EP1119353A1

EP1119353A1 - Compositions and methods of inhibiting neoplastic diseases with compounds related to limocitrin and 5-desmethyl sinensetin

Info

Publication number: EP1119353A1
Application number: EP99950209A
Authority: EP
Inventors: Najla Guthrie; John A. Manthey; Robert M. Horowitz
Original assignee: KGK Synergize Inc; US Department of Agriculture USDA
Current assignee: KGK Synergize Inc; US Department of Agriculture USDA
Priority date: 1998-10-06
Filing date: 1999-10-05
Publication date: 2001-08-01
Also published as: AU6291699A; CA2346333A1; WO2000019998A1; EP1119353A4; JP2005015469A; JP2003509334A

Abstract

Compositions and methods for the prevention and treatment of neoplastic diseases and atherosclerosis are described. Individuals at a high risk of developing or having neoplasia or atherosclerosis undergoing conventional therapies may be treated with an effective dose of limocitrin compounds including, but not limited to, 3,5,7,4'-tetramethoxylimocitrin, limocitrin 3,5,7,4'-trimethyl ether 5-acetate, 3,5,7,4'-tetramethoxylimocitrin or 3,7,4'-trimethoxylimocitrin, and 5-desmethyl sinensetin.

Description

COMPOSITIONS AND METHODS OF INHIBITING

NEOPLASTIC DISEASES WITH COMPOUNDS RELATED

TO LIMOCITRIN AND 5-DESMETHYL SINENSETIN

1. INTRODUCTION

The present invention relates to compositions and methods for the prevention

and treatment of neoplastic cells and diseases, especially breast cancer, with compounds

derived from limocitrin compounds. The present invention also relates to compositions and methods for the prevention and treatment of atherosclerosis, thrombosis, inflammatory diseases, allergies or viral diseases. These compounds are a group of naturally occurring

and/or synthetic flavonoids including, but not limited to 5-desmethyl sinensetin. and the

limocitrin-derived compounds, 3,5,7,4'-tetramethoxylimocitrin, 3,7,4'-trimethoxylimocitrin,

3,5,7,4'-tetraethoxylimocitrin or limocitrin-3,5,7,4'-trimethyl ether 5-acetate.

2. BACKGROUND

Limocitrin analogues are a group of citrus-derived flavonoids that are naturally

occurring in the plant or are chemically synthesized. 5-desmethoxy sinensetin is chemically

synthesized from sinensetin (Tatum, J.H. et al., 1972, Phytochemistry II: 2283-2288),.

although it has been reported to occur in trace quantities in mandarin leaves (Sugiyama, S. et

al., 1993, chem. Pharm. Bull. 41 :714-719. Flavonoids are polyphenolic compounds that

occur ubiquitously in foods of plant origin. The major dietary sources of flavonoids are

vegetables, fruits and beverages such as tea and red wine (Hertog, M.G.L. et al., 1993, J.

Agric. Food Chem 41 :1242-1246).

Plants have evolved flavonoids as protection against parasites, herbivores,

pathogens and oxidative cell injury. In addition, flavonoids contribute to the color of fruits

and vegetables. Cook, N.C. et al., 1996, J. Nutr. Biochem. 7:66-76. The average intake of

dietary flavonoids in the United States has been estimated at lg/day expressed as glycosides, of which about 170 mg expressed as aglycones, consist of flavonols, flavonones and flavones (Kuhnau, J., 1976, World Rev. Nutr. Diet 24:fl 17-191). Flavonol and flavone intake thus exceeds that of other dietary antioxidants such as beta-carotene (2-3mg/day) and vitamin E (7-10mg/day) and equals approximately one-third of vitamin C (70-100mg/day) (Nutrient intakes. Individuals in 48 states. Year 1977-1978. Report No. 1-2. Consumer Nutrition

Division, Human Nutrition Information Service. Hyattsville, M.D.: U.S.D.A. 1984). Flavonoids have been demonstrated to be the most potent dietary antioxidants and in light of

the large dietary consumption, flavonoids make a major contribution to the antioxidant

potential of the human diet. The main food sources of flavonols and flavones are black tea,

onions, apples, herbs and spices, cloves or black pepper (Hertog, M.G.L., et al., 1992, J.

Agric. Food Chem. 40:2379-2383).

