WO2002102814A1 - Apoptosis inducer - Google Patents
Apoptosis inducer Download PDFInfo
- Publication number
- WO2002102814A1 WO2002102814A1 PCT/KR2002/001123 KR0201123W WO02102814A1 WO 2002102814 A1 WO2002102814 A1 WO 2002102814A1 KR 0201123 W KR0201123 W KR 0201123W WO 02102814 A1 WO02102814 A1 WO 02102814A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- apoptosis
- cells
- present
- cell
- leukemia
- Prior art date
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- 239000000411 inducer Substances 0.000 title claims abstract description 19
- 230000006907 apoptotic process Effects 0.000 title claims description 69
- GOLCXWYRSKYTSP-UHFFFAOYSA-N Arsenious Acid Chemical compound O1[As]2O[As]1O2 GOLCXWYRSKYTSP-UHFFFAOYSA-N 0.000 claims abstract description 47
- 229910017013 As4O6 Inorganic materials 0.000 claims abstract description 28
- 208000032839 leukemia Diseases 0.000 claims abstract description 14
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 claims abstract description 10
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 3
- 201000005787 hematologic cancer Diseases 0.000 claims description 9
- 230000006698 induction Effects 0.000 claims description 8
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 6
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- 238000011282 treatment Methods 0.000 abstract description 12
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- HRNLUBSXIHFDHP-UHFFFAOYSA-N N-(2-aminophenyl)-4-[[[4-(3-pyridinyl)-2-pyrimidinyl]amino]methyl]benzamide Chemical compound NC1=CC=CC=C1NC(=O)C(C=C1)=CC=C1CNC1=NC=CC(C=2C=NC=CC=2)=N1 HRNLUBSXIHFDHP-UHFFFAOYSA-N 0.000 description 1
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- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 1
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- 230000005522 programmed cell death Effects 0.000 description 1
- 108010006693 promyelocytic leukemia-retinoic acid receptor alpha fusion oncoprotein Proteins 0.000 description 1
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 1
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- UYVQXFOBKTWZCW-UHFFFAOYSA-N tetraarsenic Chemical compound [As]12[As]3[As]1[As]32 UYVQXFOBKTWZCW-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/66—Arsenic compounds
- C07F9/68—Arsenic compounds without As—C bonds
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01G—COMPOUNDS CONTAINING METALS NOT COVERED BY SUBCLASSES C01D OR C01F
- C01G28/00—Compounds of arsenic
- C01G28/005—Oxides; Hydroxides; Oxyacids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/36—Arsenic; Compounds thereof
Definitions
- the present invention generally relates to new uses and pharmaceutical compositions of solid As 4 0 6 as apoptosis inducers and anti-cancer drugs. More specifically, a novel use of solid As 4 0 6 known as "Chunjisan” is disclosed which induces active cell death by apoptosis to treat blood cancer such as leukemia.
- Anti-cancer drugs have been developed before the study of apoptosis. And it is recently suggested that an anti-cancer drug induces apoptosis of cancer cells.
- necrosis is a passive cell degradation process by stimuli such as burn, ischemia and poisonous substance and generates inflammation by releasing cytosolic materials caused by swelling and lysis of cell.
- apoptosis is a active cell suicide mechanism which results in cell shrinkage, nuclear condensation and DNA fragmentation and does not generate inflammatory response in vivo because apoptotic body eliminates these materials by phagocytosis.
- the apoptosis is essential for development;, differentiation and homeostasis of multicellular organism.
- arsenic compound had been used as anti-cancer drugs in Oriental medicine. In 1970s, it was confirmed that principal component of arsenical compounds is As203. Recently, it is shown that injection of arsenic trioxide with clinically applicable density (0.5-2 ⁇ mol/L) had an excellent effect in treating acute promyelocytic leukemia resistive to all-trans retinoic acid (Gallagher, R. E. Arsenic: New life for an old potion. N. Engl. J. Med., 339: 1389-1391, 1998, Shen, S. X., Chen, G. Q, Ni, J. PL, Li, X. S., Xiong, S. M., Qui, Q.
- arsenic compound is tried to apply in other blood cancers and solid cancers.
- Tetraarsenic hexaoxide(solid As 4 O 6 ) has been used as basic material to produce other arsenic compounds like As203, for insecticides, decolorizers in glass manufacture or preservatives of leathery.
- solid As 4 0 6 has been known as toxic substance to induce cancers, and chemists focus on only its chemical structure study(Becker K. A., Plieth K., and Stranski I. N. The polymorphic modifications of arsenic trioxide. Prog. Inorg. Chem. 4, 1-72 (1962), Pupp C, Lao R. C, Murray J. J., pottie R.
