CN100471493C - Liposome preparation containing Oxaliplatin - Google Patents
Liposome preparation containing Oxaliplatin Download PDFInfo
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- CN100471493C CN100471493C CNB2003101247766A CN200310124776A CN100471493C CN 100471493 C CN100471493 C CN 100471493C CN B2003101247766 A CNB2003101247766 A CN B2003101247766A CN 200310124776 A CN200310124776 A CN 200310124776A CN 100471493 C CN100471493 C CN 100471493C
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Abstract
The invention discloses a liposome preparation containing antineoplastic metallic complex which is derived by hydrophilic polymers and aglycones, in one embodiment, the antineoplastic metallic complex includes Oxaliplatin, the hydrophilic polymers include polyethylene glycol, the aglycone is transferrin. In accordance with the invention, the transferring expressed on the surface of the tumor cell can facilitate the absorption of agent in the liposome by the tumor cells. The invention also provides the pharmaceutical composition containing the liposome and method for application.
Description
Invention field
The present invention relates to a kind of Liposomal formulation as antineoplastic agent.
Background technology
Following background of the present invention only offers reader understanding the present invention, and is not described in detail or makes up prior art of the present invention.
Cisplatin is a kind of antitumor drug, be widely used as treating multiple cancer, comprise testicular tumor, bladder tumor, the renal pelvis tumor, the ureter tumor, carcinoma of prostate, ovarian cancer, the cancer of head and cervical region, non-small cell type pulmonary carcinoma, esophageal carcinoma, cervical cancer, yet neuroblastoma and gastric cancer., because cisplatin toxicity is accompanied by greatly and usually such as the kidney disorder that comprises acute renal failure, the inhibition of marrow function, feel sick, vomiting and these disadvantageous side effect of loss of appetite, so it has very big shortcoming. in order to overcome these shortcomings, the derivant of having developed cisplatin is carboplatin and oxaliplatin for example. the therapeutical effect of oxaliplatin and nephrotoxicity identical and to cause telling property relatively low with cisplatin.
Yet liposome often is used to reduce the toxicity of some drugs, and chemical compound lot can not be wrapped in the liposome effectively, but also is accompanied by the stability problem of Liposomal formulation. Liposomal formulation also must be able to make it be sent to target cell effectively.
Therefore, can improve combination and the method that the storage stability of the Liposomal formulation that contains antineoplastic agent and they are sent to the effective degree of target tumor cell great value is arranged.
Summary of the invention
The invention provides a kind of Liposomal formulation that contains the antitumor metal complex at liposome. this liposome hydrophilic polymer and aglucon derivatization. in various embodiments, the antitumor metal complex is selected from: gold, copper, platinum, nickel, palladium, ferrum, ruthenium and osmic complex. in preferred embodiments, the antitumor metal complex is an oxaliplatin. in various embodiments, aglucon is selected from: transferrin, folic acid, hyaluronic acid, sugar chain-for example galactose or mannose, monoclonal antibody, pyridoxal phosphate, vitamin B12, sialic acid lewis X (sialyl Lewis X), the epidermal growth factor of peptide-for example, basic fibroblast growth factor, VEGF, the bonding molecule of vascular cell (VCAM-1), the bonding molecule of iuntercellular (ICAM-1), the bonding molecule of platelet endothelium (PECAM-1), arginine-glycine-aspartic acid (RGD) peptide, or aspartic acid-glycine-arginine (NGR) peptide, Fab ' segment with monoclonal antibody. in various embodiments, hydrophilic polymer is selected from: Polyethylene Glycol (PEG), poly-Propylene Glycol, poly-hydroxyl propylene glycol, polypropylene glycol, poly-methyl propanediol and poly-hydroxyl expoxy propane. in one embodiment, hydrophilic polymer is that Polyethylene Glycol and aglucon are transferrins.
" polymer " formed by two or more less molecules (monomer) are covalently bound together. and " hydrophilic polymer " is diffluent polymer in aqueous solution. in various embodiments, the concentration of the contained oxaliplatin of Liposomal formulation is 1-20mg/ml, 1-10mg/ml, 5-10mg/ml, 10-15mg/ml, 15-20mg/ml, or about 8mg/ml, 7.5-8.5mg/ml, 7-9mg/ml, " approximately " in 6-10mg/ml or the 7-10mg/ml. this paper context refers to fluctuate 10%. in other embodiments, can use the oxaliplatin of any concentration, for example, about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% or 25% (w/v). in one embodiment, Liposomal formulation in sucrose solution is provided. in various embodiments, concentration of sucrose is approximately 5% in the solution, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% (w/v). in various embodiments, about 9% (w/v) arranged in the solution, 8.5-9.5% (w/v), the sucrose of 8-10% (w/v) or 7-11% (w/v). " derivatization " means covalent bond between liposome and hydrophilic polymer and the aglucon. can carry out derivatization by aglucon being connected on the hydrophilic polymer that links to each other with molecule in the double-layer of lipoid structure that stably is in liposome or other molecules. for example, aglucon can be connected the far-end of hydrophilic polymer, and the near-end of hydrophilic polymer is connected on the polar end group of the phospholipid molecule in the double-layer of lipoid structure that stably is in liposome. also can be by carrying out derivatization on the molecule in the double-layer of lipoid structure that is in liposome with aglucon being advanced to be connected on homeostasis. for example, aglucon can directly be connected on the double-deck phospholipid member of liposome. in another embodiment, can carry out derivatization by aglucon is linked to each other with connector covalency on being connected hydrophilic polymer, and on the covalently bound molecule in being embedded in the double-layer of lipoid structure of liposome of this hydrophilic polymer.
