CN103040764B - Bleomycin hydrocloride lipidosome injection - Google Patents
Bleomycin hydrocloride lipidosome injection Download PDFInfo
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Abstract
The invention discloses a bleomycin hydrocloride lipidosome injection and a preparation method thereof. The lipidosome injection is prepared by using bleomycin hydrocloride, cholesteryl succinate, distearyl phosphatidyl ethanolamine, polyoxyethylene 40 hydrogenated castor oil, trehalose and mannitol. The bleomycin hydrocloride lipidosome injection disclosed by the invention has the advantage of appropriate particle size, uniform distribution, good stability, higher encapsulation efficiency and lower leaking rate; and the preparation method disclosed by the invention has the advantage of good reproducibility and is suitable for large-scale industrial production.
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 Bleomycin A5 hydrochloride. lipidosome injection and method for making thereof, belong to medical technical field.
Background technology
Hemangioma is the modal benign tumors of the mankind, and the hemangioma of liver is one of modal benign tumor.Bleomycin A5 belongs to the antitumor antibiotic of wide spectrum, by Pingyang mycete, is produced, and main component is bleomycin A
5, can suppress thymidine and enter DNA and discharge free core alkali, and make it to decompose and destroy, thereby suppress the synthetic of DNA and cut off DNA chain, the schizogamy of interference cell, affect cellular metabolism function, the necrosis of promotion cellular change.Bleomycin A5 is that one is safer, and side effect is little, to hemopoietic system and function of immune system substantially without the new type antineoplastic medicine of injury.
Bleomycin A5 hydrochloride. is white crystalline powder, easily molten in water or in methanol, and slightly soluble in ethanol is almost insoluble in acetone, chloroform/chloroform or ether.Bleomycin A5 hydrochloride. chemical name is N ＇-[3-[(4-aminobutyl) amino] propyl group] bleomycin amide hydrochloride, molecular formula: C
57h
89n
19o
21s
2nHCl, structural formula is as follows:
Bleomycin A5 hydrochloride. be the quality inspection standard recorded in bis-of version < < pharmacopeia > > in 2005 kind (with octadecylsilane chemically bonded silica be filler; Take sodium pentanesulfonate solution-methyl alcohol-acetonitrile (76: 16: 8) as mobile phase, content by external standard method with Bleomycin A5 hydrochloride. in calculated by peak area test sample), the Bleomycin A5 hydrochloride. dosage form of listing is mainly freeze-dried powder at present, the Bleomycin A5 hydrochloride. injection 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, brings toxic and side effects, has left hidden danger to clinical use.
Patent CN1391901A discloses a kind of angiomatous injection for the treatment of containing Bleomycin A5, and it is comprised of Bleomycin A5,0.5% lignocaine and dexamethasone, and wherein Bleomycin A5 accounts for 3～10mg, 0.5% lignocaine 3～10ml, dexamethasone 1～5mg.At compatibility of drugs, mix under service condition, its stability is bad, and bioavailability is also not high enough.
The open one of CN101284127A is treated angiomatous injection and preparation method thereof.Described compositions is organic formulated by Bleomycin A5, interferon, dexamethasone, epinephrine, lignocaine and water for injection, but the safety issue of multiple medicines thing can not be guaranteed, and causes cost to increase.
CN101015504A discloses a kind of suppository and preparation process thereof for the treatment of adenomyosis.Said preparation preparation process is complicated and use inconvenience, can only in operation, use, and safety is not high.
CN101536987A discloses a kind of containing anti-angiogenic tumor medicine sodium alginate micro ball vascular embolism agent and its preparation method and application, it is characterized by the described anti-angiogenic tumor medicine of sodium alginate parcel, ratio is 5: 1-60: 1, described anti-angiogenic tumor medicine is Bleomycin A5, sodium morrhuate, carbamide or bleomycin, the stability of this medicine is good not, and particle diameter and encapsulation ratio be difficult to measure and control, and causes bioavailability low, and preparation process is too complicated, be not suitable for the large production of industry.
CN200980155850A discloses a kind of vesicle formation, utilize serum composition vitamin E to be transported to the character of hepatic parenchymal cells, by making vitamin E absorption or chemical bond in vesicle surface, thereby biological active substances contained in vesicle effectively and specifically can be transported to hepatic parenchymal cells.
