WO2001078701A2

WO2001078701A2 - Method and compositions for preventing hormone induced adverse effects

Info

Publication number: WO2001078701A2
Application number: PCT/IL2001/000291
Authority: WO
Inventors: Joseph Levy; Yoav Sharoni
Original assignee: Lycored Natural Products Industries Ltd
Current assignee: Lycored Ltd
Priority date: 2000-03-29
Filing date: 2001-03-28
Publication date: 2001-10-25
Anticipated expiration: 2002-09-29
Also published as: IL135335A0; JP2003530428A; DE60120659D1; KR20020093858A; DE60120659T2; US20030148946A1; US20010027216A1; EP1267851A2; NO20024586D0; SE0202857D0; US7144586B2; AU4451101A; JP5020455B2; US20030004146A1; RU2002125508A; EP1267851B1; ATE329586T1; US20040198674A1; AU2001244511B2; WO2001078701A3

Abstract

A method for preventing the adverse effects which may be associated with the administration of at least one hormone to a subject without detectable cancer comprising administering to such subject at least one carotenoid. The method of the instant invention can be utilized to prevent a variety of adverse effects associated with the administration of hormones, including for example, an increased risk for developing cancer. The instantly claimed method prevents such adverse effects without inhibiting the beneficial activity of the hormone. Further provided by the present invention are compositions which are useful for preventing the adverse effects associated with the administration of hormones. The compositions of the instant invention may be in unit dosage form suitable for daily administration to a human.

Description

METHOD AND COMPOSITIONS FOR PREVENTING HORMONE INDUCED

ADVERSE EFFECTS

Field of the Invention

The present invention provides a method and compositions for preventing adverse effects

associated with the administration of hormones such as phytoestrogens and steroidal estrogens.

Background of the Invention Hormone intake by humans can occur through, wter alia, consumption of pharmaceutical

compositions, foodstuffs, nutritional supplements, and nutraceuticals. Such hormones

include phytoestrogens, or nonsteroidal estrogens, steroidal estrogens and progestins. Phytoestrogens comprise, for example, genistein, daidzein and glycitein, and their respective glucoside, malonylglucoside and acetylglucoside derivatives. Estrogens and

progestins are known to be used for hormone replacement therapy (HRT) and in

contraceptive medications. HRT with estrogens or with estrogen/progestin combinations has been the standard method for treating symptoms associated with menopause (Ernster NL et al. (1988) Benefits and Risks of Menopausal Estrogen and/or Progestin Hormone

Use, Prev. Med. 17:201-223). The onset of menopause in mature adult women, which is

accompanied by reduced estrogen production, is associated with an array of symptoms. These symptoms include hot and cold flashes, palpitations, dizziness, headaches, altered

secretions as well as weight loss and gain. Reduced levels of circulating estrogen in

post-menopausal women are also associated with increased risks of osteoporosis and coronary heart disease. Treatment protocols using estrogen alone significantly reduce the risks of cardiovascular disease and osteoporosis, if treatment begins at menopause. The

protective effect of estrogen against heart disease is related to its ability to raise levels of

circulating HDL and lower levels of LDL.

In contrast with this beneficial effect, long-term use of estrogens is positively correlated with an increased risk for endometrial cancer development. This risk may be reduced by

simultaneous administration of a progestin, which prevents overgrowth of endometrial cells. Hence, an estro gen/pro gestin combined HRT protocol is recommended for a woman with an intact uterus. This form of combination therapy however, apparently diminishes

the beneficial effects of estrogen on the plasma lipid profile (Lobo R. 1992. The Role of Progestins in Hormone Replacement Therapy; Am. J Obstet. Gynecol. 166:1997-2004).

Furthermore, some progestins are associated with an increased risk of mammary cancer

development (Staffa J. A. et al. 1991. Progestins and Breast Cancer: An Epidemiologic Review, 57: 473-491; King R. J. B. 1991. A Discussion of the Roles of Estrogen and

Progestin in Human Mammary Carcinogenesis, J. Ster. Biochem. Molec. Bio.

39:8111-8118).

