CN101190188A

CN101190188A - Anthracene nucleus medicinal liposome injection and preparation method

Info

Publication number: CN101190188A
Application number: CNA2007100010588A
Authority: CN
Inventors: 刘全志; 杨文斌
Original assignee: BEIJING TIANHENG MEDICINE INST
Current assignee: BEIJING TIANHENG MEDICINE INST
Priority date: 2006-11-30
Filing date: 2007-01-23
Publication date: 2008-06-04

Abstract

The invention discloses an anthracycline liposome injection, including the injection and a frozen powder injection and a preparation process of the injection. The frozen liposome consists of the anthracycline or an anthracycline hydrochloride, compound neutral phospholipids, surfactant, negative-charge phospholipids, buffers, PH regulators and freezing protection agent. The preparation process includes the following steps: preparation of a hollow liposome, a homogenized liposome, an anthracycline lopsome and an anthracycline lopsome suspension; adding the freezing protection agent; constant volume; sterilization; sub-package; freezing and drying; storage, etc. The liposome injection can be preserved for 12 months under the room temperature with better stability. And the encapsulation rate can be above 95 percent and a granule diameter is between 30 and 300mm. The side effect is low and the technique is simple, thus being convenient for industrial production.

Description

Anthracene nucleus medicinal liposome injection and preparation method

Technical field

The present invention relates to the preparation of anthracene nucleus medicament and hydrochlorate thereof, relate to a kind of injectable anthracene nucleus medicament thermal sensitive liposome specifically, the invention still further relates to the preparation technology of this pharmaceutical preparation.

The present invention adopts long circulating liposomes and thermal sensitive liposome technology, with special surfactant (hydrolytic phosphatide, high molecular polymer etc.) in the phospholipid bilayer structure of the liposome of Jia Ruing, liposome is enlarged markedly, owing to add the storage-stable that negative charge phospholipid has enlarged markedly liposome in the immobilized artificial membrane in its phase transition temperature and the release medicine speed more than the phase transition temperature.

Background technology

The mechanism of action of anthracene nucleus class broad-spectrum anti-cancer drug and hydrochlorate thereof mainly is: 1. by intercalation of DNA two strands and DNA high-affinity is arranged, blocking dna and RNA's is synthetic, makes the DNA chain interruption by influencing topoisomerase II; 2. be attached to and change membrane fluidity and ion transport on the film; 3. the reduction process by the enzyme mediation produces semiquinone free radical and oxygen-derived free radicals.Cardiac toxicity is relevant with the membrane lipid peroxidating of Mediated by Free Radicals.For example epirubicin and hydrochlorate thereof are applicable to breast carcinoma, malignant lymphoma, ovarian cancer, digestive tract cancer, pulmonary carcinoma, leukemia, incidence cancer, soft tissue sarcoma, bladder cancer, renal carcinoma, malignant melanoma etc., it is wide to have the antitumor spectrum, good effect, and cardiac toxicity is little than amycin, but bigger by toxic and side effects after the intravenous injection, comprises nauseating, vomiting, bone marrow depression, serious alopecia, and anorexia, gastritis even ulcer, oral mucosa inflammation etc.In addition, long-term clinical application anthracene nucleus class broad-spectrum anti-cancer drug, owing to destroying cardiac muscle fiber, it makes the damaged accumulation cardiac toxicity that causes of cardiac muscle fiber, show as tachycardia, arrhythmia and congestive heart failure etc., and current research finds that the cardiac toxicity of anthracene nucleus medicament can't disappear because of drug withdrawal.The normal injection agent of anthracene nucleus medicament is because short (T of interior half-life of body _1/2＜10h), tissue distribution is wide, general toxicity is bigger, especially the patient usually can because of wicked etc. seriously toxic reaction discontinue medication, antitumous effect reduces greatly, has seriously limited the clinical practice of common anthracene nucleus medicament preparation.

Liposome (Liposome) is dispersed in phospholipid by Britain scholar Bangham in nineteen sixty-five at first and finds when carrying out electron microscopic observation in the water.Phospholipid is dispersed in and forms multilamellar vesicle, every layer of bilayer structure that is lipid in the water naturally; Separated by water between vesicle central authorities and each layer, bilayer thickness is about 4nm, and this bilayer folliculus with similar biofilm structure is called liposome.Liposome is a kind of novel targeting microgranule drug administration carrier, it comprises at least one vesicle that comprises interior water that is made of the phospholipid bilayer film, liposome generally is divided into according to the type and the size of its film: its particle diameter of small unilamellar vesicle (SUV) is at 20nm～100nm, big unilamellar liposome (LUV), its particle diameter is greater than 100nm, multivesicular liposome and multilamellar liposome (MLV) particle diameter is all bigger, generally greater than 1 μ m.

The Lay of Britain in 1971 is graceful to wait the people to begin liposome is used for pharmaceutical carrier; the main mechanism of action is drug microparticles or solution are wrapped in interior aqueous phase that the liposome bilayer lipid membrane sealed or embed in the liposome phospholipid bilayer film with certain proportion, and this microgranule has the class cellularity.Conventional liposome enters mainly to be engulfed by reticuloendothelial system (RES) in the human body and activates the autoimmune function of body, and the interior distribution of the body that changes encapsulated medicine, drug main will be put aside in histoorgans such as liver, spleen, lung and bone marrow, thereby improve the therapeutic index of medicine, reduce the toxicity of the therapeutic dose and the reduction medicine of medicine.Liposome as pharmaceutical carrier mostly is large unilamellar vesicle at present, and particle diameter is generally between 100-1000nm.The seventies middle and late stage, people begin one's study liposome with being the effective carrier of making anthracycline anticancer drug (for example doxorubicin and hydrochlorate thereof), the end of the eighties, existing clinical experiment began to carry out, to American-European existing liposome medicament listing of the mid-90.For example, after doxorubicin hydrochloride was wrapped in and enters human body by intravenous injection again in the specific liposome, the clinical experiment result showed that its toxic and side effects obviously reduces, the drug half-life significant prolongation, but its drug effect does not weaken on the contrary and is strengthened.The doxorubicin product that has gone on the market at present has three kinds, is respectively the Doxil of U.S. Alza company development

And Caelyx

, the MyocetTM of U.S. Elan company.Doxil wherein

And Caelyx

Be that " stealthy (stealth) technology is wrapped in epirubicin hydrochloride in the liposome microgranule; mix the phospholipid derivative that PEG modifies in the phospholipid bilayer structure; it sterically hinderedly can effectively be avoided the identification of RES system and engulf what surface of liposome formed; prolong its circulating half-life in vivo significantly, their half-life can reach 66 hours in utilization.Clinical test results shows, Doxil

Can obviously reduce the wicked and the bone marrow depression effect of doxorubicin hydrochloride, significantly be increased in accumulating of specific paries such as solid tumor.

Bibliographical information and experimentation show, no matter be conventional liposome or long circulating liposomes, all lack effectively initiatively release mechanism in vivo, though medicine half-life significant prolongation in vivo, valid density at specific part significantly increases, but its release amount of medicine is but very little or it is slow to discharge, circulation time is long in vivo to cause liposome, the terminal deposition of blood capillary is discharged medicine after the biodegradation, cause the distinctive side effect of liposome, as brothers' effect, oral mucositis has brought great misery to the patient, greatly reduces the compliance of patient's medication.According to pharmacodynamic study, wrapping kmedicine by liposome can not make medicine reach synergic purpose for a long time, and its meaning is main to be aspect attenuation.

For attenuation and potentiation, the tumor heat therapy has obtained using widely.Clinical studies show, the vascular permeability of heating region increases greatly, velocity of blood flow obviously increases, effectively improve the infiltration of liposome at heating region, character with responsive to temperature liposome makes its its double-decker when being higher than normal body temperature (39～42 ℃) defective occur, and quick and a large amount of oozes out from liposome, effectively the drug level in the focus zone of Ti Gaoing, and other position burst sizes of whole body seldom, reduced general toxicity.The problem of this technology existence is main phospholipid composition such as the DPPC that adopts in the present thermal sensitive liposome prescription at present, its phase transition temperature is 42 ℃, in 39～42 ℃ phase transition temperature scope, the liposome double-decker has the glue crystalline state to change liquid crystal state into, and the integrity of liposome does not destroy, though the permeability of phospholipid bilayer structure increases, the actual free drug concentration in the focus zone is not high, initiatively the DeGrain of release.

The stability of liposome is to limit the major issue of liposome extensive use for a long time, comprises chemical stability, physical stability and plasma stability.Because prior art is guaranteeing to exist aspect anthracene nucleus medicinal liposome stable not enough, often wants the ultralow temperature storage, and the shelf-life weak point.

Summary of the invention:

The purpose of this invention is to provide a kind of good stability, envelop rate height, anthracene nucleus medicament thermal sensitive liposome injection that toxic and side effects is few, comprise injection and lyophilized injectable powder.Another object of the present invention provides the preparation technology of this thermal sensitive liposome injection.

The new prescription of anthracene nucleus medicinal liposome injection provided by the invention (comprising injection and lyophilized injectable powder), adopted phospholipid with specific phase transition temperature, character with responsive to temperature liposome makes its its double-decker when being higher than normal body temperature (39～42 ℃) defective occur, medicine oozes out from liposome, the drug level in the focus zone of Ti Gaoing effectively, solved anthracene nucleus lipoid plastid release mechanism problem in vivo well, the local heat instrument that is used clinically can more effectively be sent to target position with medicine, and discharge, the targeting and the local drug concentration of liposome have been increased greatly, and circulation time is long in vivo can to avoid drug-loaded liposome effectively, effectively avoids brothers' effect.Simultaneously because after adding surfactant-based (as hydrolytic phosphatide), in the phase transition temperature zone, outer phospholipid undergoes phase transition fusion and becomes liquid crystal state, have the hydrolytic phosphatide of certain solubility from the phospholipid bilayer desorption this moment in water, thixotropy and permeability increase, destroyed the arrangement architecture of double-layer of lipoid, the integrity of liposome is damaged, the medicine that inner bag carries discharges rapidly, the concentration of free drug enlarges markedly in the heating focal zone, reach the treatment concentration of medicine rapidly in the focus zone, and other position burst sizes of whole body seldom, reduced general toxicity.Shown in accompanying drawing 11～18, not moisture prescription burst size under 41 ℃ of conditions of separating phospholipid is starkly lower than and contains hydrolytic phosphatide and the identical prescription of other component.

