CN103120645B - Irinotecan or irinotecan hydrochloride lipidosome and preparation method thereof - Google Patents
Irinotecan or irinotecan hydrochloride lipidosome and preparation method thereof Download PDFInfo
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- CN103120645B CN103120645B CN201310028546.3A CN201310028546A CN103120645B CN 103120645 B CN103120645 B CN 103120645B CN 201310028546 A CN201310028546 A CN 201310028546A CN 103120645 B CN103120645 B CN 103120645B
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Abstract
The invention discloses an irinotecan or irinotecan hydrochloride lipidosome and a preparation method thereof. The lipidosome contains irinotecan or irinotecan hydrochloride, neutral phospholipid and cholesterol, wherein the weight ratio of the cholesterol to the neutral phospholipid is 1:(3-5), and the irinotecan or irinotecan hydrochloride lipidosome is prepared by an ion gradient method.
Description
The application is application number is 200980154026.9, and the applying date is December in 2009 3 days, and denomination of invention is the divisional application of the Chinese patent application of " irinotecan or irinotecan hydrochloride lipidosome and preparation method thereof ".
Technical field
The present invention relates to a kind of irinotecan or irinotecan hydrochloride lipidosome and preparation method thereof, and contain injection of this liposome and preparation method thereof.
Background technology
Irinotecan is the semi-synthetic derivant of camptothecine.Camptothecine can be combined with topoisomerase I specifically, and the latter's inducing reversible single-strand ruptures, thus DNA double chain structure is untwisted; Irinotecan and active metabolite SN-38 thereof can be combined with topoisomerase I-DNA complex, thus stop connecting again of fracture strand.Existing research prompting, the cytotoxicity of irinotecan is owing in DNA building-up process, and replicative enzyme and topoisomerase I-DNA-irinotecan (or SN-38) three complex interact, thus cause DNA double chain interruption.
Irinotecan hydrochloride pharmacological action is obvious, clinical efficacy is definite, be widely used in treating malignant tumor, but, it is the same with other camptothecines, and the same problem existed is exactly that the saturated lactonic ring in its structure has pH dependency, in physiological conditions can be reversible change its carboxylate form into, and anti-tumor activity is weakened.The existing commercial preparation of irinotecan hydrochloride is Irinotecan hydrochloride injection and freeze-dried powder thereof, clinically after intravenously administrable, free drug is directly in the physiological environment of meta-alkalescence, easily there is hydrolysis and change carboxylate form in the lactonic ring in its structure, thus lose activity, indirectly reduce the curative effect of medicine.And the toxic and side effects of preparation is comparatively large, and main manifestations is Neutrophilic granulocytopenia and late-onset diarrhea.
Liposome is as Recent study a kind of pharmaceutical carrier comparatively widely, and its main feature to protect encapsulated medicine, increases medicine stability, changes medicine distribution behavior in vivo, and the passive or active targeting of carrying medicaments is to diseased region.Liposome effectively can improve curative effect of medication as the good carrier of antitumor drug, reduces toxic and side effects.
International application WO2005/117878 discloses prescription and the preparation method of irinotecan liposome, contains irinotecan or irinotecan hydrochloride, is selected from phospholipid, the cholesterol such as HSPC, PHOSPHATIDYL ETHANOLAMINE, lecithin, cuorin in said preparation.Equally, Chinese patent application CN1994279A also discloses prescription and the preparation method of irinotecan liposome, and wherein the use phosphatidylcholine of embodiment 5 has prepared liposome as phospholipid.
Although the prescription recorded in above-mentioned patent documentation has better effects, if be prepared into the preparation of applicable human body use, this liposome still can not be satisfactory in stability, particle diameter etc.
Summary of the invention
The object of this invention is to provide a kind of drug loading higher, and it is high to meet envelop rate simultaneously, good stability, is applicable to the irinotecan or the irinotecan hydrochloride lipidosome that are prepared into preparation.
Although describe liposome composition and the preparation method of irinotecan at present in some documents (such as international application WO2005/117878 and CN1994279A), wherein the part index number of individual program is better.But do not provide any information for the control of the aspect such as stability, particle diameter.Applicant is through studying further liposome, wonderful discovery is when the adjuvant and consumption relation of selecting prescription meet some condition, the consumption of special cholesterol has a certain impact to liposomal particle size and stability, the basis of property phosphorus and cholesterol in selecting controls ratio therebetween, the particle diameter of liposome can be made to become little and even, and improve the stability of liposome.Compared with other prescriptions, the indexs such as the stability of the application's liposome significantly improve.The present invention compares with technology such as international application WO2005/117878 with CN1994279A in addition, alkali-free functional compounds and cation lipid in product, prescription composition is simple, and drug loading is high, good stability, liposome of the present invention has good antitumous effect.
Liposome of the present invention contains irinotecan or irinotecan hydrochloride, neutral phospholipid and cholesterol, and the part by weight of wherein said cholesterol and neutral phospholipid is 1:3 ~ 5, and preferred proportion is 1:3.5 ~ 4.5, most preferably is 1:4.
Neutral phospholipid used in the present invention can select the materials such as hydrogenated soy phosphatidyl choline (HSPC), egg yolk lecithin (EPC), soybean phospholipid (SPC), and particularly when neutral phospholipid uses hydrogenated soy phosphatidyl choline, its effect is best.When the weight proportion relation controlled between medicine and phospholipid is following relation further, the drug loading of liposome greatly improves:
Irinotecan or irinotecan hydrochloride 1 part
Neutral phospholipid 2 ~ 5 parts, preferred 2.5-4 part.
