WO2020242218A1 - Glutathione precursor drug-delivery carrier comprising glutathione-s-transferase and protein, which has binding capacity for target cell or target protein, and use thereof - Google Patents

Glutathione precursor drug-delivery carrier comprising glutathione-s-transferase and protein, which has binding capacity for target cell or target protein, and use thereof Download PDF

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WO2020242218A1
WO2020242218A1 PCT/KR2020/006906 KR2020006906W WO2020242218A1 WO 2020242218 A1 WO2020242218 A1 WO 2020242218A1 KR 2020006906 W KR2020006906 W KR 2020006906W WO 2020242218 A1 WO2020242218 A1 WO 2020242218A1
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glutathione
protein
transferase
receptor
target
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PCT/KR2020/006906
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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 WO2020242218A1 publication Critical patent/WO2020242218A1/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
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
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    • 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
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    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
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    • 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
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
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    • 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
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
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    • 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
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    • 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
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    • 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
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    • C12Y—ENZYMES
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    • 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
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    • C07K2318/00—Antibody mimetics or scaffolds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • a glutathione precursor drug comprising a protein having glutathione-S-transferase and a target cell or target protein-binding ability as a delivery vehicle and a pharmaceutical composition.
  • anticancer drugs include chemical anticancer drugs, targeted anticancer drugs, and immune anticancer drugs.
  • Chemical anticancer agents are also called cytotoxic anticancer agents and chemical anticancer agents. Although other anticancer drugs are not without side effects, chemotherapy drugs, which are the first-generation anticancer drugs, are the main culprit of white blood cell reduction, hair loss, vomiting, middle age, and diarrhea, which are commonly known side effects of chemotherapy. Using chemical anticancer drugs does not destroy all normal cells. It mainly affects the hematopoietic stem cells, hair follicle cells, oral and intestinal mucosa through which food passes, and reproductive organs of the bone marrow. This is because Hwaak anticancer drugs are designed to find and attack the characteristics of cancer cells that proliferate in a short period of time.
  • CAR-T cell therapy refers to a method of inducing reprogrammed T cells to attack cancer cells by inserting artificially designed genes into T cells extracted from patients.
  • CAR-T therapy shows high therapeutic effect in patients with intractable cancer who do not work well with anticancer drugs, but the toxicity is very strong, and when cytokine release syndrome appears as a side effect, the patient dies in many cases, making it unstable. many.
  • the cost of a single administration reaches 150 million US standards, and the treatment effect is not great for solid cancer.
  • 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 precursor drug combined with the glutathione-S-transferase.
  • 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 pharmaceutical composition for preventing or treating cancer comprising a glutathione precursor drug combined with the glutathione-S-transferase.
  • 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 precursor drug conjugated to the glutathione-S-transferase 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 glutathione precursor drug conjugated to the glutathione-S-transferase 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 provides a drug delivery system containing the glutathione precursor drug combined with the glutathione-S-transferase.
  • 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 proper separation between functional moieties or to maintain the necessary inter-moiety interaction.
  • 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.
  • a 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, the vector 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 binding of the glutathione-S-transferase and the glutathione precursor drug may be caused by GSH (Glutathione).
  • the glutathione precursor drug may be any one or more selected from the group consisting of any one of the following Formulas 1 to 7 or a pharmaceutically acceptable salt thereof. Since the glutathione precursor drug contains glutathione, it may be combined with glutathione-S-transferase as described above.
  • 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 pharmaceutical composition for preventing or treating cancer comprising a glutathione precursor drug combined with the glutathione-S-transferase.
  • 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
  • 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 method for preventing or treating cancer comprising administering a composition containing the glutathione precursor drug conjugated to the glutathione-S-transferase to an individual in need thereof.
  • the glutathione precursor drug may be any one or more selected from the group consisting of any one of Formulas 1 to 7 or a pharmaceutically acceptable salt thereof.
  • GST glutathione-S-transferase
  • a protein having a target cell or target protein binding ability a protein having a target cell or target protein binding ability
  • a glutathione precursor drug bound to the glutathione-S-transferase are as described above.
  • subject preferably a mammal, more preferably a human.
  • Mammals include, but are not limited to, murines, monkeys, humans, farm animals, sports animals and pets. Tissues, cells and their progeny of biological entities obtained in vivo or cultured in vitro are also included.
  • 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.
  • treatment or “treating” or “relaxing” or “improving” are used interchangeably. These terms refer to methods of obtaining beneficial or desired results, including but not limited to therapeutic benefits and/or prophylactic benefits.
  • a therapeutic benefit refers to any therapeutically significant improvement or effect thereon of one or more diseases, disorders or symptoms under treatment.
  • the composition may be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more physiological symptoms of the disease, even if the disease, condition, or symptom is not yet present.
  • an effective amount refers to an amount of an agent sufficient to produce an advantageous or desired result.
  • the therapeutically effective amount may vary according to one or more of the subject and condition to be treated, the weight and age of the subject, the severity of the condition, the mode of administration, and the like, which can be easily determined by those skilled in the art. Further, the term applies to the capacity to provide an image for detection by any of the imaging methods described herein.
  • the specific dosage may vary depending on one or more of the particular agent selected, the dosage regimen that follows, whether it is administered in combination with other compounds, the timing of administration, the tissue being imaged, and the body delivery system carrying it.
  • 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 by mixing 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.
  • Another aspect provides a method for preparing a drug delivery system comprising the following steps i) to iii):
  • step i) linking a linker (eg, GSH) to the drug; ii) binding the GST fusion protein to the drug surface bonded to the linker in step i).
  • a linker eg, GSH
  • the delivery system of the glutathione precursor drug 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. 1A is a result of confirming that the GST-Afb fusion protein does not decrease cell viability with respect to Afb target cells; And FIG. 1B is a result of confirming the cell targeting ability that the GST-Afb fusion protein exhibits against Afb target cells.
  • Figure 2 is a schematic diagram of the synthesis of entry 2, a type of glutathione precursor drug.
  • entry 3 is a schematic view of the synthesis of entry 3, which is a type of glutathione precursor drug.
  • Figure 4 is a schematic diagram of the synthesis of entry 5, a type of glutathione precursor drug.
  • Figure 5 is a schematic diagram of the synthesis of entry 7, a type of glutathione precursor drug.
  • FIG. 6 is a schematic diagram of the synthesis of entry 9, which is a type of glutathione precursor drug.
  • FIG. 7 is a schematic diagram of the synthesis of Entry 11, a type of glutathione precursor drug.
  • Figure 8 is a schematic diagram of the synthesis of entry 12, a type of glutathione precursor drug.
  • 9 is a graph showing binding affinity when entry 2 binds to a fusion protein.
  • 10 is a graph showing binding affinity when entry 3 binds to a fusion protein.
  • 11 is a graph showing binding affinity when Entry 5 binds to a fusion protein.
  • 12 is a table showing binding affinity values when entries 2,3,5,7,9,11 and 12 bind to a fusion protein.
  • 13 is a graph showing the cytotoxicity of the complex in which the fusion protein and Entry 2 are bound.
  • 15 is a graph showing the cytotoxicity of the complex in which the fusion protein and Entry 5 are bound.
  • 16 is a graph showing the cytotoxicity of the complex in which the fusion protein and Entry 7 are bound.
  • 17 is a graph showing the cytotoxicity of the complex in which the fusion protein and entry 9 are bound.
  • 19 is a graph showing the cytotoxicity of the complex in which the fusion protein and Entry 12 are bound.
  • FIG. 20 is a table showing IC50 values indicating cytotoxicity of complexes in which fusion proteins and entries 2,3,5,7,9,11 and 12 are bound.
  • Example 1 Expression of a fusion protein having glutathione-S-transferase and target cell or target protein binding ability
  • a fusion protein having a target function 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 for a total of 10 minutes at 1 minute intervals. After crushing, centrifugation was performed 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 separated GST-Afb (GST-HER2 Afb and GST-EGFR Afb were dialyzed overnight in PBS (pH 7.4) and concentrated.)
  • the concentrated GST-HER2 Afb and GST-EGFR Afb were analyzed by SDS-PAGE and ESI-TOF. The purity and molecular weight of the separated protein were analyzed through electrospray ionization time-of-light mass spectrometry (MS) analysis.
  • MS electrospray ionization time-of-light mass spectrometry
  • 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 does not show toxicity to cells, but can 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. ), 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. 1B). 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.
  • the synthesis of the glutathione precursor drug of the present invention was prepared in the same scheme as in FIGS. 2 to 8.
  • the entries described below refer to the glutathione precursor drug.
  • 2-bromoethyl bis(2-chloroethyl)carbamate which is entry 1
  • is beet (2-chloroethyl) amine (Bis( 2-chloroethyl)amine) (20 mg, 0.1408 mmol) and sodium hydroxide (8.448 mg, 0.2112 mmol) were first mixed with 5 mL of methyl chloride, followed by 2-bromoethyl carbonochloridate (2-bromoethyl carbonochloridate) (26.39 mg, 0.1408 mmol) was added.
  • Entry 3 was cisplatin (10mg, 0.0333 mmol) mixed with GSH ((glutathione), 15mg, 0.05mmol) pH 7.4 PBS solution, reacted at room temperature for 24 hours, and separated through HPLC.
  • GSH (glutathione)
  • Figure 2 is a schematic diagram of the synthesis of entry 2, a type of glutathione precursor drug.
  • entry 3 is a schematic view of the synthesis of entry 3, which is a type of glutathione precursor drug.
  • Figure 4 is a schematic diagram of the synthesis of entry 5, a type of glutathione precursor drug.
  • Figure 5 is a schematic diagram of the synthesis of entry 7, a type of glutathione precursor drug.
  • 6 is a schematic view of the synthesis of entry 9, which is a type of glutathione precursor drug.
  • FIG. 7 is a schematic diagram of the synthesis of Entry 11, a type of glutathione precursor drug.
  • Figure 8 is a schematic diagram of the synthesis of entry 12, a type of glutathione precursor drug.
  • the binding between the glutathione precursor drug and the fusion protein was confirmed using an isothermal titration calorimetry (ITC) method.
  • ITC isothermal titration calorimetry
  • a control sample was filled with distilled water, and then a plastic syringe was connected to the injection port of the injection syringe using a tube.
  • the injection syringe was rinsed with distilled water and then rinsed with a buffer solution.
  • the injection syringe was completely emptied by aspirating air through the system.
  • the needle of the injection syringe was put into the glutathione-S-transferase solution to which the HER2 target affibody was attached, and the fusion protein was drawn into the syringe until the entire syringe was full.
  • the injection port of the syringe was immediately closed and the tube and the plastic syringe were separated.
  • the injection syringe was discharged two more times and filled to 300 uL again. Since the syringe was removed from the fusion protein solution and the volume could be removed from the syringe, the drop was removed by wiping the side with Kimwipe, taking care not to touch the syringe tip with Kimwipe. In addition, care was taken not to tap or bottle the syringe as it may cause loss of volume at the syringe tip.
  • the injection syringe was placed in a PBS buffer solution in which entries 2, 3, 5, 7, 9, 11 and 12 were dissolved, respectively.
  • the system is given time to equilibrate and wait 5 minutes for the heat signal to return to the baseline before the next injection occurs. Thereafter, the subsequent injection was maintained at 300 uL, and the experimental temperature was selected at 25°C. After setting the parameters as above, the experiment was started. The experiment was repeated 3 times to reduce the occurrence of errors.
  • the data were analyzed. Specifically, you can easily perform data fitting using macros in any data fitting program (usually provided by the manufacturer with the instrument). Select the data fitting model (single binding site, two/multiple binding sites, cooperative bonds, etc.) to be used to fit the data.
  • the data can be suitable for initial guessing of the fitting parameters, stoichiometry (n), enthalpy ( ⁇ H) and binding affinity (Ka) and ITC enthalpy values can be compared to the enthalpy of the van't Hoff plot. It may be helpful to use different concentrations of ligands or macromolecules because the absolute value of the heat signal must increase as the macromolecule concentration increases. If the buffers of the cells and syringes do not match, noise is likely to occur. Another possibility for noise arises in samples with impurities.
  • 9 is a graph showing binding affinity when entry 2 binds to a fusion protein.
  • 10 is a graph showing binding affinity when entry 3 binds to a fusion protein.
  • 11 is a graph showing binding affinity when Entry 5 binds to a fusion protein.
  • 12 is a table showing binding affinity values when entries 2,3,5,7,9,11 and 12 bind to a fusion protein.
  • Cytotoxicity was analyzed to observe the anticancer effect of the complex of glutathione precursor drug (entry) and fusion protein combined.
  • the cell viability of the complex in which Her2-oriented fusion protein and entries 2, 3, 5, 7, 9, 11, and 12 are respectively bound to SKBR3 cells, which are human breast cancer cells is 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT)
  • MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
  • 13 is a graph showing the cytotoxicity of the complex in which the fusion protein and Entry 2 are bound.
  • 15 is a graph showing the cytotoxicity of the complex in which the fusion protein and Entry 5 are bound.
  • 16 is a graph showing the cytotoxicity of the complex in which the fusion protein and Entry 7 are bound.
  • 17 is a graph showing the cytotoxicity of the complex in which the fusion protein and entry 9 are bound.
  • 19 is a graph showing the cytotoxicity of the complex in which the fusion protein and Entry 12 are bound.
  • FIG. 20 is a table showing IC50 values indicating cytotoxicity of complexes in which fusion proteins and entries 2,3,5,7,9,11 and 12 are bound.