The stability of liposome is to limit the major issue of liposome extensive use for a long time, at present, the anthracene nucleus medicinal liposome of document and patent report mostly is injection, because the hydrolysis of phospholipid and the gathering of liposome can have a strong impact on the stability of liposome, liposome is increased in preparation and storage Chinese medicine slip, and particle diameter increases.Liposome of the present invention comprises negative charge phospholipid, has increased the absolute value of the Zeta potential of surface of liposome effectively, and the electrostatic repulsion of surface of liposome is increased, and suppresses the gathering of liposome effectively.In the freeze-dried powder preparation process, the pre-freeze stage, liposome constantly was concentrated along with the outer water ice of liposome is separated out, and accumulative trend increases, and adding the gathering that negative charge phospholipid can stop liposome this moment effectively, the liposome particle diameter is stable within the specific limits after the assurance lyophilizing rehydration.What is particularly worth mentioning is that, the adding of the hydrolytic phosphatide of negative charge such as MPPG and MPPA, not only make liposome have the surface and be electronegativity, simultaneously also make liposome have the characteristic of temperature-sensitive release, and the adding of MSPE-PEG can make liposome have temperature-sensitive drug release feature and macrocyclic double grading.In addition; also added hydrophilic high molecular polymer (as the PVP class) in the prescription, with disaccharide compound together as freeze drying protectant, can be in the pre-freeze process; effectively bag is stated from surface of liposome, prevent because of the liposome aqueous crystallization to the phospholipid bilayer structural damage.

Existing result of study shows, the phospholipid hydrolysis is to cause the unsettled main cause of liposome always, the hydrolysis of lecithin, saturated soybean phospholipid and phosphatidyl glycerol etc. all is subjected to the influence (equal facile hydrolysis under acidity and the alkali condition) of pH value, and hydrolyzate can make the pH value of liposome turbid liquor descend, quickening the further hydrolysis of liposome. these phospholipid compositions are all the most stable when pH6.5, hydrolysis rate constant minimum.Therefore among the present invention, make the lipidosome injection pH6-7 that makes, guarantee preparation stabilization by the pH regulator agent.In addition, the present invention carries out lyophilization to anthracene nucleus medicament after being wrapped in the blank liposome effectively, make freeze-dried powder, and the water in the weeding of grease plastid has further strengthened the stability of anthracene nucleus medicament hydrochlorate liposome.

The invention provides the preparation technology of above-mentioned anthracene nucleus medicinal liposome injection, it is characteristic according to liposome, select for use the mixture of phospholipids of specific proportions to prepare the blank liposome that can wrap up anthracene nucleus medicament and hydrochlorate thereof, adopt pH gradient method medicine carrying method, entrapment efficiency is up to more than 95%, and be prepared into preparation by special processing technique, can after being injected into human body, use said preparation auxiliary heating instrument to control the speed that anthracene nucleus medicament discharges in human body, not only reduced the toxic and side effects of anthracene nucleus medicament, and control medicine distribution in vivo effectively, its drug effect is increased.

In the prior art, the anthracene nucleus medicament hydrochlorate can adopt " ion gradient method " to be wrapping in the blank liposome, the foreign minister produced the pH gradient in it was induced by the difference of ion concentration inside and outside the liposome phospholipid bilayer structure, made that medicine is easier to be wrapping in the liposome, and these ions comprise NH ₄ ⁺, H ⁺Deng, use H ⁺The time method be known as " pH gradient method ". anthracene nucleus medicinal liposome injection of the present invention, can be according to prior art, adopt in " ion gradient method " effect preferably " pH gradient method " wrap up anthracene nucleus medicament and hydrochlorate thereof.This method makes the outer anthracene nucleus medicament of liposome or its hydrochlorate more easily enter in the liposome by the pH difference of regulating the liposome internal and external environment, and intravital anthracene nucleus medicament of lipid or hydrochlorate exist with ionic forms, be difficult to penetrate into the liposome phospholipid bilayer, thereby make entrapment efficiency very high, generally can reach more than 95%.Concrete measure is by regulating blank liposome foreign minister's pH value, the foreign minister produces the pH gradient in making, add anthracene nucleus medicament or its hydrochlorate solution in the foreign minister, exist with molecular forms, can pass phospholipid bilayer enters in the inner phase, enter the anthracene nucleus medicament of inner phase or its hydrochlorate and exist, be difficult to pass phospholipid bilayer, therefore effectively raise the encapsulation efficiency of liposome with the form of ion or complex.Liposome can prepare by high pressure homogenizer, also can come the controlling particle size scope to the blank liposome suspension pushes through respective aperture under pressure microporous membrane by extrusion equipment.Behind packaging medicine, the free anthracene nucleus medicament of foreign minister is removed by diafiltration.Then anthracene nucleus medicament and hydrochlorate liposome thereof are placed and Liposomal dispersion like the Human Physiology environmental classes.The preparation of blank liposome can adopt low temperature spray drying-aquation method, film dispersion method and solvent dilution method to prepare.In the preparation process exact temperature with the difference of liposome component difference.

Purpose of the present invention can realize by following measure:

Prescription (pressing the 50ml volumeter)

Anthracene nucleus medicament 50mg～600mg

Synthetic neutral phospholipid 25mg～10000mg

Negative charge phosphatidase 12 5mg～900mg

Surfactant 25mg～900mg

PEGization phospholipid derivative 0～900mg

Buffer agent 300mg～3000mg

PH regulator agent 150mg～1500mg

Cryoprotective agent (saccharide) 0.5～10g

Hydrophilic polymer 0～10g

Water for injection is settled to 50ml

Be preferably:

Anthracene nucleus medicament 50mg～300mg

Synthetic neutral phospholipid 500mg～4000mg

Negative charge phosphatidase 15 0mg～500mg

Surfactant 50mg～500mg

PEGization phospholipid derivative 0～500mg

Buffer agent 300mg～3000mg

PH regulator agent 150mg～1500mg

Cryoprotective agent (saccharide) 0.5～10g

Hydrophilic polymer 0～5g

Water for injection is settled to 50ml

Satisfy simultaneously: the part by weight of anthracene nucleus medicament and phospholipid is 1: 99～50: 50, its phospholipid bilayer structure is made up of synthetic neutral phospholipid, negative charge phospholipid, surfactant and/or PEGization phospholipid derivative, its part by weight is 98: 1: 1: 0～40: 20: 20: 20, further preferred range was 98: 1: 1: 0～70: 10: 10: 10.Described anthracene nucleus medicament comprises various than star class medicine, especially epirubicin, daunorubicin, and doxorubicin, pirarubicin, darubicin and they are the salt of organic acid or mineral acid separately, the preferred salt hydrochlorate; Described synthetic neutral phospholipid can be dipalmitoyl phosphatidyl choline (DPPC), distearoyl phosphatidylcholine (DSPC), two Laurel phosphatidyl cholines (DLPC) or two nutmeg phosphatidyl cholines (DMPC), preferred dipalmitoyl phosphatidyl choline (DPPC), distearoyl phosphatidylcholine (DSPC) or their mixture; Wherein negative charge phospholipid can be distearyl phosphatidyl glycerol (DSPG), two palmityl phosphatidyl glycerols (DPPG), two palmityl phosphatidic acid (DPPA); Surfactant wherein, comprise single lauroyl phosphatidylcholine (MLPC), single tridecanoyl phosphatidylcholine (MTPC), single myristoyl phosphatidylcholine (MMPC), single pentadecanoyl phosphatidylcholine (MPePC), single palmitoylphosphatidyl choline (MPPC), single heptadecanoyl phosphatidylcholine (MHPC), single stearoyl phosphatidylcholine (MSPC), single oleoyl phosphatidylcholine (MOPC), Dan Shijiu phosphatidyl choline (MNPC), Dan Ershi phosphatidyl choline (MAPC), single myristoyl phosphatidic acid (MMPA), single palmityl phosphatidic acid (MPPA), single stearoyl phosphatidic acid (MSPA), single oleoyl phosphatidic acid (MOPA), single myristoyl PHOSPHATIDYL ETHANOLAMINE (MMPE), single palmityl phospholipid PHOSPHATIDYL ETHANOLAMINE (MPPE), single stearoyl PHOSPHATIDYL ETHANOLAMINE (MSPE), single oleoyl PHOSPHATIDYL ETHANOLAMINE (MOPE), single myristoyl phosphatidyl glycerol (MMPG), single palmityl phosphatidyl glycerol (MPPG), single stearoyl phosphatidyl glycerol (MSPG), single oleoyl phosphatidyl glycerol (MOPG), single oleoyl Phosphatidylserine (MOPS) or their mixture, preferred single palmitoylphosphatidyl choline (MPPC), single palmityl phosphatidyl glycerol (MPPG), single palmityl phospholipid PHOSPHATIDYL ETHANOLAMINE (MPPE), single oleoyl Phosphatidylserine (MOPS) and single myristoyl phosphatidic acid (MMPA); Wherein synthetic neutral phospholipid is the main component of phospholipid bilayer structure, adds negative charge phospholipid and can make bilayer more stable, behind the adding surfactant, has significantly increased the release of anthracene nucleus medicament when phase transition temperature; Wherein buffer agent can be citric acid-citrate, phosphate, tartrate, acetate, oxalates, lactate, the buffer pH value scope of preparing during the preparation liposome is 3.0～5.0, be preferably 3.5～4.5, further preferred pH=4, molar concentration 50～500mM.Described pH regulator agent can be sodium carbonate, sodium bicarbonate, sodium hydrogen phosphate or sodium hydroxide etc., preferred sodium carbonate.Described cryoprotective agent comprises saccharide, hydrophilic polymer or their mixture, and wherein saccharide comprises trehalose, lactose, sucrose, mannitol, glucose, maltose, glycerol, and hydrophilic polymer comprises polyvinylpyrrolidone (PVP).Be preferably lactose, sucrose and trehalose add the PVP better effects if, and the concentration of above-mentioned cryoprotective agent (W/V) is 1%～20%.Described PEGization phospholipid derivative, can be two polyethyleneglycol modified palmityl PHOSPHATIDYL ETHANOLAMINE (PEG-DSPE), preferred two palmityl PHOSPHATIDYL ETHANOLAMINE-Macrogol 2000s (DSPE-PEG2000), consumption are thermal sensitive liposome when being zero, are long circulation thermal sensitive liposome when non-vanishing.

Described anthracene nucleus medicament thermal sensitive liposome, its dosage form are injection and lyophilized injectable powder.

The lipidosome injection of anthracene nucleus medicament of the present invention and lyophilized injectable powder can be by prior art for preparing, preferably according to following method preparation.

