CN102357076B - Preparation method of protein nanoparticles coating insoluble drug - Google Patents

Preparation method of protein nanoparticles coating insoluble drug Download PDF

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CN102357076B
CN102357076B CN201110299807.6A CN201110299807A CN102357076B CN 102357076 B CN102357076 B CN 102357076B CN 201110299807 A CN201110299807 A CN 201110299807A CN 102357076 B CN102357076 B CN 102357076B
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protein
drug
insoluble drug
water
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CN102357076A (en
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周建平
霍美蓉
崔蓓
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China Pharmaceutical University
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Abstract

The invention belongs to the field of pharmacy, and discloses a preparation method of protein nanoparticles coating an insoluble drug, which comprises the following steps: preparing an insoluble drug and a water-soluble carrier material into a water-soluble carrier solid dispersion containing the insoluble drug; adding the solid dispersion into a water-based medium containing protein substances, evenly mixing, and processing the mixture under high shear conditions to obtain the protein nanoparticle suspension coating the insoluble drug; and preparing the required preparation formulation. The method does not need to introduce any other organic solvent except ethanol, and even does not need to introduce any organic solvent, thereby preventing organic solvent residues in the preparation, and greatly enhancing the safety of clinical application. The method has the advantages of simple technique, low cost and strong operability.

Description

A kind of preparation method of the protein nano granule that wraps up insoluble drug
Technical field
The invention belongs to pharmaceutical field, relate to a kind of its preparation method of the protein nano granule that wraps up insoluble drug.
Background technology
Insoluble drug involved in the present invention is taking taxone as example.Taxone (as paclitaxel, Docetaxel etc.) is one of effective antitumour medicine being used clinically at present.Paclitaxel is that the bark from Ramulus et folium taxi cuspidatae or its kind found of the seventies or needle, to separate the natural product Docetaxel that obtains be semi-synthetic product, finds that afterwards this is the antitumor agent that a class has 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 in stable non-functional state, thereby reach the object that stops tumor cell mitosis and propagation.And preclinical study shows that Docetaxel is compared with paclitaxel, to the affinity of microtubule is stronger, plasma half-life is longer and cell in the holdup time longer.
Although taxone has good antitumor action, but its water solublity is lower (about 10ug/ml) all, the paclitaxel injection that therefore used clinically and Docetaxol injection all need to use respectively surfactant polyoxyethylene Oleum Ricini and polyoxyethylene sorbitan monoleate (tween 80) and cosolvent ethanol to come the object of dissolving taxol drug.Paclitaxel injection and Docetaxol injection are respectively successfully to develop the product of listing by Bristol-Myers Squibb Co. of the U.S. and French Sanofi-Aventis company the earliest.
Although use surfactant polyoxyethylene Oleum Ricini and Polysorbate is after 80s can be mixed with injection, but its toxic and side effects in clinical practice is larger, easily cause more complication, comparatively common comprise the untoward reaction such as anaphylaxis, bone marrow depression (main manifestations is that neutrophilic granulocyte reduces), fluid retention, neurotoxicity, alopecia.
A large amount of polyoxyethylene castor oils that studies show that can promote a large amount of histamine releasings in vivo, and then produce anaphylaxis, also can cause nerve conduction to postpone sensory nerve pathological changes.In addition, polyoxyethylene castor oil can also form molecule encapsulation paclitaxel molecule in blood, has affected the diffusion of drug molecule to tissue, thereby has affected its Graft Versus Tumor.Also there are some researches show the divinyl hexyl phthalate (Diethylehexy Phthalate) in polyoxyethylene castor oil solubilized PVC transfusion device.And polyoxyethylene sorbitan monoleate (tween 80) has hemolytic, and viscosity is large, and clinical application is very inconvenient.All there is certain problem at aspects such as toxic and side effects, convenient drug administration and stability in above-mentioned injection formulation, therefore develops new taxone form of administration and necessitate.
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 for surrounding's formation one deck disulfide bond crosslinking shell of water-fast pharmacologically active medicine actually.The protein that contains cysteine and/or disulfide bond is pharmaceutically acceptable biological carriers, and this patent is taking human serum albumin as example.
Human serum albumin (human serum albumin, the protein of the strand sugar based HSA) being formed by 585 aminoacid, molecular mass 66.5kDa, be rich in protein (accounting for the 50%-60% of human plasma total protein) in human plasma, there is the feature of long circulation (half-life reaches 19 days) in stable chemical performance, safety non-toxic, non-immunogenicity, good biocompatibility, biodegradable, body.
HSA, as antineoplastic drug carrier material, has lot of advantages, mainly comprises:
1. superior drug loading potentiality: on HSA polypeptide chain with more polar group and hydrophobic amino acid, small-molecule drug, particularly insoluble drug to 70% has the affinity of height, and can there is Reversible binding, for its payload and release provide possibility; In albumin secondary structure containing having an appointment 48% α-helixstructure, 15% beta-pleated sheet structure, all the other are 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, the tumor of Fast Growth has very strong picked-up and deposit albumin ability.HSA, as antineoplastic drug carrier, is combined by the Albumin receptor on endothelial cellular membrane, the caveolin on active cell film, thus antitumor drug is transferred in tumor cell interstitial.Tumor cell interstitial and tumor cell surface are rich in a large amount of aminothiopropionic acid acidic secretion albumen, this protein function is similar to Albumin receptor, can attract specially and adhere to albumin, and aminothiopropionic acid acidic secretion albumen is in all overexpressions of nearly all tumor tissues (bladder, liver, ovary, kidney, digestive tract, mammary gland etc.), this has just formed the medicine storage pond in tumor tissues, for the wide spectrum target killing of the albumin nanometer carrier of loaded with anti-tumor medicine provides may.
3. structure modifiability: contain a large amount of reaction active groups in HSA structure, as free amine group, carboxyl, sulfydryl etc., can, with antitumor drug by chemical bond coupling, improve drug solubility, improve body-internal-circulation time and tumor-targeting; Also can with antibody or part etc. " target molecule " coupling, further improve the cancer target performance of HSA nano-carrier.In recent years, also have and report macromolecular material phase couplings such as HSA and glucosan, aldohexose or PNIPAM 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, by 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 drug solubility, to improve body-internal-circulation time and tumor-targeting, existing more report abroad.
But by by HSA and the direct chemical coupling of medicine, there is following problem: 1) medicine selectable range is narrow: medicine need have certain reaction active groups, and easily lost efficacy at chemical coupling process Chinese medicine; 2) ambiguity of curative effect performance: between medicine and HSA, the easy degree of chemical bond rupture has determined the performance of curative effect of medication, too fast fracture, HSA loses the advantage as carrier material, crosses fracture slowly, and medicine cannot be brought into play curative effect; 3) cost is high, and drug loading is low: in coupling process, medicine feeding amount is far longer than HSA, and coupling drug amount is limited, causes preparation cost large (particularly antitumor drug expensive raw material price), the defect that system drug loading is low.
2. nanometer is passed medicine system: 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 technique mainly contain: emulsifying coacervation, desolvation, pH coacervation and Nab technology.
Although first three methods research Applicative time is the longest, report is maximum, but still has a lot of problems:
1) particle diameter is large.Nanoparticle particle diameter 0.5-10 μ m, and be subject to operating condition impact, the albumin nano granular that is less than 200nm is several without report, and excessive particle diameter is unfavorable for that nano-carrier accumulates in tumor by the EPR effect of tumor;
2) need to solidify.Because HSA is highly water-soluble, prepared nanoparticle needs curing operation, adds chemical cross-linking agent to make albumin occur to be cross-linked or heat and makes albumen generation degeneration, and the former toxicity is larger, and easily make medicine occur crosslinked simultaneously and lose curative effect, latter is not suitable for the load of responsive to temperature medicine; In addition, 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 complexity.Owing to having used the poisonous additives such as surfactant, oil, chemical cross-linking agent in technique, need clean purification with a large amount of organic solvents, but residually will bring hidden danger to drug safety on a small quantity; Adopt high speed centrifugation to separate (16000-20000g) and collect nanoparticle, not only high to instrument requirement, and in water, weigh bad dispersibility;
4) applicable medicine scope is little.Only be suitable for the load of water soluble drug, and quite a few antitumor drug is insoluble drug.
Nab technology is the medicine albumin-binding nanotechnology that America Biological Science Co., Ltd sets up in recent years.This technology is only suitable for the parcel of the fat-soluble medicine that plasma protein binding rate is higher.2005, the paclitaxel albumin-binding nanoparticle of preparing with this technology was (triumphant ) obtain FDA approval listing, after being mainly used in the failure of metastatic breast cancer combined chemotherapy or the breast carcinoma of recurrence in adjuvant chemotherapy 6 months.
Said preparation is with respect to traditional polyoxyethylene castor oil preparation (Thailand
Figure BDA0000096074710000041
) there is lower toxicity, better curative effect, but also there are many defects, comprising: the 1) use of toxic solvents.Employing chloroform is solvent, though there is vacuum evaporation operation, increased technology difficulty, and product may have 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, and therefore preparation pH value need be controlled within the scope of pH5.4-5.8 and (also only stablize 24h in this scope).And human internal environment's pH value is 7.4, after said preparation enters blood circulation, the specificity picked-up of tumor will be separated out from carrier and be unfavorable for to the expectation medicine short time, and dosage is safe clinically at present 1.5 times of preparation.
Because the preparation methoies such as ultrasonic technology all can not be used for the production of industrially scalable, and its diameter of particle obtaining is too large, and this makes it improper and can not be used for patient's administration and use.Therefore America Biology Science Co., Ltd has recorded and Docetaxel/paclitaxel of requiring protection high pressure homogenizing method respectively to prepare can to remake and human serum albumin's freeze-dried preparation in patent US2007082838 and CN98808225 and CN97199720, and after gained reconstruct, the stability of suspension exceedes 24 hours.
