CN100496609C - Stable liposome composition - Google Patents
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- CN100496609C CN100496609C CNB2006101487072A CN200610148707A CN100496609C CN 100496609 C CN100496609 C CN 100496609C CN B2006101487072 A CNB2006101487072 A CN B2006101487072A CN 200610148707 A CN200610148707 A CN 200610148707A CN 100496609 C CN100496609 C CN 100496609C
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Abstract
A stable liposome composition used as the carrier of anticancer taxol is prepared from saturated hydrogenated phosphate, cholesterol, sodium (or potassium) cholesterol sulfate and cane sugar through thin-film evaporating-freeze-drying process.
Description
Technical field
The invention belongs to field of pharmaceutical preparations, be specifically related to a kind of stable liposome composition, and application and the preparation method of this liposome composition aspect envelope paclitaxel, and the purposes of this Paclitaxel liposome.
Background technology
Anti-cancer medicine paclitaxel (paclitaxe) is the natural product that extracts from the bark of yewtree (Taxus brevifolia Nutt.), is used for the treatment of ovarian cancer, breast carcinoma and nonsmall-cell lung cancer.But atomic, only solvable in organic solvents such as methanol, ethanol, dimethyl sulfoxide because of its dissolubility in water, thereby bring very big difficulty for the paclitaxel intravenously administrable.Solve one of its deliquescent known method, be to adopt surfactant polyoxyethylene Oleum Ricini and ethanol as the dissolution with solvents paclitaxel, paclitaxel injection (U.S. Bristol-Myers Squibb Co. production as FDA (Food and Drug Adminstration) (FDA) in 1992 approval, commodity are called Taxol, at the commodity of China's registration taxol by name).Though polyoxyethylene castor oil can increase the water solublity of paclitaxel, can discharge histamine during owing to its degradation in vivo, thereby the serious quick reaction that surpasses may take place.In addition, paclitaxel itself also has haematics toxicity, bone marrow depression, leukopenia, thrombocytopenia, anemia and other toxicity.
Therefore, we expect to prepare a kind of stable preparation that can reduce the paclitaxel toxic and side effects, avoid on the one hand the anaphylaxis that brought by polyoxyethylene castor oil and ethanol double solvent, reduce the system toxicity of paclitaxel on the one hand.
Liposome (liposome) be can yet be regarded as the carrier of paclitaxel administration reach a kind of method of this purpose.The filmogen that liposome is commonly used has amphiphilic species such as phospholipid, cholesterol, sphingomyelins class, and wherein phospholipid is usually as the main composition composition of bimolecular lipid membrane, and cholesterol plays an important role to flowability, the pliability of adjusting film.
The basic research of the Paclitaxel liposome that just carries out since nineteen ninety generation shows that Paclitaxel liposome preparation is compared with injection, and curative effect is similar, but toxicity reduces, and toleration improves.At present in the Paclitaxel liposome research field, a plurality of disclosed technical schemes are arranged, as Sharma-A at " Int-J-Cancer " 1997,71, the Paclitaxel liposome that report is made with three kinds of synthetic phospholipids among the 103-107, the cationic-liposome of mentioning among the CN1378443A, the taxol nano magnetic target preparation of the ultra-fine magnetic fluid preparation of the employing of mentioning among the CN1399958A, the charged Paclitaxel liposome of mentioning among the W09513053 with phosphatidyl glycerol ester (PG) preparation, WO9318751, US5424073, US5648090, CN1275076A, mention among the CN1714094A with special phospholipid--the liposome of cuorin (cardiolipid) preparation, that mentions among the CN1291474A contains amino acid whose Paclitaxel liposome, the taxol hidden liposome of mentioning among the CN1391891A with the film preparation of amphipathic ethylene glycol-DSPE (PEG-DSPE) modified liposome.
Produce (the accurate word H20030357 of traditional Chinese medicines at the injection Paclitaxel liposome that China has gone on the market by Nanjing Sike Pharmaceutical Co., Ltd, commodity power by name is pounced on element), claim according to its description, still there is the risk that allergy takes place, need inject dexamethasone etc. in advance and carry out the antianaphylaxis pretreatment, compare with paclitaxel injection, aspect toleration, there is no obvious improvement.