Liposome of the present invention can prepare according to the method for preparing lipidosome of this area routine, for liposome of the present invention, preferably uses ion gradient legal system standby.When using ion gradient method, there is between described intraliposomal aqueous phase and outer aqueous phase the ion gradient that buffer agent is formed, preferred described intraliposomal aqueous phase has the high ion gradient of ion concentration than outer aqueous phase, this particle diameter that can improve lay up period liposome is stablized, better maintenance drug effect, it is little and even that this can control liposome mean diameter, and liposomal particle size can be made to be reduced to minimum level in the change of storage period.
The present invention can make liposomal particle size be reduced to minimum level in the change of storage period by the lipid derivate adding hydrophilic macromolecule in formula, can extend liposome circulation time in vivo adding of polyethyleneglycol derivative simultaneously.Polyethyleneglycol derivative is selected from the hard ester acyl PHOSPHATIDYL ETHANOLAMINE (DSPE-PEG of Macrogol 2000-two
2000) Polyethylene Glycol 5000-DSPE, Macrogol 2000-DPPE, Polyethylene Glycol 5000-DPPE.Therefore in order to improve the long-lasting of medicine, the application preferably adds the lipid derivate of hydrophilic macromolecule in liposome, on this prescription proportional basis, uses DSPE-PEG
2000that effect is the most obvious.The weight ratio of preferred lipid derivate and irinotecan or irinotecan hydrochloride is 0.2 ~ 0.4.
Liposome can contain electrically charged phospholipid further, electrically charged phospholipid be selected from PE, DPPG, DSPG, GLYCEROL,DIMYRISTOYL PHOSPHATIDYL, two oleic acid Phosphatidylserine, DOPG, dilaurylphosphatidic acid, two myristoyl phosphatidic acid or G 12S3P one or more, and the part by weight of electrically charged phospholipid and neutral phospholipid is 1:5 ~ 1:100.
The preferred liposome of the present invention contains the composition of following weight proportion:
And the ratio of cholesterol and hydrogenated soy phosphatidyl choline is 1:4.
Present invention also provides the preparation method of irinotecan or irinotecan hydrochloride lipidosome, liposome of the present invention can select common method for preparing lipidosome to prepare.Those skilled in the art can, according to the prescription of liposome provided by the invention, select various method to prepare.For the prescription selected by liposome of the present invention, preferred preparation method is ion gradient method.This preparation method comprises following step:
1) blank liposome is prepared by any one method in following A to D:
A, select neutral phospholipid according to formula, cholesterol is dissolved in dehydrated alcohol or in dehydrated alcohol-tert-butyl alcohol mixed solvent, mix with buffer agent, obtain blank liposome crude product after removed under reduced pressure ethanol, adopt high pressure homogenizer afterwards or/and extrusion equipment prepares blank liposome to required granularity;
B, select neutral phospholipid according to formula, cholesterol is dissolved in chloroform or chloroform-methanol mixed solvent, rotary evaporation forms lipid film, add buffer agent aquation and obtain blank liposome crude product, adopt high pressure homogenizer afterwards or/and extrusion equipment prepares blank liposome to required granularity;
C, select neutral phospholipid according to formula, cholesterol mixes with buffer agent, adopts high pressure homogenizer afterwards or/and extrusion equipment prepares blank liposome to required granularity;
D, select neutral phospholipid according to formula, cholesterol is dissolved in dehydrated alcohol or in dehydrated alcohol-tert-butyl alcohol mixed solvent, mix with buffer agent, adopts high pressure homogenizer afterwards or/and extrusion equipment prepares blank liposome to required granularity;
2) generation of aqueous ion gradient inside and outside liposome membrane: the external aqueous phase of displacement blank liposomes, makes intraliposomal aqueous phase and outer aqueous phase produce ion gradient;
3) pastille liposomal preparation: preparation irinotecan hydrochloride aqueous solution, joins and have in the blank liposomes dispersion liquid of ion gradient, heated and stirred, hatch certain hour and get final product.
After step 3) " pastille liposomal preparation " step, also can comprise following steps:
4) removal of free drug and sample is concentrated: in irinotecan hydrochloride lipidosome, add buffer medium, adopt tangential flow apparatus to remove non-encapsulated medicine, simultaneously by sample concentration to suitable volume.
Present invention also provides the lipidosome injection containing above-mentioned liposome.When becoming to be applicable to the injection used by liposomal preparation, it is useful for adding stabilizing agent.Stabilizing agent used in the present invention also can select normally used stabilizing agent, such as vitamin E, ethylenediaminetetraacetic acid etc., and these stabilizing agents are all helpful to the stability of preparation.For above-mentioned prescription, by the research of stabilizing agent is found ethylenediaminetetraacetic acid or its salt best relative to other stabilizing agent effects, the most beneficial for the stability improving liposome, therefore can select in ethylenediaminetetraacetic acid, disodium EDTA, ethylenediaminetetraacetic acid two calcium salt one or more, and the additional proportion of stabilizing agent is 0% ~ 0.5w/v% and lower limit is not 0%.
Preferably containing antioxidant in compositions of the present invention, described antioxidant can be selected from water soluble antioxidants or oil-soluble antioxidants, described oil-soluble antioxidants is selected from alpha-tocopherol, α-tocopheryl acid succinate, α-tocopheryl acetate or its mixture, described water soluble antioxidants is selected from ascorbic acid, sodium sulfite, sodium sulfite, sodium pyrosulfite, Cys or its mixture, and the additional proportion of antioxidant is 0% ~ 0.5w/v% and lower limit is not 0%.
Injection can be injection or freeze-dried powder form.Osmotic pressure regulator can be contained in preparation, described osmotic pressure regulator be selected from glucose, sucrose, sorbitol, mannitol, sodium chloride, glycerol, histidine and hydrochloride thereof, glycine and hydrochloride, lysine, serine, glutamic acid, arginine or valine one or more, and the additional proportion of osmotic pressure regulator is 0% ~ 5w/v% and lower limit is not 0%.