High shear technology has the functions such as superpower mixing, pulverizing, dispersion, emulsifying, has significant especially crushing effect for the macromolecular material such as SBS, SBR, can effectively solve pulverizing, an emulsifying difficult problem for all kinds of materials, has replaced traditional colloid mill equipment.Compared with traditional production technology, there is the advantages such as energy consumption is low, production cost is low, product quality is high, super-refinement.
In CN98808225 and CN97199720 patent, record also claimed delivery system that contains water-insoluble drug and protein-coated granule and preparation method thereof, wherein said average diameter of particles is 10-200nm.Described in it, the preparation method of drug regimen system is that the mixture of the organic facies containing described water-insoluble drug dispersion therein and proteinaceous aqueous medium composition is placed 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 the embodiment never obtaining in this patent and claim.The inventor has prepared disclosed mixture, in the pressure limit of then recommending at it, with Avestin high pressure homogenizer, mixture is processed, obtain the nano-emulsion of pH=6.8, and according to evaporating and removing desolventizing by rotary evaporator described in patent, can produce the nano-particle that mean diameter is about 220nm, after carrying out adding normal saline after lyophilization, be easy to recasting and become suspension, but its nano-particle size compared with lyophilization before obviously increase, mean diameter is increased to 390nm by the 220nm before lyophilization, and in 4h, there is the precipitation of nano-particle.In addition, filter with the microporous filter membrane of being recorded in patent CN97199720, be easy to occur stopping up, and medicine productive rate is lower than 30% but result is filter, these are different from the yield results of the 70-100% stating in patent.
In US2007082838 patent, record and the claimed albumin nano granular that comprises Docetaxel medicine that adds stabilizing agent sodium citrate and/or sodium chloride; its mean diameter < 200nm; and described in patent; these use the nano-emulsion obtaining higher than homogenizing method to it is said very high stability; the meaning of term " stability " had both represented that mean diameter was not in time or freezing dry process and changing herein; also represent not occur the drug precipitation (US200708283, embodiment 12) of nano-particle.
The inventor once repeatedly repeated the embodiment 11,16 of the embodiment, particularly US200708283 of above-mentioned patent, and 18, never obtain disclosed result in this patent working example and claims.The inventor has prepared disclosed mixture, then in the pressure limit of its recommendation, with Avestin high pressure homogenizer, it is processed, obtain the nano-emulsion of pH=7.2, utilize rotary evaporator evaporation to remove after organic solvent, produce the nano-particle of the about 280nm of mean diameter.But there is very soon the precipitation of nano-particle, carrying out described filtering with microporous membrane (1.2um, 0.8um, 0.45um and 0.22um) time more difficult, easily there is situation about stopping up in filter membrane, and the nanosuspension that its lyophilized products forms after remaking in physiological solution is unstable, occurs macroscopic precipitation in approximately 8 hours, it is completely different that the stability of claiming in these and patent is greater than the result of 24 hours.
Summary of the invention
The object of the invention is the problems referred to above and shortcoming for prior art, the invention provides the protein nano granule of the clinical practice character that a kind of preparation technology is simpler, character is more stable and better.
Another object of the present invention is to provide the preparation method of above-mentioned protein nano granule.
The inventor stumbles in the process of preparing protein nano granule, when by water-solubility carrier material (as, Polyethylene Glycol, mannitol, PVP, etc.) add after protein system, the protein nano granule that bag carries insoluble drug more easily forms, particle diameter is even simultaneously, and stability improves.In order to investigate this effect of water-solubility carrier material in system, the inventor studied water-solubility carrier material and protein-based between relation, surprisingly, the result of transmission electron microscope and means of differential scanning calorimetry all reflects, this the two be not simple mixed system, but formed a kind of new compound system by interaction.We infer the formation due to this new compound system, insoluble drug can better be carried by bag, thereby preparation technology's operability is significantly promoted, and make nano-particle more stable and more even simultaneously.
Due to the significant advantage of this new compound system, the inventor attempts this water-solubility carrier first to mix with insoluble drug then, insoluble drug is present in water-solubility carrier with the form of molecular dispersion, the two adds in protein-based solution with the form of solid dispersion, mixes and pass through the protein nano granule of the effect formation insoluble drug of high shear force.Found that protein nano granule more easily forms, stability is better; Meanwhile, this nano-particle has the long circulation of obvious blood and tissue, organ targeting, has better pharmacodynamic properties.
The invention provides a kind of by slightly solubility material (as paclitaxel, Docetaxel, cyclosporin, hydroxy camptothecin, etc.) join the preparation method of a Nano grade carrier with the form of molecular dispersion state.The method more easily obtains insoluble drug nano-particle (average diameter is less than 200 nanometers) than nab technology, and do not need to add any poisonous organic solvent (as, dichloromethane, chloroform, etc.), avoid residual in preparation of organic solvent, greatly improved the safety of clinical application.
Meanwhile, the invention provides a kind of protein nano granule, this protein nano granule is high to insoluble drug 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 insoluble drug that the method obtains has the long circulation of obvious blood and tissue, organ targeting, therefore possesses better pharmacodynamic properties.In sum, the invention provides the protein nano granule of the clinical practice character that a kind of preparation technology is simpler, character is more stable and better.This protein nano granule have particle diameter evenly, good stability, the good high feature of safety.
The object of the invention is to realize by following technical proposal:
A preparation method of wrapping up the protein nano granule of insoluble drug, the method comprises the following steps:
A. insoluble drug and water-solubility carrier material are made to the water-solubility carrier solid dispersion that contains insoluble drug;
B. the solid dispersion of gained is added in the aqueous medium containing protein substance, mix homogeneously, mixture has obtained wrapping up the suspension of the protein nano granule of insoluble drug through shear conditions processing;
C. suspension is dried or is first dried again through aseptic filtration, make solid preparation, semi-solid preparation or the gas preparation of the protein nano granule that has wrapped up insoluble drug by required dosage form;
Or
Step b is obtained to suspension directly as liquid preparation or be further prepared into the liquid preparation of other types or by dry suspension or first redissolve and make liquid preparation again after aseptic filtration is dry again.
Described preparation method, wherein said insoluble drug refers to that in every ml water dissolubility is less than the material of 1mg or 1 μ l; Preferably one or more in pharmacological active substance, diagnostic reagent or nutrient substance.
Described preparation method, wherein pharmacological active substance is selected from analgesic, febrifuge, anesthetics, anti-asthmatic, antibiotic, antidepressants, antidiabetic drug, antifungal agent, antihypertensive, anti-inflammatory agent, antineoplastic agent, antianxiety drugs, immunosuppressant, anti-migraine medicine, tranquilizer, sleeping pill, anti-anginal drug, psychosis, antimanic drugs, anti-arrhythmic, anti-arthritic, antigout drug, anticoagulation, Thrombolytic Drugs, anti-fibrinolytic medicine, hemorheology reagent, antiplatelet drug, anticonvulsant, anti-Parkinson medicine, antihistaminic, antipruritic, calcium regulating, antimicrobial drug, antiviral agents, anti-infective, bronchodilator, hormone, antidiabetic drug, lipid lowerers, nucleic acid, promoting erythrocyte generates medicine, antiulcer, anti-reflux medicine, antinauseant/Bendectin, mitotane, ganciclovir knot propylhomoserin ester, nitroso ureas salt, one or more in anthracycline antibiotics and ellipticine, diagnostic reagent is selected from one or more in ultrasonic contrast reagent, pneumoradiography reagent or magnetic radiography reagent, nutrient substance is selected from one or more in aminoacid, sugar, protein, carbohydrate, fatsoluble vitamin or fat.
Described preparation method, wherein antineoplastic agent is selected from cyclophosphamide, D actinomycin D, bleomycin, rubidomycin, amycin, mitomycin, methotrexate, fluorouracil, carboplatin, carmustine, methyl-carmustine, cisplatin, etoposide, interferon, camptothecin analogues, phenesterin, paclitaxel and derivant thereof, Docetaxel and derivant thereof, amycin and derivant thereof, vinblastine, vincristine, tamoxifen, A-20968; Described immunosuppressant is selected from cyclosporin, azathioprine, mizoribine or tacrolimus; Described antiviral drugs is selected from gamma interferon, azidothymidine AZT, amantadine, acycloguanosine; Described antifungal agent is selected from griseofulvin, ketoconazole, amphotericin B, nysfungin class, candicidin; Described antihypertensive is selected from Propranolol, Propafenone, nimodipine, oxprenolol, nifedipine, reserpine, pargyline hydrochloride, deserpidine, diazoxide, Minoxidil, rauwolfine, sodium nitroprusside, ajmaline, alseroxylon, phentolamine mesylate.
Described preparation method, wherein water-solubility carrier material is selected from one or more in 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 refers to can be by 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, or their mixture.
Described preparation method, wherein naturally occurring protein comprises that albumin, immunoglobulin, casein, lipoprotein, hemoglobin, lysozyme, α-2-macroglobulin, fibronectin, glass connect element, Fibrinogen, lipase; Described synthetic protein polymer is selected from one or more in the following material that contains free sulfhydryl groups and/or disulfide group modification: PVOH, poly-ethyl oxazoline, polyacrylamide, polyvinylpyrrolidone, polyglycols, PGA, polycaprolactone or its copolymer, synthetic polyamino acid.
Described preparation method, wherein aqueous medium is selected from water for injection, pure water, mannitol solution, aqueous phosphatic, dextran solution, D/W, sodium-chloride water solution, Freamine Ⅲ, vitamin solution, carbohydrate solutions, or their any two or more mixture.