In one embodiment, can be by hydrophilic polymer is covalently bound in lipophilic molecule (for example, phospholipid molecule or fatty acid molecule) on carry out derivatization, this lipophilic molecule is the double-deck part of liposome, and aglucon covalently bound on hydrophilic polymer, (directly connect or connect) by connector carry out derivatization. in one embodiment, one end of hydrophilic polymer covalently bound in liposome on the head group of lipid (for example, the PEG molecule is covalently bound on DSPE-DSPE), and the other end is connected on the aglucon. and " aglucon " refers to the material that links to each other with the receptor or the surface antigen of mammalian cell surface. and " sugar chain " refers to monosaccharide, disaccharidase, oligosaccharide and polysaccharide. in various embodiments, sugar chain refers to galactose or mannose, or their polymer. but in other embodiments, sugar chain refers to rhamnose, trehalose, xylose, arabinose, or glucose molecule, or the chain that constitutes of two or more these or other monosaccharide molecules. in other embodiments, sugar chain refers to disaccharidase, for example, sucrose, lactose, maltose, dextrinose, trehalose, cellobiose, or the polymer of these disaccharide arbitrarily, or oligosaccharide or polysaccharide chain.
On the other hand, the invention provides a kind of Liposomal formulation that is included in the intravital antitumor metal complex of lipid that contains. hydrophilic polymer stably is present in the liposome and aglucon links to each other with hydrophilic polymer. and hydrophilic polymer is by linking to each other and stable existence with phospholipid as liposome double-decker member's a part. and " stable existence " is meant that the liposome that links to each other with polymer still has at least 75% polymer to link to each other with liposome in preservation under 4 ℃ the temperature after 90 days. in various embodiments, under 4 ℃ temperature, preserved 30 days, 60 days, 90 days, 120 days, still have at least 75% after 150 days or 180 days, 80%, 85%, 90% or 98% polymer links to each other with liposome. in one embodiment, polymer by covalently bound (directly connect or indirectly connect by connector) on as the phospholipid of the double-decker member's of liposome a part and stable existence.
On the other hand, the invention provides the pharmaceutical composition of treatment tumor. this pharmaceutical composition comprises preparation of the present invention and pharmaceutically acceptable carrier.
The present invention also provides the method for treatment tumor. and these methods comprise that the patient to this treatment of needs takes preparation of the present invention or pharmaceutical composition. and the people of this treatment of needs comprises trouble tumor or cancered people, but also comprising the people who manages to prevent or stop tumor development. said composition can be used to dwindle or kill already present tumor, can be used for also preventing that already present tumor from becoming big or further diffusion. in various embodiments, tumor refers to colon cancer, gastric cancer, hepatocarcinoma, pulmonary carcinoma, breast carcinoma, ovarian cancer, cancer of pancreas, esophageal carcinoma, or the cancer of other type.
General introduction to summary of the invention is non-limiting above, other features and advantages of the present invention will be below to embodying in the specific descriptions of preferred implementation and the claim.
Description of drawings
Fig. 1 is the flow chart that shows the preparation process of the liposome that combines transferrin of the present invention.
Fig. 2 is that the Cytotoxic chart .y axle that shows oxaliplatin is illustrated in survival cells after the oxaliplatin treatment of 1 μ g/ml, 5 μ g/ml, 100 μ g/ml. the percent of the untreated control cells number of this value representation (concentration that is oxaliplatin is 0 o'clock). and data show LD
50(i.e. the concentration of oxaliplatin during 50% cell death) is 8 μ g/ml.
Fig. 3 is the table that shows the physical characteristic of not modified liposome, PEG liposome and transferrin-PEG liposome
Fig. 4 is the figure of transferrin receptor number that shows the cell surface of normal leukocyte and the deutero-cell line of all kinds tumor.
Fig. 5 is the table that shows the incidence rate of the bloody ascites of the rat take not modified liposome, PEG liposome and transferrin-PEG liposome and tumor nodule.
Fig. 6 shows Cytotoxic the illustrating of oxaliplatin liposome of the present invention to Colon 26 cells. this diagram has shown the LD of oxaliplatin solution, Bare-Liposomal formulation, PEG liposome and transferrin-PEG liposome to Colon 26 cells
50Value is respectively 2 μ g/ml, 60 μ g/ml, 18 μ g/ml and 8 μ g/ml.
Fig. 7 is that the liposome that shows oxaliplatin of the present invention illustrates the Cytotoxic of AsPC-1 cell. this diagram has shown the LD of oxaliplatin solution, Bare-Liposomal formulation, PEG liposome and transferrin-PEG liposome to the AsPC-1 cell
50Value is respectively 5 μ g/ml, 45 μ g/ml, 75 μ g/ml and 8 μ g/ml.
