WO2012130141A1 - 一种增强靶细胞摄取治疗剂的方法和药物组合物 - Google Patents

一种增强靶细胞摄取治疗剂的方法和药物组合物 Download PDF

Info

Publication number
WO2012130141A1
WO2012130141A1 PCT/CN2012/073202 CN2012073202W WO2012130141A1 WO 2012130141 A1 WO2012130141 A1 WO 2012130141A1 CN 2012073202 W CN2012073202 W CN 2012073202W WO 2012130141 A1 WO2012130141 A1 WO 2012130141A1
Authority
WO
WIPO (PCT)
Prior art keywords
cancer
use according
tumor
endostatin
endocytic pathway
Prior art date
Application number
PCT/CN2012/073202
Other languages
English (en)
French (fr)
Inventor
罗永章
陈阳
付彦
贾琳
常国栋
Original Assignee
清华大学
北京普罗吉生物科技发展有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 清华大学, 北京普罗吉生物科技发展有限公司 filed Critical 清华大学
Priority to CA2837122A priority Critical patent/CA2837122C/en
Priority to EP12765089.3A priority patent/EP2702997B1/en
Priority to AU2012237786A priority patent/AU2012237786B2/en
Priority to US14/008,428 priority patent/US9364493B2/en
Priority to JP2014501425A priority patent/JP6114940B2/ja
Publication of WO2012130141A1 publication Critical patent/WO2012130141A1/zh