As disclosed in US Patent No. 5,516,528, HRT formulations have been developed which include phytoestrogens such as the soy-derived isoflavones genistein and daidzein. The health benefits of these plant products was first suggested by epidemiologic data indicating

that Asian populations in which soy is a dietary staple suffer relatively low incidences of breast, uterine and other hormone-dependent cancers, ostensibly due to a high intake of

soy and soy-derived products. Although soy isofiavones have been shown to exert anti-pro liferative effects in human

adenocarcinoma and breast cancer cell lines in vitro, these effects occur only at relatively high, i.e. 15 molar (" ") concentrations (Constantino u, A. I. et al. 1998. Genistin Induces

Maturation of Cultured Human Breast Cancer Cells and Prevents Tumor Growth in Nude Mice, Am. J. Clin. Nutr. 68:1426s-1430s; Le BaU, J. C. et al 1998. Estrogenic and

Antipro liferative Activities on MCF-7 Human Breast Cancer Cells by Flavenoids, Cancer

Lett. 130:209-216). The anti-pro liferative effects on cancer cells in vitro caused by phytoestrogens at such high concentrations may not have clinical relevance because the

IC50 values are at least one order of magnitude greater than the blood concentrations

achievable from soy-based diets.

A phytoestrogen concentration range of approximately 0.1 to 10 μM is representative of

that found in healthy humans, both Asian and European, with soy-based diets. (Adlercreutz, H. et al. 1993. Plasma Concentrations of Phyto-oestrogens in Japanese Men,

Lancet 342:1209-1210; Gooderham et al, 1996. A Soy Protein Isolate Rich in Genistein and Daidzein and its Effects on Plasma Isoflavone Concentrations, Platelet Aggregation, Blood Lipids and Fatty Acid Composition of Plasma Phospholipid in Normal Men, J. Nutr.

125:2000-2006). At these lower concentrations, various phytoestrogens, including genistein, counestrol, biochanin A, apigenin, luiolin, kaempferl and enterolactone, were

shown to induce cell proliferation in estrogen receptor-positive, but not in estrogen

receptor negative human breast cancer cell lines, thus demonstrating the estrogenic effects

of these compounds (Wang, C. and Kurtzer, M. S. 1997. Phytoestrogen concentration

Determines Effects on DNA synthesis in Human Breast Cancer Cells, Nutr. Cancer

28:236-247). Hence, although phytoestrogens have been disclosed as beneficial components for HRT

formulations, it has been found that the presence of phytoestrogens at levels normally

found in healthy humans increases the risk for development of hormone-dependent

cancers.

The carotenoid astaxanthin has been demonstrated to have anti-tumorigenic effects in vivo in rodent models (Tanaka, T. et al. 1995. Suppression of azoxymethane-induced rat colon

carcinogenesis by dietary administration of naturally occurring xanthophylls astaxanthin and canthaxanthin during the postinitiation phase. Carcinogenesis 16: 2957-2963; Tanaka,

T. et al. 1995. Chemoprevention of rat oral carcinogenesis by naturally occurring xanthophylls, astaxanthin and canthaxanthin. Cane. Res. 55:4059-4064).

The carotenoid phytoene has also demonstrated anti-cancer activity. The cancer

preventive activity of phytoene was demonstrated following introduction of the gene encoding phytoene synthase into mammalian cells normally lacking this gene. These cells acquired resistance against malignant transformation imposed by transfection of activated

oncogenes (Nishino, H. 1998. Cancer prevention by carotenoids. Mutat. Res.

402:159-163).

β-carotene was the first carotenoid suggested to have anti-cancer properties (Peto et al.

1981. Carotenoids and cancer: an update with emphasis on human intervention studies,

Nature 290:201-208). Epidemio logical studies of β-carotene's effect on cancer, however, have produced conflicting results. Although some studies have showed that β-carotene

increases the risk for developing cancer (Omenn et al., 1996. Effects of a combination of

beta-carotene and vitamin A on lung cancer and cardiovascular disease, N. Engl. J. Med

334:1150-1155), other cell culture and animal studies have indicated quite consistently

that β-carotene has an anti-carcinogenic effect.

Lycopene, a carotenoid found in tomatoes, is strongly associated with anti-oxidant and anti-cancer activities. The anti-proliferative effects of lycopene on breast cancer cells in

vitro has been shown to be mediated through interference with the IGF-1 receptor

signaling pathway and cell cycle progression (Karas et al. 2000. Lycopene interferes with

cell cycle progression and insulin-like growth factor I signaling in mammary cancer cells. Nutr. Cancer, 36:101-11). IGF-I is a growth factor obligatory for malignant transformation of breast tissue, and its concentration in plasma determines risk factor for

cancers of both the breast (LeRoith, D., Werner, H., Beitner- Johnson, D. and Roberts, C.T., Jr. 1995. Molecular and cellular aspects of the insulin-like growth factor I receptor.