The preparation method of the lipidosome injection of described anthracene nucleus medicament comprises the following step: 1) preparation blank liposome: select phospholipid for use, be dissolved in mix homogeneously in chloroform or the chloroform-methanol mixed solvent according to prescription; With Rotary Evaporators solvent decompression in the solution is removed, form maybe will the write out a prescription phospholipid of composition of lipid membrane and be dissolved in the organic solvent, low temperature spray drying obtains the mixture of phospholipid; Buffer agent is mixed with the buffer (pH=4) of 50～500mM, comes aquation lipid membrane or mixture of phospholipids with buffer, hydration temperature generally between 40 ℃～70 ℃, blank liposome suspension; 2) liposome that homogenizes: aquation back fully prepares liposome to the required particle diameter and the uniformity with high pressure homogenizer, also can reach the blank liposome suspension pushes through respective aperture under pressure microporous membrane by extrusion equipment, the particle diameter of liposome is controlled at 10nm～1000nm, be preferably 30～300nm, 50～250nm more preferably, can effectively avoid liposome to be caught by reticuloendothelial system (RES), help to improve the stability of liposome in blood plasma, prolong circulation time in vivo, liposome particle diameter and particle size distribution can detect with nanometer laser particle size analyzer or transmission electron microscope; 3) liposome of preparation anthracycline-containing drug salts hydrochlorate: it is 1%～20% sugar aqueous solution that the anthracene nucleus medicament hydrochlorate is dissolved in water for injection or concentration, is heated to 30 ℃～40 ℃; The aqueous solution of the pH regulator agent of preparation 10mM～1000mM adds in the blank liposome suspension with certain proportion, regulates pH value to 7～9; With anthracene nucleus medicament hydrochlorate solution and blank liposome suspension mix homogeneously and at 30 ℃～45 ℃ insulation 10～100min down, once by a certain percentage every the 5min jolting; 4) preparation anthracene nucleus medicament hydrochlorate liposome turbid liquor, be added to the weight percent proportion by subtraction outside and be 3%～20% sugar or sugar aqueous solution, mix homogeneously, make the concentration of foreign minister's sugar in the liposome equal, get Liposomal dispersion, through the microporous filter membrane filtration of 0.22 μ m near obtaining, fill, 5) gland, sealing is preserved, and gets anthracene nucleus medicament thermal sensitive liposome injection.Finished product can be saved to use under-20 ℃.

The liposome of described injection anthracene nucleus medicament (being freeze-dried powder) preparation method comprises the following step: 1) preparation blank liposome: select phospholipid for use, be dissolved in mix homogeneously in chloroform or the chloroform-methanol mixed solvent according to prescription; With Rotary Evaporators solvent decompression in the solution is removed, form maybe will the write out a prescription phospholipid of composition of lipid membrane and be dissolved in the organic solvent, low temperature spray drying obtains the mixture of phospholipid; Buffer agent is mixed with the buffer (pH=4) of 50～500mM, comes aquation lipid membrane or mixture of phospholipids with buffer, hydration temperature generally between 40 ℃～70 ℃, blank liposome suspension; 2) liposome that homogenizes: aquation back fully prepares liposome to the required particle diameter and the uniformity with high pressure homogenizer, also can reach the blank liposome suspension pushes through respective aperture under pressure microporous membrane by extrusion equipment, the particle diameter of liposome is controlled at 10nm～1000nm, be preferably 30～300nm, 50～250nm more preferably, can effectively avoid liposome to be caught by reticuloendothelial system (RES), help to improve the stability of liposome in blood plasma, prolong circulation time in vivo, liposome particle diameter and particle size distribution can detect with nanometer laser particle size analyzer or transmission electron microscope; 3) liposome of preparation anthracycline-containing drug salts hydrochlorate: it is 1%～20% sugar aqueous solution that the anthracene nucleus medicament hydrochlorate is dissolved in water for injection or concentration, is heated to 30 ℃～40 ℃; The aqueous solution of the pH regulator agent of preparation 10mM～1000mM adds in the blank liposome suspension with certain proportion, regulates pH value to 7～9; With anthracene nucleus medicament hydrochlorate solution and blank liposome suspension mix homogeneously and at 30 ℃～45 ℃ insulation 10～100min down, once by a certain percentage every the 5min jolting; 4) preparation anthracene nucleus medicament hydrochlorate liposome turbid liquor, be added to the weight percent proportion by subtraction outside and be 3%～20% the sugar or the PVP of sugar aqueous solution and 1～10%, mix homogeneously, make the concentration of foreign minister's sugar in the liposome equal near obtaining, get Liposomal dispersion, microporous filter membrane through 0.22 μ m filters fill, lyophilizing, the pre-freeze temperature should preserved 2 hours below-45 ℃, first section dry employing :-50 ℃ (1～5h) ,-35 ℃ (8～20h) ,-25 ℃ (5～10h),-10 ℃ (2～6h), 0 ℃ (20 ℃ of 1～3h), second section dryings (5～15h), obtain injection anthracene nucleus medicament hydrochlorate liposome; 5) gland, sealing is preserved, and gets finished product.Finished product can be saved to use under 2 ℃～25 ℃.

Advantage of the present invention: the particle diameter of lipidosome injection of the present invention and Injectable liposomal further can be controlled in 50～250nm between 30nm～300nm, entrapment efficiency can reach more than 95%.Simultaneously, owing to adopt Freeze Drying Technique that the stability of liposome is improved greatly, preservation condition is also more wide in range.In addition, basic identical before the various physicochemical properties of liposome and the lyophilizing after the rehydration, because crucial homogenizing step can be finished by high pressure homogenizer efficiently in the production process of anthracene nucleus medicament hydrochlorate liposome, and said preparation also can be made freeze-dried formulation by lyophilizing and preserves, this not only makes the stability of anthracene nucleus medicinal liposome further improve, and makes the suitability for industrialized production of said preparation become possibility.Six month stability experiment results of lyophilized powder of the present invention under 2 ℃～25 ℃ show its mean diameter rate of change less than 10%, and the rate of change of entrapment efficiency has shown the stability of anthracene nucleus medicament hydrochlorate Liposomal formulation excellence of the present invention less than 3%.Added thermo-sensitive material in the injection anthracene nucleus medicament hydrochlorate lipidosome injection of the present invention, make liposome have release mechanism more initiatively, can be used with microwave heating equipment, improved liposome greatly the accumulating and discharge of targeting moiety, improved drug effect significantly and reduced toxic and side effects.

Preparation technology of the present invention meets the suitability for industrialized production requirement, has effectively solved the problem of liposome stability by Freeze Drying Technique, and its preparation can be preserved under the drying at room temperature condition more than 6 months, and its every physicochemical property has no significant change.

Description of drawings

Fig. 1 liposome structure sketch map

The release profiles of Fig. 2 epirubicin hydrochloride liposome in the PBS buffer

The release profiles of Fig. 3 daunorubicin hydrochloride liposome in the PBS buffer

The release profiles of Fig. 4 NSC 654509 liposome in the PBS buffer

The release profiles of Fig. 5 hydrochloric acid darubicin liposome in the PBS buffer

Fig. 6 blank liposome DSC collection of illustrative plates (DPPC: MPPC: DSPG: DSPE-PEG=80: 5: 5: 10)

The different chilling rates of Fig. 7 to the freeze-dried lipidosome rehydration after the influence of particle diameter

The different reserve temperature anthracene nucleus medicinal liposome of Fig. 8 envelop rate

Fig. 9 negative charge phospholipid is to the influence of particle diameter before and after the lyophilizing of daunorubicin thermal sensitive liposome

The stability (20 ℃) of the epirubicin lipidosome injection that the different phospholipid of Figure 10 are formed

The release of Figure 11 epirubicin hydrochloride liposome in 50% blood plasma-PBS buffer

The release of Figure 12 daunorubicin hydrochloride liposome in 50% blood plasma-PBS buffer

The release of Figure 13 NSC 654509 liposome in 50% blood plasma-PBS buffer

The release of darubicin liposome in 50% blood plasma-PBS buffer that softens of Figure 14 hydrochloric acid

The release of Figure 15 epirubicin hydrochloride liposome in 50% blood plasma-PBS buffer

The release of Figure 16 daunorubicin hydrochloride liposome in 50% blood plasma-PBS buffer

The release of Figure 17 NSC 654509 liposome in 50% blood plasma-PBS buffer

The release of Figure 18 hydrochloric acid darubicin liposome in 50% blood plasma-PBS buffer

Figure 19 Solid-Phase Extraction flow chart

Technical scheme of the present invention can specify by following embodiment, but protection scope of the present invention is not limited only to this.

Embodiment 1:

Preparation prescription (50ml capacity)

Epirubicin hydrochloride 100mg citric acid 365mg

DPPC 1700mg sodium citrate 380mg

MPPC 100mg sodium carbonate 340mg

DSPG 100mg lactose 2500mg

PEG-DSPE 100mg water for injection is settled to 50ml

Preparation technology:

In the prescription ratio phospholipid is joined in the round-bottomed flask, add the dissolving of chloroform-methanol (3: 1) mixed solvent, under 45 ℃ of conditions, the decompression rotary evaporation removes and desolvates.Phospholipid forms thin film at the round-bottomed flask inwall, with the thin film dry 20h under vacuum condition for preparing, guarantees that organic solvent removes fully.With the citric acid-sodium citrate buffer (pH4.0) that adds in the thin film for preparing, include 5% lactose, 45～60 ℃ of aquation 1h, obtain multilamellar liposome (MLVs), temperature greater than 45 ℃ of condition of high voltage under with the MLVs that makes successively by the aperture at 200nm, 100nm, the polycarbonate membrane of 80nm, obtain particle diameter and be the big unilamellar liposome (LUVs) about 50～250nm, the blank liposome that makes is cooled to 50 ± 5 ℃, add sodium carbonate liquor, pH value is adjusted to 7.5 ± 0.5, add 35 ℃ of epirubicin hydrochloride solution that preheating is good, 35 ± 5 ℃ of water 30～60min, intermittent jolting, the lactose of adding recipe quantity, dissolving, the content that makes lactose in the prescription is 5%, water for injection is settled to the filtering with microporous membrane of 50ml through 0.22 μ m, and embedding is preserved under-20 ℃ of conditions.

Embodiment 2:

Preparation prescription (50ml capacity)

Epirubicin hydrochloride 100mg sodium citrate 380mg

DPPC 1600mg sodium carbonate 340mg

DSPG 200mg trehalose 5000mg

PEG-DSPE 200mg PVP 500mg

Citric acid 365mg

Preparation technology:

In the prescription ratio phospholipid is joined in the round-bottomed flask, add the dissolving of chloroform-methanol (3: 1) mixed solvent, under 45 ℃ of conditions, the decompression rotary evaporation removes and desolvates.Phospholipid forms thin film at the round-bottomed flask inwall, with the thin film dry 20h under vacuum condition for preparing, guarantees that organic solvent removes fully.Citric acid-sodium citrate buffer (pH4.0) will be added in the thin film that prepare, include 10% trehalose, 45～60 ℃ of aquation 1h, obtain multilamellar liposome (MLVs), temperature greater than 45 ℃ of condition of high voltage under with the MLVs that makes successively by the aperture at 200nm, 100nm, the polycarbonate membrane of 80nm, obtain particle diameter and be the big unilamellar liposome (LUVs) about 50～250nm, the blank liposome that makes is cooled to 50 ± 5 ℃, add sodium carbonate liquor, pH value is adjusted to 7.5 ± 0.5, add 35 ℃ of epirubicin hydrochloride solution that preheating is good, 35 ± 5 ℃ of water 30～60min, intermittent jolting, the trehalose and the PVP of adding recipe quantity, dissolving, the concentration that makes trehalose in the prescription is 10%, through the filtering with microporous membrane of 0.22 μ m, fill, add half plug, pre-freeze is adopted middling speed cooling (1.0～1.5 ℃), and sample is stablized 2～6h at-50 ℃, opening program control is dry, first section drying-50 ℃ 3h ,-35 ℃ of 16h ,-10 ℃ of 4h, 0 ℃ of 1h, 20 ℃ of 8h of second section drying, lyophilizing finishes, tamponade, the sealing of jewelling lid, 20 ℃ of drying conditions are preserved down.

