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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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• • 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

• 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.
• 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) .
• 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) .
• 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.
• 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
• 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-;
• 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.
• 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.

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• 1998
  • 1998-02-04 EP EP98903921A patent/EP0981337A4/de not_active Withdrawn
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