CN103040753B - Ginkgolide lipidosome injection - Google Patents
Ginkgolide lipidosome injection Download PDFInfo
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Abstract
The invention discloses ginkgolide lipidosome injection and a preparation method thereof. The lipidosome injection is prepared by ginkgolide, cholesteryl hydrogen succinate, soyabean phosphatidylinositol, DSPE (distearoyl phosphatidyl ethanolamine), trehalose and poloxamer 188. The provided lipidosome injection has small and even particle sizes; ring opening of the ginkgolide of the injection is avoided; the toxic reaction is reduced; the irritation is small; and the efficacy time is prolonged.
Description
Technical field
The present invention relates to a kind of lipidosome injection and method for making thereof, be specifically related to a kind of bilobalide lipidosome injection and method for making thereof, belong to medical technical field.
Background technology
Cardiovascular and cerebrovascular disease is in the world today, to threaten one of disease that the mankind are the most serious, and its M & M has exceeded neoplastic disease and leapt to the first in the world.Along with the quickening of the acceleration of the raising of living standards of the people, aged tendency of population, rhythm of life, dietary habit are to high heat, high esterified development, factors causes the diseases such as coronary heart disease, hypertension, hyperlipidemia to become the most serious disease of threat health, and its M & M also has the trend rising year by year.
Folium Ginkgo extract and preparation thereof have now become the treatment that is widely used in heart and brain association control disease, in American-European medical herbs is sold, seniority among brothers and sisters is the first, its curative effect to cardiovascular and cerebrovascular vessel obtains generally acknowledging of the world, and the annual sales volume of ginkgo agent that only Germany is permitted pharmaceutical factory watt hereby and French Yi Pu crude drug factory just exceedes 1,000,000,000 dollars.Bilobalide is one of main active of Folium Ginkgo.Bilobalide comprises bilobalide (Ginkgolides) A, B, C, M, J etc. and bilobalide (Bilobalid).1932, Ginkgolid was separated first by Furukawa from Semen Ginkgo bitter principle; Nineteen sixty, Fujise successfully isolates several crystallizations; Then 1967, by Marayama etc., by chemical method, derive its structure, and determine that its structure is Ginkgolide A. B. C, M.Press IHB nomenclature, above-mentioned bilobalide is named as respectively BN52020, BN52021, BN52022, BN52023, BN52024.
Bilobalide is platelet activating factor (PAF) receptor antagonist, and cardiovascular and cerebrovascular disease is had to unique pharmacological action, is widely used in treatment and prevention cardiovascular and cerebrovascular disease.Bilobalide belongs to terpene lactone constituents, and polarity is little, and poorly water-soluble is made in traditional injection process and found that this clarity of injection is poor, easy crystallize out after placement a period of time, and stability is difficult for reaching the requirement of injection.The preparation technology of bilobalide injector at present, take Portugal's potassium amine as cosolvent, bilobalide is unstable, and the easy open loop of lactonic ring, causes reduced activity.
The bilobalide dosage form of listing is mainly injection at present, the bilobalide injector that common process is prepared, physics and chemistry stable in properties is poor, long-term storage drug quality can decline but also can generate some impurity, bring toxic and side effects, leave hidden danger to clinical use, because the compatibility research of the reason Chinese medicine injections such as technology of preparing can not show a candle to Western medicine, and easily cause allergy.Bilobalide is salify under alkali condition, can cause part open loop, and after open loop, lactone dissolubility increases, but the version of closed loop is only main activity form, for example, referring to US5798342.The injection of ginkgolide B of France Beaufour-Ipsen company exploitation, code name: BN52021, it is exactly the freeze-dried powder of part open loop form, clinical efficacy is with regard to fall flat [Brochet B, etal:J Neurology, Neurosurgery and Psychiatry.1995,58:360-362].
CN1594319A discloses a kind of bilobalide extraction process and bilobalide injection and preparation method thereof, this injection is the sterile water solution that adopts HP-β-CD increase-volume to make, in medicinal liquid, add again a certain amount of isoosmotic adjusting agent and chelating agen, although the inclusion technique adopting increases dissolving, but because bag mediating recipe amount ratio is less, bag and rate are not high, and stability is good not.CN1813825A and CN1830981A disclose ginkgolide emulsion of a kind of injection and preparation method thereof, it adopts conventional emulsifying agent preparation method to be prepared from, comprise bilobalide, oil for injection, emulsifying agent and isoosmotic adjusting agent composition, but the stability of Emulsion is good not, the consumption of vegetable oil is on the high side, and bioavailability is low.CN1709239A discloses a kind of gingko lactone lyophilized powder for injection and preparation method thereof.This freeze-dried powder is by mannitol disodiumedetate (calcium), sodium metabisulfite, and sodium hydroxide, citric acid preparation, because bilobalide is insoluble, the redissolution of lyophilized injectable powder is good not, and the bioavailability of medicine is not high.CN1686111A discloses a kind of ginkgo lactone injection agent and preparation method thereof, and this injection is also the lyophilized injectable powder by adding certain cosolvent and excipient to prepare, and also there is no well to solve the redissolution problem of medicine, and bioavailability is low.CN101084889A discloses a kind of freeze-dried ginkgolide powder injection and preparation method thereof, and this injectable powder is mainly comprised of bilobalide, metal base or meglumine, polyvidone.Although added alkaline matter to increase dissolubility, but the stability of said preparation is bad, easily produces impurity, bioavailability is not high yet.
