CN102327230A - Protein nanometer granules wrapped with taxane medicaments and preparation method for nanometer granules - Google Patents
Protein nanometer granules wrapped with taxane medicaments and preparation method for nanometer granules Download PDFInfo
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Abstract
The invention belongs to the field of pharmacy, and discloses protein nanometer granules wrapped with taxane medicaments and a preparation method for the nanometer granules. In a formula, the granules comprise the following substances in percentage by weight: 0.1 to 10 percent of taxane medicaments, 0.1 to 40 percent of water-soluble carrier material and 50 to 90 percent of protein substances. The method comprises the following steps of: preparing water-soluble carrier solid dispersion containing the taxane medicaments from the taxane medicaments and the water-soluble carrier material; adding the obtained solid dispersion into an aqueous medium containing the protein substances, mixing uniformly, and performing high-shear treatment on the mixture to obtain suspension of the protein nanometer granules wrapped with slightly-soluble medicaments; and further preparing required formulations. The protein nanometer granules have the advantages of large medicine-carrying quantity, uniform grain diameters, high stability and safety and the like; and by the method, toxic organic solvent residues are avoided, the safety of clinical administration is improved, and a process is simple, low in cost and high in operability.
Description
Technical field
The invention belongs to pharmaceutical field, relate to a kind of particulate its preparation method of protein nano that wraps up taxone.
Background technology
Taxone (like paclitaxel, Docetaxel etc.) is one of employed clinically at present effective antitumour medicine.Paclitaxel be the seventies find from the bark of Ramulus et folium taxi cuspidatae or its kind or needle, to separate the natural product Docetaxel that obtains be semi-synthetic product, find that afterwards this is one type of antitumor agent with special Anticancer Effect and Mechanism.
The Anticancer Effect and Mechanism of taxone is to promote microtubule polymerization, reduces the depolymerization speed of microtubule, thereby makes microtubule be in stable non-functional state, thereby reach the purpose that stops tumor cell mitosis and propagation.And preclinical study shows that Docetaxel compares with paclitaxel, stronger to the affinity of microtubule, plasma half-life is longer and cell in the holdup time longer.
Although taxone has good antitumor action; But its water solublity all lower (about 10ug/ml), therefore employed clinically paclitaxel injection and Docetaxel injection all need use surfactant polyoxyethylene Oleum Ricini and polyoxyethylene sorbitan monoleate (tween 80) and cosolvent ethanol to come the purpose of dissolving taxol drug respectively.Paclitaxel injection and Docetaxel injection then are respectively successfully to develop the product of listing by U.S. Bristol-Myers Squibb Co. and French Sanofi-Aventis company the earliest.
Though use the surfactant polyoxyethylene Oleum Ricini and Polysorbate is after 80s can be mixed with injection; But its toxic and side effects in clinical practice is bigger; Cause more complication easily, comparatively common comprise untoward reaction such as anaphylaxis, bone marrow depression (mainly showing as neutrophilic granulocyte reduces), fluid retention, neurotoxicity, alopecia.
A large amount of research shows that polyoxyethylene castor oil can promote a large amount of histamines to discharge in vivo, and then produces anaphylaxis, also can cause nerve conduction to postpone the sensory nerve pathological changes.In addition, polyoxyethylene castor oil can also form molecule encapsulation paclitaxel molecule in blood, has influenced the diffusion of drug molecule to tissue, thereby has influenced its GVT.Also have research to show the divinyl hexyl phthalate (Diethylehexy Phthalate) in the polyoxyethylene castor oil solubilized PVC transfusion device.And polyoxyethylene sorbitan monoleate (tween 80) has hemolytic, and viscosity is big, and clinical application is very inconvenient.All there is certain problem in above-mentioned injection formulation at aspects such as toxic and side effects, convenient drug administration property and stability, therefore develops new taxone form of administration and necessitates.
The polymer of many biocompatibility can be used for preparing polymer shell, is wrapped in the outside of water-fast pharmacologically active medicine.In fact any polymer, natural or synthetic, as long as can contain sulfydryl or disulfide bond in its structure as required, just can be used for around in fact water-fast pharmacologically active medicine, forming one deck disulfide bond crosslinking shell.The protein that contains cysteine and/or disulfide bond is pharmaceutically acceptable biological vehicle, and this patent is example with human serum albumin.
Human serum albumin (human serum albumin; The protein of the strand sugar basedization of HSA) forming by 585 aminoacid; Molecular mass 66.5kDa; Be rich in protein (accounting for the 50%-60% of human plasma total protein) in the human plasma, have the characteristics of long circulation (half-life reaches 19 days) in stable chemical performance, safety non-toxic, non-immunogenicity, good biocompatibility, biodegradable, the body.
HSA has lot of advantages as the antitumor drug carrier material, mainly comprises:
1. superior drug loading potentiality: have more polar group and hydrophobic amino acid on the HSA polypeptide chain; Small-molecule drug to 70%, particularly insoluble drug have the affinity of height; And reversible combination can take place, for its payload and release provide possibility; Contain 48% the α-Luo Xuanjiegou of having an appointment in the albumin secondary structure, 15% beta-pleated sheet structure, all the other are the random coil structure, therefore have a lot of netted spaces, have created favourable steric requirements for carrying medicine.
2. broad-spectrum tumor targeting: albumin is the important nitrogenous source of tumor cell, and growing tumors has very strong picked-up and deposit albumin ability fast.HSA is as the antitumor drug carrier, through with endothelial cellular membrane on the albumin receptors bind, the caveolin on the active cell film, thus antitumor drug is transferred between tumor cell in the matter.Matter and tumor cell surface are rich in a large amount of NAC acidic secretion property albumen between tumor cell; This protein function is similar to the albumin receptor; Can attract and adhere to albumin specially; And NAC acidic secretion property albumen is in all overexpressions of nearly all tumor tissues (bladder, liver, ovary, kidney, digestive tract, mammary gland etc.), and this has just formed the medicine storage pond in the tumor tissues, for the wide spectrum target killing of the albumin nanometer carrier of loaded with anti-tumor medicine provides maybe.
3. structure modifiability: contain a large amount of reaction active groups in the HSA structure, like free amine group, carboxyl, sulfydryl etc., can with antitumor drug through the chemical bond coupling, improve medicine dissolution property, improve body-internal-circulation time and tumor-targeting; Also can with " target molecule " couplings such as antibody or part, further improve the cancer target performance of HSA nano-carrier.In recent years, also have report with HSA and macromolecular materials such as glucosan, aldohexose or PNIPAM mutually coupling to improve the physicochemical property of HSA as drug carrier material.
In view of HSA has unique advantage as drug carrier material, at present both at home and abroad to mainly concentrating on both direction based on albuminous drug delivery system research:
1. prodrug: utilize a large amount of reaction active groups of HSA; With medicine, especially antitumor drug (amycin, paclitaxel, Docetaxel, methotrexate, mitomycin, 5-fluorouracil) chemical coupling in free amine group, carboxyl or the sulfydryl of HSA; To improve medicine dissolution property, to improve body-internal-circulation time and tumor-targeting, existing abroad more report.
