[go: up one dir, main page]

WO2018093065A1 - Pharmaceutical composition for cancer prevention and treatment comprising tubulosine - Google Patents

Pharmaceutical composition for cancer prevention and treatment comprising tubulosine Download PDF

Info

Publication number
WO2018093065A1
WO2018093065A1 PCT/KR2017/012129 KR2017012129W WO2018093065A1 WO 2018093065 A1 WO2018093065 A1 WO 2018093065A1 KR 2017012129 W KR2017012129 W KR 2017012129W WO 2018093065 A1 WO2018093065 A1 WO 2018093065A1
Authority
WO
WIPO (PCT)
Prior art keywords
cancer
lymphoma
tubulosine
jak3
composition
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/KR2017/012129
Other languages
French (fr)
Korean (ko)
Inventor
김병학
예상규
이충기
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cytus H&b Co Ltd
Original Assignee
Cytus H&b Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cytus H&b Co Ltd filed Critical Cytus H&b Co Ltd
Publication of WO2018093065A1 publication Critical patent/WO2018093065A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration

Definitions

  • the present invention relates to a pharmaceutical composition for preventing and treating cancer, including tubulosine.
  • One of the important signals regulating this biological process is JAKs (Janus family tyrosine kinases) and their substrate transcription factors, substrate transcription factors and signal transducer and activator of transcription (STAT) proteins.
  • Mammals encode four JAKs (JAK1, JAK2, JAK3, and TYK2) and seven STATs (STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6), and many cytokines and growth factors are JAK or STAT proteins Activating signaling pathways by specific combinations of O'Shea JJ et al., Annu Rev Med. 2015; 66: 311-328; Springuel L et al., Haematologica 2015; 100: 1240-1253.
  • JAK3 In pediatric pre-B cell acute lymphocytic leukemia (ALL), the expression of BLNK was completely lost or rapidly decreased. Somatic mutations of the JAK3 allele have been identified in AML and acute megakaryotic leukemia (AMKL) patients and cell lines, Down syndrome acute lymphoblastic and myeloid leukemia, T cell ALL, cutaneous T cell lymphomas (CTCLs) for high risk children ALL. In this case, patients continued to activate JAK3 signals by gain-of-function. These results suggest that abnormally activated JAK3 signaling contributes to the pathogenesis of certain partial hematopoietic stem cells, and that JAK3 is a therapeutic target for the treatment of patients with this disease.
  • tubulosin directly blocks the catalytic activity of JAK3 and is a small-molecule substance capable of treating various diseases caused by abnormal activity of JAK3.
  • Another object of the present invention is to provide a method for treating cancer comprising administering the above-described composition of the present invention to a subject in need thereof.
  • Another object of the present invention is to provide a composition comprising tubulosine as a use for treating cancer.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising (a) a therapeutically effective amount of tubulosine represented by the formula (1); And (b) a pharmaceutically acceptable carrier, the pharmaceutical composition for preventing, ameliorating or treating cancer, comprising:
  • NCI National Cancer Institute
  • tubulosine inhibited the catalytic activity of JAK3 and strongly inhibited constitutively-active and cytokine-induced JAK3 signaling in leukemia and lymphoma cell lines, thereby inhibiting active JAK3-mediated cancer cell survival and proliferation. Accordingly, the present inventors have confirmed that tubulin (NSC131547) is a small-molecule substance that can directly block catalytic activity of JAK3, thereby treating various diseases caused by abnormal activity of JAK3.
  • tubulosine (NSC131547) was found to directly block the catalytic activity of JAK3.
  • Tubulosine binds mainly to the kinase domain of JAK3 in the JAK family, effectively inhibiting JAK3-mediated signal transduction at low concentrations.
  • Tubulosine did not inhibit the activation of other tumor cell kinases in various cancer cells.
  • tubulin was observed to reduce cell survival and proliferation in cancer cells expressing JAK3, which is sustained-active.
  • tubulosine selectively inhibits the activity of JAK3, demonstrating that it is a potential small-molecule candidate capable of treating various diseases caused by abnormal activity of JAK3.
  • composition of the present invention inhibits the catalytic activity of JAK3 (Janus Kinase 3).
  • composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered through various routes such as intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, and transdermal administration.
  • Cancer of the present invention is hematologic cancer, breast cancer, brain cancer, neuroendocrine tumor, gastric cancer, lung cancer, prostate cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, adrenal cancer, colon cancer, colon cancer, cervical cancer , Bone cancer, skin cancer, thyroid cancer, parathyroid cancer and ureter cancer.
  • Lymphoma refers to Non-Hodgkins Lymphoma (NHL); Diffuse large B-cell lymphoma (DLBCL); Follicular lymphoma (FL); Hodgkin disease; Burkitt's Lymphoma; Cutaneous T cell lymphoma; Primary central nervous system lymphoma and lymphomatous metastases.
  • T cell lymphomas of the invention are selected from the group consisting of, but not limited to: lymphoblastic lymphomas in which malignant tumors develop in primitive lymphoid progenitor cells from the thymus; Mature or peripheral T cell neoplasms include T-cell prolymphocytic leukemia, T-cell granular lymphocytic leukemia; Aggressive NK-cell leukemia; Cutaneous T cell lymphoma; Adult T-cells associated with anaplastic large cell lymphoma, enteropathy-type T cell lymphoma, and human T-lymphotropic virus-1 (HTLV-1) Adult T-cell leukemia / lymphoma, angioimmunoblastic T cell lymphoma, subcutaneous periculitic T cell lymphoma and initially peripheral lymph node paracortex Peripheral T cell lymphoma that contains and does not grow in a true follicular pattern.
  • the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.
  • solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose, lactose in the pharmaceutical composition. , Gelatin and the like are mixed and formulated.
  • lubricants such as magnesium stearate, talc and the like may also be used in addition to simple excipients.
  • Oral liquid preparations include suspensions, solvents, emulsions, syrups, and the like.
  • various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be used. May be included.
  • preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilizers, suppositories, and the like.
  • Non-aqueous solvents and suspending agents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like.
  • injectables may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, preservatives, and the like.
  • compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container.
  • the formulation may be in the form of a solution, suspension or emulsion in an oil or an aqueous medium, or may be in the form of extracts, powders, granules, tablets or capsules, and may further include a dispersant or stabilizer.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising (a) a therapeutically effective amount of tubulosine represented by the following formula (1); And (b) a pharmaceutically acceptable carrier; administering to a subject in need thereof a composition comprising:
  • the cancer treatment method of the present invention uses the above-described pharmaceutical composition for treating cancer of the present invention, and the common content between the two is omitted in order to avoid excessive complexity of the present specification.
  • “individual” is not particularly limited, but includes, for example, humans, monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, mice, rabbits or guinea pigs. do.
  • the invention provides a composition comprising tubulosine represented by the following formula (1) as a use for treating cancer:
  • tubulosine Since tubulosine selectively inhibits JAK3 activity among JAK family members, it can be used as a therapeutic agent for treating various diseases (especially cancer) caused by non-ideal JAK3 activity.
  • 1A-1C Schematic diagram of a structure-based virtual database screening model.
  • 1a Chemical structure of tubulosine (C 29 H 37 N 3 O 3 ; MW 475.6).
  • 1b Predictive binding model of tubulosine and JAK3 kinase domains (JAK3-JH1, PDB ID: 1YVJ). Only side chains contacted with tubulosine within 3.5 ⁇ s were labeled.
  • Tubulosine selectively inhibits JAK3 kinase activity in vitro.
  • Each JAK immunoprecipitate was pretreated for 1 hour with DMSO (vehicle), tubulosine (25, 50, 75, and 100 nM) or pan-JAK kinase inhibitor AG490 (150 ⁇ M), respectively. Then, as a kinase reaction, His-tagged recombinant STAT3 ⁇ protein (2 ⁇ g) and ATP (2 ⁇ M) were added and reacted at 30 ° C. for 30 minutes.
  • JAK3 immunoprecipitates were performed by ATP-dependent (2, 10, 50, 500, and 1000 ⁇ M) in vitro kinase assays, with or without tubulosine (100 nM). Reaction products were confirmed by western blotting using appropriate antibodies.
  • Tubulosine is an ATP-competitive kinase inhibitor against JAK3.
  • 4A-4D Tubulosine completely blocks active JAK2 signaling intrinsically in cancer cells.
  • 4a-4d Human cancer cell lines L540 (4a), HDLM-2, MDA-MB-468, DU145 and A431 (4b), mouse leukemia cell lines BKO-84, BaF3 / JAK3V674A and BaF3 / TEL-JAK3 (4c) and BaF3 / TEL-JAK2 (4d) was treated with DMSO (vehicle), tubulosine (25, 50, 75, and 100 nM) or pan-JAK inhibitor AG490 (150 ⁇ M) for 24 hours. Western blotting was performed with whole cell lysates of the cells. GAPDH was used as loading control.
  • FIGS. 5A-5D Tubulosine suppresses JAK3 / STAT signaling in cancer cells.
  • 5a-5d Tubulosine (100) in human Hodgkin's lymphoma cell line L540 (5a) and mouse leukemia cell lines BKO-84 (5b), BaF3 / JAK3V674A (5c), and BaF3 / TEL-JAK3 (5d). nM) was treated for some time. Western blotting was performed with whole cell lysates of the cells. GAPDH was used as loading control.
  • FIGS. 6a-6b High concentrations of tubulosine inhibit JAK1, JAK2, and / or TYK2-mediated signals in cancer cells.
  • 6a-6b Human Hodgkin's lymphoma HDLM-2 cells (6a) and mouse leukemia BaF3 / TEL-JAK2 cells (6b) in DMSO (vehicle), tubulosine or pan-JAK inhibitor AG490 (150 ⁇ M) was reacted for 24 hours. Western blotting was performed with whole cell lysates of the cells. GAPDH was used as loading control.
  • FIGS 7A-7C Tubulosine blocks IL-2-induced JAK3 / STAT5 signals.
  • 7a-7b Rat pre-T lymphoma Nb2 cells were starved for 16 hours by treatment with DMSO (vehicle), tubulosine (25, 50, 75, and 100 nM) or pan-JAK inhibitor AG490 (150 ⁇ M). Prolactin (PRL, 100 ng / mL, A) or IL-2 (100 ng / mL, B) was treated for 10 minutes.
  • DMSO vehicle
  • tubulosine 25, 50, 75, and 100 nM
  • pan-JAK inhibitor AG490 150 ⁇ M
  • Prolactin PRL, 100 ng / mL, A
  • IL-2 100 ng / mL, B
  • Tubulosine does not affect other tumorigenic kinase signal activation in cancer cells.
  • FIGS 9A-9B Tubulosine selectively reduces the survival of cancer cells expressing constitutively-active JAK3.
  • 9a-9b DMSO (vehicle), tubulosine (LSO, BKO-84 and BaF3 / JAK3V674A cells (9a) and HDLM-2, MDA-MB-468, DU145, and BaF3 / TEL-JAK2 cells (9b).
  • 25, 50, and 100 nM) or pan-JAK inhibitor AG490 (150 ⁇ M) was reacted.
  • Western blotting was performed with whole cell lysates of the cells. GAPDH was used as loading control.
  • FIGS. 10A-10B Tubulosine inhibits the proliferation of cancer cells that selectively express constitutively-active JAK3.
  • 10a-10b DMSO (vehicle), tubulosine (L), BKO-84 and BaF3 / JAK3V674A cells (10a) and HDLM-2, MDA-MB-468, DU145, and BaF3 / TEL-JAK2 cells (10b).
  • 25, 50, and 100 nM) or pan-JAK inhibitor AG490 (150 ⁇ M) was reacted.
  • FIG. 11 is a schematic diagram illustrating that tubulosin selectively targets JAK3 and JAK3-mediated subtarget signals.
  • protein coordinates from the complex structure between JAK3-JH1 and its inhibitor, the staurosporine analog AFN941, were selected as a template.
  • hydrogen atoms were added with AMBER software, which was then added to the PDB2PQR-determined ionizable states at the aspartic acid (Asp), glutamic acid (Glu), histidine (His) and lysine (Lys) residues.
  • Asp aspartic acid
  • Glu glutamic acid
  • His histidine
  • Lys lysine residues.
  • the final structures were clustered based on structural similarity quantified by root mean square deviation (RMSD) values between the structures.
  • the center of the most dominant cluster was chosen as the representative.
  • the values of 100 and 500,000 represent the parameters (ga_pop_size) for each number in the population and the maximum number of generations (ga_num_evals) for the general algorithm in AutoDock, respectively.
  • ga_pop_size the parameters for each number in the population
  • ga_num_evals the maximum number of generations
  • docking was repeated using the six structures of JAK1-JH1 or JAK2-JH1 as templates in the same procedure.
  • the PDB code of the structure is 3EYG and 3EYH for JAK1-JH1 and JAK2-JH1, respectively; And 2B7A, 2W1I, 3E62 and 3FUP.
  • Tubulosine (NSC131547) is one of the compounds identified in the NCI Diversity Set Compound, and has been deposited with the Developmental Therapeutics Program (DTP) / NCI and provided to researchers for nonclinical purposes. Information on tubulosine synthesis and purity can be found on the DTP / NCI website.
  • DTP Developmental Therapeutics Program
  • Human Hodgkin's lymphoma cell lines HDLM-2 and L540, and B-cell derived multiple myeloma cell line U266 is a German Collection of Microorganisms and Cell Cultures DSMZ, Germany).
  • Human breast cancer cell lines MDA-MB-468, prostate cancer cell line DU145 and epidermal cancer cell line A431 were obtained from the American Type Culture Collection (ATCC) Rockville, MD, USA, and were cultured in DMEM medium containing 10% FBS.
  • Pre-B leukemia cell line BKO-84 Bone marrow-derived, derived from BLNK ⁇ / ⁇ mice from Daisuke Kitamura and consistently expressing mutant JAK3 (BaF3 / JAK3V674A), TEL-JAK2 (BaF3 / TEL-JAK2) and TEL-JAK3 (BaF3 / TEL-JAK3) pro-B cell line BaF3 was determined by Dr. Hiroyuki Mano and Dr.
  • the rat pre-T lymphoma cell line Nb2 was obtained from Olivier A. Bernard. Obtained from Charles V. Clevenger.
  • Antibodies specific for phospho-JAK1 (Tyr1022 / 1023), JAK1, phospho-JAK2 (Tyr1007 / 1008), JAK2, phospho-JAK3 (Tyr980 / 981), JAK3, phospho-TYK2 (Tyr1054 / 1055), TYK2, phospho-STAT1 (Tyr701), STAT1, phospho-STAT3 (Tyr705), phospho-STAT5 (Tyr694), phospho-Src (Tyr416), Src, phospho-Lyn (Tyr507), Lyn, phospho-Akt (Ser473), Akt, phospho-ERK1 / 2 (Thr202 / Tyr204), ERK1 / 2, PARP, caspase 3, caspase 9, Bcl-2, Bcl-xL, Mcl-1, survivin, Bax, Bad, p21, p27, RIP3, Beclin 1, LC3B and
  • Cells were plated at 1 ⁇ 10 4 per well in 96-well plates and incubated until 70-80% confluence. Cells were then treated with DMSO (vehicle), tubulosine, or AG490 (150 ⁇ M) at regular time intervals. Cell viability was further reacted at 37 ° C for 3 hours, followed by addition of 10 ⁇ L assay reagent (EZ-CyTox Enhanced Cell Viability Assay Reagent, Daeil Lab Service, Seoul, Korea) and microreaders (Molecular Devices, Sunnyvale, USA) at 450 nm. On the other hand, surviving cells were counted through trypan blue analysis and cell proliferation was measured.
  • DMSO vehicle
  • JAKs in vitro kinase assays were performed by conventional methods (Kim BH et al, MS-1020 is a novel small molecule that selectively inhibits JAK3 activity.Br J Haematol. 2010; 148: 132-143 / Kim BH et al, NSC114792, a novel small molecule identified through structure-based computational database screening, selectively inhibits JAK 3. Mol Cancer 2010; 9: 36.).
  • lysis buffer (20 mM Tris-HCl, pH 7.4, 500 mM NaCl, 0.25% Triton X-100, 1 mM EDTA, 1 mM EGTA, 10 mM ⁇ -glycerophosphate, 1 mM DTT) in HDLM-2 or L540 cells , 300 ⁇ Na 3 VO 4, 1 mM PMSF and phosphatase inhibitor cocktail).
  • Cell lysates were pre-cleared with protein A / G-sepharose at 4 ° C. for 2 hours, and anti-JAK1, anti-JAK2, anti-JAK3, or anti-TYK2 antibodies were washed at 4 ° C. for 1 day. (overnight) treatment.
  • Immune complexes were precipitated with protein A / G-sepharose beads. The precipitate was washed twice with kinase buffer (25 mM Tris / HCl, pH 7.5, 20 mM ⁇ -glycerophosphate, 10 mM MgCl 2, 2 mM DTT, 1 mM Na 3 VO 4, and protease inhibitor cocktail).
  • kinase buffer 25 mM Tris / HCl, pH 7.5, 20 mM ⁇ -glycerophosphate, 10 mM MgCl 2, 2 mM DTT, 1 mM Na 3 VO 4, and protease inhibitor cocktail.
  • DMSO vehicle
  • tubulosine or pan-JAK inhibitor AG490 150 ⁇ M
  • the kinase reaction occurred with addition of His-tagged STAT3 ⁇ protein (2 ⁇ g) under the condition of ATP (2-1000 ⁇ M) addition or no addition at 0 ° C. for 30 minutes.
  • SDS-PAGE was performed as a reaction product, and antibodies against phospho-STAT3, STAT3, JAK1, JAK2, JAK3, or TYK2 were used.
  • JH1 Janus homology domains that share important structural homology.
  • JH1 is a kinase domain, which is important for JAK catalytic activity.
  • AutoDock Version 4.2 for the kinase domain of JAK3 was used and virtual screening was performed with NCI diversity set compounds. Ensemble docking with various early ligand conformers improved the reliability of AutoDock. The result was better in accuracy and energy than the result obtained by simply increasing the number of runs with the same conformer.
  • FIG. 1A The model structure of tubulosine in complex with JAK3-JH1 (FIG. 1A) is shown in Val-812, Ala-829, Glu-847, Met-878, Leu-881, Leu-932, and Asp-943 (FIG. 1B). It appeared to be in contact with the side chain atom.
  • the AutoDock-calculated binding free energy between JAK3-JH1 and tubulosine is -11.79 kcal / mol, whereas the binding free energies of AFN941, CP-690550 and CMP-6 are -10.64, -9.23 and -10.32 kcal / mol, respectively.
  • FIG. 1C The binding mode is similar to AFN941, CP-690550, and CMP-6 in the region around two -OCH 3 , but different in the region in contact with Asp-847.
  • Tubulosine directly blocks JAK3 kinase activity by binding to the ATP-binding site
  • JAK immunoprecipitates were obtained from cell lysates of Hodgkin's lymphoma HDLM-2 or L540 cells. Each JAK is active in these cells. Each immunoprecipitate was reacted with recombinant STAT3 ⁇ protein under different concentrations of tubulosine.
  • Ki and Km values indicating JAK3 kinase activity were measured and found to be 6.3 ⁇ 0.6 nM with tubulosine and 106.4 ⁇ 5.7 ⁇ M with ATP addition (FIG. 3C).
  • Staurosporine (STS) was used as a positive control of the ATP-competitive inhibitor against JAKs.
  • tubulosine As mentioned above, we confirmed the selectivity of tubulosine for JAK3 inhibition. To assess this selectivity, we tested whether tubulosine could inhibit activated JAK3 signals in various cancer cell lines.
  • JAK3 signals are constitutively-active in cell lines L540, BKO-84, BaF3 / JAK3V674A, and BaF3 / TEL-JAK3, and other JAKs are not activated.
  • Tubulosine inhibits tyrosine phosphorylation of JAK3 and its substrate STAT5 concentration-dependently (FIGS. 4A and 4C).
  • the cell line was observed over time by treatment with 100 nM tubulosine.
  • tubulosine effectively inhibited tyrosine phosphorylation of JAK3 and its substrates STAT3 and STAT5 within 2 hours (FIGS. 5A-5D).
  • tubulin concentrations that inhibit other JAKs were treated with tubulosine in a concentration range of 100 nM to 1 ⁇ M for 16 hours.
  • Tyrosine phosphorylation of JAKs including JAK1, JAK2, and TYK2, and their substrates, STAT3 and STAT5
  • tubulosine was inhibited by tubulosine at concentrations above 400 nM (FIGS. 6A and 6B).
  • tubulosine for inhibition of JAK3 signal by IL-2 or prolactin (PRL) stimulation in rat pre-T lymphoma Nb2 cells that were used in cytokine-dependent activity studies of JAK signals.
  • Cells were treated with DMSO (vehicle), tubulosine or AG490 for 16 hours, followed by 10 minutes with IL-2 or PRL.
  • IFN interferon
  • IFN- ⁇ -stimulation markedly increased tyrosine phosphorylation of JAK1, TYK2, STAT1 and STAT3 in multiple myeloma U266 cells as compared to cells not treated with IFN- ⁇ .
  • Tyrosine phosphorylation was not affected up to 100 nM tubulosine concentration (FIG. 7C).
  • the pan-JAK inhibitor AG490 non-selectively inhibited tyrosine phosphorylation of all JAKs, STAT3 and STAT5 in cells treated with IL-2-, PRL-, or IFN- ⁇ .
  • Tubulosine increases ERK activity but does not affect other tumorigenic signals
  • tubulosine can influence the signals of other tumorigenic factors such as the non-receptor tyrosine kinase Src family, serine / threonine kinase Akt, and extracellular signal-regulated kinase (ERK).
  • tumorigenic factors such as the non-receptor tyrosine kinase Src family, serine / threonine kinase Akt, and extracellular signal-regulated kinase (ERK).
  • tubulosine levels in the active levels of Src family tyrosine kinases did not show a significant inhibitory effect (Figs. 8a and 8b, lanes 3 and 4).
  • Akt phosphorylation levels after treatment with tubulosine in the cells did not show a significant inhibitory effect (Figs. 8a and 8b, lanes 3 and 4).
  • the ERK1 / 2 phosphorylation level was significantly increased in tubulosine concentration only in L540 cells expressing constitutively-active JAK3.
  • Activated JAK / STAT signaling is associated with increased survival and proliferation of cancer cells, and small molecule inhibitors targeting this signaling have been reported to inhibit cell survival in various solid cancers and hematologic malignancies. These evidences show that tubulosine may affect cell survival only in cancer cells that exhibit sustained-active JAK3 signals. To demonstrate this possibility, cell survival assays were performed by treating various cancer cell lines with DMSO (vehicle), tubulosine or AG490 at various times.
  • tubulosine significantly reduced the survival of various cancer cells showing time- and concentration-dependent, sustained-active JAK3 signals rather than cells with other JAKs-mediated signals (FIGS. 9A and 9B). Cancer cell viability was confirmed by treatment with the pan-JAK inhibitor AG490 and significantly reduced cell viability in all cell lines.
  • the proliferation rate of cancer cells was confirmed by counting the number of cells using trypan blue assay.
  • tubulosine effectively inhibited the proliferation of cancer cells showing sustained-active JAK3 signals in a concentration and time dependent manner (FIG. 10A).
  • proliferation of other JAK-activated cancer cells had minimal effect of tubulosine (FIG. 10B).
  • AG490 effectively inhibited the proliferation of cancer cells in all cell lines.
  • the present invention relates to a pharmaceutical composition for treating cancer comprising tubulosine as an active ingredient.
  • tubulosine inhibits JAK catalytic activity in leukemia and lymphoma cell lines, thereby strongly inhibiting both structurally activated cytokine-induced JAK3 signaling, thereby preventing survival and proliferation of active JAK-mediated cancer cells. It can be usefully used as an inhibitor.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Nutrition Science (AREA)
  • Physiology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The present invention relates to a pharmaceutical composition for cancer treatment comprising tubulosine as an active ingredient. In the composition of the present invention, tubulosine inhibits the catalytic activity of a JAK in leukemia and lymphoma cell lines, thereby entirely and strongly inhibits structurally activated cytokine-induced JAK3 signaling, and thus can be usefully employed as a preparation for inhibiting the survival and proliferation of active JAK-mediated cancer cells.