Endocr. Rev. 16:143-59; Hankinson S.E. et al. 1998. Circulating concentrations of insulin-like growth factor-I and risk of breast cancer. Lancet 351:1393-6) and prostate (Chan, J. M., Stampfer, MJ. Giovannucci, E., Gann, P.H., Ma, J. 1998 Plasma insulin-like

growth factor-I and prostate cancer risk: a prospective study. Science 279:563-66).

U.S. Patent No. 5,827,900 discloses the use of lycopene for inhibiting the growth of cancers in vitro and in vivo, including hormone-dependent endometrial and breast cancers.

U.S. 5,827,900 requires very high carotenoid dosage levels. The '900 Patent discloses 7 mg/Kg to 20 mg/Kg per day as illustrative lycopene dosages. The method of the '900 Patent would thus require 490 mg - 1400 mg of lycopene per day for a person weighing

70 kg (154 lbs.).

The combination of lycopene and soy isoflavones in dietary supplements has been

disclosed in U.S. 5,904,924. The '924 patent discloses a nutritional powder composition

comprising soy isoflavones (phytoestrogens) and lycopene. The '924 patent only refers to

the ability of phytoestrogens to decrease the risk of estrogen dependent cancers. Nowhere

does the '924 patent disclose that dietary intake of phytoestrogens incurs a risk for

adverse health affects, and that such risk can be reduced by the concomitant consumption

of carotenoids.

The use of phytoestrogens in dietary supplements has been disclosed in U.S. Patent Nos. 5,830,887 and 5,807,586. The combination of carotenoid mixtures, vitamin A and

phytoestrogens in dietary supplements for women was disclosed in U.S. 5,807,586. The dosage amounts of vitamin A and mixed carotenoids disclosed in the '586 Patent range

from about 400 to about 2000 μg retinol equivalents ("RE"). 1 RE is equivalent to about

6 μg beta-carotene and about 12 μg alpha-carotene or cryptoxanthin. The dosage range

disclosed in the '586 Patent is thus approximately equivalent to about 2.4 - 12 mg of beta-carotene and about 4.8 - 24 mg of alpha-carotene or cryptoxanthin. Since lycopene

and lutein do not exhibit substantial provitamin A activity, the RE for these carotenoids

cannot be calculated. The dosage levels disclosed in the '586 Patent, however, are

expressed only in RE units. The disclosure of the '586 Patent thus does not limit dosage

levels of carotenoids which do not exhibit substantial provitamin A activity, such as

lycopene and lutein. The instant invention is directed to a method and compositions useful for preventing

adverse effects which may be associated with the intake of pharmaceutical compositions,

foodstuffs, nutritional supplements, or nutraceuticals comprising hormones such as

estrogens, phytoestrogens and progestins. Such adverse effects include, but are not limited to, the increased risk of various types of cancer. The administration of

phytoestrogens has been previously disclosed as beneficial in decreasing the risks for

developing cancer. It has been found, however, that intake of phytoestrogens can incur an

increased risk for developing hormone-dependent cancers.

There is thus a long felt need for a method and a composition useful for preventing the adverse effects which may be associated with the intake of foodstuffs, pharmaceutical compositions, nutritional supplements, and nutraceuticals comprising phytoestrogens,

steroidal estrogens, and/or progestins. A need also exists for compositions which can be used to prevent the adverse effects associated with the adrninistration of pharmaceutical

compositions containing hormones such as, for example, steroidal estrogens and progestins, without inhibiting the beneficial activity of such hormones.

It is therefore a purpose of the present invention to provide a method for inhibiting the

adverse effects which may be associated with the consumption of food stuff or dietary

supplements comprising phytoestrogens and progestins . It is another purpose of the present invention to provide a composition useful for

preventing the adverse effects which may be associated with the consumption of food stuff

or dietary supplements comprising phytoestrogens and progestins.

It is yet a further purpose of the present invention to provide a novel hormone-comprising

pharmaceutical composition with reduced side effects relating to adverse effects that the hormones may have, thus overcoming the disadvantages of the pharmaceutical

composition known from the prior art.

Other objects of the invention will become apparent as the description proceeds.

Summary of the Invention

The present invention provides a method for preventing the adverse effects which may be associated with the administration of at least one hormone to a subject without detectable

cancer comprising administering to such subject at least one carotenoid. The method of

the instant invention can be utilized to prevent a variety of adverse effects associated with

the adrninistration of hormones, including, for example, an increased risk for developing cancer. The instantly claimed method prevents such adverse effects without inhibiting the

beneficial activity of the hormone.