Liposome has good targeting and biocompatibility in vivo.B.C.Keller equals [J] .Trends in Food science and Technology, 2001, in 12:25-31., summarize the liposome research overview of natural constituent, thought that liposome can improve the bioavailability of Folium Ginkgo extract, assisted medicine to pass through blood brain barrier.Along with the continuous progress of biotechnology; liposome preparation technology gradual perfection; liposome mechanism of action is further illustrated; in addition liposome is applicable to vivo degradation, avirulence and non-immunogenicity; particularly great number tested data proves that liposome has the protective effect to active pharmaceutical ingredient as pharmaceutical carrier; can improve Drug therapy index, reduce drug toxicity and reduce drug side effect, and reduce the advantages such as drug dose.
There is oral Folium Ginkgo extract liposome listing abroad; for the protection of old people's cardiovascular and cerebrovascular vessel; [the J.Biochemical Engineering Journal such as such as Shinjiro Yamamoto; 2002; 12:125-301] ginkgo leaf component of different solvents and temperature extraction is made to Coliposomes with dimyristoyl phosphatidyl choline and Tween-20 with ultrasonic method parcel; find that it has the activity of stronger melanoma, and the extract anti-tumor activity of parcel is very not low.
Gold leaf, the Study on Preparation of Folium Ginkgo extract lipoid plastid, the master thesis > > of < < Jilin University 2009, get Tween80, Span80, the cholesterol of recipe quantity, add the mixed organic solvents of Er Lv Jia Wan ﹕ methanol (1 ﹕ 3), make to dissolve, 37 ℃ of rotary evaporations form thin film.Dry thin film is dissolved in 60mL ether, add in the solution (using dissolve with ethanol medicine) and PBS solution of 10mL medicine, ultrasonic 10min, will add 20mL PBS solution, hydration 10min again in the solution after ultrasonic, be placed in 30 ℃ of water bath with thermostatic control agitator shaken overnight, make lipoid plastid suspension, wherein surfactant and cholesterol molar ratio are 1:1, and HLB value is 11, GbE and carrier are 15:100, and hydration temperature is 60 ℃.
Liu Ting, the preparation of ginkalide B flexible nano-liposomes and transdermal test in vitro research, < < CHINA JOURNAL OF CHINESE MATERIA MEDICA > > the 34th volume the 17th phase and Liu Ting, the transdermal administration research of ginkalide B flexible lipidosome, < < Northwest University 2010 years of master thesis > >, by film dispersion method, prepare flexible lipidosome, disclose: take 500mg soybean lecithin, 100mg cholesterol, 50mgGB, 20mgVitE, add 100mL dehydrated alcohol to make it to dissolve, be transported in 500mL round-bottomed flask, the round-bottomed flask that above-mentioned solution is housed is connected with rotating pressure-decreasing evaporimeter, on round-bottomed flask, form uniform thin film.Take 100mg sodium cholate simultaneously, add dielectric solvent to make it to dissolve, its solution is dropped to eluting film material in round-bottomed flask, and continue aquation 1h.The liposome turbid liquor obtaining, with the ultrasonic 10min of water-bath type Ultrasound Instrument, then with the filtering with microporous membrane of 0.22 μ m, is obtained to GB flexible nano-liposomes.In preparation process, do not add sodium cholate, obtain common GB nanometer liposome.The GB flexible lipidosome outward appearance of preparation is ball shape, and mean diameter is in 200nm left and right, and particle size distribution is good; Recording average envelop rate is 75.91%.
How to develop a kind of integrity that can keep bilobalide structure, do not use again organic solvent as its solvent, the injection of making is stable again, has become the task of top priority.Due to the deficiency of bilobalide injector, for bilobalide lipidosome injection, there is demand at present.Oral liposome and bilobalide lipidosome injection technical problem to be solved and standard thereof are incomparable, the present invention is intended to overcome bilobalide dissolubility in water of above-mentioned injection little and snperoxiaized problem and the open loop of lactone structure facile hydrolysis and the problem that lost efficacy in vivo, a kind of bilobalide lipidosome injection is provided, the neither open loop of its bilobalide, toxic reaction and zest are little, and effective drug duration is long.
Summary of the invention
The stability of liposome is to limit for a long time the major issue of liposome extensive use, liposome ubiquity is easily assembled, is merged, cause entrapped drug to leak, therefore in industrial preparation, meet the real non-easy thing of medicinal liposome of stability requirement, the technical staff that pharmaceutical field has a universal experience knows clearly and faces various technical difficulties preparing aspect medicinal liposome, all these existing absolutely not theories can be expected solution need to overcome many technical barriers.Therefore the bilobalide liposome that obtains excellent in stability need to be paid creative practice.
