WO2002062349A1 - Procede et composition pour inhiber l'incidence et la proliferation des cellules cancereuses du systeme nerveux et du cerveau - Google Patents

Procede et composition pour inhiber l'incidence et la proliferation des cellules cancereuses du systeme nerveux et du cerveau Download PDF

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Publication number
WO2002062349A1
WO2002062349A1 PCT/US2002/002828 US0202828W WO02062349A1 WO 2002062349 A1 WO2002062349 A1 WO 2002062349A1 US 0202828 W US0202828 W US 0202828W WO 02062349 A1 WO02062349 A1 WO 02062349A1
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WO
WIPO (PCT)
Prior art keywords
cancer cells
cells
analogues
nervous system
brain
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Application number
PCT/US2002/002828
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English (en)
Inventor
Tom Slaga
Addanki Kumar
William Alworth
Original Assignee
Oncology Sciences Corporation
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
Priority claimed from US09/777,151 external-priority patent/US20020068724A1/en
Priority claimed from US09/777,559 external-priority patent/US20030027803A1/en
Priority claimed from US09/780,269 external-priority patent/US6518261B2/en
Application filed by Oncology Sciences Corporation filed Critical Oncology Sciences Corporation
Publication of WO2002062349A1 publication Critical patent/WO2002062349A1/fr

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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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
    • 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
    • A61K31/075—Ethers or acetals
    • A61K31/085—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
    • A61K31/567—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in position 17 alpha, e.g. mestranol, norethandrolone

