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WO2004098601A1 - Inhibiteur de la pkc, inhibiteur de l'angiogenese contenant de la 6-anilinoquinoline-5,8-quinone comme ingredient actif - Google Patents

Inhibiteur de la pkc, inhibiteur de l'angiogenese contenant de la 6-anilinoquinoline-5,8-quinone comme ingredient actif Download PDF

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WO2004098601A1
WO2004098601A1 PCT/KR2003/000900 KR0300900W WO2004098601A1 WO 2004098601 A1 WO2004098601 A1 WO 2004098601A1 KR 0300900 W KR0300900 W KR 0300900W WO 2004098601 A1 WO2004098601 A1 WO 2004098601A1
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comr2029
angiogenesis
cells
pkc
activity
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Inventor
Dae Yoon Chi
Hwan Mook Kim
Oh Young Kwon
Si Hwan Song
Sung Hee Cho
Han Young Choi
Eun Young Yoon
Dong Ho Hong
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CHEMON Inc
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CHEMON Inc
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    • 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

Definitions

  • the present invention relates to an angiogenesis inhibitor containing 6- anilinoquinoline-5, 8-quinone (COMR2029) as an effective ingredient.
  • the angiogenesis inhibitor of the present invention can be effectively used for the prevention or treatment of the angiogenesis-related diseases such as cancer, rheumatoid arthritis and diabetic retinopathy.
  • Solid tumor is supplied with growth factors, oxygen and nutrients by diffusion until it grows 1 —2 mm 3 and, from then on, it should be supplied with them by new blood vessels for further growth.
  • PKC inhibitor suppresses angiogenesis caused by an angiogenesis inducer, VEGF.
  • PKC is a kind of serine/threonine kinases and is activated most in the presence of calcium ion and DAG (Diacylglycerol) . At least 10 classes of PKC have been found so far and their formation depends specifically on intracellular expression and activation mediated by lipid and calcium. PKC is detected during the process of signal transduction by hormones, growth factors and neurotransmitters . PKC participates in phosphorylation of various proteins, meaning that PKC regulates the protein activity. On the special occasion, phosphorylation of EGF receptor caused by PKC has a function of down-regulation of the activity of tyrosine kinase of the receptor. PKC family is divided into three major subgroups.
  • the first subgroup is classical PKC that PKC ⁇ , ⁇ I , ⁇ II and Y belongs to.
  • the members of the classical PKC are Ca 2+ /lipid-dependent .
  • the second group is novel PKC exemplified by PKC ⁇ , ⁇ , r ⁇ and ⁇ . Even though the members of the second group are Ca 2+ - independent, they need lipid as they are activated.
  • the third group is atypical PKC that is not Ca 2+ , lipid-dependent and PKC ⁇ and ⁇ belongs to. PKC ⁇ seems similar to the members of novel PKC, but it is classified into another group since it has membrane-spanning domain.
  • PKC is activated by various agonists, growth factors, antigens, cytokines and neurotransmitters .
  • phorbol ester like TPA directly stimulates classical and novel PKC.
  • PKC plays an important role in intracellular actions such as secretion, differentiation, tumorigenesis, mitogenesis, signal transduction, gene expression and cytoskeletal regulation.
  • PKC has continuously been reported to be important for integrin-mediated adhesion and signaling event.
  • adhesion, spreading and cell migration in extracellular matrix increase as TPA and some of other cellular forms are treated with PKC.
  • PKC when PKC is suppressed, cell adhesion, cell spreading, cell migration, FAK phosphorylation and focal adhesion formation are all inhibited.
  • PKC ⁇ and PKC ⁇ relate to focal adhesion.
  • PKC ⁇ and PKC ⁇ relate to integrin activation and locate on membrane.
  • PLC activity, diacylglycerol level and arachidonic acid production increase as PKC is activated.
  • Integrin receptor antagonist inhibits the actions related to integrin and indirectly affects various diseases and pathological condition by working on integrin receptor.
  • Integrin receptor antagonist is believed to be a good candidate for many useful medicines. Integrin receptor is a heterodimer transmembrane receptor where cells are attached and communicate with an extracellular matrix and other cells .
  • a v ⁇ 3 ligand can be effectively used for the treatment and/or the suppression of recurrent stenosis (recurrence of stenosis after correctional operation of valvular disease), arteriosclerosis, diabetic retinopathy, degeneration of yellow spot, angiogenesis
  • a v ⁇ 3 antagonist inhibiting angiogenesis can be effectively used for the treatment of cancers by suppressing the growth of tumors.
  • angiogenesis is a major cause for the development and the progress of arthritis and integrin a v ⁇ 3 is the primary target of inflammatory arthritis.
  • a new approach to treat arthritis such as rheumatoid arthritis can be made by a v ⁇ 3 antagonist inhibiting angiogenesis.
  • the antagonist is able to inhibit angiogenesis by working as an antagonist a v ⁇ s, a integrin receptor.
  • the monoclonal antibody against a v ⁇ 5 has been confirmed to inhibit VEGF-induced angiogenesis in cornea of rabbit and chorion of chick. Therefore, it is for sure that the compounds antagonizing a v ⁇ 5 can be successfully used for the treatment and the prevention of degeneration of yellow spot, diabetic retinopathy, viral diseases, cancers, and metastatic tumor growth .