For the preparation of lyophilized powder form, injection, further containing freeze drying protectant, obtains liposome freeze-drying powder injection after carrying out lyophilization.Freeze drying protectant be selected from glucose, sucrose, trehalose, mannitol, dextran or lactose one or more.
The present invention's preferred injection liposome contains the composition of following weight proportion:
And the ratio of cholesterol and hydrogenated soy phosphatidyl choline is 1:4.
The preparation method of above-mentioned injection comprises following step:
1) blank liposome is prepared by any one method in following A to D:
A, select neutral phospholipid according to formula, cholesterol is dissolved in dehydrated alcohol or in dehydrated alcohol-tert-butyl alcohol mixed solvent, mix with buffer agent, obtain blank liposome crude product after removed under reduced pressure ethanol, adopt high pressure homogenizer afterwards or/and extrusion equipment prepares blank liposome to required granularity;
B, select neutral phospholipid according to formula, cholesterol is dissolved in chloroform or chloroform-methanol mixed solvent, rotary evaporation forms lipid film, add buffer agent aquation and obtain blank liposome crude product, adopt high pressure homogenizer afterwards or/and extrusion equipment prepares blank liposome to required granularity;
C, select neutral phospholipid according to formula, cholesterol mixes with buffer agent, adopts high pressure homogenizer afterwards or/and extrusion equipment prepares blank liposome to required granularity;
D, select neutral phospholipid according to formula, cholesterol is dissolved in dehydrated alcohol or in dehydrated alcohol-tert-butyl alcohol mixed solvent, mix with buffer agent, adopts high pressure homogenizer afterwards or/and extrusion equipment prepares blank liposome to required granularity;
2) generation of aqueous ion gradient inside and outside liposome membrane: the external aqueous phase of displacement blank liposomes, makes intraliposomal aqueous phase and outer aqueous phase produce ion gradient;
3) pastille liposomal preparation: preparation irinotecan hydrochloride aqueous solution, joins and have in the blank liposomes dispersion liquid of ion gradient, heated and stirred, hatch certain hour and get final product.
After step 3) " pastille liposomal preparation " step, also can comprise following steps:
4) removal of free drug and sample is concentrated: in irinotecan hydrochloride lipidosome, add buffer medium, adopt tangential flow apparatus to remove non-encapsulated medicine, simultaneously by sample concentration to suitable volume.
After obtaining liposome, also by adjustment liposomal body substrate concentration, standardize solution, filtration sterilization, embedding, in bottle, obtains lipidosome injection; Or add freeze drying protectant in liposomal body matter sample, adjustment drug level, standardize solution, filtration sterilization, embedding is in bottle, and lyophilization, obtains freeze-dried powder.
Beneficial effect of the present invention:
Irinotecan hydrochloride is made Liposomal formulation, overcome the deficiency of existing product and technology, pharmaceutical pack is wrapped in intraliposomal aqueous phase, improve medicine stability, medicine is existed with the state of lactonic ring in vivo, the concentration of active metabolite SN-38 in blood plasma can be maintained for a long time, increase preparation curative effect to reach, reduce the toxic and side effects of medicine.
Irinotecan of the present invention or irinotecan hydrochloride lipidosome preparation are by controlling the proportionate relationship of specific medicine, phospholipid and cholesterol, can reach and solve the low problem of liposome drug loading, medicine fat ratio (w/w) can reach more than 0.25, entrapment efficiency can reach more than 90% simultaneously, and preferred prescription can reach more than 95%; The present invention is by selecting the use magnitude relation of cholesterol and phospholipid further, and the liposomal particle size of preparation is less, improves the stability of liposome.The present invention also by the screening to stabilizing agent, optimizes and adds the stability that a certain proportion of edetate significantly improves liposome; The particle diameter of liposome, between 10nm-220nm, is evenly distributed; Stable in properties, irinotecan or irinotecan hydrochloride lipidosome injection influence factor experimental result show, place 10 days under 40 DEG C of degree conditions, particle diameter and envelop rate are all without significant change, and indices meets the requirements; Irinotecan or irinotecan hydrochloride lipidosome injection are compared with commercial preparation, and tumour inhibiting rate significantly improves, and toxicity significantly reduces.
Accompanying drawing explanation
Fig. 1 shows the particle size distribution of irinotecan of the present invention or irinotecan hydrochloride lipidosome injection.
Fig. 2 shows the aspect graph of irinotecan of the present invention or irinotecan hydrochloride lipidosome injection.
Fig. 3 shows the result of the anti-tumor in vivo test of pesticide effectiveness of irinotecan of the present invention or irinotecan hydrochloride lipidosome injection.
Detailed description of the invention
Embodiment is used for further illustrating the present invention below, but the present invention is not limited to this embodiment.
Embodiment 1
Prescription
Preparation method:
The hydrogenated soy phosphatidyl choline (HSPC) of recipe quantity, cholesterol (CHOL) are dissolved in appropriate dehydrated alcohol and obtain lipid soln, and mix with 100ml ammonium sulfate, removed under reduced pressure ethanol obtains blank liposome crude product.Adopt 5 circulations of high pressure homogenizer 1000bar homogenizing afterwards, extrude liposome by extrusion equipment afterwards and control its particle diameter (extruder spreads 2 0.1 μm of extruded films, extrudes 5 times).Add the DSPE-PEG prepared afterwards
2000aqueous solution, stirs and hatches 20 minutes.Adopt tangential flow ultra-filtration unit dialysis blank liposome, middle continual supplementary water for injection, obtains blank liposome.
With water for injection preparation irinotecan hydrochloride aqueous solution, be that 1:3.5 joins and above-mentionedly has in the blank liposomes dispersion liquid of ion gradient according to the part by weight of irinotecan hydrochloride and HSPC, 60 DEG C of heated and stirred, hatch and namely obtain drug-loaded liposome in 20 minutes.Adopt tangential flow ultra-filtration unit to remove non-encapsulated medicine, simultaneously by sample concentration extremely about 50ml, add 0.45g sodium chloride and regulate osmotic pressure.Adjustment drug level, standardize solution, 0.22 μm of membrane filtration is degerming, and inflated with nitrogen embedding, in bottle, obtains irinotecan hydrochloride lipidosome injection.