In above-mentioned these technical schemes of mentioning, the not use of clear and definite saturated phospholipid.And as known in those skilled in the art, contain undersaturated fatty acid-based in matrix material commonly used such as natural phosphatidyl choline, the phospholipid of natural soybean, easily oxydrolysis becomes peroxide, malonaldehyde, fatty acid and lysophosphatide, and the latter can further be hydrolyzed into phosphoglycerol complex and fatty acid etc.The oxydrolysis of phospholipid also can make the flowability of film reduce, and promotes drug leakage.These unstable factors are unfavorable for preparation, storage and the safe handling clinically of liposome product.In addition, the compatibility of charged liposome and serum or blood, cell, tissue or the compatibility often can not expect, the large-scale aggregation that electrically forms in blood as strong cation causes the formation of organic thrombus in vivo.Simultaneously, as known to those skilled in the art, electric charge can influence the stability of bimolecular film with expecting, the liposome that charged phospholipid forms increases the repulsive force between the lipid somatocyst, make it to be difficult for coagulative precipitation, but simultaneously, the phospholipid of lotus like charges can cause the unstability of membrane structure in the bimolecular film, and therefore too high electric charge is not always useful to the stable entrapped drug of liposome.In addition, hidden liposome can make liposome stable, effectively prolong liposome circulation time in vivo, but it adopts amphipathic ethylene glycol-DSPE
(PEG-DSPE) cost an arm and a leg.In addition, there is the problem of selling at exorbitant prices equally in this special matrix material of cuorin (cardiolipid).
In liposome, cholesterol is attached in the bimolecular film of phospholipid formation, can realize the stability and the suitable flowability and the pliability of film.Content of cholesterol all has appreciable impact to medicine clad ratio, particle diameter.The derivant of cholesterol is also through being commonly used for the preparation of liposome.Be used to mix with preparation pH sensitive lipid body as the Cholesteryl hemisuccinate of mentioning among the US4891208 with PHOSPHATIDYL ETHANOLAMINE.This liposome has negative charge completely, and the efficient that is absorbed by cell is very low.Disclosed amphoteric compound among the CN1492876A based on sterol skeleton, the 3-position of ring is replaced by the amphiprotic group of one or more isoelectric point, IPs between 4 to 9, according to the technical scheme of this patent disclosure, need complicated synthesis technique to produce this cholesterol derivative.Multiple factor affecting pH value in the body, but just as recognized by those skilled in the art, the release of controlling medicine based on pH value in the body is always not reliable.
Therefore, if can prepare stabilized liposomes with the common widely matrix material in source, thereby control simultaneously its carrying capacity make its in vivo process more can expect, to be expected to remedy the deficiency of the above-mentioned technical scheme of mentioning, provide a kind of novel, stay-in-grade, be suitable for industrialized pharmaceutical carrier.
Summary of the invention
The invention provides a kind of liposome composition, described liposome does not adopt special charged phospholipid, also avoid using expensive Polyethylene Glycol-DSPE (PEG-DSPE) and cuorin (cardiolipid), and adopt the sodium salt of common hydrogenation saturated phospholipid, cholesterol, cholesterol sulfate or potassium salt as main carrier, the coating active composition, gained liposome composition carrying capacity of the present invention is low, quality is highly stable, possess industrial prospect, remedied the deficiency of existing liposome technology.
The present invention also provides a kind of preparation method of liposome composition, is that employing thin film evaporation-cryodesiccated method has obtained stable liposome composition.
The present invention also provides a kind of method that improves liposome stability and drug encapsulation stability simultaneously, promptly, the zeta current potential of liposome dispersion is remained in the scope of-15 to-35 millivolts (mV) near the sodium salt or the potassium salt that add micro-cholesterol sulfate in the neutral film forming matrix material.
The present invention especially provides a kind of stable Paclitaxel liposome composition and method of making the same, with and in the application of preparation aspect the antitumor drug.