  • angiogenesis factors such as VEGF (vascular endothelial growth factor) , bFGF (basic fibroblast growth factor) , HGF (hepatocyte growth factor) , EGF (epithelial growth factor), angiopoietin, etc., receptors of angiogenesis factor having tyrosine kinase activity such as FGFR (fibroblast growth factor receptor), Flk-1/KDR, Flt-1, Flt-3, Tie-1, Tie-VEGF (vascular endothelial growth factor) , bFGF (basic fibroblast growth factor) , HGF (hepatocyte growth factor) , EGF (epithelial growth factor), angiopoietin, etc., receptors of angiogenesis factor having tyrosine kinase activity such as FGFR (fibroblast growth factor receptor), Flk-1/KDR, Flt-1, Flt-3, Tie-1, Tie-
  • angiogenesis inhibitors such as angiostatin and endostatin.
  • angiogenesis factors VEGF draws a special attention since VEGF seems to be closely related to the extent of tumor progression and treatment in most cases.
  • Flk-1/KDR Flk-1/KDR is expressed in endothelial cells of blood vessels specifically, making VEGF a useful candidate for the development of a specific angiogenesis inhibitor.
  • Angiostatin a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis Lung Carcinoma, Cell , 79, 315-328, 1994) and endostatin (M. S. O'Reilly, T. Boehm, C. Chen, et al . , Endostatin: an endogeneous inhibitor of angiogenesis and tumor growth. Cell , 88, 277-285 , 1997), recently found intra angiogenesis inhibitors, were proved to be very effective anticancer agents, which was confirmed by test animal model, and contributed to the development of angiogenesis inhibitors as anticancer agents.
  • an angiogenesis inhibitor has more benefits than conventional anticancer agents. Particularly, since angiogenesis is indispensable for tumorigenesis or metastasis, inhibition of angiogenesis results in the prevention of both tumorigenesis and metastasis. Besides, since angiogenesis inhibitor targets not aneuploid cancer cells but normal diploid endothelial cells of blood vessels, the inhibitor does not cause side effects such as resistance induced by heterogeneity and genetic instability of cancer cells.
  • the conventional anticancer agents only work against specific or some kinds of cancers. On the contrary, angiogenesis inhibitors affect every kinds of cancer requiring angiogenesis. Angiogenesis is hardly occurring but some special occasions such as wound healing, menstruation, etc.
  • angiogenesis inhibitors as anticancer agents might be very effective to treat cancers with less side effects .
  • Angiogenesis has also been known to induce angiogenic diseases like rheumatoid arthritis, diabetic retinopathy, etc., in addition to tumor growth and metastasis. Therefore, angiogenesis inhibitors are expected to be effectively used for the treatment and the prevention of angiogenic diseases along with cancers.
  • the present inventors have worked hard on finding a novel angiogenesis inhibitor and finally found the fact that 6-anilinoquinoline-5, 8-quinone has a function to inhibit angiogenesis without cytotoxicity .
  • the present inventors have accomplished this invention by confirming that the said 6-anilinoquinoline-5 , 8-quinone can be effectively used for the treatment or the prevention of angiogenesis-related diseases
  • FIG. 1 is a set of photographs showing the inhibition of angiogenesis by COMR2029 after hypodermically injecting matrigel in mouse abdomen;
  • FIG. 2 is a set of electrophoreses photographs showing the apoptosis-inducing capability of COMR2029, which was analyzed by agarose gel electrophoresis; A : B16(F10), Lane 1 and 8 :
  • Lane 3 COMR2029 0.03 ⁇ g/ml
  • Lane 4 COMR2029 0.1 ⁇ g/ml
  • Lane 5 COMR2029 0.3 ⁇ g/ml
  • Lane 6 COMR2029 1 ⁇ g/ml
  • Lane 7 COMR2029 3 ⁇ g/ml
  • Lane 2 Solvent control (VH) Lane 3 Camptothecin 0.3 ⁇ g/ml, Lane 4 COMR2029 0.1 ⁇ g/ml, Lane 5 COMR2029 0.3 ⁇ g/ml, Lane 6 : COMR2029 1 ⁇ g/ml,
  • FIG. 3 is a graph showing the changes of intracellular Ca 2+ concentration by treating COMR2029, resulted from the analysis using Fura- 2 /AM;
  • FIG. 4 is a set of photographs showing the inhibition of invasion after treating Bl ⁇ (F10) cells with 3 ⁇ g/ml, 1 ⁇ g/ml or 0.3 ⁇ g/ml of COMR2029 respectively;
  • FIG. 5 is a set of photographs showing the inhibition of invasion after treating LOX-IMVI cells with 0.03 ⁇ g/ml , 0.1 ⁇ g/ml or 0.3 ⁇ g/ml of COMR2029 respectively;
  • FIG. 6 is a graph showing the tumor volumes of nude mice transplanted with human skin cancer cell line LOX-IMVI after treating COMR2029 or solvent control (VH) ;
  • FIG. 7 is a graph showing the body weights of nude mice transplanted with human skin cancer cell line LOX-IMVI after treating COMR2029 or solvent control (VH) ;
  • FIG. 8 is a graph showing the tumor volumes of nude mice transplanted with human skin cancer cell line LOX-IMVI after treating COMR2029, positive control (adriamycin) or solvent control
  • FIG. 9 is a graph showing the body weights of nude mice transplanted with human skin cancer cell line LOX-IMVI after treating COMR2029, positive control (adriamycin) or solvent control (VH) ;
  • FIG. 10 is an electrophoresis photograph showing the inhibition activity of COMR2029 to phosphorylation of MAPK (ERK 1/2);
  • FIG. 11a - FIG. lie are electrophoresis photographs showing the inhibition activity of COMR2029 to transcription regulating elements, in which each test results were obtained using LOX- IMVI (FIG. 11a), B16 (F10) (FIG. lib) and HUVEC (FIG. lie) cells;
  • FIG. lid is a set of electrophoresis photographs showing the result of confirmation test for FIG. lla-Fig. lie, in which the inhibition activity of COMR2029 to phosphorylation of AP-1 and NF-kB in LOX-IMVI is revealed;
  • FIG. 12 is a set of graphs showing the effect of COMR2029 on PKC activity
  • FIG. 13 is a graph showing the effect of
  • FIG. 14 is a graph showing the effect of COMR2029 on uPA activity as time passes;
  • FIG. 15 is a diagram showing the mechanism of uPA protein working on cell migration and invasion
  • the present invention provides an agent for the prevention or the treatment of angiogenesis- related diseases containing 6-anilinoquinoline- 5, 8-quinone (referred “COMR2029” hereinafter) as an effective ingredient. Further features of the present invention will appear hereinafter.