Described preparation method, the method for the water-solubility carrier solid dispersion that wherein preparation contains insoluble drug 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 ethanol; Preferably adopt solvent-fusion method.(method such as fusion method, solvent method, solvent-fusion method is to prepare the conventional method of solid dispersion.For example, by dissolve with ethanol for medicine (solvent), water-solubility carrier heating and melting (melting), both mix, and this is solvent-fusion method.Again for example, by medicine heating and melting, water-solubility carrier heating and melting, both mix, and this is fusion method.If solvent-fusion method, for " not needing to introduce any other organic solvents except ethanol ", only use ethanol.If fusion method is " not needing to 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 melting of some medicine, can cause excess Temperature, degrade, stability can not ensure, therefore this patent mainly adopts solvent-fusion method, specifically adopts which kind of method to determine according to the character of the medicine of selecting and carrier.)
In described preparation method, shear conditions refers to that application possesses the equipment of high pressure and high shearforce simultaneously, makes mixture reach the objects such as efficient mixing, pulverizing, dispersion and emulsifying; Wherein, high pressure refers to that pressure limit is at 5000 to 30000 pounds/inch 2, preferably 6000～25000 pounds/inch 2.
Described preparation method, wherein the mean diameter of this protein nano granule is 20～1000nm, is preferably 20～200nm.
Described preparation method, wherein drying means adopts lyophilization or spraying to be dried; Aseptic filtration adopts 0.22 μ m filter to filter.
Wrap up a protein nano granule for insoluble drug, the formula of this protein nano granule contains the material of following percentage by weight: 0.1～10% insoluble drug, 0.1～40% water-solubility carrier material, 50～90% protein substances.This protein nano granule adopts said method to prepare.
In detail, the present invention is to provide a kind of preparation method that slightly solubility material (as paclitaxel, Docetaxel, cyclosporin, amycin etc.) is joined to a Nano grade carrier with the form of molecular dispersion state.The method more easily obtains the little nano-particle of insoluble drug (average diameter is less than 200 nanometers) than nab technology, and does not need to add any poisonous organic solvent (as, dichloromethane, chloroform, etc.).The nano-particle of the insoluble drug that the method obtains is more stable, and drug loading is high, has the long circulation of obvious blood and tissue, organ targeting, therefore possesses better pharmacodynamic properties.
The method provides a kind of method that forms the nano-particle of slightly solubility material by desolventizing technology, such as, high shear force (as ultrasonic, high-pressure homogenization effect or conditions of similarity) can be used to form the substrate of nano-particle under the condition that lacks any conventional surfactants and any polymerization core substance.
The method provides the preparation method of a kind of reusable little nano-particle (diameter is less than 200 nanometers), and this granule can pass through 0.22 micron of filter aseptic filtration.This certain size the preparation of nano-particle of filtering by 0.22 micron of filter is significant, because the preparation that contains a large amount of any protein (as albumin) all can not carry out disinfection as autoclaving by conventional method, because heat can make albuminous degeneration.Certainly, if adopt the larger filter in aperture also the mean diameter of granule can be controlled to 1000nm left and right, but the present invention is preferably controlled at 20～200nm by the mean diameter of granule.
In aqueous medium, add the concentration range of protein substance (for example, human serum albumin) be about 0.05-25% (w/v, g/ml, lower with), scope among 0.5%-10% (w/v) better.These aqueous mediums have aqueous medium, Freamine Ⅲ, vitamin solution, carbohydrate solutions or the similar medium of normal saline, buffer saline, water, buffering, and the mixture of any two or more these medium.Different from conventional nano-particle formation method, in mixture, do not need to 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.).
By slightly solubility material (for example, paclitaxel, Docetaxel etc.) and water-solubility carrier (as, Polyethylene Glycol, polyvinylpyrrolidone, poloxamer etc.) be prepared into a kind of solid dispersion (powder) containing slightly solubility material and water-solubility carrier by methods such as fusion method, solvent method or melting solvent methods.The formation of this solid dispersion does not need to introduce any other organic solvents except ethanol, even do not need to introduce organic solvent, avoid residual in preparation of organic solvent (as chloroform, dichloromethane etc.), greatly improved the safety of clinical application.
Under low-shearing force, form a kind of mixture being formed by micron and nanometer droplet through homogenization.This can be by well known to a person skilled in the art that mode completes, and for example, adopts a kind of opereating specification at the Routine Test Lab pressure-even pulp crusher of about 2,000 to about 15,000 revs/min.
Medicament-carried nano granule is under high shear condition to form through homogenization.This homogenization is carried out conventionally in high-pressure homogenate device, 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 2in scope, carry out better.The Emulsion generating is containing imperceptible aqueous carrier nanometer droplet (containing the pharmacological active substance of dissolving etc.) and imperceptible protein nano droplet.Acceptable homogenization process comprises can give high shear and cavitation, as high-pressure homogenate device, ultrasonic processor, high-shear mixer, and similar devices.
Liquid suspended matter can drying obtains the powder of the protein nano granule that wraps up insoluble drug.The powder generating can in any suitable time and in suitable aqueous medium redispersion obtain can be to the suspension of mammal administration.These aqueous mediums have aqueous medium, Freamine Ⅲ, vitamin solution, carbohydrate solutions or the similar medium of normal saline, buffer saline, water, buffering, and the mixture of any two or more these medium.Comprise that for obtaining method that this powder adopts lyophilization, spraying are dry, and similar technology.
The moisture including by removing it for example, can further convert powder type to vacuum freeze-drying method in suitable temperature-time range.Protein (for example; human serum albumin) itself play cryoprotective agent effect; do not need to use conventional freezing protective agent as mannitol, sucrose, glycerol, and similar compound, and this powder is by adding water, normal saline or buffer easily to recombinate.Although do not need, certainly can understand into as very necessary, these conventional cryoprotective agents can join in formula of the present invention.
According to an embodiment of the invention, provide a submicron particles (nano-particle) that formation is very tiny, diameter is less than the method for the granule of 200 nanometers.This granule can carry out aseptic filtration before using in liquid suspension mode.Process for preparation gained final products of the present invention (being drug particles) can aseptic filtration be significant, because can not be by conventional method if autoclave be to for example, carrying out sterilizing containing the suspension of high concentration protein (, human serum albumin).
The application of the protein nano granule of described parcel insoluble drug in pharmacy, this protein nano granule can be used for gastrointestinal administration and parenteral administration.The dosage form of this protein nano granule 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.
Being designed for insoluble drug of the invention process comprises: pharmacologically active medicine, diagnostic reagent, one or more in nutrient substance etc.
Described pharmacologically active agent is selected from analgesic, febrifuge, anesthetics, anti-asthmatic, antibiotic, antidepressants, antidiabetic drug, antifungal agent, antihypertensive, anti-inflammatory agent, antineoplastic agent, antianxiety drugs, immunosuppressant, anti-migraine medicine, tranquilizer, sleeping pill, anti-anginal drug, psychosis, antimanic drugs, anti-arrhythmic, anti-arthritic, antigout drug, anticoagulation, Thrombolytic Drugs, anti-fibrinolytic medicine, hemorheology reagent, antiplatelet drug, anticonvulsant, anti-Parkinson medicine, antihistaminic, antipruritic, calcium regulating, antimicrobial drug, antiviral agents, antimicrobial drug, anti-infective, bronchodilator, hormone, antidiabetic drug, lipid lowerers, protein, nucleic acid, promoting erythrocyte generates medicine, antiulcer/anti-reflux medicine, antinauseant, Bendectin, fatsoluble vitamin, mitotane, ganciclovir knot propylhomoserin ester, nitroso ureas salt, anthracycline antibiotics and ellipticine.
