WO2020242217A1 - Liposome comprising glutathione-s-transferase and protein having capacity to bind to target cell or target protein, and use thereof - Google Patents

Liposome comprising glutathione-s-transferase and protein having capacity to bind to target cell or target protein, and use thereof Download PDF

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WO2020242217A1
WO2020242217A1 PCT/KR2020/006905 KR2020006905W WO2020242217A1 WO 2020242217 A1 WO2020242217 A1 WO 2020242217A1 KR 2020006905 W KR2020006905 W KR 2020006905W WO 2020242217 A1 WO2020242217 A1 WO 2020242217A1
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protein
liposome
glutathione
transferase
target
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PCT/KR2020/006905
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French (fr)
Korean (ko)
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김채규
유자형
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울산과학기술원
주식회사 퓨전바이오텍
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Publication of WO2020242217A1 publication Critical patent/WO2020242217A1/en

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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
    • A61K47/69—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
    • A61K47/6905—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 colloid or an emulsion
    • A61K47/6911—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 colloid or an emulsion the form being a liposome
    • 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
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • 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
    • A61K47/62—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
    • 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
    • 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
    • A61K47/6905—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 colloid or an emulsion
    • A61K47/6911—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 colloid or an emulsion the form being a liposome
    • A61K47/6913—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 colloid or an emulsion the form being a liposome the liposome being modified on its surface by an antibody
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C12N9/1088—Glutathione transferase (2.5.1.18)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
    • C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
    • C12Y205/01018—Glutathione transferase (2.5.1.18)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • It relates to a liposome comprising a protein having glutathione-S-transferase and a target cell or a protein having a target protein binding ability, and its use as a drug delivery system and pharmaceutical composition.
  • Nanotechnology is capable of diagnosing and treating new concepts that were not possible with existing technologies, showing the infinite possibilities of nanotechnology.
  • DDS Drug Delivery System
  • a nanotechnology-based drug delivery system (DDS, Drug Delivery System) is widely applied in the field of disease diagnosis and treatment.
  • DDS Drug Delivery System
  • These nanoparticle-based drug delivery systems have been proven through animal experiments to minimize side effects of drugs and to increase therapeutic efficacy by delivering drugs more effectively.
  • the nanoparticle-based drug delivery system can maximize the therapeutic effect by optimizing the in vivo kinetics and distribution of drugs by first, controlling the size and physical properties of the nanoparticles, and secondly, the broad spectrum due to the non-selective biodistribution of existing anticancer drugs. It has the advantage of reducing toxicity and side effects, drug resistance, and facilitating combination therapy.
  • Nanopharmaceutical delivery system-based products that have been approved by the US FDA to date are based on highly biocompatible nanoparticles such as Doxyl® (nanoliposomes containing doxorubicin) and Abraxane® (albumin nanoparticles containing paclitaxel). Since it does not have the function of selectively targeting and delivering anticancer drugs, it is evaluated that its efficacy is limited because it is simply passive delivery by EPR effect. Passive targeting depending on the EPR effect can show excellent efficacy in animal cancer models, but in actual human cancers, it is rare that EPR effects are relatively rare, so treatment efficacy is limited. In particular, when there are few new blood vessels such as prostate cancer or pancreatic cancer tissue, there is a disadvantage that it is difficult to expect effective drug delivery according to the EPR effect.
  • GST glutathione-S-transferase
  • a protein having a target cell or target protein binding ability A linker connecting the glutathione-S-transferase and a protein having binding ability to a target cell or a target protein; And it is to provide a drug delivery system containing the glutathione-S-transferase and a liposome carrying a drug are bound.
  • GST glutathione-S-transferase
  • a protein having a target cell or target protein binding ability A linker connecting the glutathione-S-transferase and a protein having the ability to bind to a target cell or a target protein;
  • GST glutathione-S-transferase
  • a linker connecting the glutathione-S-transferase and a protein having the ability to bind to a target cell or a target protein and it is to provide a pharmaceutical composition for preventing or treating cancer comprising the glutathione-S-transferase and a liposome carrying an anticancer agent are bound.
  • GST glutathione-S-transferase
  • a protein having a target cell or target protein binding ability A linker connecting the glutathione-S-transferase and a protein having the ability to bind to a target cell or a target protein; And administering a composition containing the glutathione-S-transferase and a liposome carrying the drug to an individual in need thereof.
  • GST glutathione-S-transferase
  • a protein having a target cell or target protein binding ability A linker connecting the glutathione-S-transferase and a protein having the ability to bind to a target cell or a target protein;
  • GST glutathione-S-transferase
  • a linker connecting the glutathione-S-transferase and a protein having the ability to bind to a target cell or a target protein and it is to provide a method for preventing or treating cancer comprising administering a composition containing a liposome carrying the glutathione-S-transferase and an anticancer agent to an individual in need thereof.
  • GST glutathione-S-transferase
  • a protein having a target cell or target protein binding ability A linker connecting the glutathione-S-transferase and a protein having the ability to bind to a target cell or a target protein; And it is to provide a drug delivery system containing the glutathione-S-transferase and a liposome carrying a drug are bound.
  • a protein having the ability to bind to a target cell or a target protein refers to a receptor or a target protein of a cell. It may mean a protein that is specifically recognized or specifically binds to a receptor or a target protein of a cell.
  • the protein that specifically binds to the receptor or target protein of the cell is any one selected from the group consisting of an antibody, an antigen-binding fragment, an affibody, a diabody, and an aptamer. Can be.
  • the term "affibody molecule” may refer to an antibody mimic capable of binding to a specific target protein (receptor).
  • the Affibody molecule is composed of 20 to 150 amino acid residues, and may be composed of 2 to 10 alpha helixes.
  • the affibody molecule may include an anti-ErbB affibody molecule (ab31889), a HER2-specific affibody molecule (ZHER2:342), an anti-EGFR affibody molecule (ZEGFR:2377), and the like.
  • the present invention is not limited thereto, and includes all affibody molecules capable of recognizing a specific receptor or target protein of a cell.
  • target receptors or target proteins examples include amyloid beta peptide, synuclein (e.g., alpha-synuclein), apolipoprotein (e.g., apolipoprotein A1), complement Complement factor (e.g., C5), carbonic anhydrase (e.g. CAIX), interleukin-2 receptor alpha chain (IL2RA; CD25), CD antigen on the cell surface (e.g.
  • the target cell or protein having the ability to bind to the target protein may specifically bind to receptor tyrosine kinases (RTKs). More specifically, the receptor tyrosine kinase is an epidermal growth factor receptor, insulin receptor, platelet-derived growth factor receptor, vascular endothelial growth factor receptor, fibroblast growth factor receptor, cholecystokinin (CCK) receptor, neurotrophic factor (NGF). ) Receptor, hepatocyte growth factor (HGF) receptor, ephrin (Eph) receptor, angiopoietin receptor, and related to receptor tyrosine kinase (RYK) receptor. .
  • RTKs receptor tyrosine kinases
  • the GST and the target cell or a protein having a target protein binding ability may be linked through a linker.
  • the linker may be a polypeptide consisting of 1 to 400, 1 to 200, or 2 to 200 arbitrary amino acids.
  • the peptide linker may include Gly, Asn and Ser residues, and neutral amino acids such as Thr and Ala may also be included.
  • Amino acid sequences suitable for peptide linkers are known in the art. It is also possible to adjust the copy number "n" by taking into account the optimization of the linker to achieve adequate separation between functional moieties or to maintain the necessary inter-moiety interactions.
  • the linker may be a flexible linker including G, S, and/or T residues.
  • linkers include (GGGGS) n (SEQ ID NO: 2), (SGGGG) n (SEQ ID NO: 3), (SRSSG) n (SEQ ID NO: 4), (SGSSC) n (SEQ ID NO: 5), (GKSSGSGSESKS) n (SEQ ID NO: 6), (RPPPPC) n (SEQ ID NO: 7), (SSPPPPC) n (SEQ ID NO: 8), (GSTSGSGKSSEGKG) n (SEQ ID NO: 9), (GSTSGSGKSSEGSGSTKG) n (SEQ ID NO: 10), (GSTSGSGKPGSGEGSTKG) n (SEQ ID NO: 11), or (EGKSSGSGSESKEF) n (SEQ ID NO: 12), wherein n is an integer of 1 to 20, or 1 to 10.
  • Another aspect provides a polynucleotide encoding the fusion protein.
  • polynucleotide refers to a deoxyribonucleotide or a polymer of ribonucleotides present in a single-stranded or double-stranded form. It encompasses RNA genomic sequences, DNA (gDNA and cDNA) and RNA sequences transcribed therefrom, and unless specifically stated otherwise, includes natural polynucleotides as well as their analogs with modified sugar or base sites.
  • the polynucleotide is a single chain polynucleotide.
  • Another aspect provides a vector comprising the polynucleotide.
  • the term "vector” refers to a vector capable of expressing a protein of interest in a suitable host cell, and refers to a genetic construct comprising a regulatory element operably linked to express a gene insert.
  • the vector may include an expression control element such as a promoter, an operator, an initiation codon, a stop codon, a polyadenylation signal, and/or an enhancer, and the promoter of the vector may be constitutive or inducible.
  • the vector may be an expression vector capable of stably expressing the fusion protein in a host cell.
  • the expression vector may be a conventional one used in the art to express foreign proteins in plants, animals or microorganisms.
  • the recombinant vector can be constructed through various methods known in the art.
  • the vector may include a selectable marker for selecting a host cell containing the vector, and in the case of a replicable vector, may include an origin of replication.
  • the vector can be self-replicating or introduced into the host DNA, and the vector is selected from the group consisting of plasmid, lentivirus, adenovirus, adeno-associated virus, retrovirus, herpes simplex virus, and basinia virus. Can be.
  • the vector includes a promoter operable in animal cells, for example, mammalian cells.
  • Suitable promoters include promoters derived from mammalian virus and promoters derived from the genome of mammalian cells, such as CMV (Cytomegalovirus) promoter, U6 promoter and H1 promoter, MLV (Murine Leukemia Virus) LTR (Long terminal repeat) promoter, adenovirus early promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, RSV promoter, EF1 alpha promoter, metallotionine promoter, beta-actin promoter, Promoter of human IL-2 gene, promoter of human IFN gene, promoter of human IL-4 gene, promoter of human lymphotoxin gene, promoter of human GM-CSF gene, human phosphoglycerate kinase (PGK) promoter, mouse force Phoglycerate kina
  • the fusion protein described above may be operably linked to a promoter.
  • operably linked refers to a functional linkage between a nucleic acid expression control sequence (eg, a promoter, a signal sequence, or an array of transcriptional regulatory factor binding sites) and another nucleic acid sequence, thereby The regulatory sequence controls the transcription and/or translation of the other nucleic acid sequence.
  • Another aspect provides a host cell comprising the fusion protein, polynucleotide, or vector.
  • the cells are yeast, fungi, protozoa, plants, higher plants and insects, or cells of amphibians, or cells of mammals such as CHO, HeLa, HEK293, and COS-1.
  • yeast fungi, protozoa, plants, higher plants and insects, or cells of amphibians, or cells of mammals such as CHO, HeLa, HEK293, and COS-1.
  • the organism may be yeast, fungi, protozoa, plants, higher plants and insects, amphibians, or mammals.
  • the cells may be animal cells or plant cells.
  • the liposome may be a nano liposome.
  • the liposome may have a particle size of 10 nm to 250 nm.
  • the liposome may contain one or more lipids selected from the group consisting of phospholipids, steroids and PEG-containing phospholipids.
  • liposome includes an aqueous compartment surrounded by at least one lipid bilayer.
  • lipids containing hydrophilic head groups When lipids containing hydrophilic head groups are dispersed in water, they can spontaneously form a bilayer film called a lamellar.
  • the lamellar consists of two monolayer sheets of lipid molecules with their non-polar (hydrophobic) surface facing each other and their polar (hydrophilic) surface facing the aqueous medium.
  • the term liposome includes monolayered vesicles consisting of a single lipid bilayer and generally has a diameter in the range of about 10 to about 400 nm, about 50 to about 300 nm, or about 100 to 200 nm.
  • Liposomes may also be multilayered, generally having a diameter in the range of 1 to 10 ⁇ m and anywhere from 2 to hundreds of concentric lipid bilayers alternating with the layers of the aqueous phase.
  • liposomes may include multilayered vesicles (MLV), large monolayered vesicles (LUV) and small monolayered vesicles (SUVs).
  • LUV multilayered vesicles
  • LUV large monolayered vesicles
  • SUVs small monolayered vesicles
  • Liposomal lipids may be cationic, amphoteric, neutral or anionic, or any mixture thereof.
  • Liposomes or nanoliposomes herein may contain any suitable lipid, including cationic lipids, amphoteric lipids, neutral lipids or anionic lipids as described above.
  • Suitable lipids may include fats, waxes, steroids, cholesterol, fat-soluble vitamins, monoglycerides, diglycerides, phospholipids, sphingolipids, glycolipids, cationic or anionic lipids, derivatized lipids, and the like.
  • Suitable phospholipids include, but are not limited to, phosphatidylcholine (PC), phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), and phosphatidylinositol (PI), dimyristo Il phosphatidyl choline (DMPC), distearoyl phosphatidyl choline (DSPC), dioleoyl phosphatidyl choline (DOPC), dipalmitoyl phosphatidyl choline (DPPC), dimyristoyl phosphatidyl glycerol (DMPG), distearoyl phosphatidyl Glycerol (DSPG), dioleoyl phosphatidyl glycerol (DOPG), dipalmitoyl phosphatidyl glycerol (DPPG), dimyristoyl phosphatidyl serine (DMPS
  • Lipid extracts such as egg PC, heart extract, brain extract, liver extract and soybean PC are also useful in the present invention.
  • the soybean PC may comprise Hydro Soy PC (HSPC).
  • the lipid may comprise a derivatized lipid, such as a PEGylated lipid.
  • Derivatized lipids may include, for example, DSPE-PEG2000, cholesterol-PEG2000, DSPE-polyglycerol, or other derivatives generally known in the art.
  • the liposome or nano-liposome may contain a steroid characterized by the presence of a fused tetracyclic hard ring system.
  • steroids include, but are not limited to, cholesterol, cholic acid, progesterone, cortisone, aldosterone, estradiol, testosterone, and dehydroepiandrosterone. Synthetic steroids and derivatives thereof are also contemplated for use in the present invention.
  • Cationic lipids contain positively charged functional groups under physiological conditions.
  • Cationic lipids include, but are not limited to, N,N-dioleyyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-( 1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleyyloxy)propyl)-N,N,N -Trimethylammonium chloride (DOTMA), N-[1-(2,3-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N-[1-( 2,3-dioleyyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide (
  • the phospholipids are HSPC and DSPE-PEG (2000), and the steroid may utilize cholesterol.
  • any suitable combination of lipids may be used to provide liposomes or nanoliposomes herein.
  • the lipid composition can be tailored to influence characteristics such as leakage rate, stability, particle size, zeta potential, protein binding, circulation in vivo, and/or accumulation in tissues or organs.
  • the molar percentage (mol%) of a particular type of lipid present is typically about 0% to about 10%, about 10% to about 30%, about 30 of the total lipids present in the liposome or lipid nanoparticle.
  • the liposome or nano liposome comprises 56 mol% HSPC, 39 mol% cholesterol, and 5 mol% DSPE-PEG (2000).
  • the binding of the glutathione-S-transferase and liposome may be caused by GSH (Glutathione). That is, the liposome may be a liposome to which GSH (Glutathione) is bound.
  • the GSH may act as a binding site of glutathione-S-transferase to link liposomes with glutathione-S-transferase.
  • the glutathione-S-transferase, a protein having the ability to bind to a target cell or a target protein, and a linker-containing protein may be two or more bound to the one liposome. That is, two or more fusion proteins may be bound to one liposome to more easily bind to a target cell or a target protein.
  • Types of active pharmaceutical ingredients that can be delivered into an individual using a drug delivery system include anticancer agents, contrast agents (dyes), hormones, anti-hormones, vitamins, calcium agents, inorganic agents, saccharides, organic acids, protein amino acids, antidote, Enzyme preparations, metabolic preparations, diabetes combination preparations, tissue revitalization preparations, chlorophyll preparations, pigment preparations, tumor preparations, tumor treatments, radioactive medicines, tissue cell diagnostics, tissue cell treatments, antibiotic preparations, antiviral preparations, complex antibiotic preparations, Chemotherapeutic agents, vaccines, toxins, toxoids, antitoxins, leptospira serum, blood products, biological agents, analgesics, immunogenic molecules, antihistamines, drugs for allergies, nonspecific immunogenic agents, anesthetics, stimulants, psychotropic agents, small molecule compounds, nucleic acids , Aptamers, antisense nucleic acids, oligonucleotides, peptides, siRNAs and micro RNAs.
  • the drug may be an anticancer agent.
  • the anticancer drugs are camptothecin, doxorubicin, cisplatin, verapamil, fluorouracil, oxaliplatin, daunorubicin, daunorubicin, irinotecan, and irinotecan.
  • GST glutathione-S-transferase
  • a protein having a target cell or target protein binding ability A linker connecting the glutathione-S-transferase and a protein having the ability to bind to a target cell or a target protein;
  • GST glutathione-S-transferase
  • a linker connecting the glutathione-S-transferase and a protein having the ability to bind to a target cell or a target protein and it provides a pharmaceutical composition for preventing or treating cancer comprising the glutathione-S-transferase and liposome carrying an anticancer agent are bound.
  • GST glutathione-S-transferase
  • a protein having a target cell or target protein binding ability A linker connecting the glutathione-S-transferase and a protein having binding ability to a target cell or a target protein;
  • GST glutathione-S-transferase
  • a linker connecting the glutathione-S-transferase and a protein having binding ability to a target cell or a target protein and it provides a method for preventing or treating cancer comprising administering a composition containing a liposome carrying the glutathione-S-transferase and an anticancer agent to an individual in need thereof.
  • GST glutathione-S-transferase
  • a protein having a target cell or target protein binding ability A linker connecting the glutathione-S-transferase and a protein having the ability to bind to a target cell or a target protein;
  • a composition containing a liposome carrying the glutathione-S-transferase and an anticancer agent is provided for use in cancer prevention or treatment.
  • GST glutathione-S-transferase
  • a protein having a target cell or target protein binding ability a linker connecting the glutathione-S-transferase and a protein having a target cell or target protein binding ability
  • the Liposomes bound to glutathione-S-transferase are as described above.
  • therapeutic agent or “pharmaceutical composition” refers to a molecule or compound that imparts several beneficial effects upon administration to a subject.
  • the beneficial effect is to enable diagnostic decisions; Improvement of a disease, symptom, disorder or condition; Reducing or preventing the onset of a disease, symptom, disorder or condition; And the response of a disease, symptom, disorder or condition in general.
  • the cancer may be lung cancer (eg, non-small cell lung cancer), pancreatic cancer, gastric cancer, liver cancer, colon cancer, brain cancer, breast cancer, thyroid cancer, bladder cancer, esophageal cancer, or uterine cancer.
  • the cancer may be any one or more selected from the group consisting of gastric cancer, breast cancer, lung cancer, liver cancer, esophageal cancer, and prostate cancer having resistance to anticancer drugs (eg, multi-drug resistance).
  • the pharmaceutical composition can be administered parenterally during clinical administration and can be used in the form of a general pharmaceutical formulation.
  • Parenteral administration may mean administration through a route other than oral administration such as rectal, intravenous, peritoneal, muscle, arterial, transdermal, nasal, inhalation, ocular and subcutaneous.
  • the pharmaceutical composition of the present invention may further contain one or more active ingredients exhibiting the same or similar functions.
  • the pharmaceutical composition When formulating the pharmaceutical composition, it is prepared by using a diluent or excipient such as a commonly used filler, extender, binder, wetting agent, disintegrant, and surfactant.
  • a diluent or excipient such as a commonly used filler, extender, binder, wetting agent, disintegrant, and surfactant.
  • Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories.
  • the non-aqueous solvent and the suspension solvent propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used.
  • a base for the suppository Witepsol, Macrogol, Tween 61, cacao butter, liurinji, glycerogelatin, and the like may be used.
  • the pharmaceutical composition may be used in combination with various carriers (Carriers) allowed as drugs such as physiological saline or organic solvents, and carbohydrates such as glucose, sucrose or dextran, ascorbic acid in order to increase stability or absorption.
  • Carriers allowed as drugs such as physiological saline or organic solvents, and carbohydrates such as glucose, sucrose or dextran, ascorbic acid in order to increase stability or absorption.
  • Antioxidants such as (Ascorbic acid) or glutathione, chelating agents, small molecule proteins or other stabilizers can be used as drugs.
  • the effective dose of the pharmaceutical composition is 0.01 to 100 mg/kg, preferably 0.1 to 10 mg/kg, and may be administered once to three times a day.