Accordingly, an object of the instant method is to prevent the adverse effects associated with administration of estrogen and/or progestin in hormone replacement therapy without

inhibiting the beneficial activity of such hormone. Another object of the present invention is to provide a method for preventing the adverse

effects associated with the adrninistration of phytoestrogens. It is yet another object of the

presently claimed method to prevent adverse effects associated with the administration of

phytoestrogens without inhibiting the beneficial activity of such hormone.

Further provided by the present invention are compositions which are useful for

preventing the adverse effects associated with the administration of hormones. The

compositions of the instant invention may be in unit dosage form suitable for once daily

administration to a human.

An object of the instant invention is to provide compositions for preventing adverse effects

associated with the administration of phytoestrogens.

A further object of the present invention is to provide compositions for preventing adverse

effects associated with the adrninistration of steroidal hormones or progestins.

Yet another object of the instant invention is to provide compositions that are useful for preventing adverse effects associated with the adrninistration of phytoestrogens, steroidal

hormones or progestins, and that do not inhibit the beneficial activity of such hormones.

Description of the Drawings

Fig. 1 - Stimulation of ECC-1 endometrial cancer cell proliferation by isoflavenoids and

soy extract. Fig. 2 - Lycopene inhibits both genistein- and estradiol-induced stimulation of hormone

dependent malignant cells.

Fig. 3 - Effect of lycopene on mammary cancer cells cultured in the presence of genistein

and IGF- 1.

Fig. 4 - Various carotenoids inhibit estradiol- and genistein-induced proliferation of ECC-1

endometrial cancer cells.

Fig. 5. - Lycopene inhibits in a dose dependent manner estradiol- and genistein-induced

proliferation of ECC-1 endometrial cancer cells.

Fig. 6. - Combined effect of lycopene and phytoene on genistein-induced proliferation of ECC cells.

Detailed Description of a Preferred Embodiment of the Invention

The following description is illustrative of preferred embodiments of the invention. The following description is not to be construed as limiting, it being understood that the skilled

person may carry out many obvious variations to the invention.

As used throughout this specification, "hormone" refers to steroidal estrogens, progestins,

and nonsteroid estrogens (phytoestrogens) derived from higher plants, as well as

chemically modified derivatives, synthetic equivalents, and mixtures thereof. As used throughout this specification, "steroidal estrogen" or "estrogen" refers to

estradiol, estrone, estriol, synthetic equivalents, chemically modified derivatives, and

mixtures thereof.

As used throughout this specification, "progestin" refers to agents that cause progesterone

effects, such as, for example, progesterone, medroxyprogesterone, norethindrone,

norethisterone, norgestrel, synthetic equivalents, chemically modified derivatives, and

mixtures thereof.

As used throughout this specification, "phytoestrogen" refers to soy protein isoflavones,

flavones, as well as the glucoside, malonylglucoside and acetylglucoside derivatives, synthetic equivalents, chemically modified derivatives, and mixtures thereof. Illustrative

phytoestrogens include, for example, daidzein, genistein, and glycitein.

As used throughout this specification, "administration" or "adrninistering" refers to the introduction to a subject by one or more of various routes, including oral ingestion,

dermal, vaginal, intrauterine, intramuscular or intravenous injection.

Carotenoids useful for the instant invention comprise, but are not limited to, lycopene,

alpha-carotene, beta-carotene, zeta-carotene, phytoene, phytofluene, lutein, zeaxanthin,

cryptoxanthin astaxantine, and mixtures thereof. The carotenoids referred to throughout the specification are from natural or synthetic sources or from genetically modified

organisms. The present invention provides a method for preventing the adverse effects which may be

associated with the administration of at least one hormone to a subject without detectable

cancer comprising administering to such subject at least one carotenoid in an amount

effective to prevent such adverse effects. The carotenoid can be administered in a

composition separate from the hormone or in a composition further comprising the

hormone.

A single carotenoid as well as combinations and mixtures thereof can be administered in

the method of the present invention. It has surprisingly been found that the various combinations of lycopene, phytoene and phytofluene demonstrate a beneficially synergistic

effect in preventing the adverse effects associated with the administration of hormones. Accordingly, carotenoid mixtures of lycopene and phytoene; lycopene and phytofluene; and lycopene, phytoene, and phytofluene can be administered in the presently claimed

method.