Description

튜불로신을 포함하는 암 예방 및 치료용 약제학적 조성물Pharmaceutical composition for the prevention and treatment of cancer containing tubulosine

본 발명은 튜불로신을 포함하는 암 예방 및 치료용 약제학적 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for preventing and treating cancer, including tubulosine.

사이토카인 및 성장인자는 세포 성장, 분화, 면역 항상성, 자가면역 및 염증성 질환과 같은 생물학적 반응을 조절하는데 중요한 역할을 한다. 이러한 생물학적 과정을 조절하는 중요한 시그널 중 하나는 JAKs(Janus family tyrosine kinases) 및 이들의 기질 전사인자(substrate transcription factors), 신호 전달자(substrate transcription factors) 및 STAT(signal transducer and activator of transcription) 단백질이다. 포유류는 4 개의 JAKs(JAK1, JAK2, JAK3, 및 TYK2) 및 7 개의 STATs(STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, 및 STAT6)를 코딩하고, 많은 사이토카인 및 성장인자들은 JAK 또는 STAT 단백질의 특이한 조합에 의해 신호전달 경로를 활성화시킨다[O'Shea JJ et al., Annu Rev Med. 2015;66:311-328; Springuel L et al., Haematologica 2015;100:1240-1253].Cytokines and growth factors play an important role in regulating biological responses such as cell growth, differentiation, immune homeostasis, autoimmunity and inflammatory diseases. One of the important signals regulating this biological process is JAKs (Janus family tyrosine kinases) and their substrate transcription factors, substrate transcription factors and signal transducer and activator of transcription (STAT) proteins. Mammals encode four JAKs (JAK1, JAK2, JAK3, and TYK2) and seven STATs (STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6), and many cytokines and growth factors are JAK or STAT proteins Activating signaling pathways by specific combinations of O'Shea JJ et al., Annu Rev Med. 2015; 66: 311-328; Springuel L et al., Haematologica 2015; 100: 1240-1253.

JAK3는 조혈 세포(hematopoietic cells)에서 주로 발현되고, 인터루킨(IL)-2, IL-4, IL-7, IL-9, IL-15, 및 IL-21의 사이토카인 수용체인 일반적인 감마 사슬(γc) 서브패밀리를 이용한 신호전달을 매개하며, 이는 T-세포 발달 및 면역 시스템 항상성에서 JAK3가 중요한 역할을 하는 것을 의미한다. 그러나, JAK3/STAT 시그널링의 비정상적인 활성화로 인해 조혈성 종양(hematopoietic malignancy)이 나타난다. 조혈모줄기세포 이식을 이용한 상염색체 열성 SCID(severe combined immunodeficiency)의 유전자치료는 인간 환자 및 마우스 모델에서 γc-매개 JAK3/STAT5 시그널의 직접적인 활성화를 통하여 T-세포 루케미아의 위험성을 증가시킨다. 급성 골수성 백혈병(acute myeloid leukemia, AML) 환자 약 70%에서의 백혈병 발아세포[Birkenkamp KU et al., Leukemia 2001;15:1923-1931], 인간 혈액암에서 유래한 다양한 세포주, 예컨대 mantle-cell lymphoma[Yared MA et al., Arch Pathol Lab Med. 2005;129:990-996], Burkitt's lymphoma[Gee K et al., Cell Immunol. 2001;211:131-42], anaplastic large cell lymphoma(ALCL)[Lai R et al., Hum Pathol. 2005;36:939-944], 및 enteropathy-associated T-cell lymphoma[Malamut G et al., J Clin Invest. 2010;120:2131-2143]에서 JAK3-매개 STAT 시그널링이 지속적으로 활성화되었다. 또한, 자가분비화(autocrine) JAK3/BKL의 억제제인 종양 억제인자 B 세포 링커(BLNK)의 기능 상실에 의해 확립된 pre-B-세포 백혈병 마우스 모델에서, 지속적으로 활성화된 JAK3/STAT 신호 전달이 나타났다. 소아 전 B 세포 급성 림프 구성 백혈병(ALL)에서 BLNK의 발현은 완전히 소실되거나 급격히 감소되었다. JAK3 대립 유전자의 체세포 돌연변이는 AML 및 급성 거핵성 백혈병(AMKL) 환자 및 세포주, 고위험 소아 ALL의 경우 다운증후군 급성 림프 구성 및 골수성 백혈병, T 세포 ALL, 피부 T 세포 림프종(CTCLs) 등에서 확인되었다. 이 경우 환자들은 기능 보강(gain-of-function)에 의해 JAK3 신호가 지속적으로 활성화되었다. 이러한 결과는 비정상적으로 활성화된 JAK3 신호가 조혈모세포의 특정 부분 병인에 기여하며, JAK3은 이러한 질환을 가진 환자의 치료를 위한 치료 표적임을 시사한다.JAK3 is a common gamma chain (γc) that is expressed primarily in hematopoietic cells and is a cytokine receptor for interleukin (IL) -2, IL-4, IL-7, IL-9, IL-15, and IL-21 Mediates signaling with subfamily, which means that JAK3 plays an important role in T-cell development and immune system homeostasis. However, hematopoietic malignancy appears due to abnormal activation of JAK3 / STAT signaling. Gene therapy of autosomal recessive combined immunodeficiency (SCID) using hematopoietic stem cell transplantation increases the risk of T-cell leuemia through direct activation of γc-mediated JAK3 / STAT5 signals in human patients and mouse models. Leukemia germ cells (Birkenkamp KU et al., Leukemia 2001; 15: 1923-1931) in about 70% of patients with acute myeloid leukemia (AML), various cell lines derived from human hematologic cancers, such as mantle-cell lymphoma Yared MA et al., Arch Pathol Lab Med. 2005; 129: 990-996, Burkitt's lymphoma [Gee K et al., Cell Immunol. 2001; 211: 131-42], anaplastic large cell lymphoma (ALCL) [Lai R et al., Hum Pathol. 2005; 36: 939-944], and enteropathy-associated T-cell lymphoma [Malamut G et al., J Clin Invest. 2010; 120: 2131-2143, JAK3-mediated STAT signaling was constantly activated. In addition, in a pre-B-cell leukemia mouse model established by loss of function of tumor suppressor B cell linker (BLNK), an inhibitor of autocrine JAK3 / BKL, sustained activation of JAK3 / STAT signaling is appear. In pediatric pre-B cell acute lymphocytic leukemia (ALL), the expression of BLNK was completely lost or rapidly decreased. Somatic mutations of the JAK3 allele have been identified in AML and acute megakaryotic leukemia (AMKL) patients and cell lines, Down syndrome acute lymphoblastic and myeloid leukemia, T cell ALL, cutaneous T cell lymphomas (CTCLs) for high risk children ALL. In this case, patients continued to activate JAK3 signals by gain-of-function. These results suggest that abnormally activated JAK3 signaling contributes to the pathogenesis of certain partial hematopoietic stem cells, and that JAK3 is a therapeutic target for the treatment of patients with this disease.