  • the present invention provides an agent for the prevention or the treatment of angiogenesis- related diseases containing COMR2029 as an effective ingredient.
  • the PKC inhibitor of the present invention that inhibits angiogenesis induced by angiogenic factors can be used for the prevention or the treatment of a disease selected from a group consisting of diabetic retinopathy, macular degeneration, retinopathy of immature infants, angiogenic glaucoma, arthritis and solid tumors, but not always limited thereto (D'Amato, R.J. and Adamis, A, P. (1995) Ophthalmol . , 102:1261-1262; Arbiser, J.L. (1996) J. Am . Acad . Derm . , 34 (3) :486-497; O'Brien, K.D. et al . (1996) Circula tion , 93 (4 ): 672-682; Hanahan, D. and Folkman, (1996) Cell , 86:353-364).
  • a disease selected from a group consisting of diabetic retinopathy, macular degeneration, retinopathy of immature infants,
  • COMR2029 showed cytotoxicity against almost all the tumor cell lines and especially, very strong cytotoxicity against skin cancer cell line (see Table 3) . Even it's strong cytotoxicity against skin cancer cell line, COMR2029 did not affect cell cycle, apoptosis, caspase-3 activity that was related to apoptosis, and intracellular Ca 2+ concentration (see FIG. 2 and FIG. 3) .
  • the present inventors performed in vi tro invasion assay. As a result, COMR 2029 functioned to inhibit membrane transmission in B16(F10) cells that have been widely used for the experiments testing the ability to inhibit membrane transmission in general (see FIG. 4) .
  • COMR2029 In LOX-IMVI cells that have not been commonly used for invasion assay, COMR2029 also inhibited membrane transmission dose-dependently (see FIG. 5).
  • COMR2029 injected adriamycin, a sort of anticancer agent, in positive control and compared the tumor sizes between the two groups. Resultingly, the extent of diminution of tumor size was not very different. Thus, COMR2029 was confirmed to have strong anticancer activity (see FIG. 8 and FIG. 9) .
  • the present inventors also investigated the effect of COMR2029 on MAPK regulation. As a result, the inventors confirmed that COMR2029 dose-dependently inhibited the activity of ERKl/2 among MAPKs, intracellular transmitters (see FIG.
  • a signal transduction system by calcium is one of many important signal transduction systems. Calcium induces the activation of transcription factors related to gene expression, which is regulated by various signal transduction enzyme system and those enzymes are exemplified by PKC (protein kinase C) , CaM kinase (calcium/calmodulin dependent protein kinase) and ERK (extracellular signal regulated kinase) , a kind of MAP (mitogen-activated protein) kinase.
  • PKC protein kinase C
  • CaM kinase calcium/calmodulin dependent protein kinase
  • ERK extracellular signal regulated kinase
  • ERK has been known to be the most important enzyme for the expression of a gene relating to long-term memory. Activated ERK moves into nucleus and activates transcription factors directly or do indirectly by activating other enzyme systems.
  • COMR2029 of the present invention inhibits the expression of ERK, meaning that one of the signal transduction systems is blocked and the activation of transcription factor and gene expression are all affected.
  • the ERK protein is the last of signal transduction system Ras-Raf-MEK-ERK, a signal transduction system of angiogenesis inhibitor.
  • the present inventors also investigated the effect of COMR2029 on the expressions of various transcription regulating factors with 4 transcription regulating factors in 3 cell lines.
  • FIG lid From the results of RT-PCR, it was also confirmed that p21 and uPA were decreased just like AP-1, suggesting that AP-1 binding sites were located in promoter region, and thus COMR2029 of the present invention related to angiogenesis inhibitors .
  • the present inventors further investigated the effect of COMR2029 on the intracellular activity of PKC (protein kinase C) . As a result,
  • COMR2029 inhibited the PKC activity (see FIG. 12) .
  • PKC is a kind of serine/threonine kinases and is activated most in the presence of calcium ion and DAG (Diacylglycerol) .
  • DAG Diacylglycerol
  • At least 10 kinds of PKC have been known and they change their forms specifically according to the level of their expression and activation caused by lipid and calcium in tissues.
  • PKC can be observed during the signal transduction process induced by hormones, growth factors and neurotransmitters .
  • PKC induces phosphorylation of various proteins, resulting in the variation of protein activity.
  • phosphorylation of EGF receptor induced by PKC functions to down-regulate the activity of tyrosine kinase of the receptor.
  • COMR2029 along with MAPK and transcription regulating factor, inhibits PKC activity, which is the same result as others.
