WO2015032207A1 - 主动靶向型抗肿瘤药物及其制备方法 - Google Patents

主动靶向型抗肿瘤药物及其制备方法 Download PDF

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WO2015032207A1
WO2015032207A1 PCT/CN2014/075727 CN2014075727W WO2015032207A1 WO 2015032207 A1 WO2015032207 A1 WO 2015032207A1 CN 2014075727 W CN2014075727 W CN 2014075727W WO 2015032207 A1 WO2015032207 A1 WO 2015032207A1
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nanocarrier
composite
pro
npy
composition
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PCT/CN2014/075727
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English (en)
French (fr)
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李娟�
吴爱国
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中国科学院宁波材料技术与工程研究所
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Priority to CH00295/16A priority Critical patent/CH710313B1/de
Priority to DE112014004133.5T priority patent/DE112014004133T5/de
Priority to US14/917,256 priority patent/US20160213788A1/en
Publication of WO2015032207A1 publication Critical patent/WO2015032207A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to the field of pharmaceutical technology, and in particular to an active targeted antitumor drug and a preparation method thereof. Background technique
  • Active targeted drugs by modifying active target molecules (such as antibodies or ligands, etc.) on the surface of a drug or drug carrier, and binding to specific antigens or receptors on certain tissues or cells to achieve drug-specific cell and tissue initiative.
  • active target molecules such as antibodies or ligands, etc.
  • the function of targeting Due to the high specificity, high selectivity and high affinity between antigen-antibody and receptor-ligand, active targeting has higher targeting efficiency than passive targeting, and thus active targeted drug delivery system Research is also very active at home and abroad.
  • Actively targeted drugs based on the principle of specific binding of antigens to antibodies have many problems, such as low effective concentration of target drugs, strong racial specificity, high immunogenicity, and high R&D production costs.
  • active targeted drugs based on ligand- and receptor-specific binding principles are currently targeted for tumors due to their high selectivity, non-racial specificity, non-immunogenicity, high stability and low cost.
  • the focus and hotspot of drug delivery system design include tumor-targeted drug mediated by folate receptors, transferrin receptors, integrin receptors, and peptide receptors.
  • peptide receptor-mediated tumor targeting drugs have received increasing attention.
  • Neuropeptide Y is a hormone that is widely present in the central and peripheral regions and maintains homeostasis.
  • Six NPY receptors have been discovered and identified, namely ⁇ ⁇ ⁇ 2 , ⁇ 3 , ⁇ 4 , ⁇ 5 and ⁇ 6 , which are widely present in the mammalian central nervous system and peripheral nervous system.
  • the function of cockroaches is inseparable from its receptors, and the diversity of receptors causes functional diversity.
  • drugs for neuropeptide receptors are used to treat diseases associated with physiological disorders, including: obesity, cardiovascular disease, high Blood lipids, epilepsy, anxiety and other diseases.
  • anti-tumor drugs targeting NPY receptors are rare, especially anti-tumor drugs for kidney cancer, stomach cancer, breast cancer and ovarian cancer have not been reported.
  • the object of the present invention is to provide an active targeted antitumor drug and a preparation method thereof.
  • a first aspect of the invention provides a composite comprising:
  • the target molecule being coupled to a surface of the nanocarrier
  • the target molecule is selected from the group consisting of: [D-Arg 25 ]-NPY, [D-His 26 ]-NPY, [D-Arg 25 ,
  • the nanocarrier has a particle diameter of 200 nm or less and a polydispersity index (PDI) of less than 0.5.
  • PDI polydispersity index
  • the target molecule is present in an amount of from 1.11 to 22.2% by weight based on the total weight of the composite. In another preferred embodiment, the target molecule is present in an amount of from 5.60 to 11. lwt%, based on the total weight of the composite. In another preferred embodiment, the composite has one or more of the following characteristics:
  • the nanocarrier has a particle diameter of 10 to 200 nm.
  • the nanocarrier is selected from the group consisting of: protein nanoparticles, oligopeptide nanoparticles, phospholipid nanoliposomes, polysaccharide nanoparticles, polyether nanoparticles, polyester nanoparticles, Polyester polymer micelles.
  • the protein nanoparticle is selected from the group consisting of: human serum albumin nanoparticles, bovine serum albumin nanoparticles.
  • the phospholipid nanoliposome is selected from the group consisting of: phosphatidylcholine nanoliposomes, dipalmitoylphosphatidylcholine nanoliposomes, distearoylphosphatidylcholine nanoliposomes , dipalmitoylphosphatidylethanolamine nanoliposomes, distearoylphosphatidylethanolamine nanoliposomes, dipalmitoylphosphatidylglycerol nanoliposomes.
  • polyester-based nanoparticles are selected from the group consisting of: polyethylene glycol-polylactic acid nanoparticles, polyethylene glycol-polylactide glycolide nanoparticles, polyethylene glycol-polycaprolactone Nanoparticles.
  • the polysaccharide nanoparticles comprise: chitosan-based nanoparticles.
  • the polyester-based polymer micelle is selected from the group consisting of: polyethylene glycol-polylactic acid micelles, polyethylene glycol-polycaprolactone micelles, polyethylene glycol-distearoyl Phosphatidylethanolamine micelles, polyethylene glycol-polyethyleneimine micelles.
  • a second aspect of the invention provides a composition comprising:
  • the antitumor drug is selected from the group consisting of: doxorubicin, paclitaxel, docetaxel, cisplatin, mitoxantrone, daunorubicin, vincristine, all-trans retinoic acid, epirubicin , letoticon, irinotecan, 2-methoxyestradiol, gemcitabine, vinorelbine, 5-fluorouracil, methotrexate, capecitabine, lomustine, etoposide or a combination thereof .
  • the anti-tumor drug is embedded in the complex nanocarrier.
  • the encapsulation efficiency of the nanocarrier against the tumor drug is 80% or more in the composition. (preferably 90% or more).
  • the concentration of the antitumor drug in the composition is 5-l (g/mL
  • the killing rate of the composition on the tumor cells is > 60%, preferably > 70%.
  • the tumor cells include breast cancer, ovarian cancer, kidney cancer or gastric cancer tumor cells.
  • the antitumor drug is present in an amount of from 1.0 to 3.0% by weight based on the total weight of the composition. It is preferably 1.5 to 2.7 wt%.
  • the target molecule is present in an amount of from 1.11 to 22.2% by weight based on the total weight of the composition. Preferably it is 5.60-l l. lwt%.
  • a third aspect of the present invention provides a method for preparing the composition of the second aspect, comprising the steps of: (1) providing a nanocarrier, wherein the nanocarrier is loaded with an antitumor drug;
  • the nanocarrier of the step (1) is subjected to a coupling reaction with a target molecule to obtain the composition.
  • the nanocarrier has a particle diameter of 200 nm or less, preferably 10 to 200 nm.
  • the method for preparing the nanocarrier comprises the following steps:
  • step (b) mixing the aqueous solution of step (a) with an organic solution to obtain an emulsion;
  • step (c) curing the emulsion of step (b) to obtain the nanocarrier.
  • step 0 (b) mixing the aqueous solution of step 0) with an organic solution to obtain an emulsion;
  • step (c) curing the emulsion of step (b) to obtain the drug nanocarrier.
  • step (b) mixing the aqueous solution of step (a) with an organic solution to obtain a first emulsion
  • step (c) mixing the first emulsion of step (b) with an aqueous solution in which an emulsifier is dissolved to obtain a second emulsion;
  • step (d) curing the second emulsion of step (C) to obtain the nanocarrier.
  • step (a) separately providing a suspension comprising an antitumor drug, a nanocarrier and an organic solvent; (b) curing the suspension of step (a) to obtain the nanocarrier;
  • the hydrophilic membrane is selected from the group consisting of polyethylene glycol (PEG), polyoxyethylene (PEO), polyvinylpyrrolidone (PVP) or polyvinyl alcohol (PVA).
  • the hydrophobic membrane is selected from the group consisting of polyoxypropylene (PPO), polystyrene (PS), polyamino acid, polylactic acid (PLA), spermine or short chain phospholipids.
  • the emulsifier is selected from the group consisting of Pluronic F68, Dextran 70 or sodium cholate.
  • the coupling reaction is selected from the group consisting of:
  • a fourth aspect of the invention provides the use of the complex of the first aspect for the preparation of a medicament for treating cancer Things.
  • the cancer comprises: breast cancer, ovarian cancer, kidney cancer, and gastric cancer. More preferably, the cancer includes kidney cancer and gastric cancer.
  • a fifth aspect of the invention provides the use of the composition of the second aspect, the composition for the preparation of a medicament for treating cancer.
  • a sixth aspect of the invention provides a medicament comprising:
  • a pharmaceutically acceptable carrier is selected from:
  • the dosage form of the medicament is selected from the group consisting of: a solid preparation, a liquid preparation, or an injection.
  • the drug is administered to a mammal, preferably a human.
  • the pharmaceutical dosage form is an injection.
  • the administration of the injection includes: intravenous injection, intramuscular injection, subcutaneous injection, intraluminal injection.
  • a seventh aspect of the invention provides a method of treating cancer, the method comprising the steps of: administering a safe and effective amount of the composition of the second aspect or the medicament of the sixth aspect to a subject in need thereof.