Yet pass through HSA and the coupling of medicine direct chemical, have following problem: 1) the medicine selectable range is narrow: medicine need have the certain reaction active group, and is prone to lose efficacy at chemical coupling process Chinese medicine; 2) ambiguity of curative effect performance: the easy degree of chemical bond rupture has determined the performance of curative effect of medication between medicine and the HSA, too fast fracture, and HSA loses the advantage as carrier material, crosses fracture slowly, and medicine then can't be brought into play curative effect; 3) cost is high, and drug loading is low: in the coupling process, the medicine inventory is far longer than HSA, and the coupling drug amount is limited, causes preparation cost big (particularly antitumor drug expensive raw material price), the defective that the system drug loading is low.
2. nanometer is passed the medicine system: the physical encapsulation medicine can significantly improve the load of carrier material to medicine.At present, the albumin nano granular of report preparation both at home and abroad or medicine carrying technology mainly contain: emulsifying coacervation, desolvation method, pH coacervation and Nab technology.
Though the first three methods Application Research time is the longest, report is maximum, but still has a lot of problems:
1) particle diameter is big.Nanoparticle particle diameter 0.5-10 μ m, and be subject to operating condition influence, less than several nothings reports of the albumin nano granular of 200nm, excessive particle diameter is unfavorable for that nano-carrier accumulates in tumor through the EPR effect of tumor;
2) need to solidify.Because HSA is highly water-soluble; Prepared nanoparticle needs curing operation, promptly adds chemical cross-linking agent and makes albumin take place crosslinked or heat to make albumen generation degeneration, and the former toxicity is bigger; And be prone to make medicine to take place crosslinked simultaneously and lose curative effect, the latter then is not suitable for the load of responsive to temperature medicine; In addition, the somebody thinks that employing method crosslinked or that be heating and curing can reduce the hydrophilic on HSA nanoparticle surface, thereby reduces circulation time in blood, is unfavorable for cancer target;
3) aftertreatment technology is complicated.Owing to used poisonous additives such as surfactant, oil, chemical cross-linking agent in the technology, need to clean purification, but small amount of residual will have been brought hidden danger to drug safety with a large amount of organic solvents; Adopt high speed centrifugation to separate (16000-20000g) and collect nanoparticle, not only high to instrument requirement, and in water, weigh bad dispersibility;
4) suitable medicine scope is little.Only be suitable for the load of water soluble drug, and quite a few antitumor drug is an insoluble drug.
The Nab technology is the medicine albumin-binding nanotechnology that America Biological Science Co., Ltd sets up in recent years.This technology only is suitable for the parcel of plasma protein binding rate than higher fat-soluble medicine.2005; Paclitaxel albumin-binding nanoparticle (triumphant
) with the preparation of this technology obtains FDA approval listing, is mainly used in the breast carcinoma of recurrence in metastatic breast cancer combined chemotherapy failure back or the NACT 6 months.
Said preparation has lower toxicity, better therapeutic with respect to traditional polyoxyethylene castor oil preparation (safe
); But also there are many defectives, comprise: the 1) use of toxic solvents.The employing chloroform is a solvent, though the vacuum evaporation operation is arranged, increased technology difficulty, and product has dissolvent residual; 2) drug loading is low.Only can be controlled at 2-10wt%, need to consume a large amount of HSA; 3) inside and outside poor stability.Said preparation is under pH＞5.8 conditions, and medicine was very easily separated out in several hours, so the preparation pH value need be controlled in the pH5.4-5.8 scope (also only stablizing 24h in this scope).And human internal environment's pH value is 7.4; Said preparation gets into after the blood circulation; Estimate that the medicine short time will separate out and be unfavorable for the specificity picked-up of tumor from carrier, dosage is 1.5 times of safe
preparation clinically at present.
Because method for preparing such as ultrasonic technology all can not be used for the production of industrially scalable, and its diameter of particle that obtains is too big, this make its improper with can not be used for patient's administration and use.Therefore America Biology Science Co., Ltd has put down in writing in patent US2007082838 and CN98808225 and CN97199720 and has required protection respectively to prepare the Docetaxel/paclitaxel that can remake and human serum albumin's freeze-dried preparation with the high pressure homogenizing method, and after the gained reconstruct stability of suspension above 24 hours.
The high shear technology has functions such as superpower mixing, pulverizing, dispersion, emulsifying, for macromolecular materials such as SBS, SBR significant especially crushing effect is arranged, and can effectively solve all kinds of material grinding, an emulsifying difficult problem, has replaced traditional colloid mill equipment.Compare with traditional production process, have advantages such as energy consumption is low, production cost is low, product quality is high, super-refinement.
Record and requirement protection contain water-insoluble drug and the particulate delivery system of protein-coated and preparation method thereof in CN98808225 and CN97199720 patent, and wherein said average diameter of particles is 10-200nm.The mixture that the method for preparing of its said drug regimen system is formed for the organic facies that will contain said water-insoluble drug and dispersed therein and proteinaceous aqueous medium places in the high pressure homogenizer of 3000-30000Psi; Make it accept high shear treatment; Produce above-mentioned granule, and said composition does not contain surfactant.
The inventor once repeatedly repeated the embodiment 1,5 and 6 of the embodiment, particularly CN97199720 of above-mentioned patent, illustrated result in embodiment that never obtains in this patent and the claim.The inventor has prepared disclosed mixture; In its pressure limit of recommending, mixture is handled then with the Avestin high pressure homogenizer; Obtain the nano-emulsion of pH=6.8, and desolvate, can produce the nano-particle that mean diameter is about 220nm according to removing described in the patent with the rotary evaporator evaporation; After carrying out lyophilization, be easy to recasting behind the adding normal saline and become suspension; But its nano-particle size obviously increases before than lyophilization, and it is 390nm that mean diameter is increased by the 220nm before the lyophilization, and in 4h, the deposition of nano-particle promptly occurs.In addition, filter with the microporous filter membrane of being put down in writing among the patent CN97199720, be easy to occur to stop up but the result is a filter, and the medicine productive rate is lower than 30%, these are different with the yield results of the 70-100% that stated in the patent.
Record and requirement protection add the albumin nano granular that comprises the Docetaxel medicine of stabilizing agent sodium citrate and/or sodium chloride in the US2007082838 patent; Its mean diameter＜200nm; And described in patent; These it is said very high stability with being higher than nano-emulsion that homogenizing method obtains; The meaning of term " stability " had both been represented mean diameter not in time or freezing dry process and changing here, and the drug precipitation (US200708283, embodiment 12) of nano-particle does not appear in expression yet.
The inventor once repeatedly repeated the embodiment 11,16 and 18 of the embodiment, particularly US200708283 of above-mentioned patent, never obtained disclosed result in this patent working example and claims.The inventor has prepared disclosed mixture; In the pressure limit of its recommendation, it is handled then with the Avestin high pressure homogenizer; Obtain the nano-emulsion of pH=7.2, utilize rotary evaporator evaporation to remove organic solvent after, produce the nano-particle of the about 280nm of mean diameter.But the deposition that occurs nano-particle very soon; Difficult when carrying out described filtering with microporous membrane (1.2um, 0.8um, 0.45um and 0.22um); Situation about stopping up appears in filter membrane easily; And its lyophilized products nanometer suspension liquid that the back forms of in physiological solution, remaking is unstable, macroscopic deposition occurs in about 8 hours, and the stability of claiming in these and the patent is completely different greater than 24 hours result.
Summary of the invention
The objective of the invention is the problems referred to above and shortcoming to prior art, the present invention provides that a kind of preparation technology is simpler, the character protein nano granule of stable and better clinical practice character more.
Another object of the present invention provides the particulate method for preparing of above-mentioned protein nano.
