CN104622807A - Vinorelbine tartrate long-circulating liposome and preparation method thereof - Google Patents
Vinorelbine tartrate long-circulating liposome and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a vinorelbine tartrate long-circulating liposome comprising the following components in parts by weight: 1 weight part of vinorelbine tartrate, 5-50 weight parts of phospholipid substances, 1-20 weight parts of cholesterol and 1-20 weight parts of a long-circulating membrane material. By adopting the liposome disclosed by the invention, the problems that an existing preparation is inconvenient to use clinically, is easy to cause medicine leakage, and is poor in stability and low in encapsulation efficiency can be solved, the anti-tumor effects and stability of vinorelbine tartrate can be greatly improved, and the blood vessel stimulation and other adverse reactions can be reduced. The invention also relates to a preparation method of the vinorelbine tartrate long-circulating liposome, and the preparation method is simple in preparation process and easy to control quality, and is suitable for industrial production.
Description
Technical field
The invention belongs to medicine preparation field, be specifically related to a kind of vinorelbine tartrate long circulating liposomes and preparation method thereof.
Background technology
Vinorelbine is a kind of semisynthetic vinca anti-cancer agent, and active anticancer is high, and antitumor spectra is wide.Research shows, the pharmacological action of vinorelbine is formed by the microtubule in blocks cellular mitosis process to realize, thus make cell division stop at mitosis metaphase, and then stops the division growth of cancerous cell.Clinically, vinorelbine is mainly used in treatment nonsmall-cell lung cancer, metastatic breast cancer and refractory non-Hodgkin's lymphoma, ovarian cancer, tumor of head and neck etc.But the injection of this medicine has larger zest when Clinical practice, as too high in local concentration or medicinal liquid exosmoses and can produce larger toxicity stimulation to transfusion vein, and can cause phlebitic generation.There are some researches show, its phlebitis incidence rate is 47.2%.Therefore the injection used clinically at present all requires must first use normal saline flushing vein, and must confirm that injection needle can start injection at intravenous, once medicine liquid leakage should stop injection immediately, remaining medicine separately changes vein and injects.Untoward reaction main manifestations is have stronger neurotoxicity, bone marrow depression, leukopenia, and gastrointestinal reaction, peripheral nervous toxicity and alopecia incidence rate all increase.This brings a lot of misery to patient undoubtedly, brings a lot of inconvenience in use, limits vinorelbine popularity clinically and application.
Liposome has the structure being similar to cell membrane, and the hydrophilic head of lipoid forming bilayer forms the inner surface of film, and lipophilic afterbody is then in the centre of film.This membranelike structure just because of liposome can carry various material that is hydrophilic, hydrophobic and amphiphilic, and they are typically entrapped within liposome interior aqueous phase, insert the surface that class lipid bilayer or absorption are connected to liposome.Liposome, as pharmaceutical carrier, had both served the protective effect to medicine, turn improved the targeting of medicine to body specific part, therefore in raising drug effect, had much superior characteristic.But conventional liposome enters very soon by reticuloendothelial system phagocytic after in body, and main targeting at reticuloendothelial systems such as liver, spleen, bone marrow compared with in the organ of horn of plenty, poor to the targeting at other positions.And it is subject to the effect of albumen, enzyme etc. in vivo and seepage occurs, affect the treatment and toxicity is increased.Opsonic combination in liposome and blood plasma can be reduced by adding the macromole such as the derivant (as PEG-DSPE (PEG-DSPE)) of the distearyl acid phospholipid amide of amphipathic PEG in liposome, increase its blood stability, prolong half-life and the medicine time in blood.
Therefore in view of above-mentioned characteristic, for making vinorelbine play clinical efficacy better, reduce it to the zest of blood vessel and untoward reaction, improve anti-tumor activity, domestic and international pharmacy worker is devoted to the research and development of vinorelbine Novel Drug Delivery Systems.Chinese patent application CN101933904 discloses vinorelbine lipoplast preparation and preparation method, and said preparation comprises three subpackage unit: blank liposome, sodium radio-phosphate,P-32 solution and vinorelbine tartrate; Before Clinical practice, three subpackage unit need be mixed and can inject by vein after suitably heating medicine carrying.This employs the higher sodium citrate buffer solution of concentration in applying for and other multiple buffer salts carry out adjust ph, and the Clinical practice safety of these buffer salts exists larger hidden danger.The Liposomal formulation of this point of packaging brings very big inconvenience to production, transport and Clinical practice in addition.
Chinese patent application CN100998562A discloses the preparation method of vinorelbine lipoplast, and pH gradient method medicine carrying can cause medicine to be revealed along with the prolongation of standing time, is difficult to the inside and outside stability ensureing preparation.Chinese patent application CN1839800A discloses vinorelbine lipoplast and freeze-dried powder thereof.Because preparing vinorelbine tartrate is water soluble drug, this application adopts Passive loading legal system for liposome, and envelop rate is difficult to reach requirement.Chinese patent application CN101138548A discloses vinorelbine nano-micelle, this micelle adopts polyglycol derivatization phospholipid to make carrier parcel vinorelbine, this micellar preparation medicine after blood infinite dilution is easily revealed, and thus glue bundle body internal stability exists some problems.