  • the cancer comprises: breast cancer, ovarian cancer, kidney cancer, and gastric cancer. More preferably, the cancer includes kidney cancer and gastric cancer. It is to be understood that within the scope of the present invention, the various technical features of the present invention and the technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here. DRAWINGS
  • Figure 1 is a transmission electron micrograph and a DLS particle size distribution of the composition [D-Arg 25 ]-NPY-ANP-TXT.
  • Figure 2 is a graph showing the change in particle size of the composition [D-Arg 25 ]-NPY-ANP-TXT in aqueous NaCl solution, aqueous PBS and serum for 1-15 days.
  • Figure 3 is a graph comparing the uptake of the composition [D-Arg 25 ]-NPY-ANP-TXT by tumor cells MCF-7 and HEC-1B-Y5. detailed description
  • compositions prepared by coupling certain target molecules to the surface of a nanocarrier loaded with an antitumor drug are highly specific to neuropeptides on specific tumor cells.
  • the body binds, and the anti-tumor drug can be targeted to these cells, increasing the effective concentration of the drug in the tumor cells, and having almost no toxic side effects on normal tissues and cells.
  • the composition of the present invention has a strong killing effect on tumor cells, particularly on breast cancer, ovarian cancer, renal cancer and gastric cancer cells, and thus can be used for the preparation of a medicament for treating the above tumor.
  • the present invention has been completed on this basis.
  • biotin refers to vitamin H, or vitamin B7 or coenzyme R, having a molecular weight of 244.31 Da.
  • avidin is a glycoprotein having a molecular weight of about 60 kDa. It mainly includes: egg white avidin (also known as natural avidin, egg white avidin or avidin), streptavidin, egg yolk avidin and avidin. Target molecule
  • the target molecule of the present invention refers to a polypeptide agonist molecule or a non-peptide antagonist molecule which can specifically and efficiently bind to a neuropeptide receptor, thereby causing various biological activities, wherein the polypeptide agonist molecule includes (but does not Limited to): [D-Arg 25 ]-NPY, [D-His 26 ]-NPY, [D-Arg 25 , D-His 26 ]-NPY, [Arg 6 , Pro 34 ]pNPY, [Asn 6 , Pro 34 pNPY, [Cys 6 , Pro 34 ]pNPY, [Phe 6 , Pro 34 ]pNPY, [Arg 7 , Pro 34 ]pNPY, [D-His 26 , Pro 34 ]NPY, [Phe 7 , Pro 34 ]pNPY, [Pro 30 , Nle 31 , Bpa 32 , Leu 34 ] NPY (28-36), [Pro 30 , Nal 32 , Leu 34
  • non-peptide antagonist molecules include, but are not limited to: BIBO3304, PD160170, LY366258, J-104870, LY 357897, J-115814.
  • the inventors have unexpectedly discovered through research that the above target molecules of the present invention can bind specifically to breast cancer, ovarian cancer, renal cancer and gastric cancer cells, but do not specifically bind to brain tumors and endometrial tumor cells.
  • the high specific binding described herein means that, under the same conditions, after the complex of the present invention is combined with tumor cells and non-tumor cells (i.e., normal cells), the ratio of uptake ratio to the complex satisfies the following conditions: Bi/ Bc 2.5 , preferably B /BQ 3 , more preferably B /BQ 4; wherein, in each of 10,000 tumor cells, the uptake rate of the tumor cells to the complex is expressed; Bo is expressed in every 10,000 normal cells, The uptake rate of normal cells to the complex.
  • the complex of the present invention is a binary complex composed of a biodegradable nanocarrier and a target molecule, wherein the target molecule is coupled to the surface of the nanocarrier.
  • the excess of the target molecule causes precipitation or agglomeration of the composite nanoparticles, which in turn increases the particle size of the composite nanoparticles (>200 nm).
  • the target molecule is contained in an amount of from 1.11 to 22.2% by weight, preferably from 5.60 to 11.1% by weight, based on the total weight of the composite, and the balance is a biodegradable nanocarrier.
  • the complex of the present invention has good dispersibility and stability in an aqueous solution of NaCl, PBS or serum, and no precipitation or agglomeration occurs.
  • the nanocarrier may be selected from the group consisting of: oligopeptide nanoparticles, phospholipid nanoliposomes, polysaccharide nanoparticles, polyether nanoparticles, polyester nanoparticles, polyester polymer micelles or Its combination. Among them, albumin-based nanoparticles, phospholipid-based nanoparticles, and polysaccharide-based nanoparticles are preferable.
  • a preferred class of proteinaceous nanoparticles includes: human serum albumin nanoparticles (HSA), bovine serum albumin nanoparticles (BSA), or combinations thereof.
  • a preferred class of phospholipid nanoliposomes include: phosphatidylcholine (PC) nanoliposomes, dipalmitoylphosphatidylcholine (DPPC) nanoliposomes, distearoylphosphatidylcholine (DSPC) Nanoliposomes, dipalmitoylphosphatidylethanolamine (DPPE) nanoliposomes, distearoylphosphatidylethanolamine (DSPE) nanoliposomes, dipalmitoylphosphatidylglycerol (DPPG) nanoliposomes or combinations thereof .
  • PC phosphatidylcholine
  • DPPC dipalmitoylphosphatidylcholine
  • DSPC distearoylphosphatidylcholine
  • Nanoliposomes dipalmitoylphosphatidylethanolamine (DPPE) nanoliposomes
  • DPPE dipalmitoylphosphatidylethanolamine
  • DSPE dipalmitoyl
  • a preferred class of polyester nanoparticles includes: polyethylene glycol-polylactic acid (PEG-PLA) nanoparticles, polyethylene glycol-polylactide glycolide (PEG-PLGA) nanoparticles, polyethylene glycol- Polycaprolactone (PEG-PCL) nanoparticles or a combination thereof.
  • PEG-PLA polyethylene glycol-polylactic acid
  • PEG-PLGA polyethylene glycol-polylactide glycolide
  • PEG-PCL polyethylene glycol- Polycaprolactone
  • a preferred class of polysaccharide nanoparticles includes: chitosan-based nanoparticles.
  • polyester polymer micelles include: polyethylene glycol-polylactic acid (; PEG-PLA) micelles, polyethylene glycol-polycaprolactone (PEG-PCL) micelles, polyethylene glycol - distearoylphosphatidylethanolamine (PEG-DSPE) micelles, polyethylene glycol-polyethyleneimine (PEG-cl-PEI) micelles or combinations thereof.
  • PEG-PLA polyethylene glycol-polylactic acid
  • PEG-PCL polyethylene glycol-polycaprolactone
  • PEG-DSPE polyethylene glycol - distearoylphosphatidylethanolamine
  • PEG-cl-PEI polyethylene glycol-polyethyleneimine
  • the preparation method of the composite of the present invention mainly comprises the steps of: (1) preparation of a nanocarrier and (2) reaction of the nanocarrier with a target molecule.
  • the preparation method of the nano carrier can be prepared by a method well known to those skilled in the art, and the reaction between the nano carrier and the target molecule can be carried out by chemical coupling.
  • a preferred type of coupling method is as follows:
  • the nanocarrier contains a carboxyl group (such as polyglutamic acid, polyaspartic acid, polypeptides and proteins containing glutamic acid and aspartic acid, and polysaccharides containing a carboxyl group)
  • a target molecule containing an amino group may be selected.
  • EDAC and NHS N-hydroxysuccinimide
  • the target molecule solution with the terminal amino group is added dropwise, so that the activated carboxyl group reacts with the amino group of the target molecule to form a stable reaction.
  • a target molecule containing a carboxyl group may be selected, and then the carboxyl group of the target molecule is activated by the above method to graft onto the surface of the nanocarrier; for a biodegradable nanocarrier containing neither a carboxyl group nor an amino group (such as poly
  • the ether and the polyester polymer may be subjected to a copolymerization method to carry an amino group or a carboxyl group, and after the nanocarrier is formed, the target molecule may be grafted on the surface of the nanocarrier by the same method as above.
  • a thiol group is introduced on the target molecule, and a maleimide group is introduced on the surface of the drug-loading system, and then the addition reaction is carried out at room temperature in a neutral or alkaline aqueous environment such as a phosphate buffer solution.
  • the thiolation of the target molecule is mainly carried out by reacting an amino group on the target molecule with a thiolation reagent (such as 2-IT, SPDP, SATP and SSDD) to form a thiolated product;
  • a thiolation reagent such as 2-IT, SPDP, SATP and SSDD
  • Lipid molecules and maleimide (MAL) modified polymeric materials such as MAL-PEG-PLA, MAL-PEG-PLGA, MAL-PEG-PCL) when maleimide is introduced into the drug delivery system ,
  • MAL-PEG-DSPE MAL-PEG-DSPE
  • the hydrophobic end such as PLA, PLGA, PCL and DSPE
  • PLA, PLGA, PCL and DSPE can be prepared and embedded in the lipid bilayer.
  • PEG-maleimide end maleimide liposomes on the surface of the lipid membrane.
  • maleimide can also be introduced directly onto the surface of the drug delivery system. If a bifunctional linker capable of forming a maleimide (such as a bifunctional propionic acid linker, a reactive ester in the molecule reacts with an amino group to form a maleimide group), a maleimide group is introduced on the nanocarrier. Imine group.
  • the biotin is introduced into the target molecule and the drug-loading system, respectively, and the avidin is used as a bridging agent to realize the construction of the targeted drug delivery system. That is, the avidin is first mixed with the biotinylated drug-loading system, and the unbound site is then combined with the biotinylated target molecule.
