CN104758942A - Protein-based pharmacological active substance composition, and preparation method and applications thereof - Google Patents

Protein-based pharmacological active substance composition, and preparation method and applications thereof Download PDF

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CN104758942A
CN104758942A CN201410001419.9A CN201410001419A CN104758942A CN 104758942 A CN104758942 A CN 104758942A CN 201410001419 A CN201410001419 A CN 201410001419A CN 104758942 A CN104758942 A CN 104758942A
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active substance
pharmacological active
protein
water
organic solvent
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梁兴杰
安菲菲
柳娟
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National Center for Nanosccience and Technology China
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5169—Proteins, e.g. albumin, gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K9/00—Medicinal preparations characterised by special physical form
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    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5052—Proteins, e.g. albumin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The invention relates to a protein-based pharmacological active substance composition, and a preparation method and applications thereof. The pharmacological active substance delivery system comprise particles composed of solid or liquid pharmacological active substances and protein coating; the protein coating comprises free protein associated with the protein coating; one part of the pharmacological active substances are coated with the protein coating, and the other part of the pharmacological active substances are associated with the free protein; average diameter of the particles ranges from 10 to 200nm, and the maximum dynamic hydrodynamic size of the particles when the particles are dispersed in a water solution is larger than 10nm. The pharmacological active substance delivery system can not be rapidly dissociated to be smaller than 10nm because of dissolving of injections, so that enrichment effect at tumor sites is improved, and biological distribution is improved.

Description

Based on pharmacological active substance composition and method of making the same and the application of protein
Technical field
The present invention relates to delivery system, particularly relate to pharmacological active substance induction system, particularly based on pharmacological active substance composition and method of making the same and the application of protein.
Background technology
Intravenous drug conveying can rapidly, directly with carry the blood flow of medicine to all the other positions of health and reach balance.There is medicine peak concentration in order to avoid injecting rear short time Ink vessel transfusing, medicine being carried in stable carrier, can after aggressive dosage intravenous injection, medicine being discharged gradually at Ink vessel transfusing.
But on the one hand, many common clinical medicine hydrophobicitys are comparatively large, are difficult to be dispersed in waterborne liquid environment, cause the difficulty of administering mode and drug absorption.During by intravenously administrable, need to use the cosolvents such as Cremphor, and the use of these cosolvents likely causes serious clinical side effects.And, limited at the concentration effect of diseased tissue and target spot by the pharmaceutically-active ingredients using the administering mode of cosolvent to cause.On the other hand, in the body of the clinical medicine molecule of many good water solubility, removing speed is fast, and blood drug level decrease speed is fast, and its treatment curative effect needs to be improved further.
Nanotechnology has outstanding value to raising curative effect of medication.On the one hand, nanotechnology can improve entrained pharmacological component medicine in vivo for medicine moves process and moves process for medicine by improving nano-particle medicine in vivo itself.Nanotechnology effectively can improve dissolubility and the rate of dissolution of hydrophobic pharmacological active substance, improves the bioavailability of pharmacological active substance.And nanotechnology can extend the blood circulation time of pharmacological component effectively, improve the enrichment degree of pharmacological component at diseased tissue and target spot.
The clinical value using nanotechnology to improve pharmacological active substance must consider following problem: the material used must be clinical safety, and the composition of preparation-obtained nano-composition is also all clinical safety; The nanostructured of the pharmacological active substance compositions prepared should keep stable, in the clinical acceptable injection of at least one, serious reunion does not occur or dissociates.
In the compositions preparation process of pharmacological active substance, the material of clinical approval should be selected as raw material, such as albumin, protamine, antibody, immunoglobulin etc. as far as possible.With the material of these clinical approvals for raw material, assembling the pharmacological active substance compositions that can obtain composition safety through carrying out nanometer with pharmacological active substance, can ensure that prepared pharmacological active substance compositions has higher clinical safety as much as possible.The nanostructured that the nanostructured of the pharmacological active substance compositions prepared should keep relative stability through the dilution of clinical acceptable injection, particularly maximum particle diameter distribution (transmission electron microscope or dynamic scattering analysis (Number% adds up, Malvern)) should still remain within the scope of 10-200nm.On the one hand, if there is serious reunion after being injected liquid dissolved dilution in pharmacological active substance compositions, when maximum particle diameter distribution rises to more than 10-15 micron order, blood capillary may be caused to block or infraction, cause local hemorrhage, ischemia or anoxia and possible tissue die.On the other hand, for the treatment of some diseases, as the treatment of tumor, the blood capillary at diseased tissue place has permeability and the anelasticity (EPR effect) of enhancing, nanoparticle can be enriched in tumor tissues effectively by passive target mode, this requires that the particle diameter of nanoparticle is less than certain size, literature research is had to show, the nano-particle that size is less than 400nm can be enriched in tumor locus (Cancer research by EPR effect, 1995, 55(17): 3752-3756.), also there are some researches show, the nano-particle that size is less than 200nm can more effectively be enriched in tumor locus (Naturenanotechnology, 2007, 2(12): 751-760.).And there are some researches show, the Nano medication of diameter about 100nm can be enriched in tumor tissues (Int.J.Pharm., 121(1995), 195-203 most effectively; Nature nanotechnology, 2011,6(12): 815-823), and particle diameter is less than the nanoparticle of 10nm easily by the blood capillary filtration discharge of glomerule in blood, passive target effect is subject to weakening (Nature Reviews Drug Discovery, 2008,7(9): 771-782.).Therefore, the nanostructure size of pharmacological active substance compositions keeps relative stability very important within the scope of 10-200nm, safety in the body that not only can ensure nano material, realizes effective enrichment of the diseased tissue such as tumor, and can be degerming by 200nm or 220nm membrane filtration.
The example of a pharmacological active substance is paclitaxel.It is a kind of natural product, is first to separate (J.Am.Chem.Soc.93:2325(1971) from Pacific yew tree (Taxus brevifolia)).In antimitotic drug, the paclitaxel containing a diterpene carbon skeleton presents unique binding mode to the microtubular protein causing mitosis spindle to be formed.Differ widely as vinblastine or colchicine stop tubulin to assemble with other antimitotic drug, paclitaxel is a kind of known plant product that can suppress tubulin depolymehzation process, thus stop reproduction process of cell.
This naturally occurring diterpenoids compound of paclitaxel shows obvious antitumor and antitumaous effect to kinds of tumors.Paclitaxel to the anti-tumor activity as excellent in B16 melanoma, L1210 leukemia, MX-1 mammary neoplasms and CS-1 colon tumor xenografts have of far-ranging tumor model, and is widely used in clinical cancer therapy.But low the making of the water solubility of paclitaxel becomes problem to people's administration.Really, the medicine of conveying insoluble or indissoluble inherently in an aqueous medium, if oral delivery is invalid may be subject to serious infringement.For this reason, paclitaxel formula used at present needs cremphor to make medicament solubilization.
In Clinical practice, paclitaxel itself does not show excessive toxic action, but causes serious anaphylaxis for the emulsifying agent/cosolvent of solubilize drugs.Before current dosing schedules is included in injectable drug, give patient's antihistaminic and steroid to weaken the irritated side effect of cremphor.
For improving the water solubility of paclitaxel, several research worker can give the modified with functional group of higher water solubility its chemical constitution.Wherein, sulfonated derivative (Kingston etc., United States Patent (USP) 5,059,699(1991)) and amino-acid ester (Mathew etc., J.Med.Chem.35:145-151(1992)) show obvious biological activity.The soluble derivative of modified generation is conducive to intravenous conveying and is dissolved in harmless carrier as the paclitaxel in normal saline.But this modification makes the cost of pharmaceutical preparation increase, and may bring out unwanted side reaction and/or anaphylaxis, may reduce the curative effect of medicine.
Document was once reported with the carrier of protein microsphere as pharmacological active substance or diagnostic products.Albumin microsphere has been prepared by the method for thermal denaturation or chemical crosslinking.Thermal denaturation microsphere prepares between temperature 100 DEG C to 150 DEG C from a kind of mixture (the material albumin that such as, will mix and a kind of suitable oil) of emulsifying.Then, with a kind of suitable solvent microsphere washed and store for future use.Leucuta etc. (International Journalof Pharmaceutics41:213-217(1988)) describe the preparation method of thermal denaturation microsphere.