Links

Classifications

    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • 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
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/065—Diphenyl-substituted acyclic alcohols
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A61K31/718—Starch or degraded starch, e.g. amylose, amylopectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61K47/56—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • 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
    • A61K9/0012—Galenical forms characterised by the site of application
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • the present invention relates to a novel use of an existing drug, and in particular, the present invention relates to a method and composition for improving the therapeutic effect of a target cell by ingesting a therapeutic agent, and in particular to a lipid raft-membrane-dependent type A pharmaceutical composition consisting of an endocytic pathway modulator and certain drugs, such as anti-tumor drugs.
  • the present invention also relates to a method of screening for a lipid-membrane-dependent endocytic pathway modulator having a synergistic effect against a tumor drug. Background technique
  • tumor neovascularization and lymphatic endothelial cells provide new targets for cancer therapy (Folkman, J. N Engl J Med 1971; 285: 1182-1186, Witte MH et al. Lymphology. 1987; 20(4): 257 -66
  • obesity, diabetic retinopathy, persistent vitreous hyperplasia, psoriasis, atopic dermatitis, arthritis, arteriosclerosis, endometriosis, asthma, ascites, peritoneal adhesions, etc. have also been shown to be associated with neovascular abnormalities. The production is closely related (Carmeliet P., Nature Medicine 2003; 9(6): 653-660).
  • Endostatin (hereinafter referred to as ES) is a 20 kDa cleavage product at the carboxyl terminus of collagen XVI. It is an endogenous vascular/lymphangiogenesis inhibitor that inhibits vascular/lymphatic endothelial cell proliferation. Migration, and inhibition of neovascular/lymphangiogenesis activity. Further experiments have shown that recombinant endostatin can inhibit the growth and metastasis of a variety of tumors in animal models without drug resistance (Folkman J. et al. Cell 1997; 88:277-285, Folkman J. et al Nature 1997; 390: 404-407, Zhuo W.
  • EGFR Epithelial Growth Factor Receptor
  • the anti-EGFR monoclonal antibody cetuximab is an antibody-based antitumor drug that specifically binds to EGFR on the surface of tumor cells.
  • Nystatin is a polyene antibiotic with a broad-spectrum antifungal effect and can be safely used in buccal or topical applications without interaction with other drugs.
  • NT has high antibacterial activity against Candida, and Cryptococcus neoformans, Aspergillus, Mucor, Microsporum, Bacillus capsulatum, Dermatitis buds and Dermatophytes are also generally sensitive to NT.
  • NT is also commonly used in the prevention of patients susceptible to fungal infections, such as AIDS patients or patients receiving chemotherapy.
  • the antifungal principle of this class of drugs is: It binds to the main component of the fungal cell membrane, ergosterol, and eventually causes perforation of the fungal cell membrane and potassium ion leakage until the fungus dies. Since ergosterol is a lipid substance unique to fungal cell membranes, NT and AMB do not cause accidental killing of eukaryotic cell membranes when applied to human or animal bodies.
  • NT affects cholesterol-related endocytosis at the cellular level.
  • NT is widely used as an inhibitor of the lipid raft-caveolae-dependent endocytic pathway.
  • the principle of action is: Lipid-cavity-dependent endocytosis relies on the important substance of cholesterol (cholesterol) on the cell membrane. NT can inhibit this endocytic pathway by binding to cholesterol on the membrane.
  • Agents similar to the principle of action of NT include amphotericin B, methyl-p-cyclodextrin and filipin, which inhibit the binding of cholesterol to the cell membrane. Lipid-membrane endocytic pathway.
  • Amphotericin B (hereinafter referred to as AMB) has the same mechanism of action as NT, and is an effective polyene broad-spectrum antifungal drug for the treatment of fungal infections such as cryptococcus and Aspergillus.
  • Wickstrom et al (2002, 2003) reported ES and alpha5betal on endothelial cell membranes. Integrin alpha5betal and caveolin, which is a constituent protein of the cavern, interact, and ES interacts with lipid rafts, revealing the interrelationship between ES and lipid raft-membrane on the cell membrane. (Wickstrom et al, Cancer Res. 2002; 62: 5580-9, Wickstrom et al, J. Biol. Chem. 2003; 278(39): 37895-37901).
  • lipid raft-endocytosis may be the route of administration for targeted tumor-targeted drugs (Kojic et al, PLoS ONE 2008; 3: e3597).
  • Migalovich et al (2009) reported that NT increased the endocytosis of daidzein-bovine serum albumin (daidzein-BSA) by ovarian cancer cells by regulating cell lipid raft-endocytosis.
  • Daidzein-bovine serum albumin is a potential tumor developer, and NT increases the endocytosis of daidzein-bovine serum albumin in tumor cells, which may make this tumor developer play a better practical effect (Migalovich Et al, Cancer Res. 2009; 69: 5610-5617).
  • lipid raft-cavity-dependent endocytosis may be involved in the endocytosis of many important substances such as cytokines, foreign proteins, and even drug molecules. By regulating and controlling such endocytosis, it may affect the body at the cellular level. Ingestion, with application potential. Summary of invention
  • the present invention is based on the discovery that ES can utilize both the caveola/lipid raft pathway and the clathrin-coated pits pathway. Endocytosis by endothelial cells, if the inhibitor NT interferes with the lipid raft-cave-dependent endocytic pathway, an unexpected effect can be obtained to increase the level of endocytic uptake of the drug in vascular/lymphatic endothelial cells, and further promote the ES pair. Inhibitory effects of endothelial cell activity, tumor angiogenesis, and tumor growth.
  • the reason for this finding may be that the clathrin-coated vesicle-efficient endocytic pathway is compensatoryly active in the case of a lipid-membrane-dependent endocytic pathway that is inhibited. Increased overall endocytic uptake.
  • EGFR ligands have been reported to have an endocytic mechanism similar to ES.
  • EGFR monoclonal antibody is a kind of effective anti-tumor drug, which inhibits the related signal transduction and tumor proliferation by binding to EGFR specific to tumor cells.
  • the present inventors have found that NT can also significantly enhance the endocytosis and accumulation of the anti-tumor drug EGFR monoclonal antibody in cancer cells by affecting lipid-dependent endocytosis, and promote the therapeutic effect of EGFR monoclonal antibody, which is further applied to tumor treatment and Potential in areas such as tumor imaging.
  • endothelin receptor type A cholera toxin
  • transforming growth factor- ⁇ receptors B cell antigen receptor
  • bone morphogenetic protein receptor HM1.24
  • integrins which have similar endocytic mechanisms to ES
  • the invention provides the use of a modulator of a lipid raft-cave-dependent endocytic pathway for the preparation of a pharmaceutical composition for increasing uptake of a therapeutic agent by a target cell in a subject.
  • the invention provides a method of increasing uptake of a therapeutic agent by a target cell in a subject, comprising administering to the individual a modulator of a lipid raft-membrane-dependent endocytic pathway.
  • the invention provides a modulator of a lipid raft-cave-dependent endocytic pathway for use in increasing the uptake of a therapeutic agent by a target cell in a subject.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a lipid raft-cave-dependent endocytic pathway modulator and a medicament, such as an anti-tumor drug.
  • the modulator of the lipid raft-cave-dependent endocytic pathway is an inhibitor of a lipid raft-cave-dependent endocytic pathway, for example, the inhibitor is An olefinic antifungal (such as nystatin or amphotericin B), or the inhibitor is, for example, methylated ⁇ -cyclodextrin or Philippine.
  • the inhibitor is An olefinic antifungal (such as nystatin or amphotericin B), or the inhibitor is, for example, methylated ⁇ -cyclodextrin or Philippine.
  • the therapeutic agent can pass through a lipid raft-membrane-dependent endocytic pathway and a cage
  • the two pathways of protein-coated vesicle endocytic pathway are taken up by target cells.
  • the subject has an angiogenesis-related disease or a lymphangiogenesis-related disease.
  • the subject has a tumor, such as lung cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, esophageal cancer, nasopharyngeal cancer, malignant melanoma tumor, bone cancer, lymphoma, breast cancer, cervical cancer, prostate cancer , hemangioma, neuroendocrine tumor, oral cancer, sarcoma, kidney cancer or cholangiocarcinoma.
  • a tumor such as lung cancer, pancreatic cancer, liver cancer, gastric cancer, colorectal cancer, esophageal cancer, nasopharyngeal cancer, malignant melanoma tumor, bone cancer, lymphoma, breast cancer, cervical cancer, prostate cancer , hemangioma, neuroendocrine tumor, oral cancer, sarcoma, kidney cancer or cholangiocarcinoma.
  • the therapeutic agent is an inhibitor of blood vessel or lymphangiogenesis, such as endostatin or a derivative thereof, wherein the endostatin is, for example, natural endostatin or recombinant endostatin, such as recombination Human endostatin.
  • the endostatin has the amino acid sequence of SEQ ID NO. 1, SEQ ID N0.2, SEQ ID N0.3 or SEQ ID N0.4.
  • the endostatin derivative is polyethylene glycol (PEG)-modified endostatin.
  • the polyethylene glycol (PEG) is a monomethoxy polyethylene glycol having an average molecular weight of 5-40 kD, such as monomethoxy polyethylene glycol propionaldehyde.
  • the monomethoxypolyethylene glycol propionaldehyde has an average molecular weight of 20 kD
  • the polyethylene glycol (PEG) modified site is the N-terminus of endostatin. A-amino group.
  • the therapeutic agent is an antibody capable of inhibiting the growth of tumor cells.
  • the antibody is an antibody against epidermal growth factor receptor (EGFR), such as an EGFR monoclonal antibody, and an example of an EGFR monoclonal antibody is cetuximab.
  • EGFR epidermal growth factor receptor
  • the modulator of the lipid raft-cave-dependent endocytic pathway is selected from the group consisting of intravenous injection, intravenous drip, subcutaneous injection, intramuscular injection, intraperitoneal injection, subcutaneous embedding , transdermal absorption, parenteral administration of hepatic artery injection.
  • the modulator of the lipid raft-cave-dependent endocytic pathway is formulated in a liposomal-embedded form.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the aforementioned endostatin or antibody, and an inhibitor of the lipid raft-cave-dependent endocytic pathway described above.
  • the invention also relates to the screening of lipids having synergistic effects against anti-tumor drugs A method of ⁇ -cave-dependent endocytic pathway modulators.
  • the present invention also relates to a drug or a reagent similar to the principle of action of NT (such as amphotericin B), and a combination method, a combined preparation and a drug combination between a pharmaceutical agent similar to the action mechanism or endocytic pathway of ES or EGFR monoclonal antibody. Things. DRAWINGS
  • Figure 1A-E NT and its counterparts (amphotericin B, methylated beta-cyclodextrin and filipin) promote endocytic uptake of ES and its derivatives and modifications in endothelial cells in a dose-dependent manner .
  • ⁇ Using Western Blotting to detect NT promotes endocytosis of ES in vascular endothelial cells, and this promotion is positively correlated with NT concentration. This promotion is manifested by a significant increase in the cytoplasm, nuclear fraction, and whole cell content of ES after endocytosis.
  • Amphotericin B was detected by Western Blotting. Methylated ⁇ -cyclodextrin and Filipin also promoted endocytosis of ES in endothelial cells, and this promotion was positively correlated with NT concentration.
  • FIG. 2 Western Blotting method was used to detect the combination of ES and NT to increase the inhibitory effect of ES on endothelial cell ERK and p38 MAPK signaling pathway by increasing ES endocytosis in endothelial cells.
  • Figure 3A-B ES combined with NT to inhibit cell migration
  • A Cell migration experiments confirmed that the combination of ES and NT enhanced the inhibitory effect of ES on endothelial cell activity, resulting in a further reduction in the number of endothelial cells that migrated than in the absence of NT.
  • Figure 4A-E Combination of ES and NT inhibits tumor growth and improves ES distribution in tumor tissues
  • A The combination of ES and NT can further enhance the inhibitory effect of ES on tumor growth in the A549 lung cancer animal model.
  • the combination of ES and NT can further enhance the inhibitory effect of ES on tumor growth in the H22 liver cancer animal model.
  • E Using an animal fluorescence imaging system, a combination of ES and NT was found to enhance the uptake and distribution of ES (with rhodamine fluorescent labeling) in tumor-bearing tumor tissues.
  • Figure 5A-B NT promotes entry of EGFR monoclonal antibody into tumor cells
  • A Using Western Blotting to detect NT promotes endocytosis of EGFR monoclonal antibodies in cancer cells.
  • ES and EGFR monoclonal antibodies are generated by means of the lipidola-lipid raft pathway and the clathrin-coated pits pathway.
  • Endocytic protein drug provides a kind of New pharmaceutical compositions and new methods of enhancing efficacy.
  • Lipid raft is a microdomain rich in cholesterol and sheath pity on the plasma membrane.
  • the saturated fat chain of the sheath pity and the glycosphingolipids are tightly packed, and the gap between the saturated aliphatic chains is filled with cholesterol as a spacer molecule to form a liquid-ordered phase, which is about 50 nm in diameter. about.
  • Lipid rafts are a dynamic structure that is closely related to signal transduction and protein sorting of cell membranes.
  • Membrane also known as caveolae, plasma membrane microcapsule, caveolae
  • lipid rafts which has the same membrane lipid composition as lipid rafts, and does not contain clathrin (also known as clathrin, clathrin).
  • caveolin a small molecular weight protein, 21KD. It is abundant in fat cells, endothelial cells, epithelial cells, and smooth muscle cells, and is associated with endocytosis.
  • the membrane cavity is also rich in certain signal molecules, indicating that it is involved in cell signal transduction.
  • Clathrin-coated pits are a membrane transport dynamic structure formed by clathrin-mediated endocytosis.
  • the endocytic mode that relies on this structure is called the clathrin-coated vesicle-dependent endocytic pathway.
  • Lymphangiogenesis related diseases are diseases closely related to the abnormal formation of blood vessels and lymph nodes, including tumors, arthritis and psoriasis. Disease, diabetes, obesity and a variety of eye diseases.
  • the present invention shows that the addition of an inhibitor of the lipid raft-cave-dependent endocytic pathway, NT, to ES into a liquid culture environment of vascular endothelial cells can significantly enhance the endocytosis of ES in vascular endothelial cells, and this enhancement The effect is positively correlated with the concentration of NT. At the same time, we observed the same results in lymphatic epithelial cells.
  • an inhibitor of the lipid raft-cave-dependent endocytic pathway, NT an inhibitor of the lipid raft-cave-dependent endocytic pathway