Each prescription change of size of preparation is as shown in the table, and result shows, when phospholipid and cholesterol weight ratio are 4:1, sample particle diameter is minimum.
Sample prepared by different phospholipid and cholesterol weight ratio is placed under 25 DEG C of conditions, investigates stability.Result, as following table, is placed 60 days under 25 DEG C of conditions, and phospholipid and cholesterol weight ratio are that 4:1 sample particle diameter and envelop rate are without significant change, the sample particle diameter of other ratio increases, envelop rate also declines to some extent, visible, and phospholipid and cholesterol weight ratio are that the sample stability of 4:1 is better.
Conclusion: comprehensive indices, result proves that the ratio between C/PL controls can reach reasonable result of use in 1:3 ~ 5, and optimum proportioning is 1:4.
Embodiment 2
Prescription
Preparation method:
The hydrogenated soy phosphatidyl choline of recipe quantity, cholesterol are dissolved in appropriate dehydrated alcohol and obtain lipid soln, and mix with 100ml ammonium sulfate, removed under reduced pressure ethanol obtains blank liposome crude product.Adopt 5 circulations of high pressure homogenizer 1000bar homogenizing afterwards, extrude liposome by extrusion equipment afterwards and control its particle diameter (extruder spreads 2 0.1 μm of extruded films, extrudes 5 times).Add the DSPE-PEG prepared afterwards
2000aqueous solution, stirs and hatches 20 minutes.Adopt tangential flow ultra-filtration unit dialysis blank liposome, middle continual supplementary water for injection, obtains blank liposome.
With water for injection preparation irinotecan hydrochloride aqueous solution, be that 1:3.5 joins and above-mentionedly has in the blank liposomes dispersion liquid of ion gradient according to the part by weight of irinotecan hydrochloride and HSPC, 60 DEG C of heated and stirred, hatch and namely obtain drug-loaded liposome in 20 minutes.Adopt tangential flow ultra-filtration unit to remove non-encapsulated medicine, simultaneously by sample concentration extremely about 50ml, add 0.45g sodium chloride and regulate osmotic pressure.Adjustment drug level, standardize solution, 0.22 μm of membrane filtration is degerming, and inflated with nitrogen embedding, in bottle, obtains irinotecan hydrochloride lipidosome injection.
Embodiment 3
Blank liposome prescription and preparation method identical with embodiment 2, be respectively as 1:1.5,1:2,1:3.5,1:4 and 1:5 are prepared liposome unlike the part by weight according to irinotecan hydrochloride and HSPC, irinotecan hydrochloride lipidosome sample envelop rate and particle diameter see the following form.
Result explanation, when the ratio of irinotecan hydrochloride and HSPC is 1:1.5, envelop rate significantly reduces, and drug loading declines obviously when its ratio is 1:5, be not suitable for the preparation being prepared into clinical practice, when its ratio is between 1:2 and 1:4 envelop rate and drug loading all higher.
Embodiment 4
Blank liposome and drug-loaded liposome is prepared according to the consumption of prescription component in embodiment 2, preparation method with method described in embodiment 2, difference be adopt high-purity egg yolk lecithin (EPC) respectively, prepared by HSPC that high-purity soybean phospholipid (SPC) is replaced in prescription.Stability is investigated under the liposomal samples of preparation being placed in 25 DEG C of conditions.The results are shown in following table.Result of the test shows, the liposomal samples stability adopting HSPC to prepare is best, places 2 months leading indicators without significant change under 25 DEG C of conditions.
Embodiment 5
Prescription
Preparation method <1>:
Alcohol injection: by the hydrogenated soy phosphatidyl choline of recipe quantity, DSPE-PEG
2000, cholesterol is dissolved in appropriate dehydrated alcohol and obtains lipid soln, be injected in the normal saline solution of irinotecan hydrochloride, removed under reduced pressure ethanol obtains liposome crude product.Adopt 5 circulations of high pressure homogenizer 1000bar homogenizing afterwards, extrude liposome by extrusion equipment afterwards and control its particle diameter (extruder spreads 2 0.1 μm of extruded films, extrudes 5 times).Adjustment drug level, standardize solution, 0.22 μm of membrane filtration is degerming, and inflated with nitrogen embedding, in bottle, obtains irinotecan hydrochloride lipidosome injection.
Preparation method <2>:
Film dispersion method: by the hydrogenated soy phosphatidyl choline of recipe quantity, DSPE-PEG
2000, cholesterol is dissolved in appropriate chloroform and obtains lipid soln, by lipid soln rotary evaporation film forming, eliminate the normal saline solution aquation that then chloroform add irinotecan hydrochloride and hatch about 1h.Adopt 5 circulations of high pressure homogenizer 1000bar homogenizing afterwards, then extrude liposome by extrusion equipment and control its particle diameter (extruder spreads 2 0.1 μm of extruded films, extrudes 5 times).Adjustment drug level, standardize solution, 0.22 μm of membrane filtration is degerming, and inflated with nitrogen embedding, in bottle, obtains irinotecan hydrochloride lipidosome injection.
Irinotecan hydrochloride lipidosome envelop rate prepared by mensuration preparation method <1>, <2> and embodiment 2 and particle diameter.
Result shows, adopt Passive loading method as alcohol injection and film evaporation method prepare irinotecan hydrochloride lipidosome time, object product can be prepared.But its sample envelop rate is lower, and most of medicine does not all enter into liposome; And the sample adopting active loading method to prepare (embodiment 2) envelop rate is high, drug loading is high, and particle diameter is little and even, so adopt active loading method.Namely ion gradient legal system is adopted to have extraordinary effect for irinotecan hydrochloride lipidosome in the present invention.
