WO1998034583A2 - Activite anti-angiogenique amelioree des derives constamment charges d'hormones steroidiennes - Google Patents

Activite anti-angiogenique amelioree des derives constamment charges d'hormones steroidiennes Download PDF

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WO1998034583A2
WO1998034583A2 PCT/US1998/002176 US9802176W WO9834583A2 WO 1998034583 A2 WO1998034583 A2 WO 1998034583A2 US 9802176 W US9802176 W US 9802176W WO 9834583 A2 WO9834583 A2 WO 9834583A2
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group
carbon atoms
alkyl
radical selected
aryl
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PCT/US1998/002176
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WO1998034583A3 (fr
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Anat Biegon
Marcus E. Brewster
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Pharmos Corporation
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Priority claimed from IL12018497A external-priority patent/IL120184A/xx
Priority claimed from US08/833,074 external-priority patent/US6083990A/en
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Priority to EP98903921A priority Critical patent/EP0981337A4/fr
Priority to CA002280181A priority patent/CA2280181A1/fr
Priority to JP53487698A priority patent/JP2001511182A/ja
Priority to AU60562/98A priority patent/AU746007B2/en
Publication of WO1998034583A2 publication Critical patent/WO1998034583A2/fr
Publication of WO1998034583A3 publication Critical patent/WO1998034583A3/fr

