Antiangiogenic activity of betulinic acid and its derivatives

Details Antiangiogenic activity of betulinic acid and its derivatives Antiangiogenic activity of betulinic acid and its derivatives

1998-10-06

2001-05-08

Krass, Frederick (Department: 1614)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Cyclopentanohydrophenanthrene ring system doai

C514S177000, C514S178000, C514S179000, C514S180000, C514S181000, C514S182000, C514S569000, C514S570000, C514S908000, C514S510000

Reexamination Certificate

active

06228850

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to use of betulinic acid and/or its derivatives for inhibiting and/or preventing angiogenesis. This invention also relates to novel betulinic acid derivatives and compositions containing betulinic acid derivatives with or without betulinic acid.
BACKGROUND OF THE INVENTION
Betulinic acid is a pentacyclic triterpene. It has several botanical sources, but can also be chemically derived from betulin, a substance found in abundance in the outer bark of white birch trees (
Betula alba
). Betulinic acid has been found to selectively kill human melanoma cells (Nature Medicine, Vol.1 (10), 1995, WO 96/29068). The cytotoxic potential of betulinic acid was tested using three human melanoma cell lines, Mel-1, -2, and 4. The growth of all of the cell lines was inhibited significantly by treatment with betulinic acid. The effectiveness of betulinic acid against melanoma cancer cells was also tested using athymic mice. It seems to work by inducing apoptosis in cancer cells.
The anti-cancer activity of betulinic acid and some of its derivatives has also been demonstrated using mouse sarcoma 180 cells implanted s.c. in nude mice (JP 87,301,580), inhibition of growth of P388 lymphocytic leukemia cells in vitro (Choi.Y-H et al., Planta Medica Vol.XLVII,511-513,1988) and inhibiting growth of cancer cells, particularly by inhibiting ornithine decarboxylase (Yasukawa, K et al, Oncology 48:72-76,1991; WO 95/04526).
Recently, we reported the anti-leukemia and anti-lymphoma activity of betulinic acid and its derivatives with ED
50
values in the range of approximately 0.5 to 4.0 &mgr;g/ml. and also reported anti-prostate, anti-lung and anti-ovarian cancer activity of betulinic acid and betulinic acid derivatives with ED
50
values in the range of approximately 0.6 to 6.8 &mgr;g/ml, to 1.3 to 7.7 &mgr;g/ml, and 0.4 to 8.1 &mgr;g/ml respectively (see U.S. application Ser. No. 09/040,856 filed on Mar. 18, 1998 now U.S. Pat. No. 6,048,847, the subject matter of which is incorporated by reference).
The lupanes, and specifically betulinic acid, have been reported to be effective anti-inflammatory agents. (Sotomatsu s., et al; Skin and Urology 21:138,1959 and Inoue H. et al; Chem.Pharm.Bull 2:897-901, 1986). The anti-inflammatory activity of betulinic acid is, at least in part, due to its capacity to inhibit enzymes involved in leukotriene biosynthesis, including 5-lipoxygenase. Betulinic acid and its derivatives have been tested for their ability to inhibit 5-lipoxygenase activity by monitoring their ability to inhibit conversion of linoleic acid to 5-HETE. On pre-incubating with the enzyme, betulinic acid inhibits the production of 5-HETE by approximately 40%. (WO 95/04526).
Angiogenesis is the growth of new microvessels, a process that depends mainly on locomotion, proliferation, and tube formation by capillary endothelial cells. During angiogenesis, endothelial cells emerge from their quiescent state and can proliferate as rapidly as bone marrow cells, but unlike the bone marrow, angiogenesis is usually focal and of brief duration. Pathologic angiogenesis, while still a focal process, persists for months or years. The angiogenesis that occurs in diseases of ocular neovascularisation, arthritis, skin diseases, and tumors rarely terminates spontaneously and has until recently, been difficult to suppress therapeutically. Therefore, the fundamental goal of all antiangiogenic therapy is to return foci of proliferating microvessels to their normal resting state, and to prevent their regrowth (Cancer: Principles & Practice of Oncology, Fifili Edition, edited by Vincent T. DeVita, Jr., Samuel Hellman, Steven A. Rosenberg. Lippincott-Raven Publishers, Philadelphia© 1997).