본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술 분야의 수준 및 본 발명의 내용이 보다 명확하게 설명된다.Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

본 발명자들은 JAK3(Janus family tyrosine kinases)의 비정상적인 활성으로 인해 발생하는 다양한 질병을 치료할 수 있는 조성물을 개발하기 위해 예의 연구 노력하였다. 이에, NCI(National Cancer Institute) 다양성 세트 화합물을 사용하여 구조-기반 전산 데이터베이스 스크리닝을 수행한 결과, 강력하고 선택적인 JAK3 억제제로서 튜불로신(tubulosine, NSC131547)을 동정하였다. 튜불로신은 JAK3의 촉매 활성을 차단하여 백혈병 및 림프종 세포주에서 지속적-활성형(constitutively-active) 및 사이토카인-유도성 JAK3 신호 전달을 강력히 억제하여 활성 JAK3-매개 암세포 생존 및 증식을 억제하였다.The present inventors have made diligent research efforts to develop compositions capable of treating various diseases caused by abnormal activity of JAK3 (Janus family tyrosine kinases). Thus, structure-based computational database screening was performed using the National Cancer Institute (NCI) diversity set compound to identify tubulosine (NSC131547) as a potent and selective JAK3 inhibitor. Tubulosine inhibited the catalytic activity of JAK3 and strongly inhibited constitutively-active and cytokine-induced JAK3 signaling in leukemia and lymphoma cell lines, thereby inhibiting active JAK3-mediated cancer cell survival and proliferation.

이에, 본 발명자들은 튜불로신(NSC131547)이 JAK3의 촉매 활성을 직접적으로 차단하여, JAK3의 비정상적인 활성으로 인해 발생하는 다양한 질병을 치료할 수 있는 저분자(small-molecule) 물질임을 확인함으로써 본 발명을 완성하게 되었다.Accordingly, the present inventors completed the present invention by confirming that the tubulosin (NSC131547) directly blocks the catalytic activity of JAK3 and is a small-molecule substance capable of treating various diseases caused by abnormal activity of JAK3. Was done.

따라서, 본 발명의 목적은 튜불로신(tubulosine)을 포함하는 암 예방, 개선 또는 치료용 약제학적 조성물을 제공하는 것이다.Accordingly, it is an object of the present invention to provide a pharmaceutical composition for preventing, ameliorating or treating cancer comprising tubulosine.

본 발명의 다른 목적은 상술한 본 발명의 조성물을 이를 필요로 하는 개체에게 투여하는 단계를 포함하는 암 치료방법을 제공하는 것이다.Another object of the present invention is to provide a method for treating cancer comprising administering the above-described composition of the present invention to a subject in need thereof.

본 발명의 또 다른 목적은 암을 치료하기 위한 용도로서 튜불로신(tubulosine)을 포함하는 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition comprising tubulosine as a use for treating cancer.

본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

본 발명의 일 양태에 따르면, 본 발명은 (a) 화학식 1로 표시되는 튜불로신(tubulosine)의 치료학적 유효량; 및 (b) 약제학적으로 허용가능한 담체(carrier)를 포함하는 암 예방, 개선 또는 치료용 약제학적 조성물을 제공한다:According to one aspect of the present invention, the present invention provides a pharmaceutical composition comprising (a) a therapeutically effective amount of tubulosine represented by the formula (1); And (b) a pharmaceutically acceptable carrier, the pharmaceutical composition for preventing, ameliorating or treating cancer, comprising:

[화학식 I][Formula I]

Figure PCTKR2017012129-appb-I000001
Figure PCTKR2017012129-appb-I000001

본 발명자들은 JAK3(Janus family tyrosine kinases)의 비정상적인 활성으로 인해 발생하는 다양한 질병을 치료할 수 있는 조성물을 개발하기 위해, NCI(National Cancer Institute) 다양성 세트 화합물을 사용하여, 구조-기반 전산 데이터베이스 스크리닝을 수행하였고, 그 결과, 강력하고 선택적인 JAK3 억제제로서 튜불로신(tubulosine, NSC131547)을 동정하였다.We perform structure-based computational database screening using National Cancer Institute (NCI) diversity set compounds to develop compositions that can treat a variety of diseases caused by abnormal activity of Janus family tyrosine kinases (JAK3). As a result, tubulosine (NSC131547) was identified as a potent and selective JAK3 inhibitor.

튜불로신은 JAK3의 촉매 활성을 차단하여 백혈병 및 림프종 세포주에서 지속적-활성형(constitutively-active) 및 사이토카인-유도성 JAK3 신호 전달을 강력히 억제하여 활성 JAK3-매개 암세포 생존 및 증식을 억제하였다. 이에 본 발명자들은 튜불로신(NSC131547)이 JAK3의 촉매 활성을 직접적으로 차단하여, JAK3의 비정상적인 활성으로 인해 발생하는 다양한 질병을 치료할 수 있는 저분자(small-molecule) 물질임을 확인하였다.Tubulosine inhibited the catalytic activity of JAK3 and strongly inhibited constitutively-active and cytokine-induced JAK3 signaling in leukemia and lymphoma cell lines, thereby inhibiting active JAK3-mediated cancer cell survival and proliferation. Accordingly, the present inventors have confirmed that tubulin (NSC131547) is a small-molecule substance that can directly block catalytic activity of JAK3, thereby treating various diseases caused by abnormal activity of JAK3.

돌연변이를 가진 환자에서 JAK3 매개 신호전달의 활성화는 T 세포 발달 및 면역계 항상성 감소로 인한 SCID (severe combined immunodeficiency) 및 조혈 악성 종양과 같은 면역 병인을 유발한다. 이러한 증거는 조혈 악성 종양을 비롯한 다양한 인간 질병을 치료하는데 있어 JAK3가 매력적인 치료 목표임을 시사한다.Activation of JAK3-mediated signaling in patients with mutations leads to immune etiologies such as Severe combined immunodeficiency (SCID) and hematopoietic malignancies due to reduced T cell development and immune system homeostasis. This evidence suggests that JAK3 is an attractive therapeutic target for treating a variety of human diseases, including hematopoietic malignancies.

이에, JAK3의 새로운 소분자 억제제를 확인하기 위해, JAK3 키나제 도메인의 3D 구조와 National Cancer Institute의 다양성 세트 화합물을 사용하여 구조 기반의 가상 스크리닝을 수행하였다. 튜불로신(NSC131547)은 JAK3의 촉매 활성을 직접적으로 차단하는 것으로 확인되었다. 튜불로신은 JAK 패밀리 중 주로 JAK3의 키나아제 도메인에 결합하여 저농도에서 효과적으로 JAK3 매개 신호 전달을 억제하였다. 튜불로신은 다양한 암 세포에서 다른 종양 세포 키나아제의 활성화를 억제하지는 못했다. 마지막으로, 튜불로신이 지속적-활성인 JAK3을 발현하는 암 세포에서 세포 생존과 증식을 감소시키는 것을 관찰하였다.Thus, to identify new small molecule inhibitors of JAK3, structure-based virtual screening was performed using the 3D structure of the JAK3 kinase domain and the diversity set compound of the National Cancer Institute. Tubulosine (NSC131547) was found to directly block the catalytic activity of JAK3. Tubulosine binds mainly to the kinase domain of JAK3 in the JAK family, effectively inhibiting JAK3-mediated signal transduction at low concentrations. Tubulosine did not inhibit the activation of other tumor cell kinases in various cancer cells. Finally, tubulin was observed to reduce cell survival and proliferation in cancer cells expressing JAK3, which is sustained-active.

본 발명에서는 튜불로신이 JAK3의 활성을 선택적으로 억제함을 보여주었으며, 이는 JAK3의 비정상적인 활성으로 인해 발생하는 다양한 질병을 치료할 수 있는 잠재적인 저분자(small-molecule) 후보 물질이라는 사실을 입증한다.The present invention has shown that tubulosine selectively inhibits the activity of JAK3, demonstrating that it is a potential small-molecule candidate capable of treating various diseases caused by abnormal activity of JAK3.

본 발명의 일 실시예에 있어서, 본 발명의 조성물은 JAK3(Janus Kinase 3)의 촉매 활성을 억제한다.In one embodiment of the present invention, the composition of the present invention inhibits the catalytic activity of JAK3 (Janus Kinase 3).

본 발명의 조성물은 경구 또는 비경구로 투여할 수 있고, 비경구 투여인 경우에는 정맥내 주입, 피하 주입, 근육 주입, 복강 주입, 경피 투여 등 다양한 경로를 통해 투여할 수 있다.The composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered through various routes such as intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, and transdermal administration.

본 발명의 암은 혈액암, 유방암, 뇌암, 신경내분비종양, 위암, 폐암, 전립선암, 난소암, 간암, 기관지암, 비인두암, 후두암, 췌장암, 방광암, 부신암, 대장암, 결장암, 자궁경부암, 골암, 피부암, 갑상선암, 부갑상선암 및 요관암으로 구성된 군에서 선택될 수 있다.Cancer of the present invention is hematologic cancer, breast cancer, brain cancer, neuroendocrine tumor, gastric cancer, lung cancer, prostate cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, adrenal cancer, colon cancer, colon cancer, cervical cancer , Bone cancer, skin cancer, thyroid cancer, parathyroid cancer and ureter cancer.

본 발명의 혈액암은 림프종, 백혈병 또는 골수종이다.Hematological cancer of the present invention is lymphoma, leukemia or myeloma.

본 명세서에서 용어 “림프종 (lymphoma)”은 비-호지킨 림프종(Non-Hodgkins Lymphoma, NHL); 확산 거대 B 세포 림프종(diffuse large B-cell lymphoma, DLBCL); 여포성 림프종(follicular lymphoma, FL); 호지킨병(Hodgkin disease); 버킷림프종(Burkitt's Lymphoma); 피부 T 세포 림프종(cutaneous T cell lymphoma); 원발성 중추 신경계 림프종(primary central nervous system lymphoma) 및 림프계 전이(lymphomatous metastases)를 포함한다.As used herein, the term “lymphoma” refers to Non-Hodgkins Lymphoma (NHL); Diffuse large B-cell lymphoma (DLBCL); Follicular lymphoma (FL); Hodgkin disease; Burkitt's Lymphoma; Cutaneous T cell lymphoma; Primary central nervous system lymphoma and lymphomatous metastases.

본 발명의 T 세포 림프종은 다음으로 구성된 군에서 선택되며, 이에 한정되는 것은 아니다: 악성 종양이 흉선으로부터의 원시 림프성 전구 세포에서 발생하는 림프모구 림프종(lymphoblastic lymphomas); 성숙 또는 말초 T 세포 신 생물은 T 세포 전림프세포성 백혈병(prolymphocytic leukemia), T 세포 과립 림프세포성 백혈병(T-cell granular lymphocytic leukemia); 공격적 NK-세포 백혈병(aggressive NK-cell leukemia); 피부 T 세포 림프종(cutaneous T cell lymphoma); 퇴행성 대 세포 림프종(anaplastic large cell lymphoma), 장폐색형 T 세포 림프종(enteropathy-type T cell lymphoma), 인간 T- 림프성 바이러스(Human T-lymphotropic virus-1, HTLV-1)와 연관된 성인 T-세포 백혈병/림프종(Adult T-cell leukemia/lymphoma), 혈관면역모세포성 T세포 림프종(angioimmunoblastic T cell lymphoma), 피하 지방층염 T 세포 림프종(subcutaneous parmiculitic T cell lymphoma) 및 최초에는 말초 림프절 곁피질(paracortex)을 포함하고 진정한 난포 패턴으로 성장하지 않는 말초 T 세포 림프종.T cell lymphomas of the invention are selected from the group consisting of, but not limited to: lymphoblastic lymphomas in which malignant tumors develop in primitive lymphoid progenitor cells from the thymus; Mature or peripheral T cell neoplasms include T-cell prolymphocytic leukemia, T-cell granular lymphocytic leukemia; Aggressive NK-cell leukemia; Cutaneous T cell lymphoma; Adult T-cells associated with anaplastic large cell lymphoma, enteropathy-type T cell lymphoma, and human T-lymphotropic virus-1 (HTLV-1) Adult T-cell leukemia / lymphoma, angioimmunoblastic T cell lymphoma, subcutaneous parmiculitic T cell lymphoma and initially peripheral lymph node paracortex Peripheral T cell lymphoma that contains and does not grow in a true follicular pattern.

본 발명의 B 세포 림프종은 다음으로 구성된 군에서 선택되며, 이에 한정되는 것은 아니다: 광범위 거대 B 세포 림프종(diffuse large B cell lymphoma, DLBCL), 여포성 림프종(follicular lymphoma), MALT (mucosa-associated lymphatic tissue lymphoma), 만성 림프구성 백혈병(chronic lymphocytic leukemia), 맨틀세포 림프종(mantle cell lymphoma, MCL), 버킷림프종(burkitt lymphoma), 종격 거대 B 세포 림프종(mediastinal large B cell lymphoma), 원발성 고분자글로불린혈증(waldenstrom macroglobulinemia), 변연부 B 세포 림프종(nodal marginal zone B cell lymphoma, NMZL), 비장 변연 림프종(splenic marginal zone lymphoma, SMZL), 혈관내 거대 B-세포 림프종, 원발성 삼출액 림프종(primary effusion lymphoma), 림프종모양육아종증(lymphomatoid granulomatosis), 및 AIDS-연관 림프종.B cell lymphoma of the present invention is selected from the group consisting of, but not limited to: diffuse large B cell lymphoma (DLBCL), follicular lymphoma, mucosa-associated lymphatic (MALT) tissue lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma (MCL), burkitt lymphoma, mediastinal large B cell lymphoma, primary polyglobulinemia ( waldenstrom macroglobulinemia, nodal marginal zone B cell lymphoma (NMZL), splenic marginal zone lymphoma (SMZL), vascular giant B-cell lymphoma, primary effusion lymphoma, lymphoma Lymphomatoid granulomatosis, and AIDS-associated lymphoma.

본 발명에 따르면, 골수종 (myeloma)은 다발성 골수종 (multiple myeloma)이다.According to the present invention, myeloma is multiple myeloma.

본 발명의 약제학적 조성물은 약제학적으로 허용되는 담체 (carrier)를 포함한다. 상기 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 약제학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다.The pharmaceutical composition of the present invention comprises a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is conventionally used in the preparation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, Polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

한편, 경구 투여를 위한 고형 제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형 제제는 상기 약제학적 조성물에 적어도 하나 이상의 부형제, 예를 들면, 전분, 탄산칼슘, 수크로오스, 락토오스, 젤라틴 등을 혼합하여 제형화한다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 등과 같은 윤활제가 사용될 수도 있다.On the other hand, solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose, lactose in the pharmaceutical composition. , Gelatin and the like are mixed and formulated. In addition, lubricants such as magnesium stearate, talc and the like may also be used in addition to simple excipients.

경구용 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 예시될 수 있으며, 흔히 사용되는 단순 희석제인 물, 액체 파라핀 이외에 여러 가지 부형제, 예를 들면, 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다.Oral liquid preparations include suspensions, solvents, emulsions, syrups, and the like. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be used. May be included.