Definitions

  • TITLE METHOD AND COMPOSITION FOR INHIBITING THE
  • the present invention relates to non-surgical intervention of brain and nervous system cancer, as well as prophylactic use of herein identified compounds in the prevention of brain and nervous system cancer.
  • NS cancer Metastatic and primary brain and nervous system
  • Brain and NS cancer can arise in various forms in the diverse structures of the brain and nervous system.
  • the most prevalent forms of the disease are: primitive neuroectodermal tumors/medulloblastoma (ages 0-9), astrocytoma (ages 10-14), pilocytic astrocytoma (ages 15-19), pituitary tumors (ages 20-34), meningiomas (ages 35-44), glioblastoma (ages 45-74), and meningioma (ages 75 and older).
  • the end result of the disease is death or not, the disease can have profound and catastrophic effects on individuals and their ability to perceive and function in the world.
  • the disease often robs people of their intelligence, memory, consciousness-, emotions, and/or basic motor functions.
  • the disease leaves people limited or unable to perceive the world around them, unable to perform or remember simple tasks, otherwise grossly impaired, and/or dead.
  • Radiotherapy which often attempts to deliver highly destructive doses of ionizing radiation through the normal tissues of the body in an attempt to preferentially kill highly specific and often imperfectly defined areas of cancerous tissue, can have serious and significant side effects due to the destruction of normal nervous system or other tissues of the body.
  • Chemotherapy which attempts to preferentially kill cancerous cells instead of normal cells through the diverse administration of chemical agents or drugs to the tissues of the body, is limited in efficacy by the chemical agents
  • Angiogenesis refers to the process by which new blood vessels are formed in the body. Without a dedicated blood supply, solid tumors have only limited growth potential - perhaps 2 mm in diameter maximum. However, angiogenesis often occurs in cancerous tissues and tumors, thus enabling solid tumors to sequester greater amounts of nutrients from the body and allowing them to proliferate rapidly, even spreading to other parts of the body. Angiogenesis is a critical means by which solid tumors grow rapidly and metastasize, hastening the process of death or disfigurement.
  • a novel approach for the treatment of brain and nervous system cancer would be the development of pharmacological agents that have dual roles as anti-angiogenic as well as pro-apoptotic agents.
  • Such an agent will have the ability to target both components of a cancer: kill the tumor cell by induction of apoptosis and cut off the blood supply to the tumor cell so that it will not grow.
  • 2-ME 2- methoxyoestradiol
  • 2-ME is an endogenous non-toxic metabolic byproduct of estrogens that is present in human urine and blood.
  • a potential role for 2-ME as a chemopreventive agent has been reported in the mammary and pancreatic models.
  • 2-ME has also been shown to inhibit endothelial cell proliferation implicating its potential role in angiogenesis.
  • apoptosis has been implicated as a mechanism for 2-ME's cytostatic and anti-angiogenic effect.
  • 2-ME is an anti-tumorigenic agent with a significant therapeutic advantage since it can preferentially inhibit actively proliferating cells (characteristic of tumor cells) without affecting the growth of normal cycling cells. Additionally, 2-ME appears to also inhibit the formation of new blood vessels. To the best of our knowledge, this is the first compound that targets two components of cancer: the tumor cells and their blood supply. From these facts, the present inventors postulated that 2-ME may aid people with brain and/or nervous system tumors, either by shrinking the tumors or by slowing their spread (metastasis) or by cutting the blood flow to the tumor (angiogenesis). The work herein demonstrates that 2-ME is a chemical compound with a significant role as an antitumorogenic agent with broad efficacy in brain and nervous system cancer.
  • analogues of 2- ME may aid people with brain and/or nervous system tumors, either by shrinking the tumors or by slowing their spread (metastasis) or by cutting the blood flow to the tumor (angiogenesis).
  • analogues of 2-ME are chemical compound with a significant role as an antitumorogenic agent with broad efficacy in brain and nervous system cancer.
  • the present invention provides both a composition and method for inhibiting the proliferation of brain and nervous system cancer cells.
  • the composition is, and the method is based on the use of a composition consisting (among active ingredients) substantially of 2-methoxyestradiol and/or a number of analogues thereof.
  • the present inventors have demonstrated beyond serious doubt that these compounds have a pronounced effect in inhibiting the proliferation of cancerous brain and nervous system cells and, therefore, provide a critically needed stepping stone for advancing toward meaningful treatment of brain and nervous system cancer.
  • 2-ME 2-methoxyestradiol
  • DAOY medulloblastoma
  • T98-G and U97-MG astrocytoma
  • U87 non-tumorigenic cell line U87 for the same purposes.
  • the cell lines DAOY, U87, and T98-G were obtained from Dr. Victor Levin at The University of Texas M.D. Anderson Cancer Center in Houston, Texas.
  • FIGURE 1 (A & B . Cell Growth
  • the assay is based on the principle that actively growing cells generate reducing equivalents such as NADH that is necessary for the cells to reduce the tetrazolium compound to a formazan product. This was detected by measuring the absorbance at 570 nm using Molecular Devices SpectraMaxPlus plate reader. An increase in the conversion of the MTS tetrazolium compound to the colored formazan product indicated by an increased absorbance provides a relative measure of viable cell number, while a decrease provides a relative measure of cell death.
  • the present inventors examined the effect of 2-ME or 16-episteriol on cell proliferation of DAOY cells using the CellTiter96 Aqueous One solution assay as described earlier.
  • 16-ES 16-episteriol