Described medicine is exemplified below:
Analgesics/antipyretic (as: aspirin, acetaminophen, ibuprofen, naproxen sodium, codeine phosphate, dihydrocodeine bitartrate, Diflunisal, mefenamic acid, the ring fourth alcohol tartrate of muttering, choline salicylate, butalbital, methotrimeprazine, miltown etc.); Anesthetis (as: cyclopropane, enflurane, halothane, isoflurane, methoxiflurane, nitric oxide, diprivan see propofol (the third phenol), etc.);
Anti-asthmatic (as: azelastine, ketotifen, Traxanox, etc.);
Antibiotic (as: neomycin, streptomycin, chloromycetin, cephalosporin, ampicillin, penicillin, tetracycline etc.);
Antidepressants (as: nefopam, oxypertine, amoxapine, maprotiline hydrochlorate, two hydroxyl salicylic acid miboplatin are bright, nortriptyline, isocarbossazide, etc.);
Antidiabetic drug (as: biguanides, hormone, sulfonyl urea derivative class, etc.);
Antifungal agent (as: griseofulvin, ketoconazole, amphotericin B, nysfungin class, candicidin, etc.);
Antihypertensive (as, Propranolol, Propafenone, oxprenolol, nifedipine, reserpine, Trimetaphan Camphorsulfonate, deserpidine, diazoxide, Minoxidil, rauwolfine, sodium nitroprusside, ajmaline, alseroxylon, phentolamine mesylate, reserpine, etc.);
Anti-inflammatory agent (as: (nonsteroidal) antibiotic medicine, naproxen, ibuprofen, piroxicam, (steroid) corticosterone, dexamethasone ， fluazacort, hydrocortisone, meticortelone, prednisone, etc.);
Antineoplastic agent (as: cyclophosphamide, unwrapping wire mycin, bleomycin, rubidomycin, amycin, mitomycin, methotrexate, 5-fluorouracil, carboplatin, carmustine (BCNU), Semustine, cisplatin, etoposide, interferon, camptothecin analogues, phenesterin, paclitaxel (taxol) and derivant thereof, Docetaxel and derivant thereof, vinblastine, vincristine, tamoxifen, A-20968, etc.);
Antianxiety drugs (as: L0, prazepam, oxazepam, Tranxene, stable, hydroxyzine pamoate, alprazolam, droperidol, halazepam, chlomezanone, dantrolene, etc.);
Immunosuppressant (as: cyclosporin, azathioprine, mizoribine, tacrolimus [FK506 (tacrolimus)] etc.); Antimigraine (as: isometheptene mucate, bonadorm, etc.);
Tranquilizer/hypnotic (as: barbiturates (as pentobarbital, pentobarbital sodium, barbose), Benzodiazepines (as flurazepam hydrochloride, triazolam, Damiano Tommasi dissolves, midazolam hydrochloride, etc.));
Anti-anginal drug (as: β-adrenergic antagonist, calcium channel blocker (as: nifedipine, etc.));
Psychosis (as: haloperidol, thioridazine, tiotixene, fluphenazin decanoate, chlorine Perphenazine Enanthate, perphenazine, Lithium Citrate de, prochlorperazine, lithium carbonate, etc.);
Anti-arrhythmic (as: esmolol, atlansil, digoxin, etc.);
Anti-arthritic (as: Phenylbutazone, sulindac, penicillamine, disalicylic acid, piroxicam, azathioprine, indometacin, meclofenamate sodium, Kidon (Ono), the acid of benzophenone benzo, auranofin, aurothioglucose, Tolmentin Sodium, etc.);
Antigout drug (as: colchicine, allopurinol, etc.);
Anticoagulant (as: heparin, heparin sodium, warfarin sodium, etc.);
Thrombolytic agent (as: urokinase, streptokinase, recombinant plasminogen activator, etc.);
Antifibrinolytic agent (as aminocaproic acid);
Hemorheologic agent (as pentoxifylline);
Antiplatelet drug (as: aspirin, empirin, etc.);
Anticonvulsant (as: valproic acid, divalproex sodium, phenytoin, phenytoin Sodium, clonazepam, primoline, phenobarbital, sodium phenobarbital, carbamazepine, amobarbital sodium, mesuximide, MET, mebaral, mephenytoin phensuximide, paradione, ethotoin, phenacal, barbose, dipotassium clorazepate, trimethadione, etc.);
Anti-Parkinson medicine (as: ethosuximide etc.);
Antihistaminic/pruritus (as: chlorphenamine, teldane, Pyribenzamine, etc.);
The medicine (as: calcitonin, parathyroid hormone, etc.) regulating for calcium;
Antibiotics (as: aztreonam, chloromycetin, metronidazole, etc.);
Antiviral drugs (as: IFN-γ, azidothymidine AZT, acycloguanosine, etc.);
Antimicrobial agents (as: cephalosporins (as: Cefazolin sodium, cefradine, Cefaclor, cefapirin sodium, ceftizoxime sodium, one hydration cefadroxil, ceftazidime, cefalexin, cephalothin sodium, hydrochloric acid one hydration cefalexin, cefamandole sodium, cefoxitin sodium, cefonicid sodium, ceforanide, ceftriaxone sodium, ceftazidime, cefadroxil, cefradine, Cefuroxime Sodium, Deng), penicillins (as: ampicillin, amoxicillin, benzathine penicillin G, cloxacillin, sodium ampicillin, scotcil, potassium v calcium, piperacillin sodium, azlocillin sodium, carbenicillin indanyl sodium carindacillin, scotcil, neoproc, methicillin sodium, nafcillin sodium, Deng), erythromycin series (as: erythromycin ethylsuccinate, erythromycin, erythromycin propionate lauryl sulfate, erythromycin stearate, erythromycin ethylsuccinate, Deng)),
Anti-infective (as: GM-CSF);
Bronchodilator (as: sympathomimetic nerve class (as: neoisuprel, salbutamol, epinephrine, tartaric acid epinephrine), anticholinergic (as: ipratropium bromide), xanthine (as: aminophylline, diprophylline, metaproterenol sultate, aminophylline), mast cell stabilizers (as: sodium cromoglicate), suck corticosteroids (as: flunisolide beclometasone, one hydration beclomethasone), salbutamol, beclomethasone (BDP), ipratropium bromide, breathe heavily happy peaceful, ketotifen, Sha meter Te Luo, terbutaline sulphate, omcilon, aminophylline, Nedocromil Na, metaproterenol sultate, salbutamol, flunisolide, Deng),
Hormone (as: androgen class (as: danazol, depo-testosterone, fluoxymesterone, ethyl testosterone, testosterone enanthatas, methyltestosterone, fluoxymesterone, depo-testosterone), estrogens (as: estradiol, estrone, conjugated estrogen hormone), cortical steroid (as: omcilon, betamethasone, dexamethasone, prednisone, methyl meticortelone suspension, TA, methyl meticortelone, chlordene triamcinolone acetonide, hydrocortisone, meticortelone, prednisolone 21-tertbutylacetate, meticortelone acetate, prednisolone phosphate sodium, Deng), thyroid hormones (as: levothyroxine sodium, Deng),
Hypoglycemic medicine (as: insulin human, purification bovine insulin, Purification of Pig insulin, glyburide, chlorpropamide, glipizide, tolbutamide, tolazamide, etc.);
Fat-reducing medicament (as: clofibrate, dextrothyroxine sodium, probacol, lovastatin, nicotinic acid, etc.);
Protein (as: deoxyribonuclease, alginase, superoxide dismutase, lipase, etc.);
Nucleic acid (as: justice or the antisensenucleic acids of any treatment protein of encoding, comprise any protein of mentioning here, etc.);
For stimulating the medicine (as: erythropoietin) of erythrocyte growth;
Antiulcer/anti-reflux medicine (as: famotidine, first eyeball miaow guanidine, etc.);
Nausea/antiemetic (as: nabilone, prochlorperazine, dimenhydrinate, torecan, scopolamine, etc.);
Fatsoluble vitamin (as: vitamin A, D, E, K, etc.);
Also have other drug as mitotane, ganciclovir valine ester (visadine), Nitrosourea salt, anthracycline antibiotics, NSC-264137, etc.
Plan to comprise that with the example of the invention process diagnostic medicament preparation is if contrast medium for ultrasonic diagnosis, radiopaque contrast medium (as iodate octane, halocarbon, renograph element etc.), mri contrast agent (as carbon fluoride, fat-soluble paramagnetic compound etc.) are and if change without physics or chemical modification the diagnostic medicament that its characteristic that is in fact insoluble in water is just difficult to conveying.
The example that is designed for trophism medicine of the invention process comprises the mixture of aminoacid, sugar, protein, carbohydrate, fatsoluble vitamin (such as vitamin A, C, D, K etc.) or fat or their any two or more materials.
In the enforcement of this invention, the protein substance of many biocompatibility can be used for preparing the carrier shell of protein nano granule, is wrapped in the outside of insoluble drug.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 for formation one deck disulfide bond crosslinking shell around insoluble drug.Sulfydryl or disulfide bond can preexist in polymer architecture maybe and can import by suitable chemical modification, such as naturally occurring polymer as protein, peptide, polynucleotide, polysaccharide (as starch, cellulose, glucosan, Algin, chitosan, pectin, hyaluronic acid etc.), proteoglycan, lipoprotein etc. are all the candidates with this modification.
Protein-based albumin (containing 35 cysteine), immunoglobulin, casein, lipoprotein, hemoglobin (each α of comprising in 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 protein substances of the conventional application of the present invention.
Albumin for preferred protein substance of the present invention at present.For example α-2-macroglobulin (a kind of known opsonin), can, for strengthening huge picked-up of biting the insoluble drug of like cell to carrier parcel, maybe can promote the insoluble drug of this carrier parcel to enter into liver and spleen.
Equally, the synthetic polypeptide that contains cysteine residues is also the good candidate that forms the protein substance of shell in the periphery of insoluble drug.In addition, polyvinyl alcohol, poly hydroxyethyl methylacrylate, polyacrylic acid, PEOz, polyacrylamide and polyvinylpyrrolidone etc. are all chemical modification (as introduced sulfydryl and/or disulfide bond), and form the good candidate of shell (as causing crosslinked).So the example that is designed for these materials of the invention process is, the synthetic amino acids that contains cysteine residues and/or disulfide bond; Polyvinyl alcohol, contains free sulfhydryl group and/or disulfide bond after modified; Poly hydroxyethyl methylacrylate, contains free sulfhydryl group and/or disulfide bond after modified; Polyacrylic acid, contains free sulfhydryl group and/or disulfide bond after modified; Ju ethoxazole quinoline, contains free sulfhydryl group and/or disulfide bond after modified; Polyacrylamide, contains free sulfhydryl group and/or disulfide bond after modified; Polyvinylpyrrolidone, contains free sulfhydryl group and/or disulfide bond after modified; Polyglycols, contains free sulfhydryl group and/or disulfide bond after modified; Polyactide, PGA, polycaprolactone, or their copolymers, contain free sulfhydryl group and/or disulfide bond after modified; And any two or more mixture of above-mentioned material.
Beneficial effect of the present invention
The invention provides a kind of preparation method of the protein nano granule that wraps up insoluble drug, the method is encapsulated in the insoluble drug granule of suspendible a kind of by can biocompatible composition polymer housing, and its diameter is nanoscale.The method does not need to introduce any other organic solvents except ethanol, even do not need to introduce organic solvent, avoid organic solvent (as chloroform, dichloromethane etc.) residual in preparation, and can not produce the anaphylaxis causing because adding cosolvent or emulsifying agent, greatly improve the safety of clinical application; The method does not need to use conventional surfactant yet.The method provides a kind of and more easily forms and more stable insoluble drug nano-particle, and the method technique is simple, cost is low, strong operability.The method the present invention provides the preparation method of the nano-particle of a kind of energy aseptic filtration simultaneously.