The process of preparative chemistry crosslinked microsphere to comprise with glutaraldehyde process Emulsion to be joined by protein adhesive, then washs and stores for future use.Lee etc. (Science213:233-235(1981)) and U.S. Patent No. 4,671,954 describe this preparation method.
Protein microsphere size prepared by said method is greater than 10 microns, and the clinical safety risk of intravenous approach administration is comparatively large, limits the probability of its further clinical practice.
A kind of conventional method preparing pastille nano-particle is dissolved in in the solvent of water immiscibility (as dichloromethane or other chlorination, fatty or aromatic solvent) by the insoluble polymer of polylactic acid or other biological close capacitive, pharmacological active substance is dissolved in this polymer solution, surfactant is added in oil phase or aqueous phase, oil in water emulsion is formed, by Emulsion slow evaporation under vacuum by suitable method.As enough little in oil droplet and evaporate time stablize, just obtain a kind of aqueous suspension of polymer.Because medicine is just present in polymer solution when starting, so a kind of compositions may be obtained in this way, wherein drug molecule within it catch by the granule that formed by a kind of polymeric matrices.Research worker is had to report with solvent Evaporation preparation containing the microsphere of various medicine and the method for nano-particle, for example, see Tice and Gilley, Journal of controlled Release2:343-352(1985); Bodmeier and McGinity, Int.J.Pharmaceutics43:179(1988); Cavalier etc., J.Pharm.Pharmacol.38:249(1985); And d ' Souza etc., WO94/10980.
Clinical marketed drug Abraxane prepares in conjunction with high pressure homogenize the albumin paclitaxel nano medicine that particle diameter is about 130nm by emulsion method.Its preparation process must use immiscible organic solvent dissolution pharmacological active substance with water, mixes form biphase organic facies and aqueous phase with albumin aqueous solution, then is less than the granule of 200nm (with reference to US005916596A through high pressure homogenize formation size; CN1515244A).But Abraxane powder dissociates rapidly in Clinical practice, formation size is less than the granule of the single albumin size of 10nm, do not improve bio distribution and the blood circulation time (Chem.Soc.Rev. of taxane molecule, 2012,41,2971-3010).Therefore, nanostructured stable albumin/pharmacological active substance compositions is prepared by changing technique, improve pharmacological active substance at the concentration effect of tumor locus by the EPR effect of tumor locus, improve bio distribution, these are all the problems that this area needs solution badly.
Summary of the invention
For the deficiencies in the prior art, the invention provides a kind of pharmacological active substance compositions based on protein, described compositions does not need to add emulsifying agent and solubilizing agent, therefore avoid causing allergic reaction, and can not dissociate rapidly to 10nm size because of the dissolved dilution of injection, therefore improve the concentration effect at tumor locus, improve bio distribution.The present invention also provides preparation method and the application thereof of described compositions.
In first aspect, the invention provides a kind of pharmacological active substance induction system, comprise the pharmacological active substance of solid or liquid and the granule of protein-coated formation, wherein said protein-coated has the free protein with its association, a part for described pharmacological active substance is included in described protein-coated, and another part of described pharmacological active substance and free protein associate; The average diameter of described granule is 10 to 200nm, and maximum power hydration particle diameter when it is scattered in aqueous solution is at more than 10nm.
The pharmacological active substance induction system with above-mentioned feature is entered after in blood of human body by intravenous injection, the part pharmacological active substance associated with free protein can discharge fast, but the part pharmacological active substance be included in described protein-coated can not discharge at short notice fast, but slowly stably discharge within longer time, therefore improve blood circulation time.The most significant difference of pharmacological active substance induction system of the present invention and the such composition to report before this is that the average diameter of the granule of pharmacological active substance induction system of the present invention is 10 to 200nm, maximum power hydration particle diameter when it is scattered in aqueous solution is at more than 10nm, and the blood capillary avoiding glomerule filters discharges and the passive target effect that causes weakens; When the such composition reported before this is scattered in aqueous solution, its maximum power hydration particle diameter is reduced to below 10nm rapidly, and the blood capillary due to glomerule filters discharges and causes passive target effect to weaken.
In the present invention, so-called " aqueous solution " refers to the biological acceptable aqueous solutions such as water, normal saline, buffer solution, glucose solution, vitamin solution, Freamine Ⅲ or their mixture.The present invention have studied normal saline especially as aqueous solution for diluting the situation of described pharmacological active substance induction system, and in the normal saline of 0.1mg/mL, the maximum power hydration particle diameter of described granule is at about 100nm.Therefore, as preferably of the present invention, the maximum power hydration particle diameter of described Granular composite when the normal saline of 0.1mg/mL is at more than 10nm, preferably such as, at more than 20nm, more preferably 50-180nm, 60nm, 70nm, 80nm, 100nm, 120nm, 150nm, 160nm, 170nm, 175nm, 60-150nm, 70-100nm, 80-120nm, 120-150nm or 130-170nm.
Pharmacological active substance induction system of the present invention can be exist with the form of granule dry powder, also the granule of described pharmacological active substance induction system can be suspended in biocompatibility aqueous solution.Wherein, described biocompatibility aqueous solution can be acceptable injection, is more preferably normal saline, glucose injection or buffer.
In second aspect, the invention provides a kind of method preparing pharmacological active substance induction system described in first aspect, comprising:
(1) by protein, pharmacological active substance, biocompatibility aqueous solution be mixed and made into mixed solution with the organic solvent that water dissolves each other;
(2) organic solvent that removing is dissolved each other with water carries out high pressure homogenize again, or carries out high pressure homogenize and remove the organic solvent dissolved each other with water again, obtains nano suspending liquid, then by the dry pharmacological active substance induction system obtaining powdered.
The most significant difference of method of the present invention and prior art is to adopt the organic solvent dissolved each other with water to replace organic solvent immiscible with water of the prior art.Inventor finds that the pharmacological active substance induction system that method of the present invention obtains can not be dissociated rapidly to 10nm size because of the dissolved dilution of injection unexpectedly.
In method of the present invention, pharmacological active substance can be water-insoluble (hydrophobicity) pharmacological active substance, water solublity (hydrophilic) pharmacological active substance or amphiphilic pharmacological active substance.
For water insoluble pharmacologically active material, mixed solution can be prepared by following steps:
(1a) protein is dissolved in biocompatibility aqueous solution, forms protein solution; Water insoluble pharmacologically active material is dissolved in the organic solvent dissolved each other with water, forms pharmacological active substance solution;
(1b) described pharmacological active substance solution is mixed with described protein solution.
For water solublity pharmacological active substance, mixed solution can be prepared by following steps:
(1a) protein and water solublity pharmacological active substance are dissolved in biocompatibility aqueous solution jointly, form proteins/water dissolubility pharmacological active substance mixed solution;
(1b) mix with the organic solvent that water dissolves each other with described proteins/water dissolubility pharmacological active substance mixed solution.
For amphiphilic pharmacological active substance, one of said two devices can be selected.
In method of the present invention, described protein is selected from albumin, protamine, antibody, immunoglobulin, casein, insulin, lysozyme, Fibrinogen, lipase, collagen protein, fibronectin, glass connect element, apoferritin, ferritin, one or more in hemoglobin, preferred human serum albumin, recombination human serum albumin, protamine, antibody, ferritin or apoferritin, most preferably human serum albumin.
In method of the present invention, described pharmacological active substance is selected from antineoplastic agent, analgesics/antipyretic, anesthetis, anti-asthmatic, antibiotic, antidepressants, antidiabetic drug, antifungal, antihypertensive, antibiotic medicine, tumor complication auxiliary treatment biotech drug, antianxiety drugs, immunosuppressant, antimigraine, tranquilizer/sleeping pill, anti-anginal drug, psychosis, antimanic drugs, anti-arrhythmic, anti-arthritic, gout agent, anticoagulant, thrombolytics, anti-fibrinolytic medicine, hemorheology agent, antiplatelet drug, anticonvulsant, anti-Parkinson medicine, antihistaminic/antipruritic, calcium regulator, antibacterial, antiviral agents, antimicrobial drug, anti-infective, bronchodilator, hormone, blood sugar lowering, hypolipidemic, protein, nucleic acid, erythrocyte generates stimulant, antiulcer/anti-reflux dose, antinauseant/antiemetic, vitamin or preparation.
In method of the present invention, described biocompatibility aqueous solution is acceptable injection, is more preferably normal saline, glucose injection or buffer.