Detailed description to invention
Liposome or lipid capsule are the spherical double-layer of lipoid structures that the water-based kernel is arranged. it onion spline structure that comprises a series of bimolecular lipid layers is separated by this by the aqueous solution, outermost layer is lipid. the bioactivator that liposome is used for various uses at parcel have an enormous advantage liposome can be individual layer or multilayer. multilamellar liposome is made of many bilayers that are inserted with aqueous medium. they have comprise a series of by the onion spline structure of aqueous solution bimolecular lipid layer separated from one another, its outermost layer is lipid. the capsule of individual layer has the spherical double-layer of lipoid of the single parcel aqueous solution. according to their size, it is divided into little individual layer capsule (SUV), its diameter is 250nm or less, with large individual layer capsule (LUV), no matter its diameter is that individual layer capsule (little or large) or multilamellar liposome all can be applied to the present invention greater than 250nm.. in one embodiment of the invention, liposome is little individual layer capsule, and its diameter is less than 200nm.
When forming liposome, the molecule in the aqueous solution is wrapped into the aqueous core of liposome in the heart, therefore it is protected the impact of avoiding external environment. because the lipid physical efficiency merges with cell membrane, the lipid physical efficiency of injecting in the body is delivered to cytoplasm effectively.
Oxaliplatin, anti-form-1-1, the platinum of 2-DACH (II) is suitable-oxalate base complex compound (platinum (II) cis-oxalato complex), be a kind of complex compound of platinum, represent with following formula:
Oxaliplatin is a kind of effective antitumour or cancer therapy drug, its therapeutic activity and renal toxicity identical with cis-platinum and to cause telling property lower. it is used in particular for treating colon cancer, but also effectively be used for the treatment of cancer of the stomach, liver cancer, lung cancer, breast cancer, oophoroma, cancer of pancreas, cancer of the esophagus and other cancer. the preparation technology of oxaliplatin in this area be known (for example, referring to Japanese Patent Publication JP-9-40685). in composition of the present invention, oxaliplatin may reside in the aqueous solution, solution is wrapped in the liposome, so that the concentration of oxaliplatin in liposome is 1-20mg oxaliplatin/ml solution. in one embodiment, Liposomal formulation of the present invention contains 1-20 μ g oxaliplatin/mg lipid, and 100-300 μ g aglucon/mg lipid.
In one embodiment; liposome contains oxaliplatin in 9% sucrose (w/v), 8-10% sucrose (w/v), 8.5-9.5% sucrose (w/v) or about 9% sucrose (w/v) solution. find that this sucrose concentration can make the more oxaliplatin of volume of liposome. because 9% sucrose solution etc. ooze; contain the lipid physical efficiency of oxaliplatin by normal saline dilution; and stable in the blood of circulation. be surprised to find that this lipid physical efficiency is transported to tumor locus (damage) and significant oxaliplatin does not occur leaks from liposome. in addition; the general utility functions of sugar juice can be protected liposome membrane, and a kind of like this Liposomal formulation can freeze drying and can long preservation and have no adverse effects.
Liposomal formulation
In one embodiment, liposome of the present invention is by phospholipid is dissolved in the appropriate organic solvent, then the solution that generates is dispersed in the aqueous solution that contains therapeutic component, then the dispersion that forms being carried out ultrasonic or anti-phase evaporation. many phospholipid can be used for the present invention. for example, lecithin, PHOSPHATIDYL ETHANOLAMINE, the stearic bicine diester PHOSPHATIDYL ETHANOLAMINE, Phosphatidylserine, phosphatidylinositols, LYSOLECITHIN SUNLECITHIN A, phosphatidyl glycerol, sphingomyelins or phosphatidic acid all can be used for the present invention. for stability and the permeability that improves lipid film, can add other low-polarity component, for example, cholesterol or other steroid, stearic hydramine, phosphatidic acid, the dotriacontane phosphate ester, tocopherol or lanoline extract.
In other embodiments, can with phospholipid, neutral lipid, surfactant or other relevant similar amphipathic compound lipid capsule of the present invention arranged. can be by formula A-B with these materials classifications, A is hydrophilic, normally polar group, for example, carboxylic group; B is hydrophobic, promptly lipophilic, non-polar group, long-chain fat hydrocarbyl group for example. as previously mentioned, compositions that it should be noted that lipid components can alter a great deal and can obviously not reduce the parcel performance, also can use above-mentioned other lipid outside listed.