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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
    • A61K31/13—Amines
    • A61K31/14—Quaternary ammonium compounds, e.g. edrophonium, choline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/24—Drugs for disorders of the endocrine system of the sex hormones
    • A61P5/32—Antioestrogens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • This invention relates to pharmaceutical compositions that are useful for the treatment or prevention of pathological angiogenesis or conditions requiring prevention of angiogenesis. More particularly, this invention relates to the use of quaternary derivatives of steroid agonists or antagonists having improved anti-angiogenic properties.
  • Angiogenesis is a complex process in which capillary blood vessels grow in an ordered sequence of events (Folkman and Klagsbrun, Science 235, 442-447, 1987; Folkman and Shing, J. Biol. Chem. 267, 10931-10934, 1992) .
  • a substantial body of evidence supports the hypothesis that tumor angiogenesis is fundamental for the growth and metastasis of solid tumors (Folkman and Klagsbrun ibid . , 1987; eidner et al . Amer. J. Pathol. 143, 401-409, 1993; O'Reilly et al . Cell 79, 316-328, 1994) .
  • the majority of clinical tumors are not even clinically detectable until after the occurrence of neovascularization, whose induction in solid tumors is mediated by one or more angiogenic factors.
  • angiogenesis is also important in a number of other pathological processes, including, but not limited to, arthritis, psoriasis, diabetic retinopathy, retinopathy of prematurity, macular degeneration, scleroderma, hemangioma, retrolental fibroplasia, abnormal capillary proliferation in hemophiliac joints, prolonged menstruation and other disorders of the female reproductive system.
  • arthritis psoriasis
  • diabetic retinopathy retinopathy of prematurity
  • macular degeneration scleroderma
  • hemangioma hemangioma
  • retrolental fibroplasia abnormal capillary proliferation in hemophiliac joints, prolonged menstruation and other disorders of the female reproductive system.
  • angiogenesis may be outlined briefly as follows. When a new capillary sprouts from the side of a venule, endothelial cells degrade the basement membrane, migrate toward an angiogenic source, proliferate, form a lumen, join the tips of two sprouts to generate a capillary loop, and manufacture a new basement membrane (Folkman, Perspectives in Biology and Medicine, 29, 1-36, 5 1985) .
  • ECM extracellular matrix
  • endothelial cells i.e., collagens, laminin, thrombospondin, 0 fibronectin and SPARC
  • Bovine aortic endothelial cells BAE
  • type I collagen may be 5 involved in directing the migration and assembly of BAE cells (Iruela-Arispe et al . Lab. Invest. 64, 174-186, 1991).
  • angiogenesis mechanism In order to treat angiogenesis related disorders, several inhibitors of the angiogenesis mechanism are being studied, including platelet factor 4, the fumagillin 0 derivative AGH 1470, Interferon ( ⁇ 2 a, thrombospondin, angiostatic steroids, and angiostatin (Folkman ibid . , 1995; O'Reilly et al . , ibid . , 1994).
  • anti-estrogens have also been shown to inhibit angiogenesis (Garliardi and Collins, Cancer Res. 53, 533-535, 1993). Unfortunately, many 5 of these inhibitors all share the property of being relatively non-specific in their effects and, therefore, potentially toxic. A more specific inhibitor would be most useful, particularly an inhibitor that would selectively block an underlying mechanism of angiogenesis without 0 adversely affecting other physiological functions.
  • proteins e . g . , antibodies, thrombospondin, angiostatin and platelet factor IV
  • proteins e . g . , antibodies, thrombospondin, angiostatin and platelet factor IV
  • they must be administered in high doses and frequencies.
  • an inhibitor of angiogenesis which specifically blocks the proliferation of vascular structures without substantially affecting other physiological processes — including an inhibitor of angiogenesis associated with tumor growth or progression.
  • compositions for breast cancer currently consists of hormonal and cytotoxic agents. Hormonal therapy was developed because, in many women, breast cancer cells have receptors for the steroid hormone estrogen. The growth of these estrogen receptor-positive cancer cells can be stimulated by estrogen. Anti-estrogen therapy attempts to reduce or stop the synthesis of estrogen or to block the action of estrogen on the cancer cell.
  • tamoxifen (U.S. Patent No. 4,536,56) holds a prevalent position. Originally used as an anti-estrogen to treat breast cancer in patients with estrogen receptor-positive tumors, the drug was also found to slow the growth of breast cancer in women with estrogen receptor-negative tumors. Tamoxifen is, therefore, useful in most patients.