Embodiment 6
Preparation method:
Blank liposome: by lipid ethanol injection.Homogenizing 1000bar, 6 times; 200nm extrudes 3 times, and 100nm extrudes 5 times; Add PEG
2000-DSPE, gives 30min for 60 DEG C.Tangential flow dialysis 3 times, adds 50ml at every turn.Wherein VE is added in phospholipid organic solvent, and EDTA is added in ammonium sulfate.
Drug-loaded liposome: the irinotecan hydrochloride aqueous solution preparing about 10mg/ml, adds in blank liposome, gives 15min for 60 DEG C.With slipstream concentrating sample to about 50ml, be 5mg/ml sample.
Stability result sees the following form, visible, adds separately disodiumedetate sample indices without significant change, can significantly improve the stability of liposome, and the stability of other stabilizing agent to liposome does not have clear improvement.
Embodiment 7
Prescription (1)
Preparation method:
The hydrogenated soy phosphatidyl choline of recipe quantity, cholesterol are dissolved in appropriate dehydrated alcohol and obtain lipid soln, and mix with 100ml manganese sulfate solution, removed under reduced pressure ethanol obtains blank liposome crude product.Extrude liposome by extrusion equipment afterwards and control its particle diameter (extruder spreads 2 0.1 μm of extruded films, extrudes 5 times).Adopt tangential flow ultra-filtration unit dialysis blank liposome, middle continual supplementary water for injection, obtains blank liposome.With water for injection preparation irinotecan hydrochloride aqueous solution, join and have in the blank liposomes dispersion liquid of ion gradient, 50 DEG C of heated and stirred, hatch and namely obtain drug-loaded liposome in 20 minutes.Adopt tangential flow ultra-filtration unit to remove non-encapsulated medicine, simultaneously by sample concentration extremely about 50ml, add 2.5g mannitol and regulate osmotic pressure.Adjustment drug level, standardize solution, 0.22 μm of membrane filtration is degerming, and inflated with nitrogen embedding, in bottle, obtains irinotecan hydrochloride lipidosome injection.Recording liposome particle size through nano particle size analyzer is 89.3nm, and envelop rate is 97.5%.
Prescription (2)
Preparation method:
The hydrogenated yolk lecithin of recipe quantity, cholesterol are dissolved in appropriate dehydrated alcohol and obtain lipid soln, mixes with 100ml Adlerika.Extrude liposome by extrusion equipment afterwards and control its particle diameter (extruder spreads 2 0.1 μm of extruded films, extrudes 5 times).Adopt tangential flow ultra-filtration unit dialysis blank liposome, middle continual supplementary water for injection, obtains blank liposome.With water for injection preparation irinotecan hydrochloride aqueous solution, join and have in the blank liposomes dispersion liquid of ion gradient, 50 DEG C of heated and stirred, hatch and namely obtain drug-loaded liposome in 20 minutes.Adopt tangential flow ultra-filtration unit to remove non-encapsulated medicine, simultaneously by sample concentration extremely about 50ml, add 2.5g histidine and regulate osmotic pressure.Adjustment drug level, standardize solution, 0.22 μm of membrane filtration is degerming, and inflated with nitrogen embedding, in bottle, obtains irinotecan hydrochloride lipidosome injection.Recording liposome particle size through nano particle size analyzer is 87.6nm, and envelop rate is 98.1%.
Prescription (3)
Preparation method:
The hydrogenated soy phosphatidyl choline of recipe quantity, cholesterol are dissolved in appropriate dehydrated alcohol and obtain lipid soln, and mix with 100ml ammonium sulfate, removed under reduced pressure ethanol obtains blank liposome crude product.After adopting 5 circulations of high pressure homogenizer 1000bar homogenizing afterwards, add the DSPE-PEG prepared afterwards
2000aqueous solution, stirs and hatches 20 minutes.Adopt tangential flow ultra-filtration unit dialysis blank liposome, middle continual supplementary water for injection, obtains blank liposome.With water for injection preparation irinotecan hydrochloride aqueous solution, join and have in the blank liposomes dispersion liquid of ion gradient, 60 DEG C of heated and stirred, hatch and namely obtain drug-loaded liposome in 20 minutes.Adopt tangential flow ultra-filtration unit to remove non-encapsulated medicine, simultaneously by sample concentration extremely about 50ml, add 0.45g sodium chloride and regulate osmotic pressure.Adjustment drug level, standardize solution, 0.22 μm of membrane filtration is degerming, and inflated with nitrogen embedding, in bottle, obtains irinotecan hydrochloride lipidosome injection.Recording liposome particle size through nano particle size analyzer is 87.3nm, and envelop rate is 99.2%.
Embodiment 8
Prescription
Preparation method:
By the hydrogenated soy phosphatidyl choline of recipe quantity, cardiolipin, DSPE-PEG
5000, cholesterol, alpha-tocopherol be dissolved in appropriate dehydrated alcohol and obtain lipid soln, mix with 100ml citric acid solution, removed under reduced pressure ethanol obtains blank liposome crude product.Adopt high pressure homogenizer 1000bar afterwards, homogenizing 5 times.Adopt tangential flow ultra-filtration unit dialysis blank liposome, middle continual supplementary 0.9% sodium chloride solution 400ml, obtains blank liposome.With water for injection preparation irinotecan hydrochloride solution, join and have in the blank liposomes dispersion liquid of ion gradient, 60 DEG C of heated and stirred, hatch and namely obtain drug-loaded liposome in 20 minutes.Tangential flow ultra-filtration unit is adopted to remove non-encapsulated medicine, simultaneously by sample concentration extremely about 50ml.Adjustment drug level, standardize solution, 0.22 μm of membrane filtration is degerming, and inflated with nitrogen embedding, in bottle, obtains irinotecan hydrochloride lipidosome injection.Recording liposome particle size through nano particle size analyzer is 85.8nm, and envelop rate is 98.6%.