Generally speaking, for most liposomal pharmaceutical preparation, the pharmaceutical properties of wrapped medicine is comprised of lipid and concentration is controlled.The inventor is through studying with keen determination unexpected discovery, by selecting bilobalide, cholesterol succinate, soybean phospholipid acyl inositol, DSPE, trehalose and the PLURONICS F87 of specified weight proportioning, can form the bilobalide lipidosome injection of excellent quality, thereby complete the present invention.
The object of this invention is to provide a kind of bilobalide lipidosome injection, it is made by the medicine and the excipient composition that comprise following weight proportion:
Condition is: the weight of the weight of DSPE and soybean phospholipid acyl inositol and cholesterol succinate and between weight ratio be 2:1-6:1, be preferably 4:1.
Preferably, bilobalide lipidosome injection according to the present invention is made by the medicine and the excipient composition that comprise following weight proportion:
Condition is: the weight of the weight of DSPE and soybean phospholipid acyl inositol and cholesterol succinate and between weight ratio be 2:1-6:1, be preferably 4:1.
Further preferably, bilobalide lipidosome injection according to the present invention is made by the medicine and the excipient composition that comprise following weight proportion:
Condition is: the weight of the weight of DSPE and soybean phospholipid acyl inositol and cholesterol succinate and between weight ratio be 4:1.
Further preferably, according to bilobalide lipidosome injection of the present invention, wherein between the weight of DSPE and the weight of soybean phospholipid acyl inositol than being about 10:1.
Another object of the present invention is to provide the preparation method of above-mentioned bilobalide lipidosome injection, and the method comprises the following steps:
(1) under nitrogen protection, cholesterol succinate, DSPE, soybean phospholipid acyl inositol, PLURONICS F87 and bilobalide are dissolved in organic solvent, stir and make its dissolving; Above-mentioned solution is placed in to eggplant-shape bottle, and organic solvent is removed in 45 ℃ of water-bath decompressions, on bottle wall, forms uniformly transparent film;
(2) under nitrogen protection, in bottle, add buffer salt solution, stir, make immobilized artificial membrane eluting abundant swelling hydration, after aquation is complete, at 600bar, to 800bar, do gradient homogenizing 4～6 times preferably 5 times, 0.22 μ m filtering with microporous membrane, makes bilobalide liposome;
(3), under aseptic condition, in the liposome of solution state, under constantly stirring, add trehalose, 400W-600W supersound process twice, each 15～30 minutes, injects water standardize solution, then through 0.22 μ m filtering with microporous membrane, fill, obtains bilobalide lipidosome injection.
Lipidosome injection of the present invention has reduced toxic and side effects, improved formulation products quality, had good preparation stability, in preparation and storage process, liposome can not break because of dehydration, fusion etc., after long term storage, liposome keeps good envelop rate and lower slip equally.
The bilobalide lipidosome injection making by the inventive method, has improved the dissolubility of bilobalide, has improved the quality of formulation products, reduced toxic and side effects, increased the retention time of medicine in body circulation, improved the bioavailability of medicine, curative effect obviously improves; And preparation method is simple, be suitable for industrialized great production.
Accompanying drawing explanation
Fig. 1 is the blood drug level-time graph of bilobalide lipidosome injection, illustrated the injection of preparing in embodiment 1-3 and comparative example 1-3 after administration respectively at the average blood drug level of 0.5h, 1h, 2h, 4h, 6h, 8h, 10h, 12h, 16h and 24h and the relation curve of time.
Wherein:
The specific embodiment
By specific embodiment the present invention, be further described below, the features and advantages of the invention will become more clear along with these explanations.
In order to form colory bilobalide lipidosome injection, thereby importantly find, can good compatible with bilobalide it well be sealed and non-leakage filmogen, and find the excipient composition that can make liposome form stable injectable agent.
To achieve these goals, large quantity research and realization that the inventor carries out, find the bilobalide of specified weight proportioning, cholesterol succinate, soybean phospholipid acyl inositol, DSPE, trehalose and poloxamer can be made bilobalide lipidosome injection, wherein, compared with bilobalide injector of the prior art or its liposome, envelop rate as the bilobalide of active constituents of medicine is high, liposome particle diameter is little and be evenly distributed, the retention time significant prolongation of the active constituents of medicine of preparation of the present invention in body circulation, the biocompatibility of medicine is high, bioavailability obviously improves, curative effect obviously improves.
On the one hand, the invention provides a kind of bilobalide lipidosome injection, it is made by the medicine and the excipient composition that comprise following weight proportion:
Condition is: the weight of the weight of DSPE and soybean phospholipid acyl inositol and cholesterol succinate and between weight ratio be 2:1-6:1, be preferably 4:1.
Preferably, bilobalide lipidosome injection according to the present invention is made by the medicine and the excipient composition that comprise following weight proportion:
Condition is: the weight of the weight of DSPE and soybean phospholipid acyl inositol and cholesterol succinate and between weight ratio be 2:1-6:1, be preferably 4:1.