  • epiestriol is an inactive analog of 2-ME that lacks the methoxy group at the second
  • FIGURE 2 Morphological Response
  • FIGURE 3 Flow Cvtometrv
  • FIGURE 4 Apoptosis/Activation Of Caspase-3
  • Figure 4A Induction of apoptosis by 2-ME was detected using the ApoTarget Quick Apoptotic DNA ladder detection kit from BioSource International, Inc. (Camarillo, CA). Genomic DNA was isolated from the cells treated with 3 ⁇ M 2-ME for 24 hours, following the manufacturer's directions. DNA concentration was estimated by measuring the absorbance at 260 nm and equal amounts of DNA were loaded onto a 1% agarose gel.
  • Apoptosis is an active, genetically controlled process by which cells self-destruct and is accompanied by characteristic morphological and biochemical changes. (7)
  • One of the earliest events during this process is the activation of a calcium-dependent endonuclease associated with nuclear DNA fragmentation.
  • chromosomal DNA is degraded primarily into large DNA fragments followed by subsequent formation of smaller oligonucleosomal fragments resulting in the
  • Genomic DNA was prepared from DAOY cells following treatment with 2-ME using the ApoTarget DNA ladder detection kit according to the manufacturer's protocol.
  • Figure 4B To corroborate the findings with regards to apoptosis, the present inventors also utilized the CaspACE Assay System (Promega Corporation, Madison, WI) to measure the DEVDase activity. Increase in the activity of DEVDase (caspase 3) is an early regulatory event in the induction of apoptosis.
  • the caspase activity system measures the release of p-nitroaniline (hereinafter "p-NA”), a yellow colored product from the substrate Ac-DEVD-pNA colorimetrically.
  • p-NA p-nitroaniline
  • caspases Activation of a series of cytosolic proteases called caspases has been shown to be involved in the induction of apoptosis by a wide variety of agents and extracellular signals resulting in the cleavage of various cellular protein substrates leading to impairment of tissue homeostasis and ultimate destruction of the cell. (10) Among these proteases, caspase-3 has been implicated as a key protease that is activated during the early stages of apoptosis and is detected only in cells undergoing apoptosis. As shown in Figure 4B, no caspase activity was detected in the extracts prepared from
  • FIGURE 5 p53's Involvement
  • cell extracts were prepared by lysing the cells in a buffer
  • Equal amounts of extracts were fractioned on a 10% SDS-polyacrylamide gel.
  • the tumor suppressor protein p53 is a key regulator of cell cycle check
  • p53 function of p53 is mediated by accumulation of wild-type p53 in response to extracellular signals, with the sequential induction of either cell cycle arrest or
  • tumor cells with a selective growth advantage, compared to normal tissues.
  • apoptosis following 2-ME treatment may not be mediated through p53.
  • 2-ME treatment may not be mediated through p53.
  • 2-ME can be used as a potential therapeutic agent for the treatment of brain and nervous system cancer with numerous advantages: (i) it is a natural endogenous estrogen metabolite that reaches micromolar levels during pregnancy, indicating it's non-toxic nature; (ii) it has a dual advantage of being able to inhibit cell proliferation and formation of new blood vessels necessary for tumor cell proliferation and metabolism. The latter property is especially beneficial in the treatment of brain tumors, as one of the limitations of treating brain tumors is the blood brain barrier.
  • the sensitivity of primary or normal cells to 2-ME is not know, the differential sensitivity of 2-ME towards the different cell lines used in this study suggests that it may have no effect on normal cells. Unraveling the mechanisms of 2-ME induced apoptosis may lead to more effective medical therapy for brain tumors, reduction in mortality and morbidity following conventional therapy.
  • analogues of 2-ME to be prepared as described below are designed (1) to determine which components of the 2-ME molecule in addition to the 2-methoxy group are required for the observed chemopreventive effects and (2) to determine if growth- inhibitory 2-ME analogues can be created that are effective in the treatment of brain and nervous system cancer.
  • the initial compounds to be synthesized will be 2 alkoxy substituted analogues of estrone shown in figure 6. These compounds will then be converted into the 2-ME analogues as shown in figure 3 (analogues 19-21, 23-25, and 27-29).
  • Figure 6 illustrates how the A ring of the E 2 steroidal nucleus will be modified to generate 2-alkoxy substituted analogues of estrone (analogues 8-10) and a 2-ethyl substituted estrone analogue (analogue 14).
  • the key reactions in this figure are the synthesis of compound 2, 2,4-diiodoestrone, and its conversion to compound 3, the 2- iodoestrone derivative.
  • the iodination and diodination of the estrone starting material (analogue 1) will be carried out as described by Ikegawa et al in their synthesis of catecholic equilin and equilin derivatives.
  • the intermediate 4 will be coupled with trimethylsilylacetylene in 9:1 CH 3 CN/H 2 0 catalyzed with Pd(AcO) 2 /PPh 3 /CuI.
  • the present inventors have carried out a model
  • analogues prepared as outlined in figures 6 and 7 above will be converted into (i) 2- methoxyestrone and its analogues and (ii) 2, 3 -methylenedioxy estrone analogues modified at position C-17.
  • the preparation of these structures will not only allow us
  • 17-ethynyl-2-ME derivative, 23 will decrease the rate of metabolism and deactivation of 2-ME and its analogues. As outlined in figures 8 and 9 below, the present
  • the 2-ethynyl intermediate shown in figure 6 (analogue 12) will also be converted into 2-ethynylestrone and 2- ethynylestradiol for testing.
  • the 2-ethynylestrone derivative 11 shown in figure 6 will also be converted into 2- ethynylestrone and 2-ethynylestradiol as shown in figure 7 for the other intermediates. This will generate two additional 2-ME analogues for biological testing.
  • derivative of 2-ME may have a longer effective half-life both in vitro and in vivo.
  • p73 is a human p-53 related protein that can induce apoptosis. Nature. 389:191-194.