  • the anti-estrogen tamoxifen is particularly effective in delaying recurrence in breast cancer patients and in the palliative treatment of advanced metastatic breast cancer. It is also useful in the treatment of gliomas and hepatomas as well as endo etrial, uterine, ovarian and prostatic neoplasms (Litherland, S. et al . Cancer Treatment Reviews, 15, 183, 1988; Jordan, C. , Br. J. Pharmacol., 110, 507, 1993) .
  • Anti-estrogens including tamoxifen, compete with estrogen for receptor sites in cancerous tissues. Occupancy of the receptor site by an anti-estrogen fails to elicit the full spectrum of transcriptional actions generated by estrogens and, thus, blocks their activity. It is generally believed that estrogens function by first binding to the target cell cytosolic receptors, and then moving into the cell nucleus, where they affect DNA transcription.
  • Hydrophilic compounds and particularly compounds with ionic charges are often very poorly distributed into the CNS and brain since a lipophilic barrier (the blood-brain barrier) exists.
  • One method for creating a permanent charge on a drug is the incorporation of a quaternary ammonium salt (nitrogen with four hydrocarbon groups attached) . Tamoxifen and other anti-estrogens that contain an amino group can be quaternized (converted to a quaternary ammonium group) . Such quaternization results in imparting a permanent positive charge to the parent molecule which should effectively reduce the molecule's penetration across physiological membranes which are inherently lipophilic and resistant to penetration of ions, particularly large ions.
  • TMI tumor regression that began almost immediately upon dose initiation and which resulted in complete regression of the implanted cancer in 40% of animals tested.
  • the parent compound, tamoxifen merely slowed tumor growth in that study (Cancer Res. 56, 4238, 1996) .
  • permanently charged steroid agonists and antagonists are unexpectedly potent anti-angiogenic agents. It is further disclosed that permanently charged anti-estrogens may mediate their anti-angiogenic effects by inhibiting the transcription of metalloproteases, including collagenases that are required for the restructuring of the extracellular matrix.
  • the methods of the invention will be useful with a wide variety of steroid agonists and antagonists including, but not limited to, charged derivatives of glucocorticoids, estrogens, androgens and progestins or their respective antagonists.
  • These anti-angiogenic anti-estrogens would possess estrogen antagonist activity, and may possess partial estrogen agonist or mixed activity, but would be limited in biodistribution by being permanently charged, thereby exhibiting reduced side effects and being beneficial for clinical use.
  • Another object of this invention and clinical benefit is the comparatively rapid elimination from circulation of these agents due to the fact that they are not sequestered in fat tissue, thereby reducing toxicity and allowing for precise control of dosing.
  • Yet another aspect of this invention is to provide for the formulation and drug delivery of the aforementioned anti-angiogenic anti-estrogen agents.
  • DRUG is a radical selected from the group consisting of a steroid agonist or antagonist, a mixed agonist-antagonist, and a partial agonist;
  • Y may be exemplified by, but is not limited to, the following anions: phosphate, sulfate, chloride, bromide, iodide, an alkyl or aryl sulfonate, or an organic anion such as acetate, citrate or oxalate.
  • Y is a non-toxic pharmaceutically acceptable anion
  • anti-estrogen is a radical selected from the group consisting of an estrogen antagonist, a mixed agonist-antagonist, and a partial agonist
  • X is a direct bond or a radical selected from the group consisting of -0-; -NH-; -NR-, wherein R is an alkyl or aryl group with less than ten carbons; -P0 3 -; -S-; -SO-; and -S0 2 -;
  • R_ and R 2 are the same or different and may be a radical selected from the group consisting of H, an alkyl of 1-10 carbon atoms, an arylalkyl of 7-16 carbon atoms, and an aryl;
  • R 3 , R 4 and R 5 are independently a radical selected from the group consisting of a branched or unbranched, cyclic or noncyclic alkyl of 1-10 carbon atoms; an alkyl of up to 10 carbon atoms substituted by a carboxy, hydroxy, alkoxy, halo, or nitro group; a branched or unbranched, cyclic or noncyclic arylalkyl of 7-16 carbon atoms; and an aryl; and n is 0-12.
  • a most preferred embodiment according to the present invention comprises an anti-angiogenic compound of the general formula III:
  • X is a direct bond or a radical selected from the group consisting of -0-, -NR-, -S-, -SO-, -S0 2 -, and -P0 3 -;
  • R, R x and R 2 are independently a radical selected from the group consisting of H, an alkyl of 1-10 carbon atoms; an aralkyl of 7-16 carbon atoms; and an aryl; n is 0-12;
  • G is a cationic radical selected from the group consisting of -N (R' ) (R" ) (R" ' ) , -(O) N (R')(R"), -S (R' ) (R” ) , and -P (R' ) (R" ) (R" ' ) ;