In addition, the method that routine prepares vinorelbine lipoplast is pH gradient method, ammonium sulphate gradient, the liposome stability wherein prepared with pH gradient method is poor, usual need are designed to three subpackage unit to improve preparation stability, but the Liposomal formulation of this point of packaging brings very big inconvenience to production, transport and Clinical practice; Prepare in liposome process with ammonium sulphate gradient, aqueous phase ammonium sulfate except the methods such as usual employing dialysis, column chromatography, ultrafiltration, these methods exist that quantity of sample handling is little, consuming time, diluted sample, fenestra easily block the problems such as ultrafiltration efficiency is low, thus be only suitable for the preparation of a small amount of sample, be not suitable for industrialized great production.
Summary of the invention
Technical problem
Present inventor finds: published liposome adopts the preparation of pH gradient method, because inside and outside liposome, aqueous pH values difference is larger, liposome instability in storage process is caused to cause medicine to be revealed, therefore need to be designed to three subpackage unit (blank liposome, sodium radio-phosphate,P-32 solution and vinorelbine tartrate), before Clinical practice, three subpackage unit need be mixed and can inject by vein after suitably heating medicine carrying, though this design can improve the stability of drug-loaded liposome at storage process, bring very big inconvenience to production, transport and Clinical practice.In addition, need use the citric acid buffer salt of high concentration when adopting pH gradient method to prepare liposome, there is larger hidden danger in Clinical practice safety.Because vinorelbine tartrate is water soluble drug, Passive loading method is adopted to prepare its liposome encapsulation lower.When preparing drug-loaded liposome with ammonium sulphate gradient, usually all adopt the outer aqueous phase ammonium sulfate of method removing liposome such as dialysis, column chromatography, ultrafiltration to set up ammonium sulphate gradient for medicine carrying, these conventional methods are all only suitable for laboratory scale preparation, be not suitable for industrialized great production, therefore, for this specific medicine of vinorelbine tartrate, must for the requirement of its clinical practice and suitability for industrialized production, find specific preparation and preparation technology, to realize the object improving curative effect and stability, reduction vein blood vessel zest and other toxic and side effects.
Technical scheme
For solving the above-mentioned technical problem existed in prior art, present inventor has performed research extensively and profoundly, finally obtaining the present invention.
An object of the present invention is to provide a kind of stable vinorelbine tartrate long circulating liposomes for clinical, the problems such as described liposome solves the inconvenience of existing preparation Clinical practice, medicine easily leaks, poor stability, envelop rate are low, greatly can improve stability and the antitumor action thereof of vinorelbine tartrate, reduce blood vessel irritation and other untoward reaction.
Another object of the present invention is to provide a kind of preparation method of above-mentioned vinorelbine tartrate long circulating liposomes.
In order to realize foregoing invention object, the invention provides a kind of vinorelbine tartrate long circulating liposomes, it comprises:
In the present invention, preferably, described vinorelbine tartrate long circulating liposomes can comprise the acceptable buffer of pharmacy further, and its concentration range is 10-50mM, pH is 5.5-8.In the present invention, described buffer, for maintaining the pH of liposome in certain limit, increases the stability of liposome.In the present invention, preferably, the acceptable buffer of described pharmacy is selected from histidine buffering liquid, phosphate buffer, HEPES buffer.
In the present invention; preferably; described vinorelbine tartrate long circulating liposomes can comprise freeze drying protectant further; described freeze drying protectant is used for the lyophilization of gained liposome to be prepared into its lyophilized powder; its consumption is pressed phospholipid substance weight ratio and is calculated, and the phospholipid substance of 1 weight portion adds the freeze drying protectant of 0.1-5 weight portion.In the present invention, preferably, described freeze drying protectant be selected from sucrose, lactose, mannitol, trehalose, maltose etc. one or more.
In the present invention, described phospholipid substance is the Main Ingredients and Appearance forming liposome, can be the pharmaceutically acceptable phospholipid of any one that can be used in preparing liposome, preferably be selected from soybean phospholipid, hydrogenated soya phosphatide (HSPC), Ovum Gallus domesticus Flavus lecithin, hydrolecithin, sphingomyelins, cuorin, distearoyl phosphatidylcholine (DSPC), dipalmitoyl phosphatidyl choline (DPPC), dimyristoyl phosphatidyl choline (DMPC), DOPC (DOPC), DSPE (DSPE), DPPE (DPPE), DMPEA (DMPE), DOPE (DOPE), DSPG (DSPG), DPPG (DPPG), one or more in GLYCEROL,DIMYRISTOYL PHOSPHATIDYL (DMPG) and DOPG (DOPG), are more preferably and are selected from soybean phospholipid, hydrogenated soya phosphatide, Ovum Gallus domesticus Flavus lecithin, hydrolecithin, one or more in DSPC.
In the present invention, described cholesterol is as the composition of liposome, and its consumption has appreciable impact to the stability of preparation and release behavior.