The present invention forms and achieves the above object by optimizing liposome.One of essential component of preparation liposome of the present invention is the hydrogenation saturated phospholipid.Usually, the carbon number of the saturated fat acidic group that contains of this saturated phospholipid is 14～18.Carbon number is less than 14, and the ability of water reduced in liposome held, and after giving, the stability of liposome in blood reduces.On the other hand, carbon number is too much, and biocompatibility reduces, and because its carbochain increases, and phase transition temperature raises, producing in the liposome process needs very high temperature, and this stability to medicine and liposome all is adverse factors.Therefore, saturated phospholipid used in the present invention for example is selected from: soybean phospholipid that hydrogenation is saturated or lecithin, dipalmitoyl-phosphate ester choline, distearoyl phosphatidylcholine.The saturated phospholipid that the present invention uses is not limited to above phospholipid kind, and the carbon number of all saturated fat acidic groups that contains is 14～18 phospholipid, all is applicable to purpose of the present invention.
The preferred saturated phospholipid of the present invention is selected from the saturated soybean phospholipid of hydrogenation, saturated lecithin, dipalmitoyl-phosphate ester choline, the distearoyl phosphatidylcholine of hydrogenation, the more preferably saturated saturated lecithin of soybean phospholipid, hydrogenation of hydrogenation.The preferred purity of the saturated soybean phospholipid of hydrogenation is more than 97%, and its main component is a phosphatidylcholine, contains the phosphatidyl ethanolamine and the lipositol of trace simultaneously, because its aliphatic chain hydrogenation is saturated, thereby prevents hydrolysis oxidation.
Another of preparation liposome of the present invention must component be cholesterol.Cholesterol becomes membrane lipid, the flowability that can regulate bimolecular lipid membrane with the saturated phospholipid conduct.
The 3rd of preparation liposome of the present invention must component be 3 derivant of a class cholesterol, the sodium salt or the potassium salt of preferred cholesterol sulfate.The cholesterol sulfate sodium salt is a kind of amphiphilic species, and the amphotericin colloid dispersion (trade name AMPHOCIL) of U.S.'s listing promptly adopts it as matrix material, forms a kind of tabular dispersion with medicine.At US4822777, US5032582, US5194266 this compositions has been described.In the present invention, creatively with the sodium salt or the potassium salt of cholesterol sulfate, see the molecular structure shown in formula I and the formula II, be added in the above-mentioned matrix material micro-ly, obtain beyond thought effect, obtained the liposome that stable mean diameter is 100-300nm.
Formula I cholesterol sulfate sodium formula II cholesterol sulfate potassium
The sodium salt of cholesterol sulfate or potassium salt are a kind of amphiphilic species of bear electricity, its role is to: (1) joins in the membrane lipid micro-ly, can not influence the formation of film because of the repulsion between electric charge; (2) because the affinity interaction of its water-wet side and phospholipid water-wet side makes the bimolecular film of liposome more stable, increase the pliability of film, formed particle diameter and concentrate on the interior liposome of 100-300nm scope, simultaneously, reduced the leakage of medicine; (3) but there is not application risk in the derivant of this cholesterol safe disposal in vivo.
A technical scheme that realizes liposome of the present invention is as follows:
The one-tenth membrane lipid of liposome bilayer is made up of the material of following ratio: the molar ratio of hydrogenation saturated phospholipid, cholesterol, cholesterol sulfate sodium salt or potassium salt is: 100:20-50:0.2-5, more preferably the molar ratio of this three classes material is: 100:35-45:0.5-2, as active component, the molar ratio of itself and hydrogenation saturated phospholipid is: 2-8:100 with paclitaxel.In this scheme, also can further comprise natural type or synthesising complex E as antioxidant, the weight ratio of itself and hydrogenation saturated phospholipid is 0.3-1:100.Above-mentioned substance is prepared in proportion, can be obtained liposome of the present invention, for example adopt high pressure homogenize method, micropore squeezing and pressing method, ultrasonic dispersion, preferably adopt high pressure homogenize method with method for preparing lipidosome commonly used.The mean diameter of the liposome that the employing method for preparing obtains is in 100 nanometer to 300 nanometer range.Preparation method in the above-mentioned list of references of mentioning all is incorporated herein by reference.