  • R' is a radical selected from the group consisting of an alkyl of 1-10 carbon atoms; an alkyl of up to 10 carbon atoms substituted by a carboxy, hydroxy, alkoxy, halo, or nitro group; a cycloalkyl of 4-8 carbon atoms; a cycloalkyl-alkyl of 5-18 carbon atoms; and an aralkyl of 7-16 carbon atoms;
  • R'' and R' ' ' are independently a radical selected from the group consisting of an alkyl of 1-7 carbon atoms and a 4- to 8-membered nitrogen containing ring;
  • B is a radical selected from the group consisting of an alkyl of 1-7 carbon atoms, a halogen, a nitrogen, and a moiety which is linked to the 2-position of the phenyl that is neither the phenyl linked to the same ethylene carbon as B, nor the phenyl substituted by the radical containing the permanently ionic group G, and which is selected from the group consisting of -CH 2 C (R (R 2 ) - and -CH 2 -0-;
  • L and M are independently 0-3 ; 1 and m are independently 1-7; and
  • Y is a pharmaceutically acceptable anion
  • FIG. 1 Effects of estradiol, tamoxifen, TMI or a placebo on the growth of MCF-7 tumors implanted in the ventral fat pad of nude mice.
  • the left panel gives data for tamoxifen, TMI and the placebo, while the right panel adds the group treated with estradiol.
  • Figure 2 Percent change in total necrosis relative to day 0 induced by TMI or placebo in MCF-7 human breast tumors implanted in nude mice.
  • FIG. 1 Effect of TMI on endothelial cell area in GSL-1 labeled tumor sections. Animals were treated for three days with TMI or with a placebo pellet.
  • Figure 6. Effect of Tamoxifen or TMI on Gelatinase A activity (72 kDa isoform system) determined at 6 hours (left) or 24 hours (right) after exposure of cells.
  • Figure 7. Effect of 20 ⁇ M Tamoxifen or TMI on collagenase activity for both the 72 kDa and 92 kDa isoform systems determined using zymography at 6 and 24 hours after drug administration.
  • FIG. 8 Effect of TMI on Gelatinase A activity in bovine endothelial cells.
  • Figure 9 Effect of TMI or TBB on collagenolytic activity (Gelatinase A, MMP-2 and Gelatinase B, MMP-9) in human fibrosarcoma cells (HT-1018) .
  • FIG. 10 Effect of TMI on the MMP-2 (Gelatinase A) related CAT activity in transfected bovine endothelial cells.
  • permanently charged anti-estrogens may be used to prepare medicaments effective in the treatment of angiogenesis.
  • anti-estrogens derived from triphenylethylene such as tamoxifen, toremifene and clomiphene
  • anti-estrogens derived from diphenyl naphthalene such as nafoxidine
  • anti-estrogens derived from triphenyl ethanol such as ethamoxytriphetol
  • the modification of drugs to form permanently charged derivatives may be most conveniently accomplished by the preparation of quaternary salts. Such compounds may be prepared by a variety of chemical reactions. In the case of anti-estrogens containing an amino group, one such method is to react the anti-estrogen with an alkylating agent.
  • the alkylating agent can be an alkyl halide, tosylate, alkyl or dialkyl sulfate or any other appropriate moiety.
  • the alkylation may be performed with or without addition of organic solvents, as appropriate, and may be carried out under cooling or at room temperature or with heating, as appropriate, to ensure that the reaction proceeds satisfactorily to completion.
  • the reaction may be monitored by standard analytical methods known to one skilled in the art including thin layer chromatography, high pressure liquid chromatography, nuclear magnetic resonance spectroscopy or any other suitable method.
  • the resulting quaternary salt may be purified by standard methods, known to the artisan, usually including at least one step involving recrystallization.
  • the associated anion may be changed if desired by standard procedures such as ion-exchange columns.
  • the compounds provided can be formulated by any required method to provide pharmaceutical compositions suitable for administration to a patient.
  • the novel compositions contain, in addition to the active ingredient, conventional pharmaceutically acceptable carriers, diluents and the like.
  • Solid compositions for oral administration such as tablets, pills, capsules or the like, may be prepared by mixing the active ingredient with conventional, pharmaceutically acceptable ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate and gums, with pharmaceutically acceptable diluents.
  • the tablets or pills can be coated or otherwise compounded with pharmaceutically acceptable materials known in the art to provide a dosage form affording prolonged action or sustained release.
  • Other solid compositions can be prepared as microcapsules for parenteral administration.
  • Liquid forms may be prepared for oral administration or for injection, the term including subcutaneous, intramuscular, intravenous, and other parenteral routes of administration.
  • the liquid compositions include aqueous solutions (with or without organic cosolvents) , aqueous or oil suspensions, emulsions with edible oils, as well as similar pharmaceutical vehicles.