Although the molecular mechanisms responsible for transition of a cell to angiogenic phenotype are not known, the sequence of events leading to the formation of new vessels has been well documented (Hanahan, D., Science 277, 48-50, 1997). The vascular growth entails either endothelial sprouting (Risau, W., Nature 386, 671-674, 1997) or intussusception (Patan, S., et al; Microvasc. Res. 51, 260-272, 1996). In the first pathway, the following sequence of events may occur: (a) dissolution of the basement of the vessel, usually a postcapillary venule, and the interstitial matrix; (b) migration of endothelial cells toward the stimulus; (c) proliferation of endothelial cells trailing behind the leading endothelial cell (s); (d) formation of lumen (canalization) in the endothelial array/sprout; (e) formation of branches and loops by confluencelanastomoses of sprouts to permit blood flow; (f) investment of the vessel with pericytes; and (g) formation of basement membrane around the immature vessel. New vessels can also be formed via the second pathway: insertion of interstitial tissue columns into the lumen of preexisting vessels. The subsequent growth of these columns and their stabilization result in partitioning of the vessel lumen and remodelling of the local vascular network.
The rationale for antiangiogenic therapy is that progressive tumor growth is angiogenesis-dependent (Folkman, J.; N.Engl.J.Med., 285, 1182, 1971). The switch to the angiogeneic phenotype appears to be an independent event that occurs during the multistage progression to neoplasia. The angiogenic switch itself, while relatively sudden and well localized, is nonetheless a complex process. This phenotype is currently understood in terms of a shift in the net balance of stimulators and inhibitors of angiogenesis, during which inhibitors are downregulated.
Endothelial cell survival and growth are driven by tumor derived mitogens and motogens. These findings have led to a model of tumor growth in which the endothelial cell compartment and the tumor cell compartment interact with each other. They not only stimulate each other's growth, but if the endothelial cells are made unresponsive to angiogenic stimuli from the tumor cells, by administration of a specific endothelial inhibitor, both primary tumors and metastatic tumors can be held dormant at a microscopic size (O'Reilly M S, et al; Cell, 19, 315, 1994 and O'Reilly, MS et al; Nature Med, 2, 689, 1996). One could take advantage of this difference between endothelial cells and tumor cells by administering an angiogenesis inhibitor together with conventional cytotoxic chemotherapy up to the point at which the cytotoxic therapy would normally be discontinued because of toxicity or drug resistance. The angiogenesis inhibitor(s) could then be continued for years, to maintain either stable disease or tumor dormancy. Such combinations of antiangiogenic and cytotoxic therapy in tumor-bearing animals have been curative, whereas either agent alone is merely inhibitory.
Eicosapentanoic acid (EPA), a n-3 polyunsaturated fatty acid found in fish and marine animals is a precursor for eicosanoids. EPA taken by man competes with arachidonic acid for inclusion in cycloxygenase and lipoxygenase pathways. Exposure of endothelial cells to EPA in vitro inhibited their capacity to form tubes—an essential aspect of the angiogenic process and substantially decreases their ability to break through an artificial basement membrane (Matrigel) in vitro (Kanayasu T et al, Lipids 1991;26:271-276). Repeated topical application of EPA in eye drops significantly suppressed the resulting neovascularisation and associated inflammation in induced immunogenic keratitis in rabbit corneas (Verbey N L J, et al, Curr Eye Res 1986; 7:549-557).
A similar effect is observed with gamma linolenic acid (GLA). A number of studies demonstrate that GLA slows cancer growth in animals. GLA also slows angiogenesis. (Ormerod L D et al, Am.J. Pathol. 1990; 137:1243-1252 and Verbey N L J, et al, Curr Eye Res 1986; 7:549-557). The mechanism of this effect is not clear, although GLA supplementation increases certain lipoxygenase products which potently inhibit 5-lipoxygenase enzyme as well as 12-lipoxygenase. (Vanderhoek J Y et al, Biochem. Pharmacol. 1982, 31:3463-3467; Miller C C et al, Prostaglandins 1988; 35: 917-938; Mill

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