  • biotinylated PEG-PLGA can be prepared by dissolving PLGA-COOH (molecular weight 20 kDa) in methylene chloride, stirring at room temperature, and adding 8 times the amount of NHS and EDC to activate.
  • PLGA-COOH molecular weight 20 kDa
  • NH 2 -PEG-biotin (molecular weight: 3400 Da) was mixed and dissolved in chloroform, and an appropriate amount of hydrazine, hydrazine-diisopropylethylamine was added thereto, and the reaction was continued overnight. Unreacted PEG molecules were washed away with methanol, diethyl ether precipitated and dried in vacuo to give PLGA-PEG-biotin.
  • a certain proportion of PLGA-PEG-COOH and PLGA-PEG-biotin are mixed and dissolved in acetone, slowly added dropwise to deionized water, and the acetone is removed by rotary evaporation at room temperature, and the organic solvent is removed by ultrafiltration to obtain biotinylation.
  • PEG-PLGA nanoparticles The biotinylated PEG-PLGA nanoparticles were incubated with the avidin solution at room temperature for a certain time, and the free avidin was removed by centrifugation.
  • the biotinylated target molecule is stirred at room temperature, and then centrifuged to remove the free biotinylated target molecule, thereby obtaining a target molecularized PEG-PLGA nanoparticle targeted delivery system.
  • the composition of the present invention comprises the complex of the present invention and an antitumor drug loaded on the complex nanocarrier.
  • the antitumor drug is usually present in an amount of from 1.5 to 3.0% by weight based on the total weight of the composition. It is preferably 2.0 to 3.0 wt%.
  • the content of the target molecule was 1.11 to 22.2 wt%. It is preferably 5.60 to 11.1 wt%.
  • the particle size of the nanocarrier in the composite in the composition of the present invention is preferably 200 nm or less, preferably It is 10 to 200 nm.
  • a class of preferred anti-tumor drugs include, but are not limited to, doxorubicin, paclitaxel, docetaxel, cisplatin, mitoxantrone, daunorubicin, vincristine, all-trans retinoic acid, epirubicin , letotecan, irinotecan, 2-methoxyestradiol, gemcitabine, vinorelbine, 5-fluorouracil, methotrexate, capecitabine, lomustine, etoposide or a combination thereof.
  • Preferred are doxorubicin, paclitaxel, docetaxel, mitoxantrone, daunorubicin, irinotecan, gemcitabine, vinorelbine, capecitabine, etoposide.
  • the preparation method of the composition of the invention mainly comprises the steps of:
  • the preparation of the nanocarrier can be produced by ultrasonic emulsification.
  • the nanocarrier can be prepared by the following three methods:
  • aqueous solution in which a hydrophilic antitumor drug and a hydrophilic membrane material (such as polyethylene glycol) are dissolved as an aqueous phase, and an organic solvent (such as two) in which an oil-soluble emulsifier (such as sodium cholate) is dissolved.
  • an oil phase As an oil phase, the aqueous phase and the oil phase are mixed and stirred for coarse dispersion, and then emulsified by an ultrasonic cell crusher to obtain a water-in-oil type nanoemulsion, and cross-linking is added to the obtained nanoemulsion under magnetic stirring.
  • the agent such as glutaraldehyde
  • excess cross-linking agent and emulsifier are removed to obtain a biodegradable nanocarrier embedded with an anti-tumor drug.
  • an organic solvent such as dichloromethane
  • a hydrophobic antitumor drug and a hydrophobic membrane material such as PACA
  • a water-soluble emulsifier such as Pluronic F68, Dextran 70
  • a crosslinking agent is added to the obtained nanoemulsion under magnetic stirring (for example).
  • Glutaraldehyde is cross-linked and cured, and excess cross-linking agent and emulsifier are removed to obtain an anti-tumor A biodegradable nanocarrier for the drug.
  • PEG-PLA PEG-PLA
  • an organic solvent of an oil-soluble emulsifier such as sodium cholate
  • the aqueous phase and the oil phase are mixed and stirred for coarse dispersion, and then emulsified by an ultrasonic cell crusher to obtain a water-in-oil type nanometer.
  • the emulsion is then emulsified by adding the obtained water-in-oil nanoemulsion to an aqueous phase in which a water-soluble emulsifier is dissolved, thereby obtaining a W/O/W type nanoemulsion, and then obtaining the W/O/W type under magnetic stirring.
  • a cross-linking agent such as glutaraldehyde
  • glutaraldehyde is added to the nanoemulsion to crosslink and cure, and excess cross-linking agent and emulsifier are removed to obtain a biodegradable nanocarrier embedded with an antitumor drug.
  • the above nanocarrier of the present invention can also be produced by a solvent removal method.
  • a preferred method comprises: dissolving a water-soluble antitumor drug and a nanocarrier oligopeptide or protein in an aqueous solution of NaCl, then adding ethanol dropwise, and the process of dropping continues the magnetic force. Stirring, when the solution becomes a milky white suspension, the glutaraldehyde cross-linked solidified nanocarrier is added, and the excess cross-linking agent is removed to obtain biodegradable nanoparticles embedded with the anticancer drug.
  • reaction method in the above step (2) is the same as the method of reacting the nanocarrier with the target molecule in the complex of the present invention. It is to be understood that the above composition can also be prepared by incorporating an antitumor drug into the prepared complex of the present invention.
  • composition of the present invention can be used for the preparation of antitumor drugs, particularly for the preparation of a medicament for the treatment of breast cancer, ovarian cancer, kidney cancer and gastric cancer.
  • the medicament of the present invention contains an effective amount of the composition of the present invention, a pharmaceutically acceptable carrier or excipient.
  • the term “comprising” or “including” as used herein includes “including”, “consisting essentially of”, and “consisting of.”
  • the term “pharmaceutically acceptable” ingredient is suitable for use in humans and/or animals without excessive adverse side effects (eg, toxicity, irritation, and allergies;), ie, a reasonable benefit/risk ratio substance.
  • the term “effective amount” means a function or activity that can be exerted on a human and/or animal and which can be accepted by humans and/or animals
  • the term “pharmaceutically acceptable carrier” is used to mean A carrier for administration of a therapeutic agent, including various excipients and diluents.
  • the term refers to pharmaceutical carriers which are not themselves essential active ingredients and which are not excessively toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences, Mack Pub. Co., N. 1991.
  • the pharmaceutical dosage form of the present invention comprises: a solid preparation, a liquid preparation or an injection. It is preferably an injection.
  • the administration of the medicament of the present invention is a mammal, preferably a human.
  • the medicament or composition of the invention is administered one or more times per day, for example
  • the routes of administration include, but are not limited to, oral administration, injection administration, intracavitary administration, and transdermal administration; preferred administrations for injection include: intravenous injection, intramuscular injection, subcutaneous injection, intracavitary injection.
  • the specific dose should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • the safe and effective amount of the compositions of the present invention will generally be at least about 10 mg or at least 85 mg / kg body weight per day, and in most cases no more than about 200 or no more than about 115 mg / kg body weight / day.
  • a preferred dosage is about 100 mg / kg body weight / day.
  • the complexes and compositions of the present invention have good dispersibility and stability in an aqueous solution of NaCl, PBS or serum, and no precipitation or agglomeration occurs.
  • the complexes and compositions of the present invention bind highly specifically to breast cancer, ovarian cancer, renal cancer and gastric cancer cells, and have a strong targeting effect on tumor tissues.
  • composition and the medicament of the invention can target the anti-tumor drug to the tumor cells, effectively increase the concentration of the drug in the cells, have a strong killing effect on the tumor cells, and have almost no normal tissues and cells. toxic side effect.
  • the aqueous solution was washed twice, and finally freeze-dried for 48 h to obtain a BSA nanocarrier.
  • the TXT solution was dispersed in a 20 mg/mL aqueous solution of BSA nanocarrier, and the nanocarrier ANP-TXT embedded with the antitumor drug was prepared by the same method as above.
  • the composite nanoparticles have uniform particle size, good dispersibility, and are stable between 110 and 120 nm.
  • the composite nanoparticles have a PDI index of less than 0.5 as determined by a nanoparticle size analyzer method.
  • Example 2 Activity test of composition [D-Arg 25 ]-NPY-ANP-TXT on tumor cells MCF-7 and HEC-1B-Y5
  • the cells selected in the cell experiment include: human breast tumor MCF-7 cells, human endometrial tumor HEC-1B-Y5 cells. (purchased from the American Standard Biological Collection Center ATCC and American Sciencell Company)
  • docetaxel (TXT) has excellent killing effect on both MCF-7 cells and HEC-1B-Y5 cells, but the composition [D-Arg 25 ]-NPY-ANP -TXT has an excellent killing effect on MCF-7 cells, but the killing effect on HEC-1B-Y5 cells is not obvious, so the above results indicate that the composition [D-Arg 25 ]-NPY-ANP-TXT can The MCF-7 cells were killed unexpectedly and significantly, with high selectivity and had an excellent killing effect.
  • Example 3 Activity test of composition [D-Ar g 25 ]-NPY-ANP-TXT on different tumor cells
  • the tumor cells selected in the cell assay include: human ovarian tumor UWB 1.289 cells, human gastric tumor GIST-H1 cells, human kidney tumor SW-13 cells, human brain tumor SMS-KAN cells.