The inventor stumbles in preparation protein nano particulate process, when with the water-solubility carrier material (like, Polyethylene Glycol; Mannitol, PVP, etc.) add protein system after; The protein nano granule of bag loaded taxane class medicine more is prone to form, and particle diameter is even simultaneously, and stability improves.In order to investigate this effect of water-solubility carrier material in system; The inventor studied the water-solubility carrier material and protein-based between relation; Surprisingly; Transmission electron microscope and differential scanning calorimetric result all reflect, this two be not simple mixed system, but formed a kind of new compound system through interaction.We infer because the formation of this new compound system makes taxone better to be carried by bag, thereby makes preparation technology's operability significantly promote, and make nano-particle more stable and more even simultaneously.
Because the significant advantage of this new compound system; The inventor attempts this water-solubility carrier is mixed earlier with taxone then; Taxone is present in the water-solubility carrier with the form of molecular dispersion; The two form with solid dispersion adds in the protein-based solution, and the effect of mixing and process high shear force forms the protein nano granule of taxone.The result finds that the protein nano granule more is prone to form, and stability is better; Simultaneously, this nano-particle has the long circulation of tangible blood and tissue, organ targeting property, has better pharmacodynamic properties.
The present invention provides a kind of method for preparing that taxone (like paclitaxel, Docetaxel, etc.) is joined a Nano grade carrier with the form of molecular dispersion state.This method more be prone to obtain taxone nano-particle (average diameter is less than 200 nanometers) than nab technology, and need not add any deleterious organic solvent (like, dichloromethane; Chloroform; Or the like), avoided organic solvent residual in preparation, improve clinical application safety greatly.
Simultaneously, the present invention provides a kind of protein nano granule, and this protein nano granule is high to the taxone drug loading, can significantly reduce the consumption of protein substance; This protein nano granule also possesses good inside and outside stability, and liquid preparation can be stablized preservation at ambient temperature for a long time, improves medication convenience and safety.The nano-particle of the taxone that this method obtained has the long circulation of tangible blood and tissue, organ targeting property, therefore possesses better pharmacodynamic properties.In sum, the present invention provide that a kind of preparation technology is simpler, the character protein nano granule of stable and better clinical practice character more.This protein nano granule have particle diameter evenly, good stability, the good high characteristic of safety.
The objective of the invention is to realize through following technical proposal:
A kind of protein nano granule that wraps up taxone, the particulate prescription of this protein nano contains the material of following percentage by weight:
0.1～10% taxone, 0.1～40% water-solubility carrier material, 50～90% protein substances.
Described protein nano granule, wherein taxone is selected from one or more in the following material: paclitaxel, Docetaxel, perhaps their derivant.
Described protein nano granule, wherein said water-solubility carrier material is selected from one or more in the following material: Polyethylene Glycol, polyvinylpyrrolidone, poloxamer, polyethylene oxide, mixed aliphatic ester, mannitol, carbamide, sodium alginate, HPMC, polyacrylic resin, gelatin, sodium carboxymethyl cellulose.
The alleged protein substance of the present invention is meant can be through the following material of sulfydryl and/or disulfide bond crosslinking: natural existence or synthetic protein and derivant thereof, synthetic protein polymer and derivant thereof, natural or synthetic albuminoid and derivant thereof, perhaps their mixture.
Said naturally occurring protein comprises that albumin, immunoglobulin, casein, lipoprotein, hemoglobin, lysozyme, α-2-macroglobulin, fibronectin, glass connect element, Fibrinogen, lipase; Said synthetic protein polymer is selected from and contains in the following material that free sulfhydryl groups and/or disulfide group modify one or more: gather ethanol, gather ethyl oxazoline, polyacrylamide, polyvinylpyrrolidone, polyglycols, gather Acetic acid, hydroxy-, bimol. cyclic ester, PCL or its copolymer, synthetic polyamino acid.
The particulate method for preparing of described protein nano, this method comprises the following steps:
A. taxone and water-solubility carrier material are processed the water-solubility carrier solid dispersion that contains taxone;
B. the solid dispersion adding with gained contains in the aqueous medium of protein substance, and mix homogeneously, mixture are handled the particulate suspension of protein nano that has obtained wrapping up taxone through shear conditions;
C. suspension is carried out drying or dry again through aseptic filtration earlier, process the particulate solid preparation of the protein nano that has wrapped up taxone, semi-solid preparation or gas preparation by required dosage form;
Perhaps
Step b is obtained suspension directly as liquid preparation or further be prepared into the liquid preparation of other types or suspension is dry or redissolve after the drying through aseptic filtration earlier again again and process liquid preparation.
Described method for preparing, wherein aqueous medium is selected from water for injection, pure water; Mannitol solution, aqueous phosphatic, dextran solution; D/W, sodium-chloride water solution, Freamine; Vitamin solution, carbohydrate solutions, or their any two or more mixture.
Described method for preparing, the method that wherein prepares the water-solubility carrier solid dispersion that contains taxone is fusion method, solvent method, solvent-fusion method, solvent-spraying (freezing) seasoning, polishing or Double helix squeezing and pressing method; And these methods are not introduced any organic solvent or any other organic solvents except that ethanol; Preferred solvent-the fusion method that adopts; (methods such as fusion method, solvent method, solvent-fusion method are the conventional methods of preparation solid dispersion.For example, with medicine with dissolve with ethanol (solvent), water-solubility carrier heating and melting (fusion), both mix, this is solvent-fusion method.Again for example, with the medicine heating and melting, the water-solubility carrier heating and melting, both mix, and this is a fusion method.If solvent-fusion method, then for " need not introduce any other organic solvents except that ethanol " promptly, only use ethanol.If fusion method then is " need not introduce any organic solvent ".What the present invention preferably adopted is solvent-fusion method; The advantage that solvent-fusion method is compared fusion method is: the direct fusion of some medicine; Can cause temperature too high, degrade, stability can not guarantee; Therefore this patent mainly adopts solvent-fusion method, specifically adopts which kind of method to determine according to the character of medicine of selecting for use and carrier.)
Said shear conditions is meant that application possesses the equipment of high pressure and high shearforce simultaneously, mixture is reached efficiently mix, pulverizing, dispersion and emulsifying purpose; Wherein, high pressure is meant that pressure limit is at 5000 to 30000 pounds/inch
2, preferred 6000～25000 pounds/inch
2
Described method for preparing, wherein the particulate mean diameter of this protein nano is 20～1000nm, is preferably 20～200nm.
Described method for preparing, wherein drying means adopts lyophilization or spray drying; Aseptic filtration adopts 0.22 μ m filter to filter.
Say that at length the present invention provides a kind of method for preparing that taxone (like paclitaxel, Docetaxel, etc.) is joined a Nano grade carrier with the form of molecular dispersion state.This method more be prone to obtain the little nano-particle of taxone (average diameter is less than 200 nanometers) than nab technology, and need not add any deleterious organic solvent (like, dichloromethane, chloroform, etc.).The nano-particle of the taxone that this method obtained is more stable, and drug loading is high, has long circulation of tangible blood and tissue, organ targeting property, therefore possesses better pharmacodynamic properties.
This method provides a kind of method that forms the nano-particle of taxone through the technology of desolvating; Such as; High shear force (as ultrasonic, high-pressure homogenization effect or conditions of similarity) can be used under the condition that lacks any conventional surfactants and any polymerization core substance, form the substrate of nano-particle.