  • the anticancer agent abarelix [abarelix (Plenaxis depot ® )]; Aldesleukin (Prokine ® )]; Aldesleukin [Aldesleukin (Proleukin ® )]; Alemtuzumabb (Campath ® )]; Alitretinoin (Panretin ® )]; Allopurinol [allopurinol (Zyloprim ® )]; Altretamine (Hexalen ® )]; Amifostine [amifostine (Ethyol ® )]; Anastrozole (Arimidex ® )]; Arsenic trioxide (Trisenox ® )]; Asparaginase [asparaginase (Elspar ® )]; Azacitidine (Vidaza ® )]; Bevacuzimab [bevacuzimab (Avastin ® )]; Bevacuzim
  • the present invention provides a method for producing a liposomal drug delivery system having an outer layer of protein corona, comprising steps i) to iii):
  • the liposome having the protein corona shield first produced a protein corona outer layer that is pre-coated with a fusion protein, a corona layer surrounded by serum proteins in the body environment is not formed. The immune response can be avoided and thus can have a significant stealth effect. Therefore, the liposome having the outer corona protein layer can not only sustain the remaining time in vivo, but also can be effectively delivered to the target cell by improving the targeting ability to the target cell, and thus can be usefully used as a target therapeutic agent.
  • the drug delivery system composed of liposomes not only can the remaining time in the living body be sustained, but also the targeting ability to the target cells is improved and can be effectively delivered to the target cells, so it can be usefully used as a target therapeutic agent. There is an effect.
  • FIG. 1 is a schematic diagram of the structure of a fusion protein forming the outer layer of the protein corona of the liposome in the present invention.
  • FIG. 2A is a result of confirming that the GST-Afb fusion protein does not decrease cell viability with respect to Afb target cells;
  • FIG. 2B is a result of confirming the cell targeting ability of the GST-Afb fusion protein to Afb target cells.
  • FIG. 3 is a schematic diagram of the synthesis of GSH-PEG-DSPE, one of the components of a liposome carrying a drug.
  • FIG. 4 is a schematic diagram of the components of a liposome carrying a drug.
  • 5 is an image of the shape of Dox-liposome-GSH observed with a transmission electron microscope.
  • Figure 7 is a drug-bearing liposome with glutathione-S-transferase (GST) and a protein having the ability to bind to a target cell or a target protein, that is, a target-directed fusion protein (Dox-liposome-fusion protein ) Is a schematic diagram.
  • GST glutathione-S-transferase
  • Dox-liposome-fusion protein a target-directed fusion protein
  • Figure 8 is a graph observed by dynamic light scattering of the size of Dox-liposome-fusion protein.
  • 10 is a graph showing the zeta potential of Dox-liposome-GSH.
  • 11 is a graph showing the zeta potential of Dox-liposome-fusion protein.
  • 13 is a photograph and graph showing the anti-tumor effect of Dox-liposome-fusion protein.
  • Example 1 Expression of outer layer fusion protein for use in liposomes with protein corona outer layer
  • a fusion protein capable of constituting the outer layer of the protein corona was prepared.
  • the fusion protein was expressed in a form in which affibody (Afb) capable of specifically binding to a receptor on the surface of cancer cells and GST were combined.
  • HER2 Afb specifically binding to HER2 and EGFR Afb specifically binding to EGFR were used as Afb.
  • a gene encoding an additional linker domain (SEQ ID NO: 1: GGGLVPRGSGGGCGGGGTGGGSGGG) was coupled to the end of the gene encoding each HER2 Afb or EGFR Afb, and then inserted into the pETduet plasmid.
  • a GST-encoding gene designed to connect a 6 ⁇ His tag to the N-terminus of GST was inserted into the pETduet plasmid, and a plasmid for overexpression of the fusion protein (GST-Afb) in which Afb and GST were linked by a linker was prepared.
  • GST-Afb the fusion protein
  • the constructed plasmid was inserted into the E. coli BL21 (DE3) strain and cultured, and then treated with IPTG and cultured at 30° C. for 16 hours to induce overexpression of GST-Afb.
  • the overexpression-induced E was inserted into the E. coli BL21 (DE3) strain and cultured, and then treated with IPTG and cultured at 30° C. for 16 hours to induce overexpression of GST-Afb.
  • coli cells were centrifuged at 4°C for 10 minutes at 5000 ⁇ g to obtain precipitated cells, and the cells were suspended in a phosphate buffer solution (50 mM sodium phosphate and 100 mM sodium chloride, pH 6.5). The cell suspension was treated with lysozyme and incubated for 20 minutes at room temperature, followed by disruption for 30 seconds and ultrasonic disruption at 1 minute intervals for a total of 10 minutes. After crushing, centrifugation at 12000 ⁇ g at 4° C. for 1 hour to obtain a supernatant as a fraction containing GST-Afb.
  • phosphate buffer solution 50 mM sodium phosphate and 100 mM sodium chloride, pH 6.5
  • the supernatant was purified by immobilized metal affinity chromatography (1mL HisTrap FF column, GE HealthCare) using FPLC to separate GST-Afb.
  • the isolated GST-Afb (GST-HER2 Afb and GST-EGFR Afb were concentrated by dialysis overnight in PBS (pH 7.4)).
  • FIG. 1 is a schematic diagram of the structure of a fusion protein forming the outer layer of the protein corona of the liposome in the present invention.
  • GST-Afb fusion protein expressed in the present invention can be applied as a protein corona shield (PCS) in a drug delivery system, cytotoxicity and intracellular absorption capacity were examined.
  • PCS protein corona shield
  • SK-BR-3 cells a human breast cancer cell line
  • the prepared SK-BR-3 cells were cultured in DMEM medium (11995065, Invitrogen, S. Korea). 10% fetal bovine serum (FBS), 100 ⁇ g/ml streptomycin and 100 U/ml penicillin were added to the medium, and the medium was changed once daily during the culture period.
  • FBS fetal bovine serum
  • the culture environment was maintained in a 5% CO2 incubator at 37°C. When the cells proliferated to 85% saturation after cell inoculation, adherent cultured cells were separated and used in experiments.
  • SK-BR-3 cells were isolated, each cell was inoculated into a 96-well plate (Thermo Scientific Inc. Korea) at a concentration of 5x10 3 cells/well, and cultured in a 5% CO 2 incubator at 37° C. for 24 hours. Then, the GST-HER2 Afb obtained in Example ⁇ 1-1> was treated with the SK-BR-3 cells at a concentration of 0.3 ⁇ M to 10 ⁇ M, and further cultured for 24 hours. After completion of the culture, cell viability was confirmed using alamar blue dye (DAL 2015, Invitrogen, Korea).
  • the excitation wavelength for the fluorescent dye was set to 565 nm, and the monitoring emission was set to 590 nm, and fluorescence analysis was performed with a fluorescent plate reader (Tecan Infinite Series, Germany).
  • GST among GST-HER2 Afb was labeled with fluorescence-5-maleimide (F5M) to confirm the location of GST-HER2 Afb absorbed into cells (cell uptake).
  • F5M fluorescence-5-maleimide
  • normal epithelial cell line MCF-10A cells were used to perform the same method to confirm cell viability and intracellular absorption.
  • GST-HER2 Afb did not show toxicity to cells, but could be specifically absorbed by cancer cells.
  • GST-HER2 Afb was treated and cultured on SK-BR-3 cells and MCF-10A cells, it was confirmed that the degree of apoptosis was not exhibited regardless of the treatment concentration, so that cytotoxicity was not exhibited by GST-Afb (FIG. 2A ), GST-HER2 Afb showed binding ability to bind only to SK-BR-3 cells, which is a breast cancer cell line, and it was confirmed that GST-Afb exhibited a specific targeting ability for cancer cells (FIG. 2B).
  • the GST-Afb fusion protein expressed for use as an outer layer of protein corona in the present invention does not exhibit toxicity to normal cells and can exhibit targeting ability against cancer cells.
  • a liposome As the basic structure of the drug delivery system, a liposome was prepared. The liposome was manufactured in a nano size to obtain an EPR (Enhanced Permeability and Retention) effect. As a component of the liposome, DSPE-PEG (2000) in which HSPC (L- ⁇ -phosphatidylcholine, hydrogenated (Soy)), Cholesterol, and GSH were bound was used. It was synthesized as shown in FIGS. 3 and 4.
  • DSPE-PEG-maleimide (32.7 mg, 0.01635 mmol) and glutathione (7.5 mg, 0.0244 mmol) were added to 3 mL of 1M pH 7.4 HEPES buffer and stirred at 22° C. for 15 hours. In the dark condition, nitrogen gas was injected and reacted. Glutathione remaining after the reaction was removed by washing with HEPES buffer, and the reacted DSPE-PEG-GSH was separated with a centrifuge. The isolated DSPE-PEG-GSH was confirmed by NMR using DMSO-d6.
  • HSPC phosphatidiylcholine
  • cholesterol 15.079 mg, 0.039 mmol
  • doxorubicin 10 mg
  • DSPE-PEG-GSH 14 uL ethanol
  • doxorubicin-supported liposomes Dox-liposome-GSH
  • the shape and size of liposomes were observed by Transmission Electron Microscopy (TEM) and Dynamic Light Scattering (DLS), and the size of the observed liposomes was about 106 nm (FIGS. 5 and 6).
  • FIG. 3 is a schematic diagram of the synthesis of GSH-PEG-DSPE, one of the components of a liposome carrying a drug.
  • FIG. 4 is a schematic diagram of the components of a liposome carrying a drug.
  • 5 is an image of the shape of Dox-liposome-GSH observed with a transmission electron microscope.
  • a liposome having an outer layer of protein corona to be used as a drug delivery system in the present invention a liposome to which GST-Afb was applied as an outer layer of protein corona was prepared. Since the liposome is used as a carrier capable of carrying a substance through chemical functional groups on the inside and on the surface, in the present invention, it was prepared by binding GSH to the surface of the liposome, and thus formed by forming an outer layer of protein corona bound through GST (Fig. 7).
  • the Dox-liposome-GSH (104.4 mg) obtained in Example ⁇ 2-1> was prepared in 10 ml of a 10 mM tris-HCl pH 7.5 buffer solution, and then the Example ⁇ 1-1> at a concentration of 20 mg/ml PBS. After 10 ul of the fusion protein obtained in was added, the mixture was stirred at 65° C. for 10 minutes. Then, dialysis was performed twice using 10 mM tris-HCl pH 7.5 buffer to synthesize doxorubicin-supported liposomes (Dox-liposome-fusion protein) having a fusion protein as an outer layer.
  • the shape and size of the Dox-liposome-fusion protein were observed by Transmission Electron Microscopy (TEM) and Dynamic Light Scattering (DLS).
  • the size of the Dox-liposome-fusion protein is about 150 nm, and the surface charge is ⁇ 0 mv, indicating that the fusion protein formed a complex with Dox-liposome-GSH (FIGS. 8 to 11 ).
  • Figure 7 is a drug-bearing liposome with glutathione-S-transferase (GST) and a protein having the ability to bind to a target cell or a target protein, that is, a target-directed fusion protein (Dox-liposome-fusion protein ) Is a schematic diagram.
  • GST glutathione-S-transferase
  • Dox-liposome-fusion protein a target-directed fusion protein
  • Figure 8 is a graph observed by dynamic light scattering of the size of Dox-liposome-fusion protein.
  • 10 is a graph showing the zeta potential of Dox-liposome-GSH.
  • 11 is a graph showing the zeta potential of Dox-liposome-fusion protein.
  • the liposome (Dox-liposome-fusion protein) having the outer layer of the GST-Afb protein corona has cytotoxicity to the target cells.
  • the cell viability of Her2-oriented fusion protein + Dox-liposome-GSH for SKBR3 cells, human breast cancer cells is 3-(4,5-dimethylthiazol-2-yl) It was evaluated by observing the reduction of -2,5-diphenyltetrazolium bromide (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) to insoluble formazan (formazan). .
  • DMEM fetal calf serum
  • cells were seeded in a 96-well plate at a density of 5 ⁇ 10 3 cells per well and incubated overnight, and then the cells were treated with Dox-liposome-fusion protein in a concentration range of 0 to 25 ug/ml. Incubated for 24 hours. Control experiments were performed with fusion protein and Dox-liposome-GSH. Then, the cells were converted to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium).
  • Balb/c nude female mice were purchased from Orient Bio, Korea, and all animal experiments were performed according to a protocol approved by the Institutional Animal Care and Use Committee of Ulsan Institute of Science and Technology (UNIST).
  • mice to be injected with Dox-liposome-fusion protein, Dox-liposome-GSH, fusion protein, and PBS were selected, respectively, and mice with tumors larger or smaller than that were selected. I didn't choose.
  • 100 ⁇ L of Dox-liposome-fusion protein, 50 mg/kg, 100 ⁇ L of Dox-liposome-GSH, 50 mg/kg, 100 ⁇ L of fusion protein, 5 mg/kg, 1X PBS as a control 100 ⁇ L was injected intravenously every day for 3 weeks. Thereafter, tumor size and body weight of all groups were measured and compared.
  • 13 is a photograph and graph showing the anti-tumor effect of Dox-liposome-fusion protein.

Abstract

The present invention relates to a liposome comprising glutathione-S-transferase and a protein having the capacity to bind to a target cell or a target protein, and use thereof as a drug carrier and a pharmaceutical composition. A drug carrier comprising the liposome according to an aspect can not only maintain residence time in vivo, but can also be effectively delivered to target cells due to an enhanced ability to target the target cells, and thus can be effectively used as a targeted therapeutic agent.

Description

글루타치온-Ｓ-전이효소 및 표적 세포 또는 표적 단백질 결합능을 갖는 단백질을 포함하는 리포좀 및 이의 용도Liposomes containing glutathione-S-transferase and a protein having the ability to bind to target cells or target proteins, and uses thereof
글루타치온-Ｓ-전이효소 및 표적 세포 또는 표적 단백질 결합능을 갖는 단백질을 포함하는 리포좀 및 이의 약물 전달체 및 약학적 조성물로서의 용도에 관한 것이다. It relates to a liposome comprising a protein having glutathione-S-transferase and a target cell or a protein having a target protein binding ability, and its use as a drug delivery system and pharmaceutical composition.
최근 나노기술의 발전과 더불어 새로운 물리적, 화학적, 광학적 특성을 가지는 나노크기의 소재들을 암 진단 및 치료분야에 응용하는 연구가 활발히 진행되고 있다. 나노기술은 기존기술로는 가능하지 않던 신개념의 진단 및 치료가 가능해 나노기술의 무한한 가능성을 보여주고 있다. 그 중에서도 나노기술기반 약물전달시스템(DDS, Drug Delivery System)이 질병 진단 및 치료 분야에 다양하게 적용되고 있다. 이러한 나노입자 기반 약물전달시스템은 약물의 부작용을 최소화하며, 보다 효과적으로 약물을 전달하여 치료 효능을 높일 수 있는 것으로 동물실험 등을 통해 검증되어 왔다. 나노입자 기반 약물전달시스템은 첫째, 나노입자의 크기 및 물성을 조절하여 약물의 생체 내 동역학과 분포를 최적화함으로써 치료효과 극대화 할 수 있다는 점과 둘째, 기존 항암제의 비선택적인 생체 분포에 기인한 광범위한 독성 및 부작용, 약제 내성을 줄이고, 병용투여요법을 용이하게 한다는 장점이 있다.Along with the recent development of nanotechnology, researches on applying nano-sized materials having new physical, chemical, and optical properties to the field of cancer diagnosis and treatment are being actively conducted. Nanotechnology is capable of diagnosing and treating new concepts that were not possible with existing technologies, showing the infinite possibilities of nanotechnology. Among them, a nanotechnology-based drug delivery system (DDS, Drug Delivery System) is widely applied in the field of disease diagnosis and treatment. These nanoparticle-based drug delivery systems have been proven through animal experiments to minimize side effects of drugs and to increase therapeutic efficacy by delivering drugs more effectively. The nanoparticle-based drug delivery system can maximize the therapeutic effect by optimizing the in vivo kinetics and distribution of drugs by first, controlling the size and physical properties of the nanoparticles, and secondly, the broad spectrum due to the non-selective biodistribution of existing anticancer drugs. It has the advantage of reducing toxicity and side effects, drug resistance, and facilitating combination therapy.
현재까지 미국 FDA 허가를 받은 나노약물전달시스템 기반 제품은 Doxyl®(독소루비신 함유 나노리포좀)과 Abraxane®(패클리탁셀 함유 알부민 나노입자)과 같이 공히 생체적합성이 탁월한 나노입자에 기반한 것으로, 특정 암을 선택적으로 표적하여 항암제를 전달하는 기능을 가지고 있지는 않아 단순히 EPR 효과에 의한 수동적인 전달이라 효능이 제한적이라는 평가를 받고 있다. EPR 효과에 의존한 수동적 표적지향은 동물 암 모델에서는 우수한 효능을 보일 수 있으나 실제 인간 암에서는 상대적으로 EPR 효과를 보이는 경우가 드물어 치료 효능이 제한적이다. 특히 전립선암이나 췌장암 조직과 같이 신생혈관이 적을 경우에는 EPR 효과에 따른 효과적인 약물전달을 기대하기 어렵다는 단점이 있다. Nanopharmaceutical delivery system-based products that have been approved by the US FDA to date are based on highly biocompatible nanoparticles such as Doxyl® (nanoliposomes containing doxorubicin) and Abraxane® (albumin nanoparticles containing paclitaxel). Since it does not have the function of selectively targeting and delivering anticancer drugs, it is evaluated that its efficacy is limited because it is simply passive delivery by EPR effect. Passive targeting depending on the EPR effect can show excellent efficacy in animal cancer models, but in actual human cancers, it is rare that EPR effects are relatively rare, so treatment efficacy is limited. In particular, when there are few new blood vessels such as prostate cancer or pancreatic cancer tissue, there is a disadvantage that it is difficult to expect effective drug delivery according to the EPR effect.
일 양상은 글루타치온-S-전이효소(glutathione-S-transferase, GST); 표적 세포 또는 표적 단백질 결합능을 갖는 단백질; 상기 글루타치온-S-전이효소와 표적 세포 또는 표적 단백질 결합능을 갖는 단백질을 연결하는 링커; 및 상기 글루타치온-S-전이효소와 약물을 담지하는 리포좀이 결합된 것을 함유하는 약물 전달체를 제공하는 것이다.One aspect is glutathione-S-transferase (GST); A protein having a target cell or target protein binding ability; A linker connecting the glutathione-S-transferase and a protein having binding ability to a target cell or a target protein; And it is to provide a drug delivery system containing the glutathione-S-transferase and a liposome carrying a drug are bound.
다른 양상은 글루타치온-S-전이효소(glutathione-S-transferase, GST); 표적 세포 또는 표적 단백질 결합능을 갖는 단백질; 상기 글루타치온-S-전이효소와 표적 세포 또는 표적 단백질 결합능을 갖는 단백질을 연결하는 링커; 및 상기 글루타치온-S-전이효소와 항암제를 담지하는 리포좀이 결합된 것을 포함하는 암 예방 또는 치료용 약학적 조성물을 제공하는 것이다. Another aspect is glutathione-S-transferase (GST); A protein having a target cell or target protein binding ability; A linker connecting the glutathione-S-transferase and a protein having the ability to bind to a target cell or a target protein; And it is to provide a pharmaceutical composition for preventing or treating cancer comprising the glutathione-S-transferase and a liposome carrying an anticancer agent are bound.
또 다른 양상은 글루타치온-S-전이효소(glutathione-S-transferase, GST); 표적 세포 또는 표적 단백질 결합능을 갖는 단백질; 상기 글루타치온-S-전이효소와 표적 세포 또는 표적 단백질 결합능을 갖는 단백질을 연결하는 링커; 및 상기 글루타치온-S-전이효소와 약물을 담지하는 리포좀을 함유하는 조성물을 그를 필요로 하는 개체에 투여하는 단계를 포함하는 개체 내 약물을 전달하는 방법을 제공하는 것이다.Another aspect is glutathione-S-transferase (GST); A protein having a target cell or target protein binding ability; A linker connecting the glutathione-S-transferase and a protein having the ability to bind to a target cell or a target protein; And administering a composition containing the glutathione-S-transferase and a liposome carrying the drug to an individual in need thereof.
또 다른 양상은 글루타치온-S-전이효소(glutathione-S-transferase, GST); 표적 세포 또는 표적 단백질 결합능을 갖는 단백질; 상기 글루타치온-S-전이효소와 표적 세포 또는 표적 단백질 결합능을 갖는 단백질을 연결하는 링커; 및 상기 글루타치온-S-전이효소와 항암제를 담지하는 리포좀을 함유하는 조성물을 그를 필요로 하는 개체에 투여하는 단계를 포함하는 암을 예방하거나 치료하는 방법을 제공하는 것이다.Another aspect is glutathione-S-transferase (GST); A protein having a target cell or target protein binding ability; A linker connecting the glutathione-S-transferase and a protein having the ability to bind to a target cell or a target protein; And it is to provide a method for preventing or treating cancer comprising administering a composition containing a liposome carrying the glutathione-S-transferase and an anticancer agent to an individual in need thereof.
일 양상은 글루타치온-S-전이효소(glutathione-S-transferase, GST); 표적 세포 또는 표적 단백질 결합능을 갖는 단백질; 상기 글루타치온-S-전이효소와 표적 세포 또는 표적 단백질 결합능을 갖는 단백질을 연결하는 링커; 및 상기 글루타치온-S-전이효소와 약물을 담지하는 리포좀이 결합된 것을 함유하는 약물 전달체를 제공하는 것이다.One aspect is glutathione-S-transferase (GST); A protein having a target cell or target protein binding ability; A linker connecting the glutathione-S-transferase and a protein having the ability to bind to a target cell or a target protein; And it is to provide a drug delivery system containing the glutathione-S-transferase and a liposome carrying a drug are bound.