  • Normal cells include: human mammary epithelial cells MCF-10a, human ovarian surface epithelial cells HOSEpiC, human renal cortical epithelial cells HRCEpiC, human gastric mucosal cells GES-1, human brain glial cells HA, human endometrial epithelial cells
  • composition of the present invention [D-Arg 25 ]-NPY-ANP-TXT has the same UWB1.289 cells, SW-13 cells and GIST-H1 cells as MCF-7 cells. Excellent killing effect, but the killing effect on SMS-KAN cells is not obvious, so the above results indicate that the composition
  • [D-Arg 25 ]-NPY-ANP-TXT can selectively kill UWB 1.289 cells, SW-13 cells and
  • GIST-H1 cells also have excellent killing effects.
  • the composition [D-Arg 25 ]-NPY-ANP-TXT can be highly specific to breast cancer, ovarian cancer, renal cancer and gastric cancer cells.
  • the combination of ground and cancer has a strong targeting effect on tumor cells, and can target anti-tumor drugs to tumor cells, effectively increase the concentration of drugs in cells, and have a strong killing effect on tumor cells, and at the same time, normal Tissue and cells have almost no toxic side effects.
  • Example 4 Activity test of different compositions of docetaxel in MCF-7 and UWB1.289 cells
  • MCF-7 cells and UWB 1.289 cells are produced by MCF-7 cells and UWB 1.289 cells.
  • Example 5 Activity testing of different compositions of docetaxel on GIST-H1 and SW-13 cells The preparation of different compositions and cytotoxicity tests were carried out in accordance with the procedure of Example 4. The test results are shown in Table 6. Table 6 Comparison of the killing effects of different compositions on GIST-H1 and SW-13 cells
  • the composition coupled with the target molecule of the present invention has strong killing effect on MCF-7, UWB 1.289, GIST-Hl and SW-13 cells.
  • C TXT was 5 g/mL
  • the survival rate of MCF-7, GIST-Hl and SW-13 cells was basically less than 30%
  • the survival rate of UWB 1.289 cells was 60%.
  • the killing effect on SMS-KAN and HEC-1B-Y5 cells was not obvious, and the cell survival rate was basically above 80%. Therefore, it can be seen that the composition of the present invention has excellent selectivity, has strong targeting effect on breast cancer, ovarian cancer, renal cancer and gastric cancer tumor cells, and has a strong killing effect on tumor cells.

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Abstract

本发明公开了一种复合物，所述复合物包括纳米载体以及偶联于纳米载体表面的靶分子。所述纳米载体的粒径在200mn以下，且多分散指数（PDI）小于0.5。本发明还公开了包含所述复合物以及装载于所述复合物纳米载体中的抗肿瘤药物、其制备方法和用途。所述组合物能将抗肿瘤药物输送至肿瘤细胞内，对肿瘤细胞有很强的杀灭作用。

Description

主动靶向型抗肿瘤药物及其制备方法
技术领域
本发明涉及制药技术领域， 具体地涉及一种主动靶向型抗肿瘤药物及其制备 方法。 背景技术
主动靶向药物即通过在药物或药物载体表面修饰活性靶分子 （如抗体或配体 等） 后， 与某些组织或细胞上特异性的抗原或受体结合， 实现药物对特定细胞和 组织主动寻靶的功能。 由于抗原-抗体、 受体-配体之间高特异性、 高选择性和高亲和 性的特点,主动靶向较被动靶向具有更高的靶向效率，因而主动靶向释药系统的研究在 国内外也都非常活跃。
基于抗原与抗体特异性结合的原理设计的主动靶向药物存在诸多问题，例如， 靶点药物有效浓度低、 种族特异性强、 免疫原性高和研发生产成本高等。
然而基于配体和受体特异性结合原理所设计的主动靶向药物， 由于具有高选 择性、 无种族特异性、 无免疫原性、 高稳定性且低成本的特点， 因此是目前肿瘤 靶向递药系统设计的重点和热点。 其中包括叶酸受体、 转铁蛋白受体、 整合素受 体和多肽受体等介导的肿瘤靶向药物研究。 近年来， 多肽受体介导的肿瘤靶向药 物越来越受到人们的关注。
但迄今为止,有关抗癌药物靶向给药系统的研究，大多以靶向至肿瘤组织为目的， 如何使更多的药物到达肿瘤部位后进入肿瘤细胞，真正达到肿瘤细胞靶向给药的目的, 是近几年靶向给药系统的研究重点。尽管很多药物运载系统表现出了相当好的肿瘤靶 向性,可以将药物递送至肿瘤细胞表面,但由于载体与靶细胞结合后入胞能力较弱，相 当大一部分的药物在细胞外释放,药物作用同时也激活肿瘤细胞膜多种基因，多种分子 机制协同参与耐药表型的形成,构成耐药性,进一步降低了药物入胞效率，使胞内药物 浓度过低,无法有效抑制肿瘤细胞的生长。
神经肽 Y (NPY) 是一种广泛存在于中枢和外周并维持内环境稳态的激素。 已有 6种 NPY受体被发现和鉴定， 它们分别为 ΝΡΥ Υ^ Υ2、 Υ3、 Υ4、 Υ5和 Υ6， 这些受体广泛存在于哺乳动物的中枢神经系统和外周神经系统。 ΝΡΥ的功能与其 受体密不可分， 受体的多样性引起功能多样性。 研究表明， 目前已知的针对神经 肽受体的药物多用于治疗与生理紊乱相关的疾病， 包括： 肥胖、 心血管疾病、 高 血脂、 癫痫、 焦虑等疾病。 然而以 NPY受体为靶向的抗肿瘤治疗药物却不多见， 特别是针对肾癌、 胃癌、 乳腺癌和卵巢癌的抗肿瘤治疗药物未见报道。
目前基于 NPY不同受体的激动剂和抑制剂即受体的配体分子，虽然已有广泛 的研究， 然而由于 NPY受体的多样性以及其存在的广泛性， 因此寻找某种合适配 体来修饰药物载体， 使该载体能高特异性地与肿瘤细胞的受体结合且不影响细胞 内的其他受体的生物活性， 并且能够将载体中的抗肿瘤药物靶向输送至这些细胞 内， 从而增加药物在该肿瘤细胞中的有效浓度， 已成为主动靶向药物设计与研发 的关键。 发明内容
本发明的目的在于提供一种主动靶向型抗肿瘤药物及其制备方法。