  • the present inventors also investigated the effect of COMR2029 on the activity of sGC (soluble guanynyl cyclase) and uPA.
  • sGC soluble guanynyl cyclase
  • uPA uPA
  • the angiogenesis inhibitor of the present invention for the prevention or the treatment of the angiogenesis-related diseases contains the above COMR2029 as an effective ingredient.
  • the above COMR2029 can be administered orally or parenterally and be used in general form of pharmaceutical formulation.
  • the COMR2029 of the present invention can be prepared for oral or parenteral administration by mixing with generally used fillers, extenders, binders, wetting agents, disintegrating agents, diluents such as surfactant, or excipients.
  • Solid formulations for oral administration are tablets, pill, dusting powders and capsules.
  • Solid formulations are prepared by mixing one or more suitable excipients such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. Except for the simple excipients, lubricants, for example magnesium stearate, talc, etc, can be used.
  • Liquid formulations for oral administrations are suspensions, solutions, emulsions and syrups, and the abovementioned formulations can contain various excipients such as wetting agents, sweeteners, aromatics and preservatives in addition to generally used simple diluents such as water and liquid paraffin.
  • Formulations for parenteral administration are sterilized aqueous solutions, water-insoluble excipients, suspensions, emulsions, and suppositories.
  • Water insoluble excipients and suspensions can contain, in addition to the active compound or compounds, propylene glycol, polyethylene glycol, vegetable oil like olive oil, injectable ester like ethylolate, etc.
  • Suppositories can contain, in addition to the active compound or compounds, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerinated gelatin, etc.
  • the effective dosage of COMR2029 of the present invention is 10 mg/kg and its estimated LD50 value is much greater than 1000 mg/kg, which are confirmed by acute toxicity assay with rats tested via oral administration.
  • HUVEC Human Umbilical Vein Endothelial Cell
  • Matrigel was distributed to 96-well plate by 40 ⁇ l and polymerized in C0 2 incubator at 37 ° C for 30 minutes. In that experiment, matrigel was to provide matrix that was necessary for tube formation, that is, angiogenesis by HUVEC cells.
  • HUVEC cells cultured in EGM-2 medium were distributed into wells of a plate by 180 ⁇ l with the concentration of 1.1X10 5 cells/m ⁇ .
  • the wells of the plate were treated with COMR2029 by 0.03, 0.1, 0.3, 1, and 3 ⁇ g/ml respectively and were cultured in C0 2 incubator at 37 ° C for 18-24 hours. After cultivation, microphotographs were taken for detection .
  • VH formed more than 10 tubes.
  • tube formation was not observed in the wells treated with 3 ⁇ g/ml and 1 ⁇ g/ml of COMR 2029 each. Just a line to form a tube was seen dimly from the concentration of 0.3 ⁇ g/ml, yet the tube was not formed until the concentration reached 0.1 ⁇ g/ml . The tube began to be formed from the concentration
  • the present inventors investigated if COMR2029 had cytotoxicity against HUVEC cells, a human umbilical vein endothelial cell line, with cell growth inhibition test using SRB (SRB method, Skehan, et al., Proc . Am . Assoc . Cancer Res . , 30: 612, 1989) .
  • SRB SRB method, Skehan, et al., Proc . Am . Assoc . Cancer Res . , 30: 612, 1989
  • the above cell line was cultured in EGM-2 medium.
  • COMR2029 was dissolved in 100% dimethylsulfoxide (referred "DMSO" hereinafter) and was diluted consecutively to adjust the concentration to 0.01, 0.03, 0.1, 0.3, 1 ⁇ g/ml respectively.
  • DMSO dimethylsulfoxide
  • Tz (Time Zero) plate was fixed using 50% TCA to determine the cell concentration before cells were treated with reagents.
  • the plate that would be treated with COMR2029 was divided into two groups. One group was left for 24 hours, and then treated with 5 different concentrations of COMR2029 that were adjusted by 0.1% DMSO. The other group was treated with COMR2029 right away and was transferred into a fresh medium two hours later, which was treated with COMR2029 again after 24 hours. Tz plate was washed with tap water after an hour.
  • a plate treated with reagents was fixed by treating 50% TCA (50 ⁇ /well ) after two days, which was washed with tap water 1 hour later. The washed plate was dried at room temperature. Then, 0.4% SRB solution (dissolved in 1% acetic acid) was added by 100 ⁇ l per well. After 30 minutes since then, the plate was washed with 1% acetic acid solution, which was dried at room temperature. 10 mM tris base (pH 10.5) was added into each well by 100 ⁇ . Finally, optical ' density was measured at 540 nm using a microplate reader. The group treated with COMR2029 24 hours later was presented in the below Table 1 and the other group treated with COMR2029 right away, transferred into a fresh medium two hours later and treated with COMR2029 again after 24 hours, was presented in the below Table 2.
  • the present inventors performed in vivo angiogenesis assay after hypodermically injecting matrigel in mice.
  • COMR2029 was prepared as follows; COMR2029 5%, Cremophor 20% and 0.85% saline 75%. COMR2029 was administered into the abdominal cavity of the mice with the concentration of 10 mg/kg everyday from day 1 to day 8. Cremophor solution without COMR2029 was administered for the solvent control. On the 9 th day after implanting matrigel under the skin of the mice, the mice were sacrificed for examination and photographs were taken.
  • mice of solvent control not treated withCOMR2029 turned red by angiogenesis, while mice treated with COMR2029 colored yellow since angiogenesis was hardly occurred (FIG. 1) . Therefore, it was confirmed that COMR2029 functioned to inhibit angiogenesis.