In method of the present invention, the described organic solvent dissolved each other with water is selected from one or more the mixed solvent in ethanol, DMF, oxolane, dimethyl sulfoxine, acetone, acetonitrile, methanol, propanol, glycerol, capryl alcohol, dioxane and methyl pyrrolidone.
In method of the present invention, in described step (2), the method for the organic solvent that removing and water dissolve each other comprises: spraying dry, reduction vaporization, lyophilization, employing falling film evaporator, dialysis or ultrafiltration.
In method of the present invention, the operating pressure of described high pressure homogenize is in 3000-30000 pounds per inch 2in, more preferably in 6000-25000 pounds per inch 2in, most preferably at 9000-18000 pound/inch 2in.
In method of the present invention, the method for described drying comprises lyophilization, spraying dry, distilling under reduced pressure or their combination.
In the third aspect, the invention provides pharmacological active substance induction system described in first aspect and in preparation pharmacological active substance is transported to application in the pharmaceutical preparation of experimenter.
The present invention also provides the application of the pharmacological active substance induction system described in first aspect in the pharmaceutical preparation of preparation minimizing medicament side effect.
Beneficial effect of the present invention is: relative to prior art, the present invention does not use immiscible organic solvent with water, but use the organic solvent dissolution pharmacological active substance dissolved each other with water, prepare pharmacological active substance induction system, maximum power hydration particle diameter when it is scattered in aqueous solution is at more than 10nm, the blood capillary avoiding glomerule filters discharges and the passive target effect that causes weakens, and therefore pharmacological active substance of the present invention improves at the concentration effect of tumor locus and bio distribution.It has very strong antiproliferative effect to MCF-7 human breast cancer cell to adopt pharmacological active substance induction system of the present invention to carry out processes and displays to MCF-7 human breast cancer cell, and the test of pesticide effectiveness for the treatment of animal model for tumour shows it can the growth of remarkable Tumor suppression.
Accompanying drawing explanation
Fig. 1 is the grain size distribution (curve 1) of ABI-007 nanoparticle normal saline redissolution (1mg/mL) in the embodiment of the present invention 1 and the grain size distribution (curve 2) of commercially available Abraxane normal saline redissolution (1mg/mL).
Fig. 2 is the grain size distribution (curve 1) of ABI-007 nanoparticle normal saline redissolution (0.1mg/mL) in the embodiment of the present invention 1 and the grain size distribution (curve 2) of commercially available Abraxane normal saline redissolution (0.1mg/mL).
Fig. 3 be in the embodiment of the present invention 1 ABI-007 nanoparticle for the experimental result of external treatment MCF-7 breast cancer cell.
Fig. 4 is the experimental result that in the embodiment of the present invention 1, ABI-007 nanoparticle is used for the treatment of MCF-7 breast carcinoma tumor-bearing mice, and wherein curve 1 is treatment group result, and curve 2 is matched group result.
Fig. 5 be in the embodiment of the present invention 1 ABI-007 nanoparticle by after intravenous injection MCF-7 breast carcinoma tumor-bearing mice, in the distribution situation of Different Organs.
Detailed description of the invention
The present invention will be described in detail below.
The present invention adopts anti-solvent method to generate pharmacological active substance nano-particle in conjunction with high pressure homogenize.The combination of anti-solvent method and high pressure homogenize is very important, protein and pharmacological active substance can combine and form the reunion material being greater than 10nm by anti-solvent method, high pressure homogenize can reduce the particle size of nanoparticle to below 200nm, and the membrane filtration in 200nm or 220nm aperture easy to use is degerming.Pharmacological active substance nano-particle of the present invention is the nanometer formulation prepared according to high-shear conditions (such as supersound process or high pressure homogenize etc.); do not need to use any conventional surfactants, and do not need to use any polymer core material to form nano-particle substrate.The present invention with protein (such as human serum albumin or recombination human serum albumin etc.) as stabilizing agent.
By the particle diameter regulating the pressure of high pressure homogenize and the number of times of high pressure homogenize can control nano-particle of the present invention, to be filtered by the Filter Sterile of 0.22 or 0.2 micron pore size.The nano-particle generated is very important and significant by the metre filter of 0.22 micron pore size, and this is due to protein meeting thermocoagulation, can not by conventional method as autoclave process sterilizing.Pharmacological active substance nano-particle provided by the invention not easily dissociates to below 10nm under the dilution of acceptable injection, this accumulates on tumor locus for nanoparticle by EPR effect passive target is very important, this be due to 10nm size below nanoparticle easily excreted rapidly by glomerular filtration, weaken passive target ability (the Nature ReviewsDrug Discovery of nanoparticle, 2008,7(9): 771-782.).
Pharmacological active substance induction system provided by the invention, comprise the pharmacological active substance of solid or liquid and the granule of protein-coated formation, wherein said protein-coated has the free protein with its association, and a part for described pharmacological active substance is included in described protein-coated; Another part of described pharmacological active substance and free protein associate, can biologically effective immediately when giving mammal.
A large amount of common pharmacological active substance circulated in blood is combined with carrier protein by hydrophobic or ionic interaction, and wherein prevailing example is serum albumin.Method of the present invention and the compositions produced therefrom are the pharmacological active substancies before administration " having been combined " protein in advance by hydrophobic or hydrogen bond or polarity effect or ionic interaction or physically trapping or electrostatic interaction.
Utilize human serum albumin can improve in conjunction with the ability of paclitaxel and other medicines the ability that paclitaxel is incorporated to particle surface.Because albumin appears on colloid drug granule (being formed when removing organic solvent), because electricity repels and the amalgamation of spatial stability, facilitate the formation of the aqueous colloidal dispersion of energy long-time stable.
The present invention also provides the powder type submicron particles can recombinated in water or normal saline.This powder obtains through lyophilization after removing moisture.Human serum albumin as the structural constituent of nano-particle of the present invention, also as cryoprotective agent and restructuring adjuvant.The granule of the metre filter by 0.22 micron or 0.2 micron pore size prepared by the inventive method, producing after drying or lyophilization can for the sterile solid formula of intravenous injection.
In donor provided by the invention, the preparation method of the pharmacological active substance preparation of conveying comprises:
The aqueous medium of pharmacological active substance, the organic solvent dissolved each other with water and protein is carried out be mixed to get mixture (not containing surfactant in this mixture) and be placed in pressure limit in 3000 to 30000 pounds per inch 2high pressure homogenisers in.Organic solvent after standing shear conditions process, can remove selectively from mixture; Also first selectively can remove from mixture, then accept shear conditions process.
The process of above-mentioned formation mixture can be that organic solution is added to aqueous solution, also can be that aqueous solution is added to organic solvent, condition in mixed process can be ultrasonic, magnetic stirring, mechanical agitation, homogenate or high-shear mixer, also can be that other can promote the method for both mixing.Before mixing, water insoluble pharmacologically active material can dissolve in organic solvent, and water solublity and amphiphilic pharmacological active substance can be dissolved in albuminous aqueous solution in advance.
In research process, the present inventor is fully recognized that, although under suitable condition by pharmacological active substance, the aqueous solution of organic solvent and protein mixes the compositions that also can obtain maximum particle diameter distribution and be less than 200nm, but controllability is poor, differing greatly between different batches, be unfavorable for amplifying and produce, although the drug loading repeatedly making great efforts to improve pharmacological active substance is all difficult to simultaneously close to drug loading (10%) and the maximum power hydrated diameter (about 100-130nm) of Abraxane, in actual production, can to control of product quality, amplify production and bring great difficulty, be difficult to by 200nm or 220nm aperture membrane filtration degerming, safety and effectiveness are difficult to be protected, thus limit the application of its clinical practice.Thus, the present inventor finds to need that the mixture of pharmacological active substance, organic solvent and protein aqueous solution is carried out processed further by high pressure homogenisers through arduous experiment and technical study (typical operating pressure is in 3000-30000 pounds per inch 2, in 6000-25000 pounds per inch 2better, most preferably at 9000-18000 pound/inch 2), the hydration kinetics diameter of major part (90%, Number statistics) particle just can be made to be less than 200nm, to be conducive to product degerming by 200nm or 220nm aperture membrane filtration.
The method of the organic solvent in removing mixture comprises the methods such as spraying dry, distilling under reduced pressure, lyophilization, falling film evaporator, dialysis or ultrafiltration.