본 명세서에서 용어 "표적 세포 또는 표적 단백질 결합능을 갖는 단백질", "표적세포 인식능을 갖는 단백질", 또는 "표적 세포 또는 표적 단백질에 특이적으로 결합하는 단백질"은 세포의 수용체 또는 표적 단백질을 특이적으로 인식하는 또는 세포의 수용체 또는 표적 단백질에 특이적으로 결합하는 단백질을 의미할 수 있다. 구체적으로, 상기 세포의 수용체 또는 표적 단백질에 특이적으로 결합하는 단백질은 항체, 항원 결합 단편, 애피바디(affibody), 다이아바디(diabody) 및 앱타머(aptamer)로 이루어진 군으로부터 선택되는 어느 하나인 것일 수 있다. In the present specification, the terms "a protein having the ability to bind to a target cell or a target protein", "a protein having the ability to recognize a target cell", or "a protein that specifically binds a target cell or a target protein" refers to a receptor or a target protein of a cell. It may mean a protein that is specifically recognized or specifically binds to a receptor or a target protein of a cell. Specifically, the protein that specifically binds to the receptor or target protein of the cell is any one selected from the group consisting of an antibody, an antigen-binding fragment, an affibody, a diabody, and an aptamer. Can be.
본 명세서에서 용어 "애피바디 분자(affibody molecule)"는 특정 타겟 단백질(수용체)에 결합할 수 있는, 항체 모사체를 의미할 수 있다. 일반적으로 애피바디 분자는 20 내지 150의 아미노산 잔기로 구성되며, 2 내지 10개의 알파 헬릭스로 구성된 것일 수 있다. 더욱 상세하게는 상기 애피바디 분자는 항-ErbB 애피바디 분자(ab31889), HER2-특이적 애피바디 분자(ZHER2:342), 항-EGFR 애피바디 분자(ZEGFR:2377) 등을 포함할 수 있다. 또한, 이에 한정되지 않고, 세포의 특정 수용체 또는 표적 단백질을 인식할 수 있는 애피바디 분자를 모두 포함한다. 상기 애피바디 분자가 인식할 수 있는 표적 수용체 또는 표적 단백질의 예는, 아밀로이드 베타 펩티드, 시누클레인(예를 들면, 알파-시누클레인), 아포리포프로테인(예를 들면, 아포리포프로테인 A1), 보체 인자(Complement factor)(예를 들면, C5), 탄산무수화효소(Carbonic anhydrase)(예를 들면, CAIX), 인터루킨-2 수용체 알파 사슬(IL2RA; CD25), 세포 표면의 CD 항원(예를 들면, CD28), 또는 c-Jun, Factor VIII, 프비르노겐, GP120, H-Ras, Her2, Her3, HPV16 E7, IAPP(Human islet amyloid polypeptide), 이뮤노글로불린 A(IgA), IgE, IgM, 인터루킨(예를 들면, IL-1, IL-6, IL-8, IL-17), 인슐린, 스타필로코커스 단백질 A 도메인(Staphylococcal protein A domain), Raf-1, LOV 도메인(Light-oxygen-voltage-sensing domain), 또는 RSV G 단백질일 수 있다. 상기 애피바디에 대한 정보는 Stefan Stahl et al., Affibody Molecules in Biotechnological and Medical Applications, Trends in Biotechnology, August 2017, Vol 35, No 8에 기재되어 있으며, 상기 문헌은 그 전체가 참조로서 본 명세서에서 포함된다. In the present specification, the term "affibody molecule" may refer to an antibody mimic capable of binding to a specific target protein (receptor). In general, the Affibody molecule is composed of 20 to 150 amino acid residues, and may be composed of 2 to 10 alpha helixes. In more detail, the affibody molecule may include an anti-ErbB affibody molecule (ab31889), a HER2-specific affibody molecule (ZHER2:342), an anti-EGFR affibody molecule (ZEGFR:2377), and the like. In addition, the present invention is not limited thereto, and includes all affibody molecules capable of recognizing a specific receptor or target protein of a cell. Examples of target receptors or target proteins that can be recognized by the Affibody molecule include amyloid beta peptide, synuclein (e.g., alpha-synuclein), apolipoprotein (e.g., apolipoprotein A1), complement Complement factor (e.g., C5), carbonic anhydrase (e.g. CAIX), interleukin-2 receptor alpha chain (IL2RA; CD25), CD antigen on the cell surface (e.g. , CD28), or c-Jun, Factor VIII, pvirnogen, GP120, H-Ras, Her2, Her3, HPV16 E7, IAPP (Human islet amyloid polypeptide), immunoglobulin A (IgA), IgE, IgM, interleukin (E.g., IL-1, IL-6, IL-8, IL-17), insulin, Staphylococcal protein A domain, Raf-1, LOV domain (Light-oxygen-voltage- sensing domain), or RSV G protein. Information on the Affibody is described in Stefan Stahl et al., Affibody Molecules in Biotechnological and Medical Applications, Trends in Biotechnology, August 2017, Vol 35, No 8, the entire document is incorporated herein by reference. do.
상기 표적 세포 또는 표적 단백질 결합능을 갖는 단백질은 수용체 타이로신 카이네이즈(Receptor tyrosine kinases: RTKs)에 특이적으로 결합하는 것일 수 있다. 더욱 구체적으로, 상기 수용체 타이로신 카이네이즈는 표피 성장인자 수용체, 인슐린 수용체, 혈소판 유래 성장인자 수용체, 혈관내피 성장인자 수용체, 섬유아세포 성장인자 수용체, 콜레시스토키닌(Cholecystokinin: CCK) 수용체, 신경영양인자(Neurotrophic factor: NGF) 수용체, 간세포 성장인자 (Hepatocyte growth factor: HGF) 수용체, 에프린(Ephrin: Eph) 수용체, 안지오포이에틴 수용체, 및 RYK(related to receptor tyrosine kinase) 수용체로 이루어진 군으로부터 선택된 어느 하나일 수 있다. The target cell or protein having the ability to bind to the target protein may specifically bind to receptor tyrosine kinases (RTKs). More specifically, the receptor tyrosine kinase is an epidermal growth factor receptor, insulin receptor, platelet-derived growth factor receptor, vascular endothelial growth factor receptor, fibroblast growth factor receptor, cholecystokinin (CCK) receptor, neurotrophic factor (NGF). ) Receptor, hepatocyte growth factor (HGF) receptor, ephrin (Eph) receptor, angiopoietin receptor, and related to receptor tyrosine kinase (RYK) receptor. .
상기 GST 및 표적 세포 또는 표적 단백질 결합능을 갖는 단백질은 링커를 통해 연결되어 있을 수 있다. 예를 들어, 상기 링커는, 1 내지 400개, 1 내지 200개, 또는 2 내지 200개의 임의의 아미노산으로 이루어진 폴리펩티드일 수 있다. 상기 펩티드 링커는 Gly, Asn 및 Ser 잔기를 포함할 수 있으며, Thr 및 Ala과 같은 중성 아미노산들도 포함될 수 있다. 펩티드 링커에 적합한 아미노산 서열은 당업계에 공지되어 있다. 또한 기능적 일부분 사이의 적절한 분리를 달성하기 위하여 또는 필수적인 내부-일부분(inter-moiety)의 상호작용을 유지하기 위한　링커의 최적화를 고려하여 카피 수 "n"을 조절할 수 있다. 해당 기술분야에서 다른 가요성　링커들이 알려져 있는데, 예를 들어 수용성을 향상시키기 위하여 극성 아미노산 잔기를 추가하는 것뿐만 아니라 유연성을 유지하기 위하여 T 및 A와 같은 아미노산 잔기를 추가한 G 및 S　링커가 있을 수 있다. 따라서 일 구체예에 있어서, 상기　링커는 G, S, 및/또는 T 잔기를 포함하는 유연성　링커일 수 있다. 상기　링커는 (GpSs)n 및 (SpGs)n으로부터 선택되는 일반식을 가질 수 있고, 이 경우, 독립적으로, p는 1 내지 10의 정수이고, s = 0 내지 10의 0 또는 정수이고, p + s는 20 이하의 정수이고, 및 n은 1 내지 20의 정수이다. 더욱 구체적으로 링커의 예는 (GGGGS)n (서열번호 2), (SGGGG)n　(서열번호 3), (SRSSG)n (서열번호 4), (SGSSC)n (서열번호 5), (GKSSGSGSESKS)n (서열번호 6), (RPPPPC)n (서열번호 7), (SSPPPPC)n (서열번호 8),　 (GSTSGSGKSSEGKG)n (서열번호 9), (GSTSGSGKSSEGSGSTKG)n (서열번호 10),　(GSTSGSGKPGSGEGSTKG)n　(서열번호 11), 또는 (EGKSSGSGSESKEF)n (서열번호 12)이고, 상기 n은 1 내지 20, 또는 1 내지 10의 정수이다. The GST and the target cell or a protein having a target protein binding ability may be linked through a linker. For example, the linker may be a polypeptide consisting of 1 to 400, 1 to 200, or 2 to 200 arbitrary amino acids. The peptide linker may include Gly, Asn and Ser residues, and neutral amino acids such as Thr and Ala may also be included. Amino acid sequences suitable for peptide linkers are known in the art. It is also possible to adjust the copy number "n" by taking into account the optimization of the linker to achieve adequate separation between functional moieties or to maintain the necessary inter-moiety interactions. Other flexible linkers are known in the art, for example, G and S linkers with addition of amino acid residues such as T and A to maintain flexibility as well as adding polar amino acid residues to improve water solubility. I can. Thus, in one embodiment, the linker may be a flexible linker including G, S, and/or T residues. The linker may have a general formula selected from (G p S s ) n and (S p G s ) n , in which case, independently, p is an integer of 1 to 10, and s = 0 of 0 to 10 Or an integer, p + s is an integer of 20 or less, and n is an integer of 1 to 20. More specifically, examples of linkers include (GGGGS) n (SEQ ID NO: 2), (SGGGG) n (SEQ ID NO: 3), (SRSSG) n (SEQ ID NO: 4), (SGSSC) n (SEQ ID NO: 5), (GKSSGSGSESKS) n (SEQ ID NO: 6), (RPPPPC) n (SEQ ID NO: 7), (SSPPPPC) n (SEQ ID NO: 8), (GSTSGSGKSSEGKG) n (SEQ ID NO: 9), (GSTSGSGKSSEGSGSTKG) n (SEQ ID NO: 10), (GSTSGSGKPGSGEGSTKG) n (SEQ ID NO: 11), or (EGKSSGSGSESKEF) n (SEQ ID NO: 12), wherein n is an integer of 1 to 20, or 1 to 10.
또 다른 양상은 상기 융합 단백질을 암호화하는 폴리뉴클레오티드를 제공한다. Another aspect provides a polynucleotide encoding the fusion protein.
용어 "폴리뉴클레오티드(polynucleotide)"는 단일가닥 또는 이중가닥 형태로 존재하는 디옥시리보뉴클레오티드 또는 리보뉴클레오티드의 중합체를 의미한다. RNA 게놈 서열, DNA(gDNA 및 cDNA) 및 이로부터 전사되는 RNA 서열을 포괄하며, 특별하게 다른 언급이 없는 한 자연의 폴리뉴클레오티드뿐만 아니라 당 또는 염기 부위가 변형된 그의 유사체(analogue)도 포함한다. 일 구체예에서, 상기 폴리뉴클레오티드는 단쇄 폴리뉴클레오티드이다.The term "polynucleotide" refers to a deoxyribonucleotide or a polymer of ribonucleotides present in a single-stranded or double-stranded form. It encompasses RNA genomic sequences, DNA (gDNA and cDNA) and RNA sequences transcribed therefrom, and unless specifically stated otherwise, includes natural polynucleotides as well as their analogs with modified sugar or base sites. In one embodiment, the polynucleotide is a single chain polynucleotide.
또 다른 양상은 상기 폴리뉴클레오티드를 포함하는 벡터를 제공한다. Another aspect provides a vector comprising the polynucleotide.
본 명세서에서 사용되는 용어, "벡터"는 적당한 숙주세포에서 목적 단백질을 발현할 수 있는 벡터로서, 유전자 삽입물이 발현되도록 작동 가능하게 연결된 조절 요소를 포함하는 유전자 작제물을 지칭한다. 일 실시예에 따른 벡터는 프로모터, 오퍼레이터, 개시코돈, 종결코돈, 폴리아데닐화 시그널, 및/또는 인핸서와 같은 발현 조절 요소를 포함할 수 있으며, 벡터의 프로모터는 구성적 또는 유도성일 수 있다. 또한, 상기 벡터는, 숙주 세포 내에서 안정적으로 상기 융합 단백질을 발현시킬 수 있는, 발현용 벡터일 수 있다. 상기 발현용 벡터는 당업계에서 식물, 동물 또는 미생물에서 외래의 단백질을 발현하는 데 사용되는 통상의 것을 사용할 수 있다. 상기 재조합 벡터는 당업계에 공지된 다양한 방법을 통해 구축될 수 있다. 예를 들어, 상기 벡터는 벡터를 함유 하는 숙주세포를 선택하기 위한 선택성 마커를 포함하고, 복제 가능한 벡터인 경우, 복제 기원을 포함할 수 있다. 또한, 벡터는 자가 복제하거나 숙주 DNA에 도입될 수 있으며, 상기 벡터는 플라스미드, 렌티바이러스, 아데노바이러스, 아데노-관련 바이러스, 레트로바이러스, 헤르페스 심플렉스 바이러스, 및 배시니아 바이러스로 구성되는 군으로부터 선택되는 것일 수 있다. As used herein, the term "vector" refers to a vector capable of expressing a protein of interest in a suitable host cell, and refers to a genetic construct comprising a regulatory element operably linked to express a gene insert. The vector according to an embodiment may include an expression control element such as a promoter, an operator, an initiation codon, a stop codon, a polyadenylation signal, and/or an enhancer, and the promoter of the vector may be constitutive or inducible. Further, the vector may be an expression vector capable of stably expressing the fusion protein in a host cell. The expression vector may be a conventional one used in the art to express foreign proteins in plants, animals or microorganisms. The recombinant vector can be constructed through various methods known in the art. For example, the vector may include a selectable marker for selecting a host cell containing the vector, and in the case of a replicable vector, may include an origin of replication. In addition, the vector can be self-replicating or introduced into the host DNA, and the vector is selected from the group consisting of plasmid, lentivirus, adenovirus, adeno-associated virus, retrovirus, herpes simplex virus, and basinia virus. Can be.
상기 벡터는 동물세포, 예를 들어, 포유동물 세포에서 작동가능한 프로모터를 포함한다. 일 실시예에 따라 적합한 프로모터는 포유동물 바이러스로부터 유래된 프로모터 및 포유동물 세포의 지놈으로부터 유래된 프로모터를 포함하며, 예컨대, CMV (Cytomegalovirus) 프로모터, U6 프로모터 및 H1 프로모터, MLV(Murine Leukemia Virus) LTR(Long terminal repeat) 프로모터, 아데노바이러스 초기 프로모터, 아데노바이러스 후기 프로모터, 백시니아 바이러스 7.5K 프로모터, SV40 프로모터, HSV의 tk 프로모터, RSV 프로모터, EF1 알파 프로모터, 메탈로티오닌 프로모터, 베타-액틴 프로모터, 인간 IL-2 유전자의 프로모터, 인간 IFN 유전자의 프로모터, 인간 IL-4 유전자의 프로모터, 인간 림포톡신 유전자의 프로모터, 인간 GM-CSF 유전자의 프로모터, 인간 포스포글리세레이트 키나아제(PGK) 프로모터, 마우스 포스포글리세레이트 키나아제(PGK) 프로모터 및 설바이빈 (Survivin) 프로모터를 포함할 수 있다. The vector includes a promoter operable in animal cells, for example, mammalian cells. Suitable promoters according to an embodiment include promoters derived from mammalian virus and promoters derived from the genome of mammalian cells, such as CMV (Cytomegalovirus) promoter, U6 promoter and H1 promoter, MLV (Murine Leukemia Virus) LTR (Long terminal repeat) promoter, adenovirus early promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, RSV promoter, EF1 alpha promoter, metallotionine promoter, beta-actin promoter, Promoter of human IL-2 gene, promoter of human IFN gene, promoter of human IL-4 gene, promoter of human lymphotoxin gene, promoter of human GM-CSF gene, human phosphoglycerate kinase (PGK) promoter, mouse force Phoglycerate kinase (PGK) promoter and sulvivin promoter.
또한, 상기 벡터에서, 전술한 융합 단백질은 프로모터에 작동 가능하게 연결되어 있을 수 있다. 본 명세서에서 사용된 용어, "작동 가능하게 연결된"은 핵산 발현 조절 서열(예: 프로모터, 시그널 서열, 또는 전사조절인자 결합 위치의 어레이)과 다른 핵산 서열사이의 기능적인 결합을 의미하며, 이에 의해 상기 조절 서열은 상기 다른 핵산 서열의 전사 및/또는 번역을 조절하게 된다.In addition, in the vector, the fusion protein described above may be operably linked to a promoter. As used herein, the term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (eg, a promoter, a signal sequence, or an array of transcriptional regulatory factor binding sites) and another nucleic acid sequence, thereby The regulatory sequence controls the transcription and/or translation of the other nucleic acid sequence.
또 다른 양상은 상기 융합 단백질, 폴리뉴클레오티드, 또는 벡터를 포함하는, 숙주 세포를 제공한다. Another aspect provides a host cell comprising the fusion protein, polynucleotide, or vector.
상기 세포, 예를 들면, 진핵 세포는 효모, 곰팡이, 원생동물 (protozoa), 식물, 고등 식물 및 곤충, 또는 양서류의 세포, 또는 CHO, HeLa, HEK293, 및 COS-1과 같은 포유 동물의 세포일 수 있고, 예를 들어, 당업계에서 일반적으로 사용되는, 배양된 세포 (인 비트로), 이식된 세포 (graft cell) 및 일차 세포 배양 (인 비트로 및 엑스 비보(ex vivo)), 및 인 비보 (in vivo) 세포, 및 또한 인간을 포함하는 포유동물의 세포 (mammalian cell)일 수 있다. 또한, 상기 유기체는 효모, 곰팡이, 원생동물, 식물, 고등 식물 및 곤충, 양서류, 또는 포유 동물일 수 있다. 또한, 상기 세포는 동물 세포 또는 식물세포일 수 있다.The cells, e.g., eukaryotic cells, are yeast, fungi, protozoa, plants, higher plants and insects, or cells of amphibians, or cells of mammals such as CHO, HeLa, HEK293, and COS-1. Can be, for example, cultured cells (in vitro), transplanted cells (graft cells) and primary cell cultures (in vitro and ex vivo), and in vivo ( in vivo) cells, and also mammalian cells, including humans. In addition, the organism may be yeast, fungi, protozoa, plants, higher plants and insects, amphibians, or mammals. In addition, the cells may be animal cells or plant cells.
일 구체예에 있어서, 상기 리포좀은 나노 리포좀인 것일 수 있다.In one embodiment, the liposome may be a nano liposome.
일 구체예에 있어서, 상기 리포좀은 입자 크기가 10nm 내지 250nm인 것일 수 있다.In one embodiment, the liposome may have a particle size of 10 nm to 250 nm.
일 구체예에 있어서, 상기 리포좀은 인지질, 스테로이드 및 PEG 함유한 인지질로 구성된 군으로부터 선택되는 하나 이상의 지질을 포함하는 것일 수 있다.In one embodiment, the liposome may contain one or more lipids selected from the group consisting of phospholipids, steroids and PEG-containing phospholipids.
본 명세서에서 용어 "리포좀"은 적어도 하나의 지질 이중층에 의해 에워싸인 수성 구획을 포함한다. 친수성 헤드 기를 포함한 지질이 물에 분산될 때, 이들은 층판(lamellae)이라고 불리는 이중층 막을 자발적으로 형성할 수 있다. 층판은 서로 대면하는 그들의 비-극성 (소수성) 표면 및 수성 매질과 대면하는 그들의 극성 (친수성) 표면을 가진 지질 분자의 2개 단층 시트로 구성된다. 용어 리포좀은 단일 지질 이중층으로 이루어진 단일층상 소포를 포함하고 일반적으로 약 10 내지 약 400 nm, 약 50 내지 약 300 nm, 또는 약 100 내지 200 nm 범위의 직경을 갖는다. 리포좀은 또한 다중층상일 수 있고, 일반적으로 1 내지 10 ㎛ 범위의 직경을 갖고 어느 곳에서라도 2개 내지 수 백개의 동심 지질 이중층이 수성 상의 층과 교대한다. 일부 실시양태에서, 리포좀은 다중층상 소포 (MLV), 큰 단일층상 소포 (LUV) 및 작은 단일층상 소포 (SUV)를 포함할 수 있다. 리포좀의 지질은 양이온성, 양쪽성, 중성 또는 음이온성, 또는 이들의 어느 혼합물일 수 있다.As used herein, the term “liposome” includes an aqueous compartment surrounded by at least one lipid bilayer. When lipids containing hydrophilic head groups are dispersed in water, they can spontaneously form a bilayer film called a lamellar. The lamellar consists of two monolayer sheets of lipid molecules with their non-polar (hydrophobic) surface facing each other and their polar (hydrophilic) surface facing the aqueous medium. The term liposome includes monolayered vesicles consisting of a single lipid bilayer and generally has a diameter in the range of about 10 to about 400 nm, about 50 to about 300 nm, or about 100 to 200 nm. Liposomes may also be multilayered, generally having a diameter in the range of 1 to 10 μm and anywhere from 2 to hundreds of concentric lipid bilayers alternating with the layers of the aqueous phase. In some embodiments, liposomes may include multilayered vesicles (MLV), large monolayered vesicles (LUV) and small monolayered vesicles (SUVs). Liposomal lipids may be cationic, amphoteric, neutral or anionic, or any mixture thereof.