Upon consuming phytoestrogen-containing products, the physiological concentration of

these phytoestrogens in the subject's blood serum can reach levels of 0.4 to 4 μM. Figure

1 panel A demonstrates that incubation of ECC-1 endometrial cancer cells in the presence

of the phytoestrogens genistein or daidzein, or a mixture of the two as occurs in soy extract, at such a concentration range, induces a significant increase in cell proliferation

rate (indicated by cpm, counts per minute). Hence, subjects who reach these

phytoestrogen concentrations increase their risk of certain types of cancer, inter alia,

endometrial and mammary cancer. Figure 2 demonstrates this effect by comparing the cell proliferation rate of cancer cells

incubated in the presence of increasing concentrations of the phytoestrogen genistein and

estradiol both in the presence and absence of lycopene. It is clearly shown that the

increased rate of cell proliferation induced by genistein is substantially inhibited by the

presence of lycopene. The same effect is seen in both endometrial cancer cells (Fig. 2,

upper graphs) and mammary cancer cells (Fig. 2, lower graphs). This inhibiting effect is further demonstrated in Fig. 4 where it can be clearly seen that not only lycopene, but also

carotenoids such as astaxanthin, phytoene and beta-carotene are effective inhibitors of the

cell proliferation induced by the phytoestrogen genistein.

Physiological concentrations of phytoestrogens to levels greater than about 10 μM can

occur immediately following consumption of foodstuffs or dietary supplements comprising

phytoestrogens. Such levels are greater than 0.4 to 4 μM, which is the steady state

physiological concentration range of phytoestrogens in humans who have consumed

phytoestrogens. Figure 1, panel B demonstrates that such transiently high concentrations

of phytoestrogens can induce cell proliferation and thus increase the risk for cancer. Figure 1, panel B demonstrates that incubation of ECC-1 endometrial cancer cells with 40

μM daidzein or genistein, or a mixture of the two as occurs in soy extract, significantly

increases cell proliferation within the first day in culture. The inhibitory effect of the

phytoestrogens was significant only from the second day of incubation. The methods of

the present invention operate to prevent the adverse effects of such elevated

phytoestrogen levels which can occur immediately subsequent to consumption of

phytoestrogen-containing products or compositions. According to an embodiment of the invention, the adverse effects associated with the

administration of at least one phytoestrogen to a subject without detectable cancer are

prevented by co-administering at least one carotenoid in an amount effective to prevent

such adverse effects. The method of the present invention encompasses preventing such

adverse effects without inhibiting the beneficial activity of said phytoestrogens.

The carotenoid is administered in an amount from about 2 mg to about 10 mg per day, preferably from about 2 mg to about 6 mg per day, most preferably about 2 mg per day.

Carotenoids that do not exhibit substantial provitamin A activity such as, for example, zeta-carotene, phytoene, phytofluene, lutein, zeaxanthin, astaxantine, and lycopene, are

preferably administered in an amount of about 2 mg per day.

Where the method of the instant invention comprises administering to a subject at least one carotenoid selected from the group consisting of alpha-carotene, beta-carotene, and

cryptoxanthin, said carotenoid is preferably administered in an amount of about 2 mg per

day.

The method of the present invention can also be utilized to prevent adverse effects

associated with the administration of hormones such as estrogen and progestin to a subject

without detectable cancer. Such method can be used, for example, in hormone

replacement therapy, whereby the hormone is co -administered with at least one carotenoid

in an amount sufficient to prevent adverse effects associated with the adrninistration of such hormone. The carotenoid can be administered in a composition separate from such

hormone or in a composition further comprising such hormone.

The presently claimed method is particularly helpful for hormone replacement therapy.

Hormone replacement therapy with estrogen alone, or estrogen replacement therapy, can

incur a risk for developing endometrial cancer. In an attempt to reduce this risk, hormone

replacement therapy comprising of a combined estrogen/progestin administration is often

utilized. This form of therapy, however, can diminish the beneficial effects of estrogen.

Furthermore, some progestins are associated with an increased risk for developing mammary cancer. The method of the instant invention can prevent the adverse effects

associated with the adrninistration of estrogen and progestin, and can do so without inhibition of the beneficial activity of such hormones as occurs in conventional hormone replacement therapy. In another embodiment, the method of the present invention is

used to prevent the additive adverse effects caused by elevated levels of IGF- 1. IGF-1 occurs naturally in the serum of normal, healthy individuals. The occurrence of IGF-1 at

elevated levels, however, constitutes a significant risk factor for cancers of the breast and

prostate. The risk for developing cancer associated with the administration of hormones is thus increased in an individual who has elevated IGF-1 levels. The additive adverse effects

caused by a combination of elevated IGF-1 levels and consumption of hormones are

prevented by administering at least one carotenoid in an amount sufficient to prevent such

adverse effects.