2.1 Epidemiological Studies

Epidemiological studies consistently show an inverse association between the

consumption of fruits and vegetables and cancer risk at various sites and a positive

association with dietary factors such as fats and total calorie intake (Block, G., et al., 1992,

Nutr. Cancer 17:1-29). With numbers of diet-related cancer deaths elevated and is still rising, use of nutraceuticals have become increasingly popular in North America. "Nutraceuticals"

used herein are defined as any substance that may be considered a food or part of a food and

provides medical or health benefits, including the prevention and treatment of disease. Until

recently, natural medicine catered primarily to a limited population. Now, however, many

health care organizations are giving new consideration to alternative therapies. A potential

U.S. market for nutraceuticals has been estimated at two hundred fifty billion dollars, four

times that for conventional medicine. Presently, the share of sales for nutraceuticals used as

fortifiers accounts for nearly half of one percent of the estimated three hundred billion dollar U.S. processed food and beverage industry, and that translates into a one and one half billion

dollar market.

On the average, participants with the highest consumption of fruits and vegetables experience a fifty percent reduction in risk of cancers of the alimentary and

respiratory tract compared to participants with the lowest intakes. Fruit and vegetable consumption could be a marker for other aspects of lifestyle which are responsible for the

lower cancer rates, but it is possible that fruits and vegetables contain flavonoids such as

quercetin that prevent cancer (Steinmetz, K.A., et al., 1991, Cancer Causes Control 2:325-

357).

The association between quercetin and cardiovascular disease has been studied

in prospective cohort studies and cross-cultural ecological studies. Flavonol and flavone intake was inversely associated with mortality from coronary heart disease and to a lesser

extent with incidence of first myocardial infarction. These effects were independent of known risk factors for coronary heart disease such as serum cholesterol, body mass index,

blood pressure, smoking and intake of antioxidant vitamins, alcohol, and fat. Flavonol and flavone intake (mainly quercetin) was also inversely associated with stroke risk (Hertog,

M.G.L. et al., 1993, Lancet 324;1007-1011; Keli, S.O., et al., 1996, Arch. Inter. Med.

154:637-642). However, four thousand different types of flavonoids have been described and

it is crucial that the active components be identified not only to make a positive impact on

argiculture but also to more specifically use these nutraceuticals as anticancer agents and/or

antithrombotic, anticoronary heart disease, antimyocardial infarction and/or antistroke agents.

2.2 Cancer Growth and Chemotherapy

Cancer is a disease of inappropriate tissue accumulation. Chemotherapeutic agents share one characteristic: they are usually more effective in killing or damaging malignant cells than normal cells. However, the fact that they do harm normal cells indicates their potential toxicity. Animal tumor investigations and human clinical trials have shown that drug combinations produce higher rates of objective response and longer survival than

single agents. Combination drug therapy is, therefore, the basis for most chemotherapy employed at present (DeVita, V.T. et al., 1995, Cancer 35:98).

Cancer treatment requires inhibition of a variety of factors including tumor cell

proliferation, metastatic dissemination of cancer cells to other parts of the body, invasion, tumor-induced neovascularization, and enhancement of host immunological responses and

cytotoxicity. Conventional cancer chemotherapeutic agents have often been selected on the basis of their cytotoxicity to tumor cells. However, some anticancer agents have adverse

effects on the patient's immune system. Thus it would be greatly advantageous if a cancer

therapy or treatment could be developed that would afford non-cytotoxic protection against

factors that might lead to progression of tumors.

Because hormone therapy as well as chemotherapy is effective in controlling advanced breast cancer, it has been used as an adjuvant to mastectomy in primary breast

cancer. Patients with estrogen receptor + (ER+) or estrogen receptor - (ER-) tumors benefit

from adjuvant chemotherapy. However, tamoxifen used alone as an adjuvant to mastectomy

for breast cancer shows benefit in extending disease-free and overall survival (Cummings,

F.J. et al., 1985, Ann. Intern. Med. 103;324). While there are various methods for treating

cancerous cells and diseases, there remains a need in the art for at least reducing replication of

neoplastic cells, especially in disease states such as breast cancer. There also remains a need

in the art for methods for reducing the incidences of cardiovascular diseases. The present

invention is different from the related art and provides for methods and compositions for

using limocitrin analogues and 5-desmethyl sinensetin in treating neoplastic cells, and reducing cardiovascular disease. 3. SUMMARY OF THE INVENTION

The present invention is directed to a method for the prevention and/or

treatment of neoplastic diseases, which involves using a composition of limocitrin analogues

and 5-desmethyl sinensetin to treat an individual at high risk for, or suffering from cancer.

The present invention is also directed to a method for the prevention and/or

treatment of breast cancer, which involves using a composition of limocitrin analogues and 5-

desmethyl sinensetin in individuals at high risk for breast cancer.

The present invention is also directed to a method for the prevention and/or treatment of atherosclerosis, myocardial infarction, stroke or thrombosis, which involves

using a composition of limocitrin analogues and 5-desmethyl sinensetin in an individual in need thereof.

The present invention is further directed to a method for inducing anti-

inflammatory, anti-allergic, anti-viral and/or estrogenic activity, which involves using a composition of limocitrin analogues and 5-desmethyl sinensetin in an individual in need

thereof.