In the present invention, described long circulating film material is for realizing the long circulating function of liposome, and prolong drug circulation time in blood, increases the accumulation of medicine at tumor locus, to improve curative effect further, reduces toxicity.In the present invention, preferably, described long circulating film material is polyglycol derivatization phospholipid, it is that peg molecule is combined into by the active group on covalent bond and phospholipid molecule, preferably, be selected from mPEG2000-DSPE (PEG-PE), Polyethylene Glycol-DMPEA (PEG-DMPE), Polyethylene Glycol-DPPE (PEG-DPPE), PEG2000-DSPE (PEG-DSPE), the molecular weight of wherein said PEG is 500-5000Da, more preferably, the molecular weight of described PEG is 1000-5000Da, most preferably, described PEG molecular weight is 2000Da.
In the present invention, preferably, the particle diameter of described vinorelbine tartrate long circulating liposomes is 50-200nm, is preferably 50-120nm, more preferably 70-110nm.
In vinorelbine tartrate long circulating liposomes of the present invention, entrapment efficiency is greater than 80%, so that liposome is gathered in tumor tissues by EPR effect, reduces the distribution in other normal structure, thus improves drug effect, reduces toxicity.Entrapment efficiency in vinorelbine tartrate long circulating liposomes of the present invention is even greater than 85%, is even more greater than 90%, is even also more greater than 95%.
According to another object of the present invention, provide the preparation method of described vinorelbine tartrate long circulating liposomes, it adopts cross-flow ultrafiltration combine with technique ammonium sulphate gradient to prepare.The method can realize industrially scalable, the product that high efficiency production mass is stable.
Therefore, the invention provides the preparation method of above-mentioned vinorelbine tartrate long circulating liposomes, it comprises the following steps:
A takes the phospholipid substance of formula ratio, long circulating film material, cholesterol are dissolved in dehydrated alcohol and obtain organic facies, be injected into ammonium sulfate solution high speed (such as rotating speed the can be 5000-30000rpm) stirring that concentration is 100-400mmol/L, through high pressure (such as can at 10000-30000psi) homogenizing, ultrasonic or expressing technique, form blank liposome;
Or, take the phospholipid substance of formula ratio, cholesterol and long circulating material and be dissolved in the tert-butyl alcohol, after lyophilization, add the ammonium sulfate solution dispersion that concentration is 100-400mmol/L, form blank liposome;
B by step a gained blank liposome with pure water or aqueous sucrose solution (concentration is for 300mM) by tangential flow ultra-filtration unit (membrane molecule amount: 10-100KDa; Flow velocity: 20-400ml/min; Pressure: 0-5bar), the 5-30 of displacement liposome volume doubly, sets up ammonium sulphate gradient to remove outer aqueous phase ammonium sulfate;
Vinorelbine tartrate adds in step b process gained blank liposome by c, and at the temperature higher than lipid phase transition temperature, (preferably 37 DEG C-70 DEG C) are hatched 10min-1h and carried out medicine carrying, obtain vinorelbine tartrate long circulating liposomes.
Further, can add with buffer salt solid form after step c or be replaced into the acceptable buffer of pharmacy through cut stream ultrafiltration apparatus, its concentration range is 10-50mM, pH be 5.5-8.In the present invention, described buffer, for maintaining the pH of liposome in certain limit, increases the stability of liposome.In the present invention, preferably, the acceptable buffer salt of described pharmacy is selected from histidine buffering liquid, phosphate buffer, HEPES buffer.
Further; can add in medicine carrying long circulating liposomes and be selected from one or more in sucrose, lactose, mannitol, trehalose, maltose etc. as freeze drying protectant; for the lyophilization of gained liposome is prepared into its lyophilized powder; its consumption is pressed phospholipid substance weight ratio and is calculated, and 1 weight portion phospholipid substance adds 0.1-5 weight portion freeze drying protectant.
In preparation method of the present invention, after above-mentioned steps c or after adding freeze drying protectant, can adopt that filtering with microporous membrane is degerming obtains sterile preparation.
Ultrasonic, high pressure homogenize mentioned here or expressing technique are the particle diameters in order to reduce blank liposome, control the quality of product; Described cross-flow ultrafiltration technique is the ammonium sulfate in the external aqueous phase of removing blank liposomes, to set up ion gradient for medicine carrying.
In above-mentioned preparation process, the step of a most critical is exactly the ammonium sulfate removing outer aqueous phase, produces ammonium sulphate gradient.At present, except conventional, aqueous phase ammonium sulfate method is dialysis, column chromatography, ultrafiltration.All there is some problems in these three kinds of methods, wherein dialysis quantity of sample handling is few, and dialysis time is long; Column chromatography can cause sample greatly to dilute; There will be Pore Blocking in ultra-filtration process, ultrafiltration efficiency declines, be therefore only suitable for the process of laboratory low-volume samples, be not suitable for industrialization large-scale production.
Tangential flow filtration refers to liquid flow direction and filtering direction filtered version in vertical direction.Traditional liquid dead-end filtration (dead end) is most of microporous filter, comprise the filtered version that aseptic filtration adopts, the flow direction of its liquid is consistent with filtering direction, along with the carrying out of filtering, the cake layer that filter membrane surface is formed or gel layer thicknesses increase gradually, and flow velocity reduces gradually.When filter medium be the tiny ultrafilter membrane in aperture or micro-filtration membrane time feed liquid in solid content very high time, take dead-end filtration mode, flow velocity will reduce rapidly, and therefore dead-end filtration can only process the feed liquid of small size.Adopt tangential flow filtration mode, liquid flow produces shearing force at filter media surface, reduces the accumulation of cake layer or gel layer, ensure that the stable rate of filtration.Mainly be applied in cell harvesting, protein concentration, albumen desalination, antibiotic purification etc. at field of medicaments at present, the present invention is applied to the removal of the outer aqueous phase ammonium sulfate of liposome, has larger novelty.