  • leukemia cell lines such as MOLT-4F, RPMI8226, HL60, P388/ADR, P388, K562/ADR, K562/VIN and K562, human melanoma cell lines such as LOX- IMVI, SK-MEL-28, SK-MEL-5, UACC62, Hs 294T, Hs 695T, SK-MEL-2, RPMI7951, A253, G361, M14 and HT1080, a mouse melanoma cell line, B16(F10), a renal cancer cell line, UO-31, colon cancer cell lines such as Colo205, HCT116, HCC2998, HCT15 and SW620, lung cancer cell lines such as A549, NC1- H522 and NC1-H23, a CNS cancer cell line, SNB19, an ovarian cancer cell line, SK-OV-3, a prostate cancer cell line, PC-3, breast cancer cell lines such as Hs 5
  • COMR2029 was dissolved in 100% DMSO and was diluted consecutively to adjust the concentration to 0.01, 0.03, 0.1, 0.3 and 1 ⁇ g/ml respectively. The final concentration of sample in the plate that would be treated with COMR2029 was also adjusted to 5 different concentrations using 0.1% DMSO. Tz plate was washed with Tap water one hour later and two days later, the plate where COMR2029 was treated was fixed by treating 50 ⁇ l of 50% TCA per well, which was washed with Tap water after an hour had passed. The washed plate was dried at room temperature. 0.4% SRB solution (dissolved in 1% acetic acid) was added into each well by 100 ⁇ l , which was washed with 1% acetic acid solution after 30 minutes. The plate was dried at room temperature, again. 10 mM Tris-base (pH 10.5) was added into each well by 100 ⁇ l and dissolved.
  • GI 50 ( ⁇ g/ml ) means the concentration of a compound inhibiting cancer cell growth up to 50%.
  • COMR2029 responded sensitivity to K562, K562/VIN and K562/ADR among 8 kinds of blood cancer cell lines but did not to P388 and P388/ADR, showing over 1.0 ⁇ g/ml in GI 50 value.
  • COMR2029 showed 0.3 ⁇ g/ml in GI 50 value for kidney cancer cell line UO-31 or central nervous system cancer cell line SNB19, 0.2 ⁇ g/ml for colon cancer cell lines, HCT15 and HCT116 and 0.5 ⁇ g/ml for Colo205, HCT15 and SW620.
  • COMR2029 showed low cytotoxicity to ovarian cancer cell line SK-OV-3, while did comparatively high cytotoxicity to breast cancer cell line or prostate cancer cell line (about 0.2 ⁇ g/ml of GI 50 ) .
  • GI50 value was under 0.1 ⁇ g /ml in 9 of those 13 cell lines, SK-MEL-5, UACC62, Hs 294T, Hs 695T, SK-MEL-2, RPMI7951, A253 and G361, suggesting that COMR2029 had strong cell growth inhibiting activity.
  • GI50 value in LOX-IMVI was under 0.03 ⁇ g/ml, which meant the strongest cytotoxicity was observed in LOX-IMVI.
  • GI 50 value in lung cancer cell lines NCI-H522 and NCI-H23 was about 0.1 ⁇ g/ml . Resultingly, COMR2029 worked selectively to skin cancer
  • Example 4 the present inventors confirmed that COMR2029 had skin cancer cell line- specific cytotoxicity, and performed experiment for measuring cell cycle to investigate whether COMR2029 affected cell cycle.
  • melanoma cell line LOX-IMVI was cultured in RPMI 1640 medium supplemented with 10%
  • RNAase 1 ⁇ g/ml of RNAase and 400 ⁇ g/ml of PI for 30 minutes
  • the present inventors took advantage of several methods such as agarose gel electrophoresis, quantification of intracellular DNA, caspase-3 activity measurement and the measurement of intracellular calcium change.
  • Concerning diphenylamine method diphenylamine infiltrates in-between DNA and dyes dark blue over the apoptosis region.
  • Caspase-3 is required for the last step of apoptosis, that is, almost every cell needs this enzyme to be progressed to apoptosis. Therefore, when apoptosis is occurring, DAN is seen as ladder type on agarose gel, cells are stained dark blue by diphenylamine, resulting in the high value of optical density and the activity of caspase-3 increases .
  • lysis buffer (10 mM Tris buffer, 1 M EDTA, 0.2% Triton X-100, pH8.0
  • Lysed cells were centrifuged at 15,000 rpm for 20 minutes, resulting in the separation of DNA fragments from the precipitates.
  • the DNA fragments in supernatants were precipitated in the mixture of isoprophyl alcohol and 0.5 M NaCl at -
  • the samples were centrifuged, after which the DNA samples were dried.
  • the dried DNA samples were dissolved in 40 ⁇ l of TE solution (10 mM Tris, 1 mM EDTA, pH7.4) and 20 ⁇ l of loading buffer (15 mM EDTA, 2% SDS, 50% glycerol, 0.5% bromophenol blue, 10 unit RNase) , and then heated at 65°C for 10 minutes, followed by electrophoresis on 1.5% agarose gel containing 1.0 ⁇ g/ml of ethidium bromide at 50 V for 50-60 minutes.
  • TE solution 10 mM Tris, 1 mM EDTA, pH7.4
  • loading buffer 15 mM EDTA, 2% SDS, 50% glycerol, 0.5% bromophenol blue, 10 unit RNase
  • the present inventors analyzed DNA components in cell precipitates and supernatants using diphenylamine (referred as "DPA" hereinafter) method.