  • the compositions of the present invention may be formed as encapsulated pellets or other depots, for sustained delivery.
  • the active dose for humans is generally in the range of from 0.01 mg to about 10 g per kg body weight, in a regimen of 1-4 times a day. However, administration at longer intervals may also be possible, for compounds or formulations having prolonged action.
  • the preferred range of dosage is from 0.05 to 5 mg per kg body weight. It is evident to one skilled in the art that the dosage form and regimen would be determined by the attending physician, according to the disease to be treated, the method of administration, and the patient's general condition. It will be appreciated that the most appropriate form of administration of the pharmaceutical compositions of the present invention will depend first and foremost on the clinical indication being treated.
  • the prophylactic treatment of a healthy individual at high risk for pathological angiogenesis will necessitate a sustained maintenance dosage regimen.
  • This type of treatment might be applied to individuals at risk for diabetic retinopathy, retinopathy of prematurity, macular degeneration and other conditions that are known to afflict particular sets of patients. In contradistinction, the treatment of an existing disease might require higher doses at more frequent intervals. It is further anticipated that the treatment of certain conditions known to involve abnormal vascular smooth muscle cell proliferation, including restenosis, will be treated beneficially with compositions according to the present invention.
  • the present invention provides novel medical uses for both certain known compounds as disclosed in WO 95/25720 and for additional steroid agonists and antagonists of the general formula I as described above.
  • Certain charged derivatives of tamoxifen have been shown to possess improved anti-tumor activity when compared to tamoxifen. It is now disclosed that these derivatives display potent anti-angiogenic activity even in systems where tamoxifen itself is devoid of activity.
  • TMI tamoxifen methiodide
  • TTB tamoxifen benzyl bromide
  • TMI tamoxifen methiodide
  • TBI tamoxifen benzyl bromide
  • TMI basement membrane proteins
  • TBB tamoxifen in blocking matrix metalloprotease activity
  • Tamoxifen methiodide was prepared by reacting 2.0 g of tamoxifen (Aldrich Chemical Co., St. Louis, MO) with methyl iodide at 0°C for 24 hours. Ethyl acetate was then added to afford a white precipitate, which was recrystallized from methanol to yield >99% tamoxifen methiodide. Tamoxifen benzyl bromide was synthesized by reacting tamoxifen with benzyl bromide.
  • Cell Cultures - HT-1080 cells (CCL 121) , derived from a metastatic lesion of a human fibrosarcoma and primary bovine endothelial cells were obtained from the American Type Culture collection (Rockville, MD) . Cells were maintained under an atmosphere of 5% C0 2 , in Dulbecco's Minimal Essential Medium (DMEM) supplemented with 10% fetal calf serum, glutamine, vitamins, non-essential amino acids and antibiotics (Biological Industries, Kibbutz Beth HaEmek, Israel) .
  • DMEM Dulbecco's Minimal Essential Medium
  • Sub-confluent cell cultures were incubated for 6 to 24 hours in serum-free DMEM, either with or without various concentrations of TMI or tamoxifen, and the resultant supernatant was analyzed for collagenolytic activity.
  • the collagenolytic activity was determined using a gelatin-impregnated (1 mg/ml, Difco, Detroit MI) SDS-PAGE 8% gel, as previously described, with minor modifications. Briefly, the culture media samples were separated on the substrate-impregnated gels under non-reducing conditions, followed by 30 min of incubation of 2.5% Triton X-100 (BDH, UK) .
  • the gels were then incubated for 16 hours at 37 °C in 50 mM TRIS, 0.2 M NaCl, 5 mM CaCl 2 , 0.02% BRIJ 35 (w/v) at pH 7.6. At the end of the incubation period, the gels were stained with 0.5% Coomassie G 250 (Bio-Rad, Richmond, CA) in methanol/acetic acid/H 2 0 (30:10:60). The intensity of the stained bands was then determined using a computerized densitometer (Molecular Dynamics Model 300A) .
  • Bovine endothelial cells were harvested by brief exposure to 1 mM EDTA, washed with DMEM containing 0.1% bovine serum albumin and added to a layer of Matrigel (basement membrane components) in a 24 well plate to a density of 50,000 cells per well. After attachment, culture media (1.0 ml) was added and the plates incubated as a monolayer culture in the presence or absence of various concentrations of TMI . The plates were analyzed hourly using Hoffman optics for endothelial tube formation and growth. Photomicrographs were taken for estimation of relative inhibitory action.