In yet another embodiment, the method of the instant invention is utilized to prevent the

adverse effects associated with elevated levels of IGF-1 in the absence of hormones. The presence of IGF-1 at elevated levels constitutes a significant risk factor for certain cancers

even in the absence of hormones. These adverse effects, including increasing the risk for

developing cancer, which are caused by elevated IGF-1 levels can be prevented by

administering at least one carotenoid in an amount sufficient to prevent such adverse

effects.

The present invention provides compositions useful for preventing adverse effects

associated with the adrninistration of hormones. Such adverse effects include, for

example, an increased risk for the development of cancer. The compositions of the instant invention are in a unit dosage form suitable for administration to a human. The compositions comprise a physiologically effective amount of at least one hormone and at

least one carotenoid in an amount effective to prevent such adverse effects. The instantly

claimed compositions can prevent the adverse effects associated with the adrninistration of hormones without inhibiting the beneficial activity of such hormones.

A single carotenoid as well as combinations and mixtures thereof can be used in the compositions of the present invention. It has surprisingly been found that various combinations of lycopene, phytoene and phytofluene demonstrate a beneficially synergistic effect in preventing the adverse effects associated with the adrninistration of hormones.

Accordingly, carotenoid mixtures of lycopene and phytoene; lycopene and phytofluene;

and lycopene, phytoene, and phytofluene can be used in the presently claimed

compositions. When administering such a mixture, a mixture of lycopene and phytoene is

preferred.

\ The compositions of the present invention operate to prevent the adverse effects of

elevated phytoestrogen levels which can occur immediately subsequent to consumption of

phvtoestrogen-containing products or compositions (see Fig. 1 and 2).

Co-administering at least one carotenoid to a phytoestrogen-consuming subject in an

amount of about 2 mg per day attenuates the risk for cancer which is associated with the

consumption of phytoestrogens. The present invention encompasses a unit dosage form

suitable for once daily administration to a human comprising a physiologically effective

amount of at least one phytoestrogen and at least one carotenoid, preferably in an amount

of about 2 mg. Preferably, the carotenoid is in an amount sufficient to cause effective serum concentrations of said carotenoid of up to about 1.5 μM.

Where the composition of the present invention comprises at least one phytoestrogen, the carotenoid is preferably one which does not exhibit substantial provitamin A activity.

Carotenoids which do not exhibit substantial provitamin A activity include, for example,

lycopene, zeta-carotene, phytoene, phytofluene, lutein, zeaxanthin, and astaxantine.

Where the carotenoid comprises a mixture of carotenoids, such mixture preferably comprises lycopene and phytofluene; more preferably lycopene, phytofluene, and

phytoene; most preferably lycopene and phytoene.

A further embodiment of the instant invention is a unit dosage form suitable for once daily

administration to a human comprising a physiologically effective amount of at least one

hormone selected from the group consisting of steroidal estrogen, estradiol, estrone.

medroxyprogesterone, norethindrone, norethisterone, progestin, norgestrel, and progesterone, and at least one carotenoid present in an amount effective to prevent the

adverse effects which may result from the administration of such hormone. This unit

dosage form can prevent the adverse effects associated with the adrninistration of such

hormones without inhibiting the beneficial activity of such hormones. Preferably, the

carotenoid is in an amount from about 2 to about 10 mg, more preferably from about 2 to

about 6 mg, most preferably about 2 mg. Preferably, the carotenoid is an amount

sufficient to cause effective serum concentrations of said carotenoid of up to about 1.5

μM. Where the carotenoid comprises a mixture of carotenoids, such mixture preferably comprises lycopene phytoene and phytofluene; more preferably lycopene, phytofluene, and

phytoene.

The instantly claimed compositions that comprise at least one steroidal estrogen or

progestin are particularly helpful for hormone replacement therapies used to treat menopause in women. Conventional hormone replacement therapies used to treat

menopause can increase the risk for developing certain cancers. The compositions of the

present invention can be used in hormone replacement therapies, while also preventing adverse effects such as the risk for developing cancer which can be associated with the administration of steroidal estrogens or progestins. The presently claimed compositions

can prevent such adverse effects without inhibiting the beneficial activity of the hormones.