In addition, can be absorbed by the cell endothelial system to prevent liposome with the hydrophilic polymer modified liposome, and strengthen the absorption of tumor tissues to liposome. with hydrophilic polymer the modification of liposome has been prolonged the half-life of liposome in blood. many hydrophilic polymeies can be used for the present invention, for example comprise Polyethylene Glycol (polyethylene glycol), poly-Propylene Glycol, poly-hydroxyl propylene glycol, polypropylene glycol, poly-methyl propanediol, poly-hydroxyl expoxy propane, polyoxyalkylene (polyoxyalkylenes), polyetheramine. other polymer comprises polyvinylpyrrolidone, polyvinyl methyl ether, Ju Jia oxazolin, the Ju ethyl oxazoline, poly-Qiang Bing oxazolin, poly-hydroxypropyl methyl acrylamide, PMAm, polydimethylacrylamiin, poly-hydroxypropyl methyl acrylate, the poly-hydroxyethyl acrylate, hydroxy methocel, hydroxyethyl-cellulose, Polyethylene Glycol and poly-asparagine. polymer can be used as homopolymer or block copolymer or random copolymer.
The aglucon derivatization
The further feature of Liposomal formulation of the present invention is to the liposome derivatization with aglucon. in one embodiment, aglucon is a transferrin. transferrin is a kind of iron-binding protein, it is through being positioned at the transferrin receptor of cell surface, by the Fe that is connected with transferrin
3+Endocytosis impel ferrum to absorb in the mammiferous cell to go. therefore transferrin plays a part to cell for ferrum. transferrin receptor has the expression of bigger quantity usually in tumor cell than in normal cell. this all exists in many different types of tumors. therefore, by transferrin is connected with medicine, increase the absorption of tumor cell to medicine by transferrin receptor.
On the other hand, the invention provides a kind of pharmaceutical composition for the treatment of tumor, be Liposomal formulation of the present invention and medicine acceptable carrier. carrier comprises, sterilized water for example, buffer solution and saline. this pharmaceutical composition can further comprise various salt on demand arbitrarily, sugar, protein, starch, gelatin, vegetable oil, Polyethylene Glycol. in various embodiments, Liposomal formulation of the present invention can be through intravenous injection or the administration of Hepatic artery injecting systems. but also can use other administering mode, for example, the local injection of tumor locus, vein or intra-arterial injection, instillation to the tumor outside, or through pill injection or the continuous parenteral of injecting. the multilamellar lipid capsule also can be passed through the local injection administration, even with the form administration of ointment. can be according to route of administration, the order of severity of the state of an illness, changed dosage by the degree of the patient's that treated age and the state of an illness and side effect, but usually about 10 to 100mg/m
2In the scope in/sky. medicine composite for curing tumor of the present invention and cancer are effective.
The open of all patents that this paper quotes and document all is hereby expressly incorporated by reference, and comprises all forms, figure and claims.
Following embodiment further specifies the present invention. and the following examples are nonrestrictive, only represent aspects more of the present invention and feature.
The specific embodiment
The preparation of embodiment 1 liposome
This embodiment provides a general strategy for preparing liposome composition of the present invention. the technical staff that can certainly adopt the ability city is by with reference to getable other strategy of this disclosed strategy. this embodiment described use anti-phase evaporation (REV) preparation Liposomal formulation of the present invention embodiment (for example, referring to U.S. Pat-4,235,871 more detailed description).
In order stably to be kept at hydrophilic polymer in the double-layer of lipoid, the phospholipid derivative that can prepare hydrophilic polymer earlier, and then prepare liposome together with phospholipid derivative and phospholipid and lipid. hydrophilic polymer is synthesized derivant, wherein the phospholipid moiety chemistry is connected on the polymer. and therefore the phospholipid moiety of derivant has played the effect of stably preserving derivant in double-layer of lipoid. and the phospholipid derivative of hydrophilic polymer can prepare with a kind of like this mode, for example, as described in the U.S. Pat-5013556. the hydrophilic polymer as Polyethylene Glycol, in alkali organic solvent, handle to activate an end of hydrophilic polymer with cyanuric acid, its product and phospholipid-as the phosphatidyl ethanol synthesis, having obtained the phospholipid derivative of hydrophilic polymer thus. other end of hydrophilic polymer also can have functional group, for example carboxyl or maleimide, aglucon connects thereon.
Therefore, phospholipid (for example distearoylphosphatidylcholine (DSPC)) or (DSPE (DSPE)), the phospholipid derivative (Polyethylene Glycol-PHOSPHATIDYL ETHANOLAMINE) of lipid (cholesterol) and hydrophilic polymer mixes and is dissolved in the suitable organic solvent. and phospholipid and lipid are with the mixed between the 2:1 to 1:1, and the ratio between the 3:1 to 1:3 also is fit to. in one embodiment, having used molecular weight approximately is 2750 DSPE-PEG, wherein PEG has accounted for the phospholipid derivative that 2000. usefulness account for the hydrophilic polymer of all TL 5mol% greatly greatly and has mixed, and the phospholipid derivative weight ratio of hydrophilic polymer accounts for the 1mol% that is at least about of whole lipids, 2mol%, 3mol%, 4mol%, 5mol%, 6mol%, 7mol%, 8mol%, 9mol%, 10mol% is suitable. in another embodiment, having used the phospholipid that is at least about whole lipid 15mol%. these consumptions make the lipid physical ability retain the maximum time in blood. and the solution that makes mixes with the water buffer solution of oxaliplatin. in other embodiments, DSPC:CH:DSPE-PEG=1:1:0.1 (mol ratio), DSPE/PEG accounts for the concentration of oxaliplatin in the 5mol%. aqueous solution of TL greatly approximately from 5 to 10mg/ml (yet also being fit to from 1 to 20mg/ml concentration). and the ultrasonic back of the mixture of solvent revaporization desolvates to remove. and prepared liposome is that volume is fractionated, so that the liposome that contains oxaliplatin of the about 0.2 μ m of diameter to be provided.