本发明第一方面提供了一种复合物， 所述复合物包括：
纳米载体； 以及
靶分子， 所述靶分子偶联于所述纳米载体表面；
其中， 所述靶分子选自： [D-Arg25]-NPY、 [D-His26]-NPY、 [D-Arg25,
D-His26]-NPY、 [Arg6, Pro34]pNPY、 [Asn6, Pro34]pNPY、 [Cys6, Pro34]pNPY、 [Phe6, Pro34]pNPY、 [Arg7, Pro34]pNPY、 [D-His26, Pro34]NPY、 [Phe7, Pro34]pNPY、 [Pro30, Nle31, Bpa32, Leu34]NPY(28-36), [Pro30, Nal32, Leu34]NPY(28-36), [Pro30, Nle31, Nal32, Leu34]NPY(28-36)、 BIBO3304、 PD160170、 LY366258、 J-104870, LY 357897、 J-115814;
并且所述纳米载体的粒径在 200nm以下， 且多分散指数 （PDI) 小于 0.5。
在另一优选例中， 按复合物总重量计， 所述靶分子的含量为 1.11一 22.2wt%。 在另一优选例中， 按复合物总重量计， 所述靶分子的含量为 5.60— 11. lwt%。 在另一优选例中， 所述复合物具有以下一种或多种特征：
(a) 与乳腺癌、 卵巢癌、 肾癌或胃癌肿瘤细胞高特异性结合；
(b) 纳米载体的粒径为 10-200nm。
在另一优选例中， 所述纳米载体选自： 蛋白类纳米粒子、 寡肽类纳米粒子、 磷脂 类纳米脂质体、 多糖类纳米粒子、 聚醚类纳米粒子、 聚酯类纳米粒子、 聚酯类聚合 物胶束。
在另一优选例中， 所述蛋白类纳米粒子选自： 人血清白蛋白纳米粒子、 牛血清 白蛋白纳米粒子。 在另一优选例中， 所述磷脂类纳米脂质体选自： 磷脂酰胆碱纳米脂质体、 二 棕榈磷脂酰胆碱纳米脂质体、 二硬脂酰磷脂酰胆碱纳米脂质体、 二棕榈酰磷脂酰 乙醇胺纳米脂质体、 二硬脂酰磷脂酰乙醇胺纳米脂质体、 二棕榈酰磷脂酰甘油纳 米脂质体。
在另一优选例中， 所述聚酯类纳米粒子选自： 聚乙二醇 -聚乳酸纳米粒子、 聚 乙二醇-聚丙交酯乙交酯纳米粒子、 聚乙二醇-聚己内酯纳米粒子。
在另一优选例中， 所述多糖类纳米粒子包括： 壳聚糖类纳米粒子。
在另一优选例中， 所述聚酯类聚合物胶束选自： 聚乙二醇-聚乳酸胶束、 聚乙 二醇-聚己内酯胶束、 聚乙二醇-二硬脂酰磷脂酰乙醇胺胶束、 聚乙二醇-聚乙烯亚 胺胶束。
本发明第二方面提供了一种组合物， 所述组合物包括：
第一方面所述的复合物； 以及
装载于所述复合物纳米载体中的抗肿瘤药物。
在另一优选例中， 抗肿瘤药物选自： 阿霉素、 紫杉醇、 多西他赛、 顺铂、 米托 蒽醌、 柔红霉素、 长春新碱、 全反式维甲酸、 表阿霉素、 勒托替康、 伊立替康、 2-甲氧雌二醇、 吉西他滨、 长春瑞宾、 5-氟尿嘧啶、 甲氨蝶呤、 卡培他滨、 洛莫 司汀、 依托泊苷或其组合。
在另一优选例中， 所述抗肿瘤药物包埋于所述复合物纳米载体中。
在另一优选例中， 所述组合物中， 纳米载体对抗肿瘤药物的包封率为 80%以上。 (较佳的为 90%以上)。
在另一优选例中， 所述组合物中抗肿瘤药物浓度为 5-l(^g/mL时， 组合物对肿 瘤细胞的杀灭率为 > 60 %， 较佳地为> 70 %。
在另一优选例中， 所述肿瘤细胞包括乳腺癌、 卵巢癌、 肾癌或胃癌肿瘤细胞。 在另一优选例中， 按组合物的总重量计， 所述抗肿瘤药物的含量为 1.0— 3.0wt%。 优选为 1.5— 2.7wt%。
在另一优选例中， 按组合物的总重量计， 所述靶分子的含量为 1.11一 22.2wt%。 优选为 5.60— l l. lwt%。 本发明第三方面提供了一种第二方面所述组合物的制备方法， 包括以下步骤： (1) 提供一纳米载体， 所述纳米载体中装载有抗肿瘤药物；
(2) 将步骤 (1)的纳米载体与靶分子进行偶联反应， 得到所述组合物。 在另一优选例中， 所述纳米载体的粒径在 200nm以下， 较佳的为 10-200nm。 在另一优选例中， 所述纳米载体的制备方法包括以下步骤：
(a) 分别提供一水溶液和有机溶液， 所述水溶液包含抗肿瘤药物和亲水性膜材， 所述有机溶液包含乳化剂；
(b) 将步骤 (a)的水溶液与有机溶液混合,得到乳液；
(c) 将步骤 (b)的乳液固化， 得到所述纳米载体。
或包括以下步骤：
(a) 分别提供一水溶液和有机溶液， 所述水溶液包含乳化剂， 所述有机溶液包含 抗肿瘤药物和疏水性膜材；
(b) 将步骤 0)的水溶液与有机溶液混合,得到乳液；
(c) 将步骤 (b)的乳液固化， 得到所述药纳米载体。
或包括以下步骤：
(a) 分别提供一水溶液和有机溶液， 所述水溶液包含抗肿瘤药物， 所述有机溶液 包含疏水性膜材和乳化剂；
(b) 将步骤 (a)的水溶液与有机溶液混合,得到第一乳液；
(c) 将步骤 (b)的第一乳液与溶有乳化剂的水溶液混合， 得到第二乳液；
(d) 将步骤 (C)的第二乳液固化， 得到所述纳米载体。
或包括以下步骤：
(a) 分别提供一悬浊液， 所述悬浊液包含抗肿瘤药物、 纳米载体和有机溶剂； (b) 将步骤 (a)的悬浊液固化,得到所述纳米载体；
在另一优选例中， 所述亲水性膜材选自： 聚乙二醇（PEG) 、 聚氧乙烯（PEO) 、 聚乙烯吡咯烷酮 （PVP) 或聚乙烯醇 （PVA) 。
在另一优选例中， 所述疏水性膜材选自： 聚氧丙烯 （PPO) 、 聚苯乙烯 （PS ) 、 聚氨基酸、 聚乳酸 （PLA) 、 精胺或短链磷脂。
在另一优选例中， 所述乳化剂选自： Pluronic F68、 Dextran 70或胆酸钠。
在另一优选例中， 所述偶联反应选自：
(1) 羧基与氨基的缩合反应；
(2) 巯基与马来酰亚胺的加成反应； 或
(3) 亲和素与生物素的非共价结合。 本发明第四方面提供了一种第一方面所述复合物的用途，用于制备治疗癌症的药 物。
在另一优选例中， 所述癌症包括： 乳腺癌、 卵巢癌、 肾癌和胃癌。 更佳地， 所 述癌症包括肾癌和胃癌。
本发明第五方面提供了一种第二方面所述组合物的用途，所述组合物用于制备治 疗癌症的药物。 本发明第六方面提供了一种药物， 所述药物包括：
第一方面所述的复合物；
装载于所述复合物纳米载体中的抗肿瘤药物； 以及
药学上可接受的载体。
在另一优选例中， 所述药物的剂型选自： 固体制剂、 液体制剂或注射剂。
在另一优选例中， 所述药物的施用对象为哺乳动物， 优选人类。
在另一优选例中， 所述药物的剂型为注射剂。
在另一优选例中，所述注射剂的给药方式包括： 静脉注射， 肌肉注射， 皮下注射， 腔内注射。 本发明第七方面提供了一种治疗癌症的方法， 所述方法包括步骤： 给予需要的对 象施用安全有效量的第二方面所述的组合物或第六方面所述的药物。
在另一优选例中， 所述癌症包括： 乳腺癌、 卵巢癌、 肾癌和胃癌。 更佳地， 所 述癌症包括肾癌和胃癌。 应理解， 在本发明范围内中， 本发明的上述各技术特征和在下文 (；如实施例） 中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。 限于篇幅， 在此不再一一累述。 附图说明
图 1为组合物 [D-Arg25]-NPY-ANP-TXT的透射电镜图和 DLS粒径分布图。 图 2为组合物 [D-Arg25]-NPY-ANP-TXT在 NaCl水溶液、 PBS水溶液和血清 (serum) 中 1-15天的粒径变化图。
图 3为肿瘤细胞 MCF-7和 HEC-1B-Y5对组合物 [D-Arg25]-NPY-ANP-TXT的 摄取作用比较图。 具体实施方式
发明人经过广泛而深入的研究， 意外地发现将某些特定靶分子偶联于装载有 抗肿瘤药物的纳米载体表面所制得的组合物能高特异性地与特定肿瘤细胞上的神经 肽受体结合， 并且可将抗肿瘤药物靶向输送至这些细胞内， 增加药物在该肿瘤细胞 中的有效浓度， 对正常组织和细胞的几乎无毒副作用。 本发明的组合物对肿瘤细 胞特别是对乳腺癌、 卵巢癌、 肾癌和胃癌细胞具有较强的杀灭作用， 因此可用于 制备治疗上述肿瘤的药物。 在此基础上完成了本发明。
如本文所用， 术语 "生物素" 即指维生素 H， 或称为维生素 B7或辅酶 R ( Coenzyme R) ， 分子量为 244.31Da。
如本文所用， 术语 "亲和素"是一种糖蛋白， 分子量约为 60kDa。 主要包括： 卵白亲和素 （也称天然亲和素、 卵清亲和素或抗生物素） 、 链霉亲和素、 卵黄亲 和素及类亲和素等。 靶分子