Further preferably, bilobalide lipidosome injection according to the present invention is made by the medicine and the excipient composition that comprise following weight proportion:
Condition is: the weight of the weight of DSPE and soybean phospholipid acyl inositol and cholesterol succinate and between weight ratio be 4:1.
Preferably, the weight ratio between DSPE and soybean phospholipid acyl inositol is about 10:1.
Generally speaking, as the phospholipid that is used to form liposome, can use natural phospholipid and synthetic phospholipid, comprise PHOSPHATIDYL ETHANOLAMINE, Ovum Gallus domesticus Flavus lecithin, hydrogenation egg yolk lecithin, EPG, egg yolk lecithin acyl serine, soybean phospholipid acyl inositol, soybean lecithin, hydrogenated soya phosphatide, soybean phospholipid acyl glycerol, soy phosphatidylserine, PI, DOPC, DSPC, dipalmitoyl phosphatidyl choline, DMPC, DLPC, DOPG, DSPG, DPPG, GLYCEROL,DIMYRISTOYL PHOSPHATIDYL, PE, PEG-DSPE 2000, two soft ester acyl gallbladder phospholipid-Macrogol 2000s, HSPC-Macrogol 2000, DOPC-Macrogol 2000 etc.Inventor prepares in the test of liposome at the membrane material of the above-mentioned phospholipid of application and phospholipid and cholesterol combination, find that the liposome obtaining is like this under 40 ℃ of high temperature, relative humidity 75% ± 5% accelerated test, the stability of liposome and envelop rate are not good, therefore apply conventional phospholipid and or the liposome material of cholesterol cannot obtain meeting the object of the invention bilobalide liposome.
In the present invention, as the bilobalide of active constituents of medicine, its poorly water-soluble is fat-soluble good.For the feature of bilobalide, the inventor, by research, is surprised to find that a certain proportion of soybean phospholipid acyl inositol, cholesterol succinate and DSPE are particularly suitable for the basic phospholipid filmogen as bilobalide.And while using the combination of non-DSPE, cholesterol succinate and soybean phospholipid acyl inositol, be difficult to form colory liposome, the character such as envelop rate, stability and the percolation ratio of liposome are poor, and the liposome material of therefore applying other conventional phospholipid and/or cholesterol cannot obtain meeting the bilobalide liposome of the object of the invention.
In bilobalide lipidosome injection of the present invention, for the bilobalide of 1 weight portion, the consumption of DSPE is 20-52 weight portion.If the consumption of DSPE, lower than 20 weight portions, cannot form stable liposome; Otherwise if consumption, higher than 52 weight portions, declines as the envelop rate of the bilobalide of active constituents of medicine, the quality of injection and curative effect reduce.
In bilobalide lipidosome injection of the present invention, cholesterol succinate is for regulating the membrane stability of liposome.Cholesterol succinate is for regulating stability and the permeability of phospholipid capsule bubble.Cholesterol succinate (cholesterol hemisuccinate, CHS) is the succinum ester derivant of cholesterol, has another name called Cholesteryl hemisuccinate, and cholesterol hemisuccinate is a kind of crude drug of arteriosclerosis.Cholesterol succinate, except bear electric charge, also has good liposome membrane Stabilization.The succinic acid derivative of this cholesterol, biodegradable in body, there is not security risks, can safety as elecrtonegativity materials'use.
Cholesterol succinate is a kind of amphiphilic, combines with DSPE and soybean phospholipid acyl inositol, and its prevention is condensed into crystal structure.Cholesterol succinate mixes DSPE and soybean phospholipid acyl inositol bilayer, is similar to " buffer agent " and equally plays the effect that regulates membrane structure " mobility ".When lower than phase transition temperature, cholesterol succinate can make film reduce ordered arrangement, increases mobility; When higher than phase transition temperature, cholesterol succinate can increase the ordered arrangement of film, thereby reduces the mobility of film.Cholesterol succinate can make liposome bi-layer membrane solidify, thereby reduces the generation of free radical, reduces oxidation level, and liposome stability is significantly strengthened.
Soybean phospholipid acyl inositol easily obtains, cheap.The phase transition temperature of soybean phospholipid acyl inositol is higher, is easy to form stable liposome membrane.When DSPE and the combination of soybean phospholipid acyl inositol, under certain proportion, can form stable liposome membrane.In order to improve the character of liposome, the purity of soybean phospholipid acyl inositol used and DSPE is more than 90%, preferably more than 95%.
Negative charge phospholipid can be DSPG (DSPG), DPPG (DPPG), DPPA (DPPA), DMPG, dilaurate phosphatidyl glycerol, G 12S3P, two myristic acid phosphatidic acid, dilaurate phosphatidic acid, two palmitic acid phosphatidic acid, two oleic acid Phosphatidylserine, dilinoleic acid phosphatidylinositols, PI and soybean phospholipid acyl inositol etc.In bilobalide lipidosome injection of the present invention, preferably use negative charge soybean phospholipid acyl inositol can effectively stop gathering, further to improve the stability of lipoid microsphere.