Understand easily as those skilled in the art institute, liposome of the present invention not only can be used as the carrier of paclitaxel, is applicable to other taxanes material simultaneously, no matter and the size of its dissolubility in water.Equally, other cancer therapy drugs, as hydroxy camptothecin, too can liposome of the present invention as drug administration carrier.Other drug is no matter its water miscible difference under its cost performance acceptable prerequisite, can adopt liposome of the present invention as carrier equally.
The liposome that wherein contains reactive compound can be stablized with lyophilization, to make the suitable compositions form.In this process, need to add freeze drying protectant, rely on its stabilising membrane effect, guarantee the integrity of liposome.Known freeze drying protectant is a polyalcohols, as glycerol, glucose, sucrose, lactose, trehalose, aminoacid etc.In the present invention, preferably sucrose, lactose, maltose, glucose be as freeze-dried excipient, more preferably sucrose.According to the present invention, freeze drying protectant is 2 to 10 times of hydrogenation saturated phospholipid weight.
Drug-loaded liposome composition forms of the present invention can prepare by above-mentioned cryodesiccated method.For example, with active component, lipid material, if necessary, and antioxidant, be dissolved in proportion and become clear and bright solution in the The suitable solvent, under 50 ℃～65 ℃ constant temperature, according to conventional method, as the reduction vaporization method, if necessary, reduction vaporization in the presence of noble gas is removed the solvent in this liquid mixture, makes it film forming, add the aqueous solution that is dissolved with the freeze-dried excipient that calculates in proportion again, aquation, ultrasonic or homogenate to fineness is between 0.1～0.30 micron, with 0.22 micron filtering with microporous membrane degerming, divides in the suitable container such as the cillin bottle of packing into, remove from this mixture by lyophilization and to desolvate, obtain exsiccant pastille liposome, again packing container sealed or gland, seal with gland before can lead to noble gas.
In the preparation method of above-mentioned liposome, do not limit the amount of the employed solvent relevant, and the amount of the liquid of liposome dissolving is suited with lipid.Usually, The suitable solvent be nonpolar or low pole and can evaporate after do not stay the toxicity residue.According to conventional method,, be selected from methanol, ethanol, chloroform or their mixture as with regard to common employed organic solvent.In the present invention, preferred alcohol.The noble gas that adopts among the present invention is selected from nitrogen, helium, argon.Can steady in a long-termly store with the exsiccant pastille liposome membrane that this preparation method makes.
In the present invention, a preferred scheme is: active component is taxane and derivant thereof, more preferably paclitaxel.The present invention prepares the paclitaxel injecting and administering preparations of gained, and it does not contain Cremophor EL, has avoided containing the allergy that Cremophor EL causes, and compares with conventional paclitaxel injection, has improved toleration.
Gained Paclitaxel liposome of the present invention has been obtained useful effect:
(1) semi-finished product and stability of formulation
The Paclitaxel liposome compositions of the present invention preparation was measured its Zeta potential before lyophilizing, between-35mV-15mV, its Zeta potential is measured in room temperature placement 24 hours during this period, remains unchanged substantially.Liposome keeps suspended state in the compositions of suspendible, do not assemble.Redissolve the back according to measuring 24 hours with quadrat method, the gathering of liposome can not take place equally.The particle diameter and the later measurement result of redissolving show that its particle size distribution is at 100-250nm.And with same prescription but the liposome that does not contain the sulphuric acid cholesterol after placing two hours, sedimentation promptly takes place.
(2) effectiveness and the safety in utilization of treatment cancer
In animal experiment, Paclitaxel liposome administration group mice does not show toxicity, and every physical signs is normal, and waits dosage taxol treated animal of short duration stupor to occur after administration.Preliminary antitumor result of the test shows, liposome and the tumour inhibiting rate no difference of science of statistics that waits the dosage taxol.Hence one can see that, and Paclitaxel liposome of the present invention has improved the toleration of organism under the suitable situation of tumour inhibiting rate.