비경구 투여를 위한 제제에는 멸균된 수용액제, 비수성용제, 현탁제, 유제, 동결건조제, 좌제 등을 예시할 수 있다. 비수성용제, 현탁제에는 프로필렌글리콜, 폴리에틸렌글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 포함될 수 있다. 주사제에는 용해제, 등장화제, 현탁화제, 유화제, 안정화제, 방부제 등과 같은 종래의 첨가제가 포함될 수 있다.Examples of preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilizers, suppositories, and the like. Non-aqueous solvents and suspending agents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. Injectables may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, preservatives, and the like.

본 발명의 약제학적 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하며, 보통으로 숙련된 의사는 소망하는 치료 또는 예방에 효과적인 투여량을 용이하게 결정 및 처방할 수 있다. 본 발명의 일 구현예에 따르면, 본 발명의 약제학적 조성물의 1일 투여량은 0.001-10000 ㎎/㎏이다.Suitable dosages of the pharmaceutical compositions of the invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to reaction, Usually a skilled practitioner can easily determine and prescribe a dosage effective for the desired treatment or prophylaxis. According to one embodiment of the present invention, the daily dose of the pharmaceutical composition of the present invention is 0.001-10000 mg / kg.

본 발명의 약제학적 조성물은 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화 함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 수성 매질중의 용액, 현탁액 또는 유화액 형태이거나 엑스제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다.The pharmaceutical compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or an aqueous medium, or may be in the form of extracts, powders, granules, tablets or capsules, and may further include a dispersant or stabilizer.

본 발명의 다른 양태에 따르면, 본 발명은 (a) 다음 화학식 1로 표시되는 튜불로신(tubulosine)의 치료학적 유효량; 및 (b) 약제학적으로 허용가능한 담체(carrier);를 포함하는 조성물을 이를 필요로 하는 개체에게 투여하는 단계를 포함하는 암 치료방법을 제공한다:According to another aspect of the present invention, the present invention provides a pharmaceutical composition comprising (a) a therapeutically effective amount of tubulosine represented by the following formula (1); And (b) a pharmaceutically acceptable carrier; administering to a subject in need thereof a composition comprising:

[화학식 I][Formula I]

Figure PCTKR2017012129-appb-I000002
Figure PCTKR2017012129-appb-I000002

본 발명의 암 치료방법은 상술한 본 발명의 암 치료용 약제학적 조성물을 이용하는 것으로서, 이 둘 사이에 공통된 내용은 본 명세서의 과도한 복잡성을 피하기 위하여, 그 기재를 생략한다.The cancer treatment method of the present invention uses the above-described pharmaceutical composition for treating cancer of the present invention, and the common content between the two is omitted in order to avoid excessive complexity of the present specification.

본 발명에서 "투여"는 임의의 적절한 방법으로 환자에게 소정의 물질을 제공하는 것을 의미하며, 본 발명의 약제학적 조성물의 투여 경로는 목적 조직에 도달할 수 있는 한 일반적인 모든 경로를 통하여 경구 또는 비경구 투여될 수 있다. 또한, 본 발명의 조성물은 유효성분을 표적 세포로 전달할 수 있는 임의의 장치를 이용해 투여될 수도 있다.As used herein, "administration" means providing a patient with any substance by any suitable method, wherein the route of administration of the pharmaceutical composition of the present invention is oral or parenteral via all common routes as long as the target tissue can be reached. Oral administration. In addition, the composition of the present invention may be administered using any device capable of delivering an active ingredient to a target cell.

본 발명에서 "개체"는, 특별히 한정되는 것은 아니지만, 예를 들어, 인간, 원숭이, 소, 말, 양, 돼지, 닭, 칠면조, 메추라기, 고양이, 개, 마우스, 쥐, 토끼 또는 기니아 피그를 포함한다.In the present invention, "individual" is not particularly limited, but includes, for example, humans, monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, mice, rabbits or guinea pigs. do.

본 발명의 또 다른 양태에 따르면, 본 발명은 암을 치료하기 위한 용도로서 다음 화학식 1로 표시되는 튜불로신(tubulosine)을 포함하는 조성물을 제공한다:According to another aspect of the invention, the invention provides a composition comprising tubulosine represented by the following formula (1) as a use for treating cancer:

[화학식 I][Formula I]

Figure PCTKR2017012129-appb-I000003
Figure PCTKR2017012129-appb-I000003

본 발명의 특징 및 이점을 요약하면 다음과 같다:The features and advantages of the present invention are summarized as follows:

(a) 본 발명은 튜불로신 (tubulosine)을 포함하는 암 예방, 개선 또는 치료용 약제학적 조성물 및 이를 이용한 암 치료방법에 관한 것이다.(a) The present invention relates to a pharmaceutical composition for preventing, ameliorating or treating cancer comprising tubulosine and a method for treating cancer using the same.

(b) 튜불로신은 JAK 패밀리 멤버 중 선택적으로 JAK3 활성을 억제하므로, 비이상적인 JAK3 활성에 의한 다양한 질환 (특히, 암)을 치료할 수 있는 치료물질로서 사용될 수 있다.(b) Since tubulosine selectively inhibits JAK3 activity among JAK family members, it can be used as a therapeutic agent for treating various diseases (especially cancer) caused by non-ideal JAK3 activity.

도 1a-1c. 구조-기반 가상 데이터베이스 스크리닝 모델의 구조도. 1a) 튜불로신의 화학구조 (C29H37N3O3; M.W. 475.6). 1b) 튜불로신 및 JAK3 키나아제 도메인 (JAK3-JH1, PDB ID: 1YVJ)의 예측 결합 모델. 튜불로신과 3.5 Å 이내에서 접촉하는 곁사슬만을 라벨링하였다. 1c) JAK3-JH1 복합체에서 리간드의 중첩. JAK3 키나아제 도메인을 가진, 중첩된 튜불로신(분홍색), AFN941(청록색), CP-690550(노란색), 및 CMP-6(녹색)의 구조를 1YVJ, 3LXK 및 3LXL의 PDB 파일로부터 각각 생성하였다. 모든 구조도는 Pymol(pymol.sourceforge.net)로 생성하였다.1A-1C. Schematic diagram of a structure-based virtual database screening model. 1a) Chemical structure of tubulosine (C 29 H 37 N 3 O 3 ; MW 475.6). 1b) Predictive binding model of tubulosine and JAK3 kinase domains (JAK3-JH1, PDB ID: 1YVJ). Only side chains contacted with tubulosine within 3.5 μs were labeled. 1c) Overlapping of ligands in JAK3-JH1 complex. The structures of nested tubulosine (pink), AFN941 (blue), CP-690550 (yellow), and CMP-6 (green) with JAK3 kinase domains were generated from PDB files of 1YVJ, 3LXK, and 3LXL, respectively. All schematics were generated with Pymol (pymol.sourceforge.net).

도 2a-2c. 튜불로신은 인 비트로에서 JAK3 키나아제 활성을 선택적으로 억제한다. 2a) 각 JAK 면역침전물(immunoprecipitate)을 각각 DMSO(vehicle), 튜불로신(25, 50, 75, 및 100 nM) 또는 pan-JAK 키나아제 억제제AG490 (150 μM)로 1 시간 동안 전처리하였다. 이어, 키나아제 반응으로서, His-tagged 재조합 STAT3α 단백질(2 μg) 및 ATP(2 μM)를 첨가하여 30 ℃에서 30 분간 반응시켰다. 2b) JAK3 면역침강(immunoprecipitates)은 튜불로신(100 nM) 존재 또는 비존재 하에서, ATP-의존적(2, 10, 50, 500, 및 1000 μM) 인 비트로 키나아제 분석법으로 수행하였다. 반응생성물은 적절한 항체를 사용하여 웨스턴 블로팅으로 확인하였다. 2c) 4가지 JAKs에 대한 인 비트로 키나아제 분석은 Merck Millipore의 KinaseProfiler™ Services로 수행하였다. 튜불로신의 IC50 수치는 표에 기재하였다. 스타우로스포린(Staurosporine, STS)은 양성대조군으로 사용하였다.2A-2C. Tubulosine selectively inhibits JAK3 kinase activity in vitro. 2a) Each JAK immunoprecipitate was pretreated for 1 hour with DMSO (vehicle), tubulosine (25, 50, 75, and 100 nM) or pan-JAK kinase inhibitor AG490 (150 μM), respectively. Then, as a kinase reaction, His-tagged recombinant STAT3α protein (2 μg) and ATP (2 μM) were added and reacted at 30 ° C. for 30 minutes. 2b) JAK3 immunoprecipitates were performed by ATP-dependent (2, 10, 50, 500, and 1000 μM) in vitro kinase assays, with or without tubulosine (100 nM). Reaction products were confirmed by western blotting using appropriate antibodies. 2c) In vitro kinase analysis of four JAKs was performed with KinaseProfiler ™ Services from Merck Millipore. IC 50 values for tubulosine are listed in the table. Staurosporine (STS) was used as a positive control.

도 3a-3c. 튜불로신은 JAK3에 대한 ATP-경쟁적 키나아제 억제제이다. 3a) ATP-농도-의존적 인 비트로 JAK3 키나아제 분석을 수행하였다. 3b-3c) JAK3 키나아제 활성에 대한 튜불로신의 ATP-의존적 EC50(3b) 및 Km(3c) 값을 나타낸다.Figures 3A-3C. Tubulosine is an ATP-competitive kinase inhibitor against JAK3. 3a) JAK3 kinase assays were performed with ATP-concentration-dependent beats. 3b-3c) ATP-dependent EC 50 (3b) and Km (3c) values of tubulosine for JAK3 kinase activity.

도 4a-4d. 튜불로신은 암세포에서 본질적인 활성 JAK2 신호전달을 완전히 차단한다. 4a-4d) 인간 암 세포주 L540(4a), HDLM-2, MDA-MB-468, DU145 및 A431(4b), 마우스 백혈병 세포주 BKO-84, BaF3/JAK3V674A 및 BaF3/TEL-JAK3(4c) 및 BaF3/TEL-JAK2(4d)에 DMSO(vehicle), 튜불로신(25, 50, 75, 및 100 nM) 또는 pan-JAK 억제제 AG490(150 μM)를 처리하여 24 시간 동안 반응시켰다. 상기 세포들의 전체 세포 용해물(lysates)로 웨스턴블로팅을 수행하였다. GAPDH은 로딩 대조군으로 사용하였다. 4A-4D. Tubulosine completely blocks active JAK2 signaling intrinsically in cancer cells. 4a-4d) Human cancer cell lines L540 (4a), HDLM-2, MDA-MB-468, DU145 and A431 (4b), mouse leukemia cell lines BKO-84, BaF3 / JAK3V674A and BaF3 / TEL-JAK3 (4c) and BaF3 / TEL-JAK2 (4d) was treated with DMSO (vehicle), tubulosine (25, 50, 75, and 100 nM) or pan-JAK inhibitor AG490 (150 μM) for 24 hours. Western blotting was performed with whole cell lysates of the cells. GAPDH was used as loading control.

도 5a-5d. 튜불로신은 암세포에서 JAK3/STAT 시그널을 억제한다. 5a-5d) 인간 호지킨 림프종(Hodgkin’s lymphoma) 세포주 L540(5a) 및 마우스 백혈병 세포주 BKO-84(5b), BaF3/JAK3V674A(5c), 및 BaF3/TEL-JAK3(5d)에 튜불로신(100 nM)을 일정 시간 처리하였다. 상기 세포들의 전체 세포 용해물(lysates)로 웨스턴블로팅을 수행하였다. GAPDH은 로딩 대조군으로 사용하였다. Figures 5A-5D. Tubulosine suppresses JAK3 / STAT signaling in cancer cells. 5a-5d) Tubulosine (100) in human Hodgkin's lymphoma cell line L540 (5a) and mouse leukemia cell lines BKO-84 (5b), BaF3 / JAK3V674A (5c), and BaF3 / TEL-JAK3 (5d). nM) was treated for some time. Western blotting was performed with whole cell lysates of the cells. GAPDH was used as loading control.

도 6a-6b. 고농도의 튜불로신은 암세포에서 JAK1, JAK2, 및/또는 TYK2-매개 시그널을 억제한다. 6a-6b) 인간 호지킨 림프종(Hodgkin’s lymphoma) HDLM-2 세포(6a) 및 마우스 백혈병 BaF3/TEL-JAK2 세포(6b)에 DMSO(vehicle), 튜불로신 또는 pan-JAK 억제제 AG490(150 μM)를 처리하여 24 시간 동안 반응시켰다. 상기 세포들의 전체 세포 용해물(lysates)로 웨스턴블로팅을 수행하였다. GAPDH은 로딩 대조군으로 사용하였다. Figures 6a-6b. High concentrations of tubulosine inhibit JAK1, JAK2, and / or TYK2-mediated signals in cancer cells. 6a-6b) Human Hodgkin's lymphoma HDLM-2 cells (6a) and mouse leukemia BaF3 / TEL-JAK2 cells (6b) in DMSO (vehicle), tubulosine or pan-JAK inhibitor AG490 (150 μM) Was reacted for 24 hours. Western blotting was performed with whole cell lysates of the cells. GAPDH was used as loading control.

도 7a-7c. 튜불로신은 IL-2-유도성 JAK3/STAT5 시그널을 차단한다. 7a-7b) 래트 pre-T 림프종 Nb2 세포에 DMSO(vehicle), 튜불로신(25, 50, 75, 및 100 nM) 또는 pan-JAK 억제제 AG490(150 μM)를 처리하여 16 시간 동안 굶기고, 이어 프로락틴(PRL, 100 ng/mL, A) 또는 IL-2(100 ng/mL, B)를 10 분간 처리하였다. 7c) 인간 B-세포 유래 다발성 골수종 U266 세포에 DMSO(vehicle), 튜불로신(25, 50, 75, 및 100 nM) 또는 pan-JAK 억제제 AG490(150 μM)를 처리하여 24 시간 동안 반응시킨 후, IFN-α(1000 U/mL)를 처리하여 30 분간 반응시켰다. 상기 세포들의 전체 세포 용해물(lysates)로 웨스턴블로팅을 수행하였다. GAPDH은 로딩 대조군으로 사용하였다. Figures 7A-7C. Tubulosine blocks IL-2-induced JAK3 / STAT5 signals. 7a-7b) Rat pre-T lymphoma Nb2 cells were starved for 16 hours by treatment with DMSO (vehicle), tubulosine (25, 50, 75, and 100 nM) or pan-JAK inhibitor AG490 (150 μM). Prolactin (PRL, 100 ng / mL, A) or IL-2 (100 ng / mL, B) was treated for 10 minutes. 7c) Human B-cell derived multiple myeloma U266 cells were treated with DMSO (vehicle), tubulosine (25, 50, 75, and 100 nM) or pan-JAK inhibitor AG490 (150 μM) for 24 hours. , IFN-α (1000 U / mL) was treated and reacted for 30 minutes. Western blotting was performed with whole cell lysates of the cells. GAPDH was used as loading control.

도 8a-8b. 튜불로신은 암 세포에서 다른 종양형성 키나아제 시그널 활성화에 영향을 주지 않는다. 8a-8b) 인간 호지킨 림프종 세포주 L540 및 HDLM-2(8a) 및 인간 고형 암세포주 MDA-MB-468, DU145 및 A431(8b)에 DMSO(vehicle), 튜불로신(50 및 100 nM) 또는 pan-JAK 억제제 AG490(150 μM)를 처리하여 24 시간 동안 반응시켰다. 상기 세포들의 전체 세포 용해물(lysates)로 웨스턴블로팅을 수행하였다. GAPDH은 로딩 대조군으로 사용하였다. 8A-8B. Tubulosine does not affect other tumorigenic kinase signal activation in cancer cells. 8a-8b) DMSO (vehicle), tubulosine (50 and 100 nM) in human Hodgkin's lymphoma cell lines L540 and HDLM-2 (8a) and human solid cancer cell lines MDA-MB-468, DU145 and A431 (8b) or The pan-JAK inhibitor AG490 (150 μM) was treated and reacted for 24 hours. Western blotting was performed with whole cell lysates of the cells. GAPDH was used as loading control.