The inventor finds through research, when the weight of DSPE and the weight of soybean phospholipid acyl inositol and cholesterol succinate and between weight ratio be 2:1-6:1, while being preferably 4:1, can form stable bilobalide liposome.When weight ratio is too high, membrane stability reduces, and bilobalide is easy to seepage; When weight ratio is too low, the release of bilobalide liposome is inhomogeneous, and restive is suitable release.In addition, research finds, when the weight of DSPE and the weight of soybean phospholipid acyl inositol and cholesterol succinate and between weight ratio be 2:1-6:1, while being preferably 4:1, the liposome toxicity forming is low.
Research shows, the stability of liposome and bioavailability have close corresponding relation.Stability is higher, and bioavailability is higher.Therefore, the stability of bilobalide lipidosome injection of the present invention is high, is to cause one of factor that drug bioavailability is high.
On the other hand, the inventor studies discovery, in bilobalide lipidosome injection of the present invention, for the bilobalide of 1 weight portion, DSPE is 20-52 weight portion, the consumption of soybean phospholipid acyl inositol is 2-5 weight portion, cholesterol succinate is 3-8 weight portion, and the weight of the weight of DSPE and soybean phospholipid acyl inositol and cholesterol succinate and between weight ratio be 2:1-6:1 while being preferably 4:1, the envelop rate of the bilobalide lipidosome injection forming is high.
Research is found, when using the bilobalide, soybean phospholipid acyl inositol, cholesterol succinate of above-mentioned specified quantitative and DSPE, can obtain colory bilobalide liposome, its envelop rate and stability are all very high, toxicity is low, and bioavailability is high.
In bilobalide lipidosome injection of the present invention, use trehalose and poloxamer as excipient, be used to form stable injection.
Trehalose be trehalose be by two glucose molecules with α, α, 1; the nonreducing sugar that 1-glycosidic bond forms; self property is highly stable, and its most obvious character is under anhydrous condition, to have the biomembranous ability of protection, even if make liposome also keep complete form in the situation that of dehydration.In bilobalide lipidosome injection of the present invention, trehalose can effectively be protected form and the stability of liposome particles, further improves the stability of lipidosome injection.
Poloxamer is macromolecular compound, fusing point is low, dissolubility in water and organic solvent is all good, toxicity is low, physiological compatibility is good, has the effect of dispersion medicine and hydrotropy, because its existence can make viscosity, increases, make the medicine of sealing there is higher envelop rate, and can make medicine stable in storage.
Research finds, the size of liposome is affect that liposome distributes in vivo and the principal element of the time of staying, and the particle diameter of liposome is less, and the interior time of staying of body is longer.The bilobalide liposome particles of preparing by the inventive method is little, and particle size distribution is even, and this is one of its factor that metabolic rate is low in vivo, bioavailability is high.
Bilobalide lipidosome injection of the present invention, the specification of its bilobalide can be 10mg.
On the other hand, the present invention also provides a kind of preparation method of bilobalide lipidosome injection, specifically comprises and is prepared as follows step:
(1) under nitrogen protection, cholesterol succinate, DSPE, soybean phospholipid acyl inositol, PLURONICS F87 and bilobalide are dissolved in organic solvent, stir and make its dissolving; Above-mentioned solution is placed in to eggplant-shape bottle, and organic solvent is removed in 45 ℃ of water-bath decompressions, on bottle wall, forms uniformly transparent film;
(2) under nitrogen protection, in bottle, add buffer salt solution, stir, make immobilized artificial membrane eluting abundant swelling hydration, after aquation is complete, at 600bar, to 800bar, do gradient homogenizing 4～6 times preferably 5 times, 0.22 μ m filtering with microporous membrane, makes bilobalide liposome;
(3), under aseptic condition, in the liposome of solution state, under constantly stirring, add trehalose, 400W-600W supersound process twice, each 15～30 minutes, injects water standardize solution, then through 0.22 μ m filtering with microporous membrane, fill, obtains bilobalide lipidosome injection.
Preparation method described above, wherein said buffer salt solution is selected from the one in phosphate buffered solution, citrate buffer solution, carbonate buffer solution, borate buffer solution, is preferably pH and is 6.8 phosphate buffered solution.
Preparation method described above, wherein said organic solvent is selected from one or more in ethanol, methanol, isopropyl alcohol, the tert-butyl alcohol, n-butyl alcohol, benzyl alcohol, dichloromethane, chloroform, chloroform, and being preferably volume ratio is the ethanol of 1:1 and the mixed organic solvents of the tert-butyl alcohol.