  • DPA diphenylamine
  • COMR2029 for the test were 0.03, 0.1, 0.3, 1, and
  • the present inventors After confirming the inhibiting activity of COMR2029 to angiogenesis and cancer cell growth, the present inventors further performed in vi tro invasion assay to investigate metastasis.
  • One of the characteristics of cancer cells unlike normal cells, is invasion and as the invasion increases, the malignancy of cancer cells becomes severe.
  • in vitro invasion assay can be used to provide a prediction index of invasion that affects the formation of cancer tissues.
  • B16 cell line is used in general.
  • the membrane transmission was great in solvent control group that was not treated with COMR2029, so that lots of cells were stained, as seen in FIG. 4.
  • the COMR2029-treated group matrix invasion was inhibited regardless of the COMR2029 concentration, so that the less cells were stained than the control group.
  • the inhibiting activity to membrane transmission was also observed in LOX-IMVI cell line, which was sensitive to COMR2029, even with 10 times as low concentration as in B16(F10) cell line.
  • solvent control group as 100
  • COMR2029-treated group showed 10-30, suggesting that COMR2029 had strong inhibiting activity to tissue invasion (FIG. 5).
  • Example 8 LOX-IMVI anticancer test 1
  • the samples were prepared by adding 0.5% tween 80.
  • the dosage of COMR2029 was determined to be 1 mg/kg.
  • 0.5% tween 80 was used as a solvent control group.
  • Human melanoma cell line LOX-IMVI was used as a cell line. Particularly, adjusted the cell density to 3X10 6 cells/mouse, and implanted hypodermically at the right armpit of mice (0.2 ffll/ ouse) . Samples were administered from the next day.
  • human melanoma cell line LOX- IMVI showed very fast growth, so that it could be detected from the 3 rd day after implantation but the administration of COMR2029 with the dosage of
  • the samples were prepared by adding 0.5% tween 80.
  • the dosage of COMR2029 was determined to be 10 and 30 mg/kg.
  • 0.5% tween 80 was used as a solvent control group and adriamycin (2 mg/kg) was used as a positive control group.
  • Human melanoma cell line LOX-IMVI was used as a cell line. Particularly, adjusted the cell density to 3X10 6 cells/mouse, and implanted hypodermically at the right armpit of mice (0.3 ml/mouse) . Samples were administered from the next day.
  • the second anticancer test resulted in the confirmation of cancer cell growth inhibition by COMR2029.
  • mice of solvent control group and COMR2029-treated group showed no change in weights but mice of positive control group lost about 4 g, suggesting that the adriamycin used in the positive control group had a cytotoxicity (FIG. 8 and FIG. 9) .
  • Example 10 Measurement of immune cell
  • Treated group 1 with samples after 24 hours, for which the final concentration of sample was adjusted to 5 different concentrations by 0.1% DMSO.
  • Treated group 2 with samples right away and then transferred to fresh medium two hours later.
  • the experiment was to investigate the sensitivity of cells to drugs that went through the steps of exposing on drugs, retrieving the drugs and re-exposing on the drugs. As a result, the sensitivity of cells was not affected by the above treatment.
  • Standard material and substrate having fluorescence to MMP-2 were purchased from SIGMA (Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2) . Used a fluorescent spectrophotometer (Perkin-Elmer LS- 50B) for the experiment. Operated the machine using a computer software (Fluorescence Data Manage, Perkin-Elmer) . Set excitation and emission wavelength to 325 nm (slit 5.0 mm) and 420 nm(slit 3.0 mm) respectively, and used 20 mM Tris-HCl, 5 mM CaCl 2 and 0.15 M NaCl (reaction buffer, pH 7.5) for all the experiments.
  • reaction buffer 1 mM in DMSO, which was diluted with reaction buffer in the range of 15.6-250.0 pmoles on microplates, resulting in the making a standard curve .
  • MMP Microx MetalloProteinase
  • MMP-2 is a metastasis related enzyme, whose most sensitive and representative one is MMP-2.
  • the experiment was to measure the MMP-2 activity. Precisely, the substrate of MMP-2 was loaded and a standard curve was made, which was compared with that of experimental group to measure the activity.
  • COMR2029 was treated with the concentration of 0.03, 0.1, 0.3, 1, 3 and 10 uM respectively, the enzyme activity did not change.
  • the MMP-2 activity seemed to be decreased with the concentration of over 3 uM, which was probably because of color quenching. Thus, COMR2029 was confirmed not to inhibit the MMP-2 activity.
  • Example 13 The effect of COMR2029 on the
  • COMR2029 Used human melanoma cell line LOX-IMVI as a cell line and RPMI1640 medium supplemented with 10% FBS as a medium. Adjusted cell density to 5 X 10 6 cells/m*.
  • Pre-treated COMR2029 (30 minutes earlier) . Added 10 ng/ml of TNF- ⁇ and reacted for 2 hours. The concentration of COMR2029 for the treatment was 0.01, 0.1 and 1 ⁇ g/ml respectively.