본 명세서에서 리포좀 또는 나노 리포좀은 상기 기재된 바와 같이 양이온성 지질, 양쪽성 지질, 중성 지질 또는 음이온성 지질을 포함하여 어떠한 적절한 지질을 함유할 수 있다. 적절한 지질은 지방, 왁스, 스테로이드, 콜레스테롤, 지용성 비타민, 모노글리세리드, 디글리세리드, 인지질, 스핑고지질, 당지질, 양이온성 또는 음이온성 지질, 유도체화 지질 등을 포함할 수 있다.Liposomes or nanoliposomes herein may contain any suitable lipid, including cationic lipids, amphoteric lipids, neutral lipids or anionic lipids as described above. Suitable lipids may include fats, waxes, steroids, cholesterol, fat-soluble vitamins, monoglycerides, diglycerides, phospholipids, sphingolipids, glycolipids, cationic or anionic lipids, derivatized lipids, and the like.
적절한 인지질은, 이에 한정되지 않지만, 포스파티딜콜린 (PC), 포스파티드산 (PA), 포스파티딜에탄올아민 (PE), 포스파티딜글리세롤 (PG), 포스파티딜세린 (PS), 및 포스파티딜이노시톨 (PI), 디미리스토일 포스파티딜 콜린 (DMPC), 디스테아로일 포스파티딜 콜린 (DSPC), 디올레오일 포스파티딜 콜린 (DOPC), 디팔미토일 포스파티딜 콜린 (DPPC), 디미리스토일 포스파티딜 글리세롤 (DMPG), 디스테아로일 포스파티딜 글리세롤 (DSPG), 디올레오일 포스파티딜 글리세롤 (DOPG), 디팔미토일 포스파티딜 글리세롤 (DPPG), 디미리스토일 포스파티딜 세린 (DMPS), 디스테아로일 포스파티딜 세린 (DSPS), 디올레오일 포스파티딜 세린 (DOPS), 디팔미토일 포스파티딜 세린 (DPPS), 디올레오일 포스파티딜 에탄올아민 (DOPE), 팔미토일올레오일포스파티딜콜린 (POPC), 팔미토일올레오일-포스파티딜에탄올아민 (POPE) 및 디올레오일-포스파티딜에탄올아민 4-(N-말레이미도메틸)-시클로헥산-1-카르복실레이트 (DOPE-mal), 디팔미토일 포스파티딜 에탄올아민 (DPPE), 디미리스토일포스포에탄올아민 (DMPE), 디스테아로일-포스파티딜-에탄올아민 (DSPE), 16-O-모노메틸 PE, 16-O-디메틸 PE, 18-1-트랜스 PE, 1-스테아로일-2-올레오일-포스파티딜에탄올아민 (SOPE), 1,2-디엘라이도일-sn-글리세로-3-포스포에탄올아민 (트랜스DOPE), 및 카르디올리핀을 포함한다. 지질 추출물, 예컨대 계란 PC, 심장 추출물, 뇌 추출물, 간 추출물 및 콩 PC가 본 발명에서 또한 유용하다. 일부 실시양태에서, 콩 PC는 하이드로 소이(Hydro Soy) PC (HSPC)를 포함할 수 있다. 특정한 실시양태에서, 지질은 유도체화 지질, 예컨대 PEG화(PEGylated) 지질을 포함할 수 있다. 유도체화 지질은 예를 들어 DSPE-PEG2000, 콜레스테롤-PEG2000, DSPE-폴리글리세롤, 또는 당 기술분야에 일반적으로 공지된 기타 유도체를 포함할 수 있다.Suitable phospholipids include, but are not limited to, phosphatidylcholine (PC), phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), and phosphatidylinositol (PI), dimyristo Il phosphatidyl choline (DMPC), distearoyl phosphatidyl choline (DSPC), dioleoyl phosphatidyl choline (DOPC), dipalmitoyl phosphatidyl choline (DPPC), dimyristoyl phosphatidyl glycerol (DMPG), distearoyl phosphatidyl Glycerol (DSPG), dioleoyl phosphatidyl glycerol (DOPG), dipalmitoyl phosphatidyl glycerol (DPPG), dimyristoyl phosphatidyl serine (DMPS), distearoyl phosphatidyl serine (DSPS), dioleoyl phosphatidyl serine (DOPS) ), dipalmitoyl phosphatidyl serine (DPPS), dioleoyl phosphatidyl ethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4 -(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl- Phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), 1, 2-dielidoyl-sn-glycero-3-phosphoethanolamine (transDOPE), and cardiolipin. Lipid extracts such as egg PC, heart extract, brain extract, liver extract and soybean PC are also useful in the present invention. In some embodiments, the soybean PC may comprise Hydro Soy PC (HSPC). In certain embodiments, the lipid may comprise a derivatized lipid, such as a PEGylated lipid. Derivatized lipids may include, for example, DSPE-PEG2000, cholesterol-PEG2000, DSPE-polyglycerol, or other derivatives generally known in the art.
본 명세서에서 리포좀 또는 나노 리포좀은 융합된 테트라시클릭 고난 고리 계의 존재를 특징으로 하는 스테로이드를 함유할 수도 있다. 스테로이드의 예는 이에 한정되지 않지만 콜레스테롤, 콜산, 프로게스테론, 코르티존, 알도스테론, 에스트라디올, 테스토스테론, 데히드로에피안드로스테론을 포함한다. 합성 스테로이드 및 그의 유도체가 또한 본 발명에서 사용하기 위해 고려된다.In the present specification, the liposome or nano-liposome may contain a steroid characterized by the presence of a fused tetracyclic hard ring system. Examples of steroids include, but are not limited to, cholesterol, cholic acid, progesterone, cortisone, aldosterone, estradiol, testosterone, and dehydroepiandrosterone. Synthetic steroids and derivatives thereof are also contemplated for use in the present invention.
양이온성 지질은 생리학적 조건 하에서 양으로 하전된 작용 기를 함유한다. 양이온성 지질은, 이에 한정되지 않지만, N,N-디올레일-N,N-디메틸암모늄 클로라이드 (DODAC), N,N-디스테아릴-N,N-디메틸암모늄 브로마이드 (DDAB), N-(1-(2,3-디올레오일옥시)프로필)-N,N,N-트리메틸암모늄 클로라이드 (DOTAP), N-(1-(2,3-디올레일옥시)프로필)-N,N,N-트리메틸암모늄 클로라이드 (DOTMA), N-[1-(2,3-디테트라데실옥시)프로필]-N,N-디메틸-N-히드록시에틸암모늄 브로마이드 (DMRIE), N-[1-(2,3-디올레일옥시)프로필]-N,N-디메틸-N-히드록시 에틸암모늄 브로마이드 (DORIE), 3β-[N-(N',N'-디메틸아미노에탄)카르바모일]콜레스테롤 (DC-Chol), 디메틸디옥타데실암모늄 (DDAB) 및 N,N-디메틸-2,3-디올레일옥시)프로필아민 (DODMA)를 포함한다.Cationic lipids contain positively charged functional groups under physiological conditions. Cationic lipids include, but are not limited to, N,N-dioleyyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-( 1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleyyloxy)propyl)-N,N,N -Trimethylammonium chloride (DOTMA), N-[1-(2,3-ditetradecyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N-[1-( 2,3-dioleyyloxy)propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide (DORIE), 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol ( DC-Chol), dimethyldioctadecylammonium (DDAB) and N,N-dimethyl-2,3-dioleyyloxy)propylamine (DODMA).
일부 실시양태에서, 인지질은 HSPC 및 DSPE-PEG(2000)이고, 스테로이드는 콜레스테롤을 이용할 수 있다. In some embodiments, the phospholipids are HSPC and DSPE-PEG (2000), and the steroid may utilize cholesterol.
본 명세서에서 리포좀 또는 나노 리포좀을 제공하기 위하여 지질의 어떠한 적절한 조합이라도 사용될 수 있다. 지질 조성물은 누출 속도, 안정성, 입자 크기, 제타 포텐셜, 단백질 결합, 생체내 순환, 및/또는 조직 또는 기관 내 축적과 같은 특징에 영향을 주도록 맞춰질 수 있다. 일부 실시양태에서, 존재하는 특정한 유형의 지질의 몰 백분율 (mol%)은 전형적으로 리포좀 또는 지질 나노입자에 존재하는 총 지질의 약 0% 내지 약 10%, 약 10% 내지 약 30%, 약 30% 내지 약 50%, 약 50% 내지 약 70%, 약 70% 내지 약 90%, 약 90% 내지 100%를 구성한다. 일부 실시양태에서, 리포좀 또는 나노 리포좀은 56 mol% HSPC, 39 mol% 콜레스테롤, 및 5 mol% DSPE-PEG(2000)을 포함한다.Any suitable combination of lipids may be used to provide liposomes or nanoliposomes herein. The lipid composition can be tailored to influence characteristics such as leakage rate, stability, particle size, zeta potential, protein binding, circulation in vivo, and/or accumulation in tissues or organs. In some embodiments, the molar percentage (mol%) of a particular type of lipid present is typically about 0% to about 10%, about 10% to about 30%, about 30 of the total lipids present in the liposome or lipid nanoparticle. % To about 50%, about 50% to about 70%, about 70% to about 90%, about 90% to 100%. In some embodiments, the liposome or nano liposome comprises 56 mol% HSPC, 39 mol% cholesterol, and 5 mol% DSPE-PEG (2000).
일 구체예에 있어서, 상기 글루타치온-S-전이효소와 리포좀의 결합은 GSH(Glutathione)에 의한 것일 수 있다. 즉, 상기 리포좀은 GSH(Glutathione)가 결합된 리포좀일 수 있다. 상기 GSH는 글루타치온-S-전이효소의 결합 부위로 작용하여 리포좀과 글루타치온-S-전이효소를 연결할 수 있다.In one embodiment, the binding of the glutathione-S-transferase and liposome may be caused by GSH (Glutathione). That is, the liposome may be a liposome to which GSH (Glutathione) is bound. The GSH may act as a binding site of glutathione-S-transferase to link liposomes with glutathione-S-transferase.
일 구체예에 있어서, 상기 하나의 리포좀에 상기 글루타치온-S-전이효소, 표적 세포 또는 표적 단백질 결합능을 갖는 단백질 및 링커가 포함된 단백질이 두개 이상 결합된 것일 수 있다. 즉, 하나의 리포좀에 두개 이상의 융합 단백질이 결합하여 표적 세포 또는 표적 단백질에 더 용이하게 결합하는 것일 수 있다.In one embodiment, the glutathione-S-transferase, a protein having the ability to bind to a target cell or a target protein, and a linker-containing protein may be two or more bound to the one liposome. That is, two or more fusion proteins may be bound to one liposome to more easily bind to a target cell or a target protein.
약물 전달체를 사용하여 개체 내로 전달할 수 있는 약학적 활성 성분의 종류는 항암제, 조영제(염료), 호르몬제, 항호르몬제, 비타민제, 칼슘제, 무기질 제제, 당류제, 유기산 제제, 단백질 아미노산 제제, 해독제, 효소 제제, 대사성 제제, 당뇨 병용제, 조직 부활 용약, 클로로필 제제, 색소제제, 종양 용약, 종양 치료제, 방사성 의약품, 조직 세포 진단제, 조직 세포 치료제, 항생 물질 제제, 항바이러스제, 복합항생물질제제, 화학요법제, 백신, 독소, 톡소이드, 항독소, 렙토스피라혈청, 혈액 제제, 생물학적 제제, 진통제, 면역원성 분자, 항히스타민제, 알레르기 용약, 비특이성 면역원 제제, 마취제, 각성제, 정신 신경 용제, 저분자 화합물, 핵산, 앱타머, 안티센스 핵산, 올리고뉴클레오타이드, 펩타이드, siRNA 및 마이크로 RNA 등을 포함할 수 있다. Types of active pharmaceutical ingredients that can be delivered into an individual using a drug delivery system include anticancer agents, contrast agents (dyes), hormones, anti-hormones, vitamins, calcium agents, inorganic agents, saccharides, organic acids, protein amino acids, antidote, Enzyme preparations, metabolic preparations, diabetes combination preparations, tissue revitalization preparations, chlorophyll preparations, pigment preparations, tumor preparations, tumor treatments, radioactive medicines, tissue cell diagnostics, tissue cell treatments, antibiotic preparations, antiviral preparations, complex antibiotic preparations, Chemotherapeutic agents, vaccines, toxins, toxoids, antitoxins, leptospira serum, blood products, biological agents, analgesics, immunogenic molecules, antihistamines, drugs for allergies, nonspecific immunogenic agents, anesthetics, stimulants, psychotropic agents, small molecule compounds, nucleic acids , Aptamers, antisense nucleic acids, oligonucleotides, peptides, siRNAs and micro RNAs.
일 구체예에 있어서, 상기 약물은 항암제인 것일 수 있다.In one embodiment, the drug may be an anticancer agent.
상기 항암제는 캄토테신(camptothecin), 독소루비신(doxorubicin), 시스플라틴(cisplatin), 베라파밀(Verapamil), 플루오로우라실(fluorouracil), 옥살리플라틴(Oxaliplatin), 다우노루비신(Daunorubicin), 이리노테칸(irinotecan), 토포테칸(topotecan), 파클리탁셀(paclitaxel), 카보플라틴(carboplatin), 젬시타빈(Gemcitabine), 메소트렉세이트(Methotrexalte), 도세탁셀(Docetaxel), 아시바이신, 아클라루비신, 아코다졸, 아크로나이신, 아도젤레신, 알라노신, 알데스루킨, 알로푸리놀 소듐, 알트레타민, 아미노글루테티미드, 아모나파이드, 암플리겐, 암사크린, 안드로겐스, 안구이딘, 아피디콜린 글리시네이트, 아사레이, 아스파라기나아제, 5-아자시티딘, 아자티오프린, 바실러스 칼메테-구에린(BCG), 베이커스 안티폴, 베타-2-디옥시티오구아노신, 비스안트렌 HCl, 블레오마이신 설페이트, 불서판, 부티오닌 설폭시민, BWA 773U82, BW 502U83/HCl, BW 7U85 메실레이트, 세라세미드, 카르베티머, 카르보플라틴, 카르무스틴, 클로람부실, 클로로퀴녹살린-설포나미드, 클로로조토신, 크로모마이신 A3, 시스플라틴, 클라드리빈, 코르티코스테로이드, 코리너박테리움 파르붐, CPT-11, 크리스나톨, 사이클로사이티딘, 사이클로포스파미드, 사이타라빈, 사이템베나, 다비스 말리에이트, 데카르바진, 닥티노마이신, 다우노루바이신 HCl, 디아자유리딘, 덱스라족산, 디언하이드로갈락티톨, 디아지쿠온, 디브로모둘시톨, 디데민 B, 디에틸디티오카르바메이트, 디클라이코알데하이드, 다이하이드로-5-아자사이틴, 에치노마이신, 데다트렉세이트, 에델포신, 에플롤니틴, 엘리옷스 용액, 엘사미트루신, 에피루비신, 에소루비신, 에스트라머스틴 포스페이트, 에스트로겐, 에타니다졸, 에티오포스, 에토포사이드, 파드라졸, 파자라빈, 펜레티나이드, 필그라스팀, 피나스테라이드, 플라본 아세트산, 플록스유리딘, 플루다라빈 포스페이트, 5-플루오로우라실, Fluosol쪠, 플루타미드, 갈륨 나이트레이트, 겜사이타빈, 고세레린 아세테이트, 헤프설팜, 헥사메틸렌 비스아세트아미드, 호모하링토닌, 하이드라진 설페이트, 4-하이드록시안드로스테네디온, 하이드로지우레아, 이다루비신 HCl, 이포스파미드, 인터페론 알파, 인터페론 베타, 인터페론 감마, 인터루킨-1 알파 및 베타, 인터루킨-3, 인터루킨-4, 인터루킨-6, 4-이포메아놀, 이프로플라틴, 이소트레티노인, 류코보린 칼슘, 류프로라이드 아세테이트, 레바미솔, 리포좀 다우노루비신, 리포좀 포집 독소루비신, 로머스틴, 로니다민, 마이탄신, 메클로레타민 하이드로클로라이드, 멜팔란, 메노가릴, 메르바론, 6-머캅토푸린, 메스나, 바실러스 칼레테-구에린의 메탄올 추출물, 메토트렉세이트, N-메틸포름아미드, 미페프리스톤, 미토구아존, 마이토마이신-C, 미토탄, 미톡산트론 하이드로클로라이드, 모노사이트/마크로파아지 콜로니-자극 인자, 나빌론, 나폭시딘, 네오카르지노스타틴, 옥트레오타이드 아세테이트, 오르마플라틴, 옥살리플라틴, 파크리탁셀, 팔라, 펜토스타틴, 피페라진디온, 피포브로만, 피라루비신, 피리트렉심, 피록산트론 하이드로클로라이드, PIXY-321, 플리카마이신, 포르피머 소듐, 프레드니무스틴, 프로카르바진, 프로게스틴스, 파이라조푸린, 라족산, 사르그라모스팀, 세무스틴, 스피로게르마늄, 스피로무스틴, 스트렙토나이그린, 스트렙토조신, 술로페너르, 수라민 소듐, 타목시펜, 탁소테레, 테가푸르, 테니포사이드, 테레프탈아미딘, 테록시론, 티오구아닌, 티오테파, 티미딘 인젝션, 티아조푸린, 토포테칸, 토레미펜, 트레티노인, 트리플루오페라진 하이드로클로라이드, 트리플루리딘, 트리메트렉세이트, TNF(tumor necrosis factor), 우라실 머스타드, 빈블라스틴 설페이트, 빈크리스틴 설페이트, 빈데신, 비노렐빈, 빈졸리딘, Yoshi 864, 조루비신, 이의 약학적으로 허용가능한 염 및 이들의 혼합물 등을 포함할 수 있다. The anticancer drugs are camptothecin, doxorubicin, cisplatin, verapamil, fluorouracil, oxaliplatin, daunorubicin, daunorubicin, irinotecan, and irinotecan. (topotecan), paclitaxel, carboplatin, gemcitabine, methotrexalte, docetaxel, asibicin, aclarubicin, acodazole, acronycin, ado Gelesin, Alanosine, Aldesleukin, Allopurinol Sodium, Altretamine, Aminoglutethymide, Amonapide, Ampligen, Amsacrine, Androgens, Anguidine, Apidicholine Glycinate, Asarei , Asparaginase, 5-azacytidine, azathioprine, Bacillus calmethe-guerine (BCG), Bakers antipol, beta-2-dioxythioguanosine, bisantrene HCl, bleomycin sulfate, Bulseopan, butionine sulfoximine, BWA 773U82, BW 502U83/HCl, BW 7U85 mesylate, cerasemide, carbetimer, carboplatin, carmustine, chlorambucil, chloroquinoxaline-sulfonamide, Chlorozotosine, Chromomycin A3, Cisplatin, Cladribine, Corticosteroid, Corinerbacterium parvum, CPT-11, Crisnatol, Cyclocytidine, Cyclophosphamide, Cytarabine, Cytembena, Dabis Maleate, decarbazine, dactinomycin, daunorubicin HCl, diazauridine, dexrazoic acid, dianhydrogalactitol, diaziquone, dibromodulcitol, didemin B, diethyldithiocarbamate , Diclicoaldehyde, dihydro-5-azacytine, ethinomycin, dedatrexate, edelfosin, eflonitin, ellios solution, elsamitleucine, epirubicin, esoleubicin, estramustine phosphate , Estrogen, ethanidazole, ethiophos, etoposide, fadazole, pajarabine, fenretinide, filgrastim, finasteride, plaque Acetic acid, phloxuridine, fludarabine phosphate, 5-fluorouracil, fluosol, flutamide, gallium nitrate, gemcytabine, goserelin acetate, hefsulfam, hexamethylene bisacetamide, homoharlingtonin , Hydrazine sulfate, 4-hydroxyandrostenedione, hydrodiurea, idarubicin HCl, ifosfamide, interferon alpha, interferon beta, interferon gamma, interleukin-1 alpha and beta, interleukin-3, interleukin-4, Interleukin-6, 4-ipomeanol, iproplatin, isotretinoin, leucovorin calcium, leuprolide acetate, levamisol, liposome daunorubicin, liposome capture doxorubicin, romastin, ronidamine, mytansine, mechloretamine Hydrochloride, Melphalan, Menogaryl, Merbaron, 6-mercaptopurine, Mesna, Methanol Extract of Bacillus Calete-Guerine, Methotrexate, N-methylformamide, Mifepristone, Mitoguazone, Mitomycin-C , Mitotan, mitoxantrone hydrochloride, monosite/macrophage colony-stimulating factor, nabilon, napoxidine, neocarzinostatin, octreotide acetate, ormaplatin, oxaliplatin, paclitaxel, pala, pento Statin, piperazindione, pipebroman, pyrarubicin, pyritrexim, pyroxantrone hydrochloride, PIXY-321, plicamycin, porpimer sodium, prednimustine, procarbazine, progestins, pyrazopurine , Razox acid, Sargramostim, Semustine, Spirogermanium, Spiromustine, Streptonigreen, Streptozosine, Sulopener, Suramine Sodium, Tamoxifen, Taxotere, Tegapur, Teniposide, Terephthalamidine, Teroxirone, thioguanine, thiotepa, thymidine injection, thiazopurine, topotecan, toremifene, tretinoin, trifluoroperazine hydrochloride, trifluridine, trimetrexate, TNF (tumor necrosis factor), uracil mustard , Vinblastine sulfate, vincristine sulfate, vindesine, vinorelbine, vinzolidin, Yoshi 864, zorubicin, pharmaceutically acceptable salts thereof, and mixtures thereof And the like.