In the present invention's tangential flow filtration device used, filter membrane material is selected from polyethersulfone resin (PES), Triafol T (CT), and filter membrane molecular weight is selected from 10-100KDa, and flow velocity is 20-400ml/ minute, and pressure is 0-5Bar.
Adopt cross-flow ultrafiltration, also have following advantage:
When adopting ammonium sulfate in the outer aqueous phase of cross-flow ultrafiltration technological displacement, quantity of sample handling can reach commercial production scale, and required time is short, and work efficiency is high, and the ion concentration gradient of formation is large, and gained liposome encapsulation is high, is convenient to suitability for industrialized production, lowers production cost; Adopt cross-flow ultrafiltration technology can not change the character of liposome as particles size and distribution; Adopt cross-flow ultrafiltration, whole system can be in sealing state, and all pipelines can clean, and prevents the impact of operating process bacterial micro-organism, and for the aseptic of whole preparation process is given security, this quality control for injection is most important.
Described phase transition temperature refers to temperature during phase co-conversion between lipid gel state and liquid crystal state.Hatch at the temperature higher than lipid phase transition temperature, lipid film permeability can be made to strengthen, and vinorelbine tartrate is easier permeable membrane under the driving of ion gradient, is gathered in the interior aqueous phase of liposome.
The present invention compared with prior art has the following advantages:
The present invention just can obtain without the need to the design adopting three bottles points to pack that good stability, envelop rate are high, Clinical practice vinorelbine tartrate liposome easily.
Adopt liposome to be encapsulated in by vinorelbine tartrate in interior aqueous phase, not direct and vessel wall contact, therefore can not enter rapidly peripheral tissues gap, cause the too high generation venous injury of local drug concentration, can reduce the phlebitic generation in local; In addition, vinorelbine tartrate is encapsulated in intraliposomal aqueous phase, the contact of medicine and external environment (as light, air) can be reduced, reduce the degraded oxidation of medicine, improve the stability of medicine.
Vinorelbine tartrate long circulating liposomes energy significant prolongation medicine circulation time in blood, improves its distribution in vivo, increases the gathering of medicine at tumor locus, improves drug effect, reduce toxic and side effects, thus improve therapeutic index.
Vinorelbine tartrate long circulating liposomes particle diameter of the present invention is 50-200nm, effectively can penetrate tumor vessel, is gathered in tumor locus, realizes passive target effect by enhancing infiltration and delay effect (EPR effect).
The preparation of vinorelbine tartrate long circulating liposomes of the present invention adopts novel slipstream hyperfiltration technique in conjunction with ammonium sulphate gradient, more existing preparation method more easily realizes suitability for industrialized production, and the large and uneven problem of existing technology of preparing particle diameter can be solved, the better quality controlling product; Ammonium sulphate gradient is adopted blank liposome, vinorelbine tartrate mixing to be hatched, just can obtain the vinorelbine tartrate long circulating liposomes that envelop rate is greater than 80%, the method is simple to operate and be that the clinical practice of said preparation provides a convenient and simple method efficiently.
Accompanying drawing explanation
Fig. 1 is the grain size distribution of the vinorelbine tartrate long circulating liposomes according to the embodiment of the present invention 1 preparation.
Fig. 2 is the zeta potential diagram of the vinorelbine tartrate long circulating liposomes according to the embodiment of the present invention 2 preparation.
Fig. 3 is the vinorelbine tartrate long circulating liposomes and the release in vitro test result figure of vinorelbine tartrate free drug prepared according to the embodiment of the present invention 2.
Fig. 4 is take normal saline as contrast, according to the vinorelbine tartrate long circulating liposomes of the embodiment of the present invention 2 preparation, the efficacy testing result figure of vinorelbine tartrate injection.
Detailed description of the invention
Further illustrated the present invention below in conjunction with embodiment, following embodiment only describes the present invention by way of example.But these embodiments also do not mean that the present invention's any restriction in addition.Clearly, those of ordinary skill in the art in scope of the present invention and essence, can carry out various accommodation and amendment to the present invention.It is to be understood that this invention is intended to contain the accommodation and amendment that comprise in the dependent claims.
Reagent and medicine
Soybean phospholipid (Shanghai Taiwei Pharmaceutical Co., Ltd.); HSPC (Shanghai Advanced viecle Technology Co., Ltd.); PEG-DSPE (Shanghai Advanced viecle Technology Co., Ltd.); PEG-PE (Shanghai Advanced viecle Technology Co., Ltd.); PEG-DPPE (Shanghai Advanced viecle Technology Co., Ltd.); Sphingomyelins (the purple chemical reagent work in Shanghai); Ovum Gallus domesticus Flavus lecithin (Shanghai Taiwei Pharmaceutical Co., Ltd.); Cholesterol (Nanjing Xinbai Pharmaceutical Co); Sephadex G-50 (GE company of the U.S.); Vinorelbine tartrate (Guangzhou modern Chinese prescription Chinese medicine research company limited).