Then, aglucon is connected on the liposome. in this embodiment, aglucon is a transferrin. transferrin is the protein purification (Biocompare that commerce can get, Burlington, CA). for transferrin is connected on the liposome, can earlier an other functional group be incorporated into and be connected on the hydrophilic polymer that is present in the phospholipid derivative in the liposome membrane. therefore, a hydrophilic polymer that has imported carboxyl or maleimide groups at an end is added on the phospholipid has the liposome of carboxyl or maleimide groups to form outer surface.
Be under the situation of carboxylic group endways, the hydrochloride (EDC-HCL) and the N-hydroxysulphosuccinimide of 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide are connected on the liposome. and the liposome that connector is arranged of gained and transferrin reaction are to obtain taking off-form (anapo-form) of the bonded liposome of transferrin, and wherein transferrin is combined in outer surface. and the liposome of handling gained with ferric citrate/sodium citrate is to obtain the complete form (Fig. 1) of the bonded liposome of transferrin.
Have endways under the situation of maleimide groups, the liposome and the transferrin reaction of having introduced the SH group earlier of the connector of connection arranged, then to add ferrum, with the complete form of the bonded liposome of preparation transferrin with aforementioned same mode.
Except these embodiment, other amino, sulfydryl, acetaldehyde and carbodiimide group also can be as the connectors that is fit to.
In the selectable mode of this preparation liposome, the formation step of lipid film is to carry out in a partially filled container that the inert solid contact material arranged. important variation can be a size, volume distributed median, the composition of shape and contact material. the primary feature of contact material is: (1) contact material is inert to used material in the prescription, in other words, at contact material and used lipid, lipophilic substance, there is not unwanted reaction between organic solvent or the waterborne liquid, and (2) contact material is solid in reactions steps always, in other words, contact material should not dissolve or be cracked, and it is thin film of liquid phase to provide a suitable surface of solids to support. the tentative test of front with bead or glass bead as the inert solid contact material, proved that these materials are particularly suitable for. also expect the stainless steel ball of Metal Ball-for example, and synthetic-as plastics, also being suitable under the suitable environment. spherical contact material provides maximum surface area and has been easy to flow in whipping step under given volume, also can use other rule or irregular shapes.
The size of used in any application contact material depends on the scale of operation, stirring intensity and other factors. as an embodiment, the volume that the contact material of usually wishing to use has make container volume to the ratio of the volume of one contact material 50 to 50, between 000. in general, the diameter of spherical contact material at 1.0mm between the 100mm. what also will replenish is that the size of contact material should be distributed in certain scope. but the contact material of same volume can fully satisfy needs of the present invention. the number of used contact material is by their shape, size, the size of container, used volume of organic solvent and lipid or the amount of dissolved lipophilic substance determine. with suitable quantity to be increased in the surface area in the evaporation step, and increase the gross area of the lipid membrane that forms, but, in container, to keep sufficient volume for motion contact material in whipping step.
Can be suitable by lipid components (deutero-with hydrophilic polymer) and other any lipophilic substances and oxaliplatin are dissolved in, come together to prepare liposome in the normally nonpolar organic solvent. select organic solvent to make it from lipid, remove substantially and do not influence the lipophilic substance that other comprise in prescription by evaporation. the example of suitable solvent comprises ether, ester, alcohol, ketone and various aromatic and aliphatic hydrocarbon compound, comprising fluorocarbon. can use separately or co-solvent, 2:1 mixture as chloroform and methanol. can remove organic solvent by evaporation, usually under 20 ℃ to 60 ℃ temperature, under pressure below atmospheric pressure, to finish by rotary evaporation. evaporation conditions depends on the physical property of employed lipophilic substance in organic solvent and the prescription.
After adipose membrane forms step, with liquid, aqueous hydration lipid to form the aqueous dispersion of lipid. required stirring can realize by rotation in container or transfer-i.e. vibration. the existence of inert solid contact material makes the mechanical agitation strength-enhanced and is continued in the container, and this has promoted the formation of the lipid capsule of homogeneous volume. this hydration step is carried out under the condition of the conversion temperature that is higher than lipid components.
Waterborne liquid can be a pure water, but also can be the aqueous solution of any electrolyte or bioactive substance. for example, can use NaCl or CaCl
2Aqueous solution.
After stirring the lipid water solution mixture, make and dispersions obtainedly in the competent time, keep quite, so that lipid capsule forms and ripening. in one embodiment, container left standstill about 1 to 2 hour in room temperature. can from the container that contains the inert solid contact material, reclaim the aqueous dispersion of multilamellar liposome capsule then. if desired, can from dispersion, reclaim any uncombined active substance with known technology such as repeated centrifugation, dialysis or column chromatography. and the lipid somatocyst can be suspended in again in any suitable electrolyte buffer to use subsequently.