또 다른 양상은 글루타치온-S-전이효소(glutathione-S-transferase, GST); 표적 세포 또는 표적 단백질 결합능을 갖는 단백질; 상기 글루타치온-S-전이효소와 표적 세포 또는 표적 단백질 결합능을 갖는 단백질을 연결하는 링커; 및 상기 글루타치온-S-전이효소와 항암제를 담지하는 리포좀이 결합된 것을 포함하는 암 예방 또는 치료용 약학적 조성물을 제공한다. Another aspect is glutathione-S-transferase (GST); A protein having a target cell or target protein binding ability; A linker connecting the glutathione-S-transferase and a protein having the ability to bind to a target cell or a target protein; And it provides a pharmaceutical composition for preventing or treating cancer comprising the glutathione-S-transferase and liposome carrying an anticancer agent are bound.
또 다른 양상은 글루타치온-S-전이효소(glutathione-S-transferase, GST); 표적 세포 또는 표적 단백질 결합능을 갖는 단백질; 상기 글루타치온-S-전이효소와 표적 세포 또는 표적 단백질 결합능을 갖는 단백질을 연결하는 링커; 및 상기 글루타치온-S-전이효소와 항암제를 담지하는 리포좀을 함유하는 조성물을 그를 필요로 하는 개체에 투여하는 단계를 포함하는 암을 예방하거나 치료하는 방법을 제공한다.Another aspect is glutathione-S-transferase (GST); A protein having a target cell or target protein binding ability; A linker connecting the glutathione-S-transferase and a protein having binding ability to a target cell or a target protein; And it provides a method for preventing or treating cancer comprising administering a composition containing a liposome carrying the glutathione-S-transferase and an anticancer agent to an individual in need thereof.
또 다른 양상은 글루타치온-S-전이효소(glutathione-S-transferase, GST); 표적 세포 또는 표적 단백질 결합능을 갖는 단백질; 상기 글루타치온-S-전이효소와 표적 세포 또는 표적 단백질 결합능을 갖는 단백질을 연결하는 링커; 및 상기 글루타치온-S-전이효소와 항암제를 담지하는 리포좀을 함유하는 조성물을 암 예방 또는 치료에 사용하기 위한 용도를 제공한다.Another aspect is glutathione-S-transferase (GST); A protein having a target cell or target protein binding ability; A linker connecting the glutathione-S-transferase and a protein having the ability to bind to a target cell or a target protein; And a composition containing a liposome carrying the glutathione-S-transferase and an anticancer agent is provided for use in cancer prevention or treatment.
상기 글루타치온-S-전이효소(glutathione-S-transferase, GST), 표적 세포 또는 표적 단백질 결합능을 갖는 단백질, 상기 글루타치온-S-전이효소와 표적 세포 또는 표적 단백질 결합능을 갖는 단백질을 연결하는 링커 및 상기 글루타치온-S-전이효소와 결합된 리포좀에 대해서는 상기한 바와 같다. The glutathione-S-transferase (GST), a protein having a target cell or target protein binding ability, a linker connecting the glutathione-S-transferase and a protein having a target cell or target protein binding ability, and the Liposomes bound to glutathione-S-transferase are as described above.
용어 "치료제" 또는 "약학적 조성물"는, 대상체로의 투여 시에 몇몇 유리한 효과를 부여하는 분자 또는 화합물을 지칭한다. 유리한 효과는 진단적 결정을 가능하게 하는 것; 질병, 증상, 장애 또는 병태의 개선; 질병, 증상, 장애 또는 질환의 발병의 감소 또는 예방; 및 일반적으로 질병, 증상, 장애 또는 병태의 대응을 포함한다.The term “therapeutic agent” or “pharmaceutical composition” refers to a molecule or compound that imparts several beneficial effects upon administration to a subject. The beneficial effect is to enable diagnostic decisions; Improvement of a disease, symptom, disorder or condition; Reducing or preventing the onset of a disease, symptom, disorder or condition; And the response of a disease, symptom, disorder or condition in general.
상기 암은 폐암(예를 들면, 비소세포성 폐암), 췌장암, 위암, 간암, 대장암, 뇌암, 유방암, 갑상선암, 방광암, 식도암, 또는 자궁암일 수 있다. 또한, 상기 암은 항암제에 대한 내성(예를 들면, 다제 내성)을 갖는 위암, 유방암, 폐암, 간암, 식도암 및 전립선암으로 이루어진 군으로부터 선택되는 어느 하나 이상인 것일 수 있다. The cancer may be lung cancer (eg, non-small cell lung cancer), pancreatic cancer, gastric cancer, liver cancer, colon cancer, brain cancer, breast cancer, thyroid cancer, bladder cancer, esophageal cancer, or uterine cancer. In addition, the cancer may be any one or more selected from the group consisting of gastric cancer, breast cancer, lung cancer, liver cancer, esophageal cancer, and prostate cancer having resistance to anticancer drugs (eg, multi-drug resistance).
상기 약학적 조성물은 임상투여시 비경구로 투여가 가능하며 일반적인 의약품 제제의 형태로 사용될 수 있다. 비경구 투여는 직장, 정맥, 복막, 근육, 동맥, 경피, 비강(Nasal), 흡입, 안구 및 피하와 같은 경구 이외의 투여경로를 통한 투여를 의미할 수 있다. 본 발명의 상기 약학적 조성물을 의약품으로 사용하는 경우, 추가로 동일 또는 유사한 기능을 나타내는 유효성분을 1종 이상 함유할 수 있다.The pharmaceutical composition can be administered parenterally during clinical administration and can be used in the form of a general pharmaceutical formulation. Parenteral administration may mean administration through a route other than oral administration such as rectal, intravenous, peritoneal, muscle, arterial, transdermal, nasal, inhalation, ocular and subcutaneous. When using the pharmaceutical composition of the present invention as a pharmaceutical, it may further contain one or more active ingredients exhibiting the same or similar functions.
상기 약학적 조성물을 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 비경구투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁용제로는 프로필렌글리콜(Propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(Witepsol), 마크로골, 트윈(Tween) 61, 카카오지, 리우린지, 글리세로제라틴 등이 사용될 수 있다. When formulating the pharmaceutical composition, it is prepared by using a diluent or excipient such as a commonly used filler, extender, binder, wetting agent, disintegrant, and surfactant. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for the suppository, Witepsol, Macrogol, Tween 61, cacao butter, liurinji, glycerogelatin, and the like may be used.
또한, 상기 약학적 조성물은 생리식염수 또는 유기용매와 같이 약제로 허용된 여러 전달체(Carrier)와 혼합하여 사용될 수 있고, 안정성이나 흡수성을 증가시키기 위하여 글루코스, 수크로스 또는 덱스트란과 같은 탄수화물, 아스코르브산(Ascorbic acid) 또는 글루타치온(Glutathione)과 같은 항산화제(Antioxidants), 킬레이트화제(Chelating agents), 저분자 단백질 또는 다른 안정화제(Stabilizers)들이 약제로 사용될 수 있다.In addition, the pharmaceutical composition may be used in combination with various carriers (Carriers) allowed as drugs such as physiological saline or organic solvents, and carbohydrates such as glucose, sucrose or dextran, ascorbic acid in order to increase stability or absorption. Antioxidants such as (Ascorbic acid) or glutathione, chelating agents, small molecule proteins or other stabilizers can be used as drugs.
상기 약학적 조성물의 유효용량은 0.01 내지 100 ㎎/㎏이고, 바람직하게는 0.1 내지 10 ㎎/㎏ 이며, 하루 1회 내지 3회 투여될 수 있다.The effective dose of the pharmaceutical composition is 0.01 to 100 mg/kg, preferably 0.1 to 10 mg/kg, and may be administered once to three times a day.
일 구체예에 있어서, 상기 항암제는 아바렐릭스 [abarelix (Plenaxis depot®)]; 알데스류킨 [aldesleukin (Prokine®)]; 알데스류킨 [Aldesleukin (Proleukin®)]; 알렘투주맙 [Alemtuzumabb (Campath®)]; 알리트레티노인 [alitretinoin (Panretin®)]; 알로퓨리놀 [allopurinol (Zyloprim®)]; 알트레타민 [altretamine (Hexalen®)]; 아미포스틴 [amifostine (Ethyol®)]; 아나스트로졸 [anastrozole (Arimidex®)]; 아르세닉 트리옥사이드 [arsenic trioxide (Trisenox®)]; 아스파라기나아제 [asparaginase (Elspar®)]; 아자시티딘 [azacitidine (Vidaza®)]; 베바쿠지맙 [bevacuzimab (Avastin®)]; 벡사로텐 캡슐 [bexarotene capsules (Targretin®)]; 벡사로텐 겔 [bexarotene gel (Targretin®)]; 블레오마이신 [bleomycin (Blenoxane®)]; 보르테조밉 [bortezomib (Velcade®)]; 정맥용 부술판 [busulfan intravenous (Busulfex®)]; 경구용 부술판 [busulfan oral (Myleran®)]; 칼루스테론 [calusterone (Methosarb®)]; 카페시타빈 [capecitabine (Xeloda®)]; 카르보플라틴 [carboplatin (Paraplatin®)]; 카르무스틴 [carmustine (BCNU®, BiCNU®)]; 카르무스틴 [carmustine (Gliadel®)]; 카르무스틴과 폴리페프로산 20 임플란트 (Polifeprosan 20 Implant) (Gliadel Wafer®); 셀레콕십 [celecoxib (Celebrex®)]; 세툭시맙 [cetuximab (Erbitux®)]; 클로람부실 [chloambucil (Leukeran®)]; 시스플라틴 [cisplatin (Platinol®)]; 클라드리빈 [cladribine (Leustatin®) 2-CdA®]; 클로파라빈 [clofarabine (Clolar®)]; 사이클로포스파미드 [cyclophosphamide (Cytoxan®, Neosar®)]; 사이클로포스파미드 [cyclophosphamide (Cytoxan Injection®)]; 사이클로포스파미드 [cyclophosphamide (Cytoxan Tablet®)]; 사이타라빈 [cytarabine (Cytosar-U®)]; 사이타라빈 리포조말 [cytarabine liposomal (DepoCyt®)]; 다카르바진 [dacarbazine (DTIC-Dome®)]; 닥티노마이신 (dactinomycin), 액티노마이신 D [actinomycinD (Cosmegen®)]; 다르베포에틴 알파 [Darbepoetin alfa (Aranesp®)]; 다우노루비신 리포조말 [daunorubicin liposomal (Danuoxome®)]; 다우노루비신 (daunorubicin), 다우노마이신 [daunomycin (Daunorubicin®)]; 다우노루비신 (daunorubicin), 다우노마이신 [daunomycin (Cerubidine®)]; 데니류킨 디프티톡스 [Denileukin diftitox (Ontak®)]; 덱스라조산 [dexrazoxane (Zinecard®)]; 도세탁셀 [docetaxel (Taxotere®)]; 독소루비신 [doxorubicin (Adriamycin PFS®)]; 독소루비신 [doxorubicin (Adriamycin®, Rubex®)]; 독소루비신 [doxorubicin (Adriamycin PFS Injection®)]; 독소루비신 리포조말 [doxorubicin liposomal (Doxil®)]; 드로모스타놀론 프로피오네이트 [dromostanolone propionate (dromostanolone®)]; 드로모스타놀론 프로피오네이트 [dromostanolone propionate (masterone injection®)]; 엘리엇 B 용액 [Elliott's B Solution (Elliott's B Solution®)]; 에피루비신 [epirubicin (Ellence®)]; 이포에틴 알파 [Epoetin alfa (epogen®)]; 에를로티닙 [erlotinib (Tarceva®)]; 에스트라무스틴 [estramustine (Emcyt®)]; 에토포사이드 포스페이트 [etoposide phosphate (Etopophos®)]; 에토포사이드 (etoposide), VP-16 (Vepesid®); 엑세메스탄 [exemestane (Aromasin®)]; 필그라스팀 [Filgrastim (Neupogen®)]; 플록수리딘 [floxuridine (intraarterial) (FUDR®)]; 플루다라빈 [fludarabine (Fludara®)]; 플루오로우라실 [Fluorouracil, 5-FU (Adrucil®)]; 플루베스트란트 [folvestrant (Faslodex®)]; 게피티닙 [gefitinib (Iressa®)]; 겜시타빈 [gemcitabine (Gemzar®)]; 겜투주맙 오조가미신 [gemtuzumab ozogamicin (Mylotarg®)]; 고세렐린 아세테이트 [goserelin acetate (Zoladex Implant®)]; 고세렐린 아세테이트 [goserelin acetate (Zoladex®)]; 히스트렐린 아세테이트 [histrelin acetate (Histrelin implant®)]; 하이드록시우레아 [hydroxyurea (Hydrea®)]; 이브리투모맙 티욱세탄 [Ibritumomab Tiuxetan (Zevalin®)]; 이다루비신 [idarubicin (Idamycin®)]; 이포스파미드 [ifosfamide (IFEX®)]; 이마티닙 메실레이트 [imatinib mesylate (Gleevec®)]; 인터페론 알파-2a [interferon alfa-2a (Roferon A®)]; 인터페론 알파-2b [Interferon alfa-2b (Intron A®)]; 이리노테칸 [irinotecan (Camptosar®)]; 레날리도마이드 [lenalidomide (Revlimid®)]; 레트로졸 [letrozole (Femara®)]; 류코보린 [leucovorin (Wellcovorin®, Leucovorin®)]; 류프롤리드 아세테이트 [Leuprolide Acetate (Eligard®)]; 레바미솔 [levamisole (Ergamisol®)]; 로무스틴-CCNU [lomustine-CCNU (CeeBU®)]; 메클로레타민 (meclorethamine), 니트로겐 머스타드 [nitrogen mustard (Mustargen®)]; 메게스트롤 아세테이트 [megestrol acetate (Megace®)]; 멜파란 (melphalan), L-PAM (Alkeran®); 머캅토퓨린 [mercaptopurine, 6-MP (purinethol®)]; 메스나 [mesna (Mesnex®)]; 메스나 [mesna (Mesnex tabs®)]; 메토트렉제이트 [methotraxate (methotraxate®)]; 메톡스살렌 [methoxsalen (Uvadex®)]; 미토마이신 C [mitomycin C (Mutamycin®)]; 미토탄 [mitotane (Lysodren®)]; 미토잔트론 [mitoxantrone (Novantrone®)]; 난드롤란 펜프로피오네이트 [nandrolone phenpropionate (Durabolin-50®)]; 넬라라빈 [nelarabine (Arranon®)]; 노페투모맙 [Nofetumomab (Verluma®)]; 오프렐베킨 [Oprelvekin (Neumega®)]; 옥살리플라틴 [oxaliplatin (Eloxatin®)]; 파클리탁셀 [paclitaxel (Paxene®)]; 파클리탁셀 [paclitaxel (Taxol®)]; 파클리탁셀 단백질 결합 입자 [paclitaxel protein-bound particles (Abraxane®)]; 팔리페르민 [palifermin (Kepivance®)]; 파미드로네이트 [pamidronate (Aredia®)]; peg아데마스 [pegademase (Adagen (Pegademase Bovine)®)]; peg아스파르가아제 [pegaspargase (Oncaspar®)]; peg필그라스팀 [Pegfilgrastim (Neulasta®)]; 페멕트렉제트 디소듐 [pemetrexed disodium (Alimta®)]; 펜토스타틴 [pentostatin (Nipent®)]; 피포브로만 [pipobroman (Vercyte®)]; 플리카마이신 [plicamycin], 미트라마이신 [mithramycin (Mithracin®)]; 포르피머 소듐 [porfimer sodium (Photofrin®)]; 프로카르바진 [procarbazine (Matulane®)]; 퀴나크린 [quinacrine (Atabrine®)]; 라스뷰리카아제 [Rasburicase (Elitek®)]; 리툭시맙 [Rituximab (Rituxan®)]; 사르그라모스팀 [sargramostim (Leukine®)]; 사르그라모스팀 [Sargramostim (Prokine®)]; 소라페닙 [sorafenib (Nexavar®)]; 스트렙타조신 [streptozocin (Zanosar®)]; 수니티닙 말리에이트 [sunitinib maleate (Sutent®)]; 탈크 [talc (Sclerosol®)]; 타목시펜 [tamoxifen (Nolvadex®)]; 테모졸로마이드 [temozolomide (Temodar®)]; 테니포사이드 [teniposide, VM-26 (Vumon®)]; 테스톨락톤 [testolactone (Teslac®)]; 티오구아닌 [thioguanine, 6-TG (thioguanine®)]; 티오테파 [thiotepa (thioplex®)]; 토포테칸 [topotecan (Hycamtin®)]; 토레미펜 [toremifene (Fareston®)]; 토시투모맙 [Tositumomab (Bexxar®)]; 토시투모맙/I-131 토시투모맙 (Bexxar®); 트라스투주맙 [Trastuzumab (Herceptin®)]; 트레티노인 [tretinoin, ATRA (Vesanoid®)]; 우라실 머스타드 [Uracil Mustard (Uracil Mustard Capsules®)]; 발루비신 [valrubicin (Valstar®)]; 빈블라스틴 [vinblastine (Velban®)]; 빈크리스틴 [vincristine (Oncovin®)]; 비노렐빈 [vinorelbine (Navelbine®)]; 및 졸레드로네이트 [zoledronate (Zometa®)]로 이루어지는 군으로부터 선택되는 것일 수 있다.In one embodiment, the anticancer agent abarelix [abarelix (Plenaxis depot ® )]; Aldesleukin (Prokine ® )]; Aldesleukin [Aldesleukin (Proleukin ® )]; Alemtuzumabb (Campath ® )]; Alitretinoin (Panretin ® )]; Allopurinol [allopurinol (Zyloprim ® )]; Altretamine (Hexalen ® )]; Amifostine [amifostine (Ethyol ® )]; Anastrozole (Arimidex ® )]; Arsenic trioxide (Trisenox ® )]; Asparaginase [asparaginase (Elspar ® )]; Azacitidine (Vidaza ® )]; Bevacuzimab [bevacuzimab (Avastin ® )]; Bexarotene capsules (Targretin ® )]; Bexarotene gel (Targretin ® )]; Bleomycin [bleomycin (Blenoxane ® )]; Bortezomib (Velcade ® )]; Intravenous busulfan [busulfan intravenous (Busulfex ® )]; Busulfan oral (Myleran ® )]; Calusterone (Methosarb ® )]; Capecitabine (Xeloda ® )]; Carboplatin [carboplatin (Paraplatin ® )]; Carboxylic estramustine [carmustine (BCNU ®, BiCNU ® )]; Carmustine (Gliadel ® )]; 20 implant to carboxylic acid estramustine and poly pepeu (Polifeprosan 20 Implant) (Gliadel Wafer ®); Celecoxib (Celebrex ® )]; Cetuximab [cetuximab (Erbitux ® )]; Chlorambucil [chloambucil (Leukeran ® )]; Cisplatin (Platinol ® )]; Cladribine (Leustatin ® ) 2-CdA ® ]; Clofarabine (Clolar ® )]; Cyclophosphamide (Cytoxan ® , Neosar ® )]; Cyclophosphamide (Cytoxan Injection ® )]; Cyclophosphamide (Cytoxan Tablet ® )]; Cytarabine [cytarabine (Cytosar-U ® )]; Cytarabine liposomal (DepoCyt ® )]; Dacarbazine [dacarbazine (DTIC-Dome ® )]; Dactinomycin, actinomycin D [actinomycinD (Cosmegen ® )]; Darbepoetin alfa (Aranesp ® )]; Daunorubicin liposomal (Danuoxome ® )]; Daunorubicin, daunomycin [daunomycin (Daunorubicin ® )]; Daunorubicin, daunomycin [daunomycin (Cerubidine ® )]; Denileukin diftitox (Ontak ® )]; Dexrazoxane (Zinecard ® )]; Docetaxel (Taxotere ® )]; Doxorubicin [doxorubicin (Adriamycin PFS ® )]; Doxorubicin [doxorubicin (Adriamycin ® , Rubex ® )]; Doxorubicin [doxorubicin (Adriamycin PFS Injection ® )]; Doxorubicin liposomal (Doxil ® )]; Dromostanolone propionate (dromostanolone ® )]; Dromostanolone propionate (masterone injection ® )]; Elliott's B Solution [Elliott's B Solution ® )]; Epirubicin [epirubicin (Ellence ® )]; Ipoetin alfa [Epoetin alfa (epogen ® )]; Erlotinib [erlotinib (Tarceva ® )]; Estramustine (Emcyt ® )]; Etoposide phosphate (Etopophos ® )]; Etoposide (etoposide), VP-16 ( Vepesid ®); Exemestane [exemestane (Aromasin ® )]; Filgrastim (Neupogen ® )]; Floxuridine (intraarterial) (FUDR ® )]; Fludarabine [fludarabine (Fludara ® )]; Fluorouracil [Fluorouracil, 5-FU (Adrucil ® )]; Fluvestrant [folvestrant (Faslodex ® )]; Gefitinib [gefitinib (Iressa ® )]; Gemcitabine (Gemzar ® )]; Gemtuzumab ozogamicin (Mylotarg ® )]; Goserelin acetate (Zoladex Implant ® )]; Goserelin acetate (Zoladex ® )]; Hisstrelin acetate (Histrelin implant ® )]; Hydroxyurea (Hydrea ® )]; Ibritumomab Tiuxetan (Zevalin ® )]; Darubicin [idarubicin (Idamycin ® )]; Ifosfamide [ifosfamide (IFEX ® )]; Imatinib mesylate (Gleevec ® )]; Interferon alfa-2a [interferon alfa-2a (Roferon A ® )]; Interferon alfa-2b [Interferon alfa-2b (Intron A ® )]; Irinotecan [irinotecan (Camptosar ® )]; Lenalidomide (Revlimid ® )]; Letrozole (Femara ® )]; Leucovorin (Wellcovorin ® , Leucovorin ® )]; Leuprolide acetate [Leuprolide Acetate (Eligard ® )]; Levamisole (Ergamisol ® )]; Lomustine-CCNU (CeeBU ® )]; Meclorethamine, nitrogen mustard (Mustargen ® )]; Megestrol acetate (Megace ® )]; Mel blue (melphalan), (Alkeran ®) L-PAM; Mercapto-purine [mercaptopurine, 6-MP (purinethol ®)]; Mesna (Mesnex ® )]; Mesna [mesna (Mesnex tabs ® )]; Methotrexate [methotraxate (methotraxate ® )]; Methoxsalen (Uvadex ® )]; Mitomycin C [mitomycin C (Mutamycin ® )]; Mitotan [mitotane (Lysodren ® )]; Mitoxantrone [mitoxantrone (Novantrone ® )]; Nandrolone phenpropionate (Durabolin-50 ® )]; Nelarabine (Arranon ® )]; Nofetumomab (Verluma ® )]; Oprelvekin [Neumega ® )]; Oxaliplatin (Eloxatin ® )]; Paclitaxel [paclitaxel (Paxene ® )]; Paclitaxel [paclitaxel (Taxol ® )]; Paclitaxel protein-bound particles [paclitaxel protein-bound particles (Abraxane ® )]; Palifermin [palifermin (Kepivance ® )]; Pamidronate (Aredia ® )]; pegademas [pegademase (Adagen (Pegademase Bovine) ® )]; peg aspargase [pegaspargase (Oncaspar ® )]; pegfilgrastim (Neulasta ® )]; Pemetrexed disodium (Alimta ® )]; Pentostatin [pentostatin (Nipent ® )]; Pipebroman [pipobroman (Vercyte ® )]; Plicamycin [mithramycin (Mithracin ® )]; Porfimer sodium [Photofrin ® )]; Procarbazine (Matulane ® )]; Quinacrine (Atabrine ® )]; Rasburicase [Rasburicase (Elitek ® )]; Rituximab [Rituximab (Rituxan ® )]; Sargramostim (Leukine ® )]; Sargramostim (Prokine ® )]; Sorafenib [sorafenib (Nexavar ® )]; Streptazosin [streptozocin (Zanosar ® )]; Sunitinib maleate (Sutent ® )]; Talc (Sclerosol ® )]; Tamoxifen [tamoxifen (Nolvadex ® )]; Temozolomide [temozolomide (Temodar ® )]; I'll Forsythe [teniposide, VM-26 (Vumon ®)]; Testolactone [testolactone (Teslac ® )]; Thioguanine [thioguanine, 6-TG (thioguanine ®)]; Thiotepa [thiotepa (thioplex ®)]; Topotecan (Hycamtin ® )]; Toremifene [toremifene (Fareston ® )]; Tositumomab [Tositumomab (Bexxar ® )]; Toshio Tomorrow Thank / I-131 Toshio Thank Tomorrow (Bexxar ®); Trastuzumab [Trastuzumab (Herceptin ® )]; Tretinoin [tretinoin, ATRA (Vesanoid ® )]; Uracil Mustard [Uracil Mustard (Uracil Mustard Capsules ® )]; Valrubicin [valrubicin (Valstar ® )]; Vinblastine (Velban ® )]; Vincristine (Oncovin ® )]; Vinorelbine (Navelbine ® )]; And zoledronate [zoledronate (Zometa ® )] may be selected from the group consisting of.