(3) probability of industrialization
Liposome composition of the present invention adopts saturated phospholipid and cholesterol as becoming membrane lipid, and these are commercial available pharmaceutical formulation material.Preparation technology of the present invention is simple, and step is few, and the preparation equipment of employing is a conventional equipment, is convenient to drop into suitability for industrialized production.As the liposome composition that active component makes, avoided containing the allergy that the formulation for paclitaxel of Cremophor EL causes with paclitaxel.According to the particle size distribution and the preliminary anxious malicious result of the test of redissolving the back Paclitaxel liposome, can analyze and learn after its intravenous injection to have the passive target characteristic, directional profile is in reticuloendothelial system, thereby the general toxicity of reduction paclitaxel has improved the toleration of patient's medication.
The Paclitaxel liposome compositions of gained of the present invention can be used for mammal with reasonable dosage safety ground in a suitable manner, can be used as mammal antineoplastic therapeutic agent, especially can be applicable to the treatment of human ovarian cancer, metastatic carcinoma of ovary, breast carcinoma, nonsmall-cell lung cancer, also can with the cisplatin combined treatment for cancer that is used for.
Below term used herein is specifically noted:
Term used herein " liposome " as those skilled in the known, refers to by having the closure folliculus that amphipathic phospholipid molecule and cholesterol are polymerized.In this article, refer in general sense unilamelar liposome.
Term used herein " one-tenth membrane lipid " refers to constitute the material of liposome bilayer, and what refer to phospholipid and cholesterol and cholesterol derivative in this article and constituted is overall, herein " bilayer material " with " become membrane lipid to have same implication.
Term used herein " saturated phospholipid " refers to that fatty acid-based in the phospholipid molecule is saturated, does not contain " C=C " two keys substantially in promptly fatty acid-based." hydrogenated phospholipid ", " hydrogenation saturated phospholipid " are same implications with this term in the literary composition.
Term used herein " particle diameter " is meant the mean diameter of the liposome that records with laser particle size determination instrument.
Term used herein " cholesterol sulfate sodium salt or potassium salt " is meant the chemical compound of structure shown in formula I and the formula II, with " the sulphuric acid cholesterol " mentioned in other documents, " cholesterol acyl sulfate ester " be same implication.
The present invention is further detailed explanation below in conjunction with embodiment, and the weight of the material that is obtained by the mol ratio conversion among the embodiment feeds intake, because phospholipid mostly is mixture, so get its about molecular weight when calculating.The molecular weight of the material that relates to is as follows:
Paclitaxel 860
The saturated soybean phospholipid 760 of hydrogenation
The saturated lecithin 780 of hydrogenation
Distearoyl phosphatidylcholine 760
Two Palmic acid phosphatidylcholines 740
Cholesterol 380
Cholesterol sulfate sodium 489
Cholesterol sulfate potassium 523
Embodiment provides as the purpose of illustration, does not constitute limitation of the scope of the invention.
Description of drawings:
The electromicroscopic photograph of the liposome that Fig. 1 embodiment 1 makes
The lipidosome freeze-dried preceding particle size distribution that Fig. 2 embodiment 1 makes
Particle size distribution after the liposome that Fig. 3 embodiment 1 makes redissolves
The preparation of embodiment 1 Paclitaxel liposome
Prescription:
Taxol 0.37g
Hydrogenated soya phosphatide 4g
Cholesterol 1g
Cholesterol sulfate sodium 0.05g
Vitamin E 0.055g
Sucrose 15g
Water for injection 120ml
Take by weighing respectively recipe quantity taxol, hydrogenated soya phosphatide, cholesterol, cholesterol sulfate sodium, support one's family Plain E adds the 100ml absolute ethyl alcohol, after the dissolving, under there is situation in nitrogen, drains solvent with Rotary Evaporators, The preparation film forming is put the placement of spending the night in the vacuum desiccator, removes residual solvent. Adding is with the water-soluble sugarcane of injection Sugar juice, 55 ℃ of water-baths, magnetic agitation 1 hour, the high pressure homogenizer homogeneous is used in ultrasonic dispersion, and 0.22 is little The aseptic filtration of rice pvdf membrane is distributed into cillin bottle, freeze drying. With the logical nitrogen of cillin bottle, gland seal.