本发明的靶分子是指可特异高效地与神经肽受体相结合， 从而引起各种生物 活性的多肽类激动剂分子或非肽类拮抗剂分子，其中多肽类激动剂分子包括 (但并 不限于）： [D-Arg25]-NPY、 [D-His26]-NPY、 [D-Arg25,D-His26]-NPY、 [Arg6, Pro34]pNPY、 [Asn6, Pro34]pNPY、 [Cys6, Pro34]pNPY、 [Phe6, Pro34]pNPY、 [Arg7, Pro34]pNPY、 [D-His26, Pro34]NPY、 [Phe7, Pro34]pNPY、 [Pro30, Nle31, Bpa32, Leu34]NPY(28-36)、 [Pro30, Nal32, Leu34]NPY(28-36)、 [Pro30, Nle31, Nal32,
Leu34]NPY(28-36), 非肽类拮抗剂分子包括 (但并不限于）： BIBO3304, PD160170, LY366258, J-104870, LY 357897、 J-115814。
发明人经过研究意外发现， 本发明的上述靶分子可与乳腺癌、 卵巢癌、 肾癌 和胃癌细胞高特异性地结合， 但不与脑瘤、 子宫内膜瘤细胞特异性地结合。
其中所述的高特异性结合是指， 在相同条件下， 本发明的复合物分别与肿瘤细胞 和非肿瘤细胞（即正常细胞）结合后，对复合物的摄取率比值满足如下条件： Bi/Bc 2.5 , 较佳地 B /BQ 3， 更佳地 B /BQ 4; 式中， 表示每 10000个肿瘤细胞中， 肿瘤细胞对所述复合物的摄取率； Bo表示每 10000个正常细胞中， 正常细胞对所 述复合物的摄取率。 复合物及其制备方法
本发明的复合物是由可生物降解的纳米载体和靶分子组成的二元复合物，其中， 靶分子偶联于纳米载体的表面。 由于靶分子过量会造成复合纳米粒子沉淀或团聚， 进 而使复合纳米粒子的粒径增加 （>200nm) 。 本发明中， 按复合物的总重量计， 靶分 子的含量为 1.11一 22.2% (wt%) ， 优选为 5.60— 11.1% (wt%) ， 余量为可生物降解 的纳米载体。 本发明的复合物在 NaCl水溶液、 PBS水溶液或血清中分散性与稳定性 良好， 无沉淀或团聚现象发生。
在本发明中， 纳米载体可选自： 寡肽类纳米粒子、 磷脂类纳米脂质体、 多糖类 纳米粒子、 聚醚类纳米粒子、 聚酯类纳米粒子、 聚酯类聚合物胶束或其组合。 其中 优选为白蛋白类纳米粒子、 磷脂类纳米粒子和多糖类纳米粒子。
一类优选的蛋白类纳米粒子包括： 人血清白蛋白纳米粒子 （HSA) 、 牛血清白 蛋白纳米粒子 （BSA) 或其组合。
一类优选的磷脂类纳米脂质体包括： 磷脂酰胆碱 （PC) 纳米脂质体、 二棕榈 磷脂酰胆碱 (DPPC)纳米脂质体、 二硬脂酰磷脂酰胆碱 (； DSPC)纳米脂质体、 二棕榈 酰磷脂酰乙醇胺 (DPPE)纳米脂质体、 二硬脂酰磷脂酰乙醇胺 (DSPE)纳米脂质体、 二棕榈酰磷脂酰甘油 (DPPG)纳米脂质体或其组合。
一类优选的聚酯类纳米粒子包括： 聚乙二醇-聚乳酸 (PEG-PLA)纳米粒子、 聚乙 二醇-聚丙交酯乙交酯 (PEG-PLGA)纳米粒子、聚乙二醇-聚己内酯 (PEG-PCL)纳米粒 子或其组合。
一类优选的多糖类纳米粒子包括： 壳聚糖类纳米粒子。
一类优选的聚酯类聚合物胶束包括： 聚乙二醇-聚乳酸 (； PEG-PLA)胶束、 聚乙 二醇-聚己内酯 (PEG-PCL)胶束、 聚乙二醇-二硬脂酰磷脂酰乙醇胺 (PEG-DSPE)胶 束、 聚乙二醇 -聚乙烯亚胺 (PEG-cl-PEI)胶束或其组合。
本发明复合物的制备方法主要包括步骤： （1)纳米载体的制备和 (2)纳米载体与靶 分子反应。其中，纳米载体的制备方法可采用本领域技术人员所熟知的方法进行制备， 纳米载体与靶分子之间可采用化学偶联的方法进行反应。
一类优选的偶联方法具体如下：
当纳米载体含有羧基时 （如聚谷氨酸、 聚天门冬氨酸、 含有谷氨酸和天门冬 氨酸的多肽和蛋白质、 以及含有羧基的多糖类物质） ， 可以选择含有氨基的靶分 子， 然后用 EDAC和 NHS (N-羟基丁二酰亚胺） 活化纳米载体表面的羧基， 再滴 加带有末端氨基的靶分子溶液， 使活化的羧基与靶分子的氨基共价反应形成稳定 的酰胺键； 当纳米载体含有氨基时 （如聚赖氨酸、 组氨酸、 聚精氨酸、 含有赖氨 酸、 精氨酸和组氨酸的多肽和蛋白质、 以及含有氨基的多糖类物质） ， 可以选择 含有羧基的靶分子， 然后采取以上方法活化靶分子的羧基后使之嫁接于纳米载体 表面上； 对于既不含有羧基、 又不含有氨基的可生物降解的纳米载体 （如聚醚类 和聚酯类高分子） ， 可以采用共聚法使之带有氨基或者羧基， 制作成纳米载体之 后采用以上相同方法可使靶分子嫁接于纳米载体表面上。
另一类优选的偶联方法具体如下：
在靶分子上引入巯基， 并在载药系统表面引入马来酰亚胺基团， 然后在中性 或如碱性的水性环境中 （如磷酸盐缓冲溶液） 室温下进行加成反应。
靶分子的巯基化主要是借助巯基化试剂（如 2-IT、 SPDP、 SATP和 SSDD等） 与靶分子上的氨基反应生成巯基化产物；
在载药系统引入马来酰亚胺时， 可将脂质分子和马来酰亚胺 （MAL ) 修饰的 聚合物材料 （如 MAL-PEG-PLA、 MAL-PEG-PLGA, MAL-PEG-PCL,
MAL-PEG-DSPE) 混合溶解于有机溶媒中， 通过成膜水化、 高压乳匀处理后， 便 可制得疏水端 （如 PLA、 PLGA、 PCL禾卩 DSPE) 镶嵌在脂质双分子层内、 PEG- 马来酰亚胺端处于脂质膜表面的马来酰亚胺化脂质体。
此外， 也可在载药系统表面直接引入马来酰亚胺。 如采用可形成马来酰亚胺 的双功能连接剂 （如双功能丙酸连接剂， 其分子中的活泼酯与氨基反应可生成马 来酰亚胺基团） 在纳米载体上引入马来酰亚胺基团。
另一类优选的偶联方法具体如下：
将生物素 （biotin) 分别引入至靶分子和载药系统上， 再利用亲和素 (avidin) 作为桥连剂实现靶向递药系统的构建。即先将亲和素与生物素化的载药系统混合， 尚未结合的位点再与生物素化靶分子结合。
如生物素化 PEG-PLGA的制备， 可将 PLGA-COOH (分子量 20kDa)溶解于二 氯甲烷中， 室温下搅拌后加入 8倍量的 NHS和 EDC活化。 生成的活泼酯与
NH2-PEG-biotin (；分子量为 3400Da)混合溶解在三氯甲烷中， 加入适量 Ν,Ν-二异丙 基乙胺， 反应过夜。 用甲醇洗去未反应的 PEG分子， 乙醚沉淀并真空干燥， 得 PLGA-PEG-biotin。 然后将一定比例的 PLGA-PEG-COOH与 PLGA-PEG-biotin混 合溶解在丙酮中， 缓慢滴加至去离子水中， 室温条件下旋转蒸发除去丙酮， 超滤 浓缩除去有机溶剂， 即得生物素化 PEG-PLGA纳米粒。 将生物素化 PEG-PLGA 纳米粒与亲和素溶液室温孵育一定时间， 离心洗涤除去游离的亲和素。 将一定量 生物素化靶分子与其室温搅拌， 再离心除去游离的生物素化靶分子， 即得靶分子 化的 PEG-PLGA纳米粒靶向递药系统。 组合物及其制备方法和用途
本发明的组合物包含本发明的复合物以及装载于复合物纳米载体的抗肿瘤药 物。 按组合物的总重量计， 抗肿瘤药物的含量通常为 1.5— 3.0 wt%。 优选为 2.0— 3.0 wt%。 靶分子的含量为 1.11一 22.2 wt%。 优选为 5.60— 11.1 wt%。
为了更好地实现对抗肿瘤药物的缓释、控释作用， 并且为了防止体内调理作用 的发生， 本发明组合物中， 复合物中的纳米载体的粒径较佳地为 200 nm以下， 较佳 的为 10— 200 nm。
一类优选的抗肿瘤药物包括但不限于： 阿霉素、 紫杉醇、 多西他赛、 顺铂、 米托蒽醌、 柔红霉素、 长春新碱、 全反式维甲酸、 表阿霉素、 勒托替康、 伊立替 康、 2-甲氧雌二醇、 吉西他滨、 长春瑞宾、 5-氟尿嘧啶、 甲氨蝶呤、 卡培他滨、 洛莫司汀、 依托泊苷或其组合。 较佳地为阿霉素、 紫杉醇、 多西他赛、 米托蒽醌、 柔红霉素、 伊立替康、 吉西他滨、 长春瑞宾、 卡培他滨、 依托泊苷。
本发明的组合物的制备方法主要包括步骤：
(1) 提供一纳米载体， 纳米载体中装载有抗肿瘤药物；
(2) 将步骤 (1)的纳米载体与靶分子进行反应， 得到所述组合物。
其中， 所述纳米载体的制备可以采用超声乳化法制作。 较佳地， 所述纳米载 体可采取以下三种方法制备：
(1)用溶解了亲水性抗肿瘤药物和亲水性膜材 （如聚乙二醇） 的水溶液作为水 相， 用溶解了油溶性乳化剂 （如胆酸钠） 的有机溶剂 （如二氯甲烷） 作为油相， 将水相与油相混合搅拌进行粗分散后， 再用超声波细胞破碎机进行乳化， 获得油 包水型纳米乳液， 再在磁力搅拌下向所得纳米乳液中加入交联剂 （如戊二醛） 进 行交联固化， 除去过量的交联剂和乳化剂即可获得包埋有抗肿瘤药物的可生物降 解的纳米载体。
(2)用溶解了疏水性抗肿瘤药物和疏水性膜材 （如 PACA ) 的有机溶剂 （如二 氯甲烷） 作为油相， 用溶解了水溶性乳化剂 （如 Pluronic F68、 Dextran 70 ) 的水 溶液作为水相， 将油相与水相混合搅拌进行粗分散后， 再用超声波细胞破碎机进 行乳化， 获得水包油型纳米乳液， 再在磁力搅拌下向所得纳米乳液中加入交联剂 (如戊二醛） 进行交联固化， 除去过量的交联剂和乳化剂即可获得包埋有抗肿瘤 药物的可生物降解的纳米载体。