  • the present invention also extends the synergistic effect of NT to other drugs and agents that are similar in principle to NT.
  • a class of substances similar to the principle of action of NT such as amphotericin B (AMB), methylated ⁇ -cyclodextrin ( ⁇ -CD), and filipin, can also Enhance endocytosis of ES in endothelial cells.
  • AMB and filipin are also anti-fungal antibiotics that have already been established, and have the potential to be used in practical combination with NT.
  • amphotericin B, methylated beta-cyclodextrin (methyl-P-cyclodextrin) and filipin can significantly enhance the endocytosis of ES in vascular endothelial cells, and this enhancement effect is positively correlated with the concentration of the above-mentioned multi-disease.
  • NT can also significantly promote PEG-modified ES (PEG-ES), ES (Endu) with (M) GGSHHHHH additional amino acid at the N-terminus, and Endu (PEG-Endu) with N-terminal PEG single-point modification. Endocytosis in vascular endothelial cells, and this enhancement is positively correlated with the concentration of NT.
  • the present invention also demonstrates that the combination of NT promotes endocytosis of ES in vascular endothelial cells and further enhances ES signaling pathways related to vascular endothelial cell survival (such as ERK and p38 MAPK). Inhibitory effect.
  • the present invention demonstrates that the combination of NT and ES enhances the inhibitory effect of ES on endothelial cell migration activity. Furthermore, in animal models of A549 lung cancer and H22 liver cancer, it was demonstrated that NT can enhance the inhibitory effect of ES on tumor growth and tumor neovascularization. Further, NT can also enhance the capture and distribution of ES in tumor tissue.
  • EGFR monoclonal antibody acts as a ligand for EGFR, and its endocytosis mechanism has a certain similarity to ES, and its endocytosis in tumor cells can also be regulated by NT.
  • the present invention demonstrates by cytological experiments that endocytosis of EGFR monoclonal antibodies in cancer cells is promoted by a combination of NT. Further, the present invention also demonstrates in animal experiments that the combination of NT and EGFR monoclonal antibody promotes the distribution and uptake of EGFR monoclonal antibody in mouse transplanted tumors.
  • the present invention discloses a new use of NT, an existing antifungal antibiotic drug, which promotes the intake and efficacy of the existing antitumor drug ES or EGFR monoclonal antibody by a combination of drugs, and is further applied. Potential in areas such as cancer treatment and tumor imaging.
  • Modification of proteins with high molecular polymers is a common method for altering and controlling the kinetic properties of drugs such as half-life, immunological characteristics, and toxicological properties.
  • polyethylene glycol (PEG) is the most commonly used protein modification molecule.
  • PEG polyethylene glycol
  • This experiment demonstrates that the ES protein modified by N-terminal a-amino group by PEG (monomethoxy polyethylene glycol propionaldehyde) and Endocytosis of the derivative Endu in endothelial cells was also significantly increased by the combination of NT. This fact provides a theoretical basis for the use of NT in combination with modified, labeled ES or its derivatives for synergistic purposes.
  • the present invention also extends the scope of the combination to other drugs having similar endocytic pathways and mechanisms of action to ES, including EGFR monoclonal antibodies and modified, labeled EGFR monoclonal antibody-based molecules.
  • drugs having similar endocytic pathways and mechanisms of action to ES, including EGFR monoclonal antibodies and modified, labeled EGFR monoclonal antibody-based molecules.
  • drug intake and effects can also be promoted by a combination of polyene agents such as NT.
  • the present invention also relates to a novel dosage form and pharmaceutical composition for the combination of NT (or AMB, etc.) with ES.
  • NT is a polyene broad-spectrum antifungal antibiotic, difficult to dissolve in water, gastrointestinal tract is not easily absorbed after oral administration, direct injection medication is more toxic.
  • studies have shown that NT can be administered by intravenous infusion by liposome entrapment.
  • Aronex's NT liposome formulation Nyotran has completed Phase III clinical trial in 1999 and is expected to be launched in recent years, which will make NT The mode of administration for intravenous drip is achieved.
  • AMB and NT belong to the same broad-spectrum antifungal antibiotics, and the mechanism of action is identical.
  • three amphotericin B liposome formulations have been marketed in Europe and the United States (trade names Abelcet, Amphocil, and AmBisome) to achieve intravenous infusion of this class of poorly soluble antibiotics.
  • Another amphotericin B liposome intravenous drip was also marketed in 2003 under the trade name Feng Kesong. Therefore, it is entirely feasible to prepare a new dosage form or a new pharmaceutical composition for the combination of ES or EGFR monoclonal antibody with NT or AMB in combination with liposome dosage form and intravenous drip administration.
  • Example 1 NT promotes endocytosis and uptake of ES in vascular and lymphatic endothelial cells.
  • HMEC Human vascular endothelial cells
  • CRL 10636 Human vascular endothelial cells
  • incomplete Freund's adjuvant was used to induce the formation of peritoneal lymphangioma
  • the mouse lymphatic endothelial cells mLEC were isolated by digestion (Zuo W. et al, Zhuo W. et al, Journal of Pathology; 222: 249-260), Endu (SEQ ID NO. 3 or SEQ ID NO. 4, an ES having 9 additional amino acids (M) GGSHHHHH at the N-terminus, of which Amino acid M in the large intestine Random deletion can be performed in the expression of bacteria) (Shenzhen Mai Dejin), ES (SEQ ID NO. 1 or SEQ ID NO.
  • vascular endothelial cells When the vascular endothelial cells are adherently grown to a density of about 90%, they are replaced with DMEM medium containing NT (Hyclone): the NT stock solution is added to the medium to a certain final concentration (0 g/ml, 25 g/ml). , 50 g/ml), at 37. C, 5% C0 2 incubator was allowed to stand for 20 min for NT pretreatment; after pretreatment, a concentration of 5 mg / ml of ES stock solution was added to the medium to a final concentration of 5 ⁇ ⁇ ⁇ at 37. C, 5% C0 2 incubator was allowed to stand for 30 min to allow ES to be endocytosed by vascular endothelial cells.
  • NT Hyclone
  • mLEC cells were pre-treated with medium containing NT concentrations of 0, 75, 150 and 300 g/ml for 30 min at 37 °C, then incubated with culture medium containing ES concentration of 2 g/ml at 37 °C. After 15 min, wash three times with PBS and collect the cells.
  • ES endocytosis assays of different concentrations of NT-treated lymphatic endothelial cells were performed by Western blotting. The results showed that NT, 75, 150 and 300 g/ml increased the endocytosis of ES in lymphatic endothelial cells by about 16, 32, respectively. And 78 times, and the magnitude of ES endocytosis is positively correlated with the concentration of NT used (Fig. 1B).
  • NT can also be replaced with a drug that has the same mechanism of action as NT, such as amphotericin B (25 - 50 g/ml), methylated beta-cyclodextrin (1 - 2 mM), and the Philippines.
  • Bacteriocin 2.5 - 5 ⁇ 8 / ⁇ 1
  • Fig. 1C both promote endocytosis of ES in endothelial cells
  • the ES can also be replaced with the same drug as the ES endocytic mechanism, such as a PEGylated ES.
  • the ES was modified with a polyethylene glycol modification reagent (monomethoxypolyethylene glycol aldehyde, mPEG-ALD) with a molecular weight of 20 kDa, and its product (abbreviated as PEG-ES) was a
  • PEG-ES polyethylene glycol modification reagent
  • PEG-ES polyethylene glycol modification reagent
  • PEG-ES polyethylene glycol modification reagent
  • PEG-ES polyethylene glycol modification reagent
  • the polyethylene glycol molecule is linked to a recombinant human endostatin, which is a protein of the N-terminal a-amino group of the protein.
  • ES inhibits extracellular regulated protein kinases (ERK) and p38 mitogen-activated protein kinases (p38) in endothelial cells.
  • MAPK extracellular regulated protein kinases
  • p38 mitogen-activated protein kinases
  • MAPK extracellular regulated protein kinases
  • pl25 FAK pl25 focal adhesion kinase
  • two signal pathways, ERK and p38 MAPK were selected as detection indexes.
  • the combined use of NT further enhanced the inhibitory effect of ES on these signaling pathways.
  • endothelial cells were divided into four groups for different treatments.
  • the first group negative control group, no NT treatment, no ES treatment; the second group, NT treatment group, only NT (50 g / ml, 20 min) treatment, no ES treatment; the third group, ES treatment group, No NT processing, only ES (5 ⁇ , 30 min) treatment; the fourth group, NT and ES combination, NT (50 ⁇ , prior to ES 20 min force input) and ES (5 g/ml, 30 min) deal with.
  • the cells were subjected to NT or ES treatment according to the method of Example 1, and the cells were collected to detect ERK and p38 MAPK signaling pathways.
  • HMEC endothelial cells
  • the cells were allowed to migrate for 6 hours at 37 ° C and 5% CO 2 .
  • 5 high-magnification (400-fold) fields (Olympus 1X71) were randomly selected from each well, and the number of cells that completely migrated through the membrane to the underlying layer was calculated and averaged. The group was paralleled with three wells and the experiment was repeated twice.
  • endothelial cells were also divided into four groups for different treatments.
  • Group 1 Negative control group, no NT treatment, no ES treatment;
  • Group 2 NT treatment group, only NT (50 g/ml) treatment, no ES treatment;
  • Third group ES treatment group, no NT treatment , only ES
  • NT enhances the inhibitory effect of ES on endothelial cell migration.
  • Representative treatment fields of the migrated cells were observed in each treatment group (Fig. 3A), and the average of the migrated cell counts of each treatment group was compared (Fig. 3B).
  • Example 4 NT is able to enhance the inhibition of tumor growth and neovascularization by ES in animal models and promote the uptake and distribution of ES in tumor tissues.
  • Group 1 Negative control, saline-treated group; Group 2, NT-administered group, only NT (6 mg/kg, intraperitoneal, once daily), no ES treatment; Group III, ES In the drug-administered group, no NT treatment, only ES (12 mg/kg, intraperitoneal, once daily); Group IV, NT and ES combination, NT (6 mg/kg, intraperitoneal, daily) Once) with ES (12 mg/kg, ip, once daily). Two weeks after the administration, the nude mice were sacrificed, and the tumors were weighed and the tumor volume was compared.
  • the second group treated with NT alone had no inhibitory effect on tumor growth; the third group of ES treatment inhibited tumor growth with a tumor inhibition rate of about 40%; the fourth group of NT and ES combined drugs It enhances the inhibitory effect of ES on tumor growth, and the tumor inhibition rate is increased to about 60%.
  • NT can significantly enhance the antitumor activity of ES in animal models (Fig. 4A). There were no abnormal changes in body weight, eating and daily activities of the experimental animals in each group.
  • Detection of angiogenic levels in tumors Tumors of all four groups of nude mice were removed, fixed, and sectioned. Primary antibodies against CD31 (a marker for tumor vascular endothelial cells) and FITC-labeled secondary antibodies (Santa Cruz) were used for observation under a Nikon A1 laser scanning confocal imaging system (Nikon Inc.). The tumors of each group were randomly selected for high magnification (400 times) visual field, and the microscopic system was used to calculate the cross-sectional area of the blood vessels in each field of view and the average value was compared. The effect of NT-enhanced ES on inhibiting neovascularization activity in tumor tissues is similar to its effect on enhancing ES tumor suppressor activity. The average tumor weight of each treatment group was compared (Fig. 4A), and the average vascular area (relative unit, arbitrary unit) of each treatment group was compared (Fig. 4B).
  • NT significantly enhances the role of ES in inhibiting tumor growth and tumor angiogenesis activity.
  • the H22 liver cancer animal model has also been validated.
  • the average tumor weight of each treatment group was compared (Fig. 4C), and the average vascular area (relative unit, arbitrary unit) of each treatment group was compared (Fig. 4D). There was no abnormal change in body weight, eating and daily activities of each group of experimental animals.
  • Detection of uptake and distribution of fluorescently labeled ES in tumor tissues of tumor-bearing mice culture of human lung adenocarcinoma A549 cells in proliferative state inoculated to nude mice 6 to 8 weeks old (Beijing Vital River Laboratory Animal Technique) Ltd.) Subcutaneous. It is administered when the tumor volume reaches about 300 mm 3 .
  • the tumor-bearing nude mice were divided into two groups for different drug administration (four in each group).
  • Group 1 ES administration group, no NT treatment, only Rhodamine (Pierce) fluorescently labeled ES (dose 20 mg/kg) administered intraperitoneally; Group 2, NT and ES combination group, NT (6 Mg/kg, intraperitoneal injection and rhodamine fluorescently labeled ES (20 mg/kg, ip).
  • Group 2 NT and ES combination group, NT (6 Mg/kg, intraperitoneal injection and rhodamine fluorescently labeled ES (20 mg/kg, ip).
  • the tumor tissues were taken out, and the uptake amount of the fluorescent marker ES in the tumor was subjected to imaging and quantitative analysis using an animal fluorescence imaging system (Department of Biomedical Engineering, Tsinghua University).
  • NT enhanced the uptake and distribution of ES in animal tumor tissue by a factor of 4 (Fig. 4E).
  • Example 5 NT is able to significantly increase endocytosis of EGFR monoclonal antibody (cetuxima
  • human lung adenocarcinoma A549 cells expressing EGFR were selected, and when they were adherently grown to a density of about 90%, the cells were replaced with DMEM containing NT: the NT stock solution was added to the medium to a certain end.
  • Concentration (0 g/ml, 25 ⁇ , 50 g/ml), pre-treated for 20 min at 37 ° C in a 5% C0 2 incubator for NT pretreatment; after pretreatment, the concentration was 5 mg/ml.
  • the EGFR monoclonal antibody (cetuximab, Merck) stock solution was added to the culture medium to a final concentration of 5 g/ml, and allowed to stand at 37 ° C in a 5% C0 2 incubator for 30 min to allow the EGFR monoclonal antibody to be endothelial cells. Endocytosis; the medium was discarded at the end of endocytosis, washed 3 times with ice PBS, and endothelial cells were collected.
  • the level of EGFR monoclonal antibody in the cell fraction was measured using the Western Blotting method and compared with the amount of EGFR monoclonal antibody endocytosis in cells not co-treated with NT.
  • mice Animal Cell Cultured in a proliferative state
  • nude mice Beijing Vital River Laboratory Animal Technology Co., Ltd.
  • Tumor volume reached approximately 500 mm 3
  • one group of mice was given EGFR monoclonal antibody labeled with fluorescence (Cy5.5, GE), and the other group was given fluorescently labeled EGFR monoclonal antibody (with the previous dose) Equal) plus a combination of NT.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Biomedical Technology (AREA)
  • Dispersion Chemistry (AREA)
  • Dermatology (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicinal Preparation (AREA)