  • the lysis buffer was composed of 70 mM b-glycerophosphate (pH 7.2), 0.1 mM meta/ortho- vanadate, 2 mM MgCl 2 , 1 mM EGTA, 1 mM DTT (dithiothreitol) , 0.5% triton-X-100 , 0.2 mM PMSF
  • Example 14 The effect of COMR2029 on the
  • EMSA Electrophoretic Mobility Shift Assay
  • binding buffer 100 mM NaCl, 30 mM Hepes, 0.3 mM EDTA, 1.5 mM MgCl 2 , 10% glycerol, 1 mM DTT, 1 mM PMSF, and lg/ml each of aprotinin and leupeptin
  • binding buffer 100 mM NaCl, 30 mM Hepes, 0.3 mM EDTA, 1.5 mM MgCl 2 , 10% glycerol, 1 mM DTT, 1 mM PMSF, and lg/ml each of aprotinin and leupeptin
  • Double stranded DNA oligomer was prepared by synthesizing NF-kB (5-GGGGACTTTCC- 3) (Pierce et al . , 1988) and AP-1 (5-TGACTCA-3)
  • reaction buffer solution by dissolving 10X ATP solution, 10X biotynylated PKC pseudosubstrate solution, 10X activator solution and 10X PKC together, which was pre-treated for 30 minutes. Diluted Y - 3 P-ATP containing solution from the concentration of 10 ⁇ Ci// to 0.2 ⁇ Ci/ £ using de-ionized water, resulting in the preparation of working solution (Table 18) .
  • N represents the sample number
  • PKC reaction Put 20 ⁇ l of PKC reaction mixture in a microcentrifuge tube. Added 5 ⁇ l of sample prepared in the above Example ⁇ 15-1> thereto. In the meantime, added 5 ⁇ l of the same sample to 20 ⁇ l of control mixture without peptide substrate. Added 5 ⁇ l of de-ionized water instead of PKC sample to 20 ⁇ l of PKC reaction mixture, which would be used as a background control. Reacted each mixture at 25 ° C for 15 minutes. Terminated the reaction by adding 10 ⁇ l of stop solution. When the radioactive count of control mixture was higher than that of background control, it could not be used since it reflected that nonspecific phosphorylation of the PKC sample was too high.
  • Terminated PKC reaction by adding 3 ⁇ l of. 50% TCA and 2 ⁇ l of 1% BSA, left each reaction tube on ice for about 10 minutes and centrifuged at 14,000 g for 5 minutes. After centrifugation, transferred 25 ⁇ l of supernatants into new tubes and then added 5 ⁇ l of neutralization solution into each tube, resulting in the complete termination of reaction by neutralizing the sample.
  • reaction sample (counts per min) of the reaction sample, put 4 ⁇ l of reaction sample in the blank sample reservoir of centrifugal ultrafiltration units, which was used as a reference sample. Transferred the remaining reaction sample and reference sample in sample reservoirs to scintillation vials. After adding liquid scintillation cocktail solution into the vials with the required amount, set the channel to 32 P and measured the radioactivity.
  • PKC a kind of serine/threonine kinase, is activated most in the presence of calcium ion and DAG (Diacylglycerol) .
  • PKC is observed during the signal transduction by being induced by hormones, growth factors and neurotransmitters . PKC induces protein phosphorylation, causing the changes in protein activity. In some special cases, the phosphorylation of EGF receptor induced by PKC down-regulates tyrosine kinase activity. As seen in FIG. 12, COMR2029 plays a role in inhibiting
  • COMR2029 was confirmed to inhibit sGC activity as LY83583, which was a structural analogue of COMR2029 and known as a sGC inhibitor, did (FIG. 13) .
  • LOX-IMVI cells were loaded to a 96 well plate (200 ⁇ /well) .
  • COMR2029 was confirmed to inhibit uPA activity slightly, which was corresponding to the result of RT-PCR (FIG. 14).
  • uPA is an enzyme that plays an important role in mobility and invasion.
  • Example 18 Acute toxicity test in rats via oral
  • 6-week old SPF (specific pathogen-free) SD line rats were used in the tests for acute toxicity.
  • COMR2029 was suspended in 0.5% methylcellulose solution and orally administered once to 2 rats per group at the dosage of 1 g/kg/ ml .
  • Death, clinical symptoms, and weight change in rats were observed, hematological tests and biochemical tests of blood were performed, and any abnormal signs in the gastrointestinal organs of chest and abdomen were checked with eyes during autopsy.
  • COMR2029 did not cause any specific clinical symptoms, weight change, or death in rats. No change was observed in hematological tests, biochemical tests of blood, and autopsy. Conclusively, COMR2029 used in this experiment is evaluated to be a safe substance since it dose not cause any toxic change in rats up to the level of 1,000 mg/kg.
  • Applicable Example 1 Ophthalmic diseases such as
  • retinopathy retinopathy, glaucoma, etc.
  • ophthalmic disease induced by angiogenesis are senile macular degeneration, diabetic retinopathy, retinopathy of precocious child, angiogenic glaucoma, etc. Millions of people over the world are losing their sights because of such diseases every year (D'Amato, R.J. and Adamis, A, P. (1995) Ophthalmol . , 102:1261- 1262; Ozaki, H. et al . (1996) Ophthalmi c Res . , 28 (6) :356-60; Ciulla, T.A. et al . (1999) Expert
  • the visual cells and the pigment epithelium on the retina lose their functions and as such degeneration exceeds, senile yellow spot degeneration is developed, leading to senile macular degeneration.
  • the pigment epithelium on the retina peels off or angiogenesis is induced in the choroid which functions to supply blood to the retina, causing hemorrhage underneath nervous membrane of the retina, finally resulting in the loss of sight.
  • And diabetic retinopathy a kind of complication of diabetes, is a disease developed by invasion of capillary vessels produced by angiogenesis on the retina into corporis vitrei, causing a mal-function in circulation of capillary vessels, resulting in the loss of sight. Precisely, as oxygen shortage area on the retina becomes wider, angiogenesis is induced to supply oxygen smoothly. But the newly generated blood vessels are not normal blood vessels, so that they burst often and new fibrous materials are growing around newly generated blood vessels, which pulls the blood vessels to cause hemorrhage, leading to the loss of sight.