In order to obtain the complex of the pharmacological active substance-protein that can redissolve, need the solution dried of the protein complex of pharmacological active substance, dry method comprises rotary evaporator, falling film evaporator, spray dryer, freeze dryer or similar devices.The powder that dried obtains, can in any suitable time containing pharmacological active substance and protein, and in suitable aqueous medium, redispersion is so that obtain can to the suspension of mammal administration.
Compositions prepared by the present invention's said method, in acceptable injection dissolved dilution situation, is not easily dissociated into below 10nm size.
Compositions prepared by the present invention's said method, the nano-particle that such as paclitaxel and albumin are formed, not only toxicity is low, and does not have bone marrow inhibition.
In the present invention, " body in conveying " refer to via in oral, intravenous, subcutaneous, abdominal cavity, sheath, intramuscular, suction, external, percutaneous, rectum, vagina or similar approach conveying pharmacological active substance.
In the present invention, " aqueous solution " refers to moisture liquid, comprises water, normal saline, buffer solution, glucose solution, vitamin solution, Freamine Ⅲ or other biological acceptable aqueous solution or its mixture.
In the present invention, " biocompatibility " refers to that material can not change or affect in any harmful mode the biosystem that it is introduced into significantly.
In the present invention, " (pharmacological active substance) induction system ", " (pharmacological active substance) compositions ", " (pharmacological active substance) preparation ", " (pharmacological active substance) nano-particle " and " (pharmacological active substance-albumen) complex " and other similar concept to a certain extent implication are identical.In concrete linguistic context under the prerequisite not producing contradiction, these concepts can be replaced.
With the present invention unlike, existing Abraxane(albumin bound type paclitaxel) preparation process in clearly represent and must use immiscible organic solvent (as chloroform) with water, otherwise cannot to realize.And, Abraxane drug powder is with in physiological saline solution dilution, be very easy to dissociate to below 10nm, such as, the maximum power hydration particle size distribution of the normal saline solution of the Abraxane of 1mg/mL is about 100nm, and is less than 10nm through normal saline dilution to the maximum power hydration particle size distribution of the normal saline solution of the Abraxane of 0.1mg/mL.Method of the present invention, prepare the organic solvent only using in the process of ABI-007 conjugate and dissolve each other with water, as ethanol, DMF, oxolane, dimethyl sulfoxine, acetone, acetonitrile, methanol, propanol, glycerol, capryl alcohol, dioxane, methyl pyrrolidone or their mixed solvent.In qualitative difference, ABI-007 conjugate prepared by the present invention and Abraxane are that ABI-007 conjugate prepared by the present invention is with in physiological saline solution dilution, be not easy to dissociate to below 10nm, ABI-007 conjugate prepared by such as the present invention, when being diluted to 0.1mg/mL with physiological saline solution, still keeps the maximum power hydration particle size distribution of about 100nm diameter.It is very important that this feature plays anti-tumor in vivo ability for ABI-007 conjugate, because the nanoparticle of about 100nm kinetics hydrated diameter has best concentration effect (Cancer research at tumor locus to have had many sections of documents to prove, 1995,55(17): 3752-3756; Int.J.Pharm., 121(1995), 195-203; Nature nanotechnology, 2011,6(12): 815-823), the drug level of tumor locus effectively can be improved.And easily dissociate to the bio distribution of the Abraxane of below 10nm and blood circulation time not be improved significantly (Chem.Soc.Rev., 2012,41,2971-3010).
Can be used in pharmacological active substance of the invention process to comprise: pharmacological active agent, diagnostic reagent and nutrition material etc.Pharmacological active agent is exemplified below: analgesics/antipyretic (as, aspirin, acetaminophen, ibuprofen, naproxen sodium, buprenorphine hydrochloride, Darvon, LOMAR PWA EINECS 246-676-2 propoxyphene, pethidine hydrochloride, dihydro-morphinone hydrochloride, morphine sulfate, oxycodone hydrochloride, codeine phosphate, dihydrocodeine bitartrate, pentazocine hydrochloride, dihydrocodeinone bitartrate, levorphanol tartrate, Diflunisal, trolamine salicylate, nalbuphlne hydrochloride, mefenamic acid, ring fourth is muttered alcohol tartrate, choline salicylate, butalbital, citric acid floxamine, Diphenhydramine citrate, methotrimeprazine, cinnamyl ephedrine hydrochloride, miltown, Deng), anesthetis (e.g., cyclopropane, enflurane, halothane, isoflurane, methoxiflurane, nitric oxide, diprivan see propofol (the third phenol), ether, pentobarbital sodium, etc.), anti-asthmatic (e.g., azelastine, ketotifen, Traxanox, etc.), antibiotic (e.g., neomycin, streptomycin, chloromycetin, cephalosporin, ampicillin, penicillin, tetracycline, etc.), antidepressants (e.g., nefopam, oxypertine, adapin, amoxapine, trazodone hydrochloride, amitriptyline hydrochloride, maprotiline hydrochlorate, W-1544a, norpramin, psychostyl, tranylcypromine sulfate, fluoxetine Hydrochloride, adapin, Presamine, two hydroxyl salicylic acid miboplatin is bright, nortriptyline, amitriptyline hydrochloride, isocarbossazide, norpramin, stangyl, protriptyline hydrochloride, etc.), antidiabetic drug (e.g., insulin, biguanides, hormone, sulfonyl urea derivative class, etc.), antifungal agent (e.g., griseofulvin, ketoconazole, amphotericin B, nystatin, candicidin, etc.), antihypertensive (e.g., Propranolol, Propafenone, oxprenolol, nifedipine, reserpine, Trimetaphan Camphorsulfonate, phenoxybenzamine hydrochloride, pargyline hydrochloride, deserpidine, diazoxide, guanethidine monosulphate, Minoxidil, rauwolfine, sodium nitroprusside, ajmaline, alseroxylon, phentolamine mesylate, reserpine, etc.), antibiotic medicine (e.g., (nonsteroidal) indometacin, naproxen, ibuprofen, ramifenazone, piroxicam, (steroid) corticosterone, dexamethasone ， fluazacort, hydrocortisone, meticortelone, prednisone, etc.), antineoplastic agent (as, paclitaxel (Taxol) and derivant thereof, Docetaxel (taxotere) and derivant thereof, doxorubicin hydrochloride, amycin, epirubicin, daunorubicin, doxorubicin, zorubicin, chlorambucil (chlorambucil), phenylacetic acid chlormethine, American and French human relations (melphalan), uracil mustard (uramustine), EMP (estramustine), prednimustine (prednimustine), formylmerphalan (formylmerphalan), betamerphalan (betamerphalan), adjacent fat virtue mustard (ocaphane), cyclophosphamide (cyclophosphamide), ifosfamide (ifosfamide), trofosfamide (trofosfamide), D actinomycin D, bleomycin, rubidomycin, tretamine (tretamine), TEPA (triethylenephosphoramide, TEPA), phosphinothioylidynetrisaziridine (thiotepa), solaziquonum (solaziquone), triaziquone (triaziquone), carboquone (carboquone), mitomycin, mitoxantrone, bisantrene (bisantrene), etoposide, etoposide phosphate, teniposide, 5-fluorouracil, ftorafur (tegafur), tegadifurum (tegadifur), doxifluridine (doxifluridine), carmofur (carmofur), capecitabine (capecitabine), cytarabine hydrochloride (cytarabine hydrochloride), cytosine arabinoside (cytarabine), monophosphate cytosine arabinoside, diphosphonic acid cytosine arabinoside, ara-CTP, BH-AC (enocitabine), palmityl cytosine arabinoside (N-palmitoyl-ara-C), ancitabine (ancitabine), azacitidine (azacitidine), gemcitabine (gemcitabine), 6-purine mercaptan monohydrate, 6-T-IMP, sulfomercaprine sodium (sulfomercaprine sodium), thioguanine, azathioprine, pentostatin (pentostatin), aminopterin (aminopterin), methotrexate (methotrexate), metal platinum analog derivative (carboplatin, cisplatin, oxaliplatin, nedaplatin, Eptaplatin, Deng), homoharringtonine (homoharringtonine) and derivant thereof, busulfan (busulfan), carmustine (BCNU), lomustine (lomustine, CCNU), semustine (semustine, Me-CCNU), nimustine (nimustine, ACNU), nimustine hydrochlorate, Ranimustine (ranimustine), streptozocin (streptozotocin, streptozotocin), NSC-178248 (chlorozotocin, DCNU), Raltitrexed (raltitrexed), pemetrexed (pemetrexed), colchicine (colchicine), Demecolcine, etoposide, interferon, camptothecin analogues (camptothecine, irinotecan, irinotecan hydrochloride, 10-hydroxycamptothecine, topotecan, SN38, topotecan hydrochloride, 10-hydroxycamptothecine, Deng), phenesterin, vinblastine and derivant (vinblastine thereof, vincristine, vindesine (vindesine), vinorelbine (vinorelbine), vinorelbine