Embodiment 3:

Preparation prescription (50ml capacity)

Epirubicin hydrochloride 150mg citric acid 730mg

DPPC 1600mg sodium citrate 760mg

MSPC 100mg sodium carbonate 680mg

DSPG 100mg trehalose 7500mg

PEG-DSPE 200mg PVP 1000mg

Preparation technology:

In the prescription ratio phospholipid is joined in the round-bottomed flask, add the small amount of ethanol dissolving, under 60 ℃ of conditions, inject the good buffer of preheating, include 15% trehalose, ultrasonic 15min, use the dialysis of aquation buffer to remove foreign minister's organic solvent, 60 ℃ of aquation 1h, obtain multilamellar liposome (MLVs), temperature greater than 45 ℃ of condition of high voltage under with the MLVs that makes successively by the aperture at 200nm, 100nm, the polycarbonate membrane of 80nm, obtain particle diameter and be the big unilamellar liposome (LUVs) about 50～250nm, the blank liposome that makes is cooled to 35 ± 1 ℃, add sodium carbonate liquor, pH value is adjusted to 7.5 ± 0.5, add 35 ℃ of epirubicin hydrochloride solution that preheating is good, 35 ± 5 ℃ of water 30～60min, intermittent jolting, the trehalose of adding recipe quantity, PVP, dissolving, the concentration that makes trehalose in the prescription is 15%, microporous filter membrane through 0.22 μ m filters, and fill adds half plug, pre-freeze, adopt middling speed cooling (1.0～1.5 ℃), sample is stablized 2～6h at-50 ℃, and opening program control is dry, first section drying-50 ℃ 3h,-35 ℃ of 16h ,-10 ℃ of 4h, 0 ℃ of 1h, 20 ℃ of 8h of second section drying, lyophilizing finishes, inflated with nitrogen, tamponade, the sealing of jewelling lid, 20 ℃ of drying conditions are preserved down.

Embodiment 4:

Preparation prescription (50ml capacity)

Epirubicin hydrochloride 100mg sodium citrate 760mg

DPPC 1700mg sodium carbonate 680mg

MPPC 100mg sucrose 3750mg

DSPG 200mg PVP 1250mg

Citric acid 730mg

Preparation technology:

In the prescription ratio phospholipid is dissolved in the chloroform-methanol mixed solvent, spray drying obtains mixture of phospholipids, under 60 ℃ of conditions of liposome extracorporeal releasing experiment, inject the good buffer of preheating, include 7.5% sucrose, ultrasonic 15min, 60 ℃ of aquation 1h, obtain multilamellar liposome (MLVs), temperature greater than 45 ℃ of condition of high voltage under with the MLVs that makes successively by the aperture at 200nm, 100nm, the polycarbonate membrane of 80nm, obtain particle diameter and be the big unilamellar liposome (LUVs) about 50～250nm, the blank liposome that makes is cooled to 35 ± 1 ℃, add sodium carbonate liquor, pH value is adjusted to 7.5 ± 0.5, adds 35 ℃ of epirubicin hydrochloride solution that preheating is good, 35 ± 5 ℃ of water 30～60min, intermittent jolting, the sucrose that adds recipe quantity, PVP, dissolving makes that cane sugar content is 7.5% in the prescription, the water for injection standardize solution, through the filtering with microporous membrane of 0.22 μ m, fill adds half plug, pre-freeze, adopt middling speed cooling (1.0～1.5 ℃), sample is stablized 2～6h at-50 ℃, and opening program control is dry, first section drying-50 ℃ 4h,-35 ℃ of 12h ,-10 ℃ of 6h, 0 ℃ of 2h, 20 ℃ of 8h of second section drying, lyophilizing finishes, inflated with nitrogen, tamponade, the sealing of jewelling lid, 20 ℃ of drying conditions are preserved down.

Embodiment 5:

Preparation prescription (50ml capacity)

Daunorubicin hydrochloride 200mg citric acid 365mg

DPPC 1700mg sodium citrate 380mg

MPPC 100mg sodium carbonate 340mg

DSPA 100mg sucrose 6000mg

PEG-DSPE 100mg water for injection is settled to 50ml

Preparation technology:

In the prescription ratio phospholipid is joined in the round-bottomed flask, add the dissolving of chloroform-methanol (3: 1) mixed solvent, under 45 ℃ of conditions, the decompression rotary evaporation removes and desolvates.Phospholipid forms thin film at the round-bottomed flask inwall, with the thin film dry 20h under vacuum condition for preparing, guarantees that organic solvent removes fully.With the citric acid-sodium citrate buffer (pH4.0) that adds in the thin film for preparing, include 12% sucrose, 45～60 ℃ of aquation 1h, obtain multilamellar liposome (MLVs), temperature greater than 45 ℃ of condition of high voltage under with the MLVs that makes successively by the aperture at 200nm, 100nm, the polycarbonate membrane of 80nm, obtain particle diameter and be the big unilamellar liposome (LUVs) about 50～250nm, the blank liposome that makes is cooled to 50 ± 5 ℃, adds sodium carbonate liquor, pH value is adjusted to 7.5 ± 0.5, add 35 ℃ of daunorubicin hydrochloride solution that preheating is good, 35 ± 5 ℃ of water 30～60min, intermittent jolting, the sucrose of adding recipe quantity, dissolving, making the content of sucrose in the prescription is 12%, and water for injection is settled to 50ml, through the filtering with microporous membrane of 0.22 μ m, embedding ,-20 ℃ of drying conditions are preserved down.

Embodiment 6:

Preparation prescription (50ml capacity)

Daunorubicin hydrochloride 150mg sodium citrate 380mg

DPPC 1600mg sodium carbonate 340mg

DSPG 200mg lactose 4500mg

PEG-DSPE 200mg PVP 600mg

Citric acid 365mg

Preparation technology:

In the prescription ratio phospholipid is joined in the round-bottomed flask, add the dissolving of chloroform-methanol (3: 1) mixed solvent, under 45 ℃ of conditions, the decompression rotary evaporation removes and desolvates.Phospholipid forms thin film at the round-bottomed flask inwall, with the thin film dry 20h under vacuum condition for preparing, guarantees that organic solvent removes fully.With the citric acid-sodium citrate buffer (pH4.0) that adds in the thin film for preparing, include 9% lactose, 45～60 ℃ of aquation 1h, obtain multilamellar liposome (MLVs), temperature greater than 45 ℃ of condition of high voltage under with the MLVs that makes successively by the aperture at 200nm, 100nm, the polycarbonate membrane of 80nm, obtain particle diameter and be the big unilamellar liposome (LUVs) about 50～250nm, the blank liposome that makes is cooled to 50 ± 5 ℃, add sodium carbonate liquor, pH value is adjusted to 7.5 ± 0.5, add 35 ℃ of daunorubicin hydrochloride solution that preheating is good, 35 ± 5 ℃ of water 30～60min, intermittent jolting, the lactose that adds recipe quantity, PVP, dissolving, making the lactose content in the prescription is 9%, the water for injection standardize solution, filtering with microporous membrane through 0.22 μ m, fill adds half plug, pre-freeze, adopt middling speed cooling (1.0～1.5 ℃), sample is stablized 2～6h at-50 ℃, and opening program control is dry, first section drying-50 ℃ 3h,-35 ℃ of 16h,-10 ℃ of 4h, 0 ℃ of 1h, 20 ℃ of 8h of second section drying, lyophilizing finishes, tamponade, the sealing of jewelling lid, 2～20 ℃ of drying conditions are preserved down.

Embodiment 7:

Preparation prescription (50ml capacity)

Daunorubicin hydrochloride 300mg citric acid 730mg

DPPC 1600mg sodium citrate 760mg

MPPC 100mg sodium carbonate 680mg

DSPG 100mg trehalose 7500mg

PEG-DSPE 200mg

Preparation technology:

In the prescription ratio phospholipid is dissolved in the chloroform-methanol mixed solvent, spray drying obtains mixture of phospholipids, under 60 ℃ of conditions, inject the good buffer of preheating, include 15% trehalose, ultrasonic 15min, 60 ℃ of aquation 1h, obtain multilamellar liposome (MLVs), temperature greater than 45 ℃ of condition of high voltage under with the MLVs that makes successively by the aperture at 200nm, 100nm, the polycarbonate membrane of 80nm obtains particle diameter and is the big unilamellar liposome (LUVs) about 50～250nm, and the blank liposome that makes is cooled to 35 ± 1 ℃, add sodium carbonate liquor, pH value is adjusted to 7.5 ± 0.5, adds 35 ℃ of daunorubicin hydrochloride solution that preheating is good, 35 ± 5 ℃ of water 30～60min, intermittent jolting, the trehalose that adds recipe quantity, dissolving, the concentration that makes trehalose in the prescription is 15%, the water for injection standardize solution, through the filtering with microporous membrane of 0.22 μ m, fill adds half plug, pre-freeze, adopt middling speed cooling (1.0～1.5 ℃), sample is stablized 2～6h at-50 ℃, and opening program control is dry, first section drying-50 ℃ 3h,-35 ℃ of 16h ,-10 ℃ of 4h, 0 ℃ of 1h, 20 ℃ of 6h of second section drying, lyophilizing finishes, inflated with nitrogen, tamponade, the sealing of jewelling lid, 2～20 ℃ of drying conditions are preserved down.

Embodiment 8:

Preparation prescription (50ml capacity)

Daunorubicin hydrochloride 150mg DPPC 1700mg

MPPC 100mg sodium carbonate 680mg

DSPG 200mg sucrose 5000mg

Citric acid 730mg PVP 1250mg

Sodium citrate 760mg

Preparation technology:

In the prescription ratio phospholipid is dissolved in the chloroform-methanol mixed solvent, spray drying obtains mixture of phospholipids, under 60 ℃ of conditions of liposome extracorporeal releasing experiment, inject the good buffer of preheating, include 10% sucrose, ultrasonic 15min, 60 ℃ of aquation 1h, obtain multilamellar liposome (MLVs), temperature greater than 45 ℃ of condition of high voltage under with the MLVs that makes successively by the aperture at 200nm, 100nm, the polycarbonate membrane of 80nm, obtain particle diameter and be the big unilamellar liposome (LUVs) about 50～250nm, the blank liposome that makes is cooled to 35 ± 1 ℃, add sodium carbonate liquor, pH value is adjusted to 7.5 ± 0.5, add 35 ℃ of daunorubicin hydrochloride solution that preheating is good, 35 ± 5 ℃ of water 30～60min, intermittent jolting, the sucrose that adds recipe quantity, PVP, make that concentration of sucrose is 10% in the prescription, the water for injection standardize solution is through the filtering with microporous membrane of 0.22 μ m, fill, add half plug, pre-freeze is adopted middling speed cooling (1.0～1.5 ℃), sample is stablized 2～6h at-50 ℃, opening program control is dry, first section drying-50 ℃ 3h ,-35 ℃ of 16h,-10 ℃ of 4h, 0 ℃ of 1h, 15 ℃ of 8h of second section drying, lyophilizing finishes, inflated with nitrogen, tamponade, the sealing of jewelling lid, 2～20 ℃ of drying conditions are preserved down.