This method provides the method for preparing of a kind of reusable little nano-particle (diameter is less than 200 nanometers), and this granule can pass through 0.22 micron filter aseptic filtration.This significant through filtering a certain size the preparation of nano-particle of 0.22 micron filter, all can not carry out disinfection because contain the preparation of a large amount of any protein (like albumin), because heat can make albuminous degeneration with the method such as the autoclaving of routine.Certainly, if adopt the bigger filter in aperture also can particulate mean diameter be controlled at about 1000nm, but the present invention preferably is controlled at 20～200nm with particulate mean diameter.
The concentration range that in aqueous medium, adds protein substance (for example, the human serum albumin) is about 0.05-25% (w/v, g/ml descend together), and scope is better in 0.5%-10% (w/v).These aqueous mediums have normal saline, buffer saline, water, buffered aqueous medium, Freamine, vitamin solution, carbohydrate solutions or similar medium, and any mixture of these media more than 2 kinds.Different with the nano-particle formation method of routine, need not add surfactant (for example, sodium lauryl sulphate, lecithin, Tween 80, poly alcohol F68 and similar compound etc.) or poisonous organic solvent (for example, dichloromethane, chloroform etc.) in the mixture.
With taxone (paclitaxel, Docetaxel etc.) and water-solubility carrier (as, Polyethylene Glycol, polyvinylpyrrolidone, poloxamer etc.) be prepared into the solid dispersion (powder) of a kind of taxone and water-solubility carrier through methods such as fusion method, solvent method or fusion solvent methods.The formation of this solid dispersion need not introduced any other organic solvents except that ethanol, even need not introduce organic solvent, has avoided organic solvent (like chloroform, dichloromethane etc.) residual in preparation, improves clinical application safety greatly.
Under low-shearing force, form a kind of mixture of forming by micron and nanometer droplet through homogenization.This can known by one of skill in the art mode accomplish, for example, adopt a kind of opereating specification about 2,000 to about 15,000 rev/mins Routine Test Lab pressure-even pulp crusher.
The medicament-carried nano granule is under high shear condition to form through homogenization.This homogenization is carried out in high-pressure homogenate device usually, and typical operating pressure is at 5,000 to 30,000 pounds/inch
2Scope in, this process is at 6,000 to 25,000 pounds/inch
2Carry out better in the scope.The Emulsion that generates contains imperceptible aqueous carrier nanometer droplet (containing dissolved pharmacological active substance etc.) and imperceptible protein nano droplet.Acceptable homogenization process comprises can give high shear and cavitation, like high-pressure homogenate device, ultrasonic processor, high-shear mixer, and similar devices.
The liquid suspended matter can obtain wrapping up the particulate powder of protein nano of taxone through drying.The powder that generates can in any suitable time and in suitable aqueous medium redispersion obtain can be to the suspension of mammal administration.These aqueous mediums have normal saline, buffer saline, water, buffered aqueous medium, Freamine, vitamin solution, carbohydrate solutions or similar medium, and any mixture of these media more than 2 kinds.Comprise lyophilization, spray drying for obtaining the method that this powder adopts, and similar techniques.
Through removing the moisture that it includes, for example, in suitable temperature-time range, can further convert powder type to vacuum freeze-drying method.Protein (for example; The human serum albumin) itself plays the cryoprotective agent effect; Need not use conventional freezing protective agent such as mannitol, sucrose, glycerol, and similar compounds, and this powder can easily be recombinated through adding entry, normal saline or buffer.Although do not need, certainly can understand to necessary as very, these conventional cryoprotective agents can join in the prescription of the present invention.
According to an embodiment of the invention, a submicron particles (nano-particle) that formation is very tiny is provided, promptly diameter is less than the particulate method of 200 nanometers.This granule can carry out aseptic filtration before using with the liquid suspension mode.Process for preparation gained final products of the present invention (being drug particles) can aseptic filtration be significant, because can not sterilize to the suspension that contains high concentration protein (for example, human serum albumin) with conventional method such as autoclave.
The application of protein nano granule in pharmacy of said parcel taxone, this protein nano granule can be used for gastrointestinal administration and parenteral administration is used.The particulate dosage form of this protein nano can be solid dosage forms, semisolid dosage form, liquid dosage form and gas dosage form, comprises lyophilized powder, capsule, granule, ointment, paste, suspensoid, injection, spray, inhalant etc.
In the enforcement of this invention, the protein substance of many biocompatibility can be used for preparing the particulate carrier shell of protein nano, is wrapped in the outside of taxone.In fact any protein substance, natural or synthetic, as long as can contain sulfydryl or disulfide bond in its structure as required, just can be used for around taxone, forming one deck disulfide bond crosslinking shell.Sulfydryl or disulfide bond can preexist in the polymer architecture and maybe can import through suitable chemical modification, and for example naturally occurring polymer such as protein, peptide, polynucleotide, polysaccharide (like starch, cellulose, glucosan, Algin, chitosan, pectin, hyaluronic acid etc.), proteoglycan, lipoprotein etc. all are the candidates with this modification.
Protein-based albumin (containing 35 cysteine), immunoglobulin, casein, lipoprotein, hemoglobin (each α of comprising among the present invention
2β
2Unit contains 6 cysteine residues), lysozyme (containing 8 cysteine residues), immunoglobulin, α-2-macroglobulin, fibronectin, glass connect element, Fibrinogen, lipase etc.Wherein protein, peptide, enzyme, antibody and their mixture thereof are the conventional protein substances of using of the present invention.
Being used for preferred protein substance of the present invention at present is albumin.For example α-2-macroglobulin (a kind of known opsonin) can be used to strengthen huge picked-up of biting like cell to the taxone of carrier parcel, maybe can promote the taxone of this carrier parcel to enter into liver and spleen.
Equally, the synthetic polypeptide that contains cysteine residues also is the good candidate that forms the protein substance of shell in the periphery of taxone.In addition, polyvinyl alcohol, poly hydroxyethyl methylacrylate, polyacrylic acid, Ju ethyl oxazoline, polyacrylamide and polyvinylpyrrolidone etc. all are chemical modification (as introducing sulfydryl and/or disulfide bond) and the good candidate that forms shell (as causing crosslinked).So these examples of material that are intended for use in the present invention's enforcement are to contain the synthetic amino acids of cysteine residues and/or disulfide bond; Polyvinyl alcohol contains free sulfhydryl group and/or disulfide bond after modifying; Poly hydroxyethyl methylacrylate contains free sulfhydryl group and/or disulfide bond after modifying; Polyacrylic acid contains free sulfhydryl group and/or disulfide bond after modifying; Ju Yi oxazoline contains free sulfhydryl group and/or disulfide bond after modifying; Polyacrylamide contains free sulfhydryl group and/or disulfide bond after modifying; Polyvinylpyrrolidone contains free sulfhydryl group and/or disulfide bond after modifying; Polyglycols contains free sulfhydryl group and/or disulfide bond after modifying; Polyactide, gather Acetic acid, hydroxy-, bimol. cyclic ester, PCL, or their copolymers, after modifying, contain free sulfhydryl group and/or disulfide bond; And any two or more mixture of above-mentioned material.