tartrate, hydrochloric acid vinorelbine, Deng), imatinib mesylate (imatinib mesylate), Dasatinib (dasatinib), gefitinib (gefitinib), Erlotinib (erlotinib), BAY 43-9006 (sorafenib), Sunitinib malate (sunitinib malate), bortezomib (bortezomib), tamoxifen, etoposide, A-20968, porfimer sodium, chlorin e 6 (Chlorin e6, Ce6), 5-ALA (5-aminolaevulinic acid, ALA), Verteporfin (Verteporfin), temoporfin (temoporfin), silicon phthalocyanine, dichloro silicon phthalocyanine, Phthalocyanine Zinc, targeting CD52 recombinant humanized monoclonal antibody (Campath, MabCampath), recombinant target people epithelial growth factor receptor IgG2K monoclonal antibody (Vectibix), 111in-labelling and 90y-labels targets to CD20 Mus monoclonal antibody (Zevalin), targeting CD20 131i-labelling and non-marked mouse monoclonal antibody (BEXXAR), iodine [ 131i] (aestheticism is raw for neoplastic cell nuclei people Mus chimeric mAb, Vivatuxin), targeting CD33 is in conjunction with chemotherapeutic monoclonal antibody (Mylotarg), targeting interleukin-22: diphtheria toxin, diphtherotoxin fusion rotein (Ontak, Onzar), Kadcyla(ado-trastuzumab emtansine), natural LHRH polypeptide (buserelin, and derivant Buserelin), nafarelin (Nafarelin), leuprorelin (Leuprolide), goserelin (Goserelin), triptorelin (Triptorelin), asparaginase, Pegylation asparaginase, recombinant interleukin2, recombinant tumor necrosis factor, Interferon a2a, interferon alpha 2 b, endostatin, Deng), tumor complication auxiliary treatment biotech drug (e.g., restructuring keratinocyte growth factor Palifermin, restructuring urate oxidase Rasburicase, etc.), antianxiety drugs (e.g., L0, buspirone hydrochloride, prazepam, chlordiazepoxide hydrochloride, oxazepam, Tranxene, stable, hydroxyzine pamoate, hydroxyzine hydrochloride, alprazolam, droperidol, halazepam, chlomezanone, dantrolene, etc.), immunosuppressant (e.g., cyclosporin, azathioprine, mizoribine, FK506 (tacrolimus), etc.), antimigraine (e.g., gynergen, phloride, isometheptene mucate, bonadorm, etc.), tranquilizer/hypnotic (e.g., barbiturates (as pentobarbital, pentobarbital sodium, barbose), Benzodiazepines (as flurazepam hydrochloride, triazolam, Damiano Tommasi dissolves (tomazeparm), midazolam hydrochloride, etc.)), anti-anginal drug (e.g., β-adrenergic antagonist, calcium channel blocker (as: nifedipine, diltiazem hydrochloride, etc.), Nitrates (as: nitroglycerin, isosorbidi dinitras, nitropenthrite, erythrityl tetranitrate, etc.)), psychosis (e.g., haloperidol, loxapine succinate, loxapine hydrochloride, thioridazine, mellaril, tiotixene, fluphenazine hydrochloride, fluphenazin decanoate, Amatansol, trifluoperazine hydrochloride, chlorpromazine hydrochloride, perphenazine, Lithium Citrate de, prochlorperazine, etc.), antimanic drugs (e.g., lithium carbonate, etc.), anti-arrhythmic (e.g., bretylium tosylate toluene fulfonate, esmolol hydrochloride, verapamil hydrochloride, atlansil, encainide hydrochlorate, digoxin, digitophyllin, mexiletine hydrochloride, Dimpyramidum phosphate, procamide, quinidine sulfate, quinidine gluconate, Cardioquin (Purdue Frederick), flecainide acetate, Tocainide Hydrochloride, lidocaine hydrochloride, etc.), anti-arthritic (e.g., Phenylbutazone, sulindac, penicillamine, disalicylic acid, piroxicam, azathioprine, indometacin, meclofenamate sodium, Kidon (Ono), ketoprofen, auranofin, aurothioglucose, Tolmentin Sodium, etc.), antigout drug (e.g., colchicine, allopurinol, etc.), anticoagulant (e.g., heparin, heparin sodium, warfarin sodium, etc.), thrombolytic agent (e.g., urokinase, streptokinase, recombinant tPA, etc.), antifibrinolytic agent (e.g., aminocaproic acid, etc.), hemorheologic agent (e.g., pentoxifylline, etc.), antiplatelet drug (e.g., aspirin, empirin, ascriptin, etc.), anticonvulsant (e.g., 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 (e.g., ethosuximide, etc.), antihistaminic/pruritus (e.g., hydroxyzine hydrochloride, diphhydramine hydrochloride, chlorphenamine, brompheniramine maleate, cyproheptadine hydrochloride, teldane, tavehil, pyrrolidine hydrochloride amine, carbinoxamine maleate, diphenylpyraline hydrochloride, phenindamine tartrate, azatadine maleate, Pyribenzamine, dexbrompheniramine maleate, methdilazine hydrochloride, alimemazine, etc.), for calcium regulate medicine (e.g., calcitonin, parathyroid hormone, etc.), antibacterial (e.g., amikacin sulfate, aztreonam, chloromycetin, chloramphenicol palmitate, CAPS sodium, ciprofloxacin, Clindamycin Hydrochloride, Palmic acid clindamycin, clindamycin phosphate, metronidazole, hydrochloric acid metronidazole, gentamycin sulfate, lincomycin hydrochloride, Tobramycin Sulfate, Lyphocin (Fujisawa), aerosporin, colistimethate sodium, polymyxin E sulfate, Nano silver grain, etc.), antiviral agents (e.g., IFN-γ, azidothymidine AZT, amantadine hydrochloride, ribavirin, acycloguanosine, etc.), antimicrobial drug (as, cephalosporin (as: cefazolin sodium, cefradine, Cefaclor, cefapirin sodium, ceftizoxime sodium, cefoperazone sodium, Cefotetan Disodium, cefuroxime, cefotaxime 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, bristopen, bacampicillin hydrochloride, cloxacillin sodium, ticarcillin 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 lactobionate, erythromycin stearate, erythromycin ethylsuccinate, Deng), Tetracyclines (as: quadracycline, doxycycline hyclate, minocycline hydrochloride, Deng), Deng), anti-infective (e.g., GM-CSF, etc.), bronchodilator (as, sympathomimetic nerve class (as: adrenalin hydrochloride, metaproterenol sultate, terbutaline sulphate, neoisuprel, isoetharine mesylate, isoetharine hydrochloride, albuterol sulfate, salbutamol, methanesulfonic acid Bitolterol, isoprenaline, terbutaline sulphate, tartaric acid epinephrine, metaproterenol sultate, epinephrine, tartaric acid epinephrine), anticholinergic (as: ipratropium bromide), xanthine (as: aminophylline, diprophylline, metaproterenol sultate, aminophylline), mast cell stabilizers (as: sodium cromoglicate), inhaled hormone class (as: flunisolide beclometasone, one hydration beclomethasone), salbutamol, beclomethasone (BDP), ipratropium bromide, Chuan Le Ning, ketotifen, Sha meter Te Luo, xinafoate, terbutaline sulphate, omcilon, aminophylline, Nedocromil Na, metaproterenol sultate, salbutamol, flunisolide, Deng), 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), progestational hormone (as: acetic acid methoxy progesterone, norethindrone acetate), cortical steroid (as: omcilon, betamethasone, Betamethasone phosphate sodium, dexamethasone, DEXAMETHASONE SODIUM PHOSPHATE, dexamethasone acetate, prednisone, methyl meticortelone suspension, TA, methyl meticortelone, phosphoric acid meticortelone sodium, succinic acid methyl meticortelone sodium, succinic acid hydrocortisone sodium, succinic acid methyl meticortelone sodium, chlordene triamcinolone acetonide, hydrocortisone, cyclopentyl propionic acid hydrocortisone, meticortelone, cortisone fluoridized by acetic acid, paramethasone acetate, prednisolone 21-tertbutylacetate, meticortelone acetate, prednisolone phosphate sodium, succinic acid hydrocortisone sodium, Deng), thyroid hormones (as: levothyroxine sodium, Deng), Deng), blood sugar lowering (e.g., insulin human, purification bovine insulin, Purification of Pig insulin, glyburide, chlorpropamide, glipizide, tolbutamide, tolazamide, etc.), hypolipidemic (e.g., clofibrate, dextrothyroxine sodium, probacol, lovastatin, nicotinic acid, etc.), protein (e.g., deoxyribonuclease, alginase, superoxide dismutase, lipase, etc.), nucleic acid (e.g., the justice of any treatment protein of encoding or antisensenucleic acids, comprise any protein mentioned here, CPG, etc.), erythrocyte generation stimulant (e.g., erythropoietin, etc.), antiulcer/Anti-reflux (e.g., famotidine, cimetidine, ranitidine hydrochloride, etc.), antinauseant/antiemetic (e.g., meclozine hydrochloride, nabilone, prochlorperazine, holds dizzy peaceful, promethazine hydrochloride, torecan, scopolamine, etc.), vitamin (e.g., vitamin A, B, C, D, E, K, etc.), other drug (e.g., mitotane, visadine, Nitrosourea salt, anthracycline antibiotics, NSC-264137, etc.), preparation (as, ferriferrous oxide nano-particle (AG-USPIO, Deng), gadolinium-containing nano particles (Gadolinia. nanoparticle, Deng), coordination compound (the Gd-DTPA of metal gadolinium, Gd-EOB-DTPA, Gd-BOPTA(Multihance, Mo Disi), Mn-DPDP(Telsascan, safe happy shadow), Gd-DO3A-butrol, Gd-DTPA-BMA(Europe is shadow), Gd-HP-DO3A, Deng), containing iodine compound (Iopromide (Ultravist), iodixanol, ioversol, iohexol (Omnipaque), cardiografin, sodium amidotrizoate, amidotrizoic acid, iodized oil, Deng), golden nanometer particle, bismuth sulfide nano particle, indocyanine green and derivant thereof, fluorescein, fluorescein sodium, methylene blue, Deng).