도 9a-9b. 튜불로신은 활성형(constitutively-active) JAK3를 발현하는 암세포의 생존(survival)을 선택적으로 감소시킨다. 9a-9b) L540, BKO-84 및 BaF3/JAK3V674A 세포(9a) 및 HDLM-2, MDA-MB-468, DU145, 및 BaF3/TEL-JAK2 세포(9b)에 DMSO(vehicle), 튜불로신(25, 50, 및 100 nM) 또는 pan-JAK 억제제 AG490(150 μM)를 처리하여 반응시켰다. 상기 세포들의 전체 세포 용해물(lysates)로 웨스턴블로팅을 수행하였다. GAPDH은 로딩 대조군으로 사용하였다. 세포 생존율은 WST-1 시약으로 측정하였고, DMSO(vehicle)-처리 그룹과 비교한 대조군(control) %로 나타내었다. 결과는 3 회 독립적 실험의 평균 ± 표준편차로 나타내었다(n = 3). *p < 0.05, **p < 0.005.Figures 9A-9B. Tubulosine selectively reduces the survival of cancer cells expressing constitutively-active JAK3. 9a-9b) DMSO (vehicle), tubulosine (LSO, BKO-84 and BaF3 / JAK3V674A cells (9a) and HDLM-2, MDA-MB-468, DU145, and BaF3 / TEL-JAK2 cells (9b). 25, 50, and 100 nM) or pan-JAK inhibitor AG490 (150 μM) was reacted. Western blotting was performed with whole cell lysates of the cells. GAPDH was used as loading control. Cell viability was measured with WST-1 reagent and expressed as% control compared to DMSO (vehicle) -treated group. Results are expressed as mean ± standard deviation of three independent experiments (n = 3). * p <0.05, ** p <0.005.

도 10a-10b. 튜불로신은 선택적으로 활성형(constitutively-active) JAK3를 발현하는 암세포의 성장(proliferation)을 억제시킨다. 10a-10b) L540, BKO-84 및 BaF3/JAK3V674A 세포(10a) 및 HDLM-2, MDA-MB-468, DU145, 및 BaF3/TEL-JAK2 세포(10b)에 DMSO (vehicle), 튜불로신(25, 50, 및 100 nM) 또는 pan-JAK 억제제 AG490(150 μM)를 처리하여 반응시켰다. 세포 증식율은 트립토판 블루 분석법을 이용하여 생세포를 계수함으로써 측정하였다. 결과는 3 회 독립적 실험의 평균 ± 표준편차로 나타내었다(n = 3). *p < 0.05, **p < 0.005.Figures 10A-10B. Tubulosine inhibits the proliferation of cancer cells that selectively express constitutively-active JAK3. 10a-10b) DMSO (vehicle), tubulosine (L), BKO-84 and BaF3 / JAK3V674A cells (10a) and HDLM-2, MDA-MB-468, DU145, and BaF3 / TEL-JAK2 cells (10b). 25, 50, and 100 nM) or pan-JAK inhibitor AG490 (150 μM) was reacted. Cell proliferation was measured by counting live cells using tryptophan blue assay. Results are expressed as mean ± standard deviation of three independent experiments (n = 3). * p <0.05, ** p <0.005.

도 11은 튜불로신이 선택적으로 JAK3 및 JAK3-매개 하위 타겟 시그널을 타게팅함을 나타내는 모식도이다.FIG. 11 is a schematic diagram illustrating that tubulosin selectively targets JAK3 and JAK3-mediated subtarget signals. FIG.

이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

[실시예]EXAMPLE

실험재료 및 방법Experimental Materials and Methods

1. 구조-기반 가상 데이터베이스 스크리닝1. Structure-based Virtual Database Screening

JAK3(JAK3-JH1)의 카나아제 도메인에 결합함으로써 JAK2 활성을 억제하는 소분자를 찾아내기 위하여, AutoDock ver. 4.2를 이용하였고, 화합물의 NCI 다양성 세트로 가상 스크리닝을 수행하였다. 도킹(docing)은 2 단계로 이루어진다.To find small molecules that inhibit JAK2 activity by binding to the kinase domain of JAK3 (JAK3-JH1), AutoDock ver. 4.2 was used and virtual screening was performed with the NCI diversity set of compounds. Docking is done in two steps.

첫 번째 단계에서는, JAK3-JH1 및 이의 억제제인 스타우로스포린(staurosporine) 유사체 AFN941 간의 복합체 구조로부터의 단백질 코디네이트를 템플릿(template)으로 선정하였다. 리간드와 용매 분자를 제거한 후, AMBER software로 수소 원자를 첨가하였으며, 이는 아스파르트산(Asp), 글루탐산(Glu), 히스티딘(His) 및 리신(Lys) 잔기에서 PDB2PQR-결정된 이온화 상태(ionizable states)에 기반한 것이다(Boggon TJ, Li Y, Manley PW, Eck MJ. Crystal structure of the Jak3 kinase domain in complex with a staurosporine analog. Blood 2005;106:996-1002.) In the first step, protein coordinates from the complex structure between JAK3-JH1 and its inhibitor, the staurosporine analog AFN941, were selected as a template. After removal of the ligand and solvent molecules, hydrogen atoms were added with AMBER software, which was then added to the PDB2PQR-determined ionizable states at the aspartic acid (Asp), glutamic acid (Glu), histidine (His) and lysine (Lys) residues. (Boggon TJ, Li Y, Manley PW, Eck MJ.Crystal structure of the Jak3 kinase domain in complex with a staurosporine analog. Blood 2005; 106: 996-1002.)

두 번째 단계에서, 본 발명자들은 첫 번째 단계에서 얻어진 후보들로 상세 계산을 하였다. 추가적으로 CP-690550(PDB ID: 3LXK) 및 CMP-6(PDB ID: 3LXL)를 가진 2 가지 JAK3-JH1 구조-복합체도 포함된다. AMBER package에 의한 각 소분자의 30개 초기 구조 (conformation)를 생성하였고, 화합물 당 90 번을 실행한 앙상블 도킹(ensemble docking)을 수행하였다. In the second step, we made detailed calculations with the candidates obtained in the first step. In addition, two JAK3-JH1 structure-complexes with CP-690550 (PDB ID: 3LXK) and CMP-6 (PDB ID: 3LXL) are also included. Thirty initial conformations of each small molecule were generated by the AMBER package, followed by ensemble docking with 90 runs per compound.

최종적으로 얻은 구조는 구조 간의 RMSD(root mean square deviation) 값으로 정량화한 구조 유사성에 기반하여 클러스터링 하였다. 그리고 가장 우세한 클러스터의 중심을 대표로 선정하였다. 100 및 500,000의 값은 각각 모집단에서 각각의 수에 대한 파라미터(ga_pop_size) 및 AutoDock에서 일반 알고리즘에 대한 최대 세대 수(ga_num_evals)를 나타낸다. In-house written software 및 scripts는 모든 절차를 자동화하였다. JAK1-JH1 및 JAK2-JH1에 결합하는 튜불로신의 공통적이고 다른 특징을 이해하기 위하여, 동일 절차로 JAK1-JH1 또는 JAK2-JH1의 6가지 구조를 템플릿으로 사용하여 도킹을 반복하였다. 구조의 PDB 코드는 JAK1-JH1 및 JAK2-JH1에 대하여 각각 3EYG 및 3EYH; 및 2B7A, 2W1I, 3E62 및 3FUP 이다.The final structures were clustered based on structural similarity quantified by root mean square deviation (RMSD) values between the structures. The center of the most dominant cluster was chosen as the representative. The values of 100 and 500,000 represent the parameters (ga_pop_size) for each number in the population and the maximum number of generations (ga_num_evals) for the general algorithm in AutoDock, respectively. In-house written software and scripts automate all procedures. In order to understand the common and different features of tubulosine binding to JAK1-JH1 and JAK2-JH1, docking was repeated using the six structures of JAK1-JH1 or JAK2-JH1 as templates in the same procedure. The PDB code of the structure is 3EYG and 3EYH for JAK1-JH1 and JAK2-JH1, respectively; And 2B7A, 2W1I, 3E62 and 3FUP.

2. 튜불로신(Tubulosine)2. Tubulosine

튜불로신(NSC131547)은 NCI 다양성 세트 화합물에서 동정된 화합물 중 하나이고, Developmental Therapeutics Program(DTP)/NCI에 기탁되어 비임상 목적으로 연구자들에게 제공되어 왔다. 튜불로신 합성 및 순도(purity)에 관한 정보는 DTP/NCI 웹사이트에서 확인할 수 있다. Tubulosine (NSC131547) is one of the compounds identified in the NCI Diversity Set Compound, and has been deposited with the Developmental Therapeutics Program (DTP) / NCI and provided to researchers for nonclinical purposes. Information on tubulosine synthesis and purity can be found on the DTP / NCI website.

3. 세포주(Cell lines)3. Cell lines

인간 호지킨 림프종 세포주(human Hodgkin's lymphoma cell lines) HDLM-2 및 L540, 및 B-세포 유래 다형성 골수종 세포주(B-cell derived multiple myeloma cell line) U266는 독일 생물자원센터(German Collection of Microorganisms and Cell Cultures; DSMZ, Germany)에서 수득하였다. 인간 유방암 세포주 MDA-MB-468, 전립선암 세포주 DU145 및 유표피암 세포주 A431 는 ATCC(American Type Culture Collection; Rockville, MD, USA)에서 수득하였고, 10% FBS를 함유하는 DMEM 배지에서 배양하였다. Pre-B 백혈병 세포주 BKO-84는 Dr. Daisuke Kitamura로부터 얻은 BLNK-/- 마우스에서 유래하였고, 돌연변이 JAK3(BaF3/JAK3V674A), TEL-JAK2 (BaF3/TEL-JAK2) 및 TEL-JAK3(BaF3/TEL-JAK3)를 지속적으로 발현하는 골수-유래 pro-B 세포주 BaF3는 Dr. Hiroyuki Mano 및 Dr. Olivier A. Bernard로부터 얻었으며, 래트 pre-T 림프종 세포주 Nb2는 Dr. Charles V. Clevenger 로부터 얻었다.Human Hodgkin's lymphoma cell lines HDLM-2 and L540, and B-cell derived multiple myeloma cell line U266 is a German Collection of Microorganisms and Cell Cultures DSMZ, Germany). Human breast cancer cell lines MDA-MB-468, prostate cancer cell line DU145 and epidermal cancer cell line A431 were obtained from the American Type Culture Collection (ATCC) Rockville, MD, USA, and were cultured in DMEM medium containing 10% FBS. Pre-B leukemia cell line BKO-84 Bone marrow-derived, derived from BLNK − / − mice from Daisuke Kitamura and consistently expressing mutant JAK3 (BaF3 / JAK3V674A), TEL-JAK2 (BaF3 / TEL-JAK2) and TEL-JAK3 (BaF3 / TEL-JAK3) pro-B cell line BaF3 was determined by Dr. Hiroyuki Mano and Dr. The rat pre-T lymphoma cell line Nb2 was obtained from Olivier A. Bernard. Obtained from Charles V. Clevenger.

4. 웨스턴 블로팅(Western blotting)4. Western blotting

샘플을 SDS-PAGE로 분리하여 니트로셀롤로오스 멤브레인에 옮겼다. 멤브레인은 5% 스팀밀크를 함유하는 블로킹 버퍼로 처리하였으며, 이후 타겟 분자에 대한 1차 항체를 처리하여 4 ℃에서 하루 동안(overnight) 반응시켰다. 블롯을 세척한 후 홀스래디쉬 페록시다아제-컨쥬게이션된 2차 항체를 처리하여 상온에서 2 시간 반응시켰고, 시그널은 ECL(enhanced chemiluminescence) 시약(SurModics Inc, Eden Prairie, MN, USA)으로 확인하였다. Antibodies specific for phospho-JAK1 (Tyr1022/1023), JAK1, phospho-JAK2 (Tyr1007/1008), JAK2, phospho-JAK3 (Tyr980/981), JAK3, phospho-TYK2 (Tyr1054/1055), TYK2, phospho-STAT1 (Tyr701), STAT1, phospho-STAT3 (Tyr705), phospho-STAT5 (Tyr694), phospho-Src (Tyr416), Src, phospho-Lyn (Tyr507), Lyn, phospho-Akt (Ser473), Akt, phospho-ERK1/2 (Thr202/Tyr204), ERK1/2, PARP, caspase 3, caspase 9, Bcl-2, Bcl-xL, Mcl-1, survivin, Bax, Bad, p21, p27, RIP3, Beclin 1, LC3B 및 GAPDH에 대한 항체는 Cell Signaling Technology(Cambridge, MA, USA)에서 구입하였다. STAT3 및 STAT5 항체는 Santa Cruz Biotechnology(Santa Cruz, CA, USA)에서 구입하였다. Samples were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membrane was treated with blocking buffer containing 5% steam milk and then treated with primary antibody to the target molecule and reacted overnight at 4 ° C. After washing the blot and reacting the horseradish peroxidase-conjugated secondary antibody at room temperature for 2 hours, the signal was confirmed by an enhanced chemiluminescence (ECL) reagent (SurModics Inc, Eden Prairie, MN, USA). It was. Antibodies specific for phospho-JAK1 (Tyr1022 / 1023), JAK1, phospho-JAK2 (Tyr1007 / 1008), JAK2, phospho-JAK3 (Tyr980 / 981), JAK3, phospho-TYK2 (Tyr1054 / 1055), TYK2, phospho-STAT1 (Tyr701), STAT1, phospho-STAT3 (Tyr705), phospho-STAT5 (Tyr694), phospho-Src (Tyr416), Src, phospho-Lyn (Tyr507), Lyn, phospho-Akt (Ser473), Akt, phospho-ERK1 / 2 (Thr202 / Tyr204), ERK1 / 2, PARP, caspase 3, caspase 9, Bcl-2, Bcl-xL, Mcl-1, survivin, Bax, Bad, p21, p27, RIP3, Beclin 1, LC3B and GAPDH Antibodies to were purchased from Cell Signaling Technology (Cambridge, MA, USA). STAT3 and STAT5 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

5. 세포 증식(proliferation) 및 생존분석(viability assays)5. Cell proliferation and viability assays

96-웰 플레이트에 세포를 웰당 1 × 104로 분주하여 세포밀집도(confluence)가 70-80% 될 때까지 배양하였다. 이후 세포를 DMSO(vehicle), 튜불로신, 또는 AG490(150 μM)로 일정 시간 간격으로 처리하였다. 세포 생존(Cell viability)은 37 ℃에서 3 시간 더 반응시킨 후, 10 μL 분석시약(EZ-CyTox Enhanced Cell Viability Assay Reagent, Daeil Lab Service, Seoul, Korea)을 첨가하고 마이크로리더(Molecular Devices, Sunnyvale, USA)를 이용하여 450 nm에서 측정하였다. 한편, 생존한 세포는 트리판 블루 분석을 통하여 계수하고 세포 증식도를 측정하였다. Cells were plated at 1 × 10 4 per well in 96-well plates and incubated until 70-80% confluence. Cells were then treated with DMSO (vehicle), tubulosine, or AG490 (150 μM) at regular time intervals. Cell viability was further reacted at 37 ° C for 3 hours, followed by addition of 10 μL assay reagent (EZ-CyTox Enhanced Cell Viability Assay Reagent, Daeil Lab Service, Seoul, Korea) and microreaders (Molecular Devices, Sunnyvale, USA) at 450 nm. On the other hand, surviving cells were counted through trypan blue analysis and cell proliferation was measured.