Abstract

本发明提供一种通过促进靶细胞对治疗剂的摄取而提高其疗效的方法和组合物，所述的组合物含有脂筏-膜穴依赖型内吞通路的调节剂，所述治疗剂例如为抗肿瘤药物。本发明也涉及含有脂筏-膜穴依赖型内吞通路的调节剂和治疗剂的药物组合物。本发明还涉及筛选对抗肿瘤药物具有增效作用的脂筏-膜穴依赖型内吞通路调节剂的方法。

Description

说 明 书 一种增强靶细胞摄取治疗剂的方法和药物组合物 技术领域
本发明涉及一种已有药物的新用途， 具体而言， 本发明涉及一种通 过促进靶细胞对治疗剂的摄取而提高其疗效的方法和组合物， 并具体涉 及脂筏 -膜穴依赖型内吞通路调节剂与某些药物 (如抗肿瘤药物)组成的药 物组合物。 特别地， 本发明还涉及筛选对抗肿瘤药物具有增效作用的脂 筏 -膜穴依赖型内吞通路调节剂的方法。 背景技术
肿瘤的生长和迁移依赖于新生血管 /淋巴管的生成。 因此， 肿瘤新生 血管和淋巴管内皮细胞为癌症治疗提供了新的靶标（Folkman, J. N Engl J Med 1971; 285: 1182-1186, Witte MH et al. Lymphology. 1987; 20(4):257-66 同时， 肥胖症、 糖尿病型视网膜病、 持续性玻璃体增生、 牛皮癣、 过敏性皮炎、 关节炎、 动脉硬化、 子宫内膜异位、 哮喘、 腹水、 腹膜粘连等疾病也被证明与新生血管异常生成密切相关 （Carmeliet P., Nature Medicine 2003; 9(6): 653-660)。
血管内皮抑制素 （endostatin, 以下简称 ES) 是胶原 XVI 羧基端的 一段分子量为 20 kDa 的酶切产物， 是一种内源性血管 /淋巴管生成抑制 剂， 具有抑制血管 /淋巴管内皮细胞增殖、 迁移， 和抑制新生血管 /淋巴管 生成的活性。 进一步实验发现， 重组血管内皮抑制素在动物模型中可以 抑制多种肿瘤的生长、转移，而且不产生耐药性（Folkman J. et al. Cell 1997; 88:277-285, Folkman J. et al. Nature 1997; 390:404-407, Zhuo W. et al, Journal of Pathology; 222:249-260)。 目前， 有一种通过基因工程手段制备 的重组人血管内皮抑制素（商品名恩度， Endu) , 与天然血管内皮抑制素 相比具有 9个额外的 N-末端氨基酸 （(M)GGSHHHHH) , 已作为血管抑 制剂类抗肿瘤药物， 在以非小细胞肺癌为主要适应症的临床应用中得到 了广泛的验证。研究显示， ES会被肿瘤部位高度活跃的血管 /淋巴管内皮 细胞内吞， 但是具体机制尚不清楚。
表皮生长因子受体 ( Epithelial Growth Factor Receptor, 以下简称 EGFR)在肿瘤细胞表面异常过量表达， 并与癌细胞的增殖和转移密切相 关。 因此， 已有多个靶向 EGFR的单克隆抗体成为抗肿瘤药物。抗 EGFR 单克隆抗体西妥昔单抗是一个抗体类抗肿瘤药物， 能够特异性地结合肿 瘤细胞表面的 EGFR。
制霉菌素（nystatin, 以下简称 NT)是一种多烯类抗生素， 具有广谱 抗真菌作用， 可以安全用于口腔含服或外用， 且不与其他药物发生相互 作用。 NT对念珠菌属的抗菌活性高， 新型隐球菌、 曲菌、 毛霉菌、 小孢 子菌、 荚膜组织浆胞菌、 皮炎芽生菌及皮肤癣菌通常对 NT也敏感。 NT 也常用于易患真菌感染病人的预防， 如 AIDS 患者或接受化疗的患者。 NT这一类药物抗真菌的作用原理是： 能够结合真菌细胞膜上的主要成分 麦角固醇（ergosterol) , 并最终造成真菌细胞膜的穿孔以及钾离子泄露直 至真菌死亡。 由于麦角固醇是真菌细胞膜特有的脂类物质， 因此应用于 人体或动物体时 NT和 AMB不会对真核细胞膜造成误杀伤。
NT 可在细胞水平上影响胆固醇相关的内吞作用。 在细胞生物学领 域，尤其是哺乳类细胞内吞活动的研究中， NT被广泛用来作为脂筏（lipid raft) -膜穴 （caveolae) 依赖型内吞通路的抑制剂。 其作用原理是： 脂筏- 膜穴依赖型内吞需要依靠细胞膜上的胆固醇（cholesterol)这一重要物质， NT可以通过与膜上的胆固醇结合从而抑制这条内吞通路。 与 NT作用原 理相似的试剂还有两性霉素 B ( amphotericin B )， 甲基化 β-环糊精 (methyl-p-cyclodextrin) 和菲律宾菌素 （filipin) 等， 都会与细胞膜上的 胆固醇结合抑制脂筏-膜穴内吞通路。 两性霉素 B ( amphotericin B, 以下 简称 AMB ) 与 NT作用机理相同， 是目前临床上治疗隐球菌、 曲霉菌等 真菌感染的一种有效的多烯类广谱抗真菌药物。
Wickstrom et al(2002， 2003 )报道了在内皮细胞膜上 ES与 alpha5betal 型整合素 （integrin alpha5betal ) 以及小窝蛋白 （caveolin, 是膜穴的组成 蛋白）有相互作用， 而且 ES会与脂筏发生相互作用， 揭示出在细胞膜上 ES与脂筏-膜穴的相互关联（Wickstrom et al, Cancer Res. 2002;62: 5580-9, Wickstrom et al, J. Biol. Chem. 2003;278(39): 37895-37901 )。另夕卜， Dixelius et al (2000)， Shi et al (2007 ) Zhuo et al (2010 ) 分别报道了 ES特异性 的被血管和淋巴管内皮细胞内吞的过程 （Dixelius et al, Blood 2000, 95, 3403-3411 ; Shi et al, Blood 2007, 110, 2899-2906, Zhuo W. et al, Journal of Pathology; 222:249-260
2002年， Pike et al和 Roepstorff et al分别发现抑制胆固醇和脂筏， 可以增加 EGFR的配体（表皮生长因子， 以下简称 "EGF")在细胞表面的 结合 （Pike et al, Biochemistry 2002;41 : 10315-22, Roepstorff et al, J Biol Chem 2002;277: 18954-60)。
Kojic et al (2008 )报道了肿瘤细胞通过脂筏-膜穴依赖型内吞来摄取 自分泌运动因子 /憐酸葡糖异构酶（AMF/PGI) ,且这一内吞过程具有细胞 种属特异性， 脂筏 -膜穴内吞可能成为针对特定肿瘤细胞靶向药物的给药 途径 （Kojic et al, PLoS ONE 2008;3： e3597)。 Migalovich et al (2009 ) 报 道了 NT通过调节细胞脂筏-膜穴内吞从而增加了卵巢癌细胞对黄豆苷原- 牛血清白蛋白 （daidzein-BSA) 的内吞。 黄豆苷原-牛血清白蛋白是一种 潜在的肿瘤显影剂， NT 增加黄豆苷原 -牛血清白蛋白在肿瘤细胞的内吞 可能会使这种肿瘤显影剂发挥出更好的实用效果 （Migalovich et al, Cancer Res. 2009;69： 5610-5617)。 由此可见， 脂筏-膜穴依赖型内吞可能 参与许多重要物质如细胞因子、 外源蛋白甚至药物分子的内吞， 通过调 节和控制此类内吞可能在细胞水平上影响机体对这些物质的摄取， 具有 应用潜力。 发明概述
本发明基于以下发现： ES 可以借助脂筏 -膜穴 （caveola/lipid raft pathway)和笼形蛋白包被小泡（clathrin-coated pits pathway)这两条途径 被内皮细胞内吞， 如果用抑制剂 NT干扰脂筏-膜穴依赖型内吞通路， 可 以获得提高药物在血管 /淋巴管内皮细胞中内吞摄取水平的意想不到的效 果， 并进一步促进 ES对内皮细胞活性、肿瘤血管生成和肿瘤生长的抑制 效果。 无意于受任何理论的限制， 推测这一发现的原因可能为在脂筏-膜 穴依赖型内吞通路受到抑制的情况下， 笼形蛋白包被小泡高效内吞途径 代偿性的活跃导致了总体内吞摄取的增强。
相似地， EGFR配体被报道具有与 ES相似的内吞机制。其中， EGFR 单抗是一类有效的抗肿瘤药物， 通过结合到肿瘤细胞特有的 EGFR起到 抑制相关信号传导和肿瘤增殖的疗效。 本发明人发现， NT还可以通过影 响脂筏依赖型内吞， 显著增强抗肿瘤药物 EGFR单克隆抗体在癌细胞中 的内吞和积累， 促进 EGFR单抗的疗效， 具有进一步应用于肿瘤治疗和 肿瘤显像等领域的潜力。此外，与 ES具有相似内吞机制的还有 endothelin receptor type A, cholera toxin, transforming growth factor- β receptors, B cell antigen receptor, bone morphogenetic protein receptor, HM1.24以及 integrins 等， 都具备与 NT联用产生增效作用的条件。
因此， 在一个方面， 本发明提供脂筏-膜穴依赖型内吞通路的调节剂 在制备用于提高对象中靶细胞对治疗剂的摄取的药物组合物中的用途。
在另一个方面， 本发明提供提高对象中靶细胞对治疗剂的摄取的方 法， 其包括给所述个体施用脂筏 -膜穴依赖型内吞通路的调节剂。
在另一个方面， 本发明提供脂筏 -膜穴依赖型内吞通路的调节剂， 其 用于提高对象中靶细胞对治疗剂的摄取。
在另一个方面， 本发明提供脂筏-膜穴依赖型内吞通路调节剂与某些 药物 (如抗肿瘤药物)组成的药物组合物。
在本发明的以上各方面的具体实施方式中， 所述脂筏-膜穴依赖型内 吞通路的调节剂是脂筏 -膜穴依赖型内吞通路的抑制剂， 例如所述抑制剂 是多烯类抗真菌药 (如制霉菌素或两性霉素 B), 或者所述抑制剂例如是甲 基化 β-环糊精或菲律宾菌素。
根据本发明， 所述治疗剂可以通过脂筏 -膜穴依赖型内吞通路和笼形 蛋白包被小泡内吞通路这两条通路被靶细胞摄取。
根据本发明， 所述对象患有血管发生相关性疾病或淋巴管发生相关 性疾病。
根据本发明， 所述对象患有肿瘤， 例如肺癌、 胰腺癌、 肝癌、 胃癌、 结直肠癌、 食道癌、 鼻咽癌、 恶性黑色素肿瘤、 骨癌、 淋巴癌、 乳腺癌、 宫颈癌、 前列腺癌、 血管瘤、 神经内分泌瘤、 口腔癌、 肉瘤、 肾癌或胆 癌。
根据本发明， 所述治疗剂是血管或淋巴管生成抑制剂， 例如血管内 皮抑制素或其衍生物， 其中所述血管内皮抑制素例如是天然血管内皮抑 制素或重组血管内皮抑制素， 例如重组人血管内皮抑制素。 根据本发明 的一个具体实施方式， 所述血管内皮抑制素具有 SEQ ID NO.1、 SEQ ID N0.2、 SEQ ID N0.3或SEQ ID N0.4所示的氨基酸序列。 根据本发明的 一个具体实施方式， 所述血管内皮抑制素衍生物是聚乙二醇 （PEG) 修 饰的血管内皮抑制素。 优选地， 所述聚乙二醇 （PEG) 是平均分子量为 5-40 kD的单甲氧基聚乙二醇， 例如是单甲氧基聚乙二醇丙醛。 在本发明 的一个具体实施方式中， 所述单甲氧基聚乙二醇丙醛的平均分子量是 20 kD, 且聚乙二醇（PEG) 修饰的位点是血管内皮抑制素的 N-末端的 α-氨 基。
根据本发明， 所述治疗剂是能够抑制肿瘤细胞生长的抗体。 根据本 发明的一个具体实施方式， 所述抗体是表皮生长因子受体（EGFR) 的抗 体， 例如 EGFR单克隆抗体， EGFR单克隆抗体的实例是西妥昔单抗。
在本发明的以上各方面的具体实施方式中， 所述脂筏-膜穴依赖型内 吞通路的调节剂通过选自静脉注射、 静脉滴注、 皮下注射、 肌肉注射、 腹腔注射、 皮下包埋、 透皮吸收、 肝动脉注射的胃肠外途径施用。 优选 地，所述脂筏 -膜穴依赖型内吞通路的调节剂被配制为脂质体包埋的形式。