The preparation of embodiment 1 vinorelbine tartrate long circulating liposomes:
By hydrogenated soya phosphatide 1.2g, cholesterol 0.3g, PEG2000-PE0.4g with 1.5ml dehydrated alcohol ultrasonic dissolution, be injected into the 30ml300mM ammonium sulfate solution being preheated to 65 DEG C, (rotating speed is 20000rpm) stirs to obtain first product at a high speed, high pressure homogenize 4 times under 20000psi again, with ultra-pure water through cross-flow ultrafiltration system (membrane molecule amount 30kDa, flow velocity is 200ml/min, and pressure is 1bar) replace aqueous phase ammonium sulfate except 10 times of volumes, namely obtain blank liposome; Gained blank liposome is mixed with phospholipid weight ratio 1:10 by medicine with vinorelbine tartrate aqueous solution (concentration is 10mg/ml), hatches 30min for 65 DEG C, obtain vinorelbine tartrate long circulating liposomes.
The preparation of embodiment 2 vinorelbine tartrate long circulating liposomes:
Take soybean phospholipid 1.5g, cholesterol 0.15g, PEG2000-DSPE0.15g, with 1.5ml anhydrous alcohol solution, be injected into the 30ml200mM ammonium sulfate solution being preheated to 65 DEG C, (rotating speed is 25000rpm) stirs to obtain first product at a high speed, high pressure homogenize 4 times under 15000psi again, with 300mM aqueous sucrose solution through cross-flow ultrafiltration system (membrane molecule amount 30kDa, flow velocity 300ml/min, pressure is 1.5bar) replace aqueous phase ammonium sulfate except 20 times of volumes, namely obtain blank liposome.Gained blank liposome is mixed with phospholipid weight ratio 1:30 by medicine with vinorelbine tartrate solution (concentration is 10mg/ml), hatch 1h for 55 DEG C, obtain vinorelbine tartrate long circulating liposomes, non-encapsulated medicine is removed again through cross-flow ultrafiltration system, and its outer aqueous phase is replaced into 300mM sucrose, 10mM histidine buffering liquid (pH=6.5), 4 DEG C save backup.
The preparation of embodiment 3 vinorelbine tartrate long circulating liposomes:
Take Ovum Gallus domesticus Flavus lecithin 1.2g, cholesterol 0.4g, PEG2000-DPPE0.4g, with 1.5ml anhydrous alcohol solution, be injected into the 30ml250mM ammonium sulfate solution being preheated to 65 DEG C, (rotating speed is 20000rpm) stirs to obtain first product at a high speed, 4 times are extruded again with the poly-carbon ester film in 100nm aperture, with 300mM aqueous sucrose solution through cross-flow ultrafiltration system (membrane molecule amount 30kDa, flow velocity 100ml/min, pressure is 1.5bar) replace 5 times of volumes to remove outer aqueous phase ammonium sulfate, namely obtain blank liposome.Gained blank liposome is mixed with phospholipid weight ratio 1:5 by medicine with vinorelbine tartrate aqueous solution (concentration is 5mg/ml), hatch 10min for 60 DEG C, obtain vinorelbine tartrate long circulating liposomes, non-encapsulated medicine is removed again through cross-flow ultrafiltration system, and its outer aqueous phase is replaced into 300mM sucrose, 20mM phosphate buffer (pH=7.4), 4 DEG C save backup.
The preparation of embodiment 4 vinorelbine tartrate long circulating liposomes lyophilized powder:
Take sphingomyelins 1g, cuorin 0.2g, cholesterol 0.06g, PEG2000-DMPE0.3g dissolve with the 5ml tert-butyl alcohol, lyophilization on freeze dryer, add the hydration of 30ml200mM ammonium sulfate more ultrasonic to translucent, with ultra-pure water through cross-flow ultrafiltration system (membrane molecule amount 10kDa, flow velocity 200ml/min, pressure is 1.5bar) replace 15 times of volumes to remove outer aqueous phase ammonium sulfate, namely obtain blank liposome.Get above-mentioned blank liposome 20ml to mix with vinorelbine tartrate aqueous solution (containing vinorelbine tartrate 20mg), 60 DEG C hatch 1h after, add sucrose 0.2g, mannitol 0.5g, lactose 1g makes caffolding agent, in freeze dryer, namely lyophilizing obtains vinorelbine tartrate long circulating liposomes lyophilized powder.
The preparation of embodiment 5 vinorelbine tartrate long circulating liposomes lyophilized powder:
Take DSPC1.2g, DPPG0.12g, cholesterol 0.4g, PEG2000-DPPE0.66g with 1.5ml anhydrous alcohol solution, be injected into the 30ml250mM ammonium sulfate high speed (rotating speed is 20000rpm) being preheated to 65 DEG C to stir, under 20000psi after homogenizing 4 times, with cross-flow ultrafiltration system (membrane molecule amount 100kDa, flow velocity 200ml/min, pressure is 1.5bar) replace 15 times of volumes with except aqueous phase ammonium sulfate, namely obtain blank liposome.Get above-mentioned blank liposome 10ml to mix with phospholipid weight ratio 1:10 by medicine with vinorelbine tartrate, 60 DEG C hatch 1h after, add sucrose 0.5g, trehalose 0.5g to dissolve, on freezer dryer, namely lyophilizing obtains vinorelbine tartrate long circulating liposomes lyophilized powder.