According to method among 2005 editions two appendix XI X of the Chinese Pharmacopoeia E, the envelop rate that records sample is 93.0%.
According to method among 2005 editions two appendix XI X of the Chinese Pharmacopoeia E, measure lyophilizing proliposome sample with laser particle size determination instrument, its mean diameter is 138.6 nanometers, the mean diameter of water for injection redissolution sample is 178.5 nanometers.Its electromicroscopic photograph is seen Fig. 1, and particle size distribution figure sees Fig. 2 and Fig. 3.
The preparation of embodiment 2 Paclitaxel liposomes
Prescription:
Paclitaxel 0.09g
Hydrogenated soya phosphatide 4g
Cholesterol 0.4g
Cholesterol sulfate potassium 0.006g
Vitamin E 0.012g
Lactose 10g
Water for injection 150ml
The paclitaxel, hydrogenated soya phosphatide, cholesterol, cholesterol sulfate potassium, the vitamin E that take by weighing recipe quantity respectively add the 100ml dehydrated alcohol, after the dissolving, prepare film forming with Rotary Evaporators, put the placement of spending the night in the vacuum desiccator, remove residual solvent.Adding is with the dissolved sucrose solution of water for injection, 58 ℃ of water-baths, and magnetic agitation 1 hour, ultra-sonic dispersion adopts 0.8,0.4,0.2 micrometer polycarbonate film successively, extrudes with the extruder high pressure, divides the cillin bottle of packing into, lyophilization, gland seal.
Method is measured similarly to Example 1, and the mean diameter of sample is 164.6 nanometers before the lyophilizing, and envelop rate is 92.1%.
The preparation of embodiment 3 Paclitaxel liposomes
Prescription:
Paclitaxel 0.18g
Hydrogenated soya phosphatide 4g
Cholesterol 0.9g
Cholesterol sulfate sodium 0.13g
Vitamin E 0.04g
Sucrose 25g
Water for injection 150ml
The paclitaxel, hydrogenated soya phosphatide, cholesterol, cholesterol sulfate sodium, the vitamin E that take by weighing recipe quantity respectively add the 100ml dehydrated alcohol, after the dissolving, prepare film forming with Rotary Evaporators, put the placement of spending the night in the vacuum desiccator, remove residual solvent.Adding is with the dissolved sucrose solution of water for injection, 60 ℃ of water-baths, and magnetic agitation 1 hour, ultra-sonic dispersion is used the high pressure homogenizer homogenizing, extrudes with 0.2 micrometer polycarbonate film, and 0.22 micron pvdf membrane aseptic filtration divides the cillin bottle of packing into, lyophilization, gland seal.
Method is measured similarly to Example 1, and the mean diameter of sample is 152.6 nanometers before the lyophilizing, and envelop rate is 90.6%.
The preparation of embodiment 4 Paclitaxel liposomes
Prescription
Paclitaxel 0.2g
Hydrogenated soya phosphatide 4g
Cholesterol 0.7g
Cholesterol sulfate potassium 0.012g
Vitamin E 0.04g
Sucrose 15g
Water for injection 150ml
The paclitaxel, hydrogenated soya phosphatide, cholesterol, cholesterol sulfate potassium, the vitamin E that take by weighing recipe quantity respectively add the 100ml dehydrated alcohol, after the dissolving, prepare film forming with Rotary Evaporators, put the placement of spending the night in the vacuum desiccator, remove residual solvent.Adding is with the dissolved sucrose solution of water for injection, 50 ℃ of water-baths, and magnetic agitation 1 hour, ultra-sonic dispersion is used the high pressure homogenizer homogenizing, extrudes with 0.2 micrometer polycarbonate film, and 0.22 micron pvdf membrane aseptic filtration divides the cillin bottle of packing into, lyophilization, gland seal.