本发明还提供一种药物组合物， 其包含前面所述的血管内皮抑制素 或者抗体， 以及前面所述的脂筏 -膜穴依赖型内吞通路的抑制剂。
在另一个方面， 本发明还涉及筛选对抗肿瘤药物具有增效作用的脂 筏 -膜穴依赖型内吞通路调节剂的方法。
本发明还涉及与 NT作用原理相似的药物或试剂 （如两性霉素 B), 和与 ES或 EGFR单抗作用机理或内吞途径相似的药物试剂之间的联合用 药方法、 联合制剂和药物组合物。 附图说明
图 1A-E: NT及其同类试剂 （两性霉素 B, 甲基化 β-环糊精和菲律 宾菌素）可以剂量依赖方式促进 ES及其衍生物和修饰物在内皮细胞中的 内吞摄取。
Α: 使用 Western Blotting 方法检测出 NT促进了 ES在血管内皮细 胞中的内吞， 且该促进作用与 NT 的浓度正相关。 这种促进作用体现在 ES内吞后在细胞质、 细胞核组分， 以及全细胞中的含量较没有 NT时显 著增加。
B: 使用 Western Blotting方法检测出 NT促进了 ES在淋巴管内皮 细胞中的内吞， 且该促进作用与 NT的浓度正相关。
C: 使用 Western Blotting 方法检测出两性霉素 B， 甲基化 β-环糊精 和菲律宾菌素也能够促进 ES在内皮细胞中的内吞， 且该促进作用与 NT 的浓度正相关。
D: 使用 Western Blotting方法检测出 NT也能够促进 PEG化 ES在 内皮细胞中的内吞， 且该促进作用与 NT的浓度正相关。
E: 使用 Western Blotting 方法检测出 NT也能够促进 N-末端带有附 加氨基酸 (M)GGSHHHHH的重组人血管内皮抑制素（Endu)及 N-末端被 PEG单点修饰的 Endu (PEG-Endu)在内皮细胞中的内吞， 且该促进作用 与 NT的浓度正相关。
图 2: 使用 Western Blotting方法检测出 ES与 NT的联合用药通过 增加 ES 在内皮细胞中的内吞， 增强了 ES 对于内皮细胞 ERK与 p38 MAPK信号通路的抑制效果。
图 3A-B: ES与 NT联合应用抑制细胞迁移 A: 细胞迁移实验证实， ES与 NT的联合用药增强了 ES对于内皮细 胞活性的抑制效果，使得发生迁移的内皮细胞数目较没有 NT时进一步减 少。
B: 细胞迁移实验中迁移细胞数目的统计结果， 反映了在不同药物处 理下内皮细胞的迁移能力。
图 4A-E: ES与 NT联合应用抑制肿瘤生长并改善 ES在肿瘤组织中 的分布
A: ES与 NT的联合用药在 A549肺癌动物模型中能够进一步增强 ES对于肿瘤生长的抑制效果。
B: 使用免疫荧光方法发现 ES与 NT的联合用药能够增强 ES对于 A549肺癌动物模型中肿瘤组织新生血管生成的抑制效果。
C: ES与 NT联合用药在 H22肝癌动物模型中能够进一步增强 ES 对于肿瘤生长的抑制效果。
D: 使用免疫荧光方法发现 ES与 NT的联合用药能够增强 ES对于 H22肝癌动物模型中肿瘤组织新生血管生成的抑制效果。
E:使用动物荧光成像系统发现 ES与 NT的联合用药能够增强 ES (带 有罗丹明荧光标记） 在荷瘤动物肿瘤组织中的摄取和分布。
图 5A-B: NT促进 EGFR单抗进入肿瘤细胞
A: 使用 Western Blotting 方法检测出 NT促进了 EGFR单抗在癌细 胞中的内吞。
B: 使用活体荧光标记示踪方法发现 NT与 EGFR单抗联合用药能够 加强 EGFR单抗在小鼠移植肿瘤中的分布和摄取。 发明详述
本发明的目的是提供多烯类广谱抗生素 NT及其同类药物的一种新 用途。 同时， 为 ES 和 EGFR 单抗等借助脂筏-膜穴依赖型内吞途径 ( caveola/lipid raft pathway ) 和笼形蛋白包被小泡内吞途径 (clathrin-coated pits pathway) 这两条途径发生内吞的蛋白药物提供一种 增强疗效的新药物组合物和新用药方法。
脂筏 （lipid raft ) 是质膜上富含胆固醇和鞘憐脂的微结构域 ( microdomain) 。 鞘憐脂和鞘糖脂的饱和脂肪链紧密聚集， 饱和脂肪 链之间的空隙填满了作为间隔分子的胆固醇， 形成液态有序相 ( liquid-ordered phase, 即脂筏)， 直径大约在 50nm左右。 脂筏是一种 动态结构， 与细胞膜的信号转导、 蛋白质分选均有密切关系。
膜穴 （也称细胞膜穴样内陷， 质膜微囊， caveolae ) 是脂筏的一种 结构， 具有和脂筏一样的膜脂组成， 不含笼形蛋白 （也称网格蛋白， clathrin) ， 含有 caveolin (—种小分子量蛋白， 21KD ) 。 大量存在于 脂肪细胞、 内皮细胞、 上皮细胞和平滑肌细胞等， 与内吞有关。 另外， 膜穴中还富含某些信号分子， 说明它与细胞的信号转导有关。
笼形蛋白包被小泡 （clathrin-coated pits) 是笼形蛋白参与介导的内 吞过程中形成的一种膜转运动态结构。 依赖于该种结构的内吞方式即称 为笼形蛋白包被小泡依赖型内吞途径。
血管发生相关性疾病 (Angiogenesis related diseases) 禾口淋巴管发生 相关性疾病 ( Lymphangiogenesis related diseases ) 即与血管禾口淋巴管异常 生成密切相关的疾病， 包括肿瘤， 关节炎和牛皮癣等多种自身免疫性疾 病， 糖尿病， 肥胖症以及多种眼科疾病。
本发明显示，将脂筏-膜穴依赖型内吞通路的抑制剂 NT与 ES共同加 入到血管内皮细胞的液体培养环境中，可以显著增强 ES在血管内皮细胞 中的内吞， 且这种增强效果与 NT的浓度正相关。 同时， 我们在淋巴管内 皮细胞中也观察到了同样的结果。
基于进一步实验事实，本发明还将 NT的增效作用扩展至与 NT作用 原理相似的其他药物和试剂。如实施例 1中所述， 与 NT作用原理相似的 一类物质， 如两性霉素 B (AMB)、 甲基化 β-环糊精 （Μβ-CD) 和菲律 宾菌素 （filipin) , 也能够增强 ES在内皮细胞中的内吞。 AMB与 filipin 也是已经成药的抗真菌抗生素，与 NT同样具有应用于实际联合用药的潜 力。 与此相类似的， 两性霉素 B ( amphotericin B )， 甲基化 β-环糊精 (methyl-P-cyclodextrin) 和菲律宾菌素 （filipin) 可以显著增强 ES在血 管内皮细胞中的内吞， 且这种增强效果与上述多稀类抗生素的浓度正相 关。
同样的， NT还可以显著促进 PEG修饰的 ES (PEG-ES), N-末端带 有 (M)GGSHHHHH附加氨基酸的 ES (Endu), 以及 N-末端被 PEG单点 修饰的 Endu (PEG-Endu)在血管内皮细胞中的内吞， 且这种增强效果与 NT的浓度正相关。
在分子细胞生物学水平上，本发明还通过实验证明了通过 NT联合用 药促进 ES在血管内皮细胞中的内吞， 并进一步增强 ES对血管内皮细胞 存活相关信号通路 （如 ERK和 p38 MAPK) 的抑制效果。
在细胞水平上， 本发明证明了 NT与 ES的联合用药增强了 ES对内 皮细胞迁移活性的抑制效果。 更进一步地， 在 A549肺癌和 H22肝癌的 动物肿瘤模型中， 证明了 NT能够增强 ES对于肿瘤生长以及肿瘤新生血 管生成的抑制效果。 更进一步的， NT还能够增强 ES在肿瘤组织中的摄 取和分布。
本发明还发现， EGFR单抗药物作为 EGFR的配体，其内吞的机制与 ES有一定的相似性， 它在肿瘤细胞中的内吞也能够被 NT所调节。 在分 子细胞生物学水平上，本发明通过细胞学实验证明了通过 NT联合用药促 进 EGFR单抗在癌细胞中的内吞。 更进一步地， 本发明还通过动物实验 证明了 NT与 EGFR单抗的联合用药促进了 EGFR单抗在小鼠移植肿瘤 中的分布和摄取。
因此， 本发明公开了 NT 这个现有的抗真菌抗生素药物的一种新用 途，即通过联合用药的方式促进了现有抗肿瘤药物 ES或 EGFR单抗的摄 入和药效， 具有进一步应用于肿瘤治疗和肿瘤显像等领域的潜力。
用高分子聚合物对蛋白进行修饰， 是改变和控制药物的动力学特性 如半衰期、免疫学特征、毒理特性的常用方法。其中聚乙二醇 (polyethylene glycol, PEG)是应用最为普遍的一种蛋白修饰分子。本实验证明， 经 PEG (单甲氧基聚乙二醇丙醛） 进行 N-末端 a-氨基定点修饰的 ES蛋白及其 衍生物 Endu在内皮细胞中的内吞也因 NT联合用药而显著增加。 这一事 实为 NT与修饰、 标记的 ES或其衍生物联合用药达到增效目的的用药方 法提供了理论依据。
基于另一部分实验事实， 本发明还将联合用药的适用范围扩展至其 他与 ES具有相似内吞途径和作用机理的药物，包括 EGFR单抗和经过修 饰、 标记的基于 EGFR单抗的分子等能够靶向肿瘤或肿瘤血管并用于治 疗或显像用途的物质，也可因为 NT等多烯类试剂的联合用药而促进药物 摄取和效果。
本发明还涉及一种 NT (或 AMB等） 与 ES联合用药的新剂型和药 物组合物。 ES应用于肿瘤治疗中的给药方式为静脉滴注。 NT属多烯类 广谱抗真菌抗生素， 难溶于水， 口服后胃肠道不易吸收， 直接注射用药 毒性较大。 不过， 有研究表明， NT可通过脂质体包埋方法实现静脉滴注 给药， 例如 Aronex公司的 NT脂质体剂型 Nyotran已于 1999年完成 III 期临床试验，有望近年上市，这将使 NT实现静脉滴注的给药方式。 AMB 与 NT同属于多烯类广谱抗真菌抗生素， 作用机理完全相同。 迄今为止， 已有 3种两性霉素 B脂质体剂型药物在欧美上市（商品名分别为 Abelcet、 Amphocil和 AmBisome )， 实现了这一类难溶性抗生素的静脉滴注给药。 另一种两性霉素 B脂质体静脉滴注液也于 2003年上市，商品名为锋克松。 因此， 结合脂质体剂型和静脉滴注给药来制备 ES或 EGFR单抗与 NT或 AMB等联合用药的新剂型或新的药物组合物是完全可行的。 实施例
实施例 1 : NT促进了 ES在血管及淋巴管内皮细胞中的内吞和摄取。
人血管内皮细胞（HMEC) (ATCC保藏号分别为 CRL 10636)， 应用 不完全弗氏佐剂诱导小鼠形成腹腔淋巴管瘤，消化法分离获得鼠淋巴管内 皮细胞 mLEC ( Zhuo W. et al, Zhuo W. et al, Journal of Pathology; 222:249-260) , Endu (SEQ ID NO. 3或 SEQ ID NO. 4， 即 N末端具有 9 个额外的氨基酸 (M)GGSHHHHH的 ES， 其中第一个氨基酸 M在大肠杆 菌中表达时可发生随机删除） （先声麦得津公司）， ES ( SEQ ID NO. 1或 SEQ ID NO. 2 , 其中第一个氨基酸 M在大肠杆菌中表达时可发生随机删 除） （Protgen) , 特异性修饰蛋白 N-末端 α-氨基的 PEG试剂采用 20 kD 的单甲氧基聚乙二醇丙醛 （mPEG-ALD ) (北京键凯科技有限公司）， PEG-ES及 PEG-Endu由 Protgen公司按照所用 PEG试剂说明书提示制备， ES单克隆抗体购自 Oncogene公司， NT及其他试剂均购自 Sigma Aldrich 公司。
待血管内皮细胞贴壁生长至密度约 90%时，更换为含有 NT的 DMEM 培养基（Hyclone ) :将 NT储液加入到培养基中，至一定终浓度（0 g/ml， 25 g/ml， 50 g/ml)， 于 37。C， 5% C02 培养箱中静置 20 min进行 NT 预处理； 预处理之后将浓度为 5 mg/ml 的 ES储液加入培养基， 至终浓度 5 μ^πύ , 于 37。C， 5% C02 培养箱中静置 30 min使 ES被血管内皮细胞 内吞。 内吞结束时弃培养基， 用冰 PBS 洗 3次， 收集内皮细胞。 使用 Western Blotting方法， 检测全细胞裂解液、 细胞质及细胞核等组分中 ES 的摄入水平， 与没有 NT共同处理的细胞中 ES内吞量进行比较。 结果表 明， 在 ES剂量相同、 内吞时间相同的情况下， 全细胞裂解液、 细胞质或 细胞核组分中 ES的含量均因 NT的联合使用而显著增加， 25 μ8/ηι1及 50 g/ml的 NT分别可使 ES内吞摄取量提高约 2倍和 15倍， 且 ES内吞增 加的幅度与所用 NT的浓度正相关 （图 1A)。