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  • 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)
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Abstract

L'invention porte sur une composition et sur un procédé visant à inhiber la prolifération des cellules cancéreuses, notamment du système nerveux et du cerveau. La composition et le procédé sont basés sur l'utilisation d'une composition comprenant (parmi des ingrédients actifs) 2-méthoxyestradiol et/ou d'un nombre d'analogues de ceux-ci. Les inventeurs révèlent enfin que ces composés s'avèrent avoir un effet prononcé à inhiber la prolifération des cellules cancéreuses et, par conséquent, sont le point de départ tant attendu pour faire avancer de manière significative les recherches dans le traitement du cancer du cerveau et du système nerveux.
PCT/US2002/002828 2001-02-05 2002-02-01 Procede et composition pour inhiber l'incidence et la proliferation des cellules cancereuses du systeme nerveux et du cerveau WO2002062349A1 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US09/777,151 US20020068724A1 (en) 2000-03-17 2001-02-05 Method and composition of novel compounds for the therapy and targeting of the primary modalities of cancer cell proliferation and homeostasis
US09/777,151 2001-02-05
US09/777,559 US20030027803A1 (en) 2000-03-17 2001-02-06 Method and composition for inhibiting the incidence and proliferation of nervous system and brain cancer cells
US09/777,559 2001-02-06
US09/780,269 2001-02-09
US09/780,269 US6518261B2 (en) 2000-03-17 2001-02-09 Use of eugenol in combination with other chemopreventative agents as prophylaxis for cancers

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WO2002062349A1 true WO2002062349A1 (fr) 2002-08-15

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PCT/US2002/002828 WO2002062349A1 (fr) 2001-02-05 2002-02-01 Procede et composition pour inhiber l'incidence et la proliferation des cellules cancereuses du systeme nerveux et du cerveau
PCT/US2002/002826 WO2002062348A1 (fr) 2001-02-05 2002-02-01 Eugenol, eventuellement combine a 2-methoxyestradiol, utile comme agent de chimioprevention des cancers

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PCT/US2002/002826 WO2002062348A1 (fr) 2001-02-05 2002-02-01 Eugenol, eventuellement combine a 2-methoxyestradiol, utile comme agent de chimioprevention des cancers

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5958892A (en) * 1996-07-30 1999-09-28 Board Of Regents, The University Of Texas System 2-methoxyestradiol-induced apoptosis in cancer cells

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6136992A (en) * 1997-03-13 2000-10-24 The United States Of America As Represented By The Department Of Health And Human Services 2-alkoxy estradiols and derivatives thereof
AU1289899A (en) * 1997-10-31 1999-05-24 Arch Development Corporation Methods and compositions for regulation of 5-alpha reductase activity

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5958892A (en) * 1996-07-30 1999-09-28 Board Of Regents, The University Of Texas System 2-methoxyestradiol-induced apoptosis in cancer cells

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE HCAPLUS [online] (COLUMBUS, OH); BRUEGGEMEIER ET AL.: "Catechol estrogen analogs as probes of estrogen carcinogenesis", XP002950373, accession no. STN Database accession no. 1996:435915 *
HORM. CARCINOG. II. PROC. INT. SYMP. 2ND, 1996, pages 480 - 483 *

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