Embodiment 9
Prescription
Preparation method:
The DPPC of recipe quantity, DPPG, cholesterol are dissolved in appropriate dehydrated alcohol and obtain lipid soln, and mix with 100ml ammonium sulfate (containing disodiumedetate), removed under reduced pressure ethanol obtains blank liposome crude product.Adopt high pressure homogenizer 1000bar afterwards, homogenizing 5 times.Adopt tangential flow ultra-filtration unit dialysis blank liposome, middle continual supplementary 0.9% sodium chloride solution 400ml, obtains blank liposome.With water for injection preparation irinotecan hydrochloride solution, join and have in the blank liposomes dispersion liquid of ion gradient, 60 DEG C of heated and stirred, hatch and namely obtain drug-loaded liposome in 20 minutes.Adopt tangential flow ultra-filtration unit to remove non-encapsulated medicine, simultaneously by sample concentration extremely about 50ml, add ascorbic acid.Adjustment drug level, standardize solution, 0.22 μm of membrane filtration is degerming, and inflated with nitrogen embedding, in bottle, obtains irinotecan hydrochloride lipidosome injection.Recording liposome particle size through nano particle size analyzer is 89.4nm, and envelop rate is 97.2%.
Embodiment 10
Prescription
Preparation method:
The hydrogenated soy phosphatidyl choline of recipe quantity, cholesterol, alpha-tocopherol are dissolved in appropriate dehydrated alcohol and obtain lipid soln, and mix with 100ml ammonium sulfate, removed under reduced pressure ethanol obtains blank liposome crude product.Adopt high pressure homogenizer 1000bar afterwards, homogenizing 5 times, then extrude liposome (extruder spreads 5 100nm extruded films, extrudes 5 times) by extrusion equipment, add the DSPE-PEG prepared afterwards
5000aqueous solution, stirs and hatches 20 minutes.Adopt tangential flow ultra-filtration unit dialysis blank liposome, middle continual supplementary 0.9% sodium chloride solution 400ml, obtains blank liposome.With water for injection preparation irinotecan hydrochloride solution, join and have in the blank liposomes dispersion liquid of ion gradient, 60 DEG C of heated and stirred, hatch and namely obtain drug-loaded liposome in 20 minutes.Adopt tangential flow ultra-filtration unit to remove non-encapsulated medicine, simultaneously by sample concentration to about 50ml, add sucrose and mannitol makes mix homogeneously.Adjustment drug level, standardize solution, 0.22 μm of membrane filtration is degerming, canned in cillin bottle, and lyophilization obtains irinotecan hydrochloride lipidosome freeze-dried powder.Be 90.8nm by measuring liposome particle size after liposome freeze-drying powder injection aquation, envelop rate is 97.5%.
Test example 1
Physicochemical characteristics according to the finished product (by embodiment 2) that the present invention obtains:
It is appropriate that [particle size distribution] gets this product, to measure with dynamic light scattering method after water dilution.Measure wavelength X=633nm, measure angle 173 °, temperature 25 DEG C.Size is with intensity footpath (Intensity) data representation.Grain size distribution is shown in Fig. 1, and mean diameter is 85.9nm.
It is appropriate that [morphology investigation] draws liposomal samples, is placed in by copper mesh on clean filter paper, on copper mesh, drips sample, with 2% phosphotungstic acid dyeing, observes this product after to be dried under JEM2010 transmission electron microscope (Jeol Ltd.).Aspect graph is shown in Fig. 2.Through transmission electron microscope observing, irinotecan liposome form is typical bilayer structure, and particle diameter major part, at below 200nm, matches with the result of dynamic light scattering determination.
[entrapment efficiency determination] medicine assay method: chromatographic column: Agilent ZORBAX Eclipse XDB-C18(4.6 × 150mm, 5 μm); Mobile phase: acetonitrile-0.05M potassium phosphate buffer (pH4, containing 1% triethylamine)=20:80; Column temperature: 40 DEG C; Sampling volume: 20 μ L; Flow velocity: 1.0mL/min.
Entrapment efficiency determination method: draw 1mL sample solution in 10mL measuring bottle, add water and be diluted to scale, shake up, put 8010 type ultrafilters (MILLIPORE company) ultrafiltration, discard just filtrate, getting subsequent filtrate is need testing solution.Draw need testing solution, reference substance solution 20 μ L injection liquid chromatography respectively, record chromatogram, calculate preparation free drug content with external standard method, be designated as W; Separately calculate the total drug content of this product by method under assay item, be designated as W
0.Be calculated as follows the envelop rate of sample.
Measurement result: this product envelop rate is 99.4.
[influence factor's test] is got under this product is placed in different condition and is carried out factors influencing, and result is as shown in the table:
Result shows, sample, to light sensitive, turns yellow through strong illumination sample appearance, and content declines, and related substance obviously increases; Envelop rate and particle diameter are without significant change when high temperature 40 ° of C for sample, but related substance slightly increases; Low temperature and freeze cycle test show that sample produces macroparticle.Consider phospholipid unstability at high operating temperatures, in conjunction with influence factor's result of the test, this product answers low-temperature dark to store.
[the anti-tumor in vivo test of pesticide effectiveness]
Medicine name: the irinotecan hydrochloride lipidosome (CPT-11 liposome) prepared by embodiment 2 is provided by Hengrui Pharmaceutical Co., Ltd., Shanghai, Irinotecan hydrochloride injection (CPT-11, Hengrui Medicine Co., Ltd., Jiangsu Prov. provides)
Compound method: be all made into desired concn with normal saline.