  • MCF-7 cells serially passed as described above, were detached from the culturing flasks with 0.03% EDTA in phosphate buffered saline (PBS) and washed several times in normal saline. Cells (10 7 /innoculum) were then injected into the ventral fat pad at the level of the breast. Coincident with cell introduction, a slow-release pellet of estradiol (0.72 mg/pellet, 45-day release profile; innovative Research, Sarasota, FL) was implanted subcutaneously in the flank of the animal. Mice were anesthetized with ketamine/Rompun (i.p.) for the surgical procedures.
  • PBS phosphate buffered saline
  • XSA is the cross-sectional area of an internal slice
  • ES is the cross-sectional area of an edge slice
  • 1 is the slice thickness
  • ssd is the distance from the middle of one slice to the middle of the next.
  • Histology - Tumors from TMI-treated animals and from placebo-treated animals were removed after cervical dislocation. Tumors were treated with a biological dye (Davidson Marking Systems, Bradley Products, Bloomington, MN) so that histological sections could be oriented in the same direction as MRI slice images.
  • the histological plane corresponding to the central slice of the MRI study was established by a single cut through the tumor. This plane was based on the position of the tumor in the spectrometer, sagittal, transverse and coronal images and anatomical landmarks.
  • the bisected tumors were fixed in 10% formalin, dehydrated in 70% ethanol, blocked in paraffin.
  • hematoxylin-eosin 4 ⁇ m histological sections were cut, placed on slides and stained with either hematoxylin-eosin or a modified trichrome method. Hematoxylin-eosin was used to assess viability, necrosis and pigmentation. The modified trichrome method was applied to the identification of fibrotic regions (i.e., the dye stains for mucopolysaccharides) . Vascular density was determined using two techniques: by morphometric analysis of histological sections and by image analysis of GSL-1 lectin stained sections. For microscopic analysis, 4 ⁇ m tumor sections were stained with a modified Trichrome method. A 1 cm grid divided into 100 squares was placed in the eyepiece.
  • the grid covered an area 250 ⁇ m 2 and each grid square represented 25 ⁇ m 2 .
  • Two sets of analyses were completed: (1) an examination of the entire tumor; and (2) an evaluation of viable areas. In the latter case, three meridians were constructed, oriented perpendicular to the long axis of the section. Starting just within the tumor-capsule interface, vascular density determinations were made every mm until the opposite edge of the tumor was reached. A minimum of three meridians (i.e., nine measurements/mm/tumor) were performed. For analysis of viable tissue, areas of highest vascularity were considered as previously outlined, with 8-12 fields per tumor being examined. In addition, endothelial cytology was noted.
  • paraffin-embedded sections (4 ⁇ m) were deparafinized and rehydrated. Sections were first treated with a blocking solution containing nonimmune goat serum for 30 min at room temperature and then with a 0.1 mg/ l solution of biotinylated Griffonia simplicifolia lectin (GSL-1) for 60 min.
  • GSL-1 lectin binds specifically to ⁇ -galactosyl residues and marks the vascular endothelium in mice.
  • the sections were then washed with TRIS buffered saline (TBS) and treated for 30 min with avidin-biotin-peroxidase complex (Vector Laboratories, Burlingame, CA) , after which the peroxidase was activated by incubation of the sections with 0.1 M acetate buffer (pH 5.2) containing 3% H 2 0 2 and 3% 3-amino-9-ethylcarbazole for 5-10 min. The sections were then washed with distilled water, counterstained with hematoxylin, dehydrated and mounted on a coverslip with permount. Image analysis of the stained sections was completed using a GALAI CUE-2 system with a 50x objective.
  • Magnetic resonance imaging (MRI) of breast tumors allows a non-invasive assessment of the effects of drug treatment on tumor pathomorphology .
  • This tool was applied to examine the effect of TMI on an implanted human breast tumor in athymic nude mice.
  • three control groups were included in this study, specifically: (1) a group of animals in which the supportive estrogen pellet was removed and replaced with a placebo pellet; (2) a group in which the estrogen pellet was replaced with a second estrogen pellet; and (3) a group in which the estrogen pellet was replaced with a tamoxifen-laden pellet.
  • the breast tumor was implanted at the level of the milk line in the ventral fat pat.
  • the left panel gives data for tamoxifen, TMI and the placebo, while the right panel adds the data from the estradiol group.
  • Drugs were administered as slow-release pellets implanted (s.c.) in the flank.
  • TMI tumor necrosis that manifests a faster onset and which is more extensive than that induced by partial estrogen ablation (i.e., removal of the estrogen pellet).
  • partial estrogen ablation i.e., removal of the estrogen pellet.
  • preliminary studies suggest that TMI is more potent than tamoxifen in the induction of necrosis.