Beneficial effect of the present invention
The present invention provides a kind of particulate method for preparing of protein nano of wrapping up taxone, and this method is encapsulated in the taxone granule of suspendible a kind of by in can biocompatible composition polymer housing, and its diameter is a nanoscale.This method need not introduced any other organic solvents except that ethanol; Even need not introduce organic solvent; Avoided organic solvent (like chloroform; Dichloromethane etc.) residual in preparation, and can not produce because of adding the anaphylaxis that cosolvent or emulsifying agent cause, clinical application safety improved greatly; This method need not used conventional surfactant yet.This method provides a kind of more being prone to form and more stable taxone nano-particle, and this method technology is simple, cost is low, strong operability.The present invention of this method provides the method for preparing of the nano-particle of a kind of ability aseptic filtration simultaneously.
Simultaneously, the present invention provides a kind of protein nano granule, and this protein nano granule is high for the drug loading of taxone, can significantly reduce the consumption of protein substance; This protein nano granule also possesses good inside and outside stability, and liquid preparation can be stablized preservation at ambient temperature for a long time, improves medication convenience and safety.The nano-particle of the taxone that this method obtained has the long circulation of tangible blood and tissue, organ targeting property, therefore possesses better pharmacodynamic properties.In sum, the present invention provide that a kind of preparation technology is simpler, the character protein nano granule of stable and better clinical practice character more.This protein nano granule have drug loading height, particle diameter evenly, characteristics such as good stability, safety height.
Description of drawings
Fig. 1 is that fusion method prepares paclitaxel albumin nanometer formulation intensity particle size distribution figure.(vertical coordinate is intensity percent (%), and abscissa is particle diameter (nm), and Fig. 2, Fig. 7-Figure 11 are all roughly the same.)
Fig. 2 is that fusion-solvent evaporation method prepares paclitaxel albumin nanometer formulation intensity particle size distribution figure.
Fig. 3 is WAXD figure.Wherein: sample a: paclitaxel powder, sample b: lyophilizing human serum albumin powder, sample c: paclitaxel and albuminous physical mixture, sample d: formulation for paclitaxel sample.
Fig. 4 is DSC figure.Wherein: sample a: lyophilizing human serum albumin powder, sample b: paclitaxel powder; Sample c: paclitaxel and albuminous physical mixture; Sample d: formulation for paclitaxel sample.
Fig. 5 is a transmission electron microscope picture.Wherein: sample a: human serum albumin's freeze-dried powder, sample b: Polyethylene Glycol albumin powder, sample c: paclitaxel albumin nanometer formulation freeze-dried powder.
Fig. 6 is fluorescent scanning figure.Along with curve a → h direction, Polyethylene Glycol concentration increases among the figure.
Fig. 7 is Docetaxel albumin nanometer formulation intensity particle size distribution figure.
The specific embodiment
Below through embodiment the present invention is done further elaboration.
500 milligrams of paclitaxels are dissolved in 9.0 milliliters of ethanol.1200 milligrams of Polyethylene Glycol add paclitaxel solution wherein after complete fusion is heated in 60 ℃ of oil baths, and magnetic agitation, is cooled off rapidly under intense agitation after rotary evaporation is removed ethanol until its complete mix homogeneously.After the drying under vacuum overnight, solid dispersion is added (4.5%w/v, g/ml descend together) in 85 milliliters of human serum albumin's aqueous solutions.Mixture, is transferred in the high pressure homogenizer (EmulsiFlex-05, Canadian Avestin company) to form thick breast through high speed dispersion device (XHF-1, Shanghai gold reaches biochemical instrument factory) premix 1 minute then.High pressure homogenize is at 5000-30,000 pound/inch
2Condition under carry out, with Emulsion repetitive cycling at least 5 times, protein nano granule suspension, the product of acquisition has denseer opalescence.Measure through laser particle diameter appearance (Nano-ZS90, Britain Malvern company), the general diameter of paclitaxel albumin nanometer granule that the result obtains is in the 130-220 nanometer, and the result is as shown in Figure 1.
Protein nano granule suspension is further lyophilizing 48 hours under the situation of not adding any cryoprotective agent, and the powder that obtains can be easy to reconstitute protein nano granule suspension through adding sterilized water or normal saline.Roughly the same before particulate size and the lyophilizing after the reconstruct.
300 milligrams of paclitaxels are dissolved in 6.0 milliliters of ethanol.900 milligrams of Polyethylene Glycol are dissolved in 1.5 milliliters of dehydrated alcohol, after complete fusion is heated in 45 ℃ of oil baths, paclitaxel solution are added wherein, and magnetic agitation, is cooled off rapidly under intense agitation after rotary evaporation is removed ethanol until its complete mix homogeneously.After the drying under vacuum overnight, solid dispersion is added in 65 milliliters of human serum albumin's aqueous solutions (4.5%w/v).Mixture, is transferred in the high pressure homogenizer (EmulsiFlex-05, Canadian Avestin company) to form thick breast through high speed dispersion device (XHF-1, Shanghai gold reaches biochemical instrument factory) premix 1 minute then.Be emulsified in 5000-30,000 pound/inch
2Condition under carry out; With Emulsion repetitive cycling at least 6 times, protein nano granule suspension, the product of acquisition has denseer opalescence; Through laser particle diameter appearance (Nano-ZS90; Britain Malvern company) measure, the general diameter of paclitaxel albumin nanometer granule that the result obtains is in the 120-200 nanometer, and the result is as shown in Figure 2.
Protein nano granule suspension is further lyophilizing 48 hours under the situation of not adding any cryoprotective agent, and the powder that obtains can be easy to reconstitute protein nano granule suspension through adding sterilized water or normal saline.Roughly the same before particulate size and the lyophilizing after the reconstruct.
But embodiment 3 is less than the particulate preparation of paclitaxel albumin nanometer of the aseptic filtration of 200 nanometers
500 milligrams of paclitaxels are dissolved in 9.0 milliliters of ethanol.800 milligrams of Polyethylene Glycol are dissolved in 1.5 milliliters of dehydrated alcohol, after complete fusion is heated in 45 ℃ of oil baths, paclitaxel solution are added wherein, and magnetic agitation, is cooled off rapidly under intense agitation after rotary evaporation is removed ethanol until its complete mix homogeneously.After the drying under vacuum overnight, solid dispersion is added in 97 milliliters of human serum albumin's aqueous solutions (4.5%w/v).Mixture, is transferred in the high pressure homogenizer (EmulsiFlex-05, Canadian Avestin company) to form thick breast through high speed dispersion device (XHF-1, Shanghai gold reaches biochemical instrument factory) premix 1 minute then.Be emulsified in 10000-40,000 pound/inch
2Condition under carry out, with Emulsion repetitive cycling at least 6 times, protein nano granule suspension; The product that obtains has denseer opalescence; Measure through laser particle diameter appearance (Nano-ZS90, Britain Malvern company), the general diameter of paclitaxel albumin nanometer granule that the result obtains is in the 120-170 nanometer.
Protein nano granule suspension filters through one 0.22 micron micropore filter, and turbidness or granular size have no change.It is recyclable that the HPLC of content of taxol analyzes the paclitaxel filtration back that shows above 95%, and the result is as shown in table 1.The method can provide a kind of aseptic paclitaxel albumin nanometer suspension.
Sterile suspension is further lyophilizing 48 hours under the situation of not adding any cryoprotective agent, and the powder that obtains can be easy to reconstitute protein nano granule suspension through adding sterilized water or normal saline.Roughly the same before particulate size and the lyophilizing after the reconstruct.