The compositions of the present invention can be used to prevent the additive adverse

effects on cell proliferation incurred by elevated levels of the growth factor IGF-1

hormone intake in the presence of phytoestrogens. IGF-1 occurs naturally in the serum of

normal individuals, but its occurrence at elevated levels significantly increases the risk for developing cancers of the breast and prostate. Fig. 3 panel A demonstrates that incubation

of hormone dependent MCF-7 mammary cancer cells with both genistein (in the range

from about 0.4 to 4 μM) and IGF-1 (30 nM) significantly increases the cell proliferation

rate, as compared to control cultures stimulated only with genistein. Addition of lycopene

in the range of 3 to 5 μM inhibits the cell proliferation stimulated by the combined

presence of IGF-1 and genistein. Accordingly, a composition of the instant invention

comprising at least one phytoestrogen and at least one carotenoid, can be administered to

a subject with elevated IGF-1 levels in order to prevent the additive adverse affects caused

by such elevated IGF-1 levels.

The present invention will now be further explained in the following examples, which Further describe, but do not limit the scope of the invention.

Examples

General Procedures

Carotenoid sources and solutions

Lycopene (> 97%) was extracted from 5% tomato oleoresin as in US 5,827,900. Synthetic

lycopene was purchased from Sigma Chemical Co. (Israel), as was astaxanthin and

beta-carotene. Phytoene was extracted from tomato extract at Lycored Natural Products

Industries Ltd., Beer Sheva, Israel.

Tetrahydrofuran (THF) containing 0.25% butylated hydroxytoluene was added to purified

carotenoids as an antioxidant. Carotenoids were dissolved in THF at a concentration of 2

mM and stored at -70°C. Immediately before use, stock solutions were thawed and added to the cell culture medium under nitrogen, followed by vigorous stirring. Precipitates

formed were removed by filtration through Millex-HN filter (Millipore). Final carotenoid

concentrations in the medium were determined by spectrophotometry after extraction in 2-propanol and n-hexane:dichloronomethane, as in US patent no. 5,827,900.

The final THF concentration of 0.5% did not have any significant effect on the measured

parameters. All procedures were carried out under dim lighting.

Hormones, growth factors and other cell culture materials

Estradiol, genistein and daidzein were purchased from Sigma Chemical Co. (Israel). Soy extract containing 15% genistein, 15%o daidzein and 1% glycitein was purchased from

Kikkoman, (Chiba-ken, Japan). Human recombinant IGF-1 was purchased from GroPep, (Adelaide, Australia). Dulbecco's modified Eagle's medium (DMEM), fetal calf serum (FCS) and Ca²⁺/Mg²⁺-free phosphate buffered saline (PBS) were purchased from

Biological Industries (Beth Haemek, Israel).

Cell lines

The human endometrial cancer (estrogen dependent) cell line ECC-1 was developed by Dr. P. G. Satyaswaroop, Pennslyvania State University, PA, U.S. A, and generously

provided to us by Dr. R. Oregan, Norwestem University, Chicago, IL, U.S.A. Human

mammary cancer cell lines MCF-7 (estrogen dependent) and MDA-231 (hormone

independent) were obtained from the American Type Culture Collection (Rockville, MD,

U.S.A.). Cell culture and cell proliferation assay

Cells were cultured in DMEM containing penicillin (100 U/ml), streptomycin (0.1

mg/ml)) nystatin (12.5 μg/ml), insulin (0.6 μg/ml), and 10% FCS. Cells were stripped of

endogenous steroids according to the procedure of Nignon et al. (1987, Biochem.

Biophys. Res. Comm., 146:1502-8) by sucessive passages in medium without phenol red

containing 10% and then 3% of charcoal-stripped FCS. Cells were seeded into 96

multiwell plates (5,000 cells per well) in DMEM containing 3% of charcoal-stripped FCS.

After one day the medium was changed to one containing the solubilized carotenoid or

THF alone. The medium was changed daily to ensure a continuous supply of carotenoid.

After incubation, the number of cells and the rate of cell proliferation were estimated by the incorporation of [³H]thymidine incorporation into cellular DΝA, as described in US

patent no. 5,827,900. Cell growth was also measured by direct cell counting with a

Coulter counter after trypsinization and dilution in Isotone-II (Coulter Electronics, Luton, England). A good correlation was found between the two methods.