In targeting drug delivery system, liposome preparation technology gradual perfection, liposome mechanism of action is further illustrated, Liposomal formulation has the following advantages: (1) has slow releasing function: active component slowly discharges, delay renal excretion and metabolism, thereby extend action time, improve mass effect; (2) reduce drug toxicity; (3) dissolubility of increase medicine, improves the quality of the pharmaceutical preparations; (4) there is targeting: the contained medicine of liposome maintains high concentration in liver, spleen reticuloendothelial system internal organs part, thereby plays the effect of medicine organ targeting; (5) there is the protective effect to active pharmaceutical ingredient.
He Fuchang, Bleomycin A5 magnetic liposome is to anti-tumor effect research in the body of people's mucoepidermoid carcinoma, < < Practical oral medical journal > >, 02 phase in 1997,108-110 page, wherein disclose and passed through the Bleomycin A5 magnetic liposome of development people's mucoepidermoid carcinoma anti-tumor in vivo Functional observation, evaluate its feasibility as the novel carriers of targeted therapy tumor, but do not disclose the composition of Bleomycin A5 magnetic liposome.
Due to the deficiency that Bleomycin A5 hydrochloride. injection exists, for Bleomycin A5 hydrochloride. lipidosome injection, there is demand at present, especially need the Bleomycin A5 hydrochloride. lipidosome injection of good quality.
Summary of the invention
In fact, 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, and all these existing absolutely not theories can be expected and solved, and need to overcome many technical barriers.Therefore need to find by every means liposome prescription, to obtain the Bleomycin A5 hydrochloride. lipidosome injection of excellent in stability.
Generally speaking, for most liposomal pharmaceutical preparation, the pharmaceutical properties of wrapped medicine mainly lipid, consists of and concentration is controlled.In order to form colory Bleomycin A5 hydrochloride. lipidosome injection, thereby importantly find, can good compatible with Bleomycin A5 hydrochloride. 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 Bleomycin A5 hydrochloride. of specified weight proportioning, cholesterol succinate, DSPE, polyoxyl 40 hydrogenated castor oil, mannitol and trehalose can be made Bleomycin A5 hydrochloride. lipidosome injection, compared with Bleomycin A5 hydrochloride. injection of the prior art, wherein high as the envelop rate of the Bleomycin A5 hydrochloride. of active constituents of medicine and slip is low, 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, biocompatibility and the bioavailability of medicine obviously improve, thereby obviously improved curative effect.
On the one hand, the invention provides a kind of Bleomycin A5 hydrochloride. lipidosome injection, it is mainly made by the composition of following weight proportion:
Preferably, the weight ratio between cholesterol succinate and DSPE is 1: 4.
Further preferably, Bleomycin A5 hydrochloride. lipidosome injection of the present invention is mainly made by the composition of following weight proportion:
Preferably, the weight ratio between cholesterol succinate and DSPE is 1: 4.
Usually, as the phospholipid that is used to form liposome, can use natural phospholipid and synthetic phospholipid.Preparing the membrane material that lipidosome injection is conventional is phospholipid and additives, wherein phospholipid can be selected Ovum Gallus domesticus Flavus lecithin conventionally, hydrogenation egg yolk lecithin, EPG, egg yolk lecithin acyl serine, PI, soybean lecithin, hydrogenated soya phosphatide, soybean phospholipid acyl glycerol, soy phosphatidylserine, soybean phospholipid acyl inositol, DOPC, DSPC, dipalmitoyl phosphatidyl choline, dimyristoyl phosphatidyl choline, DLPC, DOPG, DSPG, DPPG, GLYCEROL,DIMYRISTOYL PHOSPHATIDYL, one or more in PE.The membrane material of conventional additives has cholesterol, 18-amine., phosphatidic acid etc.For the preparation of the membrane material of lipidosome injection, also have PHOSPHATIDYL ETHANOLAMINE, cholesterol second fat, paddy to carry alcohol, natrii tauroglycocholas, phosphatidyl silk amino acid, stearmide, single stearoyl phosphatidic acid, single stearoyl PHOSPHATIDYL ETHANOLAMINE, two cetyl phosphate (DCP), DPPE, single palmityl PHOSPHATIDYL ETHANOLAMINE, two myristoyl PHOSPHATIDYL ETHANOLAMINE.Additives be generally used for regulating membrane structure and or change charged character, as cholesterol can make liposome bi-layer membrane, solidify, thereby reduce the generation of free radical, reduced oxidation level, liposome stability is significantly strengthened.The inventor surprisingly finds when a large amount of screening experiment, during any membrane material outside using the combination of DSPE and cholesterol succinate, all do not obtain colory liposome, gained liposome is under the accelerated test of 40 ℃ of high temperature, relative humidity 75% ± 5%, and the character such as envelop rate, stability and percolation ratio are poor.