Method is measured similarly to Example 1, and the mean diameter of sample is 143.8 nanometers before the lyophilizing, and envelop rate is 88.2%.
The preparation of embodiment 5 Paclitaxel liposomes
Prescription
Paclitaxel 0.15g
Dipalmitoyl phosphatidyl choline 2g
Cholesterol 0.5g
Cholesterol sulfate sodium 0.005g
Vitamin E 0.025g
Sucrose 6.5g
Water for injection 75ml
The paclitaxel, dipalmitoyl phosphatidyl choline, cholesterol, the cholesterol sulfate sodium vitamin E that take by weighing recipe quantity respectively add the 100ml dehydrated alcohol, after the dissolving, prepare film forming with Rotary Evaporators, put the placement of spending the night in the vacuum desiccator, remove residual solvent.Adding is with the dissolved sucrose solution of water for injection, 58 ℃ of water-baths, and magnetic agitation 1 hour, ultra-sonic dispersion adopts 0.8,0.4,0.2 micrometer polycarbonate film successively, extrudes with the extruder high pressure, divides the cillin bottle of packing into, lyophilization, gland seal.
Method is measured similarly to Example 1, and the mean diameter of sample is 138.6 nanometers before the lyophilizing, and envelop rate is 86.4%.
The preparation of embodiment 6 Paclitaxel liposomes
Prescription
Paclitaxel 0.15g
Hydrolecithin 4g
Cholesterol 0.4g
Cholesterol sulfate potassium 0.001g
Sucrose 35g
Water for injection 200ml
The paclitaxel, hydrolecithin, cholesterol, the cholesterol sulfate potassium that take by weighing recipe quantity respectively add the 100ml dehydrated alcohol, after the dissolving, prepare film forming with Rotary Evaporators, put the placement of spending the night in the vacuum desiccator, remove residual solvent.Adding is with the dissolved sucrose solution of water for injection, 55 ℃ of water-baths, magnetic agitation 1 hour, ultra-sonic dispersion, use the high pressure homogenizer homogenizing, extrude with 0.2 micrometer polycarbonate film, 0.22 micron pvdf membrane aseptic filtration divides the cillin bottle of packing into, lyophilization passes to argon, gland seal in the cillin bottle.
Method is measured similarly to Example 1, and the mean diameter of sample is 178.5 nanometers before the lyophilizing, and envelop rate is 92.4%.
The stability test of embodiment 7 Paclitaxel liposome compositionss
This test is intended to investigate the bin stability and the stability in use of the liposomal samples for preparing among the embodiment.
The exsiccant Paclitaxel liposome compositions that makes among the embodiment 1,2,3 is stored 2～10 ℃ of long-term stabilities, observe its outward appearance, after redissolving with water for injection,, the results are shown in Table 1 with microscopic examination; Put 12 hours this solution chamber is gentle, regularly observe, the results are shown in Table 2.By the result as can be known, the taxusol-lipid thin film, is not assembled after the redissolution after 12 months 2～10 ℃ of storages, and no paclitaxel crystallization is separated out; Placed 12 hours after redissolving, do not assemble, no paclitaxel crystallization is separated out.
Table 1 taxusol-lipid body hypothermia (2-10 ℃) shelf-stability result of the test
Room temperature shelf-stability result of the test after table 2 Paclitaxel liposome redissolves
The acute toxicity test of embodiment 8 Paclitaxel liposomes
This embodiment is intended to investigate Paclitaxel liposome of the present invention instant toxic reaction after the administration and the toleration of multiple dosing in the usual way.
The Paclitaxel liposome of preparation among the embodiment 1 is redissolved with water for injection.Experimental animal: Kunming kind female mice, body weight 18～20g is divided into 4 groups at random, and 5 every group, negative group is given and the test group coordinative solvent of high volume.The 1st, 4,7 days to each the group with intravenous administration.
Result of the test: (1) instant toxicity is observed: each organizes immediate observation after the mice administration, no obvious toxicity.(2) mice body weight change: the mice body weight change is little after the administration of paclitaxel group, and the mice body weight has decline after the administration for the third time, and body weight progressively rises afterwards.Blank solvent group mice body weight progressively rises.