Laboratory animal: BALB/cA-nude nude mouse, 6-7 week,
purchased from Shanghai Slac Experimental Animal Co., Ltd..The quality certification number: SCXK(Shanghai) 2007-0005.Feeding environment: SPF level.
Test method: nude mouse subcutaneous vaccination human colon carcinoma Ls-174t cell, treats that tumor growth is to 150-300mm
3after, by animal random packet (d0).Dosage and dosage regimen see the following form.Survey 2-3 tumor volume weekly, claim Mus heavy, record data.Gross tumor volume (V) computing formula is:
V=1/2 × a × b
2wherein a, b represent length and width respectively.
D0: administration time for the first time; RTV: relative tumour volume; P value refers to compared with the control.
Matched group n=12, treatment group n=6.Vehicle: blank medium.
Experimental result:
CPT-11 liposome and CPT-11 all obviously suppress the growth of human colon carcinoma Ls-174t Nude Mice, CPT-11 liposome suppresses Ls-174t growth to have obvious dose dependent, during high dose (3mg/kg) administration, there is 4/14 tumor partial regression, suitable when curative effect during low dosage (1mg/kg) administration and CPT-1110mg/kg administration, the curative effect of prompting CPT-11 liposome likely at least improves 10 times than its injection, and concrete outcome is shown in Fig. 3.
Claims (18)
1. the preparation method of an irinotecan or irinotecan hydrochloride lipidosome, it is characterized in that described liposome contains irinotecan or irinotecan hydrochloride, neutral phospholipid and cholesterol, the part by weight of wherein said cholesterol and neutral phospholipid is 1:3 ~ 5, described neutral phospholipid is hydrogenated soy phosphatidyl choline, and described neutral phospholipid and irinotecan or irinotecan hydrochloride meet following weight proportion:
Irinotecan or irinotecan hydrochloride 1 part
Neutral phospholipid 2 ~ 5 parts,
And this preparation method comprises following step:
1) blank liposome is prepared by any one method in following A to D:
A. select neutral phospholipid according to formula, cholesterol is dissolved in dehydrated alcohol or in dehydrated alcohol-tert-butyl alcohol mixed solvent, mix with buffer agent, obtain blank liposome crude product after removed under reduced pressure ethanol, adopt high pressure homogenizer afterwards or/and extrusion equipment prepares blank liposome to required granularity;
B. select neutral phospholipid according to formula, cholesterol is dissolved in chloroform or chloroform-methanol mixed solvent, rotary evaporation forms lipid film, add buffer agent aquation and obtain blank liposome crude product, adopt high pressure homogenizer afterwards or/and extrusion equipment prepares blank liposome to required granularity;
C. select neutral phospholipid according to formula, cholesterol mixes with buffer agent, adopt high pressure homogenizer afterwards or/and extrusion equipment prepares blank liposome to required granularity;
D. select neutral phospholipid according to formula, cholesterol is dissolved in dehydrated alcohol or in dehydrated alcohol-tert-butyl alcohol mixed solvent, mix with buffer agent, adopt high pressure homogenizer afterwards or/and extrusion equipment prepares blank liposome to required granularity;
2) generation of aqueous ion gradient inside and outside liposome membrane: the external aqueous phase of displacement blank liposomes, makes intraliposomal aqueous phase and outer aqueous phase produce ion gradient;
3) pastille liposomal preparation: preparation irinotecan hydrochloride aqueous solution, join and have in the blank liposomes dispersion liquid of ion gradient, heated and stirred, hatches, and to obtain final product.
2. the preparation method of irinotecan according to claim 1 or irinotecan hydrochloride lipidosome, is characterized in that the weight proportion of described neutral phospholipid is 2.5-4 part.
3. the irinotecan according to aforementioned any one claim or the preparation method of irinotecan hydrochloride lipidosome, is characterized in that the ratio of described cholesterol and neutral phospholipid is 1:3.5 ~ 4.5.
4. the preparation method of irinotecan according to claim 3 or irinotecan hydrochloride lipidosome, is characterised in that the ratio of described cholesterol and neutral phospholipid is 1:4.
5. the preparation method of irinotecan according to claim 1 or irinotecan hydrochloride lipidosome, is characterized in that the lipid derivate of described liposome also containing hydrophilic macromolecule.
6. the preparation method of irinotecan according to claim 5 or irinotecan hydrochloride lipidosome, is characterized in that the lipid derivate of described hydrophilic macromolecule is DSPE-PEG
2000.
7. the irinotecan according to claim 5 or 6 or the preparation method of irinotecan hydrochloride lipidosome, is characterized in that the lipid derivate of described hydrophilic macromolecule and the weight ratio of irinotecan or irinotecan hydrochloride are 0.2 ~ 0.4.
8. the preparation method of irinotecan according to claim 1 or irinotecan hydrochloride lipidosome, it is characterized in that described liposome further containing electrically charged phospholipid, described electrically charged phospholipid is selected from PE, DPPG, DSPG, GLYCEROL,DIMYRISTOYL PHOSPHATIDYL, two oleic acid Phosphatidylserine, DOPG, dilaurylphosphatidic acid, one or more in two myristoyl phosphatidic acid or G 12S3P, and the part by weight of electrically charged phospholipid and neutral phospholipid is 1:5 ~ 1:100.
9. the preparation method of irinotecan according to claim 1 or irinotecan hydrochloride lipidosome, is characterized in that 3) after pastille liposomal preparation step, also comprise following steps:
4) removal of free drug and sample is concentrated: in irinotecan hydrochloride lipidosome, add buffer medium, adopt tangential flow apparatus to remove non-encapsulated medicine, simultaneously by sample concentration to suitable volume.
10. the preparation method of irinotecan according to claim 1 or irinotecan hydrochloride lipidosome, is characterized in that described buffer agent is selected from containing Na
+, K
+, Fe
2+, Ca
2+, Ba
2+, Mn
2+, Mg
2+, Li
+, NH
4 +or H
+salt in one or more.