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Abstract

The present invention relates to a glutathione precursor drug-delivery carrier comprising a glutathione-S-transferase and a protein, which has a binding capacity for a target cell or a target protein, and a use thereof as a pharmaceutical composition. A glutathione precursor drug-delivery carrier according to an aspect can prolong in vivo retention time, and also has enhanced targeting ability for a target cell so as to be effectively delivered to the target cell, thereby being effectively usable as a targeted therapeutic agent.

Description

글루타치온-Ｓ-전이효소 및 표적 세포 또는 표적 단백질 결합능을 갖는 단백질을 포함하는 글루타치온 전구체 약물의 전달체 및 이의 용도Glutathione-S-transferase and a delivery vehicle of a glutathione precursor drug containing a protein having the ability to bind to a target cell or a target protein, and use thereof
글루타치온-Ｓ-전이효소 및 표적 세포 또는 표적 단백질 결합능을 갖는 단백질을 포함하는 글루타치온 전구체 약물의 전달체 및 약학적 조성물로서의 용도에 관한 것이다.It relates to the use of a glutathione precursor drug comprising a protein having glutathione-S-transferase and a target cell or target protein-binding ability as a delivery vehicle and a pharmaceutical composition.
현재 알려져 있는 항암제의 종류는 화학항암제, 표적항암제, 면역항암제 등이 있다. Currently known types of anticancer drugs include chemical anticancer drugs, targeted anticancer drugs, and immune anticancer drugs.
화학항암제는 세포독성항암제, 화학약물항암제로도 불린다. 다른 항암제가 부작용이 없는 것은 아니지만, 세간에 흔히 알려진 항암치료 부작용인 백혈구 감소, 탈모, 구토중세, 설사 등은 대개 1세대 항암제인 화학항암제가 그 주범이다. 화학항암제를 쓴다고 모든 정상세포를 파괴되는 건 아니다. 주로 증식 속도가 빠른 골수의 조혈모세포, 모근세포, 음식물이 통과하는 구강 및 장내 점막, 생식기관 등에 영향을 준다. 화악항암제는 단기간에 증식하는 암세포의 특성을 찾아 공격하도록 설계되어있기 때문이다.Chemical anticancer agents are also called cytotoxic anticancer agents and chemical anticancer agents. Although other anticancer drugs are not without side effects, chemotherapy drugs, which are the first-generation anticancer drugs, are the main culprit of white blood cell reduction, hair loss, vomiting, middle age, and diarrhea, which are commonly known side effects of chemotherapy. Using chemical anticancer drugs does not destroy all normal cells. It mainly affects the hematopoietic stem cells, hair follicle cells, oral and intestinal mucosa through which food passes, and reproductive organs of the bone marrow. This is because Hwaak anticancer drugs are designed to find and attack the characteristics of cancer cells that proliferate in a short period of time.
대부분 정상세포는 화학항암제를 사용한 치료가 끝난 뒤 시간이 지나면 회복된다. 2~4주 간격을 두고 항암치료를 시행하는 것은 정상세포가 회복할 시간을 주는 것이다. 하지만 손발저림과 같은 말초신경독성은 완전히 회복되기까지 몇 년까지 걸릴 수도 있다고 의료계는 경고한다. 또 화학항암제로 인해 심장, 폐, 콩팥, 생식기관에 손상이 발생하면 영구적으로 지속될 수도 있다.Most of the normal cells recover over time after treatment with chemotherapy is over. Performing chemotherapy at intervals of 2 to 4 weeks gives normal cells time to recover. However, the medical community warns that peripheral neurotoxicity, such as numbness in the hands and feet, may take several years to fully recover. In addition, if damage to the heart, lungs, kidneys, and reproductive organs occurs due to chemical anticancer drugs, it may persist permanently.
최근에는 표적 치료제가 활발히 개발되고 있는데 암세포가 가진 특정 마커만을 판별하여 공격하기 때문에 정상 세포를 건드리지 않고, 암세포 살상력도 몇 배나 뛰어나다. 만성 골수성 백혈병 환자들의 희망이나 다름없는 글리벡이 그 대표적인 예. 현실적으로 정상세포, 암세포 가리지 않고 죽여버리는 전통적인 고식적 항암제의 경우에는 이미 그 효과를 더욱 끌어올리거나 부작용을 줄일 수 있는 여지가 거의 한도에 다다랐기 때문에 이러한 표적치료제의 개발이 의학의 발전에 큰 화두가 되고 있다. 다만 표적치료제도 효과가 좋은 환자군은 한정되어 있고, 내성에 취약하며, 높은 가격대에 비해 실효능이 떨어진다는 치명적인 단점이 있어 한계점이 뚜렷하다.In recent years, targeted therapeutics have been actively developed, and since they only detect and attack specific markers possessed by cancer cells, they do not touch normal cells and are several times superior in killing power of cancer cells. Gleevec, which is the hope of patients with chronic myelogenous leukemia, is a prime example. In reality, in the case of traditional anticancer drugs that kill both normal cells and cancer cells, the room for enhancing their effects or reducing side effects is almost reached, so the development of these targeted treatments is a big topic in the development of medicine. have. However, the target therapy system has a fatal disadvantage in that the patient group with good effect is limited, it is vulnerable to tolerance, and its effectiveness is inferior compared to the high price point.
화학항암제와 표적치료제의 단점을 개선한 면역항암제의 경우, CAR-T세포 치료요법이 두각을 드러내고 있다. CAR-T세포 치료요법이란 환자에게서 추출한 T 세포에 인위적으로 설계한 유전자를 삽입해, 재프로그래밍된 T세포가 암세포를 공격하게끔 유도하는 방식을 말한다. CAR-T 치료요법의 경우 항암제가 잘 듣지 않는 난치암 환자에게서 높은 치료 효과를 보이나, 독성이 매우 강력하고 부작용으로 사이토카인 방출 증후군(Cytokine Release Syndrome)이 나타날 경우 환자가 사망하는 사례가 많아 불안정한 점이 많다. 또한 1회 투여비용이 미국 기준으로 1억 5천만에 달하며, 고형암에는 치료효과가 크지 않다. In the case of immune anticancer drugs that improve the shortcomings of chemotherapy and targeted treatments, CAR-T cell therapy is emerging. CAR-T cell therapy refers to a method of inducing reprogrammed T cells to attack cancer cells by inserting artificially designed genes into T cells extracted from patients. In the case of CAR-T therapy, it shows high therapeutic effect in patients with intractable cancer who do not work well with anticancer drugs, but the toxicity is very strong, and when cytokine release syndrome appears as a side effect, the patient dies in many cases, making it unstable. many. In addition, the cost of a single administration reaches 150 million US standards, and the treatment effect is not great for solid cancer.
따라서, 현재 항암제의 단점을 보안할 수 있는 약물의 개발이 계속되고 있다.Therefore, the development of drugs that can protect against the disadvantages of anticancer drugs continues.
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 결합된 글루타치온 전구체 약물을 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 precursor drug combined with the glutathione-S-transferase.
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 결합된 글루타치온 전구체 약물을 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 a glutathione precursor drug combined with the glutathione-S-transferase.
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 결합된 글루타치온 전구체 약물을 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 precursor drug conjugated to the glutathione-S-transferase 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 결합된 글루타치온 전구체 약물을 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 glutathione precursor drug conjugated to the glutathione-S-transferase 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 결합된 글루타치온 전구체 약물을 함유하는 약물 전달체를 제공한다. 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 provides a drug delivery system containing the glutathione precursor drug combined with the glutathione-S-transferase.
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 proper separation between functional moieties or to maintain the necessary inter-moiety interaction. Other flexible linkers are known in the art, for example, G and S linkers that add amino acid residues such as T and A to maintain flexibility as well as add polar amino acid residues to improve water solubility. I can. Accordingly, 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. A 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. In addition, 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, the vector 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.
또한, 상기 글루타치온-S-전이효소와 글루타치온 전구체 약물의 결합은 GSH(Glutathione)에 의한 것일 수 있다. In addition, the binding of the glutathione-S-transferase and the glutathione precursor drug may be caused by GSH (Glutathione).
일 구체예에 있어서, 상기 글루타치온 전구체 약물은 하기 화학식 1 내지 화학식 7 중 어느 하나 또는 이의 약학적으로 허용가능한 염으로 이루어진 군에서 선택되는 어느 하나 이상인 것일 수 있다. 상기 글루타치온 전구체 약물은 글루타치온을 포함하고 있어서, 상기와 같이 글루타치온-S-전이효소와 결합되는 것일 수 있다.In one embodiment, the glutathione precursor drug may be any one or more selected from the group consisting of any one of the following Formulas 1 to 7 or a pharmaceutically acceptable salt thereof. Since the glutathione precursor drug contains glutathione, it may be combined with glutathione-S-transferase as described above.
Figure PCTKR2020006906-appb-I000001
Figure PCTKR2020006906-appb-I000001
[화학식 1][Formula 1]
Figure PCTKR2020006906-appb-I000002
Figure PCTKR2020006906-appb-I000002
[화학식 2][Formula 2]
Figure PCTKR2020006906-appb-I000003
Figure PCTKR2020006906-appb-I000003
[화학식 3][Formula 3]
Figure PCTKR2020006906-appb-I000004
Figure PCTKR2020006906-appb-I000004
[화학식 4][Formula 4]
Figure PCTKR2020006906-appb-I000005
Figure PCTKR2020006906-appb-I000005
[화학식 5][Formula 5]
Figure PCTKR2020006906-appb-I000006
Figure PCTKR2020006906-appb-I000006
[화학식 6][Formula 6]
Figure PCTKR2020006906-appb-I000007
Figure PCTKR2020006906-appb-I000007
[화학식 7][Formula 7]
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 결합된 글루타치온 전구체 약물을 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 a glutathione precursor drug combined with the glutathione-S-transferase.
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 결합된 글루타치온 전구체 약물을 함유하는 조성물을 그를 필요로 하는 개체에 투여하는 단계를 포함하는 개체 내 약물을 전달하는 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 the ability to bind to a target cell or a target protein; And it provides a method of delivering a drug in a subject comprising administering a composition containing the glutathione precursor drug conjugated to the glutathione-S-transferase to a subject 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 결합된 글루타치온 전구체 약물을 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 the ability to bind to a target cell or a target protein; And it provides a method for preventing or treating cancer comprising administering a composition containing the glutathione precursor drug conjugated to the glutathione-S-transferase to an individual in need thereof.
일 구체예에 있어서, 상기 글루타치온 전구체 약물은 화학식1 내지 화학식 7 중 어느 하나 또는 이의 약학적으로 허용가능한 염으로 이루어진 군에서 선택되는 어느 하나 이상인 것일 수 있다.In one embodiment, the glutathione precursor drug may be any one or more selected from the group consisting of any one of Formulas 1 to 7 or a pharmaceutically acceptable salt thereof.
상기 글루타치온-S-전이효소(glutathione-S-transferase, GST), 표적 세포 또는 표적 단백질 결합능을 갖는 단백질, 및 상기 글루타치온-S-전이효소와 결합된 글루타치온 전구체 약물에 대해서는 상기한 바와 같다. The glutathione-S-transferase (GST), a protein having a target cell or target protein binding ability, and a glutathione precursor drug bound to the glutathione-S-transferase are as described above.
용어 "대상체", "개체" 및 "환자"는 척추동물, 바람직하게는 포유동물, 더욱 바람직하게는 인간을 지칭하기 위해 본원에서 상호교환가능하게 사용된다. 포유동물은 쥣과, 원숭이, 인간, 농장 동물, 스포츠 동물 및 애완동물을 포함하나 이들에 한정되지 않는다. 생체내에서 수득되거나 시험관내에서 배양된 생물학적 엔티티(entity)의 조직, 세포 및 그들의 자손도 또한 포함된다.The terms “subject”, “individual” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, monkeys, humans, farm animals, sports animals and pets. Tissues, cells and their progeny of biological entities obtained in vivo or cultured in vitro are also included.
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.
본원에 사용되는 바와 같이, "치료" 또는 "치료하는" 또는 "완화하는" 또는 "개선하는"은 상호교환가능하게 사용된다. 이들 용어는 치료 이익 및/또는 예방 이익을 포함하나 이들에 한정되지 않는 유리한 또는 요망되는 결과를 수득하는 방법을 지칭한다. 치료 이익은 치료 하의 하나 이상의 질병, 질환 또는 증상의 임의의 치료적으로 유의미한 개선 또는 그에 대한 효과를 의미한다. 예방 이익에 있어서, 조성물은 특정 질병, 질환 또는 증상이 발생할 위험이 있는 대상체에게 또는 질병, 질환 또는 증상이 아직 나타나지 않을지라도, 질병의 하나 이상의 생리학적 증상을 보고하는 대상체에게 투여될 수 있다.As used herein, “treatment” or “treating” or “relaxing” or “improving” are used interchangeably. These terms refer to methods of obtaining beneficial or desired results, including but not limited to therapeutic benefits and/or prophylactic benefits. A therapeutic benefit refers to any therapeutically significant improvement or effect thereon of one or more diseases, disorders or symptoms under treatment. In a prophylactic benefit, the composition may be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more physiological symptoms of the disease, even if the disease, condition, or symptom is not yet present.