So far, as mentioned above, finished the preparation of liposome by connecting selected connecting key and aglucon.
The cytotoxicity of embodiment 3 oxaliplatins
By oxaliplatin is dissolved in 9% the sucrose solution preparation oxaliplatin solution with the concentration of 8mg oxaliplatin/ml solution.
Replenishing of the oxaliplatin solution that various concentration are arranged in RPMI 11640 culture medium of 10% hyclone, the AsPC-1 cell is used 5%CO at 37 ℃
2Cultivated 4 hours. change culture medium, cell was cultivated 48 hours again. measure cell viability with the commercial cytotoxicity testing cassete that can get, for example, APO-ALERT
TMTesting cassete (Clontech, BD, Biosciences, Clontech, Palo.Alto, CA). in cell, add culture medium and at 5%CO
2In cultivated 2 hours, measure at 450nm place and develop the color (reference wavelength: 620nm). the result as shown in Figure 2. the LD of oxaliplatin
508ug/ml.
This embodiment provides another embodiment of liposome composition of the present invention. and the liposome according to the present embodiment preparation contains following material
1, distearoylphosphatidylcholine (DSPC)
2, cholesterol (CH)
3, N-(carbamyl ylmethoxy-Macrogol 2000)-DSPE (DSPE-PEG-OMe)
4, (carboxy polyethylene glycol 3000)-DSPE (DSPE-PEG-COOH)
These components exist with following ratio:
DSPC:CH:DSPE-PEG-OMe:DSPE-PEG-COOH=2:1:0.19:0.01 (m/m). for water, use oxaliplatin solution (8mg/ml is in 9% sucrose solution).
With DSPC, cholesterol, PEG2K-OMe and PEG3K-COOH are with the mixture of the ratio of 2:1:0.19:0.01 (m/m), be dissolved in chloroform and the diisopropyl ether. in resulting solution, add oxaliplatin solution (in 9% sucrose solution) and carry out ultrasonic. this solution desolvates to remove 60 ℃ of evaporations, repeat lyophilizing 5 times. at 60 ℃ with extruder (Avestin, Ottawa, Canada) filter membrane screening products obtained therefrom (is used 400nm membrane filtration 2 times, reuse 100nm membrane filtration 5 times), centrifugal again 2 times, rotating speed 200,000g, each 30 minutes. in extrusion, lipid capsule usually is pressed through the polycarbonate leaching film that pre-determines the aperture by physics under pressure. gained be deposited in suspend again in the buffer (PH5.5) of 9% sucrose solution or 2-(N-morpholino)-ethylsulfonic acid (MES) with obtain oxaliplatin-PEG (COOH/-OMe) liposome.
The liposome that contains PEG is with transferrin (TF) derivatization. and then will (COOH/-OMe) liposome and 1-ethyl-3-(3-dimethylamino-propyl group) carbodiimide hydrochloride (EDC) (with 2.7% amount with respect to lipid components weight) and N-hydroxysulphosuccinimide (S-NHS) (with 7.3% amount with respect to lipid components weight) mix by the oxaliplatin-PEG of preceding method preparation, then mixture was left standstill 10 minutes at room temperature. gained solution and transferrin (TF) (with 20% amount) reaction with respect to lipid components weight, and under room temperature, stirring 3 hours. solution is 200, centrifugal 30 minutes of 000g, the gained precipitation is suspended in 9% sucrose solution again.
TF in as above preparation (PEG) adds ferric citrate-sodium citrate in the taking off of liposome-form, then stirring at room 15 minutes. gained solution is 200, centrifugal 30 minutes of 000g. the precipitation that obtains is suspended in 9% sucrose solution again, with the TF that obtains complete form (PEG) liposome.
As above the physical characteristic of Zhi Bei unmodified liposome, PEG-liposome and TF-PEG liposome is summarized in Fig. 3.
Embodiment 5 measures the number of cell surface transferrin receptor
This embodiment has described a kind of method of measuring cell surface transferrin receptor number. and adopt normal person's leukocyte in this embodiment and derive from the human cell of different malignant cell systems (K562, MKN45P and HL60), by the number of transferrin (TF) receptor of Scatchard assay cell surface. to cell culture adding variable concentrations
125The TF solution of I labelling and 4 ℃ of insulations 1 hour. with the concentration of protein determination assay TF, with gamma counter measure radioactivity with solution centrifugal so that cell precipitation, with ice-cooled buffer washed cell fragment, and measure to determine the concentration with the bonded TF of cell surface with gamma counter. with the number of quantification of protein algoscopy mensuration cell. from the concentration known of the TF of initial adding, deduct the concentration that combines TF, calculate the concentration of unconjugated TF. by mark concentration at the longitudinal axis in conjunction with TF, represent in conjunction with the concentration of TF and the ratio of the concentration that does not combine TF with trunnion axis, measure (promptly from Scatchard (Scatchard) plot in conjunction with the number of TF, the number of receptor). the intercept of X-axis is obtained bonded TF number (that is the number of receptor) from figure.