The preparation technology of 11. 1 kinds of irinotecans or irinotecan hydrochloride lipidosome injection, is characterized in that described technique comprises the preparation method of claim 1.
The preparation technology of 12. irinotecans according to claim 11 or irinotecan hydrochloride lipidosome injection, it is characterized in that described injection contains stabilizing agent, described stabilizing agent be selected from ethylenediaminetetraacetic acid, disodium EDTA, ethylenediaminetetraacetic acid two calcium salt one or more, the additional proportion of stabilizing agent is 0% ~ 0.5w/v% and lower limit is not 0%.
The preparation technology of 13. irinotecans according to claim 12 or irinotecan hydrochloride lipidosome injection, is characterized in that described stabilizing agent is disodiumedetate.
The preparation technology of 14. irinotecans according to claim 11 or irinotecan hydrochloride lipidosome injection, is characterized in that described injection is injection or freeze-dried powder.
The preparation technology of 15. irinotecans according to claim 11 or irinotecan hydrochloride lipidosome injection, it is characterized in that described injection contains osmotic pressure regulator, described osmotic pressure regulator be selected from glucose, sucrose, sorbitol, mannitol, sodium chloride, glycerol, histidine and hydrochloride thereof, glycine and hydrochloride, lysine, serine, glutamic acid, arginine or valine one or more, the additional proportion of osmotic pressure regulator is 0% ~ 5w/v% and lower limit is not 0%.
The preparation technology of 16. irinotecans according to claim 11 or irinotecan hydrochloride lipidosome injection, it is characterized in that described injection further containing antioxidant, described antioxidant is selected from water soluble antioxidants or oil-soluble antioxidants, described oil-soluble antioxidants is selected from alpha-tocopherol, α-tocopheryl acid succinate, α-tocopheryl acetate or its mixture, described water soluble antioxidants is selected from ascorbic acid, sodium sulfite, sodium sulfite, sodium pyrosulfite, Cys or its mixture, the additional proportion of antioxidant is 0% ~ 0.5w/v% and lower limit is not 0%.
The preparation technology of 17. irinotecans according to claim 14 or irinotecan hydrochloride lipidosome injection, is characterized in that described injection is freeze-dried powder, wherein containing freeze drying protectant, is by the obtained liposome freeze-drying powder injection of lyophilization.
The preparation technology of 18. irinotecans according to claim 17 or irinotecan hydrochloride lipidosome injection, is characterized in that described technique also comprises:
Standardize solution, degerming, subpackage: adjustment liposomal body substrate concentration, standardize solution, filtration sterilization, embedding, in bottle, obtains lipidosome injection; Or
In liposomal body matter sample, add freeze drying protectant, adjustment drug level, standardize solution, filtration sterilization, embedding is in bottle, and lyophilization, obtains freeze-dried powder.
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| CN103830182B (en) * | 2014-03-11 | 2015-12-30 | 江苏奥赛康药业股份有限公司 | A kind of long circulating irinotecan liposome composition and method of making the same |
| CN105982857B (en) * | 2015-02-09 | 2019-03-08 | 湖南科伦药物研究有限公司 | A kind of irinotecan hydrochloride lipidosome composition and preparation method thereof |
| CN105534906A (en) * | 2016-01-06 | 2016-05-04 | 青岛辰达生物科技有限公司 | Irinotecan hydrochloride lipidosome and preparation method thereof |
| CN106166139A (en) * | 2016-04-29 | 2016-11-30 | 陈西敬 | A kind of compositions liposome of palmitoyl ascorbate and hydroxy camptothecin |
| CN107456456A (en) * | 2016-06-03 | 2017-12-12 | 江苏恒瑞医药股份有限公司 | The purposes of Irinotecan or its officinal salt in the medicine for preparing treatment breast cancer |
| CN106509652B (en) * | 2016-11-08 | 2021-02-02 | 雅安职业技术学院 | Method for preparing diced pork in pot by microwave heating sterilization |
| CN108338972A (en) * | 2017-01-24 | 2018-07-31 | 江苏恒瑞医药股份有限公司 | A kind of preparation method of liposome |
| CN108338973B (en) * | 2017-01-24 | 2022-06-21 | 江苏恒瑞医药股份有限公司 | Preparation method of liposome |
| CN107898758A (en) * | 2017-11-30 | 2018-04-13 | 福建省中医药研究院(福建省青草药开发服务中心） | A kind of preparation method for the PEGylated nano liposomes for embedding rhodioside |
| CN110496103B (en) * | 2018-05-18 | 2022-05-10 | 上海维洱生物医药科技有限公司 | Docetaxel palmitate liposome and preparation method thereof |
| CN109260155B (en) | 2018-11-05 | 2021-04-06 | 杭州高田生物医药有限公司 | Irinotecan liposome preparation and application thereof |
| CN109675047B (en) * | 2019-01-07 | 2020-12-04 | 中国科学院化学研究所 | Method for carrying out liposome modification on compound with free hydroxyl |
| CN117224670B (en) * | 2023-11-13 | 2024-01-30 | 成都依思康生物科技有限公司 | Liposome adjuvant composition, vaccine composition, and preparation methods and applications thereof |
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| CN1960729A (en) * | 2004-06-01 | 2007-05-09 | 泰尔茂株式会社 | Irinotecan preparation |
| CN1994279A (en) * | 2006-12-31 | 2007-07-11 | 西安力邦医药科技有限责任公司 | Preparation process of irinotecan hydrochloride liposome for injection |
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| CN1960729A (en) * | 2004-06-01 | 2007-05-09 | 泰尔茂株式会社 | Irinotecan preparation |
| CN1994279A (en) * | 2006-12-31 | 2007-07-11 | 西安力邦医药科技有限责任公司 | Preparation process of irinotecan hydrochloride liposome for injection |
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