In the present invention, for the feature of Bleomycin A5 hydrochloride., the inventor is particularly suitable for as Bleomycin A5 hydrochloride. basis phospholipid filmogen by studying unexpected discovery DSPE together with cholesterol succinate.DSPE easily obtains, cheap, and phase transition temperature is higher, is easy to form stable liposome membrane.In Bleomycin A5 hydrochloride. lipidosome injection of the present invention, for the Bleomycin A5 hydrochloride. of 4 weight portions, the consumption of DSPE is 20-40 weight portion.If the consumption of DSPE, lower than 20 weight portions, cannot form stable liposome; Otherwise if the consumption of the consumption of DSPE, higher than 40 weight portions, declines as the envelop rate of the Bleomycin A5 hydrochloride. of active constituents of medicine, the quality of injection and curative effect reduce.
In Bleomycin A5 hydrochloride. lipidosome injection of the present invention, cholesterol succinate and polyoxyl 40 hydrogenated castor oil are worked in coordination with membrane stability and the permeability for regulating liposome, regulate the releasing properties of Bleomycin A5 hydrochloride..
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, stops it to be condensed into crystal structure.Cholesterol succinate mixes DSPE 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.
In Bleomycin A5 hydrochloride. lipidosome injection of the present invention, for the Bleomycin A5 hydrochloride. of 4 weight portions, the better consumption of cholesterol succinate is 5-10 weight portion.The inventor finds through research, when the weight ratio between cholesterol succinate and DSPE is 1: 4, can form Bleomycin A5 hydrochloride. liposome the best in quality, and the liposome toxicity forming is also minimum.
On the other hand, in liposome of the present invention, cholesterol succinate is the preferred negatively charged membrane material of the present invention, be particularly suitable for as basic phospholipid filmogen, effectively increased the absolute value of the Zeta potential of surface of liposome, the electrostatic repulsion of surface of liposome is increased, effectively suppress the gathering of liposome.In freeze-dried powder preparation process, the pre-freeze stage, liposome was constantly concentrated along with the outer water ice of liposome is separated out, and the trend of gathering increases, and adding negative charge phospholipid can effectively stop the now gathering of liposome, after assurance lyophilizing rehydration, liposome particle diameter is stable within the specific limits.In liposome of the present invention, its Zeta potential is-10～-60mV, is preferably-20～-40mV, be about-30mV of the Zeta potential of the embodiment of the present invention.
Research shows, the stability of liposome and bioavailability have close corresponding relation.Stability is higher, and bioavailability is higher.Therefore, the stability of Bleomycin A5 hydrochloride. 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 Bleomycin A5 hydrochloride. lipidosome injection of the present invention, for the Bleomycin A5 hydrochloride. of 4 weight portions, the consumption of DSPE is 20-40 weight portion, cholesterol succinate is 5-10 weight portion, polyoxyl 40 hydrogenated castor oil is 6-8 weight portion, and the weight ratio between cholesterol succinate and DSPE is 1: 4 o'clock, can obtain colory Bleomycin A5 hydrochloride. liposome, its envelop rate and stability are all very high, and toxicity is low, and bioavailability is high.
In Bleomycin A5 hydrochloride. lipidosome injection of the present invention, further improve its pharmaceutical properties with polyoxyl 40 hydrogenated castor oil.Polyoxyl 40 hydrogenated castor oil is a kind of non-ionic surface active agent, when for liposome, can improve the chemical energy between film, thereby improves its chemical stability in waterborne liquid, and then improves the stability of injection.
In Bleomycin A5 hydrochloride. lipidosome injection of the present invention, use trehalose and mannitol as excipient, be further used for forming stable injection.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 Bleomycin A5 hydrochloride. 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.Mannitol is being pharmaceutically good diuretic, reduces the diluent of intracranial pressure, intraocular pressure and treatment kidney medicine, dehydrant, sugar succedaneum, the excipient that is also used as tablet and solid, liquid, especially can support for lyophilized injectable powder provide good molding.