용어 "유효량" 또는 "치료적 유효량"은 유리한 또는 요망되는 결과를 야기하기에 충분한 작용제의 양을 지칭한다. 치료적 유효량은 치료되는 대상체 및 병태, 대상체의 체중 및 연령, 병태의 중증도, 투여 방식 등 중 하나 이상에 따라 달라질 수 있으며, 이는 당업자에 의해 용이하게 결정될 수 있다. 또한, 상기 용어는 본원에 기술된 영상화 방법 중 임의의 것에 의한 검출을 위한 이미지를 제공할 용량에 적용된다. 특정 용량은 선택된 특정 작용제, 뒤따르는 투여 요법, 그것이 다른 화합물과 병용하여 투여되는지 여부, 투여 시기, 영상화되는 조직 및 그것을 운반하는 신체 전달 시스템 중 하나 이상에 따라 달라질 수 있다.The term “effective amount” or “therapeutically effective amount” refers to an amount of an agent sufficient to produce an advantageous or desired result. The therapeutically effective amount may vary according to one or more of the subject and condition to be treated, the weight and age of the subject, the severity of the condition, the mode of administration, and the like, which can be easily determined by those skilled in the art. Further, the term applies to the capacity to provide an image for detection by any of the imaging methods described herein. The specific dosage may vary depending on one or more of the particular agent selected, the dosage regimen that follows, whether it is administered in combination with other compounds, the timing of administration, the tissue being imaged, and the body delivery system carrying it.
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 by mixing 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.
또 다른 양상은 하기 i) 내지 iii) 단계를 포함하는, 약물 전달체의 제조 방법을 제공한다:Another aspect provides a method for preparing a drug delivery system comprising the following steps i) to iii):
i) 약물에 링커(예를 들면, GSH)를 결합시키는 단계; ii) 상기 단계 i)에서 링커와 결합된 약물 표면에 GST 융합 단백질을 결합시키는 단계. i) linking a linker (eg, GSH) to the drug; ii) binding the GST fusion protein to the drug surface bonded to the linker in step i).
일 양상에 따른 글루타치온 전구체 약물의 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 delivery system of the glutathione precursor drug according to an 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 GST-Afb 융합 단백질의 세포 독성 및 세포 표적능을 확인한 결과이다:1 is a result of confirming the cytotoxicity and cell targeting ability of the GST-Afb fusion protein:
도 1a는 GST-Afb 융합 단백질이 Afb 표적 세포에 대하여 세포 생존율을 저하시키지 않음을 확인한 결과이고; 및 도 1b는 GST-Afb 융합 단백질이 Afb 표적 세포에 대하여 나타내는 세포 표적능을 확인한 결과이다.1A is a result of confirming that the GST-Afb fusion protein does not decrease cell viability with respect to Afb target cells; And FIG. 1B is a result of confirming the cell targeting ability that the GST-Afb fusion protein exhibits against Afb target cells.
도 2는 글루타치온 전구체 약물의 한 종류인 엔트리 2의 합성을 도식화 한 것이다.Figure 2 is a schematic diagram of the synthesis of entry 2, a type of glutathione precursor drug.
도 3은 글루타치온 전구체 약물의 한 종류인 엔트리 3의 합성을 도식화 한 것이다.3 is a schematic view of the synthesis of entry 3, which is a type of glutathione precursor drug.
도 4는 글루타치온 전구체 약물의 한 종류인 엔트리 5의 합성을 도식화 한 것이다.Figure 4 is a schematic diagram of the synthesis of entry 5, a type of glutathione precursor drug.
도 5는 글루타치온 전구체 약물의 한 종류인 엔트리 7의 합성을 도식화 한 것이다.Figure 5 is a schematic diagram of the synthesis of entry 7, a type of glutathione precursor drug.
도 6은 글루타치온 전구체 약물의 한 종류인 엔트리 9의 합성을 도식화 한 것이다.6 is a schematic diagram of the synthesis of entry 9, which is a type of glutathione precursor drug.
도 7은 글루타치온 전구체 약물의 한 종류인 엔트리 11의 합성을 도식화 한 것이다.7 is a schematic diagram of the synthesis of Entry 11, a type of glutathione precursor drug.
도 8은 글루타치온 전구체 약물의 한 종류인 엔트리 12의 합성을 도식화 한 것이다.Figure 8 is a schematic diagram of the synthesis of entry 12, a type of glutathione precursor drug.
도 9는 엔트리 2가 융합단백질에 결합하는 경우 결합 친화도를 나타낸 그래프이다.9 is a graph showing binding affinity when entry 2 binds to a fusion protein.
도 10은 엔트리 3이 융합단백질에 결합하는 경우 결합 친화도를 나타낸 그래프이다.10 is a graph showing binding affinity when entry 3 binds to a fusion protein.
도 11은 엔트리 5가 융합단백질에 결합하는 경우 결합 친화도를 나타낸 그래프이다.11 is a graph showing binding affinity when Entry 5 binds to a fusion protein.
도 12는 엔트리 2,3,5,7,9,11 및 12가 융합단백질에 결합하는 경우 결합 친화도 값을 나타낸 표이다.12 is a table showing binding affinity values when entries 2,3,5,7,9,11 and 12 bind to a fusion protein.
도 13은 융합단백질과 엔트리 2가 결합된 복합체의 세포 독성을 나타낸 그래프이다.13 is a graph showing the cytotoxicity of the complex in which the fusion protein and Entry 2 are bound.
도 14는 융합단백질과 엔트리 3이 결합된 복합체의 세포 독성을 나타낸 그래프이다.14 is a graph showing the cytotoxicity of the complex in which the fusion protein and Entry 3 are bound.
도 15는 융합단백질과 엔트리 5가 결합된 복합체의 세포 독성을 나타낸 그래프이다.15 is a graph showing the cytotoxicity of the complex in which the fusion protein and Entry 5 are bound.
도 16은 융합단백질과 엔트리 7이 결합된 복합체의 세포 독성을 나타낸 그래프이다.16 is a graph showing the cytotoxicity of the complex in which the fusion protein and Entry 7 are bound.
도 17은 융합단백질과 엔트리 9가 결합된 복합체의 세포 독성을 나타낸 그래프이다.17 is a graph showing the cytotoxicity of the complex in which the fusion protein and entry 9 are bound.
도 18은 융합단백질과 엔트리 11이 결합된 복합체의 세포 독성을 나타낸 그래프이다.18 is a graph showing the cytotoxicity of the complex in which the fusion protein and Entry 11 are bound.
도 19는 융합단백질과 엔트리 12가 결합된 복합체의 세포 독성을 나타낸 그래프이다.19 is a graph showing the cytotoxicity of the complex in which the fusion protein and Entry 12 are bound.
도 20은 융합단백질과 엔트리 2,3,5,7,9,11 및 12가 결합된 복합체의 세포 독성을 나타내는 IC50 값을 나타낸 표이다.FIG. 20 is a table showing IC50 values indicating cytotoxicity of complexes in which fusion proteins and entries 2,3,5,7,9,11 and 12 are bound.
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. 글루타치온-S-전이효소 및 표적 세포 또는 표적 단백질 결합능을 갖는 융합 단백질의 발현Example 1. Expression of a fusion protein having glutathione-S-transferase and target cell or target protein binding ability
<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 생체 내 환경에서도 안정성 및 표적능이 향상된 약물 전달체를 제조하고자 하였다. 이를 위해, 먼저 표적능을 갖는 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, it was intended to manufacture a drug delivery system with improved stability and targeting ability even in an in vivo environment for use as a drug delivery system. For this purpose, first, a fusion protein having a target function 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. 농축한 GST-HER2 Afb 및 GST-EGFR Afb는, SDS-PAGE 분석 및 ESI-TOF MS(electrospray ionization time-of-light mass spectrometry) 분석을 통해 분리한 단백질의 순도 및 분자량을 분석하였다.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 for a total of 10 minutes at 1 minute intervals. After crushing, centrifugation was performed 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 separated GST-Afb (GST-HER2 Afb and GST-EGFR Afb were dialyzed overnight in PBS (pH 7.4) and concentrated.) The concentrated GST-HER2 Afb and GST-EGFR Afb were analyzed by SDS-PAGE and ESI-TOF. The purity and molecular weight of the separated protein were analyzed through electrospray ionization time-of-light mass spectrometry (MS) analysis.
<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 전달체로서 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 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 1에서 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 1a), GST-HER2 Afb는 유방암 세포주인 SK-BR-3 세포에만 결합하는 결합능을 나타내어 GST-Afb가 암세포 특이적인 표적능을 나타내는 것으로 확인하였다(도 1b). 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. 1, it was confirmed that GST-HER2 Afb does not show toxicity to cells, but can 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. ), 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. 1B). 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. 글루타치온 전구체 약물의 합성Example 2. Synthesis of Glutathione Precursor Drug
본 발명의 글루타치온 전구체 약물의 합성은 도 2 내지 도 8과 같은 scheme으로 제작하였다. 하기 기재된 엔트리는 글푸타치온 전구체 약물을 의미한다.The synthesis of the glutathione precursor drug of the present invention was prepared in the same scheme as in FIGS. 2 to 8. The entries described below refer to the glutathione precursor drug.
구체적으로, 먼저 엔트리(entry) 1인 2-브로모에틸 비스(2-클로로에틸)카바메이트 (2-bromoethyl bis(2-chloroethyl)carbamate)의 합성은 비트(2-클로로에틸)아민 (Bis(2-chloroethyl)amine) (20 mg, 0.1408 mmol)과 수산화 나트륨 (Sodium hydroxide) (8.448 mg, 0.2112 mmol)을 먼저 메틸 클로라이드 (methyl chloride) 5mL에 섞은 후, 2-브로모에틸 카보노클로라이데이트 (2-bromoethyl carbonochloridate) (26.39 mg, 0.1408 mmol) 을 첨가하였다. Specifically, first, the synthesis of 2-bromoethyl bis(2-chloroethyl)carbamate, which is entry 1, is beet (2-chloroethyl) amine (Bis( 2-chloroethyl)amine) (20 mg, 0.1408 mmol) and sodium hydroxide (8.448 mg, 0.2112 mmol) were first mixed with 5 mL of methyl chloride, followed by 2-bromoethyl carbonochloridate (2-bromoethyl carbonochloridate) (26.39 mg, 0.1408 mmol) was added.
엔트리 2인 15-아미노-10-((카복시메틸)카바모일)-1-클로로-3-(2-클로로에틸)-4,12-디옥소-5-옥사-8-티아-3,11-디아자헥사데칸-16-온 산 (15-amino-10-((carboxymethyl)carbamoyl)-1-chloro-3-(2-chloroethyl)-4,12-dioxo-5-oxa-8-thia-3,11-diazahexadecan-16-oic acid)의 합성은 2-브로모에틸 비스(2-클로로에틸)카바메이트 (2-bromoethyl bis(2-chloroethyl)carbamate) (10 mg, 0.034 mmol)을 pH 7.4 PBS 용액에 넣은 후, PBS에 녹은 GSH ((glutathione), 7.683 mg, 0.025mmol)을 한방울씩 첨가하며 실온에서 24시간 교반하였다. 그 후 HPLC 과정을 통하여 분리하였다. Entry 2, 15-amino-10-((carboxymethyl)carbamoyl)-1-chloro-3-(2-chloroethyl)-4,12-dioxo-5-oxa-8-thia-3,11- Diazahexadecane-16-one acid (15-amino-10-((carboxymethyl)carbamoyl)-1-chloro-3-(2-chloroethyl)-4,12-dioxo-5-oxa-8-thia-3 ,11-diazahexadecan-16-oic acid) was prepared by using 2-bromoethyl bis(2-chloroethyl)carbamate (10 mg, 0.034 mmol) in pH 7.4 PBS. After adding to the solution, GSH ((glutathione), 7.683 mg, 0.025 mmol) dissolved in PBS was added dropwise, followed by stirring at room temperature for 24 hours. Then, it was separated through the HPLC process.
엔트리 3은 시스플라틴 (Cisplatin) (10mg, 0.0333 mmol) 을 GSH ((glutathione), 15mg, 0.05mmol) pH 7.4 PBS 용액에 섞고 실온에서 24시간 반응후, HPLC 과정을 통하여 분리하였다. Entry 3 was cisplatin (10mg, 0.0333 mmol) mixed with GSH ((glutathione), 15mg, 0.05mmol) pH 7.4 PBS solution, reacted at room temperature for 24 hours, and separated through HPLC.
엔트리 4인 2-(피리딘-2-일디술파닐)에틸 ((2S,3R,4S,6R)-6-(((1S,3S)-3-아세틸-3,5,12-트리히드록시-10-메톡시-6,11-디옥소-1,2,3,4,6,11-헥사히드로테트라센-1-일)옥시)-3-히드록시-2-메틸테트라히드로-2H-피란-4-일)카바메이트 (2-(pyridin-2-yldisulfaneyl)ethyl ((2S,3R,4S,6R)-6-(((1S,3S)-3-acetyl-3,5,12-trihydroxy-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-1-yl)oxy)-3-hydroxy-2-methyltetrahydro-2H-pyran-4-yl)carbamate)는 4-니트로페닐 (2-(피리딘-2-일디술파닐)에틸)카보네이트 (4-nitrophenyl (2-(pyridine-2-yldisulfaneyl)ethyl)carbonate) (20 mg, 0.0568 mmol)과 DMAP (18.52 mg, 0.1516 mmol)을 건조 메틸 클로라이드 (Dry methyl chloride) 7mL에 녹인 후 다우노루비신 (Daunorubicin) (20 mg, 0.079 mmol) 을 첨가 후 24시간 동안 환류시켰다. 메탄올로 DMAP을 세척한 후 컬럼크로마토그래피를 통하여 분리하였다. Entry 4, 2-(pyridin-2-yldisulfanyl)ethyl ((2S,3R,4S,6R)-6-(((1S,3S)-3-acetyl-3,5,12-trihydroxy- 10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-1-yl)oxy)-3-hydroxy-2-methyltetrahydro-2H-pyran -4-yl)carbamate (2-(pyridin-2-yldisulfaneyl)ethyl ((2S,3R,4S,6R)-6-(((1S,3S)-3-acetyl-3,5,12-trihydroxy -10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-1-yl)oxy)-3-hydroxy-2-methyltetrahydro-2H-pyran-4-yl)carbamate) Is 4-nitrophenyl (2-(pyridine-2-yldisulfaneyl)ethyl)carbonate) (20 mg, 0.0568 mmol) and DMAP (18.52 mg) , 0.1516 mmol) was dissolved in 7 mL of dry methyl chloride, and then Daunorubicin (20 mg, 0.079 mmol) was added and refluxed for 24 hours. After washing DMAP with methanol, it was separated through column chromatography.