The present invention is directed to the use of limocitrin analogues and 5-

desmethyl sinensetin and tamoxifen in an individual suffering from breast cancer.

The present invention is directed to a method for the prevention and/or

treatment of neoplastic diseases, which involves using an effective dose of a combination of

one or more limocitrin analogues and 5-desmethyl sinensetin, with or without conventional

chemotherapy or hormonal and/or radiation therapy or surgery, in a patient suffering from cancer. The present invention is also directed to a method for inhibiting the oxidation of low-density lipoproteins and platelet aggregation and adhesion, which involves using an

effective dose of a composition of limocitrin compounds.

The present invention is also directed to a method of administering an

effective amount of a limocitrin compound to cancerous cells to reduce proliferation of said

cells in vitro and in vivo.

4. DETAILED DESCRIPTION OF THE INVENTION

The method of the invention involves administering an effective dose of a one or a combination of limocitrin analogues and 5-desmethyl sinensetin, tamoxifen or a chemotherapeutic agent, in an individual who is identified as being at enhanced risk for

cancer and/or as having cancer, in order to prevent and/or treat cancer.

It may be that the ability of these compounds to inhibit tumor cell

proliferation, to inhibit the metastatic spread of tumor cells or to present immuno-suppression

induced by chemotherapeutic agents, contributes to their effectiveness in the prevention and

treatment of neoplastic diseases. These possible mechanisms of action are in no way meant

to limit the scope of the invention and are presented purely for explanatory and/or illustrative purposes.

The method of the invention also involves administering an effective dose of limocitrin analogues and 5-desmethyl sinensetin to an individual who is identified as being at

enhanced risk for atherosclerosis or thrombosis and/or as having atherosclerosis, stroke,

myocardial infarction or thrombosis, in order to prevent and treat coronary heart diseases.

It may be that the ability of the flavonoids to inhibit platelet aggregation and

adhesion, contributes to their effectiveness in the prevention and treatment of atherosclerosis,

stroke, myocardial infarction and thrombosis. These possible mechanisms of action are in no way meant to limit the scope of the invention and are presented purely for explanatory and/or

illustrative purposes.

Cancer

Cancer is the second leading cause of death in the United States, after heart

disease (Boring, CC. et al., 1993, CA Cancer J. Clin. 43:7), and develops in one in three

Americans, and one of every four Americans dies of cancer. Cancer can be viewed as a

breakdown in the communication between tumor cells and their environment, including their

normal neighboring cells. Signals, both growth-stimulatory and growth-inhibitory, are routinely exchanged between cells within a tissue. Normally, cells do not divide in the absence of stimulatory signals, and likewise, will cease dividing in the presence of inhibitory

signals. In a cancerous, or neoplastic state, a cell acquires the ability to "override' these signals and to proliferate under conditions in which normal cells would not grow.

In addition to unhindered cell proliferation, cells must acquire several traits for

tumor growth to occur. For example, early on in tumor development, cells must evade the

host immune system. Further, as tumor mass increases, the tumor must acquire vasculature to

supply nourishment and remove metabolic waste. Additionally, cells must acquire an ability

to invade adjacent tissue, and ultimately cells often acquire the capacity to metastasize to

distant sites.

Breast cancer is a major health problem in most industrialized countries. It has been estimated that 184,300 new invasive cases of breast cancer occurred among women in the United States in 1996. In North American women, characteristics that are associated with

a threefold to fourfold increase in risk for breast cancer include (1) first-degree female family

members (mother and sisters) who had breast cancer, (2) prior breast cancer, (3) nulliparity, (4) age greater than 30 years at first pregnancy and (5) early menarche or late menopause

(Sattin, R.W. et al., 1985, JAMA 253:1908). International studies have demonstrated a positive correlation between per capita consumption of fat and alcohol and the incidence of breast cancer. (Carroll and Schatazkin A. et al., 1987, N. Engl. J. Med. 316:1169). Several

studies have linked the consumption of fresh fruits and vegetables, and vitamin E with reduced risk of developing cancer, including breast cancer (Steinmetz, K.A. et al., 1991,

Cancer Causes Control 2:427-442). Although this protective effect has been generally attributed to the antioxidant capacities of vitamin C and beta-carotene present in these foods, it may be related to other phytochemical constituents such as flavonoids. The use of analogues of limocitrins and of 5-desmethyl sinensetin alone or in combination with a cancer

chemo-therapeutic agent has not been reported for the prevention and treatment of neoplastic

diseases. During recent years, conventional therapy used in prevention and treatment of cancer has become increasingly supplemented with substances that are considered as

nutraceuticals or considered as foods or parts of foods but which provide health benefits. As dietary components, the methoxylated flavones are anticipated to have very low cytotoxicity,

and accordingly as cancer treating and/or preventing agents, the methoxylated and ethoxy

flavones show very low toxicity compared with polyhydroxylated flavones. Thus, the

methoxylated and ethoxylated limocitrin analogues and 5-desmethyl sinensetin of the present invention will also have this important advantage over other dietary anti-cancer flavonoids.