Meanwhile, the invention provides a kind of protein nano granule, this protein nano granule is high for the drug loading of insoluble drug, 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 insoluble drug that the method obtains has the long circulation of obvious blood and tissue, organ targeting, therefore possesses better pharmacodynamic properties.In sum, the invention provides the protein nano granule of the clinical practice character that a kind of preparation technology is simpler, character is more stable and better.This protein nano granule has the features such as drug loading is high, particle diameter even, good stability, safety height.
Brief description of the drawings
Fig. 1 is that fusion method is prepared 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 melting-solvent evaporation method is prepared paclitaxel albumin nanometer formulation intensity particle size distribution figure.
Fig. 3 is WAXD figure.Wherein: sample a: paclitaxel powder, sample b: Lyophilized 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: Lyophilized Human serum albumin powder, sample b: paclitaxel powder; Sample c: paclitaxel and albuminous physical mixture; Sample d: formulation for paclitaxel sample.
Fig. 5 is 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.In figure, along with curve a → h direction, Polyethylene glycol increases.
Fig. 7 is Docetaxel albumin nanometer formulation intensity particle size distribution figure.
Fig. 8 is hydroxy camptothecin albumin nanometer formulation intensity particle size distribution figure.
Fig. 9 is nimodipine albumin nanometer formulation intensity particle size distribution figure.
Figure 10 is vitamin E albumin nanometer formulation intensity particle size distribution figure.
Figure 11 is iodate octane albumin nanometer formulation intensity particle size distribution figure.
Figure 12 is cyclosporin albumin nanometer formulation intensity particle size distribution figure.
Figure 13 is gamma interferon albumin nanometer formulation intensity particle size distribution figure.
Figure 14 is itraconazole albumin nanometer formulation intensity particle size distribution figure.
Detailed description of the invention
The invention will be further elaborated by the following examples.
Embodiment 1 prepares paclitaxel albumin nanometer rice grain by fusion method effect
500 milligrams of paclitaxels are dissolved in to 9.0 milliliters of ethanol.1200 milligrams of Polyethylene Glycol heat after complete melting in 60 DEG C of oil baths, and paclitaxel solution is added wherein, and magnetic agitation is until its complete mix homogeneously, and rotary evaporation is removed after ethanol, cooling rapidly under intense agitation.After vacuum drying spends the night, solid dispersion is added in 85 milliliters of human serum albumin's aqueous solutions (4.5%w/v, g/ml, lower with).Mixture, is then transferred in high pressure homogenizer (EmulsiFlex-05, Canadian Avestin company) to form thick breast through high speed disperser (XHF-1, Shanghai Jin Da biochemical instrument factory) premix 1 minute.High pressure homogenize is at 5000-30,000 pound/inch 2condition under carry out, by Emulsion repetitive cycling at least 5 times, obtain protein nano granule suspension, the product of acquisition has denseer opalescence.Measure through laser particle instrument (Nano-ZS90, Malvern company of Britain), the general diameter of paclitaxel albumin nanometer rice grain that result obtains is in 130-220 nanometer, and result as shown in Figure 1.
Protein nano granule suspension is further lyophilizing 48 hours in the situation that not adding any cryoprotective agent, and the powder obtaining can be easy to reconstitute protein nano granule suspension by adding sterilized water or normal saline.Roughly the same before the size of granule and lyophilizing after reconstruct.
Embodiment 2 prepares paclitaxel albumin nanometer rice grain by melting-solvent evaporation method
300 milligrams of paclitaxels are dissolved in to 6.0 milliliters of ethanol.900 milligrams of Polyethylene Glycol are dissolved in 1.5 milliliters of dehydrated alcohol, heat after complete melting 45 DEG C of oil baths, and paclitaxel solution is added wherein, and magnetic agitation is until its complete mix homogeneously, and rotary evaporation is removed after ethanol, cooling rapidly under intense agitation.After vacuum drying spends the night, solid dispersion is added in 65 milliliters of human serum albumin's aqueous solutions (4.5%w/v).Mixture, is then transferred in high pressure homogenizer (EmulsiFlex-05, Canadian Avestin company) to form thick breast through high speed disperser (XHF-1, Shanghai Jin Da biochemical instrument factory) premix 1 minute.Be emulsified in 5000-30,000 pound/inch 2condition under carry out; by Emulsion repetitive cycling at least 6 times; obtain protein nano granule suspension; the product obtaining has denseer opalescence; through laser particle instrument (Nano-ZS90; Malvern company of Britain) to measure, the general diameter of paclitaxel albumin nanometer rice grain that result obtains is in 120-200 nanometer, and result is as shown in Figure 2.
Protein nano granule suspension is further lyophilizing 48 hours in the situation that not adding any cryoprotective agent, and the powder obtaining can be easy to reconstitute protein nano granule suspension by adding sterilized water or normal saline.Roughly the same before the size of granule and lyophilizing after reconstruct.
Embodiment 3 be less than 200 nanometers can aseptic filtration the preparation of paclitaxel albumin nanometer rice grain
500 milligrams of paclitaxels are dissolved in to 9.0 milliliters of ethanol.800 milligrams of Polyethylene Glycol are dissolved in 1.5 milliliters of dehydrated alcohol, heat after complete melting 45 DEG C of oil baths, and paclitaxel solution is added wherein, and magnetic agitation is until its complete mix homogeneously, and rotary evaporation is removed after ethanol, cooling rapidly under intense agitation.After vacuum drying spends the night, solid dispersion is added in 97 milliliters of human serum albumin's aqueous solutions (4.5%w/v).Mixture, is then transferred in high pressure homogenizer (EmulsiFlex-05, Canadian Avestin company) to form thick breast through high speed disperser (XHF-1, Shanghai Jin Da biochemical instrument factory) premix 1 minute.Be emulsified in 10000-40,000 pound/inch 2condition under carry out; by Emulsion repetitive cycling at least 6 times; obtain protein nano granule suspension; the product obtaining has denseer opalescence; through laser particle instrument (Nano-ZS90; Malvern company of Britain) to measure, the general diameter of paclitaxel albumin nanometer rice grain that result obtains is in 120-170 nanometer.
Protein nano granule suspension filters by the micropore filter of 0.22 micron, and turbidness or granular size are without any change.The HPLC of content of taxol analyzes and shows after exceeding 95% paclitaxel filters recyclablely, and 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 in the situation that not adding any cryoprotective agent, and the powder obtaining can be easy to reconstitute protein nano granule suspension by adding sterilized water or normal saline.Roughly the same before the size of granule and lyophilizing after reconstruct.
Table 1
Figure BDA0000096074710000171
Embodiment 4 judges the physical state of paclitaxel in form of nanoparticles by X-ray powder diffraction
X-ray powder diffraction can be used for judging 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 imperfection and the crystal structure of crystal, i.e. defect and distortion etc. in size, the crystal of crystallite dimension.According to Scherrer formula (Scherrer Equation), the square root of crystallite dimension and diffraction maximum is inversely proportional to, and the size of crystallite dimension reflects its degree of crystallinity height, the i.e. wider lower degree of crystallinity of diffraction maximum correspondence.
Take a small amount of sample a-paclitaxel powder; Sample b-Lyophilized Human serum albumin powder; Sample c-paclitaxel and albuminous physical mixture; With sample d-paclitaxel albumin nanometer rice grain freeze-dried powder (by embodiment of the present invention preparation), XD-3A powder diffractometer carries out powder X-ray diffraction test.5～40 ° of the angles of diffraction, 1 °/min of scanning speed, running voltage 40kV, carries out powder X-ray diffraction test under operating current 50mA condition, and 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 °, has some little diffraction maximums 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 in addition 5.28 °, 8.84 ° and 12.36 ° of characteristic peaks of locating.Sample d formulation for paclitaxel does not demonstrate paclitaxel and has the evidence of crystallization characteristic, but very similar to the amorphous substance broadband protuberance of sample b, prompting paclitaxel is actually and exists with molecularity or unformed state in nanometer formulation, thereby shows that drug taxol is really present in albumin nanometer carrier after high pressure homogenize.
The DSC collection of illustrative plates of embodiment 5 paclitaxel albumin nanometer rice grains
Differential scanning calorimetry (Differential Scanning Calorimetry, DSC) is under temperature programmed control, to measure a kind of technology of the difference power and the temperature relation that are input to sample and reference sample.When crystal melting, to absorb energy (J) to destroy lattice, endergonic how many relevant with crystal structure, characteristic constant heat content (Δ H, the Jg of unit of molecule specific crystal formation -1) can characterize its crystal formation feature.
Take sample a-Lyophilized Human serum albumin powder; Sample b-paclitaxel powder; Sample c-paclitaxel and albuminous physical mixture; With the each 2～3mg of sample d-paclitaxel albumin nanometer rice grain freeze-dried powder (by embodiment of the present invention preparation), the heating rate heating through NETZSCH-DSC 204 analysers with 10 DEG C/min, 50～300 DEG C of temperature ranges, blanket of nitrogen is carried out dsc analysis.Result as shown in Figure 4.
As seen from the figure, curve a shows that albumin has little dehydration exothermic peak at 67.4 DEG C, and there is melting peak slowly 222.3 DEG C of left and right; In curve b, 224.2 DEG C of endothermic peaks and 244.5 DEG C of exothermic peaks are respectively melting peak and the degradation peak of paclitaxel, these two characteristic peaks be present in simultaneously paclitaxel and albuminous physical mixture (curve c) in, and be not present in paclitaxel albumin nanometer formulation (curve d) in.Above spectrum data shows that medicine is present in micelle skeleton with the form of unformed or solid solution.