엔트리 5인 N5-(3-((2-((((2S,3R,4S,6R)-6-(((1S,3S)-3-아세틸-3,5,12-트리히드록시-10-메톡시-6,11-디옥소-1,2,3,4,6,11-헥사히드로테트라센-1-일)옥시)-3-히드록시-2-메틸테트라히드로-2H-피란-4-일)카바모일)옥시)에틸)디술파닐)-1-((카복시메틸)아미노)-1-옥소프로판-2-일)글루타민 (N5-(3-((2-((((2S,3R,4S,6R)-6-(((1S,3S)-3-acetyl-3,5,12-trihydroxy-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-1-yl)oxy)-3-hydroxy-2-methyltetrahydro-2H-pyran-4-yl)carbamoyl)oxy)ethyl)disulfaneyl)-1-((carboxymethyl)amino)-1-oxopropan-2-yl)glutamine)은 2-(피리딘-2-일디술파닐)에틸 ((2S,3R,4S,6R)-6-(((1S,3S)-3-아세틸-3,5,12-트리히드록시-10-메톡시-6,11-디옥소-1,2,3,4,6,11-헥사히드로테트라센-1-일)옥시)-3-히드록시-2-메틸테트라히드로-2H-피란-4-일)카바메이트 (2-(pyridin-2-yldisulfaneyl)ethyl ((2S,3R,4S,6R)-6-(((1S,3S)-3-acetyl-3,5,12-trihydroxy-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-1-yl)oxy)-3-hydroxy-2-methyltetrahydro-2H-pyran-4-yl)carbamate) (15 mg, 0.02 mmol)을 Di water : MeOH 1:1 5mL에 녹인 후 글루타치온 (glutathione) (5.194 mg, 0.0169 mmol)을 한 방울씩 첨가하며 0 ℃에서 24시간 교반하였다. 그 후 HPLC 과정을 통하여 분리하였다.Entry 5, N5-(3-((2-((((2 S ,3 R ,4 S ,6 R )-6-(((1 S ,3 S )-3-acetyl-3,5,12) -Trihydroxy-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-1-yl)oxy)-3-hydroxy-2-methyltetra Hydro-2H-pyran-4-yl)carbamoyl)oxy)ethyl)disulfanyl)-1-((carboxymethyl)amino)-1-oxopropan-2-yl)glutamine ( N 5-(3-(( 2-((((2 S ,3 R ,4 S ,6 R )-6-(((1 S ,3 S )-3-acetyl-3,5,12-trihydroxy-10-methoxy-6,11 -dioxo-1,2,3,4,6,11-hexahydrotetracen-1-yl)oxy)-3-hydroxy-2-methyltetrahydro-2 H -pyran-4-yl)carbamoyl)oxy)ethyl)disulfaneyl)- 1-((carboxymethyl)amino)-1-oxopropan-2-yl)glutamine) is 2-(pyridin-2-yldisulfanyl)ethyl ((2S,3R,4S,6R)-6-(((1S, 3S)-3-acetyl-3,5,12-trihydroxy-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-1-yl) Oxy)-3-hydroxy-2-methyltetrahydro-2H-pyran-4-yl)carbamate (2-(pyridin-2-yldisulfaneyl)ethyl ((2S,3R,4S,6R)-6-(( (1S,3S)-3-acetyl-3,5,12-trihydroxy-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-1-yl)oxy)-3 -hydroxy-2-methyltetrahydro-2H-pyran-4-yl)carbamate) (15 mg, 0.02 mmol) was dissolved in Di water: MeOH 1:1 5 mL, and then glutathione (5.194 mg, 0.0169 mmol) was added 1 drop Each was added and stirred at 0° C. for 24 hours. Then, it was separated through the HPLC process.
엔트리 6인 (5-플루오로-2,4-디옥소-3,4-디히드로피리미딘-1(2H)-일)메틸 (2-(피리딘-2-일디술파닐)에틸) 카보네이트 ((5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl (2-(pyridin-2-yldisulfaneyl)ethyl) carbonate)는 4-니트로페닐 (2-(피리딘-2-일디술파닐)에틸)카보네이트 (4-nitrophenyl (2-(pyridine-2-yldisulfaneyl)ethyl)carbonate) (50 mg, 0.142 mmol)과 DMAP (46.23 mg, 0.3784 mmol)을 건조 DCM 15mL에 녹인 후 5-플루오로-1-(히드록시메틸)피리미딘-2,4-디온 (5-fluoro-1-(hydroxymethyl)pyrimidine-2,4-dione) (15.15 mg, 0.0946 mmol) 을 첨가 후 16시간 동안 실온에서 반응을 진행하였다. 메탄올로 DMAP을 세척한 후 DCM, 증류수로 추출한 후 MgSO4로 건조시킨 후 컬럼크로마토그래피를 통하여 분리하였다.Entry 6 (5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl (2-(pyridin-2-yldisulfanyl)ethyl) carbonate (( 5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)methyl (2-(pyridin-2-yldisulfaneyl)ethyl) carbonate) is 4-nitrophenyl (2-(pyridin-2 -Yldisulfanyl)ethyl)carbonate (4-nitrophenyl (2-(pyridine-2-yldisulfaneyl)ethyl)carbonate) (50 mg, 0.142 mmol) and DMAP (46.23 mg, 0.3784 mmol) were dissolved in 15 mL of dry DCM and 5 -Fluoro-1-(hydroxymethyl)pyrimidine-2,4-dione (5-fluoro-1-(hydroxymethyl)pyrimidine-2,4-dione) (15.15 mg, 0.0946 mmol) was added for 16 hours The reaction proceeded at room temperature. DMAP was washed with methanol, extracted with DCM and distilled water, dried over MgSO4, and separated through column chromatography.
엔트리 7인 15-아미노-10-((카복시메틸)카바모일)-1-(5-플루오로-2,4-디옥소-3,4-디히드로피리미딘-1(2H)-일)-3,12-디옥소-2,4-디옥사-7,8-디티아-11-아자헥사데칸-16-온산 (15-amino-10-((carboxymethyl)carbamoyl)-1-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,12-dioxo-2,4-dioxa-7,8-dithia-11-azahexadecan-16-oic acid)은 5-fluoro-2,4-dioxo-3,4-dihydropyrimidinyl methyl (2-(pyridine-2-yldisulfaneyl)ethyl) 카보네이트 (carbonate) (20 mg, 0.0536 mmol)를 용매 (Di water : MeOH = 1:1) 10mL에 녹인 후 글루타치온 (13.74 mg, 0.0447 mmol)을 한방울씩 첨가하며 0 ℃에서 12시간 반응 후 HPLC 과정을 통하여 분리하였다. Entry 7, 15-amino-10-((carboxymethyl)carbamoyl)-1-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)- 3,12-dioxo-2,4-dioxa-7,8-dithia-11-azahexadecane-16-onic acid (15-amino-10-((carboxymethyl)carbamoyl)-1-(5-fluoro -2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3,12-dioxo-2,4-dioxa-7,8-dithia-11-azahexadecan-16-oic acid) is 5 -fluoro-2,4-dioxo-3,4-dihydropyrimidinyl methyl (2-(pyridine-2-yldisulfaneyl)ethyl) carbonate (20 mg, 0.0536 mmol) solvent (Di water: MeOH = 1:1) After dissolving in 10 mL, glutathione (13.74 mg, 0.0447 mmol) was added dropwise, reacted at 0° C. for 12 hours, and then separated through HPLC.
엔트리 8인 (5-(4-아미노-2-옥소피리미딘-1(2H)-일)-4,4-디플루오로-3-히드록시테트라히드로푸란-2-일)메틸 (2-(피리딘-2-일디술파닐)에틸) 카보네이트 ((5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methyl (2-(pyridin-2-yldisulfaneyl)ethyl) carbonate)는 4-니트로페닐 (2-(피리딘-2-일디술파닐)에틸)카보네이트 ((4-nitrophenyl (2-(pyridine-2-yldisulfaneyl)ethyl)carbonate) (30 mg, 0.0851 mmol)과 DMAP (15.64 mg, 0.128 mmol)을 건조 DCM 15mL에 녹인 후 젬시타빈 (11.21 mg, 0.0426 mmol) 을 첨가 후 12시간 동안 실온에서 반응을 진행하였다. 테트라클로로메탄 (Tetrachloromethane)을 이용하여 재결정화시킨 후 감압필터를 이용하여 분리한 후 NMR로 확인하였다. Entry 8, (5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methyl (2- (Pyridin-2-yldisulfanyl)ethyl) carbonate ((5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methyl (2 -(pyridin-2-yldisulfaneyl)ethyl) carbonate) is 4-nitrophenyl (2-(pyridin-2-yldisulfanyl) ethyl) carbonate ((4-nitrophenyl (2-(pyridine-2-yldisulfaneyl) ethyl) carbonate) ) (30 mg, 0.0851 mmol) and DMAP (15.64 mg, 0.128 mmol) were dissolved in 15 mL of dry DCM, and gemcitabine (11.21 mg, 0.0426 mmol) was added, followed by reaction at room temperature for 12 hours. Tetrachloromethane) was used to recrystallize and then separated using a reduced pressure filter, and then confirmed by NMR.
엔트리 9인 15-아미노-1-(5-(4-아미노-2-옥소피리미딘-1(2H)-일)-4,4-디플루오로-3-히드록시테트라히드로푸란-2-일)-10-((카복시메틸)카바모일)-3,12-디옥소-2,4-디옥사-7,8-디티아-11-아자헥사데칸-16-온산 (15-amino-1-(5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-10-((carboxymethyl)carbamoyl)-3,12-dioxo-2,4-dioxa-7,8-dithia-11-azahexadecan-16-oic acid)은 (5-(4-아미노-2-옥소피리미딘-1(2H)-일)-4,4-디플루오로-3-히드록시테트라히드로푸란-2-일)메틸 (2-(피리딘-2-일디술파닐)에틸) 카보네이트 ((5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methyl (2-(pyridin-2-yldisulfaneyl)ethyl) carbonate) (15 mg, 0.0315 mmol)을 용매 (PBS 완충액) 6mL에 녹인 후 글루타치온 (6.45 mg, 0.021 mmol)을 한방울씩 첨가하며 실온에서 12시간 반응 후 원심 분리기를 이용하여 분리한 후 NMR로 반응을 확인하였다.Entry 9, 15-amino-1-(5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2- Day)-10-((carboxymethyl)carbamoyl)-3,12-dioxo-2,4-dioxa-7,8-dithia-11-azahexadecane-16-one acid (15-amino-1 -(5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)-10-((carboxymethyl)carbamoyl)-3,12-dioxo -2,4-dioxa-7,8-dithia-11-azahexadecan-16-oic acid) is (5-(4-amino-2-oxopyrimidin-1(2H)-yl)-4,4- Difluoro-3-hydroxytetrahydrofuran-2-yl)methyl (2-(pyridin-2-yldisulfanyl)ethyl) carbonate ((5-(4-amino-2-oxopyrimidin-1(2H)- Glutathione after dissolving yl)-4,4-difluoro-3-hydroxytetrahydrofuran-2-yl)methyl (2-(pyridin-2-yldisulfaneyl)ethyl) carbonate) (15 mg, 0.0315 mmol) in 6 mL of a solvent (PBS buffer) (6.45 mg, 0.021 mmol) was added dropwise and reacted for 12 hours at room temperature, separated using a centrifuge, and the reaction was confirmed by NMR.
엔트리 10인 ((2R,3S,5R)-5-(5-플루오로-2,4-디옥소-3,4-디히드로피리미딘-1(2H)-일)-3-히드록시테트라히드로푸란-2-일)메틸 (2-(피리딘-2-일디술파닐)에틸) 카보네이트 (((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)methyl (2-(pyridin-2-yldisulfaneyl)ethyl) carbonate)는 4-니트로페닐 (2-(피리딘-2-일디술파닐)에틸)카보네이트 (4-nitrophenyl (2-(pyridine-2-yldisulfaneyl)ethyl)carbonate) (20 mg, 0.057 mmol)와 DMAP (10.4 mg, 0.0851 mmol)을 건조 DMF 10mL에 녹인 후 플록스유리딘 (floxuridine) (6.99 mg, 0.0284 mmol) 을 첨가 후 16시간 동안 실온에서 반응을 진행하였다. 에탄올로 DMAP을 세척한 후 컬럼크로마토그래피를 통하여 분리하였다.Entry 10 ((2 R ,3 S ,5 R )-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hyd Roxytetrahydrofuran-2-yl)methyl (2-(pyridin-2-yldisulfanyl)ethyl) carbonate (((2 R ,3 S ,5 R )-5-(5-fluoro-2,4-dioxo -3,4-dihydropyrimidin-1( 2H )-yl)-3-hydroxytetrahydrofuran-2-yl)methyl (2-(pyridin-2-yldisulfaneyl)ethyl) carbonate) is 4-nitrophenyl (2-(pyridine- 2-yldisulfanyl)ethyl)carbonate (4-nitrophenyl (2-(pyridine-2-yldisulfaneyl)ethyl)carbonate) (20 mg, 0.057 mmol) and DMAP (10.4 mg, 0.0851 mmol) were dissolved in 10 mL of dry DMF. After addition of floxuridine (6.99 mg, 0.0284 mmol), the reaction was carried out at room temperature for 16 hours. After washing DMAP with ethanol, it was separated through column chromatography.
