Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Having -c- – wherein x is chalcogen – bonded directly to...
Reexamination Certificate
2002-04-08
2003-12-02
Reamer, James H (Department: 1614)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Having -c-, wherein x is chalcogen, bonded directly to...
C514S419000
Reexamination Certificate
active
06656963
ABSTRACT:
INTRODUCTION
1. Field of the Invention
The invention relates to indole-3-carbinols and their bioactivity.
2. Background of the Invention
More than 40,000 women die each year of metastatic breast cancer in the United States. Endocrine therapy, in particular anti-estrogen therapy, remains a major option for treatment of such patients, and results in complete plus partial response rates of 30%. This response rate results from the fact that the growth of approximately one-third of breast cancers is stimulated by estrogen, which is a natural hormone produced in women. Treating patients with anti-estrogens, such as tamoxifen, will slow the growth of these estrogen dependent tumors. Postmenopausal status, a prolonged disease-free interval, and positive estrogen and progesterone receptors are all associated with an increased response to endocrine therapy. The use of additive hormonal therapy, specifically antiestrogens, progestins, and aromatase inhibitors, have replaced surgical ablative procedures in the majority of estrogen receptor positive patients; with the most favorable therapeutic index associated with therapies that use antiestrogens (55). For patients that are estrogen receptor negative and a subset of patients that are estrogen receptor positive, the best current approach to treating breast cancers that do not require estrogen for their growth is by surgical removal of the tumors. Unfortunately, even for the patients that respond to and can benefit from tamoxifen therapy, there are many detrimental side effects including the ability of breast cancer cells to become resistant to tamoxifen, an increase in the potential for ovarian tumors and an increased risk of osteoporosis. Some of these side effects are due to tamoxifen acting as an estrogen agonist in some tissues and remaining an estrogen antagonist in other tissues.
Indole-3-carbinol (I3C) is a naturally occurring component of Brassica vegetables, such as cabbage, broccoli, and Brussels sprouts. Exposure to dietary I3C markedly reduces the incidence of spontaneous and carcinogen-induced mammary tumors in rodents and, as disclosed herein, exhibits potent growth inhibitory activity in human breast cancer cells in vitro by inducing a G1 arrest of the cell cycle. A recent screen of 90 potential chemopreventative agents in a series of 6 short term bioassays relevant to carcinogen-induced DNA damage, tumor initiation and promotion, and oxidative stress, revealed I3C to be one of only 8 compounds that tested positive in all assays (1). Indeed, I3C administered in the diet or by oral intubation prior to treatment with carcinogen reduced the incidence of 7,12-dimethyl-benz(&agr;)anthracene (DMBA)-induced mammary tumors in rodents by 70-80% (2). In another study, I3C administered to rats prior to and during DMBA or methylnitrosourea treatment reduced mammary tumor incidence by as much as 95% or 65%, respectively (3). Consistent with these results, supplementation of a purified diet with cabbage or broccoli, both vegetables are good sources of I3C, also resulted in decreased mammary tumor formation in DMBA-treated rats (4). Also, in a long term feeding experiment, in which female mice consumed synthetic diets containing I3C at 0, 500 or 2000 p.p.m., spontaneous mammary tumor incidence and multiplicity were significantly lower (ca. 50% reduction) at both doses of I3C compared to untreated control animals, and tumor latency was prolonged in the high dose group (5). I3C also has anticarcinogenic effects on other cancer types, such as hepatic derived tissues (6-9), and can reduce benzo[a]pyrene-induced neoplasia of the forestomach (2). Because of the well documented cancer protective effects of I3C, along with its low toxicity, and its wide availability, this dietary indole is currently undergoing at least two different phase I clinical trials as a cancer chemotherapeutic and preventive agent (10).
I3C has been shown to have an antiestrogenic biological activity when added to the diet. For example, oral administration of I3C to humans at doses of around 500 mg daily for one week produced an increase in estradiol 2-hydroxylation of approximately 50% in both men and women (11). I3C also increased the levels of estradiol hydroxylation activity in female rats (12). I3C can, in some systems, display an antiestrogenic growth suppressive effect. For example, long term treatment (up to six weeks) with 50 &mgr;M I3C blocked the estradiol-induced proliferation of high density cultures (confluency for 1 weeks or longer) of human MCF7 breast cancer cells (13).
A major complication in interpreting the physiological results is that I3C is extremely unstable in acidic solution and it does not completely survive exposure to gastric acid (14). I3C is converted into several natural indole derivatives with biological activities. The acid reaction mixture of I3C is composed of five major components which are resolvable by HPLC (15). Sensitive analytical methods reveal that I3C is converted to several indole derivatives in acid conditions and in the intestinal contents of rats fed on a basal diet and treated orally with I3C (15-19), suggesting that these I3C derivatives may mediate the anti-estrogenic effects of I3C. I3C is converted into biologically active components such as its dimer 3,3′-diindolylmethane (DIM) and indolo[3,2-b]carbazole (ICZ) through an acid-catalyzed reaction occurring in the low-pH environment of the stomach (17). ICZ is also produced, presumably from the nutritive indole, tryptophan, as a metabolic product of intestinal bacteria (19).
A general picture has emerged indicating that many, if not all, of the long term antiestrogenic biological activities of I3C result from the actions of one of its acid conversion products (15-19). For example, ICZ is a potent inhibitor of several estrogen-dependent responses including growth inhibition of high density cultures of human breast cancer cells (20), and ICZ inhibited [
3
H]thymidine uptake, nuclear progesterone and ER binding, and CAT activity in MCF7 cells transfected with the estrogen-responsive vit-CAT reporter plasmid. However, ICZ exhibited only a very weak affinity for the estrogen receptor suggesting that its not a direct estrogen antagonist. Further studies showed that ICZ likely mediates its antiestrogenic effects through interactions with Ah (dioxin) receptor (21). ICZ competitively binds to the Ah receptor, which then translocates to the nucleus and induces P450 CYP1A1 gene expression which has been shown to alter estrogen metabolism (21). ICZ is the most potent Ah receptor agonist among the characterized I3C derived compounds. In fact, I3C itself has a particularly low affinity for the Ah receptor (Kd of 27 &mgr;M), compared to ICZ's high affinity (Kd of 190 pM) and the relatively moderate affinity of DIM (Kd of 90 nM) for the Ah receptor(17). Thus, I3C per se does not mediate any of its activities directly through the Ah receptor, and its mechanism of signal transduction and direct target genes are unknown. Other investigators have shown that the predominant I3C conversion product DIM was highly effective in reducing DMBA-induced mammary tumors, but DIM apparently was not consistently as effective as I3C (2).
We disclose herein that treatment of a human breast cancer cells with I3C induces a reversible growth arrest in an estrogen independent manner, resulting from a G1 cell cycle arrest. The G1 arrest in cell cycle progression correlates with a significant loss of CDK6 protein, a key cyclin dependent kinase involved in progression through the G1 phase of the cell cycle. Our data indicate that I3C itself, and not its acid breakdown products, is a potent anti-tumor agent, and that stable derivatives of I3C may be used to inhibit the growth of estrogen-dependent or independent breast cancer cells and other types of cancer cells that reveal induced CDK6.
SUMMARY OF THE INVENTION
The invention provides methods and compositions relating to novel bioactive compositions. The compositions find particular use as agents for inhibiting cell grow
Bjeldanes Leonard F.
Cover Carolyn M.
Firestone Gary L.
Osman Richard Aron
Reamer James H
The Regents of the University of California
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