Embodiment 9:

Preparation prescription (50ml capacity)

NSC 654509 200mg citric acid 365mg

DPPC 1700mg sodium citrate 380mg

MPPC 100mg sodium carbonate 340mg

DSPG 100mg lactose 2500mg

PEG-DSPE 100mg water for injection is settled to 50ml

Preparation technology:

In the prescription ratio phospholipid is joined in the round-bottomed flask, add the dissolving of chloroform-methanol (3: 1) mixed solvent, under 45 ℃ of conditions, the decompression rotary evaporation removes and desolvates.Phospholipid forms thin film at the round-bottomed flask inwall, with the thin film dry 20h under vacuum condition for preparing, guarantees that organic solvent removes fully.With the citric acid-sodium citrate buffer (pH4.0) that adds in the thin film for preparing, include 5% lactose, 45～60 ℃ of aquation 1h, obtain multilamellar liposome (MLVs), temperature greater than 45 ℃ of condition of high voltage under with the MLVs that makes successively by the aperture at 200nm, 100nm, the polycarbonate membrane of 80nm, obtain particle diameter and be the big unilamellar liposome (LUVs) about 50～250nm, the blank liposome that makes is cooled to 50 ± 5 ℃, adds sodium carbonate liquor, pH value is adjusted to 7.5 ± 0.5, add 35 ℃ of NSC 654509 solution that preheating is good, 35 ± 5 ℃ of water 30～60min, intermittent jolting, the lactose of adding recipe quantity, dissolving, make that lactose concn is 5% in the prescription, water for injection is settled to 50ml, crosses the microporous filter membrane of 0.22 μ m, embedding is preserved under-20 ℃ of conditions.

Embodiment 10:

Preparation prescription (50ml capacity)

NSC 654509 150mg sodium citrate 760mg

DPPC 1700mg sodium carbonate 680mg

MPPC 100mg sucrose 3750mg

DSPG 200mg PVP 500mg

Citric acid 30mg

Preparation technology:

In the prescription ratio phospholipid is joined in the round-bottomed flask, add the small amount of ethanol dissolving, under 60 ℃ of conditions, inject the good buffer of preheating, include 7.5% sucrose, ultrasonic 15min, make the dialysis of aquation buffer remove foreign minister's organic solvent, 60 ℃ of aquation 1h, obtain multilamellar liposome (MLVs), temperature greater than 45 ℃ of condition of high voltage under with the MLVs that makes successively by the aperture at 200nm, 100nm, the polycarbonate membrane of 80nm obtains particle diameter and is the big unilamellar liposome (LUVs) about 50～250nm, the blank liposome that makes is cooled to 35 ± 1 ℃, add sodium carbonate liquor, pH value is adjusted to 7.5 ± 0.5, add 35 ℃ of NSC 654509 solution that preheating is good, 35 ± 5 ℃ of water 30～60min, intermittent jolting, the sucrose of adding recipe quantity, PVP, dissolving, make that sucrose concentration is 7.5% in the prescription, water for injection is settled to scale.Microporous filter membrane through 0.22 μ m filters, and fill adds half plug, pre-freeze is adopted middling speed cooling (1.0～1.5 ℃), and sample is stablized 2～6h at-50 ℃, opening program control is dry, first section drying-50 ℃ 3h ,-35 ℃ of 16h,-10 ℃ of 4h, 0 ℃ of 1h, 20 ℃ of 8h of second section drying, lyophilizing finishes, inflated with nitrogen, tamponade, the sealing of jewelling lid, 2～20 ℃ of drying conditions are preserved down.

Embodiment 11:

Preparation prescription (50ml capacity)

NSC 654509 100mg citric acid 730mg

DPPC 1600mg sodium citrate 760mg

MPPC 100mg sodium carbonate 680mg

DSPG 100mg trehalose 7500mg

PEG-DSPE 200mg

Preparation technology:

In the prescription ratio phospholipid is dissolved in the chloroform-methanol mixed solvent, spray drying obtains mixture of phospholipids, under 60 ℃ of conditions, inject the good buffer (containing 15% trehalose) of preheating, ultrasonic 15min, 60 ℃ of aquation 1h, obtain multilamellar liposome (MLVs), temperature greater than 45 ℃ of condition of high voltage under with the MLVs that makes successively by the aperture at 200nm, 100nm, the polycarbonate membrane of 80nm obtains particle diameter and is the big unilamellar liposome (LUVs) about 50～250nm, the blank liposome that makes is cooled to 35 ± 1 ℃, add sodium carbonate liquor, pH value is regulated 7.5 ± 0.5, add 35 ℃ of daunorubicin hydrochloride solution that preheating is good, 35 ± 5 ℃ of water 30～60min, intermittent jolting adds the trehalose of recipe quantity, makes that the trehalose mass concentration is 15% in the prescription, the water for injection standardize solution, through the filtering with microporous membrane of 0.22 μ m, fill adds half plug, pre-freeze, adopt middling speed cooling (1.0～1.5 ℃), sample is stablized 2～6h at-50 ℃, and opening program control is dry, first section drying-50 ℃ 3h,-35 ℃ of 16h ,-10 ℃ of 4h, 0 ℃ of 1h, 15 ℃ of 8h of second section drying, lyophilizing finishes, inflated with nitrogen, tamponade, the sealing of jewelling lid, 2～20 ℃ of drying conditions are preserved down.

Embodiment 12:

Preparation prescription (50ml capacity)

NSC 654509 150mg sodium citrate 380mg

DPPC 1600mg sodium carbonate 340mg

DSPG 200mg lactose 2500mg

PEG-DSPE 200mg PVP 1250mg

Citric acid 365mg

Preparation technology:

In the prescription ratio phospholipid is joined in the round-bottomed flask, add the dissolving of chloroform-methanol (3: 1) mixed solvent, under 45 ℃ of conditions, the decompression rotary evaporation removes and desolvates.Phospholipid forms thin film at the round-bottomed flask inwall, with the thin film dry 20h under vacuum condition for preparing, guarantees that organic solvent removes fully.With the citric acid-sodium citrate buffer (pH4.0) that adds in the thin film for preparing, include 5% lactose, 45～60 ℃ of aquation 1h, obtain multilamellar liposome (MLVs), temperature greater than 45 ℃ of condition of high voltage under with the MLVs that makes successively by the aperture at 200nm, 100nm, the polycarbonate membrane of 80nm, obtain particle diameter and be the big unilamellar liposome (LUVs) about 50～250nm, the blank liposome that makes is cooled to 50 ± 5 ℃, add sodium carbonate liquor, pH value is adjusted to 7.5 ± 0.5, add 35 ℃ of NSC 654509 solution that preheating is good, 35 ± 5 ℃ of water 30～60min, intermittent jolting, the lactose of adding recipe quantity, PVP, the concentration that makes lactose in the prescription is 5%, filtering with microporous membrane through 0.22 μ m, fill adds half plug, pre-freeze, adopt middling speed cooling (1.0～1.5 ℃), sample is stablized 2～6h at-50 ℃, and opening program control is dry, first section drying-50 ℃ 3h,-35 ℃ of 16h,-10 ℃ of 4h, 0 ℃ of 1h, 20 ℃ of 8h of second section drying, lyophilizing finishes, tamponade, the sealing of jewelling lid, 2～20 ℃ of drying conditions are preserved down.

Embodiment 13:

Preparation prescription (50ml capacity)

Hydrochloric acid darubicin 200mg PEG-DSPE 100mg

DPPC 1700mg citric acid 365mg

MPPC 100mg sodium citrate 380mg

DSPG 100mg sodium carbonate 340mg

Lactose 2500mg water for injection is settled to 50ml

Preparation technology:

In the prescription ratio phospholipid is joined in the round-bottomed flask, add the dissolving of chloroform-methanol (3: 1) mixed solvent, under 45 ℃ of conditions, the decompression rotary evaporation removes and desolvates.Phospholipid forms thin film at the round-bottomed flask inwall, with the thin film dry 20h under vacuum condition for preparing, guarantees that organic solvent removes fully.With the citric acid-sodium citrate buffer (pH4.0) that adds in the thin film for preparing, include 5% lactose, 45～60 ℃ of aquation 1h, obtain multilamellar liposome (MLVs), temperature greater than 45 ℃ of condition of high voltage under with the MLVs that makes successively by the aperture at 200nm, 100nm, the polycarbonate membrane of 80nm, obtain particle diameter and be the big unilamellar liposome (LUVs) about 50～250nm, the blank liposome that makes is cooled to 50 ± 5 ℃, adds sodium carbonate liquor, pH value is adjusted to 7.5 ± 0.5, add 35 ℃ of hydrochloric acid darubicin solution that preheating is good, 35 ± 5 ℃ of water 30～60min, intermittent jolting, the lactose of adding recipe quantity, dissolving, making the concentration of lactose in the prescription is 5%, and water for injection is settled to 50ml, through the filtering with microporous membrane of 0.22 μ m, embedding is preserved under-20 ℃ of conditions.

Embodiment 14:

Preparation prescription (50ml capacity)

Hydrochloric acid darubicin 150mg sodium citrate 380mg

DPPC 1600mg sodium carbonate 340mg

DSPG 200mg lactose 4500mg

PEG-DSPE 200mg PVP 500mg

Citric acid 365mg

Preparation technology:

In the prescription ratio phospholipid is joined in the round-bottomed flask, add the small amount of ethanol dissolving, under 60 ℃ of conditions, inject the good buffer of preheating, include 9% lactose, ultrasonic 15min, use the dialysis of aquation buffer to remove foreign minister's organic solvent, 60 ℃ of aquation 1h, obtain multilamellar liposome (MLVs), temperature greater than 45 ℃ of condition of high voltage under with the MLVs that makes successively by the aperture at 200nm, 100nm, the polycarbonate membrane of 80nm, obtain particle diameter and be the big unilamellar liposome (LUVs) about 50～250nm, the blank liposome that makes is cooled to 35 ± 1 ℃, adds sodium carbonate liquor, pH value is adjusted to 7.5 ± 0.5, add 35 ℃ of hydrochloric acid darubicin solution that preheating is good, 35 ± 5 ℃ of water 30～60min, intermittent jolting, the lactose of adding recipe quantity, PVP, dissolving, making the lactose content in the prescription is 9%, and the water for injection standardize solution is through the microporous filter membrane filtration of 0.22 μ m, fill, add half plug, pre-freeze is adopted middling speed cooling (1.0～1.5 ℃), sample is stablized 2～6h at-50 ℃, opening program control is dry, first section drying-50 ℃ 2h ,-35 ℃ of 18h,-15 ℃ of 5h, 0 ℃ of 2h, 20 ℃ of 8h of second section drying, lyophilizing finishes, inflated with nitrogen, tamponade, the sealing of jewelling lid, 2～20 ℃ of drying conditions are preserved down.