엔트리 11인 15-아미노-10-((카복시메틸)카바모일)-1-((2R,3S,5R)-5-(5-플루오로-2,4-디옥소-3,4-디히드로피리미딘-1(2H)-일)-3-히드록시테트라히드로푸란-2-일)-3,12-디옥소-2,4-디옥사-7,8-디티아-11-아자헥사데칸-16-온산 (15-amino-10-((carboxymethyl)carbamoyl)-1-((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)-3,12-dioxo-2,4-dioxa-7,8-dithia-11-azahexadecan-16-oic acid)은 ((2R,3S,5R)-5-(5-플루오로-2,4-디옥소-3,4-디히드로피리미딘-1(2H)-일)-3-히드록시테트라히드로푸란-2-일)메틸 (2-(피리딘-2-일디술파닐)에틸) 카보네이트 (((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)methyl (2-(pyridin-2-yldisulfaneyl)ethyl) carbonate) (33.77 mg, 0.0735 mmol)을 용매 (Di water : pH 7.4 PBS = 1:1) 10mL에 녹인 후 글루타치온 (15 mg, 0.049 mmol)을 한방울씩 첨가하며 22 ℃에서 16시간 반응 후 추출한 후 메탄올을 이용하여 원심 분리기로 분리한 후 NMR로 확인하였다.Entry 11, 15-amino-10-((carboxymethyl)carbamoyl)-1-((2 R ,3 S ,5 R )-5-(5-fluoro-2,4-dioxo-3,4 -Dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)-3,12-dioxo-2,4-dioxa-7,8-dithia-11- Azahexadecane-16-onic acid (15-amino-10-((carboxymethyl)carbamoyl)-1-((2 R ,3 S ,5 R )-5-(5-fluoro-2,4-dioxo-3, 4-dihydropyrimidin-1( 2H )-yl)-3-hydroxytetrahydrofuran-2-yl)-3,12-dioxo-2,4-dioxa-7,8-dithia-11-azahexadecan-16-oic acid) ((2 R ,3 S ,5 R )-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-3-hydroxytetrahydro Furan-2-yl)methyl (2-(pyridin-2-yldisulfanyl)ethyl) carbonate (((2R,3S,5R)-5-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin -1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)methyl (2-(pyridin-2-yldisulfaneyl)ethyl) carbonate) (33.77 mg, 0.0735 mmol) solvent (Di water: pH 7.4 PBS = 1 :1) After dissolving in 10 mL, glutathione (15 mg, 0.049 mmol) was added dropwise, reacted at 22° C. for 16 hours, extracted, separated by centrifugation using methanol, and confirmed by NMR.
엔트리 12인 N5-(1-((카복시메틸)아미노)-3-((3-(((2S)-1-(((14S,16S,33S,2R,4R,10E,12Z,14R)-86-클로로-14-히드록시-85,14-디메톡시-33,2,7,10-테트라메틸-12,6-디옥소-7-아자-1(6,4)-옥사지나나-3(2,3)-옥시라나-8(1,3)-벤젠아시클로테트라데카판-10,12-디엔-4-일)옥시)-1-옥소프로판-2-일)(메틸)아미노)-3-옥소프로필)디술파닐)-1-옥소프로판-2-일)글루타민 (N5-(1-((carboxymethyl)amino)-3-((3-(((2S)-1-(((14S,16S,33S,2R,4R,10E,12Z,14R)-86-chloro-14-hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl)oxy)-1-oxopropan-2-yl)(methyl)amino)-3-oxopropyl)disulfaneyl)-1-oxopropan-2-yl)glutamine)은 메르탄신 (Mertansine) (10 mg, 0.0135 mmol)을 용매 (DMF : pH 7.2 PBS 완충액=1:30)에 녹인 후 PBS 완충액에 녹인 글루타치온 (2.76 mg, 0.01 mmol)을 한방울씩 첨가하여 실온에서 10시간 반응시켰다. 뜨거운 DMF 소량에 녹인 후 4 ℃에서 24시간 동안 재결정화 시키고 감압필터로 생성물을 수득하였다. Entry 12, N5-(1-((carboxymethyl)amino)-3-((3-(((2S)-1-(((14S,16S,33S,2R,4R,10E,12Z,14R)- 86-chloro-14-hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(6,4)-oxaginana-3 (2,3)-oxirana-8(1,3)-benzeneacyclotetradecapan-10,12-dien-4-yl)oxy)-1-oxopropan-2-yl)(methyl)amino) -3-oxopropyl)disulfanyl)-1-oxopropan-2-yl)glutamine (N5-(1-((carboxymethyl)amino)-3-((3-(((2S)-1-((( 14S,16S,33S,2R,4R,10E,12Z,14R)-86-chloro-14-hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza -1(6,4)-oxazinana-3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl)oxy)-1-oxopropan-2-yl)( methyl)amino)-3-oxopropyl)disulfaneyl)-1-oxopropan-2-yl)glutamine) is a solvent (DMF: pH 7.2 PBS buffer = 1:30) in mertansine (10 mg, 0.0135 mmol). After dissolving, glutathione (2.76 mg, 0.01 mmol) dissolved in PBS buffer was added dropwise and reacted at room temperature for 10 hours. After dissolving in a small amount of hot DMF, it was recrystallized at 4° C. for 24 hours, and the product was obtained through a vacuum filter.
각 엔트리의 NMR 결과는 엔트리 1은 1H NMR: δ 3.47 (2H, t,　J　= 6.3 Hz), 3.56 (4H, t,　J　= 5.3 Hz), 3.83 (4H, t,　J　= 5.3 Hz), 4.34 (2H, t,　J　= 6.3 Hz), 엔트리 2는 1H NMR: δ 1.86-1.98 (2H, 1.92 (dt,　J　= 7.6, 7.4 Hz), 1.92 (dt,　J　= 7.6, 7.4 Hz)), 2.26-2.34 (2H, 2.30 (t,　J　= 7.4 Hz), 2.30 (t,　J　= 7.4 Hz)), 2.96-3.08 (4H, 3.04 (t,　J　= 6.0 Hz), 2.98 (d,　J　= 7.3 Hz), 2.98 (d,　J　= 7.3 Hz), 3.04 (t,　J　= 6.0 Hz)), 3.46 (1H, t,　J　= 7.6 Hz), 3.53-3.59 (4H, 3.56 (t,　J　= 5.3 Hz), 3.56 (t,　J　= 5.3 Hz)), 3.75-3.76 (2H, 3.75 (s), 3.75 (s)), 3.80-3.86 (4H, 3.83 (t,　J　= 5.3 Hz), 3.83 (t,　J　= 5.3 Hz)), 4.25-4.33 (2H, 4.29 (t,　J　= 6.0 Hz), 4.29 (t,　J　= 6.0 Hz)), 4.40 (1H, t,　J　= 7.3 Hz), 엔트리 3은 1H NMR: δ 1.86-1.98 (2H, 1.92 (dt,　J　= 7.6, 7.4 Hz), 1.92 (dt,　J　= 7.6, 7.4 Hz)), 2.24-2.32 (2H, 2.28 (t,　J　= 7.4 Hz), 2.28 (t,　J　= 7.4 Hz)), 3.30-3.35 (2H, 3.33 (d,　J　= 7.0 Hz), 3.33 (d,　J　= 7.0 Hz)), 3.46 (1H, t,　J　= 7.6 Hz), 3.75-3.76 (2H, 3.75 (s), 3.75 (s)), 4.56 (1H, t,　J　= 7.0 Hz), 엔트리 4는 1H NMR: δ 1.28 (3H, d,　J　= 6.7 Hz), 1.60 (1H, ddd,　J　= 14.6, 3.5, 2.9 Hz), 2.13 (3H, s), 2.30-2.49 (3H, 2.38 (ddd,　J　= 14.6, 10.2, 2.6 Hz), 2.44 (dd,　J　= 14.5, 3.8 Hz), 2.40 (dd,　J　= 14.5, 10.1 Hz)), 3.04 (1H, d,　J　= 15.8 Hz), 3.18-3.30 (2H, 3.23 (d,　J　= 15.8 Hz), 3.24 (dd,　J　= 10.3, 10.2 Hz)), 3.47-3.53 (2H, 3.50 (t,　J　= 5.2 Hz), 3.50 (t,　J　= 5.2 Hz)), 3.70 (3H, s), 3.83 (1H, td,　J　= 10.2, 3.5 Hz), 4.09 (1H, dq,　J　= 10.3, 6.7 Hz), 4.33-4.39 (2H, 4.36 (t,　J　= 5.2 Hz), 4.36 (t,　J　= 5.2 Hz)), 4.89-4.99 (2H, 4.97 (dd,　J　= 2.9, 2.6 Hz), 4.93 (dd,　J　= 10.1, 3.8 Hz)), 7.02 (1H, dd,　J　= 8.2, 1.3 Hz), 7.21 (1H, ddd,　J　= 7.4, 5.4, 1.7 Hz), 7.36 (1H, ddd,　J　= 7.8, 1.7, 0.5 Hz), 7.51 (1H, dd,　J　= 8.2, 7.8 Hz), 7.66 (1H, ddd,　J　= 7.8, 7.4, 1.9 Hz), 7.75 (1H, dd,　J　= 7.8, 1.3 Hz), 8.43 (1H, ddd,　J　= 5.4, 1.9, 0.5 Hz), 엔트리 5는 1H NMR: δ 1.28 (3H, d,　J　= 6.7 Hz), 1.60 (1H, ddd,　J　= 14.6, 3.5, 2.9 Hz), 1.86-1.98 (2H, 1.92 (dt,　J　= 7.6, 7.4 Hz), 1.92 (dt,　J　= 7.6, 7.4 Hz)), 2.13 (3H, s), 2.26-2.49 (5H, 2.38 (ddd,　J　= 14.6, 10.2, 2.6 Hz), 2.44 (dd,　J　= 14.5, 3.8 Hz), 2.40 (dd,　J　= 14.5, 10.1 Hz), 2.30 (t,　J　= 7.4 Hz), 2.30 (t,　J　= 7.4 Hz)), 3.04 (1H, d,　J　= 15.8 Hz), 3.18-3.33 (4H, 3.23 (d,　J　= 15.8 Hz), 3.24 (dd,　J　= 10.3, 10.2 Hz), 3.31 (d,　J　= 7.2 Hz), 3.31 (d,　J　= 7.2 Hz)), 3.40-3.50 (3H, 3.46 (t,　J　= 7.6 Hz), 3.43 (t,　J　= 5.1 Hz), 3.43 (t,　J　= 5.1 Hz)), 3.70 (3H, s), 3.75-3.76 (2H, 3.75 (s), 3.75 (s)), 3.83 (1H, td,　J　= 10.2, 3.5 Hz), 4.09 (1H, dq,　J　= 10.3, 6.7 Hz), 4.38-4.44 (2H, 4.41 (t,　J　= 5.1 Hz), 4.41 (t,　J　= 5.1 Hz)), 4.53 (1H, t,　J　= 7.2 Hz), 4.89-4.99 (2H, 4.97 (dd,　J　= 2.9, 2.6 Hz), 4.93 (dd,　J　= 10.1, 3.8 Hz)), 7.02 (1H, dd,　J　= 8.2, 1.3 Hz), 7.51 (1H, dd,　J　= 8.2, 7.8 Hz), 7.75 (1H, dd,　J　= 7.8, 1.3 Hz), 엔트리 6은 1H NMR: δ 3.45 (2H, t,　J　= 5.1 Hz), 4.40 (2H, t,　J　= 5.1 Hz), 5.67 (2H, s), 7.21 (1H, ddd,　J　= 7.4, 5.4, 1.7 Hz), 7.36 (1H, ddd,　J　= 7.8, 1.7, 0.5 Hz), 7.66 (1H, ddd,　J　= 7.8, 7.4, 1.9 Hz), 8.43 (1H, ddd,　J　= 5.4, 1.9, 0.5 Hz), 8.63 (1H, s), 엔트리 7은 1H NMR: δ 1.86-1.98 (2H, 1.92 (dt,　J　= 7.6, 7.4 Hz), 1.92 (dt,　J　= 7.6, 7.4 Hz)), 2.26-2.34 (2H, 2.30 (t,　J　= 7.4 Hz), 2.30 (t,　J　= 7.4 Hz)), 3.29-3.33 (2H, 3.31 (d,　J　= 7.2 Hz), 3.31 (d,　J　= 7.2 Hz)), 3.42-3.50 (3H, 3.46 (t,　J　= 7.6 Hz), 3.47 (t,　J　= 5.1 Hz), 3.47 (t,　J　= 5.1 Hz)), 3.75-3.76 (2H, 3.75 (s), 3.75 (s)), 4.42-4.49 (2H, 4.46 (t,　J　= 5.1 Hz), 4.46 (t,　J　= 5.1 Hz)), 4.53 (1H, t,　J　= 7.2 Hz), 5.66-5.67 (2H, 5.67 (s), 5.67 (s)), 8.63 (1H, s), 엔트리 8은 1H NMR: δ 3.42-3.48 (2H, 3.45 (t,　J　= 5.1 Hz), 3.45 (t,　J　= 5.1 Hz)), 3.92 (1H, d,　J　= 7.3 Hz), 4.02 (1H, dt,　J　= 7.3, 3.9 Hz), 4.42-4.48 (4H, 4.45 (t,　J　= 5.1 Hz), 4.44 (d,　J　= 3.9 Hz), 4.44 (d,　J　= 3.9 Hz), 4.45 (t,　J　= 5.1 Hz)), 5.66 (1H, s), 6.24 (1H, d,　J　= 10.2 Hz), 7.21 (1H, ddd,　J　= 7.4, 5.4, 1.7 Hz), 7.36 (1H, ddd,　J　= 7.8, 1.7, 0.5 Hz), 7.61-7.71 (2H, 7.66 (d,　J　= 10.2 Hz), 7.66 (ddd,　J　= 7.8, 7.4, 1.9 Hz)), 8.43 (1H, ddd,　J　= 5.4, 1.9, 0.5 Hz), 엔트리 9는 1H NMR: δ 1.86-1.98 (2H, 1.92 (dt,　J　= 7.6, 7.4 Hz), 1.92 (dt,　J　= 7.6, 7.4 Hz)), 2.26-2.34 (2H, 2.30 (t,　J　= 7.4 Hz), 2.30 (t,　J　= 7.4 Hz)), 3.29-3.33 (2H, 3.31 (d,　J　= 7.2 Hz), 3.31 (d,　J　= 7.2 Hz)), 3.42-3.50 (3H, 3.46 (t,　J　= 7.6 Hz), 3.47 (t,　J　= 5.1 Hz), 3.47 (t,　J　= 5.1 Hz)), 3.75-3.76 (2H, 3.75 (s), 3.75 (s)), 3.92 (1H, d,　J　= 7.3 Hz), 4.02 (1H, dt,　J　= 7.3, 3.9 Hz), 4.42-4.48 (3H, 4.44 (d,　J　= 3.9 Hz), 4.45 (t,　J　= 5.1 Hz), 4.44 (d,　J　= 3.9 Hz)), 4.45 (1H, t,　J　= 5.1 Hz), 4.53 (1H, t,　J　= 7.2 Hz), 5.66 (1H, s), 6.24 (1H, d,　J　= 10.2 Hz), 7.66 (1H, d,　J　= 10.2 Hz), 엔트리 10은 1H NMR: δ 2.06 (1H, ddd,　J　= 14.5, 7.1, 4.4 Hz), 2.27 (1H, ddd,　J　= 14.5, 8.1, 7.4 Hz), 3.31 (1H, ddd,　J　= 9.3, 7.4, 7.1 Hz), 3.42-3.48 (2H, 3.45 (t,　J　= 5.1 Hz), 3.45 (t,　J　= 5.1 Hz)), 4.12 (1H, dt,　J　= 9.3, 4.5 Hz), 4.41-4.48 (4H, 4.45 (t,　J　= 5.1 Hz), 4.43 (d,　J　= 4.5 Hz), 4.43 (d,　J　= 4.5 Hz), 4.45 (t,　J　= 5.1 Hz)), 6.13 (1H, dd,　J　= 8.1, 4.4 Hz), 7.21 (1H, ddd,　J　= 7.4, 5.4, 1.7 Hz), 7.36 (1H, ddd,　J　= 7.8, 1.7, 0.5 Hz), 7.66 (1H, ddd,　J　= 7.8, 7.4, 1.9 Hz), 8.43 (1H, ddd,　J　= 5.4, 1.9, 0.5 Hz), 8.59 (1H, s), 엔트리 11은 1H NMR: δ 1.86-1.98 (2H, 1.92 (dt,　J　= 7.6, 7.4 Hz), 1.92 (dt,　J　= 7.6, 7.4 Hz)), 2.06 (1H, ddd,　J　= 14.5, 7.1, 4.4 Hz), 2.19-2.35 (3H, 2.30 (t,　J　= 7.4 Hz), 2.27 (ddd,　J　= 14.5, 8.1, 7.4 Hz), 2.30 (t,　J　= 7.4 Hz)), 3.25-3.38 (3H, 3.31 (ddd,　J　= 9.3, 7.4, 7.1 Hz), 3.31 (d,　J　= 7.2 Hz), 3.31 (d,　J　= 7.2 Hz)), 3.42-3.50 (3H, 3.46 (t,　J　= 7.6 Hz), 3.47 (t,　J　= 5.1 Hz), 3.47 (t,　J　= 5.1 Hz)), 3.75-3.76 (2H, 3.75 (s), 3.75 (s)), 4.12 (1H, dt,　J　= 9.3, 4.5 Hz), 4.41-4.48 (3H, 4.43 (d,　J　= 4.5 Hz), 4.45 (t,　J　= 5.1 Hz), 4.43 (d,　J　= 4.5 Hz)), 4.45 (1H, t,　J　= 5.1 Hz), 4.53 (1H, t,　J　= 7.2 Hz), 6.13 (1H, dd,　J　= 8.1, 4.4 Hz), 8.59 (1H, s), 엔트리 12는 1H NMR: δ 1.08 (3H, d,　J　= 7.1 Hz), 1.32 (3H, d,　J　= 7.2 Hz), 1.45 (3H, s), 1.63 (3H, s), 1.86-1.99 (3H, 1.92 (dt,　J　= 7.6, 7.4 Hz), 1.94 (dd,　J　= 14.8, 2.6 Hz), 1.92 (dt,　J　= 7.6, 7.4 Hz)), 2.10 (1H, ddq,　J　= 9.2, 8.1, 7.1 Hz), 2.18-2.34 (3H, 2.30 (t,　J　= 7.4 Hz), 2.25 (dd,　J　= 14.8, 10.2 Hz), 2.30 (t,　J　= 7.4 Hz)), 2.37-2.41 (2H, 2.39 (t,　J　= 2.7 Hz), 2.39 (t,　J　= 2.7 Hz)), 2.59 (3H, s), 2.68-2.83 (3H, 2.72 (d,　J　= 14.0 Hz), 2.77 (dd,　J　= 15.0, 6.2 Hz), 2.73 (d,　J　= 14.0 Hz)), 2.88-3.00 (2H, 2.90 (d,　J　= 9.2 Hz), 2.95 (dd,　J　= 15.0, 2.1 Hz)), 3.14-3.17 (2H, 3.15 (t,　J　= 2.7 Hz), 3.15 (t,　J　= 2.7 Hz)), 3.29-3.33 (2H, 3.31 (d,　J　= 6.4 Hz), 3.31 (d,　J　= 6.4 Hz)), 3.41-3.50 (7H, 3.45 (s), 3.46 (t,　J　= 7.6 Hz), 3.43 (s)), 3.71 (3H, s), 3.75-3.76 (2H, 3.75 (s), 3.75 (s)), 4.23 (1H, q,　J　= 7.2 Hz), 4.30 (1H, d,　J　= 1.7 Hz), 4.54 (1H, t,　J　= 6.4 Hz), 5.17 (1H, ddd,　J　= 10.2, 8.1, 2.6 Hz), 5.68 (1H, dd,　J　= 6.2, 2.1 Hz), 5.85 (1H, dd,　J　= 9.9, 1.7 Hz), 5.97 (1H, d,　J　= 10.3 Hz), 6.66 (1H, d,　J　= 2.6 Hz), 6.77 (1H, dd,　J　= 10.3, 9.9 Hz), 7.43 (1H, d,　J　= 2.6 Hz)이다. The NMR results of each entry are: Entry 1 is 1 H NMR: δ 3.47 (2H, t, J = 6.3 Hz), 3.56 (4H, t, J = 5.3 Hz), 3.83 (4H, t, J = 5.3 Hz), 4.34 (2H, t, J = 6.3 Hz), entry 2 has 1 H NMR: δ 1.86-1.98 (2H, 1.92 (dt, J = 7.6, 7.4 Hz), 1.92 (dt, J = 7.6, 7.4 Hz)) , 2.26-2.34 (2H, 2.30 (t, J = 7.4 Hz), 2.30 (t, J = 7.4 Hz)), 2.96-3.08 (4H, 3.04 (t, J = 6.0 Hz), 2.98 (d, J = 7.3 Hz), 2.98 (d, J = 7.3 Hz), 3.04 (t, J = 6.0 Hz)), 3.46 (1H, t, J = 7.6 Hz), 3.53-3.59 (4H, 3.56 (t, J = 5.3) Hz), 3.56 (t, J = 5.3 Hz)), 3.75-3.76 (2H, 3.75 (s), 3.75 (s)), 3.80-3.86 (4H, 3.83 (t, J = 5.3 Hz), 3.83 (t , J = 5.3 Hz)), 4.25-4.33 (2H, 4.29 (t, J = 6.0 Hz), 4.29 (t, J = 6.0 Hz)), 4.40 (1H, t, J = 7.3 Hz), entry 3 is 1 H NMR: δ 1.86-1.98 (2H, 1.92 (dt, J = 7.6, 7.4 Hz), 1.92 (dt, J = 7.6, 7.4 Hz)), 2.24-2.32 (2H, 2.28 (t, J = 7.4 Hz) ), 2.28 (t, J = 7.4 Hz)), 3.30-3.35 (2H, 3.33 (d, J = 7.0 Hz), 3.33 (d, J = 7.0 Hz)), 3.46 (1H, t, J = 7.6 Hz ), 3.75-3.76 (2H, 3.75 (s), 3.75 (s)), 4.56 (1H, t, J = 7.0 Hz), entry 4 is 1 H NMR: δ 1.28 (3H, d, J = 6.7 Hz) , 1.60 (1H, ddd, J = 14.6, 3.5, 2.9 Hz), 2.13 (3H, s), 2.30-2.49 (3H, 2.38 (ddd, J = 14.6, 10.2, 2.6 Hz), 2.44 (dd, J = 14.5 , 3.8 Hz), 2.40 (dd, J = 14.5, 10.1 Hz)), 3.04 (1H, d, J = 15.8 Hz), 3.18-3.30 (2H, 3.23 (d, J = 15.8 Hz), 3.24 (dd, J = 10.3, 10.2 Hz)), 3.47-3.53 (2H, 3.50 (t, J = 5.2 Hz), 3.50 (t, J = 5.2 Hz)), 3.70 (3H, s), 3.83 (1H, td, J = 10.2, 3.5 Hz), 4.09 (1H, dq, J = 10.3, 6.7 Hz), 4.33-4.39 (2H, 4.36 (t, J = 5.2 Hz), 4.36 (t, J = 5.2 Hz)), 4.89- 4.99 (2H, 4.97 (dd, J = 2.9, 2.6 Hz), 4.93 (dd, J = 10.1, 3.8 Hz)), 7.02 (1H, dd, J = 8.2, 1.3 Hz), 7.21 (1H, ddd, J = 7.4, 5.4, 1.7 Hz), 7.36 (1H, ddd, J = 7.8, 1.7, 0.5 Hz), 7.51 (1H, dd, J = 8.2, 7.8 Hz), 7.66 (1H, ddd, J = 7.8, 7.4 , 1.9 Hz), 7.75 (1H, dd, J = 7.8, 1.3 Hz), 8.43 (1H, ddd, J = 5.4, 1.9, 0.5 Hz), entry 5 is 1 H NMR: δ 1.28 (3H, d, J = 6.7 Hz), 1.60 (1H, ddd, J = 14.6, 3.5, 2.9 Hz), 1.86-1.98 (2H, 1.92 (dt, J = 7.6, 7.4 Hz), 1.92 (dt, J = 7.6, 7.4 Hz) ), 2.13 (3H, s), 2.26-2.49 (5H, 2.38 (ddd, J = 14.6, 10.2, 2.6 Hz) , 2.44 (dd, J = 14.5, 3.8 Hz), 2.40 (dd, J = 14.5, 10.1 Hz), 2.30 (t, J = 7.4 Hz), 2.30 (t, J = 7.4 Hz)), 3.04 (1H, d, J = 15.8 Hz), 3.18-3.33 (4H, 3.23 (d, J = 15.8 Hz), 3.24 (dd, J = 10.3, 10.2 Hz), 3.31 (d, J = 7.2 Hz), 3.31 (d, J = 7.2 Hz)), 3.40-3.50 (3H, 3.46 (t, J = 7.6 Hz), 3.43 (t, J = 5.1 Hz), 3.43 (t, J = 5.1 Hz)), 3.70 (3H, s) , 3.75-3.76 (2H, 3.75 (s), 3.75 (s)), 3.83 (1H, td, J = 10.2, 3.5 Hz), 4.09 (1H, dq, J = 10.3, 6.7 Hz), 4.38-4.44 ( 2H, 4.41 (t, J = 5.1 Hz), 4.41 (t, J = 5.1 Hz)), 4.53 (1H, t, J = 7.2 Hz), 4.89-4.99 (2H, 4.97 (dd, J = 2.9, 2.6) Hz), 4.93 (dd, J = 10.1, 3.8 Hz)), 7.02 (1H, dd, J = 8.2, 1.3 Hz), 7.51 (1H, dd, J = 8.2, 7.8 Hz), 7.75 (1H, dd, J = 7.8, 1.3 Hz), Entry 6 is 1 H NMR: δ 3.45 (2H, t, J = 5.1 Hz), 4.40 (2H, t, J = 5.1 Hz), 5.67 (2H, s), 7.21 (1H , ddd, J = 7.4, 5.4, 1.7 Hz), 7.36 (1H, ddd, J = 7.8, 1.7, 0.5 Hz), 7.66 (1H, ddd, J = 7.8, 7.4, 1.9 Hz), 8.43 (1H, ddd , J = 5.4, 1.9, 0.5 Hz), 8.63 (1H, s), entry 7 is 1 H NMR: δ 1.86-1.98 (2H, 1.92 (dt, J = 7.6, 7.4 Hz), 1.92 (dt, J = 7.6, 7.4 Hz)), 2.26-2.34 (2H, 2.30 (t, J = 7.4 Hz), 2.30 (t, J = 7.4 Hz)), 3.29-3.33 (2H, 3.31 (d, J = 7.2 Hz), 3.31 (d, J = 7.2 Hz)), 3.42-3.50 (3H, 3.46 (t, J = 7.6 Hz), 3.47 (t, J = 5.1 Hz), 3.47 (t , J = 5.1 Hz)), 3.75-3.76 (2H, 3.75 (s), 3.75 (s)), 4.42-4.49 (2H, 4.46 (t, J = 5.1 Hz), 4.46 (t, J = 5.1 Hz) ), 4.53 (1H, t, J = 7.2 Hz), 5.66-5.67 (2H, 5.67 (s), 5.67 (s)), 8.63 (1H, s), entry 8 is 1 H NMR: δ 3.42-3.48 ( 2H, 3.45 (t, J = 5.1 Hz), 3.45 (t, J = 5.1 Hz)), 3.92 (1H, d, J = 7.3 Hz), 4.02 (1H, dt, J = 7.3, 3.9 Hz), 4.42 -4.48 (4H, 4.45 (t, J = 5.1 Hz), 4.44 (d, J = 3.9 Hz), 4.44 (d, J = 3.9 Hz), 4.45 (t, J = 5.1 Hz)), 5.66 (1H, s), 6.24 (1H, d, J = 10.2 Hz), 7.21 (1H, ddd, J = 7.4, 5.4, 1.7 Hz), 7.36 (1H, ddd, J = 7.8, 1.7, 0.5 Hz), 7.61-7.71 (2H, 7.66 (d, J = 10.2 Hz), 7.66 (ddd, J = 7.8, 7.4, 1.9 Hz)), 8.43 (1H, ddd, J = 5.4, 1.9, 0.5 Hz), Entry 9 is 1 H NMR : δ 1.86-1.98 (2H, 1.92 (dt, J = 7.6, 7.4 Hz), 1.92 (dt, J = 7.6, 7.4 Hz)), 2.26-2.34 (2H, 2.30 (t, J = 7 .4 Hz), 2.30 (t, J = 7.4 Hz)), 3.29-3.33 (2H, 3.31 (d, J = 7.2 Hz), 3.31 (d, J = 7.2 Hz)), 3.42-3.50 (3H, 3.46 (t, J = 7.6 Hz), 3.47 (t, J = 5.1 Hz), 3.47 (t, J = 5.1 Hz)), 3.75-3.76 (2H, 3.75 (s), 3.75 (s)), 3.92 (1H , d, J = 7.3 Hz), 4.02 (1H, dt, J = 7.3, 3.9 Hz), 4.42-4.48 (3H, 4.44 (d, J = 3.9 Hz), 4.45 (t, J = 5.1 Hz), 4.44 (d, J = 3.9 Hz)), 4.45 (1H, t, J = 5.1 Hz), 4.53 (1H, t, J = 7.2 Hz), 5.66 (1H, s), 6.24 (1H, d, J = 10.2 Hz), 7.66 (1H, d, J = 10.2 Hz), entry 10 is 1 H NMR: δ 2.06 (1H, ddd, J = 14.5, 7.1, 4.4 Hz), 2.27 (1H, ddd, J = 14.5, 8.1 , 7.4 Hz), 3.31 (1H, ddd, J = 9.3, 7.4, 7.1 Hz), 3.42-3.48 (2H, 3.45 (t, J = 5.1 Hz), 3.45 (t, J = 5.1 Hz)), 4.12 ( 1H, dt, J = 9.3, 4.5 Hz), 4.41-4.48 (4H, 4.45 (t, J = 5.1 Hz), 4.43 (d, J = 4.5 Hz), 4.43 (d, J = 4.5 Hz), 4.45 ( t, J = 5.1 Hz)), 6.13 (1H, dd, J = 8.1, 4.4 Hz), 7.21 (1H, ddd, J = 7.4, 5.4, 1.7 Hz), 7.36 (1H, ddd, J = 7.8, 1.7 , 0.5 Hz), 7.66 (1H, ddd, J = 7.8, 7.4, 1.9 Hz), 8.43 (1H, ddd, J = 5.4, 1.9, 0.5 Hz), 8.59 (1H, s), ent Li 11 is 1 H NMR: δ 1.86-1.98 (2H, 1.92 (dt, J = 7.6, 7.4 Hz), 1.92 (dt, J = 7.6, 7.4 Hz)), 2.06 (1H, ddd, J = 14.5, 7.1 , 4.4 Hz), 2.19-2.35 (3H, 2.30 (t, J = 7.4 Hz), 2.27 (ddd, J = 14.5, 8.1, 7.4 Hz), 2.30 (t, J = 7.4 Hz)), 3.25-3.38 ( 3H, 3.31 (ddd, J = 9.3, 7.4, 7.1 Hz), 3.31 (d, J = 7.2 Hz), 3.31 (d, J = 7.2 Hz)), 3.42-3.50 (3H, 3.46 (t, J = 7.6 Hz), 3.47 (t, J = 5.1 Hz), 3.47 (t, J = 5.1 Hz)), 3.75-3.76 (2H, 3.75 (s), 3.75 (s)), 4.12 (1H, dt, J = 9.3 , 4.5 Hz), 4.41-4.48 (3H, 4.43 (d, J = 4.5 Hz), 4.45 (t, J = 5.1 Hz), 4.43 (d, J = 4.5 Hz)), 4.45 (1H, t, J = 5.1 Hz), 4.53 (1H, t, J = 7.2 Hz), 6.13 (1H, dd, J = 8.1, 4.4 Hz), 8.59 (1H, s), entry 12 is 1 H NMR: δ 1.08 (3H, d , J = 7.1 Hz), 1.32 (3H, d, J = 7.2 Hz), 1.45 (3H, s), 1.63 (3H, s), 1.86-1.99 (3H, 1.92 (dt, J = 7.6, 7.4 Hz) , 1.94 (dd, J = 14.8, 2.6 Hz), 1.92 (dt, J = 7.6, 7.4 Hz)), 2.10 (1H, ddq, J = 9.2, 8.1, 7.1 Hz), 2.18-2.34 (3H, 2.30 ( t, J = 7.4 Hz), 2.25 (dd, J = 14.8, 10.2 Hz), 2.30 (t, J = 7.4 Hz)), 2.37-2.41 (2H, 2.39 (t, J = 2.7 Hz), 2.39 (t, J = 2.7 Hz)), 2.59 (3H, s), 2.68-2.83 (3H, 2.72 (d, J = 14.0 Hz), 2.77 (dd, J = 15.0, 6.2 Hz) , 2.73 (d, J = 14.0 Hz)), 2.88-3.00 (2H, 2.90 (d, J = 9.2 Hz), 2.95 (dd, J = 15.0, 2.1 Hz)), 3.14-3.17 (2H, 3.15 (t) , J = 2.7 Hz), 3.15 (t, J = 2.7 Hz)), 3.29-3.33 (2H, 3.31 (d, J = 6.4 Hz), 3.31 (d, J = 6.4 Hz)), 3.41-3.50 (7H , 3.45 (s), 3.46 (t, J = 7.6 Hz), 3.43 (s)), 3.71 (3H, s), 3.75-3.76 (2H, 3.75 (s), 3.75 (s)), 4.23 (1H, q, J = 7.2 Hz), 4.30 (1H, d, J = 1.7 Hz), 4.54 (1H, t, J = 6.4 Hz), 5.17 (1H, ddd, J = 10.2, 8.1, 2.6 Hz), 5.68 ( 1H, dd, J = 6.2, 2.1 Hz), 5.85 (1H, dd, J = 9.9, 1.7 Hz), 5.97 (1H, d, J = 10.3 Hz), 6.66 (1H, d, J = 2.6 Hz), 6.77 (1H, dd, J = 10.3, 9.9 Hz), 7.43 (1H, d, J = 2.6 Hz).
도 2는 글루타치온 전구체 약물의 한 종류인 엔트리 2의 합성을 도식화 한 것이다.Figure 2 is a schematic diagram of the synthesis of entry 2, a type of glutathione precursor drug.
도 3은 글루타치온 전구체 약물의 한 종류인 엔트리 3의 합성을 도식화 한 것이다.3 is a schematic view of the synthesis of entry 3, which is a type of glutathione precursor drug.
도 4는 글루타치온 전구체 약물의 한 종류인 엔트리 5의 합성을 도식화 한 것이다.Figure 4 is a schematic diagram of the synthesis of entry 5, a type of glutathione precursor drug.
도 5는 글루타치온 전구체 약물의 한 종류인 엔트리 7의 합성을 도식화 한 것이다.Figure 5 is a schematic diagram of the synthesis of entry 7, a type of glutathione precursor drug.
도 6은 글루타치온 전구체 약물의 한 종류인 엔트리 9의 합성을 도식화 한 것이다.6 is a schematic view of the synthesis of entry 9, which is a type of glutathione precursor drug.
도 7은 글루타치온 전구체 약물의 한 종류인 엔트리 11의 합성을 도식화 한 것이다.7 is a schematic diagram of the synthesis of Entry 11, a type of glutathione precursor drug.
도 8은 글루타치온 전구체 약물의 한 종류인 엔트리 12의 합성을 도식화 한 것이다.Figure 8 is a schematic diagram of the synthesis of entry 12, a type of glutathione precursor drug.
실시예 3. 글루타치온 전구체 약물과 융합 단백질간의 결합 확인 Example 3. Confirmation of binding between glutathione precursor drug and fusion protein
글루타치온 전구체 약물과 융합 단백질간의 결합을 ITC (isothermal titration calorimetry) 방법을 사용하여 확인하였다. The binding between the glutathione precursor drug and the fusion protein was confirmed using an isothermal titration calorimetry (ITC) method.
먼저 증류수로 대조 샘플을 채운 후, 튜브를 이용하여 플라스틱 주사기를 주입 주사기의 주입구에 연결하였다. 주입 주사기를 증류수로 헹군 뒤, 완충액으로 헹구었다. 시스템을 통해 공기를 흡입하여 주입 주사기를 완전히 비웠다. 주사 주사기의 바늘을 HER2 표적 애피바디가 부착된 글루타치온-S-전이효소 용액에 넣고 주사기 전체가 가득 찰 때까지 융합단백질을 주사기에 끌어들였다. 주입구 및 연결된 튜브에 100 uL까지 계속 끌어들인 후 주사기의 주입구를 즉시 닫고 튜브와 플라스틱 주사기를 분리하였다. 주사기에서 기포를 제거하기 위해 주입 주사기를 두 번 더 배출하고 다시 300uL까지 채웠다. 융합단백질 용액에서 주사기를 제거하고 주사기에서 용량이 제거될 수 있으므로 주사기 팁을 킴와이프에 닿지 않도록 주의하면서 킴와이프로 측면을 닦아 방울을 제거하였다. 또한 주사기 팁에서 용량이 손실될 수 있으므로 주사기를 두드리거나 병에 넣지 않도록 주의하였다. 그 다음, 주입 주사기를 엔트리 2,3,5,7,9,11 및 12를 각각 녹인 PBS 버퍼용액에 넣었다. 융합단백질을 주입 후, 시스템에 평형화 할 시간이 주어지고 다음 주입이 발생하기 전에 열 신호가 기준선으로 복귀하도록 5분 기다린다. 그 후 후속 주입을 300 uL으로 유지한 후, 실험 온도를 25 ℃로 선택한다. 위와 같이 파라미터를 설정 한 후 실험을 시작하였다. 실험은 오차발생을 줄이기 위해 3번 반복으로 진행하였다.First, a control sample was filled with distilled water, and then a plastic syringe was connected to the injection port of the injection syringe using a tube. The injection syringe was rinsed with distilled water and then rinsed with a buffer solution. The injection syringe was completely emptied by aspirating air through the system. The needle of the injection syringe was put into the glutathione-S-transferase solution to which the HER2 target affibody was attached, and the fusion protein was drawn into the syringe until the entire syringe was full. After continuing to draw up to 100 uL into the injection port and the connected tube, the injection port of the syringe was immediately closed and the tube and the plastic syringe were separated. In order to remove air bubbles from the syringe, the injection syringe was discharged two more times and filled to 300 uL again. Since the syringe was removed from the fusion protein solution and the volume could be removed from the syringe, the drop was removed by wiping the side with Kimwipe, taking care not to touch the syringe tip with Kimwipe. In addition, care was taken not to tap or bottle the syringe as it may cause loss of volume at the syringe tip. Then, the injection syringe was placed in a PBS buffer solution in which entries 2, 3, 5, 7, 9, 11 and 12 were dissolved, respectively. After injection of the fusion protein, the system is given time to equilibrate and wait 5 minutes for the heat signal to return to the baseline before the next injection occurs. Thereafter, the subsequent injection was maintained at 300 uL, and the experimental temperature was selected at 25°C. After setting the parameters as above, the experiment was started. The experiment was repeated 3 times to reduce the occurrence of errors.
그 다음, 데이터를 분석하였다. 구체적으로, 모든 데이터 피팅 프로그램 (일반적으로 제조업체에서 계측기와 함께 제공)에서 매크로를 사용하여 데이터 피팅을 쉽게 수행할 수 있다. 데이터를 피팅하는 데 사용할 데이터 피팅 모델 (한개의 결합 부위, 두개/여러개의 결합 부위, 협동 결합 등)을 선택한다. 데이터는 피팅 파라미터, 화학량론 (n), 엔탈피 (△H) 및 결합 친화도 (Ka)의 초기 추측에 적합할 수 있고 ITC 엔탈피 값을 van't Hoff 플롯의 엔탈피와 비교할 수 있다. 거대 분자 농도가 증가함에 따라 열 신호의 절대 값이 증가해야 하기 때문에 상이한 농도의 리간드 또는 거대 분자를 사용하는 것이 도움이 될 수 있다. 세포와 주사기의 완충액이 일치하지 않으면 잡음이 발생할 가능성이 높다. 잡음에 대한 또 다른 가능성은 불순물이 있는 샘플에서 발생한다.Then, the data were analyzed. Specifically, you can easily perform data fitting using macros in any data fitting program (usually provided by the manufacturer with the instrument). Select the data fitting model (single binding site, two/multiple binding sites, cooperative bonds, etc.) to be used to fit the data. The data can be suitable for initial guessing of the fitting parameters, stoichiometry (n), enthalpy (ΔH) and binding affinity (Ka) and ITC enthalpy values can be compared to the enthalpy of the van't Hoff plot. It may be helpful to use different concentrations of ligands or macromolecules because the absolute value of the heat signal must increase as the macromolecule concentration increases. If the buffers of the cells and syringes do not match, noise is likely to occur. Another possibility for noise arises in samples with impurities.