- the present inventor identified anti-cancer effect of solid As 4 0 6 separated and purified from natural arsenic tri oxides and filed a patent application in
- arsenic trioxide may cause serious gastrointestinal and hepatic side effects (Shen ZX, Chen GQ, Ni JH, Li XS, Xiong SM, Qiu QY, Zhu J, Tang W, Sun GL, Yang KQ, Chen Y, Zhou L, Fang ZW, Wagn YT, Ma J, Zhang P, Zhang TD, Chen SJ, Chen. Z, Wang ZY Use of arsenic trioxide(As2O3) in the treatment of acute promyelocytic leukemia (APL): JJ . Clinical efficacy and pharmacokinetics in relapsed patients.
- APL acute promyelocytic leukemia
- tetraarsenic hexoxide (As 4 O 6 ) does not cause gastrointestinal and hepatic side effects but minute histologic change in kidneys when about lOOmg/kg of As 4 O 6 is oral-administered to about 200g Sprague- Dawley rat.
- the present invention is disclosed that in case of oral- administration of lOmg/kg and lmg/kg of As 4 O 6 , tetraarsenic hexaoxide causes no gastrointestinal and hepatic side effects.
- the present invention has an object to provide an apoptosis inducer consisting of As 4 0 6 compound for treating diverse blood cancers including leukemia and solid tumors.
- the present inventor identifies that tetraarsenic hexaoxide As 4 0 6 shown in Fig. 1 induces apoptosis in relatively low concentration to U937 leukemia cell line.
- the present inventors identify that tetraarsenic hexaoxide As 4 0 6 induces release of Cytochrome c from mitochondrial membrane and activates caspase, thereby inducing apoptosis.
- the apoptosis mechanism is completely inhibited when generation of reactive oxygen, particularly hydrogen peroxide, is suppressed.
- As 4 0 6 of the present invention may induce apoptosis by causing generation of reactive oxygen including generation of excessive hydrogen peroxide from cancer cells, thereby treating diverse blood cancers including leukemia.
- the U937 leukemia cell is a cell line resistant to the conventional As2O3 with effect of treating leukemia as well as a cell line that do not express fusion protein PML-RAR ⁇ .
- As 4 0 6 of the present invention is effective for treatment of leukemia patients as well as PML patients.
- a method of manufacturing the As 4 0 6 of the present invention known as "Chunjisan" is disclosed in Korean Patent Application No. 1998-16486.
- the As 4 0 6 of the present invention can be manufactured through multi-step heating natural arsenic new stone and reagent arsenic.
- a detail method of manufactured As 4 0 6 of the present invention will be omitted.
- the As 4 0 6 of the present invention manufactured by other methods besides the above-described method may be used if it has the structure of Fig. 1.
- the dose of As 4 0 6 of the present invention may be changed depending on kinds of disease, age, gender, health condition of patients when applied to the human body.
- the As 4 0 6 of the present invention may be singly used or with other anti- cancer drug and complex preparations.
- the As 4 0 6 of the present invention may also be made into various oral or non-oral preparations such as powders, tablets and liquors by mixing various pharmaceutically acceptable additives such as diluent, disintegrator, air freshener and glidant.
- the As 4 0 6 of the present invention may be applied to diverse apoptosis related disease, such as blood cancers including leukemia and solid tumors.
- the As 4 O 6 of the present invention may be used with other preparations.
- Fig. 1 is a structural formula of apoptosis inducer of the present invention.
- Figs. 2a and 2b are graphs showing the effects of apoptosis inducer measured by flow cytometry of the present invention.
- Fig. 2c is a picture showing DNA fragmentation in relation to the effects of apoptosis inducer of the present invention.
- Fig. 3 is a graph showing mitochondria membrane potential depletion in relation to the effects of apoptosis inducer of the present invention.
- Fig. 4 is a Western blotting picture showing Cytochrome c release in cytoplasm in relation to the effects of apoptosis inducer of the present invention.
- Figs. 5a and 5b are graphs and pictures showing catapase-3 activation measured by flow cytometry in relation to the effects of apoptosis inducer of the present invention.
- Fig. 5c is a Western blotting analysis picture.
- Fig. 6 is a graph showing dependency of catapase-3 activation measured by flow cytometry in relation to the effects of apoptosis inducer of the present invention.
- Figs. 7a and 7b are graphs showing apoptosis inhibition effects by antioxidant measured by flow cytometry in relation to the effects of apoptosis inducer of the present invention.
- Fig. 7c is a graph showing experimental results of identifying hydrogen peroxide generation in relation to the effects of apoptosis inducer of the present invention.
- Fig. 7d is a graph showing blocking apoptosis by catalase measured by flow cytometry in accordance with the present invention.
- Example 1 Experiment of measuring apoptosis
- the experiment was performed for identifying effects inducing apoptosis of As 4 0 6 accordmg to the present invention.