Bleomycin A5 hydrochloride. lipidosome injection of the present invention, the specification of its Bleomycin A5 hydrochloride. (take Bleomycin A5) can be 4mg or 8mg, its Zeta potential is-20～-40mV.Its mean diameter of Bleomycin A5 hydrochloride. lipidosome injection of the present invention is 120nm～160nm.
On the other hand, the present invention also provides a kind of preparation method of Bleomycin A5 hydrochloride. lipidosome injection, specifically comprises and is prepared as follows step:
(1) under approximately 50 ℃ of slow magnetic agitation, cholesterol succinate, DSPE and polyoxyl 40 hydrogenated castor oil are dissolved in organic solvent, obtain lipid soln, by above-mentioned solution lyophilization, until completely dry, obtain forming loose spongiform drying solid;
(2) Bleomycin A5 hydrochloride. is dissolved in buffer solution, adds trehalose, water stirs to obtain;
(3) at approximately 25 ℃, under slow magnetic agitation, the solid of step 1 is added in the water of step 2, then at 600bar, to 900bar, do gradient homogenizing 4～6 times, 0.22 μ m filtering with microporous membrane, makes Bleomycin A5 hydrochloride. liposome;
(4) under nitrogen protection; the Bleomycin A5 hydrochloride. liposome that step (3) is made; with pH adjusting agent adjusting pH value to 7.0～9.0; 60 ℃ of water-baths 10～15 minutes; then cool the temperature to 20～30 ℃ and add mannitol, 0.22 μ m filtering with microporous membrane, fill under constantly stirring; then carry out lyophilizing, obtain Bleomycin A5 hydrochloride. lipidosome injection.
In the one side of preparation method invention, described organic solvent is selected from one or more in ethanol, chloroform, dichloromethane, methanol, n-butyl alcohol, isopropyl alcohol, acetone, benzyl alcohol, the tert-butyl alcohol, normal hexane, and being preferably volume ratio is the ethanol of 1:3 and the mixed organic solvents of isopropyl alcohol.
In the one side of preparation method invention, described in step (1), cryodesiccated process was :-50～-60 ℃ of pre-freezes 2～4 hours, then-40 ℃～-50 ℃ freezing 10～13 hours, then through 12～15 hours, sublime up into 20 ℃～25 ℃, finally 30 ℃～35 ℃ dry 6～8 hours.
In the one side of preparation method invention, described buffer solution is selected from one or more in phosphate buffer, acetate buffer, citrate buffer, borate buffer solution and carbonate buffer solution, is preferably pH value and is potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer of 5.8.
In the one side of preparation method invention, described pH adjusting agent can be potassium carbonate, potassium bicarbonate, potassium phosphate or potassium hydroxide etc., is preferably potassium hydroxide.
On the one hand preferred, the present invention also provides a kind of preparation method of Bleomycin A5 hydrochloride. lipidosome injection, specifically comprises and is prepared as follows step:
(1) cholesterol succinate, DSPE and polyoxyl 40 hydrogenated castor oil being dissolved in to volume ratio under approximately 50 ℃ of slow magnetic agitation is in the ethanol of 1:3 and the mixed organic solvents of isopropyl alcohol, obtain lipid soln, by above-mentioned solution-50～-60 ℃ of pre-freezes 2～4 hours, then freezing 10～13 hours at-40 ℃～-50 ℃, through 12～15 hours, sublime up into 20 ℃～25 ℃ again, finally at 30 ℃～35 ℃, be dried 6～8 hours, obtain forming loose spongiform drying solid;
(2) Bleomycin A5 hydrochloride. being dissolved in to pH value is, in potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer of 5.8, to add trehalose, and water stirs to obtain;
(3) at approximately 25 ℃, under slow magnetic agitation, the solid of step 1 is added in the water of step 2, then at 600bar, to 900bar, do gradient homogenizing 4～6 times, 0.22 μ m filtering with microporous membrane, makes Bleomycin A5 hydrochloride. liposome;
(4) under nitrogen protection; the Bleomycin A5 hydrochloride. liposome that step (3) is made; with potassium hydroxide adjusting pH value to 7.0～9.0; 60 ℃ of water-baths 10～15 minutes; then cool the temperature to 20～30 ℃ and add mannitol, 0.22 μ m filtering with microporous membrane, fill under constantly stirring; then carry out lyophilizing, obtain Bleomycin A5 hydrochloride. 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, by selecting suitable material composition, adopting suitable preparation technology, has obtained colory Bleomycin A5 hydrochloride. lipidosome injection, and liposome particle diameter is little, mean diameter is 120nm～160nm, particle size distribution is even, and envelop rate is high, and stability is high.