Example 1

Stimulation of ECC-1 endometrial cancer cell proliferation by isoflavonoids.

ECC-1 cells were incubated with increasing concentrations of the isoflavones genistein and

daidzein, the two primary isoflavones in soy products, and with soy extract. Cell

proliferation after three days was measured by [³H]thymidine incorporation into DΝA

(Fig. 1, panel A). Genistein, daidzein and soy extract all stimulated cell proliferation at 0.4

and 4 μM concentrations, which are in the ranges found in the plasma of soy

supplemented individuals. At higher, non-physiological concentrations (>10 μM), genistein and soy extract but not daidzein, inhibited cell proliferation after three days in culture (Fig.

1, panel A). These results demonstrate that genistein has biphasic effects on endometrial

cancer cell growth, while daidzein is only stimulatory.

ECC-1 cells were also incubated with a single high concentration (40 μM) of each of genistein, daidzein and soy extract and cell proliferation was assayed daily over the course

of three days. The results, presented in Fig. 1, panel B show that after one day of

incubation this high isoflavone concentration also stimulated cell proliferation, while

inhibitory effects of genistein were seen only by the second day in culture. These results

suggests that transiently elevated levels of isoflavones, particularily genistein, to levels normally associated with cell growth inhibition, may in fact be stimulatory in soy

supplemented individuals.

Example 2

Lycopene inhibits both genistein and estradiol stimulation of hormone-dependent

malignant cells.

The comparative effects of estradiol and genistein supplementation on the proliferation of the hormone-dependent cell lines MCF-7 mammary cancer and ECC-1 endometrial cancer

were examined (Fig. 2). In each of these cell lines, genistein exhibited biphasic effects on proliferation, stimulating at low concentrations and inhibiting at high concentrations (Fig.

2), as demonstrated in Example I. Estradiol at each of the concentrations tested (1 and 10

nM) was only stimulatory for cell growth (Fig 2). These results suggest that the

stimulatory effect of genistein may be due to its estrogenic action. Cell cultures stimulated either by genistein or estradiol, as described above, were further

supplemented with lycopene and assayed for cell proliferation after three days in culture.

As shown in Fig. 2, lycopene supplementation at 3 to 5μM significantly inhibited both

basal growth and estrogen-induced (either genistein or estradiol) growth in both of the

hormone-dependent cancer cell lines tested.

Example 3

Lycopene inhibits IGF-1 -stimulated growth in hormone-dependent and

hormone-independent mammary cancer cells. Fig. 3 shows that IGF-1 (30 nM) supplementation of both hormone-dependent MCF-7 mammary cancer cells (panel A) and hormone-independent MDA-231 mammary cancer

cells (panel B) significantly stimulates cell growth. In MCF-7 cells, the stimulatory effect

of genistein is further augmented in the presence of IGF-I (Fig. 3, panel A). MDA-231 is stimulated by IGF-I, but not by genistein. Thus genistein not only stimulates

hormone-dependent cancer cell proliferation, but IGF-I as well as other growth factors

further augment this effect.

Cell cultures supplemented as above were further supplemented with lycopene at 3 to 5

μM concentration. As shown in Fig 3, lycopene inhibits IGF stimulation in both

hormone-dependent and hormone-independent mammary cancer cell lines. In the case of

MDA-231 cells, cell proliferation was reduced to levels less than that of controls. Example 4

Various carotenoids inhibit estradiol and genistein induced proliferation of ECC-1 cells.

The ECC-1 hormone-dependent cell line was stimulated either by estradiol (10 nM, Fig 4)

or by genistein (1 μM, Fig 4) and test cultures were additionally supplemented with

various carotenoids. The results clearly demonstrate that all carotenoids tested (lycopene, beta-carotene, astaxanthin and a mixture of phytoene and phytofluene) effectively inhibited

both estradiol- and genistein-induced cell proliferation.

Example 5

Lycopene inhibits in a dose dependent manner estradiol and genistein induced proliferation

of ECC-1 endometrial cancer cells.

The hormone-dependent endometrial cancer cell line ECC-1 was stimulated either by

estradiol (10 nM, Fig 5) or by genistein (1 μM, Fig 5) and test cultures were additionally

supplemented with various concentrations of lycopene. The results clearly demonstrate that while increasing lycopene concentration resulted in greater inhibition, all lycopene

concentrations tested were effective in inhibiting both estradiol- and genistein-induced cell

proliferation. The lowest lycopene concentration tested (0.9 μM) is in the physiological

range found in human