Embodiment 15:

Preparation prescription (50ml capacity)

Hydrochloric acid darubicin 100mg citric acid 730mg

DPPC 1600mg sodium citrate 760mg

MPPC 100mg sodium carbonate 680mg

DSPG 100mg trehalose 7500mg

PEG-DSPE 200mg

Preparation technology:

In the prescription ratio phospholipid is dissolved in the chloroform-methanol mixed solvent, spray drying obtains mixture of phospholipids, under 60 ℃ of conditions of liposome extracorporeal releasing experiment, inject the good buffer of preheating, include 15% trehalose, ultrasonic 15min, 60 ℃ of aquation 1h obtain multilamellar liposome (MLVs), temperature greater than 45 ℃ of condition of high voltage under with the MLVs that makes successively by the aperture at 200nm, 100nm, the polycarbonate membrane of 80nm, obtain particle diameter and be the big unilamellar liposome (LUVs) about 50～250nm, the blank liposome that makes is cooled to 35 ± 1 ℃, add sodium carbonate liquor, pH value is adjusted to 7.5 ± 0.5, add 35 ℃ of hydrochloric acid darubicin solution that preheating is good, 35 ± 5 ℃ of water 30～60min, intermittent jolting, the trehalose that adds recipe quantity, dissolving, the concentration that makes trehalose in the prescription is 15%, crosses the microporous filter membrane of 0.22 μ m, fill, add half plug, pre-freeze is adopted middling speed cooling (1.0～1.5 ℃), sample is stablized 2～6h at-50 ℃, opening program control is dry, first section drying-50 ℃ 3h ,-35 ℃ of 16h,-10 ℃ of 4h, 0 ℃ of 1h, 20 ℃ of 8h of second section drying, lyophilizing finishes, inflated with nitrogen, tamponade, the sealing of jewelling lid, 20 ℃ of drying conditions are preserved down.

Embodiment 16:

Preparation prescription (50ml capacity)

Hydrochloric acid darubicin 150mg sodium citrate 760mg

DPPC 1700mg sodium carbonate 680mg

MPPC 100mg sucrose 3750mg

DSPG 200mg PVP 1250mg

Citric acid 730mg

Preparation technology:

In the prescription ratio phospholipid is dissolved in the chloroform-methanol mixed solvent, spray drying obtains mixture of phospholipids, under 60 ℃ of conditions of liposome extracorporeal releasing experiment, inject the good buffer of preheating, include 7.5% sucrose, ultrasonic 15min, 60 ℃ of aquation 1h, obtain multilamellar liposome (MLVs), temperature greater than 45 ℃ of condition of high voltage under with the MLVs that makes successively by the aperture at 200nm, 100nm, the polycarbonate membrane of 80nm, obtain particle diameter and be the big unilamellar liposome (LUVs) about 50～250nm, the blank liposome that makes is cooled to 35 ± 1 ℃, add sodium carbonate liquor, pH value is adjusted to 7.5 ± 0.5, adds 35 ℃ of hydrochloric acid darubicin solution that preheating is good, 35 ± 5 ℃ of water 30～60min, intermittent jolting, the sucrose that adds recipe quantity, dissolving makes that sucrose concentration is 7.5% in the prescription, the water for injection standardize solution, cross the microporous filter membrane of 0.22 μ m, fill adds half plug, pre-freeze, adopt middling speed cooling (1.0～1.5 ℃), sample is stablized 2～6h at-50 ℃, and opening program control is dry, first section drying-50 ℃ 3h,-35 ℃ of 16h ,-10 ℃ of 4h, 0 ℃ of 1h, 15 ℃ of 8h of second section drying, lyophilizing finishes, inflated with nitrogen, tamponade, the sealing of jewelling lid, 20 ℃ of drying conditions are preserved down.

Embodiment 17

Preparation prescription (50ml capacity):

Hydrochloric acid darubicin 150mg citric acid 1095mg

DPPC 1600mg sodium citrate 1140mg

MPPC 100mg sodium carbonate 1020mg

DPPG 100mg sucrose 5000mg

PEG-DSPE 200mg

Preparation technology:

In the prescription ratio phospholipid and derivant thereof are joined in the round-bottomed flask, add the dissolving of chloroform-methanol mixed solvent, under 45 ℃ of conditions, the decompression rotary evaporation removes and desolvates.Phospholipid forms thin film at the round-bottomed flask inwall, with the thin film dry 20h under vacuum condition for preparing, guarantees that organic solvent removes fully.The citric acid-sodium citrate buffer (pH4.0) of 300mM will be added in the thin film that prepare, include 10% sucrose, 45～60 ℃ of aquation 1h, obtain multilamellar liposome (MLVs), the MLVs that makes is existed under in temperature greater than 45 ℃ of condition of high voltage, successively by the aperture at 200nm, 100nm, the polycarbonate membrane of 80nm, obtain particle diameter and be the big unilamellar liposome (LUVs) about 50～250nm, the blank liposome that makes is cooled to 35 ± 1 ℃, adds sodium carbonate liquor, pH value is adjusted to 7.5 ± 0.5, add 35 ℃ of hydrochloric acid darubicin solution that preheating is good, 35 ± 1 ℃ of water-bath 30～60min, intermittent jolting adds sucrose, dissolving, making the cane sugar content in the prescription is 10%, and the water for injection standardize solution is through the microporous filter membrane filtration of 0.22 μ m, fill, add half plug, pre-freeze, adopt fast cooling (in the liquid nitrogen freezing about 40 ℃/min), sample is stablized 2～6h at-50 ℃, opening program control is dry, first section drying-50 ℃ 2h ,-40 ℃ of 5h, 35 ℃ of 15h,-10 ℃ of 2h, 0 ℃ of 2h, 20 ℃ of 8h of second section drying, lyophilizing finishes, tamponade, the sealing of jewelling lid, 20 ℃ of drying conditions are preserved down.

Embodiment 18:

Prescription: 50ml capacity

Epirubicin hydrochloride 100mg sodium citrate 1520mg

DPPC 1800mg sodium carbonate 1360mg

MPPC 50mg mannitol 7500mg

DSPG 50mg PVP 2500mg

Citric acid 1460mg water for injection is settled to 50ml

Preparation technology:

In the prescription ratio phospholipid is joined in the round-bottomed flask, add the dissolving of chloroform-methanol mixed solvent, under 45 ℃ of conditions, the decompression rotary evaporation removes and desolvates.Phospholipid forms thin film at the round-bottomed flask inwall, with the thin film dry 20h under vacuum condition for preparing, guarantees that organic solvent removes fully.With the citric acid-sodium citrate buffer (pH4.0) that adds in the thin film for preparing, include 15% mannitol, 45～60 ℃ of aquation 1h, obtain multilamellar liposome (MLVs), phospholipid concentration is, the MLVs that makes existed under greater than 45 ℃ of condition of high voltage in temperature, successively by the aperture at 200nm, 100nm, the polycarbonate membrane of 80nm, obtain particle diameter and be the big unilamellar liposome (LUVs) about 50～250nm, the blank liposome that makes is cooled to 35 ± 1 ℃, add sodium carbonate liquor, pH value is adjusted to 7～9, add 35 ℃ of epirubicin hydrochloride solution that preheating is good, 35 ± 1 ℃ of water 30～60min, intermittent jolting, add mannitol, dissolving, the content that makes mannitol in the prescription is 15%, the water for injection standardize solution, microporous filter membrane through 0.22 μ m filters, embedding, the water for injection standardize solution ,-20 ℃ of drying conditions are preserved down.

Embodiment 19:

Preparation prescription (50ml capacity)

Epirubicin hydrochloride 100mg citric acid 365mg

DPPC 1250mg sodium citrate 380mg

MPPC 250mg sodium carbonate 340mg

DSPG 250mg lactose 2500mg

PEG-DSPE 250mg water for injection is settled to 50ml

Preparation technology:

Embodiment 20:

Preparation prescription (50ml capacity)

Daunorubicin hydrochloride 100mg MPPC 250mg

DPPC 1250mg DSPG 250mg

PEG-DSPE 250mg sodium carbonate 340mg

Citric acid 365mg lactose 2500mg

Sodium citrate 380mg water for injection is settled to 50ml

Preparation technology:

In the prescription ratio phospholipid is joined in the round-bottomed flask, add the dissolving of chloroform-methanol (3: 1) mixed solvent, under 45 ℃ of conditions, the decompression rotary evaporation removes and desolvates.Phospholipid forms thin film at the round-bottomed flask inwall, with the thin film dry 20h under vacuum condition for preparing, guarantees that organic solvent removes fully.With the citric acid-sodium citrate buffer (pH4.0) that adds in the thin film for preparing, include 5% lactose, 45～60 ℃ of aquation 1h, obtain multilamellar liposome (MLVs), temperature greater than 45 ℃ of condition of high voltage under with the MLVs that makes successively by the aperture at 200nm, 100nm, the polycarbonate membrane of 80nm, obtain particle diameter and be the big unilamellar liposome (LUVs) about 50～250nm, the blank liposome that makes is cooled to 50 ± 5 ℃, add sodium carbonate liquor, pH value is adjusted to 7.5 ± 0.5, add 35 ℃ of daunorubicin hydrochloride solution that preheating is good, 35 ± 5 ℃ of water 30～60min, intermittent jolting, the lactose of adding recipe quantity, dissolving, the content that makes lactose in the prescription is 5%, water for injection is settled to the filtering with microporous membrane of 50ml through 0.22 μ m, and embedding is preserved under-20 ℃ of conditions.