Preferably, the present invention also provides a kind of preparation method of bilobalide lipidosome injection, specifically comprises and is prepared as follows step:
(1) under nitrogen protection, it is in the ethanol of 1:1 and the mixed organic solvents of the tert-butyl alcohol that cholesterol succinate, DSPE, soybean phospholipid acyl inositol, PLURONICS F87 and bilobalide are dissolved in to volume ratio, stirs and makes its dissolving; Above-mentioned solution is placed in to eggplant-shape bottle, and organic solvent is removed in 45 ℃ of water-bath decompressions, on bottle wall, forms uniformly transparent film;
(2) under nitrogen protection, to adding pH in bottle, be 6.8 phosphate buffered solution, stir, make immobilized artificial membrane eluting abundant swelling hydration, after aquation is complete, at 600bar, to 800bar, do gradient homogenizing 4～6 times preferably 5 times, 0.22 μ m filtering with microporous membrane, makes bilobalide liposome;
(3), under aseptic condition, in the liposome of solution state, under constantly stirring, add trehalose, 400W-600W supersound process twice, each 15～30 minutes, injects water standardize solution, then through 0.22 μ m filtering with microporous membrane, fill, obtains bilobalide lipidosome injection.
The challenge of preparing liposome is how to make liposome membrane to form the high vesicle of envelop rate of suitable size, appropriate configuration material.And these materials do not spill at formation liposome.The inventor is by selecting suitable material composition, adopting suitable preparation technology, obtained colory bilobalide lipidosome injection, liposome particle diameter is little, particle size distribution is even, envelop rate is high, stability is high, has obtained unexpected technique effect, synergistic result between the each composition of this possibility.
embodiment
By concrete preferred embodiment, the present invention is further described below.These embodiment are only illustrative, and should not be construed as limitation of the present invention.
the preparation of embodiment 1 bilobalide lipidosome injection
Composition used and weight thereof following (1000):
Adopt preparation technology to prepare bilobalide lipidosome injection:
(1) under nitrogen protection, it is in the ethanol of 1:1 and the mixed organic solvents of the tert-butyl alcohol that 30g cholesterol succinate, 200g DSPE, 20g soybean phospholipid acyl inositol, 30g PLURONICS F87 and 10g bilobalide are dissolved in to 1000ml volume ratio, stirs and makes its dissolving; Above-mentioned solution is placed in to eggplant-shape bottle, and organic solvent is removed in 45 ℃ of water-bath decompressions, on bottle wall, forms uniformly transparent film;
(2) under nitrogen protection, to adding 5000ml pH in bottle, be 6.8 phosphate buffered solution, stir, make immobilized artificial membrane eluting abundant swelling hydration, after aquation is complete, at 600bar, to 800bar, do gradient homogenizing 4 times, 0.22 μ m filtering with microporous membrane, makes bilobalide liposome;
(3), under aseptic condition, in the liposome of solution state, under constantly stirring, add 80g trehalose, 400W supersound process twice, each 15 minutes, injects water standardize solution, then through 0.22 μ m filtering with microporous membrane, fill, obtains bilobalide lipidosome injection.
the preparation of embodiment 2 bilobalide lipidosome injections
Composition used and weight thereof following (1000):
Adopt preparation technology to prepare bilobalide lipidosome injection:
(1) under nitrogen protection, it is in the ethanol of 1:1 and the mixed organic solvents of the tert-butyl alcohol that 55g cholesterol succinate, 360g DSPE, 35g soybean phospholipid acyl inositol, 50g PLURONICS F87 and 10g bilobalide are dissolved in to 1500ml volume ratio, stirs and makes its dissolving; Above-mentioned solution is placed in to eggplant-shape bottle, and organic solvent is removed in 45 ℃ of water-bath decompressions, on bottle wall, forms uniformly transparent film;
(2) under nitrogen protection, to adding 8000ml pH in bottle, be 6.8 phosphate buffered solution, stir, make immobilized artificial membrane eluting abundant swelling hydration, after aquation is complete, at 600bar, to 800bar, do gradient homogenizing 5 times, 0.22 μ m filtering with microporous membrane, makes bilobalide liposome;
(3), under aseptic condition, in the liposome of solution state, under constantly stirring, add 115g trehalose, 500W supersound process twice, each 23 minutes, injects water standardize solution, then through 0.22 μ m filtering with microporous membrane, fill, obtains bilobalide lipidosome injection.
the preparation of embodiment 3 bilobalide lipidosome injections
Composition used and weight thereof following (1000):
Adopt preparation technology to prepare bilobalide lipidosome injection:
(1) under nitrogen protection, it is in the ethanol of 1:1 and the mixed organic solvents of the tert-butyl alcohol that 80g cholesterol succinate, 520g DSPE, 50g soybean phospholipid acyl inositol, 60g PLURONICS F87 and 10g bilobalide are dissolved in to 2500ml volume ratio, stirs and makes its dissolving; Above-mentioned solution is placed in to eggplant-shape bottle, and organic solvent is removed in 45 ℃ of water-bath decompressions, on bottle wall, forms uniformly transparent film;
(2) under nitrogen protection, to adding 10000ml pH in bottle, be 6.8 phosphate buffered solution, stir, make immobilized artificial membrane eluting abundant swelling hydration, after aquation is complete, at 600bar, to 800bar, do gradient homogenizing 6 times, 0.22 μ m filtering with microporous membrane, makes bilobalide liposome;
(3), under aseptic condition, in the liposome of solution state, under constantly stirring, add 150g trehalose, 600W supersound process twice, each 30 minutes, injects water standardize solution, then through 0.22 μ m filtering with microporous membrane, fill, obtains bilobalide lipidosome injection.