(3)用溶解了亲水性抗肿瘤药物的水溶液作为水相， 用溶解了疏水性膜材 （如
PEG-PLA ) 和油溶性乳化剂 （如胆酸钠） 的有机溶剂作为油相， 将水相与油相混 合搅拌进行粗分散后， 再用超声波细胞破碎机进行乳化， 获得油包水型纳米乳液， 再将所得油包水型纳米乳液加入溶有水溶性乳化剂的水相中进行超声乳化， 从而 获得 W/O/W型纳米乳液， 再在磁力搅拌下向所得 W/O/W型纳米乳液中加入交联 剂 （如戊二醛） 进行交联固化， 除去过量的交联剂和乳化剂即可获得包埋有抗肿 瘤药物的可生物降解的纳米载体。
本发明的上述纳米载体也可以采用脱溶剂法制作， 一类优选的方法包括： 将 水溶性抗肿瘤药物和纳米载体寡肽或蛋白质溶于 NaCl水溶液中， 然后滴加乙醇， 滴加过程持续磁力搅拌， 当溶液变成乳白色悬浊液后加入戊二醛交联固化纳米载 体， 除去过量的交联剂即可获得包埋有抗癌药物的可生物降解纳米粒子。
上述步骤 (2)中的反应方法同本发明复合物中纳米载体与靶分子的反应方法。 应理解， 上述组合物也可通过将抗肿瘤药物包入已制备好的本发明的复合物 中来制备。
本发明的组合物可用于制备抗肿瘤药物， 特别是用于制备治疗乳腺癌、 卵巢 癌、 肾癌和胃癌的药物。 药物、 组合物及施用方法
本发明所述的药物含有有效量的本发明的组合物， 药学上可接受的载体或赋性 剂。
如本文所用，术语"含有"或"包括"包括了 "包含"、 "基本上由 ......构成"、和"由 ...... 构成"。 如本文所用， 术语"药学上可接受的"的成分是适用于人和 /或动物而无过度不 良副反应 (如毒性、剌激和变态反应;)的， 即有合理的效益 /风险比的物质。如本文所用， 术语"有效量"是指可对人和 /或动物产生功能或活性的且可被人和 /或动物所接受的 如本文所用， 术语"药学上可接受的载体"指用于治疗剂给药的载体， 包括各种赋 形剂和稀释剂。 该术语指这样一些药剂载体： 它们本身并不是必要的活性成分， 且施 用后没有过分的毒性。 合适的载体是本领域普通技术人员所熟知的。 在《雷明顿药物 科学》 （Remington's Pharmaceutical Sciences, Mack Pub.Co. , N丄 1991)中可找到关于 药学上可接受的赋形剂的充分讨论。 本发明的药物剂型包括： 固体制剂、 液体制剂或注射剂。 较佳地为注射剂。 本发明药物的施用对象为哺乳动物， 优选人类。
在本发明的另一个优选例中， 每天一次或多次施用本发明的药物或组合物， 例如
1、 2、 3、 4、 5 或 6 次。 其中给药途径包括但并不限于： 口服给药， 注射给药， 腔 内给药， 透皮给药； 优选的注射给药包括： 静脉注射， 肌肉注射， 皮下注射， 腔内注 射。 在施用本发明的药物或组合物时， 具体剂量还应考虑给药途径、 病人健康状况等 因素， 这些都是在熟练医师技能范围之内的。本发明组合物的安全有效量通常至少约 10毫克或至少 85毫克 /千克体重 /天， 而且在大多数情况下不超过约 200或不超过约 115毫克 /千克体重 /天。 较佳地的剂量是约 100毫克 /千克体重 /天。 与现有技术相比， 本发明具有以下主要优点：
(1) 本发明的复合物及组合物在 NaCl水溶液、 PBS水溶液或血清中具有良好的 分散性与稳定性， 无沉淀或团聚现象发生。
(2) 本发明的复合物和组合物能与乳腺癌、 卵巢癌、 肾癌和胃癌细胞高特异性 地结合， 对肿瘤组织具有很强的靶向作用。
(3) 本发明的组合物和药物能够将抗肿瘤药物靶向输送至肿瘤细胞内， 有效提 高细胞内的药物浓度， 对肿瘤细胞具有很强的杀灭作用， 同时对正常组织和细胞 几乎无毒副作用。 本发明提到的上述特征， 或实施例提到的特征可以任意组合。本案说明书所揭示 的所有特征可以任何组合形式并用， 说明书中所揭示的各个特征， 可以任何被提供相 同、 均等或相似目的的替代性特征取代。 因此除有特别说明， 所揭示的特征仅为均等 或相似特征的一般性例子。
下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于说明 本发明而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方法， 通常按照常规条件或按照制造厂商所建议的条件。 除非另外说明， 否则百分比和 份数按重量计算。
除非另行定义， 文中所使用的所有专业与科学用语与本领域熟练人员所熟悉 的意义相同。 此外， 任何与所记载内容相似或均等的方法及材料皆可应用于本发 明方法中。 文中所述的较佳实施方法与材料仅作示范之用。 实施例 1 组合物 [D-Arg25】-NPY-ANP-TXT的制备
(1) 包埋 TXT的 BSA纳米载体 （ANP ) 的制备
配制 pH 10.8的 10 mM NaCl水溶液，再用该溶液配制浓度为 20 mg/mL的 BSA 水溶液， 然后向 2.0 mLBSA水溶液中加入 2.0 mL无水乙醇， 磁力搅拌 lO min后 以 2.0 mL/min的滴加速率添加 4.0 mL乙醇 （总乙醇添加量与纳米载体水溶液的 体积比为 3.0 ) ， 滴加过程持续磁力搅拌， 乙醇滴加结束后立即加入 8 %的戊二醛 水溶液（戊二醛 -BSA质量比为 0.24 )交联固化 24 h， 然后加入 1.0 mL甘氨酸（40 mg/mL )来中和过量的戊二醛，反应 2.0 h后，对样品进行离心（20,000xg, 20min)， 所得样品用 lOmM NaCl水溶液洗涤两次， 最后冷冻干燥 48 h即可获得 BSA纳米 载体。将 TXT的溶液分散至 20 mg/mL的 BSA纳米载体水溶液中， 采用上述同样 方法即可制得包埋有抗肿瘤药物的纳米载体 ANP-TXT。
(2) 纳米载体表面偶联靶分子 [D-Arg25]-NPY
在 EDAC ( 1-乙基- ( 3-二甲基氨基丙基） 碳二亚胺） 的催化下， 利用靶分子 [D-Arg25]-NPY的氨基与纳米载体 ANP表面的羧基之间的化学反应， 在纳米载体 表面偶联靶分子 [D-Arg25]-NPY。 具体制备方法如下： 用磷酸缓冲液 （PBS ) 作溶 剂配制 500 g/mL的靶分子 [D-Arg25]-NPY溶液，将 50 mg EDAC溶于 10 mL 靶分 子溶液 （冰浴） ， 然后加入 90 mL溶于 PBS的 ANP-TXT悬浊液 （5.0 mg/mL) ， 将混合液置于室温下磁力搅拌，反应 4-24 h，对样品进行离心（20,000 X g, 20 min)， 所得样品用 PBS洗涤两次， 最后冷冻干燥 48 h即可获得表面偶联有靶分子， 且内 部包埋有抗肿瘤药物纳米载体的组合物 [D-Arg25]-NPY-ANP-TXT。
组合物 [D-Arg25]-NPY-ANP-TXT分别在 NaCl水溶液、 PBS水溶液和血清 ( serum) 中 1-15天的粒径变化如表 1和图 2所示。
表 1
Figure imgf000013_0001
从表 1和图 2可以看出，在三种不同溶剂中，复合纳米粒子粒径均一，分散性好， 且稳定在 110〜120nm之间。
通过纳米粒度仪方法测定， 所述复合纳米粒子的 PDI指数均小于 0.5。 实施例 2 组合物 [D-Arg25】-NPY-ANP-TXT对肿瘤细胞 MCF-7和 HEC-1B-Y5 的活性测试
(1) MTT实验 (细胞毒性试验）
1. 用含 10 %胎小牛血清得培养液配成单个细胞悬液， 以每孔 1.0 X 105个细 胞接种到 96孔板， 每孔体积 150 μL a
2. 置于 37 °C细胞培养箱内， 培养 24 h。
3. 吸弃孔内培养上清液， 加入含有 TXT的新鲜培养液 200 或含有相同浓 度 TXT的组合物 [D-Arg25]-NPY-ANP-TXT溶液 200 L。
4. 置于 37 °C细胞培养箱内， 培养 4 h后， 吸弃孔内的上清培养液， 替换为 不含任何药物或纳米粒子组合物的新鲜培养液， 继续培养 44 h。
5. 每孔加 MTT溶液 （5mg/ml用 PBS配制， pH=7.4)10 L， 置于 37 °C细胞 培养箱内， 继续孵育 4 h， 终止培养， 吸弃孔内培养上清液。
6. 每孔加 150 L DMSO , 振荡 10 min， 使结晶物充分融解。
7. 选择 550 nm波长， 在酶联免疫监测仪上测定并记录各孔的光吸收值， 测 试结果如表 2所示。
其中， 细胞实验中所选择的细胞包括： 人乳腺肿瘤 MCF-7细胞、 人子宫内膜 瘤 HEC- 1B-Y5细胞。 （购自美国标准生物品收藏中心 ATCC和美国 Sciencell公 司）
表 2 组合物 [D-Arg25]-NPY-ANP-TXT对 MCF-7和 HEC-1B-Y5的杀灭作用
Figure imgf000014_0001