The present invention provides a number of different limocitrin compounds

including but not limited to the two naturally occurring methoxylated limocitrin analogues

(3,5,7,4'-tetramethoxylimocitrin and 3,7,4'-trimethoxylimocitrin) and two synthetic limocitrin analogues (limocitrin 3,7,4'-trimethyl ether 5 acetate and limocitrin 3,5,7,4'-tetraethyl ether).

The present invention further provides the structurally-related compound, 5-desmethyl

sinensetin.

Cancers that can be prevented and/or treated by the compositions and methods

of the present invention include, but are not limited to, human sarcomas and carcinomas, e.g. carcinomas, e.g., colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chondroma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,

rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat

gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal

carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung

carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma,

craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,

oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g.,

acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic,

myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera,

lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's

macroglobulinemia, and heavy chain disease. Specific examples of such cancers are described in the sections below.

4.1 Atherosclerosis thrombosis and stroke

In the United States, the complications of artherosclerosis account for about

one half of all deaths and for about one third of deaths in persons between 35 and 65 years of

age. Atherosclerosis, or the development of atheromatous plaques in large and medium-sized

arteries, is the most common form of arteriosclerosis. Many factors are associated with the

acceleration of atherosclerosis, regardless of the underlying primary pathogenic change, for

example, age, elevated plasma cholesterol level, high arterial blood pressure, cigarette smoking, reduced high-density lipoprotein (HDL) cholesterol level, or family history of premature coronary artery disease.

The risk of death from coronary artery disease has a continuous and graded relation to total serum cholesterol levels greater than 180 mg/dl (Stamler, J. et al., 1986, JAMA 256 : 2823). Approximately one third of adults in the United States have levels that

exceed 240 mg/dl and, therefore, have a risk of coronary artery disease that is twice that of people with cholesterol levels lower than 180mg/dl. Acceleration of atherosclerosis is

principally correlated with elevation of LDL, or beta fraction, which is rich in cholesterol but

poor in triglycerides. Elevation of HDL or alpha fraction, has a negative correlation with

atherosclerosis (Castelli, W.P. et al., 1986, JAMA 256 : 2835). HDL exerts a protective

effect and the ratio of total cholesterol to HDL cholesterol is a better predictor of coronary

artery disease than the level of either alone. Total cholesterol levels are classified as being

desirable (<200 mg/dl), borderline high (200-239 mg/dl), or high (>240 mg/dl) (Report of the National Education Program Expert Panel on Detection, Evaluation, and Treatment of High

Blood Cholesterol in Adults, 1988, Arch. Intern. Med. 148 : 36).

Advances in the study of cholesterol metabolism and coronary disease have

initiated an era of increased emphasis on preventive therapy. New guidelines for the detection and treatment of high blood cholesterol in adults recommend that patients with high

cholesterol levels or with borderline-high levels and two or more additional risk factors

should have a measurement of LDL. LDL cholesterol levels are then classified as borderline-

high risk (130-159 mg/dl) or high risk (> 160 mg/dl). Dietary treatment is recommended for

those patients with high-risk levels of LDL and for those with borderline-high risk levels who

have two or more additional risk factors. Drug treatment is recommended for all patients

with LDL levels greater than 189 mg/dl and for those patients with LDL cholesterol levels between 159 and 189 mg/dl who have two or more additional risk factors. Among the many drugs that have been used to reduce serum cholesterol levels are cholestyramine, colestipol, clofibrate, gemfibrozil and lovastatin. The use of limocitrins or 5-desmethyl sinensetin alone

or in combination with a cholesterol-lowering drug has not been reported for the treatment of hypercholestrolemia.

Platelet-blood vessel interactions are implicated in the development of

thrombosis. Flavonoids inhibit platelet aggregation and adhesion ( Frankel, E.N. et al., 1993,

Lancet 341 :1103-1104). Flavonoids antagonize thromboxane formation and increase platelet

cyclic AMP levels. This is important because in addition, flavonoids scavenge free radicals

and their antioxidant actions participate in their antithrombotic action ( Gryglewski, R.J. et al., 1987, Biochem. Pharmacol. 36:317-322). Drug treatment is recommended for patients

with thrombosis and ischemic heart disease. The medical therapy includes, but is not limited

to aspirin and the combined use of beta-adrenergic blocking agents ( e.g., propranonol, nadolol, timolol, etc.), nitrates (e.g., nitroglycerin) and calcium channel blockers (e.g.,

verapamil, nifedipine, diltiazem, etc.).