그 결과, 도 9 내지 도 12에 나타낸 바와 같이 융합단백질과 각 엔트리의 결합은 0.92 내지 1.1 μM 수준으로 결합함을 나타냈다.As a result, as shown in Figs. 9 to 12, the binding between the fusion protein and each entry showed that the binding was at the level of 0.92 to 1.1 μM.
도 9는 엔트리 2가 융합단백질에 결합하는 경우 결합 친화도를 나타낸 그래프이다.9 is a graph showing binding affinity when entry 2 binds to a fusion protein.
도 10은 엔트리 3이 융합단백질에 결합하는 경우 결합 친화도를 나타낸 그래프이다.10 is a graph showing binding affinity when entry 3 binds to a fusion protein.
도 11은 엔트리 5가 융합단백질에 결합하는 경우 결합 친화도를 나타낸 그래프이다.11 is a graph showing binding affinity when Entry 5 binds to a fusion protein.
도 12는 엔트리 2,3,5,7,9,11 및 12가 융합단백질에 결합하는 경우 결합 친화도 값을 나타낸 표이다.12 is a table showing binding affinity values when entries 2,3,5,7,9,11 and 12 bind to a fusion protein.
실시예 4. 글루타치온 전구체 약물과 융합단백질이 결합된 복합체의 세포 독성 확인Example 4. Confirmation of cytotoxicity of complexes in which glutathione precursor drug and fusion protein are bound
글루타치온 전구체 약물 (엔트리)와 융합단백질이 결합된 복합체의 항암 효과를 관찰하기 위해 세포 독성을 분석하였다. Cytotoxicity was analyzed to observe the anticancer effect of the complex of glutathione precursor drug (entry) and fusion protein combined.
구체적으로, 인간 유방암 세포(human breast cancer cell)인 SKBR3 세포에 대한 Her2 지향 융합단백질(Fusion protein)과 엔트리 2, 3, 5, 7, 9, 11, 및 12가 각각 결합된 복합체의 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, 100 nM ~ 1 μM 농도 범위의 엔트리 2, 3, 5, 7, 9, 11, 및 12 를 각각 세포에 처리하여 24 시간 동안 배양하였다. 대조군 실험은 융합단백질과 각 엔트리 2, 3, 5, 7, 9, 11, 및 12 로 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 the complex in which Her2-oriented fusion protein and entries 2, 3, 5, 7, 9, 11, and 12 are respectively bound to SKBR3 cells, which are human breast cancer cells, is 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) The reduction of to insoluble formazan was observed and evaluated. 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 12 were treated on the cells, respectively, and cultured for 24 hours. Control experiments were performed with fusion proteins and entries 2, 3, 5, 7, 9, 11, and 12, respectively. 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, Her2 지향 융합단백질(Fusion protein) 또는 엔트리 2, 3, 5, 7, 9, 11, 및 12 자체의 세포독성은 50ug/ml 까지 미비한 주순이나, 융합단백질(Fusion protein)과 엔트리 2, 3, 5, 7, 9, 11, 및 12이 각각 결합된 복합체의 세포독성은 IC50 수치가 1 내지 60 ug/ml 임을 나타냈다 (도 13 내지 도 20). 이러한 결과는, 표적 단백질 결합능을 갖는 단백질을 포함하는 글루타치온―S―전이효소 (glutathione-S-transferase, GST)에 글루타치온 전구체 약물이 결합하는 복합체를 조성하고 있다는 것과, 표적세포로 글루타치온 전구체 약물을 전달하여 치료용 목적으로 적용 가능함을 나타낸다.As a result, the cytotoxicity of Her2-oriented fusion protein or entry 2, 3, 5, 7, 9, 11, and 12 itself is inadequate to 50 ug/ml, but fusion protein and entry 2, The cytotoxicity of the complexes to which 3, 5, 7, 9, 11, and 12 were each bound showed an IC50 value of 1 to 60 ug/ml (FIGS. 13 to 20 ). These results indicate that a complex of glutathione precursor drug binding to glutathione-S-transferase (GST) containing a protein having target protein-binding ability is formed, and that glutathione precursor drug is delivered to target cells. This indicates that it can be applied for therapeutic purposes.
도 13은 융합단백질과 엔트리 2가 결합된 복합체의 세포 독성을 나타낸 그래프이다.13 is a graph showing the cytotoxicity of the complex in which the fusion protein and Entry 2 are bound.
도 14는 융합단백질과 엔트리 3이 결합된 복합체의 세포 독성을 나타낸 그래프이다.14 is a graph showing the cytotoxicity of the complex in which the fusion protein and Entry 3 are bound.
도 15는 융합단백질과 엔트리 5가 결합된 복합체의 세포 독성을 나타낸 그래프이다.15 is a graph showing the cytotoxicity of the complex in which the fusion protein and Entry 5 are bound.
도 16은 융합단백질과 엔트리 7이 결합된 복합체의 세포 독성을 나타낸 그래프이다.16 is a graph showing the cytotoxicity of the complex in which the fusion protein and Entry 7 are bound.
도 17은 융합단백질과 엔트리 9가 결합된 복합체의 세포 독성을 나타낸 그래프이다.17 is a graph showing the cytotoxicity of the complex in which the fusion protein and entry 9 are bound.
도 18은 융합단백질과 엔트리 11이 결합된 복합체의 세포 독성을 나타낸 그래프이다.18 is a graph showing the cytotoxicity of the complex in which the fusion protein and Entry 11 are bound.
도 19는 융합단백질과 엔트리 12가 결합된 복합체의 세포 독성을 나타낸 그래프이다.19 is a graph showing the cytotoxicity of the complex in which the fusion protein and Entry 12 are bound.
도 20은 융합단백질과 엔트리 2,3,5,7,9,11 및 12가 결합된 복합체의 세포 독성을 나타내는 IC50 값을 나타낸 표이다.FIG. 20 is a table showing IC50 values indicating cytotoxicity of complexes in which fusion proteins and entries 2,3,5,7,9,11 and 12 are bound.

Claims (11)

  1. 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 the ability to bind to a target cell or a target protein; And
    상기 글루타치온-S-전이효소와 결합된 글루타치온 전구체 약물을 함유하는 약물 전달체. A drug delivery system containing a glutathione precursor drug bound to the glutathione-S-transferase.
  2. 청구항 1에 있어서, 상기 표적 세포 또는 표적 단백질 결합능을 갖는 단백질은 수용체 타이로신 카이네이즈(Receptor tyrosine kinases: RTKs)에 특이적으로 결합하는 것인 약물 전달체. The drug delivery system according to claim 1, wherein the target cell or protein having the ability to bind to a target protein specifically binds to receptor tyrosine kinases (RTKs).
  3. 청구항 1에 있어서, 상기 수용체 타이로신 카이네이즈(Receptor tyrosine kinases: RTKs)는 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 하나인 것인 약물 전달체.The method of claim 1, wherein the receptor tyrosine kinases (RTKs) is an epidermal growth factor receptor, an insulin receptor, a platelet-derived growth factor receptor, a vascular endothelial growth factor receptor, a fibroblast growth factor receptor, and a cholecystokinin (CCK) receptor. , Neurotrophic factor (NGF) receptor, Hepatocyte growth factor (HGF) receptor, Ephrin (Eph) receptor, angiopoietin receptor, and RYK (related to receptor tyrosine kinase) receptor. A drug delivery system that is any one selected from the group.
  4. 청구항 1에 있어서, 상기 글루타치온-S-전이효소와 글루타치온 전구체 약물의 결합은 GSH(Glutathione)에 의한 것인 약물 전달체. The drug delivery system of claim 1, wherein the binding of the glutathione-S-transferase and the glutathione precursor drug is by GSH (Glutathione).
  5. 청구항 1에 있어서, 상기 글루타치온 전구체 약물은 하기 화학식 1 내지 화학식 7 중 어느 하나 또는 이의 약학적으로 허용가능한 염으로 이루어진 군에서 선택되는 어느 하나 이상인 것인 약물 전달체. The drug delivery system of claim 1, wherein the glutathione precursor drug is any one or more selected from the group consisting of any one of the following Formulas 1 to 7 or a pharmaceutically acceptable salt thereof.
    Figure PCTKR2020006906-appb-I000008
    Figure PCTKR2020006906-appb-I000008
    [화학식 1][Formula 1]
    Figure PCTKR2020006906-appb-I000009
    Figure PCTKR2020006906-appb-I000009
    [화학식 2][Formula 2]
    Figure PCTKR2020006906-appb-I000010
    Figure PCTKR2020006906-appb-I000010
    [화학식 3][Chemical Formula 3]
    Figure PCTKR2020006906-appb-I000011
    Figure PCTKR2020006906-appb-I000011
    [화학식 4][Formula 4]
    Figure PCTKR2020006906-appb-I000012
    Figure PCTKR2020006906-appb-I000012
    [화학식 5][Formula 5]
    Figure PCTKR2020006906-appb-I000013
    Figure PCTKR2020006906-appb-I000013
    [화학식 6][Formula 6]
    Figure PCTKR2020006906-appb-I000014
    Figure PCTKR2020006906-appb-I000014
    [화학식 7][Formula 7]
  6. 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 the ability to bind to a target cell or a target protein; And
    상기 글루타치온-S-전이효소와 결합된 글루타치온 전구체 약물을 포함하는 암 예방 또는 치료용 약학적 조성물. A pharmaceutical composition for preventing or treating cancer comprising a glutathione precursor drug combined with the glutathione-S-transferase.
  7. 청구항 6에 있어서, 상기 표적 세포 또는 표적 단백질 결합능을 갖는 단백질은 표피 성장인자 수용체, 인슐린 수용체, 혈소판 유래 성장인자 수용체, 혈관내피 성장인자 수용체, 및 안지오포이에틴 수용체에 특이적으로 결합하는 것인 암 예방 또는 치료용 약학적 조성물. The method according to claim 6, wherein the target cell or protein having a target protein binding ability specifically binds to epidermal growth factor receptor, insulin receptor, platelet-derived growth factor receptor, vascular endothelial growth factor receptor, and angiopoietin receptor. A pharmaceutical composition for preventing or treating cancer.
  8. 청구항 6에 있어서, 상기 글루타치온-S-전이효소와 글루타치온 전구체 약물의 결합은 GSH(Glutathione)에 의한 것인 암 예방 또는 치료용 약학적 조성물.The pharmaceutical composition for preventing or treating cancer according to claim 6, wherein the binding of the glutathione-S-transferase and the glutathione precursor drug is by GSH (Glutathione).
  9. 청구항 6에 있어서, 상기 글루타치온 전구체 약물은 하기 화학식 1 내지 화학식 7 중 어느 하나 또는 이의 약학적으로 허용가능한 염으로 이루어진 군에서 선택되는 어느 하나 이상인 것인 암 예방 또는 치료용 약학적 조성물. The pharmaceutical composition for preventing or treating cancer according to claim 6, wherein the glutathione precursor drug is any one or more selected from the group consisting of any one of the following Formulas 1 to 7 or a pharmaceutically acceptable salt thereof.
    Figure PCTKR2020006906-appb-I000015
    Figure PCTKR2020006906-appb-I000015
    [화학식 1][Formula 1]
    Figure PCTKR2020006906-appb-I000016
    Figure PCTKR2020006906-appb-I000016
    [화학식 2][Formula 2]
    Figure PCTKR2020006906-appb-I000017
    Figure PCTKR2020006906-appb-I000017
    [화학식 3][Chemical Formula 3]
    Figure PCTKR2020006906-appb-I000018
    Figure PCTKR2020006906-appb-I000018
    [화학식 4][Formula 4]
    Figure PCTKR2020006906-appb-I000019
    Figure PCTKR2020006906-appb-I000019
    [화학식 5][Formula 5]
    Figure PCTKR2020006906-appb-I000020
    Figure PCTKR2020006906-appb-I000020
    [화학식 6][Formula 6]
    Figure PCTKR2020006906-appb-I000021
    Figure PCTKR2020006906-appb-I000021
    [화학식 7][Formula 7]
  10. 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 the ability to bind to a target cell or a target protein; And
    상기 글루타치온-S-전이효소와 결합된 글루타치온 전구체 약물을 함유하는 조성물을 그를 필요로 하는 개체에 투여하는 단계를 포함하는 개체 내 약물을 전달하는 방법.A method of delivering a drug into a subject comprising administering a composition containing the glutathione precursor drug conjugated to the glutathione-S-transferase to a subject in need thereof.
  11. 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 결합된 글루타치온 전구체 약물을 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 the glutathione precursor drug conjugated to the glutathione-S-transferase to an individual in need thereof.
PCT/KR2020/006906 2019-05-28 2020-05-28 Glutathione precursor drug-delivery carrier comprising glutathione-s-transferase and protein, which has binding capacity for target cell or target protein, and use thereof WO2020242218A1 (en)

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