the preparation of comparative example 1-3 bilobalide lipidosome injection
Inventor once applied following matrix material: dimyristoyl phosphatidyl choline and Tween-20; Soybean lecithin, cholesterol and vitamin E and/or sodium cholate; Hydrogenated soy phosphatidyl choline described in CN101912364A and 2.5: 1 compositionss of Ovum Gallus domesticus Flavus lecithin mole ratio; Patent CN101036642A nanometer liposome material, does not all obtain good bilobalide lipidosome injection.That is to say that prior art does not provide the technology enlightenment that obtains technical solution of the present invention, technique effect of the present invention is also significantly better than prior art.
The existing exemplary part comparative example of listing proved invention combination and have synergistic action effect, but should not be construed as limitation of the present invention, adopt respectively and production technology identical in embodiment 1-3, the composition in comparative example 1-3 as shown in Table 1 below made respectively to bilobalide lipidosome injection:
Composition used in table 1 comparative example 1-3
Wherein, "/" represents not use.
Particularly, comparative example's 1 use DOPE replaces DSPE, replaces cholesterol succinate with cholesterol; Comparative example's 2 use DSPGs replace soybean phospholipid acyl inositols, replace PLURONICS F87 with Tween-20; Comparative example's 3 use dimyristoyl phosphatidyl cholines replace DSPE, with PI replacement soybean phospholipid acyl inositol.
By test example, contrast below, confirmed that combination of the present invention has synergistic effect, the invention provides the bilobalide lipidosome injection of excellent quality.
the mensuration of test example 1 liposome particle diameter
Under room temperature condition, get the bilobalide lipidosome injection in embodiment 1-3 and comparative example 1-3, be placed in the sample cell of Submicron Particle Sizer Model370 particle diameter detector, measure particle size distribution and mean diameter; Observe particle shape with projection electron microscope.The results are shown in following table 2.
Table 2 liposome particle diameter testing result
As known from Table 2, the liposome particle diameter that embodiment 1-3 makes is even, aobvious spherical, big or small homogeneous; The liposome particle diameter that comparative example 1-3 makes is inhomogeneous, and shape is indefinite, not of uniform size.
Particularly, even when adopting same production technology, in embodiment 1-3, the particle appearance of gained bilobalide liposome and size thereof and distribution are obviously better than the bilobalide liposome of gained in comparative example 1-3.When the composition beyond using the present invention's composition used is described, or when composition consumption is outside the composition amount ranges of the present invention's restriction, the outward appearance of gained bilobalide liposome is inferior to the present invention, and mean diameter obviously goes out greatly a lot.
With regard to particle diameter and outward appearance aspect, in comparative example 1-3, comparative example 1 is spherical substantially but distribution is inhomogeneous, less with respect to comparative example 2-3 distribution, illustrate that soybean phospholipid acyl inositol is better than PI, PLURONICS F87 is better than Tween-20.Comparative example illustrates that DSPE is the most remarkable to particle diameter and appearance effects; The poorest material optimum that has also illustrated that the present invention limits of comparative example 3.
the mensuration of test example 2 envelop rates
Rotating speed high speed centrifugation by the bilobalide lipidosome injection of preparing in embodiment 1-3 and comparative example 1-3 with 5000r/min, centrifugal 20 minutes, get supernatant, with dissolve with methanol, HPLC method is surveyed bilobalide content, and computational envelope rate, the results are shown in following table 3.
Table 3 entrapment efficiency determination result
The envelop rate of the Liposomal formulation that as shown in Table 3, prepared by embodiment 1-3 is significantly higher than the envelop rate of the Liposomal formulation of comparative example 1-3.When the composition beyond using the present invention's composition used is described, or when composition consumption is outside the composition amount ranges of the present invention's restriction, the liposome encapsulation of gained liposome is lower than the present invention.
With regard to envelop rate, in comparative example 1-3, comparative example 2 is the poorest, has illustrated that PLURONICS F87 is the most remarkable on improving the impact of envelop rate; Comparative example 1 is best, has illustrated that cholesterol succinate and DOPE are little on its impact.
test example 3 study on the stability
Sample prepared by embodiment of the present invention 1-3 and listing bilobalide injector (lot number: 20111204, the abundant scientific and technological pharmaceutical Co. Ltd in Chengdu hundred) be placed in respectively lower 6 months of the condition of 40 ℃ of high temperature, relative humidity 75%, carry out accelerated test investigation, experimental result is shown in following table 4.
Table 4 accelerated test result
As shown in Table 4, while accelerating June, listing formulation content reduces, and related substance raises; And sample property of the present invention, content and related substance variation are all not obvious, illustrate that product stability of the present invention is good.With regard to stability, in comparative example 1-3, comparative example 1 is the poorest, and the material optimum that the present invention limits has been described, the bilobalide that lipidosome injection of the present invention is sealed can open loop not produce related substance.
test example 4 percolation ratio tests
Get sample prepared by test example 1-3 and comparative example 1-3, at ambient temperature, respectively at 0 day, 30 days, 60 days, 90 days and 180 days, make regular check on, measure envelop rate, with the dose comparison of sealing for 0 day, calculate percolation ratio, the results are shown in following table 5.