用分别含有 NT浓度为 0， 75， 150和 300 g/ml的培养基于 37°C对 mLEC细胞进行预处理 30min， 然后在 37°C条件下用含有 ES浓度为 2 g/ml 的培养液孵育 15 min, 用 PBS清洗三遍， 收细胞。 利用 Western blotting对不同浓度 NT处理的淋巴管内皮细胞进行 ES内吞测定，结果显 示 75， 150和 300 g/ml的 NT可分别使 ES在淋巴管内皮细胞中的内吞 量提高约 16， 32和 78倍， 且 ES内吞增加的幅度与所用 NT的浓度正相 关 （图 1B)。
在本实施例中， NT还可以被替换为与 NT作用机理相同的药物， 如 两性霉素 B (25 - 50 g/ml)， 甲基化 β-环糊精（1 - 2 mM )和菲律宾菌素 (2.5 - 5 μ8/ηι1), 都能够促进 ES在内皮细胞中的内吞 （图 1C)。
在本实施例中， ES还可以被替换为与 ES内吞机理相同的药物， 如 PEG化修饰的 ES。 使用分子量为 20 kDa的特异性修饰蛋白 N-末端的聚 乙二醇修饰试剂（单甲氧基聚乙二醇并醛， mPEG-ALD)来修饰 ES, 其产 物 （简称 PEG-ES) 为一个聚乙二醇分子与一个重组人内皮抑制素相连， 连接位点为蛋白的 N-末端 a-氨基的蛋白。 结果表明， 在 PEG-ES剂量相 同、 内吞时间相同的情况下， 血管内皮细胞对于 PEG-ES 的内吞也由于 NT的联合使用而显著增加， 而且 PEG-ES内吞增加的幅度与所用 NT的 浓度正相关 （图 1D)。
在另一种 ES的衍生物 Endu的内吞实验中也观察到了类似的结果， 即在 Endu及其 N-末端 α-氨基单一定点 PEG修饰产物 PEG-Endu剂量相 同、 内吞时间相同的情况下， 血管内皮细胞对于 Endu及 PEG-Endu的内 吞也由于 NT的联合使用而显著增加， 而且 Endu及 PEG-Endu内吞增加 的幅度与所用 NT的浓度正相关 （图 1E)。 实施例 2: NT增加了 ES在内皮细胞中的内吞， 从而增强了 ES对于内皮 细胞信号通路的抑制作用。
Kim et al (2002) 发现 ES在内皮细胞中可以抑制内皮细胞内细胞外 调节蛋白激酶（extracellular regulated protein kinases,简称 ERK)、 p38 促 分裂素原活化蛋白激酶 （ p38 mitogen-activated protein kinases, 简称 p38 MAPK)和 pl25 局部粘着斑激酶（pl25 focal adhesion kinase, 简称 pl25 FAK)介导的信号转导通路，从而发挥抑制内皮细胞的活性（Kim et al , J. Biol. Chem. 2002, 277, 27872-27879)。 本实施例选用 ERK和 p38 MAPK 这两条信号通路作为检测指标， NT的联合使用进一步增强了 ES对于这 些信号通路的抑制作用。
本实施例中， 内皮细胞被分成四组加以不同处理。 第一组： 阴性对 照组，无 NT处理，也无 ES处理；第二组， NT处理组，只有 NT(50 g/ml， 20 min) 处理， 无 ES处理； 第三组， ES处理组， 无 NT处理， 只有 ES (5 μ^ηή, 30 min)处理； 第四组， NT与 ES联合用药组， NT (50 μ^ηή, 先于 ES 20 min力口入） 和 ES (5 g/ml， 30 min) 联合处理。 按实施例 1 中的方法对细胞进行 NT或 ES相应处理， 收集细胞， 检测 ERK和 p38 MAPK信号通路。 结果显示， 与对照组相比， 第二组仅用 NT处理不会 影响 ERK和 p38MAPK信号的水平；第三组 ES处理抑制了 ERK和 p38 MAPK信号通路， 与 Kimetal 的报道相符； 第四组 NT和 ES联合用药 增加了 ES的内吞， 并进一步增强了 ES对 ERK和 p38 MAPK信号通路 的抑制作用。这证明了 NT的联合用药进一步增强了 ES对细胞活化信号 通路的抑制作用 （图 2)。 实施例 3: NT增强了 ES对于内皮细胞迁移的抑制效果。
细胞迁移的测定方法： 内皮细胞 （HMEC, 每孔 2xl04个细胞） 接 种到 TranswellTM板 （8 μηι孔径， Costar) 的上层含 0.5 %胎牛血清
(Hyclone)以及 10ng/mlVEGF (PeproTech EC)的 DMEM (Hyclone) 培养基中。 同时在板的上层和下层加入一定浓度的 ES (40 μ8/ηι1)或 NT
(50 g/ml)， 在 37°C 和 5% C02 中继续培养 6小时使细胞迁移。 用 戊二醛固定和结晶紫染色后， 每个孔随机选取 5个高倍放大（400倍）视 野 （Olympus 1X71)， 计算其中完全穿过膜迁移到板下层的细胞数并取平 均值， 每一组平行三个孔， 实验重复两次。
本实施例中， 内皮细胞也被分成四组加以不同处理。 第一组： 阴性 对照组， 无 NT处理， 也无 ES处理； 第二组， NT处理组， 只有 NT (50 g/ml) 处理， 无 ES处理； 第三组， ES处理组， 无 NT处理， 只有 ES
(40 g/ml) 处理； 第四组， NT与 ES联合用药组， NT (50 g/ml) 和 ES (40 μ8/ηι1) 联合处理。 结果显示， 与对照组相比较， 第二组仅用 NT 处理， 基本不影响内皮细胞的迁移能力； 第三组 ES处理能抑制内皮细胞 迁移， 减少了内皮细胞迁移到下层膜的数量， 抑制率为 61%; 第四组 NT 和 ES联合用药， 增强了 ES对内皮细胞迁移的抑制作用， 发生迁移的细 胞很少， 抑制率为 87% (相对于 ES组结果， P<0.001)。 上述结果说明， NT增强了 ES对于内皮细胞迁移的抑制作用。 各处理组迁移细胞代表性 视野 （图 3A), 各处理组迁移细胞计数平均值比较 （图 3B)。 实施例 4: NT能够在动物模型中增强 ES对于肿瘤生长和新生血管生成 的抑制作用， 并促进 ES在肿瘤组织中的摄取和分布。
培养处于旺盛增殖状态的人源肺腺癌 A549 细胞 （ATCC保藏号为 CCL-185 ) 接种至 6 ~ 8周龄的裸鼠 （北京维通利华实验动物技术有限公 司） 皮下。 当肿瘤体积达到 100 mm3左右时， 将荷瘤裸鼠分为四组进行 不同的给药处理。 第一组： 阴性对照， 生理盐水处理组； 第二组， NT给 药组， 只有 NT (6 mg/kg, 腹腔注射， 每天一次） 给药处理， 无 ES给 药处理； 第三组， ES给药组， 无 NT给药处理， 只有 ES ( 12 mg/kg, 腹 腔注射， 每天一次） 给药处理； 第四组， NT与 ES联合用药组， NT (6 mg/kg, 腹腔注射， 每天一次） 和 ES ( 12 mg/kg, 腹腔注射， 每天一次） 联合给药。 给药两周后， 裸鼠被处死， 称肿瘤并比较肿瘤体积。 结果显 示，与对照组相比较，第二组仅用 NT给药处理对肿瘤生长没有抑制作用； 第三组 ES处理能抑制肿瘤生长， 抑瘤率约 40 %； 第四组 NT和 ES联合 用药， 增强了 ES对肿瘤生长的抑制作用， 抑瘤率提高到 60 %左右。 这 说明在动物模型中， NT能够显著增强 ES的抑瘤活性 （图 4A)。 各组实 验动物的体重、 进食和日常活动均未发生异常改变。
肿瘤内血管发生水平的检测方法： 所有四组的裸鼠的肿瘤被取出、 固定、 切片。 使用抗 CD31 (肿瘤血管内皮细胞的标志分子） 的一抗以及 FITC标记的二抗 （Santa Cruz) 检测， 在 Nikon A1激光扫描共聚焦成像 系统下观察 （Nikon Inc. )。 各组肿瘤随机选取高倍放大 （400倍） 视野， 使用显微镜系统自带软件计算各个视野中血管截面积并取平均值进行比 较。 NT增强 ES抑制肿瘤组织新生血管生成活性的作用与其增强 ES抑 瘤活性的作用相类似。 各处理组肿瘤平均重量比较 （图 4A), 各处理组 肿瘤组织平均血管面积 （相对单位， arbitrary unit) 比较 （图 4B)
NT 显著增强 ES 抑制肿瘤生长和肿瘤新生血管生成活性的作用在 H22 肝癌的动物模型中也得到了验证。 各处理组肿瘤平均重量比较 （图 4C ) ， 各处理组肿瘤组织平均血管面积 （相对单位， arbitrary unit) 比较 (图 4D ) 各组实验动物的体重、 进食和日常活动均未发生异常改变。
荧光标记 ES在荷瘤小鼠的肿瘤组织中摄取和分布的检测方法：培养 处于增殖状态的人源肺腺癌 A549细胞接种至 6 〜 8周龄的裸鼠 （北京维 通利华实验动物技术有限公司） 皮下。 当肿瘤体积达到 300 mm3 左右时 给药。 荷瘤裸鼠分为两组进行不同的给药处理（每组四只）。 第一组： ES 给药组，无 NT给药处理，只有罗丹明（ Pierce )荧光标记 ES(剂量 20 mg/kg ) 腹腔注射给药； 第二组， NT与 ES联合用药组， NT ( 6 mg/kg, 腹腔注 射） 和罗丹明荧光标记 ES (20 mg/kg, 腹腔注射） 联合给药。 给药 4小 时后， 取出肿瘤组织， 利用动物荧光成像系统 （清华大学生物医学工程 系）对肿瘤中荧光标记 ES的摄取量进行显像与定量分析。 NT增强了 ES 在动物肿瘤组织中的摄取和分布， 增幅达到 4倍 （图 4E)。 实施例 5 : NT能够显著增加 EGFR单抗 （西妥昔单抗） 在肿瘤细胞中的 内吞和在动物肿瘤组织中的摄取和分布。
本实施例中选用表达 EGFR的人源肺腺癌 A549细胞，待其贴壁生长 至密度约 90%时， 更换为含有 NT的 DMEM培养基： 将 NT储液加入到 培养基中， 至一定终浓度 （0 g/ml， 25 μ^ιηΐ , 50 g/ml)， 于 37°C， 5% C02 培养箱中静置 20 min进行 NT预处理；预处理之后将浓度为 5 mg/ml 的 EGFR单抗（西妥昔单抗， Merck公司）储液加入培养基， 至终浓度 5 g/ml，于 37°C， 5% C02 培养箱中静置 30 min使 EGFR单抗被内皮细胞 内吞； 内吞结束时弃培养基， 用冰 PBS 洗 3 次， 收集内皮细胞。 使用 Western Blotting方法， 检测细胞组分中 EGFR单抗的摄入水平， 与没有 NT共同处理的细胞中 EGFR单抗内吞量进行比较。 结果表明， 在 EGFR 单抗剂量相同、 内吞时间相同的情况下， 细胞中 EGFR单抗的内吞量因 NT的联合使用而显著增加， 即 NT能够促进内皮细胞中对 EGFR单抗的 内吞， 而且 EGFR单抗内吞量增加的幅度与所用 NT的浓度正相关 （图 5A
培养处于增殖状态的人源肺腺癌 A549细胞接种至 6 〜 8周龄的裸鼠 (北京维通利华实验动物技术有限公司） 皮下， 分两组， 每组 4只。 当 肿瘤体积达到 500 mm3 左右时， 一组小鼠给以荧光（Cy5.5, GE公司）标 记的 EGFR单抗， 另一组小鼠给以荧光标记的 EGFR单抗 （与前一组剂 量相等）加上 NT的联合用药。使用荧光成像系统（清华大学生物医学工 程系） 对这两组取出的小鼠肿瘤中 EGFR单抗的荧光信号进行检测， 证 明 NT可增强 EGFR单抗在肿瘤组织中的摄取和分布， 有促进药物摄取、 提高疗效和用于肿瘤成像研究的作用 （图 5B)。