또한, 본 발명은 하기 i) 내지 iii) 단계를 포함하는, 단백질 코로나 외층을 가지는 리포좀 약물 전달체의 제조 방법을 제공한다:In addition, the present invention provides a method for producing a liposomal drug delivery system having an outer layer of protein corona, comprising steps i) to iii):
i) 리포좀 표면에 링커(예를 들면, GSH)를 결합시키는 단계; ii) 상기 단계 i)에서 링커와 결합된 리포좀의 내부 또는 표면에 약물을 담지시키는 단계; 및 iii) 상기 단계 ii)에서 약물을 담지한 리포좀 표면에 GST 융합 단백질을 코팅하여 단백질 코로나 외층(PCS)을 형성시키는 단계. i) linking a linker (eg, GSH) to the surface of the liposome; ii) loading the drug on the inside or surface of the liposome bonded to the linker in step i); And iii) forming a protein corona outer layer (PCS) by coating the GST fusion protein on the surface of the liposome carrying the drug in step ii).
상기 단백질 코로나 외층(protein corona shield)을 가지는 리포좀은 융합 단백질로 표면이 전-코팅된 단백질 코로나 외층을 먼저 생성하였기 때문에 체내 환경에서 혈청 단백질에 의해 둘러싸이는 코로나층이 형성되지 않아, 대식 세포에 의한 면역 반응을 회피할 수 있어 스텔스 효과(stealth effect)를 유의적으로 가질 수 있다. 따라서, 상기 단백질 코로나 외층을 가지는 리포좀은 생체 내 잔존 시간을 지속시킬 수 있을 뿐 아니라, 표적 세포로의 표적능이 향상되어 타겟으로 하는 세포로 효과적으로 전달될 수 있으므로, 표적 치료제로서 유용하게 사용될 수 있다.Since the liposome having the protein corona shield first produced a protein corona outer layer that is pre-coated with a fusion protein, a corona layer surrounded by serum proteins in the body environment is not formed. The immune response can be avoided and thus can have a significant stealth effect. Therefore, the liposome having the outer corona protein layer can not only sustain the remaining time in vivo, but also can be effectively delivered to the target cell by improving the targeting ability to the target cell, and thus can be usefully used as a target therapeutic agent.
일 양상에 따른 리포좀으로 구성된 약물 전달체에 의하면, 생체 내 잔존 시간을 지속시킬 수 있을 뿐 아니라, 표적 세포로의 표적능이 향상되어 타겟으로 하는 세포로 효과적으로 전달될 수 있으므로, 표적 치료제로서 유용하게 사용될 수 있는 효과가 있다. According to the drug delivery system composed of liposomes according to one aspect, not only can the remaining time in the living body be sustained, but also the targeting ability to the target cells is improved and can be effectively delivered to the target cells, so it can be usefully used as a target therapeutic agent. There is an effect.
도 1은 본 발명에서 리포좀의 단백질 코로나 외층을 이루는 융합단백질의 구조를 도식화한 것이다.1 is a schematic diagram of the structure of a fusion protein forming the outer layer of the protein corona of the liposome in the present invention.
도 2는 GST-Afb 융합 단백질의 세포 독성 및 세포 표적능을 확인한 결과이다:2 is a result of confirming the cytotoxicity and cell targeting ability of the GST-Afb fusion protein:
도 2a는 GST-Afb 융합 단백질이 Afb 표적 세포에 대하여 세포 생존율을 저하시키지 않음을 확인한 결과이고; 및 도 2b는 GST-Afb 융합 단백질이 Afb 표적 세포에 대하여 나타내는 세포 표적능을 확인한 결과이다.2A is a result of confirming that the GST-Afb fusion protein does not decrease cell viability with respect to Afb target cells; And FIG. 2B is a result of confirming the cell targeting ability of the GST-Afb fusion protein to Afb target cells.
도 3은 약물을 담지하는 리포좀의 구성요소 중 하나인 GSH-PEG-DSPE의 합성을 도식화한 것이다.3 is a schematic diagram of the synthesis of GSH-PEG-DSPE, one of the components of a liposome carrying a drug.
도 4는 약물을 담지하는 리포좀의 구성요소를 도식화한 것이다.4 is a schematic diagram of the components of a liposome carrying a drug.
도 5는 Dox-리포좀-GSH의 모양을 투과전자현미경으로 관찰한 이미지이다.5 is an image of the shape of Dox-liposome-GSH observed with a transmission electron microscope.
도 6은 Dox-리포좀-GSH의 크기를 동적 광산란으로 관찰한 그래프이다.6 is a graph of the size of Dox-liposome-GSH observed by dynamic light scattering.
도 7은 약물을 담지하는 리포좀에 글루타치온-S-전이효소(glutathione-S-transferase, GST)와 표적 세포 또는 표적 단백질 결합능을 갖는 단백질, 즉 표적지향 융합단백질이 결합한 것 (Dox-리포좀-융합단백질)을 도식화한 것이다.Figure 7 is a drug-bearing liposome with glutathione-S-transferase (GST) and a protein having the ability to bind to a target cell or a target protein, that is, a target-directed fusion protein (Dox-liposome-fusion protein ) Is a schematic diagram.
도 8은 Dox-리포좀-융합단백질의 크기를 동적 광산란으로 관찰한 그래프이다.Figure 8 is a graph observed by dynamic light scattering of the size of Dox-liposome-fusion protein.
도 9는 Dox-리포좀-융합단백질의 모양을 투과전자현미경으로 관찰한 이미지이다.9 is an image of the shape of a Dox-liposome-fusion protein observed with a transmission electron microscope.
도 10은 Dox-리포좀-GSH의 제타 전위를 나타낸 그래프이다.10 is a graph showing the zeta potential of Dox-liposome-GSH.
도 11은 Dox-리포좀-융합단백질의 제타 전위를 나타낸 그래프이다.11 is a graph showing the zeta potential of Dox-liposome-fusion protein.
도 12는 Dox-리포좀-융합단백질의 세포 독성을 나타낸 그래프이다.12 is a graph showing the cytotoxicity of Dox-liposome-fusion protein.
도 13은 Dox-리포좀-융합단백질의 항 종양 효과를 나타낸 사진 및 그래프이다.13 is a photograph and graph showing the anti-tumor effect of Dox-liposome-fusion protein.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.
실시예 1. 단백질 코로나 외층을 가지는 리포좀에 사용하기 위한 외층 융합 단백질의 발현Example 1.Expression of outer layer fusion protein for use in liposomes with protein corona outer layer
<1-1> 애피바디(affibody) 및 글루타치온-S-전이효소(glutathione-S-transferase, GST) 융합 단백질의 제조<1-1> Preparation of affibody and glutathione-S-transferase (GST) fusion protein
본 발명에서는 약물 전달체로 사용하기 위한 리포좀의 표면에 단백질 코로나의 외층을 형성시켜, 생체 내 환경에서도 안정성 및 표적능이 향상된 약물 전달체를 제조하고자 하였다. 이를 위해, 먼저 단백질 코로나 외층을 구성할 수 있는 융합 단백질(fusion protein)을 제조하였다. 융합 단백질은 암세포 표면의 수용체에 특이적으로 결합할 수 있는 애피바디(affibody, Afb)와 GST가 결합된 형태가 되도록 발현하였다.In the present invention, by forming an outer layer of protein corona on the surface of a liposome for use as a drug delivery system, it was intended to prepare a drug delivery system with improved stability and targeting ability even in an in vivo environment. To this end, first, a fusion protein capable of constituting the outer layer of the protein corona was prepared. The fusion protein was expressed in a form in which affibody (Afb) capable of specifically binding to a receptor on the surface of cancer cells and GST were combined.
구체적으로, 본 발명에서는 Afb로서 HER2에 특이적으로 결합하는 HER2 Afb 및 EGFR에 특이적으로 결합하는 EGFR Afb를 사용하였다. 각각의 HER2 Afb 또는 EGFR Afb를 암호화하는 유전자의 말단에 추가 링커 도메인(서열번호 1: GGGLVPRGSGGGCGGGGTGGGSGGG)을 암호화하는 유전자를 결합시킨 다음, pETduet 플라스미드에 삽입하였다. 또한, GST 과발현을 위해서 GST의 N-말단에 6ХHis 태그가 연결되도록 디자인한 GST 암호화 유전자를 상기 pETduet 플라스미드에 삽입하여, Afb와 GST가 링커로 연결된 융합 단백질(GST-Afb)의 과발현을 위한 플라스미드를 구축하였다. 구축된 플라스미드에서 PCR 및 DNA 서열분석을 통해 GST-Afb 암호화 서열이 정상적으로 삽입되었는지 확인하였다. 그런 다음, 구축된 플라스미드를 E. coli BL21(DE3) 균주에 삽입하여 배양한 다음, IPTG를 처리하고 30 ℃에서 16 시간 동안 배양하여 GST-Afb의 과발현을 유도하였다. 과발현 유도된 E. coli 세포는 5000Хg로 4 ℃에서 10 분간 원심분리하여 침전된 세포를 수득하고, 인산염 완충용액(50 mM 인산 나트륨 및 100 mM 염화 나트륨, pH 6.5)에 세포를 현탁하였다. 세포 현탁액에 리소자임(lysozyme)을 처리하고 20 분간 실온에서 배양한 다음, 30 초 파쇄 및 1 분 간격으로 총 10 분가 초음파 파쇄하였다. 파쇄 후, 12000Хg로 4 ℃에서 1 시간 동안 원심분리하여 상층액을 GST-Afb 포함 분획으로서 수득하였다. 상층액은 FPLC를 이용한 금속이온 친화 크로마토그래피(immobilized metal affinity chromatography; 1mL HisTrap FF 컬럼, GE HealthCare)로 정제하여 GST-Afb을 분리 하였다. 분리한 GST-Afb(GST-HER2 Afb 및 GST-EGFR Afb는 PBS(pH 7.4)에서 밤새도록 투석하여 농축하였다. Specifically, in the present invention, HER2 Afb specifically binding to HER2 and EGFR Afb specifically binding to EGFR were used as Afb. A gene encoding an additional linker domain (SEQ ID NO: 1: GGGLVPRGSGGGCGGGGTGGGSGGG) was coupled to the end of the gene encoding each HER2 Afb or EGFR Afb, and then inserted into the pETduet plasmid. In addition, for GST overexpression, a GST-encoding gene designed to connect a 6ХHis tag to the N-terminus of GST was inserted into the pETduet plasmid, and a plasmid for overexpression of the fusion protein (GST-Afb) in which Afb and GST were linked by a linker was prepared. Was built. It was confirmed whether the GST-Afb coding sequence was normally inserted through PCR and DNA sequencing in the constructed plasmid. Then, the constructed plasmid was inserted into the E. coli BL21 (DE3) strain and cultured, and then treated with IPTG and cultured at 30° C. for 16 hours to induce overexpression of GST-Afb. The overexpression-induced E. coli cells were centrifuged at 4°C for 10 minutes at 5000 Хg to obtain precipitated cells, and the cells were suspended in a phosphate buffer solution (50 mM sodium phosphate and 100 mM sodium chloride, pH 6.5). The cell suspension was treated with lysozyme and incubated for 20 minutes at room temperature, followed by disruption for 30 seconds and ultrasonic disruption at 1 minute intervals for a total of 10 minutes. After crushing, centrifugation at 12000 Хg at 4° C. for 1 hour to obtain a supernatant as a fraction containing GST-Afb. The supernatant was purified by immobilized metal affinity chromatography (1mL HisTrap FF column, GE HealthCare) using FPLC to separate GST-Afb. The isolated GST-Afb (GST-HER2 Afb and GST-EGFR Afb were concentrated by dialysis overnight in PBS (pH 7.4)).
도 1은 본 발명에서 리포좀의 단백질 코로나 외층을 이루는 융합단백질의 구조를 도식화한 것이다.1 is a schematic diagram of the structure of a fusion protein forming the outer layer of the protein corona of the liposome in the present invention.
<1-2> GST-Afb 융합 단백질의 세포 독성 및 세포 표적능 확인<1-2> Confirmation of cytotoxicity and cell targeting ability of GST-Afb fusion protein
본 발명에서 발현한 GST-Afb 융합 단백질을 약물 전달체에서 단백질 코로나 외층(protein corona shield, PCS)으로 적용할 수 있는지 확인하기 위해 세포 독성 및 세포내 흡수능을 확인하고자 하였다.In order to confirm whether the GST-Afb fusion protein expressed in the present invention can be applied as a protein corona shield (PCS) in a drug delivery system, cytotoxicity and intracellular absorption capacity were examined.
구체적으로, 먼저 GST-HER2 Afb를 대상으로 세포 독성 여부를 확인하기 위해 인간 유방암 세포주인 SK-BR-3 세포를 준비하였다. 준비한 SK-BR-3 세포는 DMEM 배지(11995065, Invitrogen, S.Korea)에서 배양하였다. 배지에는 10% 우태아혈청(FBS), 100 ㎍/㎖ 스트렙토마이신 및 100 U/㎖ 페니실린을 첨가하였고, 배양 기간 중에는 배지를 매일 한번씩 교체하였다. 배양 환경은 37 ℃의 5% CO2 배양기로 유지하였다. 세포 접종 후 세포가 85% 포화도로 증식되면, 부착 배양된 세포를 분리하여 실험에 사용하였다. SK-BR-3 세포를 분리하여, 96 웰 플레이트(Thermo Scientific Inc. Korea)에 5Х103 세포/웰의 농도로 각각의 세포를 접종하고 37 ℃의 5% CO2 배양기에서 24 시간 동안 배양하였다. 그런 다음, 상기 실시예 <1-1>에서 수득한 GST-HER2 Afb를 0.3 μM 내지 10 μM의 농도로 SK-BR-3 세포에 처리하고 24 시간 동안 추가 배양하였다. 배양 종료 후, 알라마르 블루 염색약(alamar blue dye; DAL 2015, Invitrogen, Korea)을 사용하여 세포 생존률을 확인하였다. 세포 생존률을 확인하기 위해 형광 염료에 대한 여기 파장(excitation wavelength)을 565 ㎚로 설정하였으며 이에 따른 방출 파장(monitoring emission)을 590 ㎚로 설정하여 형광 플레이트 리더기(Tecan Infinite Series, Germany)로 형광 분석하였다. 또한, GST-HER2 Afb 중 GST를 형광-5-말레이미드(fluorescein-5-maleimide, F5M)로 표지하여 세포 내로 흡수(cell uptake)된 GST-HER2 Afb의 위치를 확인하였다. 음성 대조군으로서는 정상 상피 세포주인 MCF-10A 세포를 사용하여 동일한 방법을 수행하여 세포 생존률 및 세포 내 흡수 정도를 확인하였다.Specifically, first, SK-BR-3 cells, a human breast cancer cell line, were prepared in order to determine whether or not GST-HER2 Afb is cytotoxic. The prepared SK-BR-3 cells were cultured in DMEM medium (11995065, Invitrogen, S. Korea). 10% fetal bovine serum (FBS), 100 μg/ml streptomycin and 100 U/ml penicillin were added to the medium, and the medium was changed once daily during the culture period. The culture environment was maintained in a 5% CO2 incubator at 37°C. When the cells proliferated to 85% saturation after cell inoculation, adherent cultured cells were separated and used in experiments. SK-BR-3 cells were isolated, each cell was inoculated into a 96-well plate (Thermo Scientific Inc. Korea) at a concentration of 5x10 3 cells/well, and cultured in a 5% CO 2 incubator at 37° C. for 24 hours. Then, the GST-HER2 Afb obtained in Example <1-1> was treated with the SK-BR-3 cells at a concentration of 0.3 μM to 10 μM, and further cultured for 24 hours. After completion of the culture, cell viability was confirmed using alamar blue dye (DAL 2015, Invitrogen, Korea). To check the cell viability, the excitation wavelength for the fluorescent dye was set to 565 nm, and the monitoring emission was set to 590 nm, and fluorescence analysis was performed with a fluorescent plate reader (Tecan Infinite Series, Germany). . In addition, GST among GST-HER2 Afb was labeled with fluorescence-5-maleimide (F5M) to confirm the location of GST-HER2 Afb absorbed into cells (cell uptake). As a negative control, normal epithelial cell line MCF-10A cells were used to perform the same method to confirm cell viability and intracellular absorption.
이와 함께, GST-Afb에서 GSH와 표적 수용체 간의 상호작용을 실시간으로 확인하기 위해 수정진동자저울(Quartz Crystal Microbalance, QCM) 및 표면 플라스몬 공명(surface plasmon resonance, SPR) 분석을 수행하였다.In addition, a quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) analysis were performed to confirm the interaction between GSH and the target receptor in GST-Afb in real time.