  • Retinopathy of precocious child also induces sight trouble.
  • the blood vessels in the retina of a fetus are formed in the last month of pregnancy. Therefore, in precocious child, abnormal blood vessels are developed by angiogenesis, so that scars are formed in the retina, followed by the sight trouble.
  • angiogenic glaucoma when scars are formed around newly generated blood vessels by diabetic retinopathy or other diseases, retina exfoliation follows. And as the newly generated blood vessels spread to the frontal iris, angiogenic glaucoma is developed. As explained hereinbefore, the above diseases are related to angiogenesis. Thus, the compound of the present invention having function of inhibiting angiogenesis can be effectively used for the prevention and the treatment of such diseases.
  • Arthritis is basically caused by the abnormal autoimmune response. As the disease progresses, chronic inflammation in synovial cavity induces angiogenesis and newly generated capillary vessels infiltrate through joints, resulting in the destruction of cartilage (Stupack, D.G., et al .
  • the changes caused by human defense mechanism leads the proliferation of new blood vessels and the formation of osteophytes around the target place. Joint movement is limited by the osteophytes, making the disease worse.
  • the formation of osteophytes by angiogenesis is the major cause of worsening arthritis. Therefore, the compound of the present invention having function of inhibiting angiogenesis can be successfully used for the prevention and the treatment of the mentioned disease .
  • an angiogenesis inhibitor of the present invention containing 6- anilinoquinoline-5, 8-quinone (COMR2029) as an effective ingredient can be effectively used for the treatment of angiogenesis-related diseases such as cancer, rheumatoid arthritis or diabetic retinopathy.

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Abstract

La présente invention concerne un inhibiteur de l'angiogenèse contenant de la 6-anilinoquinoline-5,8-quinone comme ingrédient actif. Cet inhibiteur d'angiogenèse peut être utilisé efficacement pour la prévention ou le traitement de maladies liées à l'angiogenèse, telles que le cancer, la polyarthrite rhumatoïde et la rétinopathie diabétique.
PCT/KR2003/000900 2003-05-06 2003-05-06 Inhibiteur de la pkc, inhibiteur de l'angiogenese contenant de la 6-anilinoquinoline-5,8-quinone comme ingredient actif Ceased WO2004098601A1 (fr)

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PCT/KR2003/000900 WO2004098601A1 (fr) 2003-05-06 2003-05-06 Inhibiteur de la pkc, inhibiteur de l'angiogenese contenant de la 6-anilinoquinoline-5,8-quinone comme ingredient actif
AU2003230309A AU2003230309A1 (en) 2003-05-06 2003-05-06 As a pkc inhibitor, inhibitor for angiogenesis containing 6-anilinoquinoline-5,8-quinone as an effective ingredient

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090318462A1 (en) * 2006-07-21 2009-12-24 The Penn State Research Foundation Protein kinase c zeta inhibition to treat vascular permeability
KR100941986B1 (ko) 2008-06-26 2010-02-11 금호타이어 주식회사 내열 특성이 향상된 타이어 카카스용 고무 조성물
CN108164491A (zh) * 2018-03-05 2018-06-15 湖北大学 具有抗炎作用的5，8-对醌式黄烷类化合物及其提取分离方法和应用

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WO1997021432A1 (fr) * 1995-12-12 1997-06-19 Laboratoire Innothera, S.A. Utilisation de derives de bicycles mono- ou dicetoniques, composes obtenus et leur application comme medicament destine au traitement des inflammations, de la migraine et des etats de chocs
JPH11335354A (ja) * 1998-03-23 1999-12-07 Kyowa Hakko Kogyo Co Ltd ５，８―キノリンジオン誘導体

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US4492704A (en) * 1982-09-30 1985-01-08 Eli Lilly And Company Quinoline quinones and anti-asthmatic use thereof
WO1997021432A1 (fr) * 1995-12-12 1997-06-19 Laboratoire Innothera, S.A. Utilisation de derives de bicycles mono- ou dicetoniques, composes obtenus et leur application comme medicament destine au traitement des inflammations, de la migraine et des etats de chocs
JPH11335354A (ja) * 1998-03-23 1999-12-07 Kyowa Hakko Kogyo Co Ltd ５，８―キノリンジオン誘導体

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CARROLL, T,JR, ET AL.: "Amodiaquine Analogs.Synthesis of 6-[[3-(N,N-di-ethylamino)methyl-4-hydroxy]anilino]-5,8-dimethoxy-2,4-dimethylquinoline and Related Compounds", JOURNAL OF MEDICINAL CHEMISTRY, vol. 17, no. 9, 1974, pages 972 - 977 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090318462A1 (en) * 2006-07-21 2009-12-24 The Penn State Research Foundation Protein kinase c zeta inhibition to treat vascular permeability
US8211893B2 (en) * 2006-07-21 2012-07-03 The Penn State Research Foundation Protein kinase C zeta inhibition to treat diabetic retinopathy
KR100941986B1 (ko) 2008-06-26 2010-02-11 금호타이어 주식회사 내열 특성이 향상된 타이어 카카스용 고무 조성물
CN108164491A (zh) * 2018-03-05 2018-06-15 湖北大学 具有抗炎作用的5，8-对醌式黄烷类化合物及其提取分离方法和应用

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