Can be used in protide of the present invention as stabilizing agent and comprise albumin (containing 35 cysteine), protamine, immunoglobulin, casein, insulin (containing 6 cysteine), hemoglobin (there are 6 cysteine residues in each α 2 β 2 unit), lysozyme (containing 8 cysteine residues), immunoglobulin, α-2-macroglobulin, fibronectin, glass connection cellulose fiber proteinogen, lipase etc.Wherein protein, peptide, enzyme, antibody and their mixture thereof are the stabilizing agents of the present invention's routine.Preferred protein is albumin, protamine and antibody.Resemble the protein that α-2-macroglobulin is such, may be used for strengthening and hugely bite the picked-up of like cell to the pharmacological active substance granule that shell wraps up, maybe can promote that this shell enwrapped granule enters into liver and spleen.Specific antibody also can be used for these nano-particle to be directed to specific part.
Can be used in organic solvents in particular of the present invention preferably easily and the organic solvent that dissolves each other of water or its mixture.Because protein is insoluble to organic solvent, along with mixing of the organic solvent dissolved each other with water and water, the protein in mixed solution is reunited, and pharmacological active substance can be wrapped in proteins agglomerate thing in proteins agglomerate process, thus forms complex.
Those skilled in the art knows, and in the scope of the invention and scope, can carry out multiple change.Organic media in polymerization shell can change, and can use multiple different pharmacological active substance in the formation of polymerization shell wall, and can use multiple proteins and other polymer that is natural or synthesis.These purposes are also quite widely, except biomedical (conveying of such as medicine, diagnostic reagent (for radiography), artificial blood and parenteral nutrition agent), this polymerization shell structure of the present invention can also join in cosmetic use, such as, use in skin nursing frost or hair products, perfume fragrance, pressure sensibility ink is medium.
Below in conjunction with embodiment, embodiment of the present invention are described in detail.It will be understood to those of skill in the art that following examples are only the preferred embodiments of the present invention, so that understand the present invention better, thus should not be considered as limiting scope of the present invention.
Preparation example 1
The object of the present embodiment proves to utilize high pressure homogenize to prepare nano-particle.30mg paclitaxel (Paclitaxel) is dissolved in 3.0ml ethanol.The aqueous solution of 27.0ml human serum albumin (1%w/v) is added in solution.Be put into by the mixture obtained in rotation (Rotary) vaporizer, under 40 DEG C of decompressions, (30mmHG) evaporates 20-30 minute removing ethanol.Then under low RPM, homogenate 5 minutes, to form rough emulsion, then is transferred to (Avestin) in high pressure homogenizer.Then in 9000-18000 pounds per inch 2(psi) carry out at least again high pressure homogenize under to circulate 5 times.The dispersion liquid obtained is translucent, and the diameter of paclitaxel particles is generally and is less than 200nm(quantity statistics, Malvern Zetasizer).Do not add any antifreezing agent, dispersion liquid lyophilization is formed dry powder.This dry powder can use sterilized water or normal saline to redissolve, and the particle diameter in the solution that redissolution obtains still remains on below 200nm.
Preparation example 2
The object of the present embodiment proves to utilize the cavitation in ultrasonic Treatment and shearing force to prepare taxol nanoparticle.20mg paclitaxel is dissolved in 2.0ml ethanol.4.0ml human serum albumin solution (5%w/v) is added in solution.By mixture homogenate 5 minutes under low RPM, form coarse emulsion, then transferred in the supersound process pond of 40kHz.At the power of 60-90%, 0 grade of lower supersound process 1 minute.Then transfer in rotary evaporator by mixture, under 40 DEG C of decompressions, (30mmHg) evaporates 20-30 minute, removing organic solvent.Obtain paclitaxel microgranule, its diameter is generally and is less than 200nm(quantity statistics, MalvernZetasizer).Do not add any antifreezing agent, dispersion liquid lyophilization is formed dry powder.This dry powder can use sterilized water or normal saline to redissolve, and the particle diameter in the solution that redissolution obtains still remains on below 200nm.
Preparation example 3
The object of the present embodiment is that introduce can the preparation process of drug particles of aseptic filtration.30mg paclitaxel is dissolved in the ethanol of 3ml.Then added by solution in the aqueous solution of 29.4ml human serum albumin (1%w/v), mixture homogenate under low RPM forms thick Emulsion in 5 minutes, then transfers in high pressure homogenizer (Avestin).Be emulsified in 9000-18000 pound/inch 2under carry out, at least carry out 6 circulations.The system obtained is put into revolution vaporizer, evaporate 40 DEG C of decompressions (30mmHg) and organic solvent was removed rapidly in 15-30 minute.The dispersion liquid finally obtained is translucent state, and the diameter of gained paclitaxel particles is generally less than 200nm(quantity statistics, Malvern Zetasizer).By dispersion liquid by 0.22 micrometer Millipore filters, obtain aseptic paclitaxel dispersion liquid thus.By dispersion liquid lyophilization, do not add any antifreezing agent.Redissolve after gained cake block adds sterilized water or normal saline, reconstruct granular size and keep below 200nm.
Preparation example 4
The object of the present embodiment is that introduce can the preparation process of drug particles of aseptic filtration.30mg paclitaxel is dissolved in the ethanol of 3ml.Then solution is added in the aqueous solution of 29.4ml human serum albumin (1%w/v), the mixture obtained is carried out spraying dry and obtain dry mixture to remove organic solvent.The mixture of drying redissolves by use solution, and under low RPM, homogenate forms thick Emulsion in 5 minutes, then transfers in high pressure homogenizer (Avestin).Be emulsified in 9000-18000 pound/inch 2under carry out, at least carry out 6 circulations.The dispersion liquid finally obtained is translucent state, and the diameter of gained paclitaxel particles is generally less than 200nm(quantity statistics, Malvern Zetasizer).By dispersion liquid by 0.22 micrometer Millipore filters, obtain aseptic paclitaxel dispersion liquid thus.By dispersion liquid lyophilization, do not add any antifreezing agent.Redissolve after gained cake block adds sterilized water or normal saline, reconstruct granular size and keep below 200nm.