Table 5 percolation ratio result of the test
As shown in Table 5, during long term storage, the bilobalide lipidosome injection percolation ratio of preparing in embodiment of the present invention 1-3 changes little, and the injection percolation ratio of preparing in comparative example 1-3 increases gradually, liposome seepage is serious, and bilobalide lipidosome injection prepared by this explanation the present invention has higher stability.With regard to percolation ratio, in comparative example 1-3, comparative example 1 is the poorest, and the material optimum that the present invention limits is described.
the mensuration of test example 5 blood drug level
63 rats are divided into 7 groups at random, every group of injection of preparing in drug administration by injection embodiment 1-3 and comparative example 1-3 respectively, and commercially available bilobalide injector (lot number: 20111204, the abundant scientific and technological pharmaceutical Co. Ltd in Chengdu hundred), injection volume is 10mg bilobalide.After administration, respectively at 0.5h, 1h, 2h, 4h, 6h, 8h, 10h, 12h, 16h and 24h, take a blood sample, blood sample after treatment, is measured blood drug level with HPLC-MS method.Draw the blood drug level of bilobalide lipidosome injection and the relation curve of time in the bilobalide lipidosome injection prepared in embodiment 1-3, comparative example 1-3, prepared, be shown in accompanying drawing 1, due to the rapid metabolism of commercially available bilobalide injector, blood drug level effective time is short, there is no slow releasing function, therefore do not provide its conventional blood distiller’s yeast line in Fig. 1.
As shown in Figure 1, the present invention has slow release effect and bioavailability is high, compared with the bilobalide lipidosome injection of preparing in comparative example 1-3, the bilobalide lipidosome injection of preparing in embodiment of the present invention 1-3 has the following advantages: release rate in vivo slows down, in body circulation, distribution time extends, reached improved slow release effect, bioavailability increases.
Result in comparative example 1-3 and experimental example 1-3 has confirmed that from different aspect the lipidosome injection of preparing embodiment of the present invention 1-3 has excellent galenic pharmacy feature, obtained collaborative windfall effect, solved galenic pharmacy technical problem, confirmed that component of the present invention has produced synergism each other.
industrial applicibility
From the result of above-described embodiment and experimental example, bilobalide liposome of the present invention has good outward appearance, and granule is little, and particle diameter is even, envelop rate is high, and stability is high, and percolation ratio is low, the time of staying is in vivo long, and bioavailability is high, has good industrial application value.Below through the specific embodiment and the embodiment the present invention is had been described in detail; but should understand; these explanations do not form any restriction to scope of the present invention; in the case of without departing from the spirit and scope of protection of the present invention; can carry out multiple modification, improvement and replacement to technical solutions and their implementation methods of the present invention, these are all because falling within the scope of protection of the present invention.
Each list of references of mentioning in the application or quoting, which is hereby incorporated by reference.
Claims (6)
1. a bilobalide lipidosome injection, it is made by the medicine and the excipient composition that comprise following weight proportion:
The weight of the weight of described DSPE and soybean phospholipid acyl inositol and cholesterol succinate and between ratio be 2:1-6:1.
2. bilobalide lipidosome injection according to claim 1, it is characterized in that the weight of described DSPE and the weight of soybean phospholipid acyl inositol and cholesterol succinate and between ratio be 4:1.
3. according to the bilobalide lipidosome injection described in any one in claim 1-2, wherein the specification of bilobalide is 10mg.
4. a method of preparing the bilobalide lipidosome injection described in any one in claim 1-3, is characterized in that comprising the steps:
(1) under nitrogen protection, cholesterol succinate, DSPE, soybean phospholipid acyl inositol, PLURONICS F87 and bilobalide are dissolved in organic solvent, stir and make its dissolving; Above-mentioned solution is placed in to eggplant-shape bottle, and organic solvent is removed in 45 ℃ of water-bath decompressions, on bottle wall, forms uniformly transparent film;
(2) under nitrogen protection, in bottle, add buffer salt solution, stir, make above-mentioned thin film eluting abundant swelling hydration, after aquation is complete, at 600bar, to 800bar, do gradient homogenizing 4～6 times, 0.22 μ m filtering with microporous membrane, makes bilobalide liposome;
(3), under aseptic condition, in the liposome of solution state, under constantly stirring, add trehalose, 400W-600W supersound process twice, each 15～30 minutes, injects water standardize solution, then through 0.22 μ m filtering with microporous membrane, fill, obtains bilobalide lipidosome injection.
5. method according to claim 4, is characterized in that described buffer salt solution is that pH is 6.8 phosphate buffered solution.
6. method according to claim 4, is characterized in that the solvent described in step (1) is that volume ratio is the ethanol of 1:1 and the mixed organic solvents of the tert-butyl alcohol.
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