Embodiment 21:

Preparation prescription (50ml capacity)

NSC 654509 100mg citric acid 365mg

DPPC 1250mg sodium citrate 380mg

MPPC 250mg sodium carbonate 340mg

DSPG 250mg lactose 2500mg

PEG-DSPE 250mg water for injection is settled to 50ml

Preparation technology:

In the prescription ratio phospholipid is joined in the round-bottomed flask, add the dissolving of chloroform-methanol (3: 1) mixed solvent, under 45 ℃ of conditions, the decompression rotary evaporation removes and desolvates.Phospholipid forms thin film at the round-bottomed flask inwall, with the thin film dry 20h under vacuum condition for preparing, guarantees that organic solvent removes fully.With the citric acid-sodium citrate buffer (pH4.0) that adds in the thin film for preparing, include 5% lactose, 45～60 ℃ of aquation 1h, obtain multilamellar liposome (MLVs), temperature greater than 45 ℃ of condition of high voltage under with the MLVs that makes successively by the aperture at 200nm, 100nm, the polycarbonate membrane of 80nm, obtain particle diameter and be the big unilamellar liposome (LUVs) about 50～250nm, the blank liposome that makes is cooled to 50 ± 5 ℃, add sodium carbonate liquor, pH value is adjusted to 7.5 ± 0.5, add 35 ℃ of NSC 654509 solution that preheating is good, 35 ± 5 ℃ of water 30～60min, intermittent jolting, the lactose of adding recipe quantity, dissolving, the content that makes lactose in the prescription is 5%, water for injection is settled to the filtering with microporous membrane of 50ml through 0.22 μ m, and embedding is preserved under-20 ℃ of conditions.

Embodiment 22:

Preparation prescription (50ml capacity)

Hydrochloric acid darubicin 100mg citric acid 365mg

DPPC 1250mg sodium citrate 380mg

MPPC 250mg sodium carbonate 340mg

DSPG 250mg lactose 2500mg

PEG-DSPE 250mg water for injection is settled to 50ml

Preparation technology:

In the prescription ratio phospholipid is joined in the round-bottomed flask, add the dissolving of chloroform-methanol (3: 1) mixed solvent, under 45 ℃ of conditions, the decompression rotary evaporation removes and desolvates.Phospholipid forms thin film at the round-bottomed flask inwall, with the thin film dry 20h under vacuum condition for preparing, guarantees that organic solvent removes fully.With the citric acid-sodium citrate buffer (pH4.0) that adds in the thin film for preparing, include 5% lactose, 45～60 ℃ of aquation 1h, obtain multilamellar liposome (MLVs), temperature greater than 45 ℃ of condition of high voltage under with the MLVs that makes successively by the aperture at 200nm, 100nm, the polycarbonate membrane of 80nm, obtain particle diameter and be the big unilamellar liposome (LUVs) about 50～250nm, the blank liposome that makes is cooled to 50 ± 5 ℃, add sodium carbonate liquor, pH value is adjusted to 7.5 ± 0.5, add 35 ℃ of hydrochloric acid darubicin solution that preheating is good, 35 ± 5 ℃ of water 30～60min, intermittent jolting, the lactose of adding recipe quantity, dissolving, the content that makes lactose in the prescription is 5%, water for injection is settled to the filtering with microporous membrane of 50ml through 0.22 μ m, and embedding is preserved under-20 ℃ of conditions.

Embodiment 23:

Preparation prescription (50ml capacity)

Epirubicin hydrochloride 100mg citric acid 365mg

DPPC 1700mg sodium citrate 380mg

MPPG 100mg sodium carbonate 340mg

DSPG 100mg lactose 2500mg

PEG-DSPE 100mg water for injection is settled to 50ml

Preparation technology:

Embodiment 24:

Preparation prescription (50ml capacity)

Daunorubicin hydrochloride 100mg citric acid 365mg

DPPC 1700mg sodium citrate 380mg

MPPE 100mg sodium carbonate 340mg

DSPG 100mg lactose 2500mg

PEG-DSPE 100mg water for injection is settled to 50ml

Preparation technology:

Embodiment 25:

Preparation prescription (50ml capacity)

NSC 654509 100mg citric acid 365mg

DPPA 1700mg sodium citrate 380mg

MPPC 100mg sodium carbonate 340mg

DSPG 100mg lactose 2500mg

PEG-DSPE 100mg water for injection is settled to 50ml

Preparation technology:

Embodiment 26:

Preparation prescription (50ml capacity)

Hydrochloric acid darubicin 100mg citric acid 365mg

DPPC 1700mg sodium citrate 380mg

MOPS 100mg sodium carbonate 340mg

DSPG 100mg lactose 2500mg

PEG-DSPE 100mg water for injection is settled to 50ml

Preparation technology:

Embodiment 27:

Preparation prescription (50ml capacity)

Epirubicin hydrochloride 100mg citric acid 365mg

DPPC 1700mg sodium citrate 380mg

MPPG 60mg sodium carbonate 340mg

PEG-DSPE 150mg lactose 2500mg

Water for injection is settled to 50ml

Preparation technology:

Embodiment 28:

Preparation prescription (50ml capacity)

Daunorubicin hydrochloride 100mg citric acid 365mg

DPPC 1800mg sodium citrate 380mg

MPPA 60mg sodium carbonate 340mg

PEG-DSPE 150mg lactose 2500mg

Water for injection is settled to 50ml

Preparation technology:

Embodiment 29:

Preparation prescription (50ml capacity)

NSC 654509 100mg citric acid 365mg

DPPC 1700mg sodium citrate 380mg

MPPE 80mg sodium carbonate 340mg

PEG-DSPE 150mg lactose 2500mg

Water for injection is settled to 50ml

Preparation technology:

Embodiment 30:

Preparation prescription (50ml capacity)

Hydrochloric acid darubicin 100mg citric acid 365mg

DPPC 1700mg sodium citrate 380mg

MPPG 60mg sodium carbonate 340mg

PEG-DSPE 150mg lactose 2500mg

Water for injection is settled to 50ml

Preparation technology:

Result and discussion

The different phosphate lipid concentration is investigated the influence of liposome medicine carrying stability

The lipidosome injection and the lyophilized injectable powder of the different phospholipid concentration for preparing among the embodiment are contrasted the change of granularity of its different samplings standing times and leak the medicine rate, the result shows, in the scope of 0～200mM/ml, increase along with phospholipid concentration, bag loading capability to medicine increases, the leakage medicine rate of medicine is more little after the freeze-dried lipidosome rehydration, but change of size becomes big with the increase of phospholipid concentration.

The release characteristics research of thermal sensitive liposome

The thermal sensitive liposome of the difference for preparing prescription is diluted to finite concentration with the PBS buffer of 10mM, be distributed under 37 ℃ and 42 ℃ of conditions and hatch 60min, respectively at 5min, 10min, 30min, the 60min point in time sampling, use fluorescence detector and measure its fluorescent value, with add a certain amount of 15%Triton X-100 with the fluorescent absorption value of the thermal sensitive liposome diluent of isoconcentration as 100% burst size, calculate the burst size of each time point.Shown in Fig. 2～5, the liposome prescription burst size that adds surfactant is apparently higher than not adding the surfactant prescription.

The phase transition temperature of liposome is measured

As shown in Figure 6, the phase transition temperature that the DSC method is measured liposome is about 42.5 ℃, and is basic identical with the phase transition temperature of DPPC phospholipid, meets the requirement of thermal sensitive liposome about phase transition temperature.

Different cryoprotective agents are to the influence of lipidosome freeze-dried stability

The lactose of experimental applications, sucrose, trehalose, PVP all demonstrate good frozen-dried protective effect, and the frozen-dried protective effect of mannitol, glucose is relatively poor, and it is better that several freeze drying protectants share the frozen-dried protective effect.

Different lyophilisation conditions are for the influence of lipidosome freeze-dried stability

In the pre-freeze process, employing cooling at a slow speed respectively (0.1～0.9 ℃/min), middling speed cooling (1.0～1.5 ℃/min), (20～50 ℃/min) mode is carried out pre-freeze to sample to fast cooling, stablizes 2h down at-50 ℃, adopt same lyophilizing program lyophilizing, change of granularity is all in 30nm after the sample rehydration that three kinds of pre-freeze modes prepare, and wherein the sample appearance for preparing in the pre-freeze mode of middling speed cooling is best, and rehydration is fastest, the change of granularity minimum, the drug leakage rate is minimum.As shown in Figure 7, particle size distribution after the freeze-dried lipidosome rehydration that three kinds of different pre-freeze modes prepare, wherein middling speed and quick freezing are less to liposome grain diameter influence, but because quick freezing is had relatively high expectations to the freeze dryer equipment performance, be unfavorable for suitability for industrialized production, so select the freezing pre-freeze condition of middling speed as liposome.

The research of freeze-dried lipidosome holding conditions

As shown in Figure 8, under the condition of different temperatures, the envelop rate after the freeze-dried lipidosome rehydration exists than big-difference, and the reserve temperature of freeze-dried lipidosome can not be greater than 20 ℃.

Contain of the influence of negative charge phospholipid to the stability of liposome

Contain during phospholipid is formed negative charge phospholipid liposome entrapment efficiency with do not contain negative charge phospholipid no significant difference, after the lyophilization rehydration liposome change of size less (± 20nm), but the negative charge liposome in vivo the half-life shorter.

As shown in Figure 9, it is less to contain before and after the prescription lyophilizing of negative charge phospholipid the change of size of liposome, and the liposome change of size is bigger before and after not containing the prescription lyophilizing of negative charge phospholipid.

As shown in figure 10, the epirubicin lipidosome injection that different phospholipid are formed was preserved 3 months under-20 ℃ of conditions, measure its mean diameter and envelop rate, the result shows that the phospholipid ratio is 85: 5: 5: it is 5: 1: 1 that 5 prescription stability obviously is better than the phospholipid ratio: 1 prescription.The experimental result of other anthracene nucleus medicinal liposome is similar to the epirubicin lipidosome injection.

Contain the PEGization phospholipid derivative for the stability influence of liposome in blood plasma

Shown in Figure 11～18, under 37 ℃ of conditions, the stability of the anthracene nucleus medicinal liposome that contains the PEGization phospholipid derivative in containing 50% blood-serum P BS buffer better, rate of release is slightly larger than the prescription that does not contain the PEGization phospholipid derivative under phase transition temperature.

Embodiment 31: anthracene nucleus medicinal liposome injection and normal injection rat body giving drugs into nose contrast for kinetic parameter

Laboratory animal

16 male SD rats, body weight 200-250g is divided into 4 groups at random, 10 every group.