6. 인 비트로 키나아제 어세이(In vitro kinase assay)6. In vitro kinase assay

JAK 패밀리 멤버의 각 단백질은 특정 1차 항체로 면역 침강시켜 수득하였고, 재조합 His-tagged STAT3α 단백질을 정체하여 인 비트로 키나아제 어세이의 기질로 사용하였다. JAKs 인 비트로 키나아제 어세이는 종래 방법으로 수행하였다(Kim BH et al,. MS-1020 is a novel small molecule that selectively inhibits JAK3 activity. Br J Haematol. 2010;148:132-143/Kim BH et al,. NSC114792, a novel small molecule identified through structure-based computational database screening, selectively inhibits JAK3. Mol Cancer 2010;9:36.). 아이스 위에서, HDLM-2 또는 L540 세포에 용해 버퍼(20 mM Tris-HCl, pH 7.4, 500 mM NaCl, 0.25% Triton X-100, 1 mM EDTA, 1 mM EGTA, 10 mM β-glycerophosphate, 1 mM DTT, 300 μM Na3VO4, 1 mM PMSF 및 포스파타아제 억제제 칵테일)를 처리하였다. 세포 용해물을 protein A/G-sepharose로 4 ℃에서 2 시간 동안 전-정제(pre-cleared)하고, 항-JAK1, 항-JAK2, 항-JAK3, 또는 항-TYK2 항체를 4 ℃에서 하루 동안(overnight) 처리하였다. 면역 복합체는 protein A/G-sepharose 비드로 침강시켰다. 침강물을 키나아제 버퍼(25 mM Tris/HCl, pH 7.5, 20 mM β-glycerophosphate, 10 mM MgCl2, 2 mM DTT, 1mM Na3VO4, 및 프로테아제 억제제 칵테일)로 2 회 세척하였다. 인 비트로 JAK 키나아제 어세이를 위하여, 면역 복합체(immune complexes)를 DMSO(vehicle), 튜불로신 또는 pan-JAK 억제제 AG490(150 μM)와 혼합하여 30 ℃에서 1 시간 반응시켰다. 0 ℃에서 30 분간 ATP(2-1000 μM) 첨가 또는 미첨가의 조건 하에서 His-tagged STAT3α 단백질(2 μg)을 첨가하면서 키나아제 반응이 일어났다. 반응생성물로 SDS-PAGE를 수행하였으며, phospho-STAT3, STAT3, JAK1, JAK2, JAK3, 또는 TYK2에 대한 항체를 이용하였다. Each protein of the JAK family member was obtained by immunoprecipitation with a specific primary antibody and the recombinant His-tagged STAT3α protein was suspended and used as a substrate for in vitro kinase assay. JAKs in vitro kinase assays were performed by conventional methods (Kim BH et al, MS-1020 is a novel small molecule that selectively inhibits JAK3 activity.Br J Haematol. 2010; 148: 132-143 / Kim BH et al, NSC114792, a novel small molecule identified through structure-based computational database screening, selectively inhibits JAK 3. Mol Cancer 2010; 9: 36.). On ice, lysis buffer (20 mM Tris-HCl, pH 7.4, 500 mM NaCl, 0.25% Triton X-100, 1 mM EDTA, 1 mM EGTA, 10 mM β-glycerophosphate, 1 mM DTT) in HDLM-2 or L540 cells , 300 μΜ Na 3 VO 4, 1 mM PMSF and phosphatase inhibitor cocktail). Cell lysates were pre-cleared with protein A / G-sepharose at 4 ° C. for 2 hours, and anti-JAK1, anti-JAK2, anti-JAK3, or anti-TYK2 antibodies were washed at 4 ° C. for 1 day. (overnight) treatment. Immune complexes were precipitated with protein A / G-sepharose beads. The precipitate was washed twice with kinase buffer (25 mM Tris / HCl, pH 7.5, 20 mM β-glycerophosphate, 10 mM MgCl 2, 2 mM DTT, 1 mM Na 3 VO 4, and protease inhibitor cocktail). For in vitro JAK kinase assay, immune complexes were mixed with DMSO (vehicle), tubulosine or pan-JAK inhibitor AG490 (150 μM) and reacted at 30 ° C. for 1 hour. The kinase reaction occurred with addition of His-tagged STAT3α protein (2 μg) under the condition of ATP (2-1000 μM) addition or no addition at 0 ° C. for 30 minutes. SDS-PAGE was performed as a reaction product, and antibodies against phospho-STAT3, STAT3, JAK1, JAK2, JAK3, or TYK2 were used.

다른 인 비트로 키나아제 어세이는 Merck Millipore의 KinaseProfiler™ Services를 이용하여 수행하였다. JAK 패밀리 멤버의 각 IC50 값은 ATP(10 mM) 및 다양한 튜불로신 농도에서 Millipore’s standard radiometric assay를 수행하여 결정하였다. JAK3의 Ki 및 Km 값은 다양한 튜불로신 및 ATP 농도에서 결정되었다. 모든 키나아제 어세이는 vehicle 및 acid blank controls 과 함께 2 회 실시되었으며, vehicle negative control activity의 %로서 나타내었다. 튜불로신의 평균 활성도(% control)는 화합물 농도에 대한 그래프로 나타내었고, Ki 값은 Cheng and Prusoff 방정식에 따른 데이터의 선형회귀 y-절편(y-intercept)으로 계산하였다. 튜불로신 없이 측정된 효소 특이활성도(enzyme specific activity, U/mg)는 ATP 농도에 따라 그래프로 나타내었고, Michaelis-Menten 방정식에 대입하여 ATP에 대한 Km(app)를 계산하였다. Another in vitro kinase assay was performed using KinaseProfiler ™ Services from Merck Millipore. Each IC 50 value of the JAK family member was determined by performing Millipore's standard radiometric assay at ATP (10 mM) and various tubulosine concentrations. Ki and Km values of JAK3 were determined at various tubulosine and ATP concentrations. All kinase assays were performed twice with vehicle and acid blank controls, expressed as% of vehicle negative control activity. The average activity (% control) of tubulosine was plotted against the compound concentration, and Ki values were calculated as the linear regression y-intercept of the data according to the Cheng and Prusoff equation. Enzyme specific activity (U / mg) measured without tubulosine was plotted according to ATP concentration, and Km (app) for ATP was calculated by substituting Michaelis-Menten equation.

7. 통계7. Statistics

데이터는 평균±표준편차 (SD)로 나타내었고, 통계적 차이는 one-way analysis of variance 및 Dunnett’s multiple comparison tests를 이용하여 결정하였다. 통계적 차이는 p < 0.05에서 유의한 것으로 판단된다.Data were expressed as mean ± standard deviation (SD), and statistical differences were determined using one-way analysis of variance and Dunnett's multiple comparison tests. Statistical differences were judged to be significant at p <0.05.

실험결과Experiment result

1. 구조-기반 가상 스크리닝을 통한 JAK 억제제로서 튜불로신의 동정1. Identification of Tubulosine as a JAK Inhibitor Through Structure-Based Virtual Screening

모든 JAK 패밀리 멤버는 중요한 구조적 상동성(homology)을 공유하는 7개 Janus homology(JH) 도메인으로 구성된다. 이러한 도메인들 중, JH1는 키나아제 도메인으로서, JAK 촉매 활성에 중요하다. JAK 활성을 억제하는 신규한 소분자를 동정하기 위하여, JAK3(JAK3-JH1)의 키나아제 도메인에 대한 AutoDock Version 4.2를 이용하였고, NCI 다양성 세트 화합물로 가상 스크리닝을 수행하였다. 다양한 초기 리간드 컨포머(conformer)를 이용한 앙상블 도킹(ensemble docking)은 AutoDock의 신뢰도를 향상시켰다. 결과는 동일한 컨포머로 러닝수(the number of runs)를 단순히 증가시켜 얻은 결과보다 정확도와 에너지 측면에서 우수하였다.All JAK family members consist of seven Janus homology (JH) domains that share important structural homology. Of these domains, JH1 is a kinase domain, which is important for JAK catalytic activity. To identify new small molecules that inhibit JAK activity, AutoDock Version 4.2 for the kinase domain of JAK3 (JAK3-JH1) was used and virtual screening was performed with NCI diversity set compounds. Ensemble docking with various early ligand conformers improved the reliability of AutoDock. The result was better in accuracy and energy than the result obtained by simply increasing the number of runs with the same conformer.

기존 분자와의 도킹은 고무적인 성능을 나타내었다. 처음에는 JAK3-JH1과 복합체를 형성하는 3 가지 소분자 AFN941, CP-690550, 및 CMP-6로 도킹을 시도하였다. 각 소분자는 JAK3-JH1-1YVJ, 3LXK, 및 3LXL의 3 가지 좌표(coordinate)로 도킹되었다. 클러스터링 이후, 가장 우세한(populated) 클러스터는 가장 낮은 에너지를 나타내었다. 가장 우수한 클러스터 중의 대표 클러스터는 1.0 Å RMSD 값 내 X-ray 구조에서 원래 발견된 구조와 유사한 구조를 나타내었다. 각 분자의 개시 컨포머(starting conformer)를 X-ray 구조가 아닌 공개 데이터베이스(pubchem.ncbi.nlm.nih.gov)의 2D 화학구조로부터 얻을 수 있다는 것은 주목할 만한 일이다.Docking with existing molecules has shown encouraging performance. Initially an attempt was made to dock with three small molecules AFN941, CP-690550, and CMP-6, which complex with JAK3-JH1. Each small molecule was docked with three coordinates: JAK3-JH1-1YVJ, 3LXK, and 3LXL. After clustering, the most prevalent clusters showed the lowest energy. The representative cluster among the best clusters showed a structure similar to that originally found in the X-ray structure within the 1.0 Å RMSD value. It is noteworthy that the starting conformer of each molecule can be obtained from the 2D chemical structure of the public database (pubchem.ncbi.nlm.nih.gov) rather than from the X-ray structure.

JAK3-JH1과의 복합체에서 튜불로신의 모델구조(도 1a)는 Val-812, Ala-829, Glu-847, Met-878, Leu-881, Leu-932, 및 Asp-943(도 1b)의 곁사슬 원자와 접촉하는 것으로 나타났다. JAK3-JH1 및 튜불로신 간의 AutoDock-계산된 결합 자유 에너지는 -11.79 kcal/mol인 반면, AFN941, CP-690550 및 CMP-6의 결합 자유에너지는 각각 -10.64, -9.23 및 -10.32 kcal/mol이다(도 1c). 결합 모드는 2 개의 -OCH3 주위 지역에서 AFN941, CP-690550, 및 CMP-6와 유사성이 있지만, Asp-847와 접촉하는 영역에서는 차이점이 있었다.The model structure of tubulosine in complex with JAK3-JH1 (FIG. 1A) is shown in Val-812, Ala-829, Glu-847, Met-878, Leu-881, Leu-932, and Asp-943 (FIG. 1B). It appeared to be in contact with the side chain atom. The AutoDock-calculated binding free energy between JAK3-JH1 and tubulosine is -11.79 kcal / mol, whereas the binding free energies of AFN941, CP-690550 and CMP-6 are -10.64, -9.23 and -10.32 kcal / mol, respectively. (FIG. 1C). The binding mode is similar to AFN941, CP-690550, and CMP-6 in the region around two -OCH 3 , but different in the region in contact with Asp-847.

2. 튜불로신은 ATP-결합 부위에 결합함으로써 JAK3 키나아제 활성을 직접적으로 차단한다2. Tubulosine directly blocks JAK3 kinase activity by binding to the ATP-binding site

JAKs의 7개 JH 도메인은 중요한 구조적 상동성이 있다. 이러한 유사성에 근거하여, JAK3 및/또는 다른 JAK3에 대한 튜불로신의 특이성(specificity)을 측정하기 위하여, 각 JAK 및 재조합 STAT3α 단백질의 면역침강을 이용한 인 비트로 키나아제 어세이를 수행하였다. JAK 면역침강물은 호지킨 림프종(Hodgkin's lymphoma) HDLM-2 또는 L540 세포의 세포 용해물에서 수득하였다. 각 JAK는 이들 세포에서 활성화되어 있다. 각 면역침강물을 농도별 튜불로신 하에서 재조합 STAT3α 단백질과 반응시켰다.The seven JH domains of JAKs have important structural homology. Based on this similarity, in vitro kinase assays were performed using immunoprecipitation of each JAK and recombinant STAT3α protein to determine the specificity of tubulosine to JAK3 and / or other JAK3. JAK immunoprecipitates were obtained from cell lysates of Hodgkin's lymphoma HDLM-2 or L540 cells. Each JAK is active in these cells. Each immunoprecipitate was reacted with recombinant STAT3α protein under different concentrations of tubulosine.

그 결과, 도 2a에서와 같이, STAT3α 단백질은 튜불로신이 없을 때 모든 JAK 면역침강물에 의해 효율적으로 인산화되었다. 이는 면역침강물이 키나아제 활성이 있음을 의미한다. AutoDock 모델링과 함께, JAK3 키나아제 활성은 튜불로신의 존재 하에서 효과적으로 억제되었으나, 튜불로신 농도 100 nM까지는 다른 JAK 활성에는 영향을 주지 않았다(도 2a). pan-JAK 억제제 AG490는 모든 JAK 의 키나아제 활성을 차단하였다. 흥미로운 것은 JAK3 키나아제 활성이 ATP 농도에 따라 점진적으로 감소한다는 것이다(도 2b 및 3a). 이는 튜불로신이 ATP-경쟁적(competitive) JAK3 억제제임을 의미한다.As a result, as in FIG. 2A, the STAT3α protein was efficiently phosphorylated by all JAK immunoprecipitates in the absence of tubulosine. This means that the immunoprecipitates have kinase activity. Along with AutoDock modeling, JAK3 kinase activity was effectively inhibited in the presence of tubulosine, but did not affect other JAK activities up to a tubulosine concentration of 100 nM (FIG. 2A). The pan-JAK inhibitor AG490 blocked the kinase activity of all JAKs. Interestingly, JAK3 kinase activity gradually decreases with ATP concentration (FIGS. 2B and 3A). This means that tubulosine is an ATP-competitive JAK3 inhibitor.

모든 JAK에 대한 튜불로신의 IC50 값을 결정하기 위하여, KinaseProfiler™ Services를 이용하여 인 비트로 키나아제 어세이를 수행하였다. 튜불로신은 IC50 4.81으로서 JAK3 키나아제 활성을 효과적으로 억제하였고, 억제 활성은 ATP에 의해 농도-의존적으로 감소되었으며, EC50 값은 2.38 μM 였다. 비록 다른 JAKs의 키나아제 활성이 억제되었지만, JAK3 억제율 보다는 낮았다. JAK1, JAK2, 및 TYK2 의 IC50 값은 각각 18.42, 25.67, 및 22.69 nM 였다(도 2c 및 3b). 이러한 결과는 튜불로신은 강력한 선택적 JAK3 억제제임을 나타낸다. JAK3 키나아제 활성을 나타내는 Ki 및 Km 값을 측정하였고, 튜불로신 첨가시 6.3 ± 0.6 nM, ATP 첨가시 106.4 ± 5.7 μM로 나타났다(도 3c). 이때, 스타우로스포린(Staurosporine, STS)은 JAKs에 대한 ATP-경쟁적 억제제의 양성 대조군으로 사용되었다.In vitro kinase assays were performed using KinaseProfiler ™ Services to determine the IC 50 value of tubulosine for all JAKs. Tubulosine effectively inhibited JAK3 kinase activity as IC 50 4.81, the inhibitory activity was reduced concentration-dependently by ATP and EC 50 value was 2.38 μM. Although the kinase activity of other JAKs was inhibited, it was lower than the JAK3 inhibition rate. IC 50 values of JAK1, JAK2, and TYK2 were 18.42, 25.67, and 22.69 nM, respectively (FIGS. 2C and 3B). These results indicate that tubulosine is a potent selective JAK3 inhibitor. Ki and Km values indicating JAK3 kinase activity were measured and found to be 6.3 ± 0.6 nM with tubulosine and 106.4 ± 5.7 μM with ATP addition (FIG. 3C). Staurosporine (STS) was used as a positive control of the ATP-competitive inhibitor against JAKs.