The preparation of embodiment 6 vinorelbine tartrate long circulating liposomes:
Take DMPC1.5g, DSPG0.3g, cholesterol 0.2g, PEG2000-DSPE0.6g with 1.5ml anhydrous alcohol solution, be injected into the 30ml250mM ammonium sulfate high speed (rotating speed is 25000rpm) being preheated to 65 DEG C to stir, under 20000psi after homogenizing 4 times, with ultra-pure water through cross-flow ultrafiltration system (membrane molecule amount 50kDa, flow velocity 200ml/min, pressure is 1.5bar) replace 8 times of volumes with except aqueous phase ammonium sulfate, namely obtain blank liposome.Gained blank liposome is mixed by medicine phospholipid weight ratio 1:40 with vinorelbine tartrate solution, hatch 20min for 60 DEG C, obtain vinorelbine tartrate long circulating liposomes, non-encapsulated medicine is removed again through cross-flow ultrafiltration system, and its outer aqueous phase is replaced into 20mM HEPES buffer (pH=7.4), 4 DEG C save backup.
The preparation of embodiment 7 vinorelbine tartrate long circulating liposomes:
Take DPPC1.5g, DOPE0.1g, cholesterol 0.2g, PEG2000-DSPE0.4g with 1.5ml anhydrous alcohol solution, be injected into the 30ml250mM ammonium sulfate high speed being preheated to 65 DEG C to stir, under 20000psi after homogenizing 4 times, with ultra-pure water through cross-flow ultrafiltration system (membrane molecule amount 30kDa, flow velocity 200ml/min, pressure is 1.5bar) replace 8 times of volumes with except aqueous phase ammonium sulfate, namely obtain blank liposome.Gained blank liposome is mixed with phospholipid weight ratio 1:10 by medicine with vinorelbine tartrate solution, hatches 20min for 60 DEG C, obtain vinorelbine tartrate long circulating liposomes.
The preparation of embodiment 8 vinorelbine tartrate long circulating liposomes:
Take DOPC1.5g, DSPE0.2g, cholesterol 0.4g, PEG2000-DSPE0.4g with 1.2ml anhydrous alcohol solution, be injected into the 30ml250mM ammonium sulfate high speed being preheated to 65 DEG C to stir, under 20000psi after homogenizing 4 times, with aqueous phase ammonium sulfate except cross-flow ultrafiltration system (membrane molecule amount 30kDa), namely obtain blank liposome.Gained blank liposome is mixed with phospholipid weight ratio 1:10 by medicine with vinorelbine tartrate solution, hatches 20min for 60 DEG C, obtain vinorelbine tartrate long circulating liposomes.
Performance test
Size and distribution is tested:
After the vinorelbine tartrate long circulating liposomes dilute with water that Example 1 is obtained, measure its size and distribution through particle size determination instrument.Result is as Fig. 1, and the equal particle diameter of its Z is 72.95nm, and polydispersity index is 0.160, and distribution of particles is comparatively even.
The particle diameter of liposome in other embodiment and distribution results is recorded in table 1 with method.
Table 1
| Embodiment 2 | Embodiment 3 | Embodiment 4 | Embodiment 5 | Embodiment 6 | Embodiment 7 | Embodiment 8 | |
| Particle diameter (nm) | 74.16 | 77.58 | 80.16 | 79.83 | 74.32 | 105.3 | 108.1 |
| Polydispersity index | 0.111 | 0.17 | 0.249 | 0.263 | 0.236 | 0.403 | 0.405 |
The mensuration of Zeta potential
The vinorelbine tartrate long circulating liposomes that Example 2 is obtained, measures its zeta current potential with nanosizer90.Result is as Fig. 2, and its zeta current potential is-10.6mv.
The mensuration of envelop rate:
The vinorelbine tartrate long circulating liposomes that Example 1-8 obtains carries out the mensuration of envelop rate.
Chromatographic condition: chromatographic column Waters
c
18post (4.6mm × 250mm, 5 μm); Mobile phase is acetonitrile-50mmol/L potassium dihydrogen phosphate (containing 0.01% triethylamine, phosphoric acid adjusts pH to 4.0) (41: 59); Flow velocity 1ml/min; Column temperature 30 DEG C; Determined wavelength 269nm; Sample size 20 μ l.
Get fully swelling Sephadex G-50 polydextran gel appropriate, prepare gel column (45cm × 1cm), precision measures vinorelbine tartrate long circulating liposomes 0.5ml upper prop, with PBS(pH=7.4) eluting, collect the stream part 16ml altogether containing liposome, put in 50ml measuring bottle, by methanol constant volume, shake up rear precision to measure 5ml and put in 10ml measuring bottle, adopt HPLC to measure the dose W wrapped up in nanometer formulation; Separately get vinorelbine tartrate long circulating liposomes 0.5ml and be placed in 50ml measuring bottle, with method operation, measure the total dose W in liposome
0.According to formula envelop rate %=(W/W
0) the * 100% average envelop rate calculating vinorelbine tartrate long circulating liposomes, result is as table 2.