- Reagents and cells used in examples were obtained according to the following methods.
- Tetraarsenic hexaoxide (As 4 0 6 ) provided from Chunjisan Co. (Seoul, Korea) was dissolved in NaOH solution (IM), diluted and. then used in the experiment.
- Z-DEVD-FMK and N-acetyl-N-cysteine (NAC) provided from Calbiochem (SanDiego, CA, USA), catalase from Sigma Co., and caspase-3 and cytochrome c antibody from Pharmingen (San Diego, CA, USA) were used in Western blotting analysis.
- U937 cell line myelogenous leukemia cell, acquired from The Korean Cell Line Bank (Cancer Research Center, Seoul National Medical College, Seoul, - Korea), were successively cultured in culture medium of 5% CO2 at 37°C humidified with RPMI-1640 culture (GIBCO) supplemented with 10% fetal calf serum (GIBCO, Grand Island, NY, USA) and penicillin /streptomycin, and then used in the experiment.
- the present inventors add constant concentration of As 4 O 6 to U937 cell line and incubate in RPMI-1640 medium supplemented with 10% fetal calf serum (GIBCO, Grand Island, NY, USA) and penicillin/streptomycin at 5% CO2 and 37°C in the examples below.
- this process will be referred to "treatment of As 4 O 6 ".
- Example 2 Measurement of mitochondria membrane potential depletion
- Example 3 Measurement of cytochrome c release in cytoplasm
- the cell pellets were dissolved in solution including 220mM mannitol, 70mM sucrose, 50mM Pipes -KOH (pH 7.4), 50mM KC1, 5mM EGTA, 2mM MgC12, lrnM EDTA, lmM dithiothreitol and various protease inhibitors, the dissolved cell pellets were homogenized and centrifuged, supernatant of the solution was obtained to perform a Western blotting analysis.
- solution including 220mM mannitol, 70mM sucrose, 50mM Pipes -KOH (pH 7.4), 50mM KC1, 5mM EGTA, 2mM MgC12, lrnM EDTA, lmM dithiothreitol and various protease inhibitors
- a secondary antibody conjugated with horse-radish peroxidase was diluted in blocking buffer solution at a ratio of 1 : 3,000 and then carried out reaction for 30 minutes. After reaction, the secondary antibody was three times washed using PBST for 20 minutes, and then stained using ECL system (Amershame, Arlington Heights, IL, USA). The result picture was shown in Fig. 4.
- FAM-DEVD-FMK, Z-DEVD-FMK derivative emitting fluorescence by covalent bond with activated catapase-3 was added in cell culture medium treated with As 4 0 6 and fluorescence was detected.
- CaspaTagTM catapase activation kit (Intergen, Purchase, NY, USA) was used as a reagent for measuring activation of catapase-3. According to protocol of manufacture, 1 X 105 cells were treated with 2.5 ⁇ M As 4 0 6 for 18 hours, and lO ⁇ 1 FAM-DEVD-FMK was added in culture solution and carried out reaction in culture medium for 1 hour. After the culture medium was washed with washing solution, the resultant material was analyzed using flow cytometry. '
- Fig. 5a is a graph that cells treated with As 4 0 6 and cells in untreated control group were labeled with FAM-DEVD-FMK using flow cytometry.
- cells which catapase-3 was not activated were shown in a first section (Ml) of the X axis while cells which catapase-3 was activated were shown in a second section(M2).
- Ml first section
- M2 cells which catapase-3 was activated
- Fig. 5a when cells treated with 2.5 ⁇ M As 4 O 6 for 18 hours, 19%) of the cells were stained in FAM-DEVD-FMK. From this result, it was shown that apoptosis induced by As 4 0 6 in U937 cells was related to activation of catapase-3.
- Example 3 The same procedure of Example 3 was performed on cell samples treated with 0, 0.5, 1, 2.5 and 5 ⁇ M As 4 0 6 for 18 hours using catapase-3 activation detection kit. The resultant picture of Western blotting analysis was shown in Fig. 5c.
- Example 3 was performed on the above three experimental groups to measure mitochondria membrane potential depletion. The result was shown in Fig. 6.
- Example 6 Experiment for identifying relevancy with reactive oxygen 1) Inhibition of apoptosis by antioxidant In order to identify whether mitochondria membrane potential depletion during the process of inducing apoptosis by As 4 0 6 in U937 cells is related to generation of reactive oxygen species in cells. N-acetyl-L-cysteine (NAC), antioxidant, was pretreated in the cells and As 4 0 6 induced apoptosis was observed. After U937 cells (1 X 105) were pretreated with NAC 30 minutes before were treated with 2.5 ⁇ M As 4 0 6 , the same procedure was performed staining with annexin-FITC V/PI to measure the number of dead cells using flow cytometry. The procedure was three times repeated, and then the death ratio of cells was shown in Fig. 7a, wherein the number of dead cells in control group was 1.