A group: common anthracene nucleus medicinal liposome;

B group: temperature-sensitive anthracene nucleus medicinal liposome;

C group: the long circulation anthracene nucleus medicinal liposome of temperature-sensitive;

D group: anthracene nucleus medicament normal saline solution

The specification of all anthracycline drug pharmaceutical preparatioies: 2mg/ml

Dosage: 5mgkg ^-1

Blood sampling mode and sample treatment:

After administration 0.5,1,2,4,6,8,12,24 and 48h eyeground vein clump get blood 0.8mL, separated plasma, put in the centrifuge tube, the centrifugal 15min of 2500rpm places the test tube of label then, get 300 μ l, join 200mg, in the anti-phase C18 solid-phase extraction column, 2000rpm is centrifugal, adds 3ml and 1mlPBS successively and carries and wash (elute), is added the 20%triton-X100 solution supersound process 1min of 100 μ l filtrate merging, the interior mark that adds about 100 μ g by second SEP, adds 1mlPBS, discard filtrate, the methanol that adds twice 1.5ml is successively carried and being washed, and decompression volatilizes organic solvent, standby, (entrapped drug sample), as the Solid-Phase Extraction flow chart, see accompanying drawing 19:

The methanol that all adds twice 1.5ml with the rinse of 1mlPBS buffer in first SEP post before every SEP post sample introduction is carried and being washed, and adds doxorubincin about 100ng as internal standard substance, and decompression volatilizes organic solvent, standby (free drug sample)

Standard curve 1: the anthracene nucleus medicament standard curve that comprises in the liposome in the isolating blood plasma

Standard curve 2: free anthracene nucleus medicament standard curve in the isolating blood plasma

The drafting of standard curve 1:

Get the anthracene nucleus medicinal liposome of measuring total content and envelop rate, add blank plasma, make the concentration of its anthracene nucleus medicament be respectively 0.5,1,2,5,10,50,100,200 μ gmL ^-1,, in the liposome extracting solution that extracts parcel, add anthracene nucleus medicament (25 μ gmL by the operation of Solid-Phase Extraction method ^-1) as interior mark, the HPLC method detects.

The drafting of standard curve 2

Get blank plasma 300 μ L, it is 0.5,1,2,5 that the anthracene nucleus medicament that adds variable concentrations respectively makes the mass concentration of anthracene nucleus medicament, 10 and 50 μ gmL ^-1, by the SEP post, adding the interior mark anthracene nucleus medicament of same concentrations, the mass concentration of anthracene nucleus medicament is 25 μ gmL ^-1The HPLC method detects, drawing standard curve 2.

The response rate and precision

It is an amount of that precision takes by weighing DXR, prepares solution and the blood sample of 0.5,10 and 200 μ gmL-1 respectively, carries out HPLC and measure, and calculates the absolute recovery of DXR in the blood sample, calculates the anthracene nucleus medicament blood sample in a few days RSD, the RSD in the daytime that contain 0.5,10 and 200 μ gmL-1.

The HPLC-fluorescence detector is measured the blood drug level method.

Chromatographic condition:

Chromatographic column: anti-phase C18,4mm Nova-Pak Radial-Pak (Waters); Mobile phase: acetonitrile/0.01molL ^-1Ammonium biphosphate/water/glacial acetic acid (42: 28: 30: 0.1); Detect wavelength: excitation wavelength 488nm, emission wavelength 560nm; Flow velocity: 1mLmin ^-1

The result is as follows, sees Table 1,2,3,4

Table 1 daunorubicin hydrochloride liposome and epirubicin injection rat body giving drugs into nose are for kinetic parameter

Table 2 epirubicin hydrochloride liposome and epirubicin injection rat body giving drugs into nose are for kinetic parameter

Gentle star liposome of table 3 hydrochloric acid pyrrole and epirubicin injection rat body giving drugs into nose are for kinetic parameter

Table 4 hydrochloric acid darubicin liposome and epirubicin injection rat body giving drugs into nose are for kinetic parameter

Claims

1. anthracene nucleus medicinal liposome injection; it is characterized in that comprising synthetic neutral phospholipid, negative charge phospholipid, surfactant, PEGization phospholipid derivative, buffer agent, pH regulator agent, cryoprotective agent, the part by weight of wherein synthetic neutral phospholipid, negative charge phospholipid, surfactant and/or PEGization phospholipid derivative is 98: 1: 1: 0～40: 20: 20: 20.

2. lipidosome injection according to claim 1 is characterized in that the part by weight of synthetic neutral phospholipid, negative charge phospholipid, surfactant and/or PEGization phospholipid derivative is 98: 1: 1: 0～70: 10: 10: 10.

3. lipidosome injection as claimed in claim 1 or 2 is characterized in that described anthracene nucleus medicament comprises epirubicin, daunorubicin, doxorubicin, pirarubicin, darubicin and their hydrochlorate.

4. lipidosome injection as claimed in claim 1 or 2 is characterized in that described synthetic neutral phospholipid comprises dipalmitoyl phosphatidyl choline, distearoyl phosphatidylcholine or their mixture.

5. lipidosome injection as claimed in claim 1 or 2 is characterized in that described negative charge phospholipid comprises two palmityl phosphatidyl glycerols, distearyl phosphatidyl glycerol, two palmityl phosphatidic acid, G 12S3P or their mixture.

6. described lipidosome injection of claim 1 and freeze-dried powder comprise hydrolytic phosphatide, comprise single lauroyl phosphatidylcholine, single tridecanoyl phosphatidylcholine, single myristoyl phosphatidylcholine, single pentadecanoyl phosphatidylcholine, single palmitoylphosphatidyl choline, single heptadecanoyl phosphatidylcholine, single stearoyl phosphatidylcholine, single oleoyl phosphatidylcholine, the Dan Shijiu phosphatidyl choline, Dan Ershi phosphatidyl choline, single myristoyl phosphatidic acid, single palmityl phosphatidic acid, single stearoyl phosphatidic acid, single oleoyl phosphatidic acid, single myristoyl PHOSPHATIDYL ETHANOLAMINE, single palmityl phospholipid PHOSPHATIDYL ETHANOLAMINE, single stearoyl PHOSPHATIDYL ETHANOLAMINE, single oleoyl PHOSPHATIDYL ETHANOLAMINE, single myristoyl phosphatidyl glycerol, single palmityl phosphatidyl glycerol, single stearoyl phosphatidyl glycerol, single oleoyl phosphatidyl glycerol, single oleoyl Phosphatidylserine or their mixture.

7. lipidosome injection as claimed in claim 1 or 2 is characterized in that described PEGization phospholipid derivative comprises two polyethyleneglycol modified palmityl PHOSPHATIDYL ETHANOLAMINE, single palmityl PHOSPHATIDYL ETHANOLAMINE.

8. lipidosome injection as claimed in claim 1 or 2, it is characterized in that described buffer agent comprises citric acid-citrate, phosphate, tartrate, acetate, oxalates, lactate or their mixture, described pH regulator agent comprises sodium bicarbonate, sodium carbonate, sodium hydrogen phosphate, sodium hydroxide or their mixture.

9. lipidosome injection as claimed in claim 1 or 2; it is characterized in that described cryoprotective agent comprises saccharide, hydrophilic polymer or their mixture; wherein saccharide comprises trehalose, lactose, sucrose, mannitol, glucose, maltose, glycerol, and hydrophilic polymer comprises polyvinylpyrrolidone PVP.

10. the preparation method of lipidosome injection as claimed in claim 1 or 2 comprises the preparation method of injection and freeze-dried powder, and its feature is as follows:

The preparation method of the lipidosome injection of anthracene nucleus medicament comprises the following step: 1) preparation blank liposome: select phospholipid for use, be dissolved in mix homogeneously in chloroform or the chloroform-methanol mixed solvent according to prescription; With Rotary Evaporators solvent decompression in the solution is removed, form maybe will the write out a prescription phospholipid of composition of lipid membrane and be dissolved in the organic solvent, low temperature spray drying obtains the mixture of phospholipid; Buffer agent is mixed with the buffer of 50～500mM pH=4, comes aquation lipid membrane or mixture of phospholipids with buffer, hydration temperature generally between 40 ℃～70 ℃, blank liposome suspension; 2) liposome that homogenizes: aquation back fully prepares liposome to the required particle diameter and the uniformity with high pressure homogenizer, also can reach the blank liposome suspension pushes through respective aperture under pressure microporous membrane by extrusion equipment, the particle diameter of liposome is controlled at 10nm～1000nm, be preferably 30～300nm, more preferably 50～250nm.3) liposome of preparation anthracycline-containing drug salts hydrochlorate: it is 1%～20% sugar aqueous solution that the anthracene nucleus medicament hydrochlorate is dissolved in water for injection or concentration, is heated to 30 ℃～40 ℃; The aqueous solution of the pH regulator agent of preparation 10mM～1000mM adds in the blank liposome suspension with certain proportion, regulates pH value to 7～9; With anthracene nucleus medicament hydrochlorate solution and blank liposome suspension mix homogeneously and at 30 ℃～45 ℃ insulation 10～100min down, once by a certain percentage every the 5min jolting; 4) preparation anthracene nucleus medicament hydrochlorate liposome turbid liquor, be added to the weight percent proportion by subtraction outside and be 3%～20% sugar or sugar aqueous solution, mix homogeneously, make the concentration of foreign minister's sugar in the liposome equal, get Liposomal dispersion, through the microporous filter membrane filtration of 0.22 μ m near obtaining, fill, 5) gland, sealing is preserved, and gets anthracene nucleus medicament thermal sensitive liposome injection;

The preparation method of freeze-dried powder comprises the following step: 1) preparation blank liposome: select phospholipid for use, be dissolved in mix homogeneously in chloroform or the chloroform-methanol mixed solvent according to prescription; With Rotary Evaporators solvent decompression in the solution is removed, form maybe will the write out a prescription phospholipid of composition of lipid membrane and be dissolved in the organic solvent, low temperature spray drying obtains the mixture of phospholipid; Buffer agent is mixed with the buffer of 50～500mM pH=4, comes aquation lipid membrane or mixture of phospholipids with buffer, hydration temperature generally between 40 ℃～70 ℃, blank liposome suspension; 2) liposome that homogenizes: aquation back fully prepares liposome to the required particle diameter and the uniformity with high pressure homogenizer, also can reach the blank liposome suspension pushes through respective aperture under pressure microporous membrane by extrusion equipment, the particle diameter of liposome is controlled at 10nm～1000nm, be preferably 30～300nm, more preferably 50～250nm.3) liposome of preparation anthracycline-containing drug salts hydrochlorate: it is 1%～20% sugar aqueous solution that the anthracene nucleus medicament hydrochlorate is dissolved in water for injection or concentration, is heated to 30 ℃～40 ℃; The aqueous solution of the pH regulator agent of preparation 10mM～1000mM adds in the blank liposome suspension with certain proportion, regulates pH value to 7～9; With anthracene nucleus medicament hydrochlorate solution and blank liposome suspension mix homogeneously and at 30 ℃～45 ℃ insulation 10～100min down, once by a certain percentage every the 5min jolting; 4) preparation anthracene nucleus medicament hydrochlorate liposome turbid liquor, be added to the weight percent proportion by subtraction outside and be 3%～20% the sugar or the PVP of sugar aqueous solution and 1～10%, mix homogeneously, make the concentration of foreign minister's sugar in the liposome equal near obtaining, get Liposomal dispersion, microporous filter membrane through 0.22 μ m filters fill, lyophilizing, the pre-freeze temperature should preserved 2 hours below-45 ℃, first section dry employing :-50 ℃ of 1～5h ,-35 ℃ of 8～20h ,-25 ℃ of 5～10h,-10 ℃ of 2～6h, 0 ℃ of 1～3h, 20 ℃ of 5～15h of second section drying obtain injection anthracene nucleus medicament hydrochlorate liposome; 5) gland, sealing is preserved, and gets finished product.