Table 1
X-ray powder diffraction can be used for judging the crystallization or the noncrystalline characteristic of paclitaxel in freeze drying powder preparations.According to X-ray diffraction (X-ray diffraction) principle, the peak shape of diffraction maximum is main relevant with crystalline imperfection and crystal structure, i.e. the size of crystallite dimension, the defective in the crystal and distortion etc.According to Scherrer formula (Scherrer Equation), the square root of crystallite dimension and diffraction maximum is inversely proportional to, and its degree of crystallinity height of the size of crystallite dimension reflection, the promptly wide more lower degree of crystallinity of diffraction maximum correspondence.
Take by weighing small amount of sample a-paclitaxel powder; Sample b-lyophilizing human serum albumin powder; Sample c-paclitaxel and albuminous physical mixture; With sample d-paclitaxel albumin nanometer granule freeze-dried powder (by embodiment of the invention preparation), the XD-3A powder diffractometer carries out the powder X-ray diffraction test.5～40 ° of the angles of diffraction, 1 °/min of scanning speed, running voltage 40kV carries out the powder X-ray diffraction test under the operating current 50mA condition, and the result is as shown in Figure 3.
Sample a show sample has been located three strong diffraction maximums at 5.28 °, 8.84 ° and 12.36 °, and some little diffraction maximums are arranged between 15～25 °.Sample b has shown the typical broadband of amorphous substance protuberance.Sample c has shown the typical broadband of amorphous substance protuberance, but can see 5.28 °, 8.84 ° and 12.36 ° of characteristic peaks of locating in addition.Sample d formulation for paclitaxel does not demonstrate the evidence that paclitaxel has the crystallization characteristic; But it is very similar with the amorphous substance broadband protuberance of sample b; The prompting paclitaxel is actually in nanometer formulation with molecularity or unformed state and exists, thereby shows that drug taxol is present in behind high pressure homogenize in the albumin nanometer carrier really.
The particulate DSC collection of illustrative plates of embodiment 5 paclitaxel albumin nanometers
(Differential Scanning Calorimetry DSC) is a kind of technology of under temperature programmed control, measuring the difference power and the temperature relation that are input to sample and reference sample to differential scanning calorimetry.To absorb energy (J) during the crystal fusion to destroy lattice, endergonic how much relevant with crystal structure, the characteristic constant heat content of molecule specific crystal formation (Δ H, the Jg of unit
-1) can characterize its crystal formation characteristic.
Take by weighing sample a-lyophilizing human serum albumin powder; Sample b-paclitaxel powder; Sample c-paclitaxel and albuminous physical mixture; With each 2～3mg of sample d-paclitaxel albumin nanometer granule freeze-dried powder (by embodiment of the invention preparation), through of the heating rate heating of NETZSCH-DSC 204 analysers with 10 ℃/min, 50～300 ℃ of temperature ranges, blanket of nitrogen is carried out dsc analysis.The result is as shown in Figure 4.
Can know that by figure curve a shows that albumin has little dehydration exothermic peak at 67.4 ℃, and melting peak is slowly arranged about 222.3 ℃; Among the curve b; 224.2 ℃ endothermic peak and 244.5 ℃ of exothermic peaks are respectively the melting peak and the degradation peak of paclitaxel; These two characteristic peaks are present in paclitaxel and the albuminous physical mixture (curve c) simultaneously, and are not present in the paclitaxel albumin nanometer formulation (curve d).Above spectrum data shows that medicine is present in the micelle skeleton with the form of unformed or solid solution.
The particulate morphology research of embodiment 6 paclitaxel albumin nanometers
It is an amount of to take by weighing sample a-human serum albumin freeze-dried powder, sample b-Polyethylene Glycol albumin powder and sample c-paclitaxel albumin nanometer granule freeze-dried powder (by embodiment of the invention preparation); With the abundant hydration of 5% glucose solution, get light blue opalescence micellar solution, the dyeing of phosphotungstic acid negative staining; Promptly getting 1 micellar solution to be measured drips in the groove of drop reaction porcelain plate; And carbon-sprayed copper net is put on the test solution, take out copper mesh behind 1～2min, inhale from the copper mesh edge with the filter paper small pieces and remove residual liquid; This copper mesh is placed on dye liquor drips (4% Salkowski's solution, pH 7.0) upward about 30s, blot unnecessary dye liquor, drying, through the H-7000 of Hitachi transmission electron microscope observing form, the result is as shown in Figure 5.
As shown in the figure, the human serum albumin can not form spherical structure, but is dispersed into the homogeneous solution state; Human serum albumin behind the adding Polyethylene Glycol demonstrates the spherical structure of rule, and particle diameter is about 100nm; And the medicine carrying albumin preparation forms regular more and intensive spherical structure, and particle diameter is about 120nm, and particle size distribution is even.Can know simultaneously that less than the particle diameter (being respectively 122nm and 138nm) of dynamic light scattering experiment gained, this possibly be because the dry run in the TEM sample preparation process causes due to the subsiding of micellar surface to the transmission electron microscope particle diameter result of sample b, sample c respectively.
The interactional fluorescent spectrometry research of embodiment 7 Polyethylene Glycol and human serum albumin
Contain trp residue among the human serum albumin, tryptophan can produce stronger fluorescence under ultraviolet excitation.Fluorescence is swept the spectrum result can know that human serum albumin's fluorescence maximum excitation wavelength is at 285nm, and maximum emission wavelength is about 350nm.
Many small-molecule drugs or water-solubility carrier can influence each other with albumin, thereby cause fluorescent quenching.According to this principle, we can do the interaction between a series of experiment other materials of proof and the albumin.
In the 10ml color comparison tube, add 1.0 * 10 of 1.0mL successively
-5Mo lL
-1Human serum albumin solution and different volumes 1.0 * 10
-4MolL
-1Polyglycol solution is diluted with water to scale, shakes up, and constant temperature 10min hatching in water bath with thermostatic control, fixedly Kex=285nm, Kem=350nm measure relative intensity of fluorescence ^F=F
0-F (F, F
0Be illustrated respectively in the Polyethylene Glycol existence and do not have Polyethylene Glycol system solution fluorescence intensity when existing), result's (IF is the unit of fluorescence intensity, and subscript F is the meaning of fluorescence fluorescent) as shown in Figure 6.
Can know that according to the fluorescence result under 25 ℃ condition, Polyethylene Glycol produces quenching effect to the endogenous fluorescence of human serum albumin.(fluorescence intensity weakens simultaneously for curve a → h), albuminous fluorescent emission wavelength generation blue shift along with the rising of Polyethylene Glycol concentration.The result shows that having produced between Polyethylene Glycol and the albumin is main interaction with the Van der Waals force, thereby makes albumin that fluorescent quenching clocklike take place, and its quenching mechanism meets dynamic quenching.
Embodiment 8 drug level are to the influence of paclitaxel albumin nanometer grain diameter size
Change the concentration of paclitaxel, keep describing among other parameters and the embodiment 2 identical, prepared a series of paclitaxel albumin nanometer formulations.Measure the lower drug level of discovery through laser particle diameter appearance and can generate the granule that diameter is approximately 160 nanometers, higher concentration then generates smaller particles, and diameter is approximately 120 nanometers.When the concentration of paclitaxel in the human serum albumin solution was 1 mg/ml, particle diameter was about 250 nanometers; When the concentration of paclitaxel in the human serum albumin solution was 2 mg/ml, particle diameter was about 180 nanometers; When the concentration of paclitaxel in the human serum albumin solution was 5 mg/ml, particle diameter was about 130 nanometers.