  • tamoxifen similar to TMI, was found to induce rapid necrosis (by day 3) as assessed by MRI, but the maximum extent of necrosis was 50% (compared to 72% for TMI) .
  • a portion of the tumors treated with tamoxifen became tolerant to the effects of the drug and began to regrow over time. This phenomenon did not occur with TMI.
  • Vascular density of murine endothelial cells was evaluated by specific staining, using the GSL-1 lectin (the equivalent of factor VIII antibody staining for human endothelial systems) followed by color image analysis of the resulting sections.
  • the mean percentage of endothelial cell areas in viable regions of control tumors was 14% and in viable areas of TMI treated tumors was 5%.
  • the three-fold reduction in endothelial cells density was highly significant (Figure 5) .
  • TBB tumor-bearing mice
  • TMI tamoxifen methiodide
  • TMI tumor necrosis factor
  • matrigel a basement membrane preparation made up of laminin, collagen type IV, heparin sulfate proteoglycan and entactin
  • TMI inhibited tube formation and growth in a concentration-dependent manner over a dose range from 1 to 20 ⁇ M.
  • a dose of 10 ⁇ M of TMI decreased tube area by more than 70%, as measured by image analysis.
  • TMI tissue necrosis factor
  • tamoxifen The effect of TMI relative to tamoxifen on collagenase activity was considered in both whole cell and cell-free systems.
  • Human fibrosarcoma cells were plated in culture and exposed to either TMI or tamoxifen at doses ranging from 0.5 to 20 ⁇ M. After 6 and 24 hours of incubation, samples of supernatant were withdrawn and assayed for collagenolytic activity associated with two collagenase isozymes (gelatinase A and B (MMP-2 and MMP-9)) by zymography (substrate impregnated non-reducing activity gels) . TMI inhibited both isozymes in a dose-dependent manner, with complete inhibition observed at the 20 ⁇ M level ( Figures 6 and 7) . Tamoxifen, on the other hand, demonstrated no significant inhibitory activity at doses up to and including 20 ⁇ M.
  • FIG. 7 shows the effect of 20 ⁇ M tamoxifen or TMI on collagenase activity for both the 72 kDa and 92 kDa isoform systems, as determined by zymography at 6 and 24 hours after drug administration.
  • TMI proved to be a potent inhibitor of collagenase (gelatinase A) activity in bovine endothelial cells with an IC 50 of between 1 and 5 ⁇ M (Figure 8).
  • TBB was also examined in this assay and directly compared with TMI .
  • TBB was more potent than TMI as an inhibitor of both the 72kDa (gelatinase A, MMP-2) and 92 kDa (gelatinase B, MMP-9) isoforms of collagenase ( Figure 9) .
  • TMI was evaluated to determine whether the inhibitory effects were manifested at the level of the enzyme or at other loci . There was no effect of TMI on enzymatic activity at any concentration examined. This data suggests that TMI inhibits gelatinase A and B biosynthesis and or gene expression in the concentration range tested.
  • TMI tumor necrosis factor-induced endothelial growth factor
  • human fibrosarcoma cells as well as bovine endothelial cells were transfected with plasmids containing the promoters for type IV collagenase (both MMP-2 and MMP-9) and the chloramphenicol acetyl transferase (CAT) construct to act as a reporter.
  • CAT chloramphenicol acetyl transferase
  • Simian virus-40 SV-40
  • TMI was added to the transfected cells at concentrations of 1, 5 and 10 ⁇ M for 2 days, at which time cell viability was confirmed by trypan blue exclusion and cell counting.
  • FIG 10 shows the effect of TMI at concentrations of 1, 5 and 10 ⁇ M on CAT activity expressed in a cell system transfected with plasmids containing the promoters for the 92 kDa isoform of collagenase (634 base-pair promoter) or the 72 kDa isoform of collagenase (basic promoter - B or promoter plus enhancer - E).
  • TMI reduced CAT activity by 45% at 1.0 ⁇ M and by 80% at 10 ⁇ M. This data suggests that the action of TMI on expression of collagenases is germane to vascular systems ( Figure 11) .
  • TMI is an inhibitor of MMP-2 in bovine endothelial cells and MMP-2/MMP-9 in human fibrosarcoma cell lines.
  • TBB is more potent than TMI in the inhibition of metalloproteases in the systems examined.
  • TMI inhibits matrix metalloproteases at the level of biosynthesis or expression. Furthermore, the lack of activity of the tamoxifen analog in cell free systems indicates that TMI does not directly inhibit the enzymes.
  • TMI and TBB therefore, exert potent anti-angiogenic action which appears to be correlated with their ability to suppress transcription or expression of metalloproteases. While the invention has been described in terms of various preferred embodiments, the skilled artisan will appreciate that various modifications, substitutions, omissions and changes may be made without departing from the spirit thereof. Accordingly, it is not intended that the scope of the present invention be limited solely by the scope of the following claims.