Concrete outcome sees Table 3.
The weight of animals changes in the acute toxicity test of table 3 Paclitaxel liposome
The test of embodiment 9 antitumor actions
Carry out preliminary antitumor test with the Paclitaxel liposome compositions of preparation among the embodiment 1.Laboratory animal adopts C
57BL/6 deceives mice, and is male, body weight 19～22g, random packet, 10 every group.Get B16 melanoma source, adopt the homogenate method, 1:6 is prepared into tumor cell suspension with normal saline, right axil subcutaneous vaccination B16 melanoma, and behind the tumor inoculation 24 hours, immediate reaction and body weight change were observed in the mouse tail vein injection administration.After experiment finishes, cut open and get tumor and weigh, be calculated as follows tumor control rate.
The average tumor of the average tumor weight-administration of tumor control rate %=[(matched group group is heavy)/the average tumor of matched group is heavy] * 100%
Result of the test: (1) immediate reaction is observed: Paclitaxel liposome does not have the reaction of instant obviously side effect after respectively organizing administration.After taxol is respectively organized the mouse vein administration (annotating speed is 1 ml/min), instant mice is the semicoma state, can recover gradually about 15 minutes.(2) each treated animal body weight has no significant change.(3) under the situation that accumulated dose equates, the tumor killing effect no significant difference of Paclitaxel liposome and taxol (seeing Table 4).
Table 4 Paclitaxel liposome different dosing regimes is to murine melanoma B16 (subcutaneous vaccination) clinical trial
Claims (10)
1. a stabilized liposomes is characterized in that, the one-tenth membrane lipid of liposome comprises 3 substitutive derivatives of hydrogenation saturated phospholipid, cholesterol and cholesterol, and active component is wrapped in the liposome; 3 substitutive derivatives of described cholesterol are selected from cholesterol sulfate sodium salt or cholesterol sulfate potassium salt; Described active component is a paclitaxel; The carbon number that described hydrogenation saturated phospholipid is selected from the saturated fat acidic group is 14～18 hydrogenation saturated phospholipid, and the molar ratio of hydrogenation saturated phospholipid, cholesterol, cholesterol sulfate sodium salt or potassium salt is: 100:20-50:0.2-5.
2. liposome as claimed in claim 1 is characterized in that, described hydrogenation saturated phospholipid is selected from the saturated soybean phospholipid of hydrogenation, the saturated lecithin of hydrogenation, dipalmitoyl phosphatidyl choline, distearoyl phosphatidylcholine.
3. liposome as claimed in claim 1 is characterized in that, the molar ratio of described hydrogenation saturated phospholipid, cholesterol, cholesterol sulfate sodium salt or potassium salt is: 100:35-45:0.5-2.
4. liposome as claimed in claim 1 is characterized in that, described liposome composition further comprises natural type or synthesising complex E as antioxidant, and the weight ratio of itself and hydrogenation saturated phospholipid is 0.3-1:100.
5. liposome as claimed in claim 1 is characterized in that, the molar ratio of described paclitaxel and hydrogenation saturated phospholipid is: 2-8:100.
6. liposome as claimed in claim 1 is characterized in that, the mean diameter of liposome is in 100 nanometer to 300 nanometer range.
7. a compositions that comprises the arbitrary described liposome of claim 1 to 6 is characterized in that, said composition further comprises the lyophilizing protective agent, and its dosage form is a lyophilized preparation.
8. liposome composition as claimed in claim 7 is characterized in that, described lyophilizing protective agent is selected from sucrose, lactose, maltose, glucose.
9. liposome composition as claimed in claim 8 is characterized in that, described lyophilizing protective agent is 2-10 a times of hydrogenation saturated phospholipid weight.
10. the arbitrary described liposome composition of claim 7 to 9 is in the purposes of preparation aspect the cancer therapy drug, and wherein the cancer kind is ovarian cancer, metastatic carcinoma of ovary, breast carcinoma, nonsmall-cell lung cancer.
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