The compatibility stability of embodiment 9 common infusion fluid agent
Investigated paclitaxel albumin nanometer lyophilized formulations (by embodiment of the invention preparation) and common infusion fluid agent (as, 5% glucose solution, 0.9% normal saline) compatibility stability, for clinical use provides reference.Generally speaking, dilution stability only need reach 8h can satisfy clinical practice, so present embodiment is a compatibility stability in its 8h of index evaluation with particle diameter, content of taxol.
Experimental result shows, all can obtain the solution of opalescence homogeneous after paclitaxel albumin nanometer lyophilized formulations redissolves with 5% glucose or 0.9% normal saline, and shows that like table 2 each index does not all have significant change in the 8h.Therefore, 5% glucose and 0.9% normal saline all can be used as the redissolution medium of paclitaxel albumin nanometer granule lyophilized formulations during clinical use.
Table 2
Paclitaxel albumin nanometer particle grain size and potential change before and after embodiment 10 lyophilizing
Zetasizer 3000HS laser particle size analyzer is through after measuring paclitaxel albumin nanometer freeze-dried powder (by embodiment of the invention preparation) redissolution; With compare mean diameter before the lyophilizing and increase slightly; Polydispersity index does not change; The basic character that the paclitaxel albumin nano granular is described does not change before and after lyophilizing, has guaranteed the stable of paclitaxel albumin nanometer formulation through cryodesiccated solid state process.
In addition, Zeta potential remains unchanged before and after the lyophilization basically, all-32～-36mV between.Generally speaking, when the absolute value of Zeta potential＞30mV,, there is bigger repulsive force between nanoparticle, and helps the stable of nanometer solution and impel nanoparticle size homogeneous because there are a large amount of electric charges in the nanoparticle surface.Paclitaxel albumin nanometer granule Zeta electric potential absolute value all＞30mV, indicate that both all have good physical stability, concrete outcome is as shown in table 3.
Table 3
The stability of paclitaxel albumin nanometer formulation after embodiment 11 reconstruct
Investigated paclitaxel albumin nanometer lyophilized formulations after 0.9% normal saline redissolves stability, for clinical use provides reference, be compatibility stability in its 8h of index evaluation with particle diameter, drug level.
Lyophilizing paclitaxel albumin nanometer formulation in the cillin bottle (by embodiment of the invention preparation) redissolved to concentration with normal saline be respectively 1,2,5 mg/ml, and be stored in room temperature state.Measure its particle diameter and medicament contg respectively in different time points, suspension was stable in 7 days at least.Wherein as shown in table 4 for 5 mg/ml preparations concrete particle diameter and content of dispersion.
Table 4
The mouse tissue distribution experiment of embodiment 12 paclitaxel albumin nanometer formulations
Choose 450 of the female white mice of healthy Kunming kind of body weight 20 ± 2g; Fasting 12h; Be divided into 75 groups at random; Press 15mg/kg dosage tail vein injection safe
(U.S. Bristol-Myers Squibb Co.) respectively; Triumphant
(America Biological Science Co., Ltd) and paclitaxel albumin nanometer formulation (by embodiment of the invention preparation); After the administration respectively at 5,15min and 0.5,1,3,8,12,24h time point get one group (n=6); After eyeball was got blood respectively, cervical vertebra dislocation was put to death, and cored, internal organs such as liver, spleen, lung, kidney, brain, tumor.Whole blood places the heparin sodium test tube, and is centrifugal, separated plasma; Internal organs are used normal saline flushing, and filter paper blots its moisture, claim to decide total amount (blood is by 8% of the mice body weight).Extract sample introduction behind the medicine in the biological sample, record medicine peak area calculates blood plasma and respectively organizes the drug level of different time.According to drug level change curve calculating in time AUC, MRT and targeting efficient.The different time drug level is as shown in the table; Its invading the exterior 5 for safe
respectively organize Chinese medicine through the time concentration; Table 6 for triumphant
respectively organize Chinese medicine through the time concentration, table 7 for paclitaxel albumin nanometer formulation respectively organize Chinese medicine through the time concentration.
Table 5 Thailand
respectively organize Chinese medicine through the time concentration (ng/ml)
Table 6 triumphant
respectively organize Chinese medicine through the time concentration (ng/ml)
Table 7 paclitaxel albumin nanometer formulation respectively organize Chinese medicine through the time concentration (ng/ml)
Can know by the result: respectively organize Chinese medicine through the time concentration
1) safe
triumphant
and the AUC value of paclitaxel albumin nanometer formulation are respectively 38.45; 75.72; 236.52; MRT is respectively 11.75,17.42,26.41;
2) the relative uptake ratio Re of tumor of safe
triumphant
and paclitaxel albumin nanometer formulation is respectively 1; 1.969; 6.151; Demonstrated this preparation tumor-targeting stronger with respect to triumphant element.
3) the cancer target efficient Te of safe
triumphant
and paclitaxel albumin nanometer formulation is respectively 0.88; 5.97; 10.76; Demonstrated the better tumor-targeting of this preparation.
The particulate rat pharmacokinetics experiment of embodiment 13 paclitaxel albumin nanometers
Get 12 of SD rats (the favorable to the people scientific and technological animal cultivation of Nanjing Xixia District field provides); Be divided into 4 groups at random; Every group 3, give Thailand
triumphant
and paclitaxel albumin nanometer formulation (by embodiment of the invention preparation) respectively.With the dosage of 7mg/kg by the tail vein injection administration, respectively at after the administration 5,15,30min, 1,2,4,6,8,12,24, the 36h eye socket is got the about 0.5mL of blood, places in the centrifuge tube that is added with heparin sodium centrifugal 10min (6000r/min) separated plasma.Add the 4mL t-butyl methyl ether behind vortex centrifugal, extract paclitaxel and internal standard substance diazepam in the blood plasma, measure the content of taxol in each time point blood plasma with high performance liquid chromatograph (LC-2010C, day island proper Tianjin), determination data is as shown in table 8.
Table 8
The result shows, but the blood circulation time of this preparation group significant prolongation paclitaxel, and this maybe be because the softish backbone of Polyethylene Glycol can significantly reduce the probability that carrier is engulfed by the endothelium reticular system.In addition; Mean residence time of this preparation (MRT) and blood medicine through the time concentration curve under area (AUC) be significantly higher than triumphant
injection (P＜0.01), be respectively triumphant
1.90 and 1.79 times.
The particulate pharmacodynamic experiment of embodiment 14 paclitaxel albumin nanometers
Get 40 of BALB/c tumor-bearing mices; Be divided into 4 groups at random; Every group 10; Gave safe
sample 2-triumphant
sample-3 paclitaxel of sample 1-albumin nanometer formulation (by embodiment of the invention preparation) and sample 4-normal saline respectively in 3,5,7,9 days; By the tail vein injection administration, it is following to investigate index with the dosage of 10mg/kg:
1. tumor growth curve: surveyed the major diameter a and the minor axis b of a tumor with slide gauge in per two days, according to formula V=0.5 * a * b
2Calculate tumor size, draw tumor growth curve;
2. inhibition rate of tumor growth: when experiment finishes, put to death mice, peel off tumor; It is heavy to take by weighing tumor; Calculate tumor growth according to following formula and suppress percentage rate: suppress percentage rate TI (%)=(1-WT/WC) * 100%, wherein WT is that the average tumor of treatment group is heavy, and WC is that the average tumor of matched group is heavy;
3. body weight change curve: measure the body weight of mice every day, draw the body weight change curve of mice.Surpassed 20% if the mice body weight reduces, can think that medicine has produced toxicity.