Claims

权 利 要 求 书
1、脂筏 -膜穴依赖型内吞通路的调节剂在制备用于提高对象中靶细胞 对治疗剂的摄取的药物组合物中的用途。
2、权利要求 1的用途，其中所述脂筏-膜穴依赖型内吞通路的调节剂 是脂筏 -膜穴依赖型内吞通路的抑制剂。
3、权利要求 2的用途，其中所述脂筏-膜穴依赖型内吞通路的抑制剂 是多烯类抗真菌药。
4、权利要求 3的用途，其中所述脂筏-膜穴依赖型内吞通路的抑制剂 选自制霉菌素和两性霉素 B。
5、权利要求 2的用途，其中所述脂筏-膜穴依赖型内吞通路的抑制剂 是甲基化 β-环糊精或菲律宾菌素。
6、 权利要求 1-5 中任一项的用途， 其中所述治疗剂可以通过脂筏- 膜穴依赖型内吞通路和笼形蛋白包被小泡内吞通路这两条通路被靶细胞 摄取。
7、 权利要求 1-6中任一项的用途， 其中所述对象患有血管发生相关 性疾病或淋巴管发生相关性疾病。
8、 权利要求 1-6中任一项的用途， 其中所述对象患有肿瘤。
9、 权利要求 7或 8的用途， 其中所述治疗剂是血管或淋巴管生成抑 制剂。
10、 权利要求 9 的用途， 其中所述血管或淋巴管生成抑制剂是选自 天然血管内皮抑制素和重组人血管内皮抑制素的一种血管内皮抑制素或 其衍生物。
11、权利要求 10的用途，其中所述血管内皮抑制素具有 SEQ ID N0.1 或 SEQ ID N0.2所示的氨基酸序列。
12、权利要求 10的用途，其中所述血管内皮抑制素是 N-末端带有附 加氨基酸序列 (M)GGSHHHHH的血管内皮抑制素， 具有 SEQ ID N0.3或 SEQ ID N0.4所示的氨基酸序列。
13、权利要求 10-12中任一项的用途，其中所述血管内皮抑制素衍生 物是聚乙二醇 （PEG) 修饰的血管内皮抑制素。
14、 权利要求 13的用途， 其中所述聚乙二醇 （PEG) 是平均分子量 为 5-40 kD的单甲氧基聚乙二醇。
15、 权利要求 14的用途， 其中所述单甲氧基聚乙二醇是单甲氧基聚 乙二醇丙醛。
16、 权利要求 15的用途， 其中所述单甲氧基聚乙二醇丙醛的平均分 子量是 20 kD。
17、 权利要求 13-16中任一项所述的用途， 其中聚乙二醇（PEG)修 饰的位点是血管内皮抑制素的 N-末端的 a-氨基。
18、 权利要求 8 的用途， 其中所述治疗剂是能够抑制肿瘤细胞生长 的抗体。
19、权利要求 18的用途，其中所述抗体是表皮生长因子受体（EGFR) 的抗体。
20、权利要求 18的用途，其中所述抗体是表皮生长因子受体（EGFR) 的单克隆抗体。
21、 权利要求 18 的用途， 其中所述表皮生长因子受体 （EGFR) 的 单克隆抗体是西妥昔单抗。
22、 权利要求 8-21中任一项的用途， 其中所述肿瘤选自肺癌、 胰腺 癌、 肝癌、 胃癌、 结直肠癌、 食道癌、 鼻咽癌、 恶性黑色素肿瘤、 骨癌、 淋巴癌、 乳腺癌、 宫颈癌、 前列腺癌、 血管瘤、 神经内分泌瘤、 口腔癌、 肉瘤、 肾癌和胆癌。
23、权利要求 1-22中任一项的用途， 其中所述脂筏-膜穴依赖型内吞 通路的调节剂通过选自静脉注射、 静脉滴注、 皮下注射、 肌肉注射、 腹 腔注射、 皮下包埋、 透皮吸收、 肝动脉注射的胃肠外途径施用。
24、权利要求 23的用途，其中所述脂筏-膜穴依赖型内吞通路的调节 剂被配制为脂质体包埋的形式。
25、 一种药物组合物， 其包含 O 2012/130141
(a) 权利要求 10-17中任一项所定义的血管内皮抑制素或者权利要求
18-21中任一项所定义的抗体， 和
(b) 权利要求 2-5 中任一项所定义的脂筏 -膜穴依赖型内吞通路的抑 制剂。
PCT/CN2012/073202 2011-03-28 2012-03-28 一种增强靶细胞摄取治疗剂的方法和药物组合物 WO2012130141A1 (zh)