Four compounds were synthesized from the lemon flavonoid limocitrin 3', 8- dimethoxy-3,5,7,4'-tetrahydroxyflavone in the present invention. Limocitrin occurs in the

peel of lemon as limocitrin-3-0-glucoside, and can be produced from the 3-glucoside by

enzymatic hydrolysis (Horwitz R.M., et al, 1960, J. Org. Chem. 25:21885-21887) or by a

chemical synthesis procedure reported by Dryer D.L., et al., 1964, Tetrahedron 20:2977-2983.

Two limocitrin analogues, 4,3,7-trimethoxylimocitrin and 3,5,6,4'-tetramethoxylimocitrin,

also occur in orange peel (Tatum J.H., et al, 1972, Phytochemistry II: 2283-2288).

The present invention provides a number of different analogues of limocitrin

including 3,5,7,4'-tetramethoxy limocitrin (3,5,7,8,3',4' hexamethoxyflavone or limocitrin- 3,5,7,4'-tetramethyl ether), 3,7,4'-trimethoxylimocitrin (or limocitrin-3,7,4' trimethyl ether),

3,5,7,4'-tetraethoxylimocitrin (or limocitrin 3,5,7,4'-tetraethyl ether), limocitrin-3,7,4' trimethyl ether 5-acetate, 3,7,4' trimethoxy limocitrin (or 5-hydro-3,7,8,3',4' pentamethoxy flavone); and 5-desmethyl sinensetin (or 5-hydroxy-6,7,3',4'-tetramehtoxy flavone).

Flavonoids share the common skeleton of diphenylpyrans, e.g., two benzene

rings A and B, linked tlirough a heterocyclic pyran or pyrone ring C in the middle. This basic

structure allows a multitude of substitution patterns and variations in the different rings,

giving rise to flavonols, flavones, catechins, flavanones, anthocyanidins and isoflavonoids

(Kϋhnau, J., 1976, World Rev. Nutr. Diet 24:117-191).

The basic structures of limocitrin analogues and 5-desmethylsinenstein are similar to the

above flavonoid structure. Specific analogues are represented below:

ensetin

ether

4.2 Dosage and Formulations.

Compounds structurally related to limocitrin and 5-desmethyl sinensetin may be formulated into pharmaceutical preparations for administration to humans for prevention and treatment of neoplastic diseases and/ or cardiovascular disease, atherosclerosis,

thrombosis, myocardial infarction or stroke.

Many of the limocitrin and 5-desmethyl sinensetin compounds may be

provided as compounds with pharmaceutically compatible counterions, a form in which they

may be soluble.

The therapeutic compounds or pharmaceutical compositions may be administered intravenously, intraperitoneally, subcutaneously, intramuscularly, intrathecally,

orally, rectally, topically or by aerosol.

Formulations suitable for oral administration include liquid solutions of the

active compound dissolved in diluents such as saline, water or PEG 400; capsules or tablets,

each containing a predetermined amount of the active agent as solid, granules or gelatin;

suspensions in an approximate medium; and emulsions.

Formulations suitable for parenteral administration include aqueous and non-

aqueous isotonic sterile solutions, which contain buffers, antioxidants and preservatives. The formulations may be in unit dose or multi-dose sealed containers.

Patient dosages for oral administration of limocitrins range from 1-1000 mg/day. commonly 1-500 mg/day, and typically from 1-100 mg/day. Stated in terms of

patient with 70 kg body weight, usual dosages range from 0.01-15 mg/kg/day, commonly

from 0.01-7.0 mg/kg/day, typically from 0.01 to 2.0 mg/kg/day.

Patient dosages for oral administration of synthetic limocitrin analogues range

from 200-5000 mg/day, commonly 1000-2000 mg/day, and typically from 500-1500 mg/day. Stated in terms of patient body weight, usual dosages range from 3-70 mg/kg/day, commonly

from 14-30 mg/kg/day, typically from 7-21 mg/kg/day.

Dosage amount and interval may be adjusted individually to provide plasma

levels of the active moiety which are sufficient to maintain the anti-proliferative, antioxidative and anti-metastatic effects.

Alternatively, one may administer the compound in a local, rather than oral manner, for example, via injection of the compound directly into a tumor, often in a depot or

sustained release formulation.

A variety of delivery systems for the pharmacological compounds may be

employed, including, but not limited to, liposomes and emulsions. The pharmaceutical

compositions also may comprise suitable solid or gel phase carriers or excipients. Examples

of such carriers or excipients include, but are not limited to, calcium carbonate, calcium

phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as

polyethylene glycols.

Furthermore, one may administer the agent in a targeted drug delivery system,

for example, in a liposome coated with tumor-specific antibody. The liposomes will be targeted to and taken up selectively by the tumor.