Table 2
| Embodiment 1 | Embodiment 2 | Embodiment 3 | Embodiment 4 | Embodiment 5 | Embodiment 6 | Embodiment 7 | Embodiment 8 | |
| Envelop rate | 91.5 | 98.6 | 99.1 | 85.9 | 90.3 | 99.7 | 89.6 | 94.3 |
Release in vitro measures:
The vinorelbine tartrate long circulating liposomes that Example 1 is obtained and vinorelbine tartrate solution (vinorelbine free drug) carry out the mensuration of release in vitro, and wherein vinorelbine tartrate solution preparation method is: take a certain amount of vinorelbine tartrate and be made into ultra-pure water the solution that concentration is 1mg/ml.
Precision measures vinorelbine tartrate liposome 1ml, add in bag filter (bag filter molecular weight 8000-14000Da), tighten two ends, be placed in the conical flask that 10ml release medium (pH7.4 phosphate buffer) is housed, constant speed vibration (100r/min) under (37.0 ± 0.5) DEG C condition.Respectively 0.5,1,2,4,8,12,24h sampling, sample introduction measures, and calculates preparation (%).Examine or check the release conditions of vinorelbine tartrate free drug simultaneously.With preparation (Q) to time (t) mapping, release profiles is shown in Fig. 3.
As seen from Figure 3, compared to free drug, vinorelbine tartrate long circulating liposomes drug release is comparatively slow, and during 24h, preparation is 51.2%, has showing sustained release effect.
Vascular stimulation test:
By Vinorelbine injection (Qilu Pharmaceutical Co., Ltd.) with carry out body surface area conversion by the vinorelbine long circulating liposomes that embodiment 3 is obtained by clinical medicine dose (30mg/ time) and draw experimental rabbit dosage (1.4mg/kg).The aseptic normal saline solution Fresh of dosage of 2ml/kg is pressed before test.Select the healthy new zealand white rabbit (source: Shanghai Experimental Animal Center) 6 of body weight 2.5-3kg, male and female have concurrently.After injection site iodine tincture and ethanol disinfection, wherein three white rabbits are in auris dextra auricular vein injection Vinorelbine injection, and the physiological saline solution injection of left ear injection same volume in contrast; Another three white rabbits are in auris dextra auricular vein injection vinorelbine tartrate long circulating liposomes suspension, and the physiological saline solution injection of left ear injection same volume in contrast.Once a day, continuous three days, after last administration 24h, reporting situations of perusal injection site.
Table 3
As shown in Table 3, the blood vessel irritation of vinorelbine long circulating liposomes is weaker than Vinorelbine injection.
Pharmacokinetics is tested:
Get healthy male SD rat (source: Shanghai Experimental Animal Center) 8 (body weight 200-220g), be divided into 2 groups at random.Tail vein is after by the dosage of 15mg/kg, tail vein injection gives vinorelbine tartrate long circulating liposomes in embodiment 1 and vinorelbine tartrate injection (Qilu Pharmaceutical Co., Ltd.) respectively, respectively at 0.083h, 0.25h, 0.5h, 1h, 2h, 4h, 6h, 8h, 24h gets blood by large rathole frame venous plexus, amount for taking blood is about 0.2ml, be placed in heparinization centrifuge tube, centrifugal 10min under 10000rpm, get 20 μ l blood plasma, add 20 μ l pure water and 460 μ l methanol, vortex 1min,-20 degree place 1h in order to protein precipitation, then in the centrifugal 10min of 20000g.Get supernatant sample introduction and measure blood drug concentration.Pharmacokinetic parameter WinNonlin Professional v6.3 (Pdayarsight, USA) software adopts non-compartment model analyzing and processing.
Table 4
Note: K
elfor elimination rate constant, t
1/2for the half-life, AUC is area under serum drug concentration
Rat Pharmacokinetic experiments result (table 4) shows, compared with injection group, vinorelbine tartrate liposome group Increased Plasma Half-life 2.63 times, AUC increases by 2.01 times, has significant difference (P<0.05) between two groups.
Efficacy testing:
Nude mice conforms 5d, makes 1 × 10 after being digested by the lewis lung carcinoma cell of exponential phase
8/ ml cell suspension, at Balb/c nude mice (source: Shanghai Experimental Animal Center) right fore subcutaneous injection 0.1ml cell suspension, sets up lotus tumor model.Treat that mouse tumor average external volume grows to 50-100mm
3during left and right, nude mice is divided into 3 groups at random, often organizes 6, be respectively the vinorelbine tartrate liposome group (10mg/kg) in normal saline group, vinorelbine tartrate injection group (purchased from Qilu Pharmaceutical Co., Ltd., dosage is 10mg/kg), embodiment 2.Adopt tail vein injection administration, be respectively administered once respectively at the 1st, 8,15 day.After administration, animal is normally raised, and measures the major diameter (R) of tumor and minor axis diameter (r) to observe the growing state of tumor, according to following formulae discovery gross tumor volume: V=R × r every 2 days
2/ 2.