Lipidosome injection of the present invention has reduced toxic and side effects, improved formulation products quality, had good preparation stability, in refrigerating process, liposome can not break because of dehydration, fusion, ice crystal etc., after long term storage, percolation ratio is low, and liposome keeps good envelop rate equally.
The Bleomycin A5 hydrochloride. lipidosome injection making by the inventive method, has increased the retention time of medicine in body circulation, has improved the bioavailability of medicine, has reduced toxic and side effects, and preparation method favorable reproducibility, is suitable for industrialized great production.
Accompanying drawing explanation
Fig. 1 is the blood drug level-time graph of Bleomycin A5 hydrochloride. lipidosome injection, for the injection of preparation in embodiment 1,3,4 and comparative example 1,3 and 4, the average blood drug level of 0.5h, 1h, 2h, 4h, 6h, 8h, 10h, 12h, 16h and 24h and the relation curve of time after administration.
Wherein:
The specific 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 Bleomycin A5 hydrochloride. lipidosome injection
Prescription (1000 bottles) composition used and weight thereof are as follows:
Adopt preparation technology to prepare Bleomycin A5 hydrochloride. lipidosome injection:
(1) under approximately 50 ℃ of slow magnetic agitation, 10g cholesterol succinate, 40g DSPE and 8g polyoxyl 40 hydrogenated castor oil being dissolved in to 200ml volume ratio is in the ethanol of 1:3 and the mixed organic solvents of isopropyl alcohol, obtain lipid soln, by above-mentioned solution-50 ℃ of pre-freezes 2 hours, then freezing 10 hours at-40 ℃, through 12 hours, sublime up into 20 ℃ again, finally at 30 ℃, be dried 6 hours, obtain forming loose spongiform drying solid;
(2) 4g Bleomycin A5 hydrochloride. being dissolved in to 1000ml pH value is, in potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer of 5.8, to add 10g trehalose, and water stirs to obtain;
(3) at approximately 25 ℃, under slow magnetic agitation, the solid of step 1 is added in the water of step 2, then at 600bar, to 900bar, do gradient homogenizing 4 times, 0.22 μ m filtering with microporous membrane, makes Bleomycin A5 hydrochloride. liposome;
(4) under nitrogen protection; the Bleomycin A5 hydrochloride. liposome that step (3) is made; with potassium hydroxide adjusting pH value to 8.0; 60 ℃ of water-baths 10 minutes; then cool the temperature to 20 ℃ and add 30g mannitol, 0.22 μ m filtering with microporous membrane, fill under constantly stirring; then carry out lyophilizing, obtain Bleomycin A5 hydrochloride. lipidosome injection.
the preparation of embodiment 2 Bleomycin A5 hydrochloride. lipidosome injections
Prescription (1000 bottles) composition used and weight thereof are as follows:
Adopt preparation technology to prepare Bleomycin A5 hydrochloride. lipidosome injection:
(1) under approximately 50 ℃ of slow magnetic agitation, 7.5g cholesterol succinate, 30g DSPE and 7g polyoxyl 40 hydrogenated castor oil being dissolved in to 200ml volume ratio is in the ethanol of 1:3 and the mixed organic solvents of isopropyl alcohol, obtain lipid soln, by above-mentioned solution-60 ℃ of pre-freezes 4 hours, then freezing 13 hours at-50 ℃, through 15 hours, sublime up into 25 ℃ again, finally at 35 ℃, be dried 8 hours, obtain forming loose spongiform drying solid;
(2) 4g Bleomycin A5 hydrochloride. is dissolved in potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer that the pH value of 1000ml is 5.8, adds 8g trehalose, water stirs to obtain;
(3) at approximately 25 ℃, under slow magnetic agitation, the solid of step 1 is added in the water of step 2, then at 600bar, to 900bar, do gradient homogenizing 6 times, 0.22 μ m filtering with microporous membrane, makes Bleomycin A5 hydrochloride. liposome;
(4) under nitrogen protection; the Bleomycin A5 hydrochloride. liposome that step (3) is made; with potassium hydroxide adjusting pH value to 9.0; 60 ℃ of water-baths 15 minutes; then cool the temperature to 30 ℃ and add 20g mannitol, 0.22 μ m filtering with microporous membrane, fill under constantly stirring; then carry out lyophilizing, obtain Bleomycin A5 hydrochloride. lipidosome injection.