3. 튜불로신은 지속적으로-활성화된 JAK3 신호전달을 완전히 차단한다3. Tubulosine completely blocks sustained-activated JAK3 signaling

앞서 언급한 결과로서 JAK3 억제에 대한 튜불로신의 선택성을 확인하였다. 이러한 선택성(selectivity)을 평가하기 위하여, 튜불로신이 다양한 암 세포주에서 활성화된 JAK3 시그널을 억제할 수 있는지 실험하였다. As mentioned above, we confirmed the selectivity of tubulosine for JAK3 inhibition. To assess this selectivity, we tested whether tubulosine could inhibit activated JAK3 signals in various cancer cell lines.

그 결과, 세포주 L540, BKO-84, BaF3/JAK3V674A, 및 BaF3/TEL-JAK3에서는 JAK3 시그널이 지속적으로 활성화되어 있고(constitutively-active), 다른 JAKs는 활성화되어 있지 않다. 튜불로신은 농도-의존적으로 JAK3 및 이의 기질 STAT5 의 티로신 인산화를 억제한다(도 4a 및 4c). As a result, JAK3 signals are constitutively-active in cell lines L540, BKO-84, BaF3 / JAK3V674A, and BaF3 / TEL-JAK3, and other JAKs are not activated. Tubulosine inhibits tyrosine phosphorylation of JAK3 and its substrate STAT5 concentration-dependently (FIGS. 4A and 4C).

이에, JAK3-매개 시그널 억제에 대해 알아보기 위해, 위 세포주에 100 nM 튜불로신을 처리하여 시간에 따라 관찰하였다. Thus, in order to investigate the inhibition of JAK3-mediated signal, the cell line was observed over time by treatment with 100 nM tubulosine.

그 결과, 튜불로신은 2 시간 이내에 JAK3 및 이의 기질인 STAT3 및 STAT5 의 티로신 인산화를 효과적으로 억제하였다(도 5a 내지 5d).As a result, tubulosine effectively inhibited tyrosine phosphorylation of JAK3 and its substrates STAT3 and STAT5 within 2 hours (FIGS. 5A-5D).

세포주 HDLM-2, MDA-MB-468, DU145, A431, 및 BaF3/TEL-JAK2는 JAK3이 아닌 다른 JAKs-매개 시그널이 활성화되어 있다. 인 비트로 키나아제 어세이와 함께, 상기 세포주는 튜불로신 100 nM 농도에 이를 때까지 JAK1, JAK2 및 STAT5의 티로신 인산화에 영향을 주지 않았다(도 4b 및 4d). pan-JAK 억제제 AG490는 모든 세포주에서 모든 JAKs 및 STAT5의 티로신 인산화를 완전히 차단하였다. 다른 JAKs를 억제하는 튜불로신 농도를 결정하기 위하여, HDLM-2 및 BaF3/TEL-JAK2 세포에 100 nM 내지 1 μM 농도 범위의 튜불로신을 16 시간 동안 처리하였다. JAK1, JAK2 및 TYK2를 포함하는 JAKs, 및 이들의 기질인 STAT3 및 STAT5의 티로신 인산화가 400 nM 농도 이상의 튜불로신에 의해 억제되었다(도 6a 및 6b). 이러한 결과는 튜불로신이 JAK3-매개 신호전달경로를 완전히 억제함을 나타낸다. Cell lines HDLM-2, MDA-MB-468, DU145, A431, and BaF3 / TEL-JAK2 have activated JAKs-mediated signals other than JAK3. With the in vitro kinase assay, the cell line did not affect tyrosine phosphorylation of JAK1, JAK2, and STAT5 until the concentration of tubulosine was 100 nM (FIGS. 4B and 4D). The pan-JAK inhibitor AG490 completely blocked tyrosine phosphorylation of all JAKs and STAT5 in all cell lines. To determine tubulin concentrations that inhibit other JAKs, HDLM-2 and BaF3 / TEL-JAK2 cells were treated with tubulosine in a concentration range of 100 nM to 1 μM for 16 hours. Tyrosine phosphorylation of JAKs, including JAK1, JAK2, and TYK2, and their substrates, STAT3 and STAT5, was inhibited by tubulosine at concentrations above 400 nM (FIGS. 6A and 6B). These results indicate that tubulosine completely inhibits JAK3-mediated signaling pathways.

4. 튜불로신은 사이토카인-유도성 JAK3 시그널링을 차단한다4. Tubulosine Blocks Cytokine-Induced JAK3 Signaling

본 발명자들은 JAK 시그널의 사이토카인-의존적 활성 연구에 사용되었던 래트 pre-T 림프종 Nb2 세포에서, IL-2 또는 프롤락틴(prolactin, PRL) 자극에 의한 JAK3 시그널 억제에 대하여 튜불로신의 선택도를 실험하였다. 세포에 DMSO(vehicle), 튜불로신 또는 AG490를 16 시간 동안 처리하고, 이어 IL-2 또는 PRL로 10 분간 처리하였다. We examined the selectivity of tubulosine for inhibition of JAK3 signal by IL-2 or prolactin (PRL) stimulation in rat pre-T lymphoma Nb2 cells that were used in cytokine-dependent activity studies of JAK signals. . Cells were treated with DMSO (vehicle), tubulosine or AG490 for 16 hours, followed by 10 minutes with IL-2 or PRL.

그 결과, JAK2, JAK3 및 STAT5에서 특정 티로신 잔기의 인산화 상태는 확인하기 어려웠고, 이들의 인산화 레벨은 IL-2 또는 PRL에 의해 현저히 증가하였다(도 7a 및 7b). 예상한 바와 같이, IL-2-자극된 세포에서는 25 nM까지의 저농도 튜불로신에 의해 JAK3 및 STAT5 인산화는 거의 완전히 사라졌다(도 7a). 그러나, 튜불로신은 100 nM 농도까지 PRL-유도된 JAK2 및 STAT5 인산화를 미미하게 억제시켰다(도 7b). As a result, the phosphorylation status of certain tyrosine residues in JAK2, JAK3 and STAT5 was difficult to identify, and their phosphorylation levels were markedly increased by IL-2 or PRL (FIGS. 7A and 7B). As expected, JAK3 and STAT5 phosphorylation was almost completely eliminated by low concentrations of tubulosine up to 25 nM in IL-2-stimulated cells (FIG. 7A). However, tubulosine slightly inhibited PRL-induced JAK2 and STAT5 phosphorylation up to 100 nM concentration (FIG. 7B).

이에, 튜불로신이 JAK1- 및/또는 TYK-2-매개성 시그널에 영향을 주는지, 인터페론(IFN)-α로 처리하여 실험하였다. Thus, it was tested by treatment with interferon (IFN) -α whether tubulosine affected JAK1- and / or TYK-2-mediated signals.

그 결과, IFN-α-자극은 다발성 골수종 U266 세포에서 IFN-α 처리하지 않은 세포와 비교하여 JAK1, TYK2, STAT1 및 STAT3의 티로신 인산화를 현저히 증가시켰다. 티로신 인산화는 튜불로신 농도 100 nM까지는 영향을 받지 않았다(도 7c). pan-JAK 억제제 AG490는 IL-2-, PRL-, 또는 IFN-α를 처리한 세포에서 모든 JAKs, STAT3 및 STAT5의 티로신 인산화를 비-선택적으로 억제하였다. 이러한 결과는 튜불로신이 다른 JAKs 보다 JAK3 에 대한 선택성이 있음을 명확히 나타낸다.As a result, IFN-α-stimulation markedly increased tyrosine phosphorylation of JAK1, TYK2, STAT1 and STAT3 in multiple myeloma U266 cells as compared to cells not treated with IFN-α. Tyrosine phosphorylation was not affected up to 100 nM tubulosine concentration (FIG. 7C). The pan-JAK inhibitor AG490 non-selectively inhibited tyrosine phosphorylation of all JAKs, STAT3 and STAT5 in cells treated with IL-2-, PRL-, or IFN-α. These results clearly show that tubulin is more selective for JAK3 than other JAKs.

5. 튜불로신은 ERK 활성을 증가시키지만, 다른 종양형성 시그널에는 영향을 미치지 않는다5. Tubulosine increases ERK activity but does not affect other tumorigenic signals

본 발명자들은 튜불로신이 비-수용체 티로신 키나아제인 Src 패밀리, 세린/트레오닌키나아제 Akt, 및 ERK(extracellular signal-regulated kinase)와 같은 다른 종양형성 인자의 시그널에 영향을 줄 수 있는지 알아보았다. We have determined whether tubulosine can influence the signals of other tumorigenic factors such as the non-receptor tyrosine kinase Src family, serine / threonine kinase Akt, and extracellular signal-regulated kinase (ERK).

그 결과, 튜불로신은 100 nM의 농도에 이를 때까지, Src 패밀리 티로신 키나아제의 활성형 레벨(L540 및 HDLM-2 세포에서의 phospho-Lyn, 및 MDA-MB-468, DU145, 및 A431 세포에서의 phospho-Src)에 대해서는 유의한 억제효과를 나타내지 않았다(도 8a 및 8b, lanes 3 및 4). 또한, 세포에 튜불로신을 처리한 후의 Akt 인산화 레벨에서도 유의적인 변화를 관찰할 수 없었다(도 8a 및 8b, lanes 5 및 6). 흥미로운 것은, 지속적-활성형(constitutively-active) JAK3을 발현하는 L540 세포에서만 튜불로신 농도의존적으로 ERK1/2 인산화 레벨이 현저히 증가되었다는 것이다. 한편, 다른 JAK 패밀리 멤버가 활성화된 세포주에서는 이를 관찰할 수 없었다(도 8a 및 8b, lanes 7 및 8). 모든 세포주에서 STAT3 인산화는 양성 대조군을 나타내며, L540 세포에서 튜불로신 처리에 의해 효과적으로 억제되었고 HDLM-2, MDA-MB-468, DU145, 및 A431와 같은 다른 세포주에서는 억제되지 않았다(도 8a 및 8b, lanes 1 및 2). 상기 결과로서, 튜불로신이 선택적으로 JAK 3 활성을 억제하고, 이의 하위 타겟 시그널 전달을 억제함을 알 수 있다.As a result, tubulosine levels in the active levels of Src family tyrosine kinases (phospho-Lyn in L540 and HDLM-2 cells, and MDA-MB-468, DU145, and A431 cells until concentrations up to 100 nM). phospho-Src) did not show a significant inhibitory effect (Figs. 8a and 8b, lanes 3 and 4). In addition, no significant changes were observed in Akt phosphorylation levels after treatment with tubulosine in the cells (FIGS. 8A and 8B, lanes 5 and 6). Interestingly, the ERK1 / 2 phosphorylation level was significantly increased in tubulosine concentration only in L540 cells expressing constitutively-active JAK3. On the other hand, it could not be observed in other JAK family member activated cell lines (FIGS. 8A and 8B, lanes 7 and 8). STAT3 phosphorylation in all cell lines represents a positive control, effectively inhibited by tubulosine treatment in L540 cells and not in other cell lines such as HDLM-2, MDA-MB-468, DU145, and A431 (FIGS. 8A and 8B). , lanes 1 and 2). As a result, it can be seen that tubulosine selectively inhibits JAK 3 activity and its downstream target signal transduction.

6. 튜불로신은 지속적-활성형 JAK3를 발현하는 암세포의 생존 및 증식을 억제한다6. Tubulosine Inhibits Survival and Proliferation of Cancer Cells Expressing Sustained-Active JAK3

활성화된 JAK/STAT 시그널링은 암세포의 생존 및 증식 증가와 연관되며, 이 시그널링을 타게팅하는 소분자 억제제는 다양한 고형암 및 혈액 악성세포에서 세포 생존을 억제하는 것으로 보고되었다. 이러한 증거들은 튜불로신이 지속적-활성형 JAK3 시그널을 나타내는 암세포에서만 세포 생존에 영향을 미칠 것이라는 가능성을 보여준다. 이러한 가능성을 입증하기 위하여, 다양한 암세포주에 DMSO(vehicle), 튜불로신 또는 AG490을 다양한 시간으로 처리하여 세포 생존 분석을 수행하였다. Activated JAK / STAT signaling is associated with increased survival and proliferation of cancer cells, and small molecule inhibitors targeting this signaling have been reported to inhibit cell survival in various solid cancers and hematologic malignancies. These evidences show that tubulosine may affect cell survival only in cancer cells that exhibit sustained-active JAK3 signals. To demonstrate this possibility, cell survival assays were performed by treating various cancer cell lines with DMSO (vehicle), tubulosine or AG490 at various times.

그 결과, 튜불로신은 시간 및 농도 의존적으로, 다른 JAKs-매개 시그널이 활성화된 세포가 아닌, 지속적-활성형 JAK3 시그널을 나타내는 다양한 암세포의 생존을 현저히 감소시켰다(도 9a 및 9b). 암 세포 생존억제는 pan-JAK 억제제 AG490 처리에 의해 확인되었으며, 모든 세포주에서 세포 생존을 현저히 감소시켰다.As a result, tubulosine significantly reduced the survival of various cancer cells showing time- and concentration-dependent, sustained-active JAK3 signals rather than cells with other JAKs-mediated signals (FIGS. 9A and 9B). Cancer cell viability was confirmed by treatment with the pan-JAK inhibitor AG490 and significantly reduced cell viability in all cell lines.

이어, 트리판 블루 분석법을 이용하여 세포 수를 계수함으로써 암 세포의 증식율을 확인하였다. Then, the proliferation rate of cancer cells was confirmed by counting the number of cells using trypan blue assay.

그 결과, 세포 생존에서와 유사하게, 튜불로신은 지속적-활성형 JAK3 시그널을 나타내는 암 세포의 증식을 농도 및 시간 의존적으로 효과적으로 억제하였다(도 10a). 그러나, 다른 JAK가 활성화된 암 세포의 증식은 튜불로신의 영향이 미미하였다(도 10b). 세포 생존율과 함께, AG490는 모든 세포주에서 암세포의 증식을 효과적으로 억제하였다. 이러한 결과들은 JAK3가 지속적-활성형 JAK3 시그널을 나타내는 암세포의 생존 및 증식에 중요한 역할을 한다는 것을 의미한다. 튜불로신에 의한 JAK3 신호전달 경로의 억제는 결과적으로 암세포 생존 및 증식 억제를 동시에 나타낸다.As a result, similar to in cell survival, tubulosine effectively inhibited the proliferation of cancer cells showing sustained-active JAK3 signals in a concentration and time dependent manner (FIG. 10A). However, proliferation of other JAK-activated cancer cells had minimal effect of tubulosine (FIG. 10B). Together with cell viability, AG490 effectively inhibited the proliferation of cancer cells in all cell lines. These results indicate that JAK3 plays an important role in the survival and proliferation of cancer cells showing sustained-active JAK3 signals. Inhibition of the JAK3 signaling pathway by tubulosine results in simultaneous inhibition of cancer cell survival and proliferation.