In cases of local administration or selective uptake, the effective local

concentration of the drug may not be related to plasma concentration.

5. EXAMPLE: EFFECT OF CITRUS LIMOCITRINS IN MDA-MB-435

CELLS AND MCF-7 ESTROGEN RECEPTOR (ER)-POSITIVE HUMAN BREAST CANCER CELLS

The effect of limocitrin analogues on the proliferation and growth of MDA- MB-435 estrogen receptor negative human breast cancer cells was studied in vitro, as

measured by the incorporation of [³H] Thymidine.

Materials

The following compounds were synthesized from the lemon flavonoid

limocitrin according to the procedures described by Horowitz, R.M. et al., J. Org.

Chem.26:2899-2902: 4'3,7-trimethoxylimocitrin (5-hydroxy-3,7,8,3'4'-pentamethoxyflavone);

3,5,7,4'-tetramethoxylimocitrin(3,5,7,8,3',4'-hexamethoxyflavone);3,5,7,4'-tetra-O- ethyllimocitrin; and limocitrin-3,7,4'-trimethyl ether-5-acetate. A total of 23 compounds were tested (See Table 1) in the MDA-MB-435 ER-ve and MCF-7 ER+ve cells. Tissue culture

medium and fetal calf serum were purchased from Gibco, Burlington, ON. [ H] Thymidine was purchased from ICN, Irvine, CA.

Cell Culture

MDA-MB-435 estrogen receptor-negative human breast cancer cells were maintained at 37°C in a minimum essential medium, supplemented with 10% (v/v) fetal bovine serum. The medium was equilibrated with a humidified atmosphere of 5% CO₂. Stock cultures were seeded at a density of 2 x 10⁴ cells/ml and allowed to multiply for 48 to 72 hours. MCF-7 estrogen receptor-positive human breast cancer cells were routinely maintained in minimum essential medium supplemented with 10% (v/v) fetal calf serum, 1 mM sodium pyruvate, lOμg/ml bovine insulin and 1% (v/v) antibiotic-antimycotic agents (10,000 units/ml penicillin G sodium, 10,000 μg/ml streptomycin sulfate and 25 μg/ml amphtericin B in 0.85% saline). Cells were grown to confluence at 37°C in a humidified atmosphere containing 5% CO in air and were passaged weekly, using 2% trypsin in citrate saline.

Incorporation of [3 HI Thymidine into DNA

MDA-MB-435 cells were plated at 2 x 10⁴ cells/well in 96-well, flat bottomed

culture plates in a total volume of 200 μl of medium and incubated at 37°C for 48 hours with

or without test compounds. [³H] Thymidine (0.5 μCi/well) was then added and after 4 hours

the cells were harvested onto a glass fiber filter paper using a semiautomatic 12-well cell

harvester. Radioactivity on the filter paper was counted using Scintiverse in a liquid

scintillation counter. The MCF-7 cells were seeded at a density of 2 x 10⁴ cells/well in 96- well, flat bottomed tissue culture plates and were incubated at 37°C for 5 days. The

compounds were then added at varying concentrations. The plates were incubated at 37°C for

2 days. Tritiated thymidine (0.5 μCi) was then added to each well to determine the number of

dividing cells at each concentration. Four hours later, the medium and excess radioactive

label were removed. Citrate saline with trypsin was added, the cells were harvested onto glass fiber filter paper and the radioactivity was counted. The percent of dividing cells was

determined by comparing the number of cpm for the treated cells to that for the control cells.

Viability of Cells

Viability of cells was measured by MTT assay (Hansen, M.B. et al., J. Imm.

Meth., 119, 203-210). In this assay a tetrazolium salt, MTT, is converted to a blue formazan

product by dehydrogenases that are active in living cells. The intensity of the blue color developed is a measure of cell viability. MDA-MB-435 cells (8xl0⁴/well) were seeded with

various concentrations of the compounds in a 96-well plate in a total volume of 200 μl of medium. MTT (25 μl of 5 mg/ml) was added to each well. After 3 hours, 100 μl of

extraction buffer consisting of 20% SDS dissolved in a DMF water (1/1) solution at pH 4.0 was added. The blue color formed was measured at 590 nm in a Dynatech MRX Microplate

Reader. A similar assay technique is used for the MCF-7 cell line. The percentage of cells

surviving was determined by comparing the absorbance of the treated cells with that of the

control.