the preparation of embodiment 3 Bleomycin A5 hydrochloride. lipidosome injections
Prescription (1000 bottles) composition used and weight thereof are as follows:
Adopt preparation technology to prepare Bleomycin A5 hydrochloride. lipidosome injection:
(1) under approximately 50 ℃ of slow magnetic agitation, 10g cholesterol succinate, 40g DSPE and 12g polyoxyl 40 hydrogenated castor oil being dissolved in to 200ml volume ratio is in the ethanol of 1:3 and the mixed organic solvents of isopropyl alcohol, obtain lipid soln, by above-mentioned solution-55 ℃ of pre-freezes 3 hours, then freezing 11 hours at-45 ℃, through 13 hours, sublime up into 23 ℃ again, finally at 33 ℃, be dried 7 hours, obtain forming loose spongiform drying solid;
(2) 8g Bleomycin A5 hydrochloride. is dissolved in potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer that the pH value of 1000ml is 5.8, adds 12g trehalose, water stirs to obtain;
(3) at approximately 25 ℃, under slow magnetic agitation, the solid of step 1 is added in the water of step 2, then at 600bar, to 900bar, do gradient homogenizing 5 times, 0.22 μ m filtering with microporous membrane, makes Bleomycin A5 hydrochloride. liposome;
(4) under nitrogen protection; the Bleomycin A5 hydrochloride. liposome that step (3) is made; with potassium hydroxide adjusting pH value to 7.0; 60 ℃ of water-baths 14 minutes; then cool the temperature to 25 ℃ and add 20g mannitol, 0.22 μ m filtering with microporous membrane, fill under constantly stirring; then carry out lyophilizing, obtain Bleomycin A5 hydrochloride. lipidosome injection.
the preparation of embodiment 4 Bleomycin A5 hydrochloride. lipidosome injections
Prescription (1000 bottles) composition used and weight thereof are as follows:
Adopt preparation technology to prepare Bleomycin A5 hydrochloride. lipidosome injection:
(1) under approximately 50 ℃ of slow magnetic agitation, 6g cholesterol succinate, 24g DSPE and 7g polyoxyl 40 hydrogenated castor oil being dissolved in to 200ml volume ratio is in the ethanol of 1:3 and the mixed organic solvents of isopropyl alcohol, obtain lipid soln, by above-mentioned solution-55 ℃ of pre-freezes 3 hours, then freezing 12 hours at-40 ℃, through 14 hours, sublime up into 25 ℃ again, finally at 35 ℃, be dried 6 hours, obtain forming loose spongiform drying solid;
(2) 4g Bleomycin A5 hydrochloride. is dissolved in potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer that the pH value of 1000ml is 5.8, adds 9g trehalose, water stirs to obtain;
(3) at approximately 25 ℃, under slow magnetic agitation, the solid of step 1 is added in the water of step 2, then at 600bar, to 900bar, do gradient homogenizing 4～6 times, 0.22 μ m filtering with microporous membrane, makes Bleomycin A5 hydrochloride. liposome;
(4) under nitrogen protection; the Bleomycin A5 hydrochloride. liposome that step (3) is made; with potassium hydroxide adjusting pH value to 7.7; 60 ℃ of water-baths 15 minutes; then cool the temperature to 30 ℃ and add 18g mannitol, 0.22 μ m filtering with microporous membrane, fill under constantly stirring; then carry out lyophilizing, obtain Bleomycin A5 hydrochloride. lipidosome injection.
the preparation of comparative example 1-4 Bleomycin A5 hydrochloride. lipidosome injection
It should be noted that, inventor is in trial test, once adopted reverse-phase evaporation one freeze-thaw method to prepare persalt Bleomycin A5 liposome: to take lecithin 9.5g, cholesterol 1.0g, vitamin E 50mg, after adding chloroform and making it to dissolve completely, to contain Bleomycin A5 hydrochloride. 0.4g, vitamin C 15mg, appropriate phosphate buffer (PBS, the pH7.4) 40ml of PVP slowly adds wherein, adds Arlacel-80 2ml simultaneously, be interrupted ultrasonic 5min complete to emulsifying, on Rotary Evaporators, remove organic facies, form gelling material, put room temperature after 4 ℃ of refrigerator freezings and melt.Repeat 3 times, obtained not good Bleomycin A5 hydrochloride. liposome, its mean diameter is 685.24nm, and envelop rate is about 70%, and percolation ratio is obviously about 8%.Use this prescription instead in embodiment 1-3 similar production technology, the character such as its envelop rate, percolation ratio, particle diameter are all undesirable.