The morphology research of embodiment 6 paclitaxel albumin nanometer rice grains
Take sample a-human serum albumin freeze-dried powder, sample b-Polyethylene Glycol albumin powder and sample c-paclitaxel albumin nanometer rice grain freeze-dried powder (by embodiment of the present invention preparation) appropriate, with the abundant hydration of 5% glucose solution, obtain light blue opalescence micellar solution, the dyeing of phosphotungstic acid negative staining, getting 1 micellar solution to be measured drips in the groove of drop reaction porcelain plate, and carbon-sprayed copper net is put on test solution, after 1～2min, take out copper mesh, suck residual liquid from copper mesh edge with filter paper small pieces; This copper mesh is placed on to dye liquor and drips (4% Salkowski's solution, pH 7.0) upper about 30s, blot unnecessary dye liquor, dry, through the H-7000 of Hitachi transmission electron microscope observing form, result as shown in Figure 5.
As shown in the figure, human serum albumin can not form spherical structure, but is dispersed into homogeneous solution state; Add the human serum albumin after Polyethylene Glycol to demonstrate more regular spherical structure, particle diameter is 100nm left and right; And medicine carrying albumin preparation forms more regular and intensive spherical structure, particle diameter is 120nm left and right, and particle size distribution is even.Simultaneously known, the transmission electron microscope particle diameter result of sample b, sample c is less than respectively the particle diameter (being respectively 122nm and 138nm) of dynamic light scattering experiment gained, and this may be because the dry run in TEM sample preparation process causes due to subsiding of micellar surface.
The interactional fluorescent spectrometry research of embodiment 7 Polyethylene Glycol and human serum albumin
In human serum albumin, contain trp residue, tryptophan can produce stronger fluorescence under ultraviolet excitation.It is known that fluorescence is swept spectrum result, and human serum albumin's fluorescence maximum excitation wavelength is at 285nm, and maximum emission wavelength is in 350nm left and right.
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 series of experiments and prove the interaction between other materials and albumin.
In 10ml color comparison tube, add successively 1.0 × 10 of 1.0mL -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, fixing Kex=285nm, Kem=350nm, measure relative intensity of fluorescence ^F=F 0-F (F, F 0system solution fluorescence intensity when being illustrated respectively in Polyethylene Glycol existence and existing without Polyethylene Glycol), result is (IF is the unit of fluorescence intensity, and subscript F is the meaning of fluorescence fluorescent) as shown in Figure 6.
Known according to fluorescence result, under the condition of 25 DEG C, Polyethylene Glycol produces quenching effect to the endogenous fluorescence of human serum albumin.Along with the rising of Polyethylene glycol, (fluorescence intensity weakens simultaneously for curve a → h), albuminous fluorescent emission wavelength generation blue shift.Result shows to have produced taking Van der Waals force as main interaction between Polyethylene Glycol and albumin, thereby makes albumin that regular fluorescent quenching occur, and its quenching mechanism meets dynamic quenching.
The impact of embodiment 8 drug level on paclitaxel albumin nanometer rice grain size
Change the concentration of paclitaxel, keep other parameters and in embodiment 2, describe identically, prepared a series of paclitaxel albumin nanometer formulations.Measure and find that lower drug level can generate diameter and be approximately the granule of 160 nanometers through laser particle instrument, higher concentration generates less granule, and diameter is approximately 120 nanometers.In the time that the concentration of paclitaxel in human serum albumin solution is 1 mg/ml, particle diameter is 250 nanometer left and right; In the time that the concentration of paclitaxel in human serum albumin solution is 2 mg/ml, particle diameter is 180 nanometer left and right; In the time that the concentration of paclitaxel in human serum albumin solution is 5 mg/ml, particle diameter is 130 nanometer left and right.
The compatibility stability of embodiment 9 common infusion fluid agent
Investigated paclitaxel albumin nanometer lyophilized formulations (by embodiment of the present 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 meet clinical practice, and therefore the present embodiment is taking particle diameter, content of taxol compatibility stability in index is evaluated its 8h.
Experimental result shows, paclitaxel albumin nanometer lyophilized formulations all can obtain the solution of opalescence homogeneous after redissolving with 5% glucose or 0.9% normal saline, and as shown in Table 2, in 8h, each index is all without significant change.Therefore, 5% glucose and 0.9% normal saline all can be used as the redissolution medium of paclitaxel albumin nanometer rice grain lyophilized formulations when clinical use.
Table 2
Particle diameter and the potential change of paclitaxel albumin nanometer rice grain before and after embodiment 10 lyophilizing
Zetasizer 3000HS laser particle size analyzer is by measuring after paclitaxel albumin nanometer freeze-dried powder (by embodiment of the present invention preparation) redissolution, mean diameter increases slightly compared with before lyophilizing, polydispersity index does not change, the basic character that paclitaxel albumin nano granular is described does not change before and after lyophilizing, has ensured the stable of paclitaxel albumin nanometer formulation by cryodesiccated solid state process.
In addition, Zeta potential substantially remains unchanged before and after lyophilization, all between-32～-36mV.Generally speaking, in the time of the absolute value > of Zeta potential 30mV, because nanoparticle surface exists a large amount of electric charges, between nanoparticle, there is larger repulsive force, and be conducive to nanometer solution stablize and impel nanoparticle size homogeneous.The equal > 30mV of paclitaxel albumin nanometer rice grain Zeta electric potential absolute value, indicates that both all have good physical stability, and 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, taking particle diameter, drug level compatibility stability in index is evaluated its 8h.
Lyophilizing paclitaxel albumin nanometer formulation in cillin bottle (by embodiment of the present invention preparation) is redissolved to concentration and is respectively 1,2,5 mg/ml with normal saline, and be stored in room temperature state.Measure respectively its particle diameter and medicament contg in different time points, suspension was at least stable in 7 days.Wherein as shown in table 4 for 5 mg/ml preparations concrete particle diameter and content of dispersion.
Table 4
The mouse tissue of embodiment 12 paclitaxel albumin nanometer formulations distributes and tests
450 of female white mice of healthy Kunming kind choosing body weight 20 ± 2g, fasting 12h, is divided into 75 groups at random, by 15mg/kg dosage tail vein injection Thailand respectively
Figure BDA0000096074710000212
(Bristol-Myers Squibb Co. of the U.S.), triumphant
Figure BDA0000096074710000213
(America Biological Science Co., Ltd) and paclitaxel albumin nanometer formulation (by embodiment of the present invention preparation), after administration respectively at 5,15min and 0.5,1,3,8,12,24h time point get one group (n=6), eyeball is got after blood respectively, cervical vertebra dislocation is put to death, and cores, the internal organs such as liver, spleen, lung, kidney, brain, tumor.Whole blood is placed in heparin sodium test tube, centrifugal, separated plasma; Internal organs normal saline flushing, filter paper blots its moisture, weighed total amount (blood is by 8% of Mouse Weight).Extract sample introduction after the medicine in biological sample, record medicine peak area, calculate blood plasma and respectively organize the drug level of different time.According to drug level temporal evolution curve calculation AUC, MRT and targeting efficiency.Different time drug level is as shown in the table, and wherein table 5 is safe
Figure BDA0000096074710000214
respectively organize Chinese medicine through time concentration, table 6 is triumphant
Figure BDA0000096074710000215
respectively organize Chinese medicine through time concentration, table 7 for paclitaxel albumin nanometer formulation respectively organize Chinese medicine through time concentration.
Table 5 Thailand
Figure BDA0000096074710000216
respectively organize Chinese medicine through time concentration (ng/ml)
Figure BDA0000096074710000217
Table 6 is triumphant
Figure BDA0000096074710000218
respectively organize Chinese medicine through time concentration (ng/ml)
Figure BDA0000096074710000219
Table 7 paclitaxel albumin nanometer formulation respectively organize Chinese medicine through time concentration (ng/ml)
Figure BDA0000096074710000221
From result: respectively organize Chinese medicine through time concentration
1) Thailand triumphant
Figure BDA0000096074710000223
be respectively 38.45,75.72,236.52 with the AUC value of paclitaxel albumin nanometer formulation; MRT is respectively 11.75,17.42,26.41;
2) Thailand
Figure BDA0000096074710000224
triumphant
Figure BDA0000096074710000225
be respectively 1,1.969,6.151 with the relative uptake ratio Re of tumor of paclitaxel albumin nanometer formulation; Demonstrate this preparation tumor-targeting stronger with respect to triumphant element.
3) Thailand
Figure BDA0000096074710000226
triumphant be respectively 0.88,5.97,10.76 with the cancer target efficiency Te of paclitaxel albumin nanometer formulation; Demonstrate the better tumor-targeting of this preparation.
The rat pharmacokinetics experiment of embodiment 13 paclitaxel albumin nanometer rice grains
Get 12 of SD rats (favorable to the people scientific and technological animal cultivation field, Prevalence In Qixia District, Nanjing City provides), be divided at random 4 groups, 3 every group, give respectively Thailand
Figure BDA0000096074710000228
triumphant
Figure BDA0000096074710000229
with paclitaxel albumin nanometer formulation (by embodiment of the present invention preparation).With the dosage of 7mg/kg by tail vein injection administration, respectively at after administration 5,15,30min, 1,2,4,6,8,12,24,36h eye socket is got the about 0.5mL of blood, is placed in the centrifuge tube that is added with heparin sodium centrifugal 10min (6000r/min) separated plasma.Add 4mL t-butyl methyl ether after vortex centrifugal, extract paclitaxel and internal standard substance diazepam in blood plasma, measure the content of taxol in each time point blood plasma with high performance liquid chromatograph (LC-2010C, Japanese Shimadzu), determination data is as shown in table 8.
