material and antioxidant, and medicine/fat ratio (w/w) is 0.05～0.2: 1;
Wherein the liposome material comprises phospholipid and cholesterol, and the mol ratio of the two is 40～60: 30～50mol%;
Antioxidant is made up of α-tocopherol and desferal, and the concentration of the two is respectively 0.010～0.030mg/ml and 0.05～0.30mg/ml.
2. a kind of anthracene nucleus medicinal liposome according to claim 1 is characterized in that: also contain poly ethylene-ethylene glycol-DSPE (DSPE-PEG2000) in the described liposome material;
Phospholipid, cholesterol and DSPE-PEG2000 three's mol ratio is 40～60: 30～50: 4～20mol%.
3. a kind of anthracene nucleus medicinal liposome according to claim 1 and 2 is characterized in that: described phospholipid is selected from a kind of in distearoyl phosphatidylcholine (DSPC), hydrogenated phospholipid phatidylcholine (HSPC) or the sphingomyelin (SM).
4. a kind of anthracene nucleus medicinal liposome according to claim 1 and 2 is characterized in that: described anthracene nucleus medicament is selected from a kind of in amycin, epirubicin or the daunorubicin.
5. the production technology of an anthracene nucleus medicinal liposome may further comprise the steps:
(1) preparation multilamellar liposome: the amount according to claim 1 or 2 described prescriptions is prepared chloroform or the chloroform/methanol solution that contains liposome material and antioxidant, and the rotary evaporation film forming adds 125～400mM, and pH value is 5～7 ammonium sulfate aquations;
(2) preparation small unilamellar vesicle: after the ammonium sulfate that contains multilamellar liposome that step (1) is obtained carries out five freeze thaws circulations, be squeezed into the small unilamellar vesicle liquid of 80～120 nanometers through extruder; Use the outer ammonium sulfate of the equilibrated Sephadex G-50 column chromatography of 5～15% sucrose liquid (pH5～7) eluting liposome subsequently.
(3) preparation anthracene nucleus medicinal liposome: it is in 5～15% the sucrose liquid that the hydrochlorate of injection stage anthracene ring antitumor medicinal is dissolved in concentration, under 50～70 ℃ of conditions, hydrochloric acid anthracene nucleus medicine sucrose liquid joined in the small unilamellar vesicle liquid and mix, in 50～70 ℃ of water-baths, place more than 10 minutes, hydrochloric acid anthracene nucleus medicine is entered in the small unilamellar vesicle, be cooled to room temperature, remove sucrose and free anthracene nucleus medicament obtains finished product.
6. the production technology of a kind of anthracene nucleus medicinal liposome according to claim 5 is characterized in that: ammonium sulfate concentrations is 125-400mM in the described step (1); PH value is 5～7.
7. the production technology of a kind of anthracene nucleus medicinal liposome according to claim 5, it is characterized in that: five freeze thaws circulation in the described step (2) be meant with multilamellar liposome with dry ice freezing after thawing naturally at room temperature, and then freezing with dry ice, so circulate five times.But in practical operation, also can omit the freeze thaw circulation step, and directly enter pressing steps.Omit the prepared liposome physicochemical property of freeze thaw circulation step and use the prepared liposome difference with insignificance of freeze thaw circulation step.
8. the production technology of a kind of anthracene nucleus medicinal liposome according to claim 5 is characterized in that: the particle diameter of small unilamellar vesicle is 80～120 nanometers in the described step (2), and the best is 100 nanometers.
9. the production technology of a kind of anthracene nucleus medicinal liposome according to claim 5, it is characterized in that: the method for removing free anthracene nucleus medicament in the described step (3) is chromatography or centrifugal separation.
10. the production technology of a kind of anthracene nucleus medicinal liposome according to claim 5 is characterized in that: use 5～15% sucrose solutions in described step (2) and (3), the best is selected 10% isotonic solution for use; Sucrose liquid pH5～7, the best is selected pH6.5 for use.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101810570A (en) * | 2010-04-16 | 2010-08-25 | 成都师创生物医药科技有限公司 | Lipid nano particle preparation containing composite formed fatty acid and anthracyclines antitumor antibiotics and preparation method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101810570A (en) * | 2010-04-16 | 2010-08-25 | 成都师创生物医药科技有限公司 | Lipid nano particle preparation containing composite formed fatty acid and anthracyclines antitumor antibiotics and preparation method thereof |
| CN101810570B (en) * | 2010-04-16 | 2012-06-20 | 成都师创生物医药科技有限公司 | Lipid nano particle preparation containing composite formed fatty acid and anthracyclines antitumor antibiotics and preparation method thereof |
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