그 결과, 도 2에서 나타난 바와 같이 GST-HER2 Afb가 세포에 대하여는 독성을 나타내지 않으나, 암 세포 특이적으로 흡수될 수 있음을 확인하였다. GST-HER2 Afb를 SK-BR-3 세포 및 MCF-10A 세포에 처리하여 배양하였을 때 처리 농도와는 관계없이 세포 사멸 정도를 나타내지 않아 GST-Afb에 의해 세포 독성을 나타내지 않음을 확인한 반면(도 2a), GST-HER2 Afb는 유방암 세포주인 SK-BR-3 세포에만 결합하는 결합능을 나타내어 GST-Afb가 암세포 특이적인 표적능을 나타내는 것으로 확인하였다(도 2b). 이를 통해, 본 발명에서 단백질 코로나 외층으로 사용하기 위해 발현한 GST-Afb 융합 단백질은 정상 세포에 대하여 독성을 나타내지 않고 암 세포에 대한 표적능을 나타낼 수 있음을 확인하였다.As a result, as shown in FIG. 2, it was confirmed that GST-HER2 Afb did not show toxicity to cells, but could be specifically absorbed by cancer cells. When GST-HER2 Afb was treated and cultured on SK-BR-3 cells and MCF-10A cells, it was confirmed that the degree of apoptosis was not exhibited regardless of the treatment concentration, so that cytotoxicity was not exhibited by GST-Afb (FIG. 2A ), GST-HER2 Afb showed binding ability to bind only to SK-BR-3 cells, which is a breast cancer cell line, and it was confirmed that GST-Afb exhibited a specific targeting ability for cancer cells (FIG. 2B). Through this, it was confirmed that the GST-Afb fusion protein expressed for use as an outer layer of protein corona in the present invention does not exhibit toxicity to normal cells and can exhibit targeting ability against cancer cells.
실시예 2. 융합 단백질 외층을 갖는 리포좀 약물 전달체의 제조Example 2. Preparation of liposome drug delivery system having outer layer of fusion protein
<2-1> 리포좀의 준비<2-1> Preparation of liposome
약물 전달체의 기본 구조로서, 리포좀(Liposome)을 준비하였다. 리포좀은 EPR(Enhanced Permeability and Retention)효과를 얻기 위해 나노 사이즈로 제작하였다. 리포좀의 구성요소로서 HSPC (L-α-phosphatidylcholine, hydrogenated (Soy)), Cholesterol, 및 GSH를 결합시킨 DSPE-PEG(2000)를 사용하였다. 도 3 및 도 4에서 나타낸 바와 같이 합성하였다.As the basic structure of the drug delivery system, a liposome was prepared. The liposome was manufactured in a nano size to obtain an EPR (Enhanced Permeability and Retention) effect. As a component of the liposome, DSPE-PEG (2000) in which HSPC (L-α-phosphatidylcholine, hydrogenated (Soy)), Cholesterol, and GSH were bound was used. It was synthesized as shown in FIGS. 3 and 4.
구체적으로, DSPE-PEG-maleimide (32.7 mg, 0.01635 mmol)와 글루타치온 (glutathione) (7.5 mg, 0.0244 mmol)을 pH 7.4 HEPES 완충액 1M 3 mL에 넣고 22 ℃에서 15시간 교반시켰다. Dark 조건에서 질소기체를 주입하며 반응시켰다. 반응 후 남은 글루타치온은 HEPES 완충액을 이용하여 세척하여 제거하고 반응한 DSPE-PEG-GSH는 원심 분리기로 분리하였다. 분리한 DSPE-PEG-GSH는 DMSO-d6를 이용하여 NMR로 반응을 확인하였다. Specifically, DSPE-PEG-maleimide (32.7 mg, 0.01635 mmol) and glutathione (7.5 mg, 0.0244 mmol) were added to 3 mL of 1M pH 7.4 HEPES buffer and stirred at 22° C. for 15 hours. In the dark condition, nitrogen gas was injected and reacted. Glutathione remaining after the reaction was removed by washing with HEPES buffer, and the reacted DSPE-PEG-GSH was separated with a centrifuge. The isolated DSPE-PEG-GSH was confirmed by NMR using DMSO-d6.
HSPC(phosphatidiylcholine) (44.25 mg, 0.056 mmol), 콜레스테롤 (15.079 mg, 0.039 mmol), 독소루비신 (10mg) 및 DSPE-PEG-GSH (16.451 mg, 0.005 mmol)을 350 uL DMSO 와 400 uL 에탄올에 넣고 60~70 ℃에서 4시간 반응시켰다. pH 7.4 PBS 완충액 7.5mL 또한 65 ℃에서 따뜻하게 데운 상태에서 지질 혼합물을 천천히 한 방울씩 넣으면서 교반시켰다. 그 후 tris-HCl pH 7.5 완충액 10mM을 이용하여 투석을 2번 진행하여, 독소루비신이 담지된 리포좀 (Dox-리포좀-GSH)을 수득하였다. 투과전자현미경(Transmission Electron Microscopy, TEM) 및 동적 광 산란 (Dynamic Light Scattering, DLS)으로 리포좀의 모양 및 크기를 관찰하였고, 관찰된 리포좀의 크기는 약 106nm이다 (도 5 및 도 6). HSPC (phosphatidiylcholine) (44.25 mg, 0.056 mmol), cholesterol (15.079 mg, 0.039 mmol), doxorubicin (10 mg) and DSPE-PEG-GSH (16.451 mg, 0.005 mmol) were added to 350 uL DMSO and 400 uL ethanol and 60~ It reacted at 70 degreeC for 4 hours. In addition, 7.5 mL of pH 7.4 PBS buffer was also stirred while warming at 65° C. while slowly adding the lipid mixture drop by drop. Then, dialysis was performed twice using 10 mM tris-HCl pH 7.5 buffer to obtain doxorubicin-supported liposomes (Dox-liposome-GSH). The shape and size of liposomes were observed by Transmission Electron Microscopy (TEM) and Dynamic Light Scattering (DLS), and the size of the observed liposomes was about 106 nm (FIGS. 5 and 6).
도 3은 약물을 담지하는 리포좀의 구성요소 중 하나인 GSH-PEG-DSPE의 합성을 도식화한 것이다.3 is a schematic diagram of the synthesis of GSH-PEG-DSPE, one of the components of a liposome carrying a drug.
도 4는 약물을 담지하는 리포좀의 구성요소를 도식화한 것이다.4 is a schematic diagram of the components of a liposome carrying a drug.
도 5는 Dox-리포좀-GSH의 모양을 투과전자현미경으로 관찰한 이미지이다.5 is an image of the shape of Dox-liposome-GSH observed with a transmission electron microscope.
도 6은 Dox-리포좀-GSH의 크기를 동적 광산란으로 관찰한 그래프이다.6 is a graph of the size of Dox-liposome-GSH observed by dynamic light scattering.
<2-2> GST-Afb 단백질 코로나 외층을 가지는 리포좀의 제조<2-2> Preparation of liposomes having an outer layer of GST-Afb protein corona
본 발명에서 약물 전달체로 사용하고자 하는, 단백질 코로나 외층을 가지는 리포좀을 제조하기 위해, 단백질 코로나 외층으로서 GST-Afb를 적용한 리포좀을 제조하였다. 리포좀은 내부 및 표면에 화학 작용기를 통해 물질을 담지할 수 있는 전달체로서 사용되므로, 본 발명에서는 리포좀 표면에 GSH를 결합하여 제조하고, 이에 GST를 통해 결합된 단백질 코로나 외층을 형성하여 제조하였다 (도 7).In order to prepare a liposome having an outer layer of protein corona to be used as a drug delivery system in the present invention, a liposome to which GST-Afb was applied as an outer layer of protein corona was prepared. Since the liposome is used as a carrier capable of carrying a substance through chemical functional groups on the inside and on the surface, in the present invention, it was prepared by binding GSH to the surface of the liposome, and thus formed by forming an outer layer of protein corona bound through GST (Fig. 7).
구체적으로, 상기 실시예 <2-1>에서 수득한 Dox-리포좀-GSH (104.4 mg) 을 10mM tris-HCl pH 7.5 완충용액 10ml에 준비한 후 20mg/ml PBS 농도의 상기 실시예 <1-1>에서 수득한 융합단백질을 10 ul 첨가한 후 65 ℃에서 10분간 교반시켰다. 그 후 tris-HCl pH 7.5 완충액 10mM을 이용하여 투석를 2번 진행하여 융합단백질을 외층으로 가지는 독소루비신이 담지된 리포좀 (Dox-리포좀-융합단백질)을 합성하였다.Specifically, the Dox-liposome-GSH (104.4 mg) obtained in Example <2-1> was prepared in 10 ml of a 10 mM tris-HCl pH 7.5 buffer solution, and then the Example <1-1> at a concentration of 20 mg/ml PBS. After 10 ul of the fusion protein obtained in was added, the mixture was stirred at 65° C. for 10 minutes. Then, dialysis was performed twice using 10 mM tris-HCl pH 7.5 buffer to synthesize doxorubicin-supported liposomes (Dox-liposome-fusion protein) having a fusion protein as an outer layer.
투과전자현미경(Transmission Electron Microscopy, TEM) 및 동적 광 산란 (Dynamic Light Scattering, DLS)으로 Dox-리포좀-융합단백질의 모양 및 크기를 관찰하였다. Dox-리포좀-융합단백질의 크기는 약 150nm 이며, 표면전하는 ~0 mv 로써, 이는 융합단백질이 Dox-리포좀-GSH와 복합체를 형성하였음을 나타낸다 (도 8 내지 도 11). The shape and size of the Dox-liposome-fusion protein were observed by Transmission Electron Microscopy (TEM) and Dynamic Light Scattering (DLS). The size of the Dox-liposome-fusion protein is about 150 nm, and the surface charge is ~0 mv, indicating that the fusion protein formed a complex with Dox-liposome-GSH (FIGS. 8 to 11 ).
도 7은 약물을 담지하는 리포좀에 글루타치온-S-전이효소(glutathione-S-transferase, GST)와 표적 세포 또는 표적 단백질 결합능을 갖는 단백질, 즉 표적지향 융합단백질이 결합한 것 (Dox-리포좀-융합단백질)을 도식화한 것이다.Figure 7 is a drug-bearing liposome with glutathione-S-transferase (GST) and a protein having the ability to bind to a target cell or a target protein, that is, a target-directed fusion protein (Dox-liposome-fusion protein ) Is a schematic diagram.
도 8은 Dox-리포좀-융합단백질의 크기를 동적 광산란으로 관찰한 그래프이다.Figure 8 is a graph observed by dynamic light scattering of the size of Dox-liposome-fusion protein.
도 9는 Dox-리포좀-융합단백질의 모양을 투과전자현미경으로 관찰한 이미지이다.9 is an image of the shape of a Dox-liposome-fusion protein observed with a transmission electron microscope.
도 10은 Dox-리포좀-GSH의 제타 전위를 나타낸 그래프이다.10 is a graph showing the zeta potential of Dox-liposome-GSH.
도 11은 Dox-리포좀-융합단백질의 제타 전위를 나타낸 그래프이다.11 is a graph showing the zeta potential of Dox-liposome-fusion protein.
실시예 3. 표적 세포에 대한 GST-Afb 단백질 코로나 외층을 가지는 리포좀의 세포 독성 확인Example 3. Confirmation of Cytotoxicity of Liposomes Having GST-Afb Protein Corona Outer Layer on Target Cells
GST-Afb 단백질 코로나 외층을 가지는 리포좀 (Dox-리포좀-융합단백질)이 표적 세포에 대하여 세포독성을 가지는지 확인하였다.It was confirmed that the liposome (Dox-liposome-fusion protein) having the outer layer of the GST-Afb protein corona has cytotoxicity to the target cells.
구체적으로, 인간 유방암 세포(human breast cancer cell)인 SKBR3 세포에 대한 Her2 지향 융합단백질(Fusion protein) + Dox-리포좀-GSH 의 세포 생존력은 3-(4,5-디메틸티아졸-2-일)-2,5-디페닐테트라졸륨 브로마이드(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT)의 불용성 포르마잔(formazan)으로의 환원을 관찰하여 평가하였다. 10% FBS가 첨가된 DMEM에서 세포를 96-웰 플레이트에 웰당 5X103 세포의 밀도로 파종하고 밤새 배양한 후, 0 ~ 25 ug/ml농도 범위의 Dox-리포좀-융합단백질을 각각 세포에 처리하여 24 시간 동안 배양하였다. 대조군 실험은 융합 단백질과 Dox-리포좀-GSH로 수행하였다. 그 다음, 세포를 3-(4,5-디메틸티아졸-2-일)-2,5-디페닐테트라졸륨 브로마이드(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT)와 배양한 뒤, 결정화된 포르마잔을 ELISA 플레이트 판독기로 595 nm에서 흡광도를 측정하여 정량화하였다. 결과는 생존력 백분율 = [(A550 (처리 된 세포)-배경값) / (A550 (처리되지 않은 세포)-배경값)] x 100으로 표현되었다. Specifically, the cell viability of Her2-oriented fusion protein + Dox-liposome-GSH for SKBR3 cells, human breast cancer cells, is 3-(4,5-dimethylthiazol-2-yl) It was evaluated by observing the reduction of -2,5-diphenyltetrazolium bromide (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) to insoluble formazan (formazan). . In DMEM supplemented with 10% FBS, cells were seeded in a 96-well plate at a density of 5 ×10 3 cells per well and incubated overnight, and then the cells were treated with Dox-liposome-fusion protein in a concentration range of 0 to 25 ug/ml. Incubated for 24 hours. Control experiments were performed with fusion protein and Dox-liposome-GSH. Then, the cells were converted to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium). bromide) (MTT) and then the crystallized formazan was quantified by measuring the absorbance at 595 nm with an ELISA plate reader. Results were expressed as percent viability = [(A550 (treated cells)-background value) / (A550 (untreated cells)-background value)] x 100.
그 결과, 대조군인 Her2 지향 융합단백질(Fusion protein) 또는 Dox-리포좀-GSH 자체의 세포독성은 80ug/ml 까지 미비한 수준이나, Dox-리포좀-융합단백질의 세포독성은 IC50 수치가 1.4 ug/ml 임을 나타냈다 (도 12). 이러한 결과는, 표적 단백질 결합능을 갖는 단백질을 포함하는 글루타치온―S―전이효소 (glutathione-S-transferase, GST)에 글루타치온이 개질화된 리포좀과의 복합체를 조성하고 있다는 것과, 표적세포로 담지된 약물인 독소루비신을 전달하여 Dox-리포좀-융합단백질을 치료 목적으로 적용 가능함을 나타낸다.As a result, the cytotoxicity of the control Her2 oriented fusion protein or Dox-liposome-GSH itself was insufficient to 80 ug/ml, but the cytotoxicity of Dox-liposome-fusion protein had an IC50 value of 1.4 ug/ml. Shown (Fig. 12). These results indicate that glutathione-S-transferase (GST), which contains a protein having a target protein-binding ability, forms a complex with liposomes modified with glutathione, and that drugs carried by target cells It shows that Dox-liposome-fusion protein can be applied for therapeutic purposes by delivering phosphorus doxorubicin.
도 12는 Dox-리포좀-융합단백질의 세포 독성을 나타낸 그래프이다.12 is a graph showing the cytotoxicity of Dox-liposome-fusion protein.
실시예 4. 표적 세포에 대한 GST-Afb 단백질 코로나 외층을 가지는 리포좀의 항종양 효과 확인Example 4. Confirmation of antitumor effect of liposomes having outer corona layer of GST-Afb protein on target cells
Dox-리포좀-융합단백질의 생체 내 항종양 효능을 분석하기 위한 실험을 수행하였다. An experiment was conducted to analyze the anti-tumor efficacy of the Dox-liposome-fusion protein in vivo.
구체적으로, Balb/c 누드 암컷 마우스는 한국의 Orient bio에서 구입했으며 모든 동물 실험은 울산과학기술원 (UNIST)의 기관 동물 관리 및 사용위원회가 승인한 프로토콜에 따라 수행하였다. SKBR3 세포를 1X PBS 에 3X106 세포 / 100 μL로 하여 0.1 mL 를 각각의 누드 마우스의 우측 측면 영역에 피하 주사하여 종양 이종 이식편 모델을 생성하였다. 디지털 캘리퍼(caliper)를 사용하여 주기적으로 종양의 성장을 모니터링 하였다. 종양 부피는 부피 = (종양 길이) x (종양 폭)2/2의 공식에 의해 계산되었다. 종양 크기가 200 mm3에 도달할 때, Dox-리포좀-융합단백질, Dox-리포좀-GSH, 융합단백질, 및 PBS 를 각각 주사할 3 마리씩의 마우스를 선택하였고, 그보다 크거나 작은 종양을 가진 마우스는 선택하지 않았다. 이어서, 각 마우스에 Dox-리포좀-융합단백질을 100 μL, 50 mg/kg, Dox-리포좀-GSH 를 100 μL, 50 mg/kg, 융합단백질을 100 μL, 5 mg/kg, 대조군으로 1X PBS를 100 μL로 하여 매일 3주간 정맥 내 주사하였다. 그 후, 모든 그룹의 종양 크기 및 체중을 측정하고 비교하였다. Specifically, Balb/c nude female mice were purchased from Orient Bio, Korea, and all animal experiments were performed according to a protocol approved by the Institutional Animal Care and Use Committee of Ulsan Institute of Science and Technology (UNIST). A tumor xenograft model was generated by subcutaneous injection of 0.1 mL of SKBR3 cells into 1X PBS at 3X10 6 cells/100 μL into the right flank region of each nude mouse. Tumor growth was monitored periodically using a digital caliper. Tumor volume volume = (tumor length) x (tumor width) was calculated by a 2/2 formula. When the tumor size reached 200 mm 3 , 3 mice to be injected with Dox-liposome-fusion protein, Dox-liposome-GSH, fusion protein, and PBS were selected, respectively, and mice with tumors larger or smaller than that were selected. I didn't choose. Then, in each mouse, 100 μL of Dox-liposome-fusion protein, 50 mg/kg, 100 μL of Dox-liposome-GSH, 50 mg/kg, 100 μL of fusion protein, 5 mg/kg, 1X PBS as a control 100 μL was injected intravenously every day for 3 weeks. Thereafter, tumor size and body weight of all groups were measured and compared.
그 결과, Dox-리포좀-GSH, 융합단백질 처리군 및 대조군과 비교하여 Dox-리포좀-융합단백질이 처리된 누드 마우스의 경우 종양 성장이 현저히 감소하였다 (도 13). 이러한 결과는, Dox-리포좀-융합단백질이 유의한 항 종양 효과를 갖는다는 것을 나타낸다. 　 As a result, tumor growth was significantly reduced in nude mice treated with Dox-liposome-fusion protein compared to the Dox-liposome-GSH, fusion protein-treated group and the control group (FIG. 13). These results indicate that the Dox-liposome-fusion protein has a significant anti-tumor effect.
도 13은 Dox-리포좀-융합단백질의 항 종양 효과를 나타낸 사진 및 그래프이다.13 is a photograph and graph showing the anti-tumor effect of Dox-liposome-fusion protein.

Claims (15)

  1. 글루타치온-S-전이효소(glutathione-S-transferase, GST); Glutathione-S-transferase (GST);
    표적 세포 또는 표적 단백질 결합능을 갖는 단백질; A protein having the ability to bind to a target cell or a target protein;
    상기 글루타치온-S-전이효소와 표적 세포 또는 표적 단백질 결합능을 갖는 단백질을 연결하는 링커; 및 A linker connecting the glutathione-S-transferase and a protein having the ability to bind to a target cell or a target protein; And
    상기 글루타치온-S-전이효소와 약물을 담지하는 리포좀이 결합된 것을 함유하는 약물 전달체. A drug delivery system containing the glutathione-S-transferase and a liposome carrying a drug are bound.
  2. 청구항 1에 있어서, 상기 리포좀은 나노 리포좀인 것인 약물 전달체.The drug delivery system according to claim 1, wherein the liposome is a nano liposome.
  3. 청구항 2에 있어서, 상기 리포좀은 입자 크기가 10nm 내지 250nm인 것인 약물 전달체.The drug delivery system of claim 2, wherein the liposome has a particle size of 10 nm to 250 nm.
  4. 청구항 1 또는 청구항 2에 있어서, 상기 리포좀은 인지질, 스테로이드 및 PEG 함유한 인지질로 구성된 군으로부터 선택되는 하나 이상의 지질을 포함하는 것인 약물 전달체.The drug delivery system of claim 1 or 2, wherein the liposome contains one or more lipids selected from the group consisting of phospholipids, steroids, and PEG-containing phospholipids.
  5. 청구항 1에 있어서, 상기 글루타치온-S-전이효소와 리포좀의 결합은 GSH(Glutathione)에 의한 것인 약물 전달체. The drug delivery system according to claim 1, wherein the glutathione-S-transferase and liposome are bound by GSH (Glutathione).
  6. 청구항 1에 있어서, 상기 하나의 리포좀에 상기 글루타치온-S-전이효소, 표적 세포 또는 표적 단백질 결합능을 갖는 단백질 및 링커가 포함된 단백질이 두개 이상 결합된 것인 약물 전달체.The drug delivery system according to claim 1, wherein two or more proteins including the glutathione-S-transferase, a protein having the ability to bind to a target cell or a target protein, and a linker are bound to the one liposome.
  7. 청구항 1에 있어서, 상기 약물은 항암제인 것인 약물 전달체. The drug delivery system of claim 1, wherein the drug is an anticancer agent.