- NAC N-acetyl-L-cysteine
- Example 2 The same procedure of Example 2 was performed on the treated cells to measure mitochondria membrane potential depletion. The degree of mitochondria membrane potential depletion was shown in Fig. 7b.
- U 937 cells were pretreated with catalase that catalyzes the decomposition of hydrogen peroxide to oxygen and water and As 4 0 6 induced apoptosis was investigated.
- U 937 cells (1 X 105) were pretreated with catalase, 30 minutes before
- Example 1-2 The same procedure of Example 1-2 was performed on the cells staining with annexin- FITC V/PI to measure the number of dead cells using flow cytometry. The experiment was three times repeated, and then relative cell death ratio was shown in Fig. 7d, wherein the number of dead cells in control group was 1.
- Fig. 7d it was shown that apoptosis induced by As 4 O 6 was completely inhibited in cells pretreated with 500U/ml catalase.
- the tetraarsenic hexaoxide of the present invention may be used for induction apoptosis mechanism to prevent and treat many apoptosis related diseases.
- the tetraarsenic hexaoxide of the present invention is useful for blood cancers including leukemia as well as various solid tumors because it may effectively induce apoptosis even at its low concentration in U937, arsenic trioxide resistant leukemia cell.
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| KR1020010034087A KR20020095835A (ko) | 2001-06-16 | 2001-06-16 | 아폽토시스 유도제 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2374463A1 (en) * | 2008-12-16 | 2011-10-12 | Chonjisan Co., Ltd | Composition for improving radiotherapy for cancer |
| AU2017360407B2 (en) * | 2016-11-18 | 2020-11-26 | Chemas Co., Ltd. | Pharmaceutical composition for preventing or treating brain cancer including crystal polymorph of tetraarsenic hexoxide, and method for preparing same |
| US11154530B2 (en) | 2016-12-05 | 2021-10-26 | Chemas Co., Ltd. | Pharmaceutical composition for treating liver cancer, comprising tetraarsenic hexoxide crystalline polymorph |
| US11191779B2 (en) | 2016-12-09 | 2021-12-07 | Chemas Co., Ltd. | Pharmaceutical composition for preventing or treating cancer, comprising tetraarsenic hexoxide crystalline polymorph |
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| KR101119587B1 (ko) * | 2011-03-10 | 2012-04-17 | 배일주 | 육산화사비소를 함유하는 용출률 및 안정성이 개선된 경구투여형 약제학적 제제 |
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Non-Patent Citations (3)
| Title |
|---|
| CHEN ZHU ET AL.: "Acute promyelocytic leukemia: Cellular and molecular basis of differentiation and apoptosis", PHARMACOLOGY & THERAPEUTICS, vol. 76, no. 1-3, 1997, pages 141 - 149 * |
| KAZUKI IWAMA ET AL.: "Apoptosis induced by arsenic trioxide in leukemia U937 cells is dependent on activation of p38, inactivation of ERK and the Ca2+-dependent production of superoxide", INTERNATIONAL JOURNAL OF CANCER, vol. 92, no. 4, 15 March 2001 (2001-03-15), pages 518 - 526 * |
| SHAO WENLIN ET AL.: "Arsenic trioxide as an inducer of apoptosis and loss of MPL/RAR alpha protein in acute promyelocytic leukemia cells", JOURNAL OF THE NATIONAL CANCER INSTITUTE, vol. 90, no. 2, 1998, pages 124 - 133 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2374463A1 (en) * | 2008-12-16 | 2011-10-12 | Chonjisan Co., Ltd | Composition for improving radiotherapy for cancer |
| EP2374463A4 (en) * | 2008-12-16 | 2012-11-07 | Chonjisan Co Ltd | COMPOSITION FOR IMPROVING RADIATION THERAPY FOR CANCER |
| AU2017360407B2 (en) * | 2016-11-18 | 2020-11-26 | Chemas Co., Ltd. | Pharmaceutical composition for preventing or treating brain cancer including crystal polymorph of tetraarsenic hexoxide, and method for preparing same |
| US11154530B2 (en) | 2016-12-05 | 2021-10-26 | Chemas Co., Ltd. | Pharmaceutical composition for treating liver cancer, comprising tetraarsenic hexoxide crystalline polymorph |
| US11191779B2 (en) | 2016-12-09 | 2021-12-07 | Chemas Co., Ltd. | Pharmaceutical composition for preventing or treating cancer, comprising tetraarsenic hexoxide crystalline polymorph |
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| KR20020095835A (ko) | 2002-12-28 |
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