Table 8
Figure BDA00000960747100002210
Figure BDA0000096074710000231
Result shows, the blood circulation time that this preparation group can significant prolongation paclitaxel, and this may be because the backbone of Polyethylene Glycol softness can significantly reduce the probability that carrier is engulfed by endothelium reticular system.In addition, the mean residence time of this preparation (MRT) and blood medicine through time concentration curve under area (AUC) be significantly higher than triumphant
Figure BDA0000096074710000232
injection (P < 0.01), is respectively triumphant
Figure BDA0000096074710000233
1.90 and 1.79 times.
The pharmacodynamic experiment of embodiment 14 paclitaxel albumin nanometer rice grains
Get 40 of BALB/c tumor-bearing mices, be divided at random 4 groups, 10 every group, gave respectively sample 1-Thailand in 3,5,7,9 days
Figure BDA0000096074710000234
sample 2-is triumphant
Figure BDA0000096074710000235
sample-3 paclitaxel albumin nanometer formulation (by embodiment of the present invention preparation) and sample 4-normal saline, with the dosage of 10mg/kg, by tail vein injection administration, investigation index is as follows:
1. tumor growth curve: survey major diameter a and the minor axis b of a tumor with slide gauge in every two days, according to formula V=0.5 × a × b 2calculate the volume of tumor, draw tumor growth curve;
2. inhibition rate of tumor growth: when experiment finishes, put to death mice, peel off tumor, take tumor weight, calculate tumor growth inhibition percentage: inhibition percentage TI (%)=(1-WT/WC) × 100% according to following formula, the average tumor weight that wherein WT is treatment group, the average tumor weight that WC is matched group;
3. body weight change curve: measure the body weight of mice every day, draw the body weight change curve of mice.Exceed 20% if Mouse Weight reduces, can think that medicine has produced toxicity.
Result is as shown in table 9 below, calculates relative tumor rate of growth by the gross tumor volume of each time point, and result shows, paclitaxel albumin nanometer formulation is with respect to Thailand with triumphant more effectively suppress the growth of MDR tumor.
Table 9
Figure BDA0000096074710000241
As shown in table 10, the heavy result of tumor after dissection shows, with respect to the average tumor weight of normal saline group, Thailand
Figure BDA0000096074710000242
the inhibition rate of tumor growth of group is 60%, triumphant
Figure BDA0000096074710000243
group is 53%, and preparation group is 66%.
Table 10
Figure BDA0000096074710000244
As shown in table 11, change known triumphant by tumor heavy (g)
Figure BDA0000096074710000245
group and this preparation group are showed no overt toxicity, but in intravenous injection Thailand
Figure BDA0000096074710000246
after, the mice phenomenon such as occur being slow in action, dull.
Table 11
Figure BDA0000096074710000247
Embodiment 15 prepares Docetaxel albumin nanometer rice grain by high pressure homogenize effect
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, heat after complete melting 45 DEG C of oil baths, and paclitaxel solution is added wherein, and magnetic agitation is until its complete mix homogeneously, and rotary evaporation is removed after ethanol, cooling rapidly under intense agitation.After vacuum drying spends the night, solid dispersion is added in 90 milliliters of human serum albumin solutions (4.5%w/v).Mixture, is then transferred in high pressure homogenizer to form thick breast through high speed disperser premix 1 minute.Be emulsified in 9000-40,000 pound/inch 2condition under carry out, by Emulsion repetitive cycling at least 5 times.The system obtaining has denseer opalescence, and the general diameter of polyenic taxusol nano granule obtaining is in 135-200 nanometer.Result as shown in Figure 7.
Protein nano granule suspension is further lyophilizing 48 hours in the situation that not adding any cryoprotective agent.The powder obtaining can be easy to reconstitute protein nano granule suspension by adding sterilized water or normal saline.Roughly the same before the size of granule and lyophilizing after reconstruct.
Embodiment 16 prepares hydroxy camptothecin albumin nanometer rice grain by high pressure homogenize effect
430 milligrams of hydroxy camptothecins are dissolved in 9.0 milliliters of ethanol.1500 milligrams of Polyethylene Glycol heat after complete melting in 60 DEG C of oil baths, and hydroxy-camptothecin aqueous slkali is added wherein, and magnetic agitation is until its complete mix homogeneously, and rotary evaporation is removed after ethanol, cooling rapidly under intense agitation.After vacuum drying spends the night, solid dispersion is added in 100 milliliters of human serum albumin solutions (4.5%w/v).Mixture, is then transferred in high pressure homogenizer to form thick breast through high speed disperser premix 1 minute.Be emulsified in 9000-40,000 pound/inch 2condition under carry out, by Emulsion repetitive cycling at least 5 times.The system obtaining has denseer opalescence, and the general diameter of hydroxy camptothecin albumin nanometer rice grain obtaining is in 180-220 nanometer, and result as shown in Figure 8.
Protein nano granule suspension is further lyophilizing 48 hours in the situation that not adding any cryoprotective agent.The powder obtaining can be easy to reconstitute protein nano granule suspension by adding sterilized water or normal saline.Roughly the same before the size of granule and lyophilizing after reconstruct.
Embodiment 17 prepares nimodipine albumin nanometer rice grain by high pressure homogenize effect
530 milligrams of nimodipine are dissolved in 9.0 milliliters of ethanol.1450 milligrams of Polyethylene Glycol heat after complete melting in 60 DEG C of oil baths, and nimodipine solution is added wherein, and magnetic agitation is until its complete mix homogeneously, and rotary evaporation is removed after ethanol, cooling rapidly under intense agitation.After vacuum drying spends the night, solid dispersion is added in 92 milliliters of human serum albumin solutions (4.5%w/v).Mixture, is then transferred in high pressure homogenizer to form thick breast through high speed disperser premix 1 minute.Be emulsified in 9000-40,000 pound/inch 2condition under carry out, by Emulsion repetitive cycling at least 6 times.The system obtaining has denseer opalescence, and the general diameter of nimodipine albumin nanometer rice grain obtaining is in 140-250 nanometer, and result as shown in Figure 9.
Protein nano granule suspension is further lyophilizing 48 hours in the situation that not adding any cryoprotective agent.The powder obtaining can be easy to reconstitute protein nano granule suspension by adding sterilized water or normal saline.Roughly the same before the size of granule and lyophilizing after reconstruct.
Embodiment 18 prepares vitamin E albumin nanometer rice grain by high pressure homogenize effect effect
1000 milligrams of Polyethylene Glycol are heated after complete melting in 50 DEG C of oil baths, vitamin E is directly added wherein, magnetic agitation is until after its complete mix homogeneously, cooling rapidly under intense agitation.After vacuum drying spends the night, solid dispersion is added in 100 milliliters of human serum albumin solutions (4.5%w/v).Mixture, is then transferred in high pressure homogenizer to form thick breast through high speed disperser premix 1 minute.Be emulsified in 9000-40,000 pound/inch 2condition under carry out, by Emulsion repetitive cycling at least 6 times.The system obtaining has denseer opalescence, and the general diameter of vitamin E albumin nanometer rice grain obtaining is in 160-260 nanometer, and result as shown in figure 10.
Protein nano granule suspension is further lyophilizing 48 hours in the situation that not adding any cryoprotective agent.The powder obtaining can be easy to reconstitute protein nano granule suspension by adding sterilized water or normal saline.Roughly the same before the size of granule and lyophilizing after reconstruct.
Embodiment 19 prepares iodate octane albumin nanometer rice grain by high pressure homogenize effect
1600 milligrams of Polyethylene Glycol heat after complete melting in 50 DEG C of oil baths, and 100 milligrams of iodate octane alcoholic solution are added wherein, and magnetic agitation is until its complete mix homogeneously, and rotary evaporation is removed after ethanol, cooling rapidly under intense agitation.After vacuum drying spends the night, solid dispersion is added in 78 milliliters of human serum albumin solutions (4.5%w/v).Mixture, is then transferred in high pressure homogenizer to form thick breast through high speed disperser premix 1 minute.Be emulsified in 9000-40,000 pound/inch 2condition under carry out, by Emulsion repetitive cycling at least 5 times.The system obtaining has denseer opalescence, and the general diameter of iodate octane albumin nanometer rice grain obtaining is in 140-220 nanometer, and result as shown in figure 11.
Protein nano granule suspension is further lyophilizing 48 hours in the situation that not adding any cryoprotective agent.The powder obtaining can be easy to reconstitute protein nano granule suspension by adding sterilized water or normal saline.After reconstruct, granular size and lyophilizing are roughly the same.
Embodiment 20 prepares cyclosporin albumin nanometer rice grain by high pressure homogenize effect
420 milligrams of Polyethylene Glycol heat after complete melting in 50 DEG C of oil baths, and 100 milligrams of cyclosporin alcoholic solution are added wherein, and magnetic agitation is until its complete mix homogeneously, and rotary evaporation is removed after ethanol, cooling rapidly under intense agitation.After vacuum drying spends the night, solid dispersion is added in 90 milliliters of human serum albumin solutions (4.5%w/v).Mixture, is then transferred in high pressure homogenizer to form thick breast through high speed disperser premix 1 minute.Be emulsified in 9000-40,000 pound/inch 2condition under carry out, by Emulsion repetitive cycling at least 5 times.The system obtaining has denseer opalescence, and the general diameter of cyclosporin albumin nanometer rice grain obtaining is in 120-220 nanometer, and result as shown in figure 12.
Protein nano granule suspension is further lyophilizing 48 hours in the situation that not adding any cryoprotective agent.The powder obtaining can be easy to reconstitute protein nano granule suspension by adding sterilized water or normal saline.Roughly the same before granular size and lyophilizing after reconstruct.