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Abstract

L'invention concerne l'utilisation d'agonistes ou d'antagonistes constamment chargés de stéroïdes comme compositions puissamment anti-angiogéniques, qui renferment comme principe actif un composé de formules générales I (médicament), II (anti-oestrogènes) ou III, dans lesquelles 'médicament' est un agoniste ou un antagoniste des stéroïdes, un mélange agoniste-antagoniste ou un agoniste partiel, et les substituants sont tels que définis dans la spécification.
PCT/US1998/002176 1997-02-09 1998-02-04 Activite anti-angiogenique amelioree des derives constamment charges d'hormones steroidiennes WO1998034583A2 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP98903921A EP0981337A4 (fr) 1997-02-09 1998-02-04 Activite anti-angiogenique amelioree des derives constamment charges d'hormones steroidiennes
CA002280181A CA2280181A1 (fr) 1997-02-09 1998-02-04 Activite anti-angiogenique amelioree des derives constamment charges d'hormones steroidiennes
JP53487698A JP2001511182A (ja) 1997-02-09 1998-02-04 ステロイドホルモンの永久に荷電された誘導体の増進された抗血管形成誘導活性
AU60562/98A AU746007B2 (en) 1997-02-09 1998-02-04 Enhanced anti-angiogenic activity of permanently charged derivatives of steroid hormones

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
IL12018497A IL120184A (en) 1997-02-09 1997-02-09 Pharmaceutical compositions comprising permanently charged derivatives of steroid agonists or antagonists for preventing angiogenesis
IL120184 1997-02-09
US08/833,074 US6083990A (en) 1997-04-02 1997-04-02 Enhanced anti-angiogenic activity of permanently charged derivatives of steroid hormones
US08/833,074 1997-04-02

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WO1998034583A3 WO1998034583A3 (fr) 1998-11-05

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FR3046792B1 (fr) * 2016-01-19 2018-02-02 Les Laboratoires Servier Nouveaux derives d'ammonium, leur procede de preparation et les compositions pharmaceutiques qui les contiennent
EP3484463B1 (fr) * 2016-08-19 2020-02-12 The United States of America, as Represented by The Secretary, Department of Health and Human Services Office of Technology Transfer Modulateurs sélectifs du récepteur des oestrogènes (serms) conférant une protection contre la dégénérescence des photorécepteurs

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WO1995026720A1 (fr) * 1994-04-04 1995-10-12 Pharmos Corporation Derives constamment ioniques d'hormones steroides et leurs antagonistes

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US2914561A (en) * 1957-08-06 1959-11-24 Wm S Merrell Co Amine derivatives of triphenylethylene
US2971001A (en) * 1959-10-21 1961-02-07 Wm S Merrell Co Quaternary salts of triphenylethanols, -ethylenes, and -ethanes
BE637389A (fr) * 1962-09-13
EP0776661A1 (fr) * 1992-10-27 1997-06-04 Nippon Kayaku Kabushiki Kaisha Préparation combiné d'un antiestrogène et d'un glucocorticoide pour le traitement des desordres autoimmunitaires

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995026720A1 (fr) * 1994-04-04 1995-10-12 Pharmos Corporation Derives constamment ioniques d'hormones steroides et leurs antagonistes

Non-Patent Citations (1)

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Title
See also references of EP0981337A2 *

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EP0981337A4 (fr) 2003-03-12
JP2001511182A (ja) 2001-08-07
AU6056298A (en) 1998-08-26
AU746007B2 (en) 2002-04-11
EP0981337A2 (fr) 2000-03-01
CA2280181A1 (fr) 1998-08-13
WO1998034583A3 (fr) 1998-11-05

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