从上表和图 3可以看出，多西他赛 (TXT)对 MCF-7细胞和 HEC-1B-Y5细胞均 具有优异的杀灭效果，但组合物 [D-Arg25]-NPY-ANP-TXT对 MCF-7细胞具有优异 的杀灭效果， 而对 HEC-1B-Y5细胞的灭杀效果却不明显， 因此上述结果表明， 组 合物 [D-Arg25]-NPY-ANP-TXT能意外并显著地、高选择性地杀灭 MCF-7细胞并且 具有优异的杀灭效果。 实施例 3 组合物 [D-Arg 25】-NPY-ANP-TXT对不同肿瘤细胞的活性测试
(1) 活性测试方法参照实施例 2， 测试结果如表 3-1和 3-2所示。 其中细胞实 验中所选择的肿瘤细胞包括： 人卵巢肿瘤 UWB 1.289细胞、 人胃肿瘤 GIST-H1细 胞、 人肾肿瘤 SW-13细胞、 人脑肿瘤 SMS-KAN细胞。 正常细胞包括： 人乳腺上 皮细胞 MCF-10a、人类卵巢表面上皮细胞 HOSEpiC、人肾皮质上皮细胞 HRCEpiC、 人胃粘膜细胞 GES-1、 人脑星型胶质细胞 HA、 人子宫内膜上皮细胞
HUM-CELL-0111。 （购自美国标准生物品收藏中心 ATCC、 美国 Sciencell公司和 中国科学院典型培养物保藏委员会细胞库） 表 3-1 组合物 [D-Arg25]-NPY-ANP-TXT对不同癌细胞的杀灭作用比较
Figure imgf000015_0001
表 3-2 组合物 [D-Arg25]-NPY-ANP-TXT对正常细胞的作用比较
Figure imgf000015_0002
从表 3-1可以看出， 本发明的组合物 [D-Arg25]-NPY-ANP-TXT对 UWB1.289 细胞、 SW-13细胞和 GIST-H1细胞如同 MCF-7细胞一样， 均具有优异的杀灭效 果， 而对 SMS-KAN细胞的灭杀效果却不明显， 因此上述结果表明， 组合物
[D-Arg25]-NPY-ANP-TXT能高选择性地杀灭 UWB 1.289细胞、 SW-13细胞和
GIST-H1细胞并且具有优异的杀灭效果。
从表 2、表 3-1和表 3-2的测试结果可以看出，组合物 [D-Arg25]-NPY-ANP-TXT 能与乳腺癌、 卵巢癌、 肾癌和胃癌细胞高特异性地结合， 对肿瘤细胞具有很强的靶 向作用， 并且能够将抗肿瘤药物靶向输送至肿瘤细胞内， 有效提高细胞内的药物 浓度， 对肿瘤细胞具有很强的杀灭作用， 同时对正常组织和细胞几乎无毒副作用。 实施例 4 包埋多西他赛的不同组合物对 MCF-7和 UWB1.289细胞的活性测 试
(1) 组合物的制备
(a) 包埋 TXT的壳聚糖纳米载体组合物的制备
(1) 包埋 TXT的壳聚糖纳米载体的制备
配制 0.2 %(w/v)的壳聚糖溶液，溶剂为 1 %(w/v)的醋酸，将多西他赛（TXT) 分散至壳聚糖溶液中，用氢氧化钠将该溶液的 pH值调至 4.7-4.8;配制 0.3 %(w/v) 的三聚磷酸钠 （TPP) 水溶液； 在磁力搅拌下， 向 0.5mL的上述壳聚糖溶液中加 入 O. lmL的 TPP溶液， 从而制得离子交联的包埋了 TXT的壳聚糖纳米载体。
(ϋ) 壳聚糖纳米载体表面偶联靶分子
将所得纳米载体表面进行靶分子偶联反应， 参照实施例 1中的步骤 (2)制备， 将靶分子 [D-Arg25]-NPY换成表 5中列举的靶分子。 (b) 包埋 TXT的 BSA纳米载体 （ANP ) 组合物的制备
参照实施例 1的步骤制备， 将靶分子 [D-Arg25]-NPY换成表 5中列举的靶分子。
(2) 细胞毒性试验
参照实施例 2中的方法进行活性测试， 其中， 试验中所选择的细胞包括：
MCF-7细胞和 UWB 1.289细胞。
测试结果如表 5所示， 其中
" + "表示对肿瘤细胞具有杀灭效果， "一"表示对肿瘤细胞基本无杀灭效果或杀灭效果弱。 具体符号含义如表 4所 示 （取某一具有代表性的浓度值， 根据数值范围取不同的符号） ： 表 4
Figure imgf000017_0001
Figure imgf000017_0002
实施例 5包埋多西他赛的不同组合物对 GIST-H1和 SW-13细胞的活性测试 不同组合物的制备方法和细胞毒性测试参照实施例 4的步骤进行。 测试结果 如表 6所示。 表 6 不同组合物对 GIST-H1和 SW-13细胞的杀灭作用比较
Figure imgf000018_0001
实施例 6 包埋多西他赛的不同组合物对 SMS-KAN和 HEC-1B-Y5细胞的活 性测试
不同组合物的制备方法和细胞毒性测试参照实施例 4的步骤进行。 测试结果 如表 7所示。
表 7 不同组合物对 SMS-KAN和 HEC-1B-Y5细胞的杀灭作用比较
Figure imgf000018_0002
[Pro30, Nal32, Leu34]NPY(28-36)
[Pro30, Me31, Nal32, Leu34]NPY(28-36)
BIBO3304
PD160170
LY366258
J-104870
LY 357897
J-115814 从表 5-7的测试结果可以看出， 偶联有本发明靶分子的组合物对 MCF-7、 UWB 1.289, GIST-Hl和 SW-13细胞均具有较强的灭杀作用， 当 CTXT为 5 g/mL 时， MCF-7、 GIST-Hl和 SW-13细胞的存活率基本在 30%以下， UWB 1.289细胞 的存活率在 60 %。 而对 SMS-KAN和 HEC-1B-Y5细胞的灭杀效果不明显， 细胞 的存活率基本在 80 %以上。 因此由此看出， 本发明的组合物具有很好的选择性， 对乳腺癌、 卵巢癌、 肾癌和胃癌肿瘤细胞具有很强的靶向作用， 并且对肿瘤细胞 具有很强的杀灭作用， 而对脑瘤和子宫内膜瘤细胞几乎没有作用。 在本发明提及的所有文献都在本申请中引用作为参考， 就如同每一篇文献被 单独引用作为参考那样。 此外应理解， 在阅读了本发明的上述讲授内容之后， 本 领域技术人员可以对本发明作各种改动或修改， 这些等价形式同样落于本申请所 附权利要求书所限定的范围。

Claims

权 利 要 求
1. 一种复合物， 其特征在于， 所述复合物包括：
纳米载体； 以及
靶分子， 所述靶分子偶联于所述纳米载体表面；
其中， 所述靶分子选自： [D-Arg25]-NPY、 [D-His26]-NPY、 [D-Arg25,
D-His26]-NPY、 [Arg6, Pro34]pNPY、 [Asn6, Pro34]pNPY、 [Cys6, Pro34]pNPY、 [Phe6 Pro34]pNPY、 [Arg7, Pro34]pNPY、 [D-His26, Pro34]NPY、 [Phe7, Pro34]pNPY、 [Pro30 Nle31, Bpa32, Leu34]NPY(28-36), [Pro30, Nal32, Leu34]NPY(28-36), [Pro30, Nle31, Nal32 Leu34]NPY(28-36), BIBO3304、 PD160170、 LY366258、 J-104870、 LY 357897、 J-115814或其组合；
并且所述纳米载体的粒径在 200nm以下， 且多分散指数 （PDI) 小于 0.5。
2. 如权利要求 1所述的复合物， 其特征在于， 按复合物总重量计， 所述靶分子 的含量为 l.l l—22.2wt%。
3. 如权利要求 1所述的复合物， 其特征在于， 所述复合物具有以下一个或多个 特征：
(a) 与乳腺癌、 卵巢癌、 肾癌或胃癌肿瘤细胞高特异性结合；
(b) 纳米载体的粒径在 10-200nm。
4. 如权利要求 1所述的复合物， 其特征在于， 所述纳米载体选自： 蛋白类纳米 粒子、 寡肽类纳米粒子、 磷脂类纳米脂质体、 多糖类纳米粒子、 聚醚类纳米粒子、 聚酯类纳米粒子、 聚酯类聚合物胶束。
5. —种组合物， 其特征在于， 所述组合物包括：
权利要求 1所述的复合物； 以及
装载于所述复合物纳米载体中的抗肿瘤药物。
6. 一种权利要求 5所述组合物的制备方法， 其特征在于， 包括以下步骤：
(1) 提供一纳米载体， 所述纳米载体中装载有抗肿瘤药物；
(2) 将步骤 (1)的纳米载体与靶分子进行偶联反应， 得到所述组合物。
7. 如权利要求 6所述的制备方法， 其特征在于， 所述偶联反应选自：
(1) 羧基与氨基的缩合反应；
(2) 巯基与马来酰亚胺的加成反应； 或 (3) 亲和素与生物素的非共价结合。
8.一种如权利要求 1所述复合物的用途， 其特征在于， 所述复合物用于制备治 疗癌症的药物。
9.一种如权利要求 5所述组合物的用途， 其特征在于， 所述组合物用于制备治 疗癌症的药物。
10. 一种药物， 其特征在于， 所述药物包括：
权利要求 1所述的复合物；
装载于所述复合物纳米载体中的抗肿瘤药物； 以及
药学上可接受的载体。
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