  8. 글루타치온-S-전이효소(glutathione-S-transferase, GST); Glutathione-S-transferase (GST);
    표적 세포 또는 표적 단백질 결합능을 갖는 단백질; A protein having the ability to bind to a target cell or a target protein;
    상기 글루타치온-S-전이효소와 표적 세포 또는 표적 단백질 결합능을 갖는 단백질을 연결하는 링커; 및 A linker connecting the glutathione-S-transferase and a protein having the ability to bind to a target cell or a target protein; And
    상기 글루타치온-S-전이효소와 항암제를 담지하는 리포좀이 결합된 것을 포함하는 암 예방 또는 치료용 약학적 조성물. A pharmaceutical composition for preventing or treating cancer comprising the glutathione-S-transferase and a liposome carrying an anticancer agent are bound.
  9. 청구항 8에 있어서, 상기 리포좀은 나노 리포좀인 것인 약물 전달체.The drug delivery system according to claim 8, wherein the liposome is a nano liposome.
  10. 청구항 9에 있어서, 상기 리포좀은 입자 크기가 10nm 내지 200nm인 것인 약물 전달체.The drug delivery system of claim 9, wherein the liposome has a particle size of 10 nm to 200 nm.
  11. 청구항 8 또는 청구항 9에 있어서, 상기 리포좀은 인지질, 스테로이드 및 PEG 함유한 인지질로 구성된 군으로부터 선택되는 하나 이상의 지질을 포함하는 것인 약물 전달체.The drug delivery system according to claim 8 or 9, wherein the liposome contains one or more lipids selected from the group consisting of phospholipids, steroids, and PEG-containing phospholipids.
  12. 청구항 8에 있어서, 상기 글루타치온-S-전이효소와 리포좀의 결합은 GSH(Glutathione)에 의한 것인 암 예방 또는 치료용 약학적 조성물.The pharmaceutical composition for preventing or treating cancer according to claim 8, wherein the binding of the glutathione-S-transferase and liposome is by GSH (Glutathione).
  13. 청구항 8에 있어서, 상기 항암제는 아바렐릭스 [abarelix (Plenaxis depot®)]; 알데스류킨 [aldesleukin (Prokine®)]; 알데스류킨 [Aldesleukin (Proleukin®)]; 알렘투주맙 [Alemtuzumabb (Campath®)]; 알리트레티노인 [alitretinoin (Panretin®)]; 알로퓨리놀 [allopurinol (Zyloprim®)]; 알트레타민 [altretamine (Hexalen®)]; 아미포스틴 [amifostine (Ethyol®)]; 아나스트로졸 [anastrozole (Arimidex®)]; 아르세닉 트리옥사이드 [arsenic trioxide (Trisenox®)]; 아스파라기나아제 [asparaginase (Elspar®)]; 아자시티딘 [azacitidine (Vidaza®)]; 베바쿠지맙 [bevacuzimab (Avastin®)]; 벡사로텐 캡슐 [bexarotene capsules (Targretin®)]; 벡사로텐 겔 [bexarotene gel (Targretin®)]; 블레오마이신 [bleomycin (Blenoxane®)]; 보르테조밉 [bortezomib (Velcade®)]; 정맥용 부술판 [busulfan intravenous (Busulfex®)]; 경구용 부술판 [busulfan oral (Myleran®)]; 칼루스테론 [calusterone (Methosarb®)]; 카페시타빈 [capecitabine (Xeloda®)]; 카르보플라틴 [carboplatin (Paraplatin®)]; 카르무스틴 [carmustine (BCNU®, BiCNU®)]; 카르무스틴 [carmustine (Gliadel®)]; 카르무스틴과 폴리페프로산 20 임플란트 (Polifeprosan 20 Implant) (Gliadel Wafer®); 셀레콕십 [celecoxib (Celebrex®)]; 세툭시맙 [cetuximab (Erbitux®)]; 클로람부실 [chloambucil (Leukeran®)]; 시스플라틴 [cisplatin (Platinol®)]; 클라드리빈 [cladribine (Leustatin®) 2-CdA®]; 클로파라빈 [clofarabine (Clolar®)]; 사이클로포스파미드 [cyclophosphamide (Cytoxan®, Neosar®)]; 사이클로포스파미드 [cyclophosphamide (Cytoxan Injection®)]; 사이클로포스파미드 [cyclophosphamide (Cytoxan Tablet®)]; 사이타라빈 [cytarabine (Cytosar-U®)]; 사이타라빈 리포조말 [cytarabine liposomal (DepoCyt®)]; 다카르바진 [dacarbazine (DTIC-Dome®)]; 닥티노마이신 (dactinomycin), 액티노마이신 D [actinomycinD (Cosmegen®)]; 다르베포에틴 알파 [Darbepoetin alfa (Aranesp®)]; 다우노루비신 리포조말 [daunorubicin liposomal (Danuoxome®)]; 다우노루비신 (daunorubicin), 다우노마이신 [daunomycin (Daunorubicin®)]; 다우노루비신 (daunorubicin), 다우노마이신 [daunomycin (Cerubidine®)]; 데니류킨 디프티톡스 [Denileukin diftitox (Ontak®)]; 덱스라조산 [dexrazoxane (Zinecard®)]; 도세탁셀 [docetaxel (Taxotere®)]; 독소루비신 [doxorubicin (Adriamycin PFS®)]; 독소루비신 [doxorubicin (Adriamycin®, Rubex®)]; 독소루비신 [doxorubicin (Adriamycin PFS Injection®)]; 독소루비신 리포조말 [doxorubicin liposomal (Doxil®)]; 드로모스타놀론 프로피오네이트 [dromostanolone propionate (dromostanolone®)]; 드로모스타놀론 프로피오네이트 [dromostanolone propionate (masterone injection®)]; 엘리엇 B 용액 [Elliott's B Solution (Elliott's B Solution®)]; 에피루비신 [epirubicin (Ellence®)]; 이포에틴 알파 [Epoetin alfa (epogen®)]; 에를로티닙 [erlotinib (Tarceva®)]; 에스트라무스틴 [estramustine (Emcyt®)]; 에토포사이드 포스페이트 [etoposide phosphate (Etopophos®)]; 에토포사이드 (etoposide), VP-16 (Vepesid®); 엑세메스탄 [exemestane (Aromasin®)]; 필그라스팀 [Filgrastim (Neupogen®)]; 플록수리딘 [floxuridine (intraarterial) (FUDR®)]; 플루다라빈 [fludarabine (Fludara®)]; 플루오로우라실 [Fluorouracil, 5-FU (Adrucil®)]; 플루베스트란트 [folvestrant (Faslodex®)]; 게피티닙 [gefitinib (Iressa®)]; 겜시타빈 [gemcitabine (Gemzar®)]; 겜투주맙 오조가미신 [gemtuzumab ozogamicin (Mylotarg®)]; 고세렐린 아세테이트 [goserelin acetate (Zoladex Implant®)]; 고세렐린 아세테이트 [goserelin acetate (Zoladex®)]; 히스트렐린 아세테이트 [histrelin acetate (Histrelin implant®)]; 하이드록시우레아 [hydroxyurea (Hydrea®)]; 이브리투모맙 티욱세탄 [Ibritumomab Tiuxetan (Zevalin®)]; 이다루비신 [idarubicin (Idamycin®)]; 이포스파미드 [ifosfamide (IFEX®)]; 이마티닙 메실레이트 [imatinib mesylate (Gleevec®)]; 인터페론 알파-2a [interferon alfa-2a (Roferon A®)]; 인터페론 알파-2b [Interferon alfa-2b (Intron A®)]; 이리노테칸 [irinotecan (Camptosar®)]; 레날리도마이드 [lenalidomide (Revlimid®)]; 레트로졸 [letrozole (Femara®)]; 류코보린 [leucovorin (Wellcovorin®, Leucovorin®)]; 류프롤리드 아세테이트 [Leuprolide Acetate (Eligard®)]; 레바미솔 [levamisole (Ergamisol®)]; 로무스틴-CCNU [lomustine-CCNU (CeeBU®)]; 메클로레타민 (meclorethamine), 니트로겐 머스타드 [nitrogen mustard (Mustargen®)]; 메게스트롤 아세테이트 [megestrol acetate (Megace®)]; 멜파란 (melphalan), L-PAM (Alkeran®); 머캅토퓨린 [mercaptopurine, 6-MP (purinethol®)]; 메스나 [mesna (Mesnex®)]; 메스나 [mesna (Mesnex tabs®)]; 메토트렉제이트 [methotraxate (methotraxate®)]; 메톡스살렌 [methoxsalen (Uvadex®)]; 미토마이신 C [mitomycin C (Mutamycin®)]; 미토탄 [mitotane (Lysodren®)]; 미토잔트론 [mitoxantrone (Novantrone®)]; 난드롤란 펜프로피오네이트 [nandrolone phenpropionate (Durabolin-50®)]; 넬라라빈 [nelarabine (Arranon®)]; 노페투모맙 [Nofetumomab (Verluma®)]; 오프렐베킨 [Oprelvekin (Neumega®)]; 옥살리플라틴 [oxaliplatin (Eloxatin®)]; 파클리탁셀 [paclitaxel (Paxene®)]; 파클리탁셀 [paclitaxel (Taxol®)]; 파클리탁셀 단백질 결합 입자 [paclitaxel protein-bound particles (Abraxane®)]; 팔리페르민 [palifermin (Kepivance®)]; 파미드로네이트 [pamidronate (Aredia®)]; peg아데마스 [pegademase (Adagen (Pegademase Bovine)®)]; peg아스파르가아제 [pegaspargase (Oncaspar®)]; peg필그라스팀 [Pegfilgrastim (Neulasta®)]; 페멕트렉제트 디소듐 [pemetrexed disodium (Alimta®)]; 펜토스타틴 [pentostatin (Nipent®)]; 피포브로만 [pipobroman (Vercyte®)]; 플리카마이신 [plicamycin], 미트라마이신 [mithramycin (Mithracin®)]; 포르피머 소듐 [porfimer sodium (Photofrin®)]; 프로카르바진 [procarbazine (Matulane®)]; 퀴나크린 [quinacrine (Atabrine®)]; 라스뷰리카아제 [Rasburicase (Elitek®)]; 리툭시맙 [Rituximab (Rituxan®)]; 사르그라모스팀 [sargramostim (Leukine®)]; 사르그라모스팀 [Sargramostim (Prokine®)]; 소라페닙 [sorafenib (Nexavar®)]; 스트렙타조신 [streptozocin (Zanosar®)]; 수니티닙 말리에이트 [sunitinib maleate (Sutent®)]; 탈크 [talc (Sclerosol®)]; 타목시펜 [tamoxifen (Nolvadex®)]; 테모졸로마이드 [temozolomide (Temodar®)]; 테니포사이드 [teniposide, VM-26 (Vumon®)]; 테스톨락톤 [testolactone (Teslac®)]; 티오구아닌 [thioguanine, 6-TG (thioguanine®)]; 티오테파 [thiotepa (thioplex®)]; 토포테칸 [topotecan (Hycamtin®)]; 토레미펜 [toremifene (Fareston®)]; 토시투모맙 [Tositumomab (Bexxar®)]; 토시투모맙/I-131 토시투모맙 (Bexxar®); 트라스투주맙 [Trastuzumab (Herceptin®)]; 트레티노인 [tretinoin, ATRA (Vesanoid®)]; 우라실 머스타드 [Uracil Mustard (Uracil Mustard Capsules®)]; 발루비신 [valrubicin (Valstar®)]; 빈블라스틴 [vinblastine (Velban®)]; 빈크리스틴 [vincristine (Oncovin®)]; 비노렐빈 [vinorelbine (Navelbine®)]; 및 졸레드로네이트 [zoledronate (Zometa®)]로 이루어지는 군으로부터 선택되는 것인 암 예방 또는 치료용 약학적 조성물.The method according to claim 8, wherein the anticancer agent Abarelix [abarelix (Plenaxis depot ® )]; Aldesleukin (Prokine ® )]; Aldesleukin [Aldesleukin (Proleukin ® )]; Alemtuzumabb (Campath ® )]; Alitretinoin (Panretin ® )]; Allopurinol [allopurinol (Zyloprim ® )]; Altretamine (Hexalen ® )]; Amifostine [amifostine (Ethyol ® )]; Anastrozole (Arimidex ® )]; Arsenic trioxide (Trisenox ® )]; Asparaginase [asparaginase (Elspar ® )]; Azacitidine (Vidaza ® )]; Bevacuzimab [bevacuzimab (Avastin ® )]; Bexarotene capsules (Targretin ® )]; Bexarotene gel (Targretin ® )]; Bleomycin [bleomycin (Blenoxane ® )]; Bortezomib (Velcade ® )]; Intravenous busulfan [busulfan intravenous (Busulfex ® )]; Busulfan oral (Myleran ® )]; Calusterone (Methosarb ® )]; Capecitabine (Xeloda ® )]; Carboplatin [carboplatin (Paraplatin ® )]; Carboxylic estramustine [carmustine (BCNU ®, BiCNU ® )]; Carmustine (Gliadel ® )]; 20 implant to carboxylic acid estramustine and poly pepeu (Polifeprosan 20 Implant) (Gliadel Wafer ®); Celecoxib (Celebrex ® )]; Cetuximab [cetuximab (Erbitux ® )]; Chlorambucil [chloambucil (Leukeran ® )]; Cisplatin (Platinol ® )]; Cladribine (Leustatin ® ) 2-CdA ® ]; Clofarabine (Clolar ® )]; Cyclophosphamide (Cytoxan ® , Neosar ® )]; Cyclophosphamide (Cytoxan Injection ® )]; Cyclophosphamide (Cytoxan Tablet ® )]; Cytarabine [cytarabine (Cytosar-U ® )]; Cytarabine liposomal (DepoCyt ® )]; Dacarbazine [dacarbazine (DTIC-Dome ® )]; Dactinomycin, actinomycin D [actinomycinD (Cosmegen ® )]; Darbepoetin alfa (Aranesp ® )]; Daunorubicin liposomal (Danuoxome ® )]; Daunorubicin, daunomycin [daunomycin (Daunorubicin ® )]; Daunorubicin, daunomycin [daunomycin (Cerubidine ® )]; Denileukin diftitox (Ontak ® )]; Dexrazoxane (Zinecard ® )]; Docetaxel (Taxotere ® )]; Doxorubicin [doxorubicin (Adriamycin PFS ® )]; Doxorubicin [doxorubicin (Adriamycin ® , Rubex ® )]; Doxorubicin [doxorubicin (Adriamycin PFS Injection ® )]; Doxorubicin liposomal (Doxil ® )]; Dromostanolone propionate (dromostanolone ® )]; Dromostanolone propionate (masterone injection ® )]; Elliott's B Solution [Elliott's B Solution ® )]; Epirubicin [epirubicin (Ellence ® )]; Ipoetin alfa [Epoetin alfa (epogen ® )]; Erlotinib [erlotinib (Tarceva ® )]; Estramustine (Emcyt ® )]; Etoposide phosphate (Etopophos ® )]; Etoposide (etoposide), VP-16 ( Vepesid ®); Exemestane [exemestane (Aromasin ® )]; Filgrastim (Neupogen ® )]; Floxuridine (intraarterial) (FUDR ® )]; Fludarabine [fludarabine (Fludara ® )]; Fluorouracil [Fluorouracil, 5-FU (Adrucil ® )]; Fluvestrant [folvestrant (Faslodex ® )]; Gefitinib [gefitinib (Iressa ® )]; Gemcitabine (Gemzar ® )]; Gemtuzumab ozogamicin (Mylotarg ® )]; Goserelin acetate (Zoladex Implant ® )]; Goserelin acetate (Zoladex ® )]; Hisstrelin acetate (Histrelin implant ® )]; Hydroxyurea (Hydrea ® )]; Ibritumomab Tiuxetan (Zevalin ® )]; Darubicin [idarubicin (Idamycin ® )]; Ifosfamide [ifosfamide (IFEX ® )]; Imatinib mesylate (Gleevec ® )]; Interferon alfa-2a [interferon alfa-2a (Roferon A ® )]; Interferon alfa-2b [Interferon alfa-2b (Intron A ® )]; Irinotecan [irinotecan (Camptosar ® )]; Lenalidomide (Revlimid ® )]; Letrozole (Femara ® )]; Leucovorin (Wellcovorin ® , Leucovorin ® )]; Leuprolide acetate [Leuprolide Acetate (Eligard ® )]; Levamisole (Ergamisol ® )]; Lomustine-CCNU (CeeBU ® )]; Meclorethamine, nitrogen mustard (Mustargen ® )]; Megestrol acetate (Megace ® )]; Mel blue (melphalan), (Alkeran ®) L-PAM; Mercapto-purine [mercaptopurine, 6-MP (purinethol ®)]; Mesna (Mesnex ® )]; Mesna [mesna (Mesnex tabs ® )]; Methotrexate [methotraxate (methotraxate ® )]; Methoxsalen (Uvadex ® )]; Mitomycin C [mitomycin C (Mutamycin ® )]; Mitotan [mitotane (Lysodren ® )]; Mitoxantrone [mitoxantrone (Novantrone ® )]; Nandrolone phenpropionate (Durabolin-50 ® )]; Nelarabine (Arranon ® )]; Nofetumomab (Verluma ® )]; Oprelvekin [Neumega ® )]; Oxaliplatin (Eloxatin ® )]; Paclitaxel [paclitaxel (Paxene ® )]; Paclitaxel [paclitaxel (Taxol ® )]; Paclitaxel protein-bound particles [paclitaxel protein-bound particles (Abraxane ® )]; Palifermin [palifermin (Kepivance ® )]; Pamidronate (Aredia ® )]; pegademas [pegademase (Adagen (Pegademase Bovine) ® )]; peg aspargase [pegaspargase (Oncaspar ® )]; pegfilgrastim (Neulasta ® )]; Pemetrexed disodium (Alimta ® )]; Pentostatin [pentostatin (Nipent ® )]; Pipebroman [pipobroman (Vercyte ® )]; Plicamycin [mithramycin (Mithracin ® )]; Porfimer sodium [Photofrin ® )]; Procarbazine (Matulane ® )]; Quinacrine (Atabrine ® )]; Rasburicase [Rasburicase (Elitek ® )]; Rituximab [Rituximab (Rituxan ® )]; Sargramostim (Leukine ® )]; Sargramostim (Prokine ® )]; Sorafenib [sorafenib (Nexavar ® )]; Streptazosin [streptozocin (Zanosar ® )]; Sunitinib maleate (Sutent ® )]; Talc (Sclerosol ® )]; Tamoxifen [tamoxifen (Nolvadex ® )]; Temozolomide [temozolomide (Temodar ® )]; I'll Forsythe [teniposide, VM-26 (Vumon ®)]; Testolactone [testolactone (Teslac ® )]; Thioguanine [thioguanine, 6-TG (thioguanine ®)]; Thiotepa [thiotepa (thioplex ®)]; Topotecan (Hycamtin ® )]; Toremifene [toremifene (Fareston ® )]; Tositumomab [Tositumomab (Bexxar ® )]; Toshio Tomorrow Thank / I-131 Toshio Thank Tomorrow (Bexxar ®); Trastuzumab [Trastuzumab (Herceptin ® )]; Tretinoin [tretinoin, ATRA (Vesanoid ® )]; Uracil Mustard [Uracil Mustard (Uracil Mustard Capsules ® )]; Valrubicin [valrubicin (Valstar ® )]; Vinblastine (Velban ® )]; Vincristine (Oncovin ® )]; Vinorelbine (Navelbine ® )]; And zoledronate [zoledronate (Zometa ® )] will be selected from the group consisting of cancer prevention or treatment pharmaceutical composition.
  14. 글루타치온-S-전이효소(glutathione-S-transferase, GST); Glutathione-S-transferase (GST);
    표적 세포 또는 표적 단백질 결합능을 갖는 단백질; A protein having a target cell or target protein binding ability;
    상기 글루타치온-S-전이효소와 표적 세포 또는 표적 단백질 결합능을 갖는 단백질을 연결하는 링커; 및 A linker connecting the glutathione-S-transferase and a protein having binding ability to a target cell or a target protein; And
    상기 글루타치온-S-전이효소와 약물을 담지하는 리포좀을 함유하는 조성물을 그를 필요로 하는 개체에 투여하는 단계를 포함하는 개체 내 약물을 전달하는 방법.A method of delivering a drug in an individual comprising administering a composition containing the glutathione-S-transferase and a liposome carrying the drug to an individual in need thereof.
  15. 글루타치온-S-전이효소(glutathione-S-transferase, GST); Glutathione-S-transferase (GST);
    표적 세포 또는 표적 단백질 결합능을 갖는 단백질; A protein having the ability to bind to a target cell or a target protein;
    상기 글루타치온-S-전이효소와 표적 세포 또는 표적 단백질 결합능을 갖는 단백질을 연결하는 링커; 및 A linker connecting the glutathione-S-transferase and a protein having the ability to bind to a target cell or a target protein; And
    상기 글루타치온-S-전이효소와 항암제를 담지하는 리포좀을 함유하는 조성물을 그를 필요로 하는 개체에 투여하는 단계를 포함하는 암을 예방하거나 치료하는 방법.A method for preventing or treating cancer comprising administering a composition containing a liposome carrying the glutathione-S-transferase and an anticancer agent to an individual in need thereof.
PCT/KR2020/006905 2019-05-28 2020-05-28 Liposome comprising glutathione-s-transferase and protein having capacity to bind to target cell or target protein, and use thereof WO2020242217A1 (en)

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