이상으로 본 발명의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현 예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

본 발명은 튜불로신(tubulosine)을 유효성분으로 포함하는 암 치료용 약제학적 조성물에 관한 것이다. 본 발명의 조성물에서 튜불로신은 백혈병과 림프종 세포주에서 JAK 촉매 활성을 억제함으로써, 구조적으로 활성화된 사이토카인-유도성 JAK3 신호전달을 모두 강력하게 억제하므로, 활성 JAK-매개 암 세포의 생존 및 증식을 억제하는 제제로서 유용하게 사용될 수 있다.The present invention relates to a pharmaceutical composition for treating cancer comprising tubulosine as an active ingredient. In the composition of the present invention, tubulosine inhibits JAK catalytic activity in leukemia and lymphoma cell lines, thereby strongly inhibiting both structurally activated cytokine-induced JAK3 signaling, thereby preventing survival and proliferation of active JAK-mediated cancer cells. It can be usefully used as an inhibitor.

Claims (19)

(a) 다음 화학식 1로 표시되는 튜불로신(tubulosine)의 치료학적 유효량; 및 (b) 약제학적으로 허용가능한 담체(carrier)를 포함하는 암 예방, 개선 또는 치료용 약제학적 조성물:(a) a therapeutically effective amount of tubulosine represented by the following formula (1); And (b) a pharmaceutically acceptable carrier, wherein the pharmaceutical composition for preventing, ameliorating or treating cancer comprises: [화학식 I][Formula I]
Figure PCTKR2017012129-appb-I000004
Figure PCTKR2017012129-appb-I000004
 제 1 항에 있어서,The method of claim 1, 상기 조성물은 JAK3(Janus Kinase 3)의 촉매 활성(catalytic activity)을 억제하는 것을 특징으로 하는 약제학적 조성물.The composition is a pharmaceutical composition, characterized in that to inhibit the catalytic activity of the JAK3 (Janus Kinase 3). 제 1 항에 있어서,The method of claim 1, 상기 조성물은 경구 또는 비경구 투여되는 것을 특징으로 하는 약제학적 조성물.The composition is a pharmaceutical composition, characterized in that oral or parenteral administration. 제 3 항에 있어서,The method of claim 3, wherein 상기 조성물은 정맥내 투여, 피하투여, 근육내 투여, 복강투여 또는 경피투여로 비경구 투여되는 것을 특징으로 하는 약제학적 조성물.The composition is administered parenterally, intravenously, subcutaneously, intramuscularly, intraperitoneally or transdermally. 제 1 항에 있어서,The method of claim 1, 상기 암은 혈액암, 유방암, 뇌암, 신경내분비종양, 위암, 폐암, 전립선암, 난소암, 간암, 기관지암, 비인두암, 후두암, 췌장암, 방광암, 부신암, 대장암, 결장암, 자궁경부암, 골암, 피부암, 갑상선암, 부갑상선암 또는 요관암인 것을 특징으로 하는 약제학적 조성물.The cancer is blood cancer, breast cancer, brain cancer, neuroendocrine tumor, gastric cancer, lung cancer, prostate cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, adrenal cancer, colon cancer, colon cancer, cervical cancer, bone cancer Pharmaceutical composition, characterized in that the skin cancer, thyroid cancer, parathyroid cancer or ureter cancer. 제 5 항에 있어서,The method of claim 5, wherein 상기 혈액암은 림프종, 백혈병 또는 다발성 골수종인 것을 특징으로 하는 약제학적 조성물. The hematologic cancer is a pharmaceutical composition, characterized in that the lymphoma, leukemia or multiple myeloma. 제 6 항에 있어서,The method of claim 6, 상기 림프종은 비호지킨 림프종(Non-Hodgkins Lymphoma, NHL); 확산 거대 B 세포 림프종(diffuse large B-cell lymphoma, DLBCL); 여포성 림프종 (follicular lymphoma, FL); 호지킨병(Hodgkin disease); 버킷림프종(Burkitt's Lymphoma); 피부 T 세포 림프종(cutaneous T cell lymphoma); 원발성 중추 신경계 림프종(primary central nervous system lymphoma) 또는 림프계 전이(lymphomatous metastases)인 것을 특징으로 하는 약제학적 조성물.The lymphomas include Non-Hodgkins Lymphoma (NHL); Diffuse large B-cell lymphoma (DLBCL); Follicular lymphoma (FL); Hodgkin disease; Burkitt's Lymphoma; Cutaneous T cell lymphoma; A pharmaceutical composition, characterized in that it is primary central nervous system lymphoma or lymphatic metastasis. (a) 다음 화학식 1로 표시되는 튜불로신(tubulosine)의 치료학적 유효량; 및 (b) 약제학적으로 허용가능한 담체(carrier);를 포함하는 조성물을 이를 필요로 하는 개체에게 투여하는 단계를 포함하는 암 치료방법:(a) a therapeutically effective amount of tubulosine represented by the following formula (1); And (b) a pharmaceutically acceptable carrier; administering a composition to a subject in need thereof;
Figure PCTKR2017012129-appb-I000005
Figure PCTKR2017012129-appb-I000005
 제 8 항에 있어서,The method of claim 8, 상기 조성물은 경구 또는 비경구 투여되는 것을 특징으로 하는 치료방법.The composition is a method of treatment characterized in that oral or parenteral administration. 제 9 항에 있어서,The method of claim 9, 상기 조성물은 정맥내 투여, 피하투여, 근육내 투여, 복강투여 또는 경피투여로 비경구 투여되는 것을 특징으로 하는 치료방법.The composition is a parenteral administration by intravenous administration, subcutaneous administration, intramuscular administration, intraperitoneal administration or transdermal administration. 제 8 항에 있어서,The method of claim 8, 상기 암은 혈액암, 유방암, 뇌암, 신경내분비종양, 위암, 폐암, 전립선암, 난소암, 간암, 기관지암, 비인두암, 후두암, 췌장암, 방광암, 부신암, 대장암, 결장암, 자궁경부암, 골암, 피부암, 갑상선암, 부갑상선암 또는 요관암인 것을 특징으로 하는 치료방법.The cancer is blood cancer, breast cancer, brain cancer, neuroendocrine tumor, gastric cancer, lung cancer, prostate cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, adrenal cancer, colon cancer, colon cancer, cervical cancer, bone cancer , Skin cancer, thyroid cancer, parathyroid cancer or ureter cancer. 제 11 항에 있어서,The method of claim 11, 상기 혈액암은 림프종, 백혈병 또는 다발성 골수종인 것을 특징으로 하는 치료방법.The hematologic cancer is lymphoma, leukemia or multiple myeloma. 제 12 항에 있어서,The method of claim 12, 상기 림프종은 비호지킨 림프종(Non-Hodgkins Lymphoma, NHL); 확산 거대 B 세포 림프종(diffuse large B-cell lymphoma, DLBCL); 여포성 림프종 (follicular lymphoma, FL); 호지킨병(Hodgkin disease); 버킷림프종(Burkitt's Lymphoma); 피부 T 세포 림프종(cutaneous T cell lymphoma); 원발성 중추 신경계 림프종(primary central nervous system lymphoma) 또는 림프계 전이(lymphomatous metastases)인 것을 특징으로 하는 치료방법.The lymphomas include Non-Hodgkins Lymphoma (NHL); Diffuse large B-cell lymphoma (DLBCL); Follicular lymphoma (FL); Hodgkin disease; Burkitt's Lymphoma; Cutaneous T cell lymphoma; A method of treatment characterized in that it is primary central nervous system lymphoma or lymphatic metastasis. 암을 치료하기 위한 용도로서 다음 화학식 1로 표시되는 튜불로신(tubulosine)을 포함하는 조성물:A composition comprising tubulosine represented by the following general formula (1) for the treatment of cancer:
Figure PCTKR2017012129-appb-I000006
Figure PCTKR2017012129-appb-I000006
 제 14 항에 있어서,The method of claim 14, 상기 조성물은 경구 또는 비경구 투여되는 것을 특징으로 하는 조성물.The composition is characterized in that the oral or parenteral administration. 제 15 항에 있어서,The method of claim 15, 상기 조성물은 정맥내 투여, 피하투여, 근육내 투여, 복강투여 또는 경피투여로 비경구 투여되는 것을 특징으로 하는 조성물.The composition is administered parenterally, by intravenous administration, subcutaneous administration, intramuscular administration, intraperitoneal administration or transdermal administration. 제 14 항에 있어서,The method of claim 14, 상기 암은 혈액암, 유방암, 뇌암, 신경내분비종양, 위암, 폐암, 전립선암, 난소암, 간암, 기관지암, 비인두암, 후두암, 췌장암, 방광암, 부신암, 대장암, 결장암, 자궁경부암, 골암, 피부암, 갑상선암, 부갑상선암 또는 요관암인 것을 특징으로 하는 조성물.The cancer is blood cancer, breast cancer, brain cancer, neuroendocrine tumor, gastric cancer, lung cancer, prostate cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, adrenal cancer, colon cancer, colon cancer, cervical cancer, bone cancer , Skin cancer, thyroid cancer, parathyroid cancer or ureter cancer. 제 17 항에 있어서,The method of claim 17, 상기 혈액암은 림프종, 백혈병 또는 다발성 골수종인 것을 특징으로 하는 조성물.The hematological cancer is a composition, characterized in that the lymphoma, leukemia or multiple myeloma. 제 18 항에 있어서,The method of claim 18, 상기 림프종은 비호지킨 림프종(Non-Hodgkins Lymphoma, NHL); 확산 거대 B 세포 림프종(diffuse large B-cell lymphoma, DLBCL); 여포성 림프종 (follicular lymphoma, FL); 호지킨병(Hodgkin disease); 버킷림프종(Burkitt's Lymphoma); 피부 T 세포 림프종(cutaneous T cell lymphoma); 원발성 중추 신경계 림프종(primary central nervous system lymphoma) 또는 림프계 전이(lymphomatous metastases)인 것을 특징으로 하는 조성물.The lymphomas include Non-Hodgkins Lymphoma (NHL); Diffuse large B-cell lymphoma (DLBCL); Follicular lymphoma (FL); Hodgkin disease; Burkitt's Lymphoma; Cutaneous T cell lymphoma; A composition characterized in that it is primary central nervous system lymphoma or lymphomatous metastases.
PCT/KR2017/012129 2016-11-18 2017-10-31 Pharmaceutical composition for cancer prevention and treatment comprising tubulosine Ceased WO2018093065A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR20160154172 2016-11-18
KR10-2016-0154172 2016-11-18

Publications (1)

Publication Number Publication Date
WO2018093065A1 true WO2018093065A1 (en) 2018-05-24

Family

ID=62146663

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2017/012129 Ceased WO2018093065A1 (en) 2016-11-18 2017-10-31 Pharmaceutical composition for cancer prevention and treatment comprising tubulosine

Country Status (2)

Country Link
KR (1) KR102100826B1 (en)
WO (1) WO2018093065A1 (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090062222A1 (en) * 2005-09-29 2009-03-05 Trustees Of Boston University Methods for Sensitizing Cancer Cells to Inhibitors
WO2015198346A1 (en) * 2014-06-24 2015-12-30 Laila Nutraceuticals A composition comprising extract of alangium salvifolium having anti-adipogenic or anti-obesic activity

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090062222A1 (en) * 2005-09-29 2009-03-05 Trustees Of Boston University Methods for Sensitizing Cancer Cells to Inhibitors
WO2015198346A1 (en) * 2014-06-24 2015-12-30 Laila Nutraceuticals A composition comprising extract of alangium salvifolium having anti-adipogenic or anti-obesic activity

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ITO, A. ET AL.: "1',2',3',4'-tetradehydrotubulosine, a Cytotoxic Alkaloid from Pogonopus Speciosus", JOURNAL OF NATURAL PRODUCTS, vol. 62, 1999, pages 1346 - 1348, XP055503386 *
TIETZE, L. F. ET AL.: "Enantioselective Total Syntheses of the Ipecacuanha Alkaloid Emetine, the Alangium Alkaloid Tubulosine and a Novel Benzoquinolizidine Alkaloid by Using a Domino Process", CHEMISTRY-A EUROPEAN JOURNAL, vol. 10, 7 June 2004 (2004-06-07), pages 2722 - 2731, XP055503389 *
TROCONIS, M. ET AL.: "Molecular Modeling Study of Tubulosine and Other Related Ipecac Alkaloids", JOURNAL OF COMPUTER-AIDED MOLECULAR DESIGN, vol. 12, 1998, pages 411 - 418, XP055503391 *

Also Published As

Publication number Publication date
KR102100826B1 (en) 2020-04-27
KR20180056366A (en) 2018-05-28

Similar Documents

Publication Publication Date Title
EP3849534B1 (en) Combination of dasatinib and adagrasib for use in the treatment of non-small cell lung cancer
Matsubara et al. Differential regulation of IL-4 expression and degranulation by anti-allergic olopatadine in rat basophilic leukemia (RBL-2H3) cells
US20220110940A1 (en) Combinations for the treatment of neoplasms using quiescent cell targeting with egfr inhibitors
WO2014056894A1 (en) Mek inhibitors in the treatment of virus diseases
US20220000870A1 (en) Treatment of cancer
US12178822B2 (en) Methods of treating hematological malignancies using 2-(2,6-dioxopiperidin-3-yl)-4-((2-fluoro-4-((3-morpholinoazetidin-1-yl)methyl)benzyl)amino)isoindoline-1,3-dion
EP2136829A1 (en) Kinase protein binding inhibitors
AU2019201169A1 (en) Pim kinase inhibitor combinations
AU2001266611A1 (en) Use of (di-substituted-phenyl)-pyrimidinyl-imidazole derivatives as jnk-inhibitors
US20250017921A1 (en) Pharmaceutical combination and tumor treatment
AU2016308704B2 (en) MDM2 inhibitors for treating uveal melanoma
WO2018093065A1 (en) Pharmaceutical composition for cancer prevention and treatment comprising tubulosine
JPWO2020059705A1 (en) Cancer combination therapy with quinoline carboxamide derivatives
TW202114687A (en) Combination of FAK inhibitor and BTK inhibitor for treating a disease
EP4497436A1 (en) Therapeutic or prophylactic medicine for fragile x syndrome
US20230226061A1 (en) Combination cancer therapy with dyrk1 inhibitors and inhibitors of the ras-raf-mek-erk (mapk) pathway
WO2025101960A1 (en) Co-administration of a co-crystal of psilocybin and psilocin with an atypical antipsychotic
WO2023011415A1 (en) Pharmaceutical composition of egfr inhibitor and use thereof
CN115429804A (en) IkZF1, ikZF3 and BTK multi-target modulators
HK40006909B (en) Treatment of cancer
HK40006909A (en) Treatment of cancer
WO2015108303A1 (en) Pharmaceutical composition for treating diabetes
WO2021262878A1 (en) Novel molecule for modulation of innate immune responses controlled by sting protein
WO2021225135A1 (en) Therapeutic agent for nakajo-nishimura syndrome
JP2007314486A (en) STAT6 activation inhibitor containing naphthalene derivative as active ingredient

Legal Events

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

Ref document number: 17872799

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17872799

Country of ref document: EP

Kind code of ref document: A1