Embodiment 21 prepares gamma interferon albumin nanometer rice grain by high pressure homogenize effect
350 milligrams of Polyethylene Glycol heat after complete melting in 50 DEG C of oil baths, and 100 milligrams of gamma interferon alcoholic solution are added wherein, and magnetic agitation is until its complete mix homogeneously, and rotary evaporation is removed after ethanol, cooling rapidly under intense agitation.After vacuum drying spends the night, solid dispersion is added in 88 milliliters of human serum albumin solutions (4.5%w/v).Mixture, is then transferred in high pressure homogenizer to form thick breast through high speed disperser premix 1 minute.Be emulsified in 9000-40,000 pound/inch 2condition under carry out, by Emulsion repetitive cycling at least 5 times.The system obtaining has denseer opalescence, and the general diameter of gamma interferon albumin nanometer rice grain obtaining is in 130-210 nanometer, and result as shown in figure 13.
Protein nano granule suspension is further lyophilizing 48 hours in the situation that not adding any cryoprotective agent.The powder obtaining can be easy to reconstitute protein nano granule suspension by adding sterilized water or normal saline.Roughly the same before granular size and lyophilizing after reconstruct.
Embodiment 22 prepares itraconazole albumin nanometer rice grain by high pressure homogenize effect
1300 milligrams of Polyethylene Glycol heat after complete melting in 50 DEG C of oil baths, and 100 milligrams of itraconazole alcoholic solution are added wherein, and magnetic agitation is until its complete mix homogeneously, and rotary evaporation is removed after ethanol, cooling rapidly under intense agitation.After vacuum drying spends the night, solid dispersion is added in 87 milliliters of human serum albumin solutions (4.5%w/v).Mixture, is then transferred in high pressure homogenizer to form thick breast through high speed disperser premix 1 minute.Be emulsified in 9000-40,000 pound/inch 2condition under carry out, by Emulsion repetitive cycling at least 5 times.The system obtaining has denseer opalescence, and the general diameter of iodate octane albumin nanometer rice grain obtaining is in 140-220 nanometer, and result as shown in figure 14.
Protein nano granule suspension is further lyophilizing 48 hours in the situation that not adding any cryoprotective agent.The powder obtaining can be easy to reconstitute protein nano granule suspension by adding sterilized water or normal saline.Roughly the same before granular size and lyophilizing after reconstruct.
Embodiment 23 drug loading data
Due to insoluble drug dissolubility in aqueous medium very low (< 1 μ g/ml), it is negligible that therefore the free drug in aqueous medium is compared (> 2mg/ml) with the medication amount that is written into carrier.By centrifuging and taking supernatant liquid filtering, measure the method for subsequent filtrate Chinese medicine content and just can calculate the medication amount being written in carrier.
Precision takes a certain amount of albumin nanometer formulation (by embodiment of the present invention preparation) freeze-dried powder, and distilled water redissolves and is settled to scale, and jolting mixes.Destroy and be diluted to finite concentration with methanol, measure the content of preparation of Chinese medicine, distinguish computational envelope rate and drug loading by following formula (1) and (2).
Figure BDA0000096074710000271
Figure BDA0000096074710000272
Figure BDA0000096074710000281
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 larger, and the paclitaxel albumin nanometer formulation that we prepare by the method has higher drug loading and envelop rate, can reduce administration volume, reduces the albuminous application of adjuvant, has good industrial prospect.
Embodiment 24
Inventor carries out single factor comparison (in table listed difference to different drug categories, water-solubility carrier, protein substance respectively with reference to the method for embodiment 2, all the other are all identical with embodiment 2), the protein nano granule of preparation after testing mean diameter in table 12, table 13, table 14.Meanwhile, DSC detects and all shows that medicine is present in protein nano granular preparation with the form of amorphous or solid solution.
Table 12
Figure BDA0000096074710000282
Figure BDA0000096074710000291
Table 13
Figure BDA0000096074710000292
Table 14
Figure BDA0000096074710000293
Figure BDA0000096074710000301

Claims (12)

1. wrap up a preparation method for the protein nano granule of insoluble drug, it is characterized in that the method comprises the following steps:
A. insoluble drug and water-solubility carrier material are made to the water-solubility carrier solid dispersion that contains insoluble drug, if adopt solvent-free method not introduce any organic solvent while preparing solid dispersion, there is the method for solvent not introduce any other organic solvents except ethanol if adopt;
B. the solid dispersion of gained is added in the aqueous medium containing protein substance, mix homogeneously, mixture has obtained wrapping up the suspension of the protein nano granule of insoluble drug through shear conditions processing;
C. suspension is dried or is first dried again through aseptic filtration, make solid preparation, semi-solid preparation or the gas preparation of the protein nano granule that has wrapped up insoluble drug by required dosage form;
Or
Step b is obtained to suspension directly as liquid preparation or be further prepared into the liquid preparation of other types or by dry suspension or first redissolve and make liquid preparation again after aseptic filtration is dry again;
Wherein:
Described water-solubility carrier material is selected from one or more in following material: Polyethylene Glycol, polyvinylpyrrolidone, poloxamer, polyethylene oxide, mannitol, carbamide, sodium alginate, HPMC, gelatin, sodium carboxymethyl cellulose; Described protein substance is the following material that can pass through sulfydryl and/or disulfide bond crosslinking: natural existence or synthetic protein, synthetic protein polymer, natural or synthetic albuminoid, or their mixture; Described insoluble drug refers to that in every ml water dissolubility is less than the material of 1mg or 1 μ l.
2. preparation method according to claim 1, is characterized in that insoluble drug is selected from one or more in pharmacological active substance, diagnostic reagent or nutrient substance.
3. preparation method according to claim 2, it is characterized in that described pharmacological active substance is selected from analgesic, febrifuge, anesthetics, anti-asthmatic, antibiotic, antidepressants, antidiabetic drug, antifungal agent, antihypertensive, anti-inflammatory agent, antineoplastic agent, antianxiety drugs, immunosuppressant, tranquilizer, sleeping pill, anti-anginal drug, psychosis, antimanic drugs, anti-arrhythmic, anti-arthritic, antigout drug, anticoagulation, Thrombolytic Drugs, anti-fibrinolytic medicine, hemorheology reagent, antiplatelet drug, anticonvulsant, anti-Parkinson medicine, antihistaminic, antipruritic, calcium regulating, antimicrobial drug, antiviral agents, bronchodilator, hormone, lipid lowerers, nucleic acid, promoting erythrocyte generates medicine, antiulcer, anti-reflux medicine, one or more in antinauseant/Bendectin, diagnostic reagent is selected from one or more in ultrasonic contrast reagent, pneumoradiography reagent or magnetic radiography reagent, nutrient substance is selected from one or more in aminoacid, protein, carbohydrate, fatsoluble vitamin or fat.
4. preparation method according to claim 3, it is characterized in that described antineoplastic agent be selected from D actinomycin D, rubidomycin,, methotrexate, methyl-carmustine, etoposide, interferon, camptothecine, phenesterin, paclitaxel, Docetaxel, amycin, vinblastine, vincristine, tamoxifen, A-20968; Described immunosuppressant is selected from cyclosporin, azathioprine, tacrolimus; Described antiviral drugs is selected from gamma interferon, amantadine; Described antifungal agent is selected from griseofulvin, ketoconazole, amphotericin B, nysfungin class, candicidin; Described antihypertensive is selected from Propafenone, nimodipine, oxprenolol, nifedipine, reserpine, deserpidine, diazoxide, Minoxidil, rauwolfine, ajmaline, alseroxylon.
5. preparation method according to claim 1, is characterized in that described naturally occurring protein comprises that albumin, immunoglobulin, casein, lipoprotein, hemoglobin, lysozyme, α-2-macroglobulin, fibronectin, glass connect element, Fibrinogen, lipase; Described synthetic protein polymer is selected from one or more in the following material that adopts free sulfhydryl groups and/or disulfide group modification: poly-ethyl oxazoline, polyacrylamide, polyvinylpyrrolidone, polyglycols, PGA, polycaprolactone or its copolymer, synthetic polyamino acid.
6. preparation method according to claim 1, it is characterized in that described aqueous medium is selected from water for injection, pure water, mannitol solution, aqueous phosphatic, dextran solution, D/W, sodium-chloride water solution, Freamine Ⅲ, vitamin solution, or their any two or more mixture.
7. preparation method according to claim 1, is characterized in that the method for water-solubility carrier solid dispersion that preparation contains insoluble drug is fusion method, solvent method, solvent-fusion method, solvent-spray drying method, solvent-freeze-drying, polishing or Double helix squeezing and pressing method; If adopt solvent-free method not introduce any organic solvent in these methods, there is the method for solvent not introduce any other organic solvents except ethanol if adopt; Described shear conditions refers to that application possesses the equipment of high pressure and high shearforce simultaneously, makes mixture reach efficient mixing, pulverizing, dispersion and emulsifying object; Wherein, high pressure refers to that pressure limit is at 5000 to 30000 lbs/in 2.
8. preparation method according to claim 7, is characterized in that the method for preparing the water-solubility carrier solid dispersion that contains insoluble drug adopts solvent-fusion method.
9. preparation method according to claim 7, is characterized in that high pressure is 6000～25000 lbs/in 2.
10. preparation method according to claim 1, the mean diameter that it is characterized in that this protein nano granule is 20～1000nm.
11. preparation methoies according to claim 10, the mean diameter that it is characterized in that this protein nano granule is 20～200nm.
12. preparation methoies according to claim 1, is characterized in that drying means adopts lyophilization or spraying to be dried; Aseptic filtration adopts 0.22 μ m filter to filter.
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