The now exemplary part comparative example that provides proved invention combination and have synergistic action effect only, but should not be construed as limitation of the present invention, adopt respectively and production technology identical in embodiment 1-4, the composition in comparative example 1-4 is as shown in Table 1 below made respectively to Bleomycin A5 hydrochloride. lipidosome injection.
Composition used in table 1 comparative example 1-4
Wherein, "/" represents not use.
Particularly, comparative example's 1 use cholesterol replaces cholesterol succinate, replaces polyoxyl 40 hydrogenated castor oil by CREMOPHORE EL; Comparative example's 2 use DOPEs replace DSPE, and with trehalose replacement mannitol and trehalose, interior foreign minister is trehalose; Comparative example's 3 use distearoyl phosphatidylcholine replace DSPE, replace polyoxyl 40 hydrogenated castor oil with Tween 80; Comparative example's 4 use soyasterols replace cholesterol succinate, and with mannitol replacement mannitol and trehalose, interior foreign minister is mannitol.
By test example, contrast below, confirmed that the combination of composition of the present invention has synergistic effect, the invention provides the Bleomycin A5 hydrochloride. lipidosome injection of excellent quality.
the mensuration of test example 1 liposome particle diameter
Under room temperature condition, get the Bleomycin A5 hydrochloride. lipidosome injection in embodiment 1-4 and comparative example 1-4, be placed in the sample cell of Submicron Particle Sizer Model 370 particle diameter detectors, 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 1-4 makes is even, aobvious spherical, big or small homogeneous; The liposome particle size distribution that comparative example 1-4 makes is inhomogeneous, and shape is indefinite, not of uniform size.
Particularly, even when adopting same production technology, in embodiment 1-4, the particle appearance of gained Bleomycin A5 hydrochloride. liposome and size thereof and distribution are obviously better than the Bleomycin A5 hydrochloride. liposome of gained in comparative example 1-4.When the composition combination 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 Bleomycin A5 hydrochloride. liposome is inferior to the present invention, and mean diameter obviously goes out greatly a lot.
the mensuration of test example 2 envelop rates
Rotating speed high speed centrifugation by the Bleomycin A5 hydrochloride. lipidosome injection of preparing in embodiment 1-4 and comparative example 1-4 with 5000r/min, centrifugal 20 minutes, gets supernatant, with dissolve with methanol, HPLC method is surveyed Bleomycin A5 hydrochloride. 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 1-4 is significantly higher than the envelop rate of the Liposomal formulation of comparative example 1-4.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.
test example 3 study on the stability
Sample prepared by embodiment of the present invention 1-4 and listing hydrochloride for injection Bleomycin A5 freeze-dried powder (lot number: 20110224, Beijing XieHe medicine Factory) 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, the content of listing preparation and comparative example 1-4 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.
test example 4 percolation ratio tests
Get sample prepared by test example 1-4 and comparative example 1-4, 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 Bleomycin A5 hydrochloride. lipidosome injection percolation ratio of preparing in embodiment of the present invention 1-4 changes little, and the injection percolation ratio of preparing in comparative example 1-4 increases gradually, liposome seepage is serious, and Bleomycin A5 hydrochloride. lipidosome injection prepared by this explanation the present invention has higher stability.
the mensuration of test example 5 blood drug level
56 rats are divided into 7 groups at random, distinguish the injection of preparation in drug administration by injection embodiment 1,3,4 and comparative example 1,3 and 4 for every group, and commercially available Bleomycin A5 hydrochloride. injection (lot number: 20110224, Beijing XieHe medicine Factory), injection volume is 0.2g Bleomycin A5 hydrochloride. (in Bleomycin A5).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.The blood drug level of Bleomycin A5 hydrochloride. lipidosome injection and the relation curve of time in drafting embodiment 1,3,4, in the Bleomycin A5 hydrochloride. lipidosome injection of preparation, comparative example 1,3-4, prepared, be shown in accompanying drawing 1, due to the rapid metabolism of commercially available Bleomycin A5 hydrochloride. injection, 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, compare with commercially available Bleomycin A5 hydrochloride. lipidosome injection with the Bleomycin A5 hydrochloride. lipidosome injection of preparation in comparative example 1,3 and 4, in the embodiment of the present invention 1,3 and 4, the Bleomycin A5 hydrochloride. lipidosome injection of preparation also has the following advantages: release rate in vivo slows down, in body circulation, distribution time extends, reach improved slow release effect, increased bioavailability.