Priority Applications (5)

Application Number Priority Date Filing Date Title
CA2837122A CA2837122C (en) 2011-03-28 2012-03-28 Methods and compositions for enhancing the uptake of therapeutic agents by target cells
EP12765089.3A EP2702997B1 (en) 2011-03-28 2012-03-28 Method and composition for enhancing target cells uptake of therapeutic agents
AU2012237786A AU2012237786B2 (en) 2011-03-28 2012-03-28 Method and composition for enhancing target cells uptake of therapeutic agents
US14/008,428 US9364493B2 (en) 2011-03-28 2012-03-28 Methods and compositions for enhancing the uptake of therapeutic agents by target cells
JP2014501425A JP6114940B2 (ja) 2011-03-28 2012-03-28 標的細胞による治療薬の取り込みを高めるための方法及び組成物

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201110085338.8A CN102698270B (zh) 2011-03-28 2011-03-28 一种增强靶细胞摄取治疗剂的方法和药物组合物
CN201110085338.8 2011-03-28

Publications (1)

Publication Number Publication Date
WO2012130141A1 true WO2012130141A1 (zh) 2012-10-04

Family

ID=46891512

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2012/073202 WO2012130141A1 (zh) 2011-03-28 2012-03-28 一种增强靶细胞摄取治疗剂的方法和药物组合物

Country Status (7)

Country Link
US (1) US9364493B2 (zh)
EP (1) EP2702997B1 (zh)
JP (1) JP6114940B2 (zh)
CN (1) CN102698270B (zh)
AU (1) AU2012237786B2 (zh)
CA (1) CA2837122C (zh)
WO (1) WO2012130141A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2015342324B2 (en) * 2014-11-03 2021-08-19 Beijing Protgen Ltd. Drug for inhibiting adipose cell differentiation and insulin resistance

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105646667A (zh) * 2016-04-06 2016-06-08 南京安吉生物科技有限公司 聚乙二醇修饰的血管生成抑制剂hm-1及其应用
AU2018310410A1 (en) 2017-07-30 2020-03-12 Beijing Protgen Ltd. Medication for inhibiting DNA-PKcs

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4812312A (en) * 1987-03-03 1989-03-14 Board Of Regents Of The University Of Texas System Liposome-incorporated nystatin
EP1743031A4 (en) * 2004-04-26 2008-05-28 Childrens Medical Center BLOOD PLATE BIOMARKERS FOR THE DETECTION OF ILLNESSES
JP2006249017A (ja) * 2005-03-11 2006-09-21 Chiba Univ 軸索再生促進剤
CN1891717A (zh) * 2005-07-08 2007-01-10 南京大学 内皮抑素的化学修饰方法及其应用
CN100475270C (zh) * 2006-01-20 2009-04-08 清华大学 一种治疗肿瘤的药物及其应用
WO2008144616A1 (en) * 2007-05-18 2008-11-27 Heidi Kay Lipid raft, caveolin protein, and caveolar function modulation compounds and associated synthetic and therapeutic methods
DE102008043724A1 (de) * 2008-11-13 2010-05-20 Biotronik Vi Patent Ag Erhöhung der Effizienz pharmazeutische Wirkstoffe-freisetzender Medizinprodukte durch Kombination mit einem Inhibitor des Transportproteins P-Glycoprotein
WO2010141097A2 (en) * 2009-06-05 2010-12-09 The Trustees Of Columbia Univerity In The City Of New York Pegylated human apoa-1 and process for production thereof
WO2013142184A1 (en) * 2012-03-19 2013-09-26 Yale University Antimicrobial compositions and methods

Non-Patent Citations (16)

* Cited by examiner, † Cited by third party
Title
CARMELIET P., NATURE MEDICINE, vol. 9, no. 6, 2003, pages 653 - 660
DIXELIUS ET AL., BLOOD, vol. 95, 2000, pages 3403 - 3411
FOLKMAN J. ET AL., CELL, vol. 88, 1997, pages 277 - 285
FOLKMAN J. ET AL., NATURE, vol. 390, 1997, pages 404 - 407
FOLKMAN, J. N ENGL J MED, vol. 285, 1971, pages 1182 - 1186
KIM ET AL., J.BIOL. CHEM., vol. 277, 2002, pages 27872 - 27879
KOJIC ET AL., PLOS ONE, vol. 3, 2008, pages E3597
MIGALOVICH ET AL., CANCER RES., vol. 69, 2009, pages 5610 - 5617
MIGALOVICH ET AL.: "Harnessing Competing Endocytic Pathways For Overcoming the Tumor-Blood Barrier:Magnetic Resonance Imaging And Near-Infrared Imaging of Bifunctional Contrast Media", CANCER RESEARCH, no. 69, 2009, pages 5610 - 5617, XP002586648 *
PIKE ET AL., BIOCHEMISTRY, vol. 41, 2002, pages 10315 - 22
ROEPSTORFF ET AL., J BIOL CHEM, vol. 277, 2002, pages 18954 - 60
SHI ET AL., BLOOD, vol. 110, 2007, pages 2899 - 2906
WICKSTROM ET AL., CANCER RES., vol. 62, 2002, pages 5580 - 9
WICKSTROM ET AL., J. BIOL. CHEM., vol. 278, no. 39, 2003, pages 37895 - 37901
WITTE MH ET AL., LYMPHOLOGY, vol. 20, no. 4, 1987, pages 257 - 66
ZHUO W. ET AL., JOURNAL OF PATHOLOGY, vol. 222, pages 249 - 260

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2015342324B2 (en) * 2014-11-03 2021-08-19 Beijing Protgen Ltd. Drug for inhibiting adipose cell differentiation and insulin resistance

Also Published As

Publication number Publication date
AU2012237786A1 (en) 2013-11-14
US20140335152A2 (en) 2014-11-13
AU2012237786B2 (en) 2015-10-15
EP2702997B1 (en) 2019-03-13
CA2837122C (en) 2018-01-02
CN102698270B (zh) 2016-02-03
US9364493B2 (en) 2016-06-14
JP6114940B2 (ja) 2017-04-19
EP2702997A1 (en) 2014-03-05
CN102698270A (zh) 2012-10-03
US20140147492A1 (en) 2014-05-29
EP2702997A4 (en) 2014-12-31
JP2014510737A (ja) 2014-05-01
CA2837122A1 (en) 2012-10-04

Similar Documents

Publication Publication Date Title
Liu et al. Secreted protein acidic and rich in cysteine mediated biomimetic delivery of methotrexate by albumin-based nanomedicines for rheumatoid arthritis therapy
Han et al. Tumor microenvironment remodeling and tumor therapy based on M2-like tumor associated macrophage-targeting nano-complexes
Guo et al. Liposomal nanoparticles carrying anti-IL6R antibody to the tumour microenvironment inhibit metastasis in two molecular subtypes of breast cancer mouse models
Hou et al. Nano-delivery of fraxinellone remodels tumor microenvironment and facilitates therapeutic vaccination in desmoplastic melanoma
Guo et al. Inhibiting metastatic breast cancer cell migration via the synergy of targeted, pH-triggered siRNA delivery and chemokine axis blockade
Wang et al. Galectin-3 expression and secretion by tumor-associated macrophages in hypoxia promotes breast cancer progression
Ju et al. Octreotide-modified liposomes containing daunorubicin and dihydroartemisinin for treatment of invasive breast cancer
He et al. Folate-linked lipoplexes for short hairpin RNA targeting claudin-3 delivery in ovarian cancer xenografts
KR20140041770A (ko) 아포토시스 유도제
Liu et al. Dual-functional nanoparticles targeting CXCR4 and delivering antiangiogenic siRNA ameliorate liver fibrosis
Hu et al. Strategies targeting tumor immune and stromal microenvironment and their clinical relevance
Salimifard et al. Codelivery of BV6 and anti-IL6 siRNA by hyaluronate-conjugated PEG-chitosan-lactate nanoparticles inhibits tumor progression
Yi et al. Sequentially targeting cancer‐associated fibroblast and mitochondria alleviates tumor hypoxia and inhibits cancer metastasis by preventing “soil” formation and “seed” dissemination
Guo et al. A CFH peptide-decorated liposomal oxymatrine inactivates cancer-associated fibroblasts of hepatocellular carcinoma through epithelial–mesenchymal transition reversion
Li et al. Liposomal Co-delivery of PD-L1 siRNA/Anemoside B4 for enhanced combinational immunotherapeutic effect
WO2012130141A1 (zh) 一种增强靶细胞摄取治疗剂的方法和药物组合物
Luo et al. Preventing acute lung injury from progressing to pulmonary fibrosis by maintaining ERS homeostasis through a multistage targeting nanomicelle
AU2013295242B2 (en) CD147 as receptor for pilus-mediated adhesion of meningococci to vascular endothelia
Jia et al. Tumor-derived PD-L1+ exosomes with natural inflammation tropism for psoriasis-targeted treatment
Lee et al. Targeted antivascular therapy with the apolipoprotein (a) kringle V, rhLK8, inhibits the growth and metastasis of human prostate cancer in an orthotopic nude mouse model
Jing et al. Enhanced antitumor efficacy using epirubicin and schisandrin B co-delivery liposomes modified with PFV via inhibiting tumor metastasis
Zhao et al. Oridonin inhibits inflammation of epithelial cells via dual-targeting of CD31 Keap1 to ameliorate acute lung injury
KR102191695B1 (ko) 전이 억제제
Li et al. cRGD peptide-modified nanocarriers for targeted delivery of angiogenesis inhibitors to attenuate advanced atherosclerosis
Li et al. Long-Circulating Liposomal Delivery System Targeting at PDGFR-β Enhances the Therapeutic Effect of IFN-α on Hepatic Fibrosis

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12765089

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2014501425

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2012765089

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2012237786

Country of ref document: AU

Date of ref document: 20120328

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2837122

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 14008428

Country of ref document: US