Preparation example 5
The object of the present embodiment is that introduce can the preparation process of drug particles of aseptic filtration.30mg paclitaxel is dissolved in the ethanol of 3ml.Then added by solution in the aqueous solution of 29.4ml human serum albumin (1%w/v), mixture homogenate under low RPM forms thick Emulsion in 5 minutes, then transfers in high pressure homogenizer (Avestin).Be emulsified in 9000-18000 pound/inch 2under carry out, at least carry out 6 circulations.The system obtained is carried out spraying dry to remove organic solvent.The dispersion liquid finally obtained is translucent state, and the diameter of gained paclitaxel particles is generally less than 200nm(quantity statistics, Malvern Zetasizer).By dispersion liquid by 0.22 micrometer Millipore filters, obtain aseptic paclitaxel dispersion liquid thus.By dispersion liquid lyophilization, do not add any antifreezing agent.Redissolve after gained cake block adds sterilized water or normal saline, reconstruct granular size and keep below 200nm.
Preparation example 6
The object of the present embodiment is that introduce can the preparation process of drug particles of aseptic filtration.30mg paclitaxel is dissolved in the ethanol of 3ml.Then solution is added in the aqueous solution of 29.4ml human serum albumin (1%w/v), the mixture obtained is carried out high pressure homogenize again spraying dry obtain dry mixture to remove organic solvent.Then, the mixture of drying redissolves by use solution, and under low RPM, homogenate forms thick Emulsion in 5 minutes, then transfers in high pressure homogenizer (Avestin).Be emulsified in 9000-18000 pound/inch 2under carry out, at least carry out 6 circulations.The dispersion liquid finally obtained is translucent state, and the diameter of gained paclitaxel particles is generally less than 200nm(quantity statistics, Malvern Zetasizer).By dispersion liquid by 0.22 micrometer Millipore filters, obtain aseptic paclitaxel dispersion liquid thus.By dispersion liquid lyophilization, do not add any antifreezing agent.Redissolve after gained cake block adds sterilized water or normal saline, reconstruct granular size and keep below 200nm.
Preparation example 7
The object of the present embodiment is that introduce can the preparation process of drug particles of aseptic filtration.65mg paclitaxel is dissolved in the ethanol of 6ml.Then solution is added in the aqueous solution of 29.4ml human serum albumin (3%w/v), the mixture obtained is carried out high pressure homogenize again spraying dry obtain dry mixture to remove organic solvent.Then, the mixture of drying redissolves by use solution, and under low RPM, homogenate forms thick Emulsion in 5 minutes, then transfers in high pressure homogenizer (Avestin).Be emulsified in 9000-18000 pound/inch 2under carry out, at least carry out 6 circulations.The dispersion liquid finally obtained is translucent state, and the diameter of gained paclitaxel particles is generally less than 200nm(quantity statistics, Malvern Zetasizer).By dispersion liquid by 0.22 micrometer Millipore filters, obtain aseptic paclitaxel dispersion liquid thus.By dispersion liquid lyophilization, do not add any antifreezing agent.Redissolve after gained cake block adds sterilized water or normal saline, reconstruct granular size and keep below 200nm.
Preparation example 8
The object of the present embodiment is that introduce can the preparation process of drug particles of aseptic filtration.The 10-hydroxycamptothecine of 65mg is dissolved in the DMF of 6ml.Then solution is added in the aqueous solution of 29.4ml human serum albumin (3%w/v), the mixture obtained is carried out high pressure homogenize again spraying dry obtain dry mixture to remove organic solvent.Then, the mixture of drying redissolves by use solution, and under low RPM, homogenate forms thick Emulsion in 5 minutes, then transfers in high pressure homogenizer (Avestin).Be emulsified in 9000-18000 pound/inch 2under carry out, at least carry out 6 circulations.The diameter of gained 10-hydroxycamptothecine granule is generally less than 200nm(transmission electron microscope).By dispersion liquid by 0.22 micrometer Millipore filters, obtain aseptic paclitaxel dispersion liquid thus.By dispersion liquid lyophilization, do not add any antifreezing agent.Redissolve after gained cake block adds sterilized water or normal saline, reconstruct granular size and keep below 200nm.
Preparation example 9
The object of the present embodiment is that introduce can the preparation process of drug particles of aseptic filtration.The vinorelbine of 65mg is dissolved in the methanol of 6ml.Then solution is added in the aqueous solution of 29.4ml human serum albumin (3%w/v), the mixture obtained is carried out high pressure homogenize again spraying dry obtain dry mixture to remove organic solvent.Then, the mixture of drying redissolves by use solution, and under low RPM, homogenate forms thick Emulsion in 5 minutes, then transfers in high pressure homogenizer (Avestin).Be emulsified in 9000-18000 pound/inch 2under carry out, at least carry out 6 circulations.The diameter of gained vinorelbine granule is generally less than 200nm(transmission electron microscope).By dispersion liquid by 0.22 micrometer Millipore filters, obtain aseptic paclitaxel dispersion liquid thus.By dispersion liquid lyophilization, do not add any antifreezing agent.Redissolve after gained cake block adds sterilized water or water for injection, reconstruct granular size and keep below 200nm.
Preparation example 10
The object of the present embodiment is that introduce can the preparation process of drug particles of aseptic filtration.The vinorelbine of 100mg is dissolved in the methanol of 10ml.Then solution is added in the aqueous solution of 90ml human serum albumin (1%w/v), the mixture obtained is carried out high pressure homogenize again spraying dry obtain dry mixture to remove organic solvent.Then, the mixture of drying redissolves by use solution, and under low RPM, homogenate forms thick Emulsion in 5 minutes, then transfers in high pressure homogenizer (Avestin).Be emulsified in 9000-18000 pound/inch 2under carry out, at least carry out 6 circulations.The diameter of gained vinorelbine granule is generally less than 200nm(transmission electron microscope).By dispersion liquid by 0.22 micrometer Millipore filters, obtain aseptic paclitaxel dispersion liquid thus.By dispersion liquid lyophilization, do not add any antifreezing agent.Redissolve after gained cake block adds sterilized water or water for injection, reconstruct granular size and keep below 200nm.
Preparation example 11
The object of the present embodiment is that introduce can the preparation process of drug particles of aseptic filtration.The indocyanine green of 100mg and the human serum albumin of 900mg are dissolved in the water of 90ml.Then add 20ml ethanol to solution, the mixture obtained is carried out high pressure homogenize again spraying dry obtain dry mixture to remove organic solvent.Then, the mixture of drying redissolves by use solution, and under low RPM, homogenate forms thick Emulsion in 5 minutes, then transfers in high pressure homogenizer (Avestin).Be emulsified in 9000-18000 pound/inch 2under carry out, at least carry out 6 circulations.The diameter of gained indocyanine green granule is generally less than 200nm.By dispersion liquid by 0.22 micrometer Millipore filters, obtain aseptic indocyanine green dispersion liquid thus.By dispersion liquid lyophilization, do not add any antifreezing agent.Redissolve after gained cake block adds sterilized water or water for injection, reconstruct granular size and keep below 200nm.
Preparation example 12
The object of the present embodiment is that introduce can the preparation process of drug particles of aseptic filtration.The doxorubicin hydrochloride of 100mg and the human serum albumin of 900mg are dissolved in the water of 90ml.Then add 20ml ethanol to solution, the mixture obtained is carried out high pressure homogenize again spraying dry obtain dry mixture to remove organic solvent.Then, the mixture of drying redissolves by use solution, and under low RPM, homogenate forms thick Emulsion in 5 minutes, then transfers in high pressure homogenizer (Avestin).Be emulsified in 9000-18000 pound/inch 2under carry out, at least carry out 6 circulations.The diameter of gained amycin granule is generally less than 200nm.By dispersion liquid by 0.22 micrometer Millipore filters, obtain aseptic amycin dispersion liquid thus.By dispersion liquid lyophilization, do not add any antifreezing agent.Redissolve after gained cake block adds sterilized water or water for injection, reconstruct granular size and keep below 200nm.