The result is as shown in table 9 below; Gross tumor volume by each time point calculates relative tumor rate of growth; The result shows that paclitaxel albumin nanometer formulation has more effectively suppressed the MDR growth of tumor with respect to safe
and triumphant
.
Table 9
As shown in table 10; Tumor after the dissection is heavy result show; Average tumor with respect to the normal saline group is heavy; The inhibition rate of tumor growth of safe
group is 60%; Triumphant
group is 53%, and the preparation group is 66%.
Table 10
As shown in table 11; Can know that by tumor heavy (g) variation triumphant
organizes and this preparation group is not all seen overt toxicity; But after intravenous injection safe
, mice phenomenon such as occur being slow in action, dull.
Table 11
500 milligrams of Docetaxels are dissolved in 7.0 milliliters of ethanol.1300 milligrams of Polyethylene Glycol are dissolved in 1.5 milliliters of dehydrated alcohol, after complete fusion is heated in 45 ℃ of oil baths, paclitaxel solution are added wherein, and magnetic agitation, is cooled off rapidly under intense agitation after rotary evaporation is removed ethanol until its complete mix homogeneously.After the drying under vacuum overnight, solid dispersion is added among 90 milliliters of human serum albumin solutions (4.5%w/v).Mixture, is transferred in the high pressure homogenizer to form thick breast through high speed dispersion device premix 1 minute then.Be emulsified in 9000-40,000 pound/inch
2Condition under carry out, with Emulsion repetitive cycling at least 5 times.There is denseer opalescence in the system that obtains, and the general diameter of polyenic taxusol nano granule that obtains is in the 135-200 nanometer.The result is as shown in Figure 7.
Protein nano granule suspension is further lyophilizing 48 hours under the situation of not adding any cryoprotective agent.The powder that obtains can be easy to reconstitute protein nano granule suspension through adding sterilized water or normal saline.Roughly the same before particulate size and the lyophilizing after the reconstruct.
Embodiment 16 drug loading data
Because taxone dissolubility in aqueous medium very low (＜1 μ g/ml), thus the free drug in the aqueous medium compare with the medication amount that is written into carrier (＞2mg/ml) can ignore.Through the centrifuging and taking supernatant liquid filtering, measure the method for subsequent filtrate Chinese medicine content and just can calculate the medication amount that is written in the carrier.
Precision takes by weighing a certain amount of albumin nanometer formulation (by embodiment of the invention preparation) freeze-dried powder, and distilled water redissolves and is settled to scale, jolting mixing.Destroy and be diluted to finite concentration with methanol, measure the content of preparation of Chinese medicine, by following formula (1) and (2) difference computational envelope rate and drug loading.
The result shows, records through HPLC, and the drug loading of paclitaxel albumin nanometer formulation lyophilized powder is (9.67 ± 0.67) %, and envelop rate is (94.02 ± 6.4) %.The clinical application amount of paclitaxel is bigger, and we have higher drug loading and envelop rate through the paclitaxel albumin nanometer formulation of the method preparation, can reduce the administration volume, reduces the albuminous application of adjuvant, has the good industrial prospect.
Embodiment 17
The inventor carries out single factor relatively (the listed difference, all the other are all identical with embodiment 2 in table) to different water-solubility carriers, protein substance respectively with reference to the method for embodiment 2, and the protein nano granule of preparation is seen table 13, table 14 through detecting mean diameter.Simultaneously, DSC detects and shows that all medicine is present in the protein nano granular preparation with the form of amorphous or solid solution.
Table 13
Table 14
Claims (9)
1. protein nano granule that wraps up taxone is characterized in that the particulate prescription of this protein nano contains the material of following percentage by weight:
0.1～10% taxone, 0.1～40% water-solubility carrier material, 50～90% protein substances.
2. protein nano granule according to claim 1 is characterized in that taxone is selected from one or more in the following material: paclitaxel, Docetaxel, perhaps their derivant.
3. protein nano granule according to claim 1 is characterized in that said water-solubility carrier material is selected from one or more in the following material: Polyethylene Glycol, polyvinylpyrrolidone, poloxamer, polyethylene oxide, mixed aliphatic ester, mannitol, carbamide, sodium alginate, HPMC, polyacrylic resin, gelatin, sodium carboxymethyl cellulose; Said protein substance is can be through the following material of sulfydryl and/or disulfide bond crosslinking: natural existence or synthetic protein and derivant thereof, synthetic protein polymer and derivant thereof, natural or synthetic albuminoid and derivant thereof, perhaps their mixture.
4. protein nano granule according to claim 3 is characterized in that said naturally occurring protein comprises that albumin, immunoglobulin, casein, lipoprotein, hemoglobin, lysozyme, α-2-macroglobulin, fibronectin, glass connect element, Fibrinogen, lipase; Said synthetic protein polymer is selected from and contains in the following material that free sulfhydryl groups and/or disulfide group modify one or more: gather ethanol, gather ethyl oxazoline, polyacrylamide, polyvinylpyrrolidone, polyglycols, gather Acetic acid, hydroxy-, bimol. cyclic ester, PCL or its copolymer, synthetic polyamino acid.
5. the particulate method for preparing of protein nano as claimed in claim 1 is characterized in that this method comprises the following steps:
A. taxone and water-solubility carrier material are processed the water-solubility carrier solid dispersion that contains taxone;
B. the solid dispersion adding with gained contains in the aqueous medium of protein substance, and mix homogeneously, mixture are handled the particulate suspension of protein nano that has obtained wrapping up taxone through shear conditions;
C. suspension is carried out drying or dry again through aseptic filtration earlier, process the particulate solid preparation of the protein nano that has wrapped up taxone, semi-solid preparation or gas preparation by required dosage form;
Perhaps
Step b is obtained suspension directly as liquid preparation or further be prepared into the liquid preparation of other types or suspension is dry or redissolve after the drying through aseptic filtration earlier again again and process liquid preparation.
6. method for preparing according to claim 5 is characterized in that said aqueous medium is selected from water for injection, pure water; Mannitol solution, aqueous phosphatic, dextran solution; D/W, sodium-chloride water solution, Freamine; Vitamin solution, carbohydrate solutions, or their any two or more mixture.
7. method for preparing according to claim 5, the method that it is characterized in that preparing the water-solubility carrier solid dispersion that contains taxone are fusion method, solvent method, solvent-fusion method, solvent-spraying (freezing) seasoning, polishing or Double helix squeezing and pressing method; And these methods are not introduced any organic solvent or any other organic solvents except that ethanol; Preferred solvent-the fusion method that adopts; Said shear conditions is meant that application possesses the equipment of high pressure and high shearforce simultaneously, mixture is reached efficiently mix, pulverizing, dispersion and emulsifying purpose; Wherein, high pressure is meant that pressure limit is at 5000 to 30000 pounds/inch
2, preferred 6000～25000 pounds/inch
2
8. method for preparing according to claim 5 is characterized in that the particulate mean diameter of this protein nano is 20～1000nm, is preferably 20～200nm.
9. method for preparing according to claim 5 is characterized in that drying means adopts lyophilization or spray drying; Aseptic filtration adopts 0.22 μ m filter to filter.
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