In the different cell types, be combined in cell surface
125The quantity of I-TF is as shown in Figure 4. find that transferrin receptor number from the cell surface of human malignant lesion's cell line is significantly higher than the number on the normal leukocyte.
This embodiment has illustrated the therapeutic effect that liposome composition of the present invention is compared with simple oxaliplatin solution. with 6 to 7 weeks big male BALB/c nu-nu nude mices as animal model, with ASPC-1 cell (from human pancreas cancer) and MKN45P cell (from people's gastric cancer) as tumor cell.
When testing the 0th day, with AsPC-1 cell (2 * 10
6Cell) or MKN45P cell (1 * 10
7Cell) peritoneal injection is gone in the nude mouse. when the 1st day and the 4th day, will be by the liposome or the oxaliplatin solution (8mg/ml of embodiment 1 preparation, in 9% sucrose solution) respectively peritoneal injection go in the nude mouse. under 2 kinds of situations, the concentration of oxaliplatin is adjusted to 5mg oxaliplatin solution/kg body weight. use Tf-PEG liposome, PEG liposome, not have the liposome .PBS administration of modification as negative control.
The nude mice of the injection AsPC-1 cell abdominal cavity that in the time of the 21st day, is opened, the nude mice of the injection MKN45P cell abdominal cavity that when the 16th day and 26 days, is opened. have or not bloody ascites and tumor nodule in the observation body. the results are shown in Fig. 5.
Discover that the nude mice that gives Tf-PEG liposome of the present invention compares with the nude mice that gives not modified liposome and PEG liposome, the incidence rate of bloody ascites and tumor nodule significantly reduces in the body.
Embodiment 7 oxaliplatins (oxaliplatin) liposome is to the cytotoxicity of tumor cell
Colon 26 cells and AsPC-1 cell are on the flat board in 96 holes, with every hole 5 * 10
3The ratio of cell is cultivated, and uses 5%CO
2Pre-overnight incubation.
Oxaliplatin (oxaliplatin) solution or various types of liposome are added in the cell with the concentration shown in the table 1. and cell is at 5%CO
2In cultivated 4 hours in 37 ℃. remove test solution, culture continues to cultivate two days, adds up viable count then. measure cell survival with the WST-1 algoscopy.
According to the WST-1 algoscopy, test solution contains 5mmol/LWST-1 reagent (10 μ L); 20mmol/LHEPES; 0.2mmol/L 1-methoxyl group-PMS; PH value is that 7.4. adds test fluid in each hole, and abundant mixing is at CO
2Cultivate in the incubator and made colour developing in two hours. survey trap at the 400-450nm place on dull and stereotyped reader, reference wavelength is greater than 600nm.
Oxaliplatin (oxaliplatin) solution and various types of Liposomal formulation compare the cytotoxicity of Colon 26 cells and AsPC-1 cell respectively shown in Fig. 6 and 7. and Fig. 6 has shown oxaliplatin (oxaliplatin) solution, Bare-liposome, PEG-liposome and the TF-PEG-liposome LD to Colon 26 cells
50Value be respectively 2 μ g/mL, 60 μ g/mL, 18 μ g/mL, 8 μ g/mL. they to the LD of AsPC-1 cell
50Value is respectively 5 μ g/mL, 45 μ g/mL, 75 μ g/mL and 8 μ g/mL.
Therefore, oxaliplatin solution shows the ED of high toxicity .TF-PEG-liposome to Colon 26 cells
50Approximately than 2.3 times of PEG-lipid heights, than 7.5 times of Bare-lipid heights. for the AsPC-1 cell, the toxicity of TF-PEG-liposome is approximately than 9.4 times of PEG-lipid heights, than 5.6 times of Bare-lipid heights. in addition, the toxic concentration of TF-PEG-liposome showed cell will be lower than other Liposomal formulation.
Table 1
| LD 50(Colon 26 cells) | LD 50(AsPC-1 cell) | |
| Oxaliplatin solution | 2μg/mL | 5μg/mL |
| Wrapped up the TF-PEG-liposome of oxaliplatin | 8μg/mL | 8μg/mL |
| Wrapped up oxaliplatin-the PEG-liposome | 18μg/mL | 75μg/mL |
| Wrapped up the Bare-liposome of oxaliplatin | 60μg/mL | 45μg/mL |
The content that the present invention describes on this illustrative ground can lack any not disclosed especially one or several element herein, to carry out under the situation of one or several qualification. used term and expression are only as descriptive but not determinate term, when using these terms and expressing, do not get rid of the feature of any demonstration and description or the equivalent of relevant portion, but it should be noted that the various modification in claim scope of the present invention are possible. therefore, though should understand by preferred embodiment and optionally feature in detail openly the present invention, those skilled in the art can adopt imagination disclosed herein, and these improvement and variation are also in incidental claims restricted portion of the present invention.
The content of the article that this paper mentioned or quoted, patent, patent application and all other files and applicable electronic information, they are hereby expressly incorporated by reference fully, as with each publication as with reference to by especially with combine individually the same. the applicant keep with this apply for practically with these articles, patent, patent application or other documents in any one piece or the bonded right of all material.