Although not being single factor in test example 1-5, comparative example 1-4 investigates its impact (description description is shown in monofactorial impact), but comparative example 1-4 has confirmed that from different aspect embodiment of the present invention 1-4, preparing lipidosome injection has unexpected effect, and combination of components of the present invention has synergism.Particularly, the result that comparative example 1-4 shows in test example 1-5 shows that comparative example 1-4 respectively has feature each other, be difficult to determine which kind of combination has comprehensive effect of optimization, but be all obviously not so good as the lipidosome injection of embodiment 1-4, the lipidosome injection that embodiment 1-4 is described has excellent galenic pharmacy feature, obtain unexpected technique effect, solved galenic pharmacy technical problem.
industrial applicibility
From the result of above-described embodiment and experimental example, Bleomycin A5 hydrochloride. liposome of the present invention has good outward appearance, and granule is little, and particle diameter is even, envelop rate is high, stability is high, and percolation ratio is low, and the time of staying is in vivo long, bioavailability is high, preparation method favorable reproducibility of the present invention, is suitable for industrialized great production, 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 (9)
1. a Bleomycin A5 hydrochloride. lipidosome injection, its composition by following weight proportion is made:
And the weight ratio between cholesterol succinate and DSPE is 1: 4.
2. Bleomycin A5 hydrochloride. lipidosome injection according to claim 1, the specification that it is characterized in that Bleomycin A5 hydrochloride. is 4mg or 8mg, its Zeta potential is-10～-60mV.
3. Bleomycin A5 hydrochloride. lipidosome injection according to claim 2, is characterized in that its Zeta potential is-20～-40mV.
4. Bleomycin A5 hydrochloride. lipidosome injection according to claim 2, is characterized in that its Zeta potential is-30mV.
5. a method of preparing the Bleomycin A5 hydrochloride. lipidosome injection described in any one in claim 1-4, is characterized in that comprising the steps:
(1) under 50 ℃ of slow magnetic agitation, cholesterol succinate, DSPE and polyoxyl 40 hydrogenated castor oil are dissolved in organic solvent, obtain lipid soln, by above-mentioned solution lyophilization, until completely dry, obtain forming loose spongiform drying solid;
(2) Bleomycin A5 hydrochloride. is dissolved in buffer solution, adds trehalose, water stirs to obtain;
(3) at 25 ℃, under slow magnetic agitation, the solid of step 1 is added in the water of step 2, then at 600bar, to 900bar, do gradient homogenizing 4～6 times, 0.22 μ m filtering with microporous membrane, makes Bleomycin A5 hydrochloride. liposome;
(4) under nitrogen protection; the Bleomycin A5 hydrochloride. liposome that step (3) is made; with pH adjusting agent adjusting pH value to 7.0～9.0; 60 ℃ of water-baths 10～15 minutes; then cool the temperature to 20～30 ℃ and add mannitol, 0.22 μ m filtering with microporous membrane, fill under constantly stirring; then carry out lyophilizing, obtain Bleomycin A5 hydrochloride. lipidosome injection.
6. method according to claim 5, is characterized in that the organic solvent described in step (1) is that volume ratio is the ethanol of 1:3 and the mixed organic solvents of isopropyl alcohol.
7. method according to claim 5, it is characterized in that described in step (1), cryodesiccated process is :-50～-60 ℃ of pre-freezes 2～4 hours, then freezing 10～13 hours at-40 ℃～-50 ℃, through 12～15 hours, sublime up into 20 ℃～25 ℃ again, finally at 30 ℃～35 ℃, be dried 6～8 hours.
8. method according to claim 5, is characterized in that the described buffer solution of step (2) is that pH value is potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer of 5.8.
9. method according to claim 5, is characterized in that described in step (4), pH adjusting agent is potassium hydroxide.
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