Test example 1
The ABI-007 nanoparticle of preparation in the embodiment of the present invention 1 and commercially available Abraxane are redissolved with normal saline, dilute respectively, its result as depicted in figs. 1 and 2, when the ABI-007 nanoparticle redissolution concentration of preparation in the embodiment of the present invention 1 is 1mg/mL, maximum power hydration particle diameter is about 100nm; When being diluted to 0.1mg/mL, maximum power hydration particle diameter still remains on about 100nm.And commercially available Abraxane redissolves concentration when being 1mg/mL, maximum power hydration particle diameter is about 100nm; But when being diluted to 0.1mg/mL, maximum power hydration particle diameter is down to below 10nm.Prove that the ABI-007 nanoparticle of preparation in the embodiment of the present invention 1 not easily dissociates rapidly to below 10nm when redissolving dilution.
Test example 2
With the ABI-007 nanoparticle of preparation in embodiment 1, external treatment is carried out to MCF-7 human breast cancer cell.By be in exponential phase the trypsinization of MCF-7 cell after, the resuspended one-tenth cell suspension of complete medium, is inoculated in 96 orifice plates thereupon, and every hole 100 μ L, is placed in 37 DEG C, 5%CO 2incubator is cultivated, and adds paclitaxel (taxol) nano-particle prepared according to embodiment 1 method of variable concentrations after 24h.MTT detection is carried out after 24h.Cultivate 4h before stopping and add the MTT of 10 μ L5mg/mL, inhale after abandoning culture fluid and add 100 μ L DMSO cessation reactions, microplate reader 570nm detects OD value.Experiment in triplicate.Experimental result is as Fig. 3, and the taxol nanoparticle prepared by result display has antiproliferative effect to MCF-7 cell, and the concentration of nanoparticle is higher, and its antiproliferative effect is stronger.
Test example 3
With the treatment of taxol nanoparticle to animal model for tumour.Adopt paclitaxel (taxol) nano-particle prepared according to embodiment 1 method.The MCF-7 HBT model of the preparation of this medicine to mouse xenografts is tested.In mice subcutaneous implantation MCF-7 breast tumor, start treatment when tumor grows to about 150-300mg size.Because tumor growth can reach These parameters in 12 days, therefore within the 13rd day, start treatment after the transfer.
Taxol nanoparticle with the dosage treatment tumor-bearing mice of 10mg/kg, gave intravenous injection at the 13rd day as the suspension in normal saline.Treatment group has 7 animals.The contrast lotus tumor group of 7 animals with pressing same method, but only accepts normal saline.Monitor the size of tumor in time.As shown in Figure 4, the display of the tumor weight of matched group increases result greatly, the animal of this group all the 28th day to 39 days between put to death.And treatment group shows significant effect, obviously much smaller than matched group at the gross tumor volume of the 33rd day all treatment group animal.
Test example 4
In ABI-007 nano-particle preparation process, fluorescent dye Cy5 labelling is carried out to albumin, thus prepare there is fluorescently-labeled ABI-007 nano-particle.After tumor-bearing mice being given to the administration of 10mg/kg dosage, undertaken quantitatively to the fluorescence intensity of the tumor-bearing mice Different Organs accepting the treatment of ABI-007 nano-particle by toy fluoroscopic imaging systems, its result as shown in Figure 5, result shows: in tumor, the concentration of ABI-007 nano-particle is organized apparently higher than other, illustrates that ABI-007 nano-particle of the present invention has obvious enrichment in tumor.
Applicant states, the present invention illustrates detailed features of the present invention and method detailed by above-described embodiment, but the present invention is not limited to above-mentioned detailed features and method detailed, namely do not mean that the present invention must rely on above-mentioned detailed features and method detailed could be implemented.Person of ordinary skill in the field should understand, any improvement in the present invention, to equivalence replacement and the interpolation of auxiliary element, concrete way choice etc. that the present invention selects component, all drops within protection scope of the present invention and open scope.

Claims (10)

1. a pharmacological active substance induction system, comprise the pharmacological active substance of solid or liquid and the granule of protein-coated formation, wherein said protein-coated has the free protein with its association, a part for described pharmacological active substance is included in described protein-coated, and another part of described pharmacological active substance and free protein associate; The average diameter of described granule is 10 to 200nm, and maximum power hydration particle diameter when it is scattered in aqueous solution is at more than 10nm.
2. pharmacological active substance induction system according to claim 1, is characterized in that, the maximum power hydration particle diameter of described Granular composite when the normal saline of 0.1mg/mL is at more than 10nm, preferably at more than 20nm, more preferably 50-180nm.
3. pharmacological active substance induction system according to claim 1 and 2, is characterized in that, described granule is suspended in biocompatibility aqueous solution;
Preferably, described biocompatibility aqueous solution is acceptable injection, is more preferably normal saline, glucose injection or buffer.
4. prepare a method for the pharmacological active substance induction system described in any one of claim 1-3, comprising:
(1) by protein, pharmacological active substance, biocompatibility aqueous solution be mixed and made into mixed solution with the organic solvent that water dissolves each other;
(2) organic solvent that removing is dissolved each other with water carries out high pressure homogenize again, or carries out high pressure homogenize and remove the organic solvent dissolved each other with water again, obtains nano suspending liquid, then by the dry pharmacological active substance induction system obtaining powdered.
5. method according to claim 4, is characterized in that, described step (1) specifically comprises:
(1a) protein is dissolved in biocompatibility aqueous solution, forms protein solution; Water insoluble pharmacologically active material is dissolved in the organic solvent dissolved each other with water, forms pharmacological active substance solution;
(1b) described pharmacological active substance solution is mixed with described protein solution.
6. method according to claim 4, is characterized in that, described step (1) specifically comprises:
(1a) protein and water solublity pharmacological active substance are dissolved in biocompatibility aqueous solution jointly, form proteins/water dissolubility pharmacological active substance mixed solution;
(1b) mix with the organic solvent that water dissolves each other with described proteins/water dissolubility pharmacological active substance mixed solution.
7. the method according to any one of claim 4-6, it is characterized in that, described protein is selected from albumin, protamine, antibody, immunoglobulin, casein, insulin, lysozyme, Fibrinogen, lipase, collagen protein, fibronectin, glass connect element, apoferritin, ferritin, one or more in hemoglobin, preferred human serum albumin, recombination human serum albumin, protamine, antibody, ferritin or apoferritin, most preferably human serum albumin;
Preferably, described pharmacological active substance is selected from antineoplastic agent, analgesics/antipyretic, anesthetis, anti-asthmatic, antibiotic, antidepressants, antidiabetic drug, antifungal, antihypertensive, antibiotic medicine, tumor complication auxiliary treatment biotech drug, antianxiety drugs, immunosuppressant, antimigraine, tranquilizer/sleeping pill, anti-anginal drug, psychosis, antimanic drugs, anti-arrhythmic, anti-arthritic, gout agent, anticoagulant, thrombolytics, anti-fibrinolytic medicine, hemorheology agent, antiplatelet drug, anticonvulsant, anti-Parkinson medicine, antihistaminic/antipruritic, calcium regulator, antibacterial, antiviral agents, antimicrobial drug, anti-infective, bronchodilator, hormone, blood sugar lowering, hypolipidemic, protein, nucleic acid, erythrocyte generates stimulant, antiulcer/anti-reflux dose, antinauseant/antiemetic, vitamin or preparation,
Preferably, described biocompatibility aqueous solution is acceptable injection, is more preferably normal saline, glucose injection or buffer;
Preferably, the described organic solvent dissolved each other with water is selected from one or more the mixed solvent in ethanol, DMF, oxolane, dimethyl sulfoxine, acetone, acetonitrile, methanol, propanol, glycerol, capryl alcohol, dioxane and methyl pyrrolidone.
8. the method according to any one of claim 4-7, is characterized in that, in described step (2), the method for the organic solvent that removing and water dissolve each other comprises: spraying dry, reduction vaporization, lyophilization, employing falling film evaporator, dialysis or ultrafiltration;
Preferably, the operating pressure of described high pressure homogenize is in 3000-30000 pounds per inch 2in, more preferably in 6000-25000 pounds per inch 2in, most preferably at 9000-18000 pound/inch 2in;
Preferably, the method for described drying comprises lyophilization, spraying dry, distilling under reduced pressure or their combination.
9. pharmacological active substance to be transported to the application in the pharmaceutical preparation of experimenter by the pharmacological active substance induction system according to any one of claim 1-3 in preparation.
10. the pharmacological active substance induction system according to any one of claim 1-3 reduces the application in the pharmaceutical preparation of medicament side effect in preparation.
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