Lacking any not detailed disclosed one or several element herein in this present invention who describes in detail, may suitably implement under the situation of one or several qualification. therefore for example term " comprises ", " comprise ", " contain " etc. and to be interpreted as open but not enclosed notion. term used herein in addition and only expressing as narrative but not determinate term, when using these terms and expressing, do not get rid of any shown in and the feature described or the equivalent of relevant portion, but will be appreciated that different modification also may be in the scope of claim of the present invention. therefore, though should understand by embodiment preferred and optionally feature the present invention is disclosed especially, improvement of the present invention and modification that those skilled in the art can adopt this paper content to be embodied, these improve and modification is considered within the scope of the invention.
At this present invention has been described generally and prevailingly. drop on each narrower kind in the general open scope and the next grouping and also constituted a part of the present invention. this has comprised general description of the present invention, and have prerequisite or the negativity of getting rid of any theme in the general scope and limit, whether the material of no matter removing was described in detail especially at this.
In addition, when describing feature of the present invention or each side, person of skill in the art will appreciate that also and can describe the present invention with any separate member of Ma Kushi group or the mode of many members subgroup in the mode of Ma Kushi group.
Other embodiments are listed in following claims.
Claims (6)
1. one kind by Polyethylene Glycol and transferrin derivatization and comprise the Liposomal formulation of oxaliplatin therein.
2. Liposomal formulation according to claim 1 is characterized in that oxaliplatin exists with the concentration of 6-10mg/ml.
3. Liposomal formulation according to claim 1 is characterized in that oxaliplatin exists with the concentration of 7-9mg/ml.
4. Liposomal formulation according to claim 1 is characterized in that oxaliplatin is present in the sucrose solution of 8-10%.
5. Liposomal formulation according to claim 1 is characterized in that oxaliplatin is present in the sucrose solution of 8-10% with the concentration of 7-9mg/m l.
6. a pharmaceutical composition for the treatment of tumor contains each described Liposomal formulation of with good grounds claim 1-5 and pharmaceutically acceptable carrier.
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| CNB2003101247766A CN100471493C (en) | 2003-12-17 | 2003-12-17 | Liposome preparation containing Oxaliplatin |
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| CN101897668B (en) * | 2009-05-27 | 2012-04-04 | 上海医药工业研究院 | Oxaliplatin liposome, preparation method and application thereof |
| CN102223456B (en) * | 2010-04-14 | 2013-09-11 | 华为终端有限公司 | Echo signal processing method and apparatus thereof |
| CN104703594B (en) * | 2012-08-10 | 2018-01-23 | 大鹏药品工业株式会社 | The aqueous dispersion and its stabilization method of the liposome of stable encapsulation oxaliplatin |
| CN103169659A (en) * | 2012-11-23 | 2013-06-26 | 杭州师范大学 | Oxaliplatin long-circulating liposome and application thereof |
| CN103181898B (en) * | 2012-11-23 | 2016-03-09 | 杭州师范大学 | A kind of oxaliplatin liposome and application thereof |
| CN108743533B (en) * | 2018-05-30 | 2021-08-06 | 温州生物材料与工程研究所 | Liposome-encapsulated ruthenium polypyridine complex and application thereof |
| CN110507613A (en) * | 2019-07-29 | 2019-11-29 | 苏州大学 | A kind of Liposomal formulation and the preparation method and application thereof |
Citations (5)
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|---|---|---|---|---|
| US5384127A (en) * | 1985-10-18 | 1995-01-24 | Board Of Regents, The University Of Texas System | Stable liposomal formulations of lipophilic platinum compounds |
| CN1154654A (en) * | 1994-08-08 | 1997-07-16 | 德彪药品股份有限公司 | Pharmaceutically stable oxaliplatinum prepn. |
| US5945122A (en) * | 1996-08-23 | 1999-08-31 | Sequus Pharmaceuticals, Inc. | Liposomes containing a cisplatin compound |
| CN1291886A (en) * | 1998-02-25 | 2001-04-18 | 圣诺菲-合成实验室公司 | Formulations |
| CN1343118A (en) * | 1999-11-05 | 2002-04-03 | 泰尼·布利卡斯 | Therapy for human cancers using cisplatin and other drugs or genes encapsulated into liposomes |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5384127A (en) * | 1985-10-18 | 1995-01-24 | Board Of Regents, The University Of Texas System | Stable liposomal formulations of lipophilic platinum compounds |
| CN1154654A (en) * | 1994-08-08 | 1997-07-16 | 德彪药品股份有限公司 | Pharmaceutically stable oxaliplatinum prepn. |
| US5945122A (en) * | 1996-08-23 | 1999-08-31 | Sequus Pharmaceuticals, Inc. | Liposomes containing a cisplatin compound |
| CN1291886A (en) * | 1998-02-25 | 2001-04-18 | 圣诺菲-合成实验室公司 | Formulations |
| CN1343118A (en) * | 1999-11-05 | 2002-04-03 | 泰尼·布利卡斯 | Therapy for human cancers using cisplatin and other drugs or genes encapsulated into liposomes |
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