As seen from Figure 4, compared with normal saline group, vinorelbine tartrate liposome and injection all have obvious growth inhibited effect to pulmonary carcinoma Nude Mice, and the vinorelbine tartrate liposome group tumor killing effect of same dose (10mg/kg) comparatively injection group is good.
Claims (10)
1. a vinorelbine tartrate long circulating liposomes, it comprises:
2. vinorelbine tartrate long circulating liposomes according to claim 1, wherein, described phospholipid substance is for being selected from soybean phospholipid, hydrogenated soya phosphatide (HSPC), Ovum Gallus domesticus Flavus lecithin, hydrolecithin, sphingomyelins, cuorin, distearoyl phosphatidylcholine (DSPC), dipalmitoyl phosphatidyl choline (DPPC), dimyristoyl phosphatidyl choline (DMPC), DOPC (DOPC), DSPE (DSPE), DPPE (DPPE), DMPEA (DMPE), DOPE (DOPE), DSPG (DSPG), DPPG (DPPG), one or more in GLYCEROL,DIMYRISTOYL PHOSPHATIDYL (DMPG) and DOPG (DOPG), preferably be selected from soybean phospholipid, hydrogenated soya phosphatide, Ovum Gallus domesticus Flavus lecithin, one or more in hydrolecithin and DSPC.
3. vinorelbine tartrate long circulating liposomes according to claim 1, wherein, described long circulating film material is polyglycol derivatization phospholipid, it is that peg molecule is combined into by the active group on covalent bond and phospholipid molecule, preferably, be selected from mPEG2000-DSPE (PEG-PE), Polyethylene Glycol-DMPEA (PEG-DMPE), Polyethylene Glycol-DPPE (PEG-DPPE) and PEG2000-DSPE (PEG-DSPE), the molecular weight of wherein said PEG is 500-5000Da, more preferably, the molecular weight of described PEG is 1000-5000Da, most preferably, described PEG molecular weight is 2000Da.
4. vinorelbine tartrate long circulating liposomes according to claim 1, wherein, the particle diameter of described vinorelbine tartrate long circulating liposomes is 50-200nm, is preferably 50-120nm, more preferably 70-110nm.
5. vinorelbine tartrate long circulating liposomes according to claim 1, wherein, the entrapment efficiency in described vinorelbine tartrate long circulating liposomes is greater than 80%, is preferably greater than 85%, more preferably greater than 90%, more preferably greater than 95%.
6. vinorelbine tartrate long circulating liposomes according to claim 1, wherein, described vinorelbine tartrate long circulating liposomes comprises the acceptable buffer of pharmacy further, and its concentration range is 10-50mM, pH is 5.5-8; Preferably, the acceptable buffer of described pharmacy is selected from histidine buffering liquid, phosphate buffer and HEPES buffer.
7. vinorelbine tartrate long circulating liposomes according to claim 1, wherein, described vinorelbine tartrate long circulating liposomes comprises freeze drying protectant further, and its consumption is pressed phospholipid substance weight ratio and calculated, and the phospholipid substance of 1 weight portion adds the freeze drying protectant of 0.1-5 weight portion; Preferably, described freeze drying protectant be selected from sucrose, lactose, mannitol, trehalose and maltose one or more.
8. the preparation method of vinorelbine tartrate long circulating liposomes according to any one of claim 1 to 5, it comprises the following steps:
A takes the phospholipid substance of formula ratio, long circulating film material, cholesterol are dissolved in dehydrated alcohol and obtain organic facies, and being injected into concentration is stir in the ammonium sulfate solution of 100-400mmol/L, through homogenizing, ultrasonic or expressing technique, forms blank liposome;
Or, take the phospholipid substance of formula ratio, cholesterol and long circulating material and be dissolved in the tert-butyl alcohol, after lyophilization, add the ammonium sulfate solution dispersion that concentration is 100-400mmol/L, form blank liposome;
Step a gained blank liposome is passed through tangential flow ultra-filtration unit with pure water or aqueous sucrose solution by b, and the 5-30 of displacement liposome volume doubly, sets up ammonium sulphate gradient to remove outer aqueous phase ammonium sulfate;
Vinorelbine tartrate adds in step b process gained blank liposome by c, hatches 10min-1h and carries out medicine carrying, obtain vinorelbine tartrate long circulating liposomes at the temperature higher than lipid phase transition temperature.
9. preparation method according to claim 7, wherein, further, add with buffer salt solid form after step c or be replaced into the acceptable buffer of pharmacy through cut stream ultrafiltration apparatus, the concentration range of the acceptable buffer of described pharmacy is 10-50mM, pH is 5.5-8; Preferably, the acceptable buffer of described pharmacy is selected from histidine buffering liquid, phosphate buffer and HEPES buffer; Or
Further; add in the medicine carrying long circulating liposomes of step c and be selected from one or more in sucrose, lactose, mannitol, trehalose and maltose as freeze drying protectant; its consumption is pressed phospholipid substance weight ratio and is calculated, and 1 weight portion phospholipid substance adds 0.1-5 weight portion freeze drying protectant.
10. preparation method according to claim 8 or claim 9, wherein, add freeze drying protectant after above-mentioned steps c or in the medicine carrying long circulating liposomes of step c after, adopts that filtering with microporous membrane is degerming obtains sterile preparation.
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