Results

Results are the average of 3 experiments. The two synthetic analogues, limocitrin 3,5,7,4'-tetraethyl ether and limocitrin 3,7,4'-trimethyl ether 5-acetate were the

most effective. These compounds have IC₅₀ of 0.5 and 0.9 ppm in estrogen receptor-negative (ER-) and 0.08 and 0.05 ppm in estrogen receptor-positive (ER+) human breast cancer cells respectively. The 3,5,7,4'-tetramethoxylimocitrin had comparable activity in the ER- cells but

was less effective in ER+ cells with IC₅₀ of 0.8 and 0.2 ppm respectively. The 3,7,4'-

trimethoxylimocitrin was the least effective having an IC₅₀ of 3.1 ppm in ER- cells and 0.5 in

ER+ cells (Table 1). Tamoxifen, a drug widely used for the treatment of ER+ tumors has an

IC5₀ of 0.04 ppm in ER+ cells but is not effective in ER- cells (IC₅₀ 90 ppm). Tamoxifen is found to be toxic with serious side effects in some patients. These results indicate that the

limocitrin compounds are potent inhibitors of both ER- and ER+ cells and are less likely to

pose the problems of toxicity and side effects posed by tamoxifen. TABLE 1

IC₅₀s in MDA-MB-435 estrogen receptor-negative and MCF-7 estrogen receptor-positive

human breast cancer cells:

ER- ER+

IC₅₀ (μg/mL)

1. Tangeretin 0.50 0.40

2. Heptamethoxyflavone 0.40 0.80 ^"

3. tetra-O-methylscutellarein 0.30 0.20

4. sinensetin 1.50 0.20

5. nobiletin 0.50 0.80

6. 5,6,7, 8,4'-pentamethoxyflavone 0.50 0.40

7. 5,6,7,8, 3',4'-tetramethoxyflavone 0.50 0.80

8 5 -hydroxy-6 , 7,8,3' ,4'-tetramethoxyflavone 0.30 0.09

9. 5,7,8,4'-tetramethoxyflavone 2.1 1.5

10. 5-hydroxy-6,7,8,3',4'-pentamethoxyflavone 0.90 0.50

11. gossypetin 3,7,8,3'4'-pentamethylether 0.80 0.20

12. 5-desmethyl sinensetin 0.02 0.01

13. quercetin tetramethylether 9.5 5.1

14. quercetin 3,5-dimethylether,7,3',4'-tribenzyl ether 2.3 1.9

15. quercetin tetramethyl ether 9.5 5.1

16. quercetin pentamethyl ether 7.4 7.0

17. quercetin 5,6,3',4'-tetramethylether 3-acetate 3.4 1.2

18. quercetin 5,7,3',4'-tetramethyl ether 9.5 5.1

19. 3,7,4'-trimethoxylimocitrin 3.1 0.5

20. 3,5,7,4'-tetramethoxylimocitrin 0.8 0.2

21. 3 ,5 ,7,4'-tetraethoxylimocitrin 0.5 0.08

22. limocitrin 3,7,4'-trimethyl ether 5-acetate 0.9 0.05

23. Tamoxifen 90 0.04

The present invention is not to be limited in scope by the embodiments

disclosed in the examples which are intended as an illustration of one aspect of the invention and any methods which are functionally equivalent are within the scope of the invention.

Indeed, various modifications of the invention in addition to those shown and described

herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims. Various publications are cited herein, the disclosures of which are incorporated their entireties.

Claims

What is claimed is:

1. A method of preventing or inhibiting the growth of a cancer in an individual comprising administering to an individual in need thereof a therapeutically

effective amount of a composition comprising a limocitrin.

2. The method according to claim 1 wherein the limocitrin comprises 3,5,7,4'-tetramethoxylimocitrin, limocitrin-3,5,7,4'-trimethyl ether-5 acetate, 3,5,7,4'-

tetramethoxylimocitrin or 3,7,4'-trimethoxylimocitrin.

3. The method according to claim 1 further comprising one or more

limocitrins.

4. The method according to claim 1 further comprising an effective

amount of a chemotherapeutic agent.

5. The method according to claim 1 further comprising an effective

amount of tamoxifen.

6. The method according to claim 3 further comprising an effective

amount of a chemotherapeutic agent.

7. The method according to claim 1 wherein the cancer is selected from

the group consisting of colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer,

prostate cancer, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic

sarcoma, chondroma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,

gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary

adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal

cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal

carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acute myelocytic leukemia; chronic leukemia; polycythemia vera, lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain

disease.

8. The method according to claim 1 wherein the composition is

administered orally, intravenously, intraperitoneally, subcutaneously or intramuscularly.

9. A composition suitable for administering to a human subject for the

prevention or treatment of cancer, said composition comprising at least two citrus limocitrins.

10. A method of preventing or inhibiting atherosclerosis in an individual

comprising administering to an individual in need thereof a therapeutically effective amount

of a composition comprising a limocitrin.

11. The method according to claim 10 wherein the limocitrin comprises

limocitrin 3,5,7,4'-tetraethyl ether, limocitrin 3,7,4'-trimethyl ether-5 acetate, 3,5,7,4'- tetramethoxylimocitrin or 3,7,4'-trimethoxylimocitrin.

12. The method according to claim 10 further comprising administering a

therapeutically effective amount of at least two limocitrins.

13. The method according to claim 10 further comprising administering a therapeutically effective amount of a cholesterol-lowering drug.

14. The method according to claim 12 further comprising administering a

therapeutically effective amount of a cholesterol-lowering drug.

15. A method of preventing or inhibiting thrombosis in an individual

comprising administering to an individual in need thereof a therapeutically effective amount of a composition comprising a limocitrin.

16. The method according to claim 15 wherein the citrus limocitrin comprises 3,5,7,4'-tetramethoxylimocitrin, limocitrin-3,5,7,4'-trimethyl ether-5 acetate,

3,5,7,4'-tetramethoxylimocitrin or 3,7,4'-trimethoxylimocitrin.

17. The method according to claim 15 further comprising administering a

therapeutically effective amount of at least two limocitrins.

18. The method according to claim 15 further comprising administering a

therapeutically effective amount of an antithrombotic drug.

19. The method according to claim 17 further comprising administering a

therapeutically effective amount of an antithrombotic drug.

20. A composition suitable for administering to a human subject for the

prevention or treatment of cancer, said composition comprising at least two limocitrins.

21. A method of preventing or inhibiting coronary heart disease in an

individual comprising administering to an individual in need thereof a therapeutically

effective amount of a composition comprising a limocitrin.

22. The method according to claim 21 wherein the citrus limocitrin

comprises 3,5,7,4'-tetramethoxylimocitrin, limocitrin-3,5,7,4'-trimethyl ether-5 acetate, 3,5,7,4'-tetramethoxylimocitrin or 3,7,4'-trimethoxylimocitrin.

23. The method according to claim 21 further comprising administering a

therapeutically effective amount of at least two limocitrins.

24. The method according to claim 21 further comprising administering a

therapeutically effective amount of a cholesterol-lowering drug.

25. The method according to claim 23 further comprising administering a

therapeutically effective amount of a cholesterol-lowering drug.

26. A composition suitable for administering to a human subject for the prevention or treatment of coronary heart disease, said composition comprising at least two

limocitrins.

27. A composition suitable for administering to a human subject for the prevention or treatment of thrombosis, said composition comprising at least two limocitrins.

28. A method comprising administering an effective amount of a limocitrin

compound to cancerous cells to reduce proliferation of said cells.

29. The method according to claim 28 wherein said limocitrin compounds

are selected from the group consisting of 3,5,7,4'-tetramethoxylimocitrin, limocitrin-3, 5,7,4'- trimethyl ether-5 acetate, 3,5,7,4'-tetramethoxylimocitrin or 3,7,4'-trimethoxylimocitrin, and

mixtures thereof.

30. The method according to claim 28 wherein said administering is in

vitro.

31. The method according to claim 1 further comprising 5

desmethylsinensetin.

32. The method according to claim 31 further comprising an effective

amount of a chemotherapeutic agent.

33. The method according to claim 31 further comprising an effective

amount of tamoxifen.

34. The method according to claim 31 further comprising an effective

amount of two or more limocitrins.

35. The method according to claim 31 wherein the cancer is selected from

the group consisting of colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic

sarcoma, chondroma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat

gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal

craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,

oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acute myelocytic leukemia; chronic leukemia; polycythemia

vera, lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease.

36. The method according to claim 31 wherein the composition is administered orally, intravenously, intraperitoneally, subcutaneously or intramuscularly.

37. A method of preventing or inhibiting atherosclerosis in an individual

of a composition comprising a limocitrin and 5-desmethyl sinensetin.

38. The method according to claim 38 further comprising administering a

therapeutically effective amount of a cholesterol-lowering drug.

39. A method of preventing or inhibiting thrombosis in an individual

of a composition comprising a limocitrin and 5 desmehtylsinensetin.

40. The method according to claim 39 further comprising administering a therapeutically effective amount of an antithrombotic drug.

41. A composition suitable for administering to a human subject for the prevention or treatment of cancer, said composition comprising at least a limocitrins and 5 desmethyl sinensetin.

42. A method of preventing or inhibiting coronary heart disease in an

effective amount of a composition comprising a limocitrin and 5 desmethyl sinensetin.

43. The method according to claim 43 further comprising administering a

therapeutically effective amount of a cholesterol-lowering drug.

44. A composition suitable for administering to a human subject for the

prevention or treatment of coronary heart disease, said composition comprising a limocitrin and 5 desmethyl sinensetin.

45. A composition suitable for administering to a human subject for the prevention or treatment of thrombosis, said composition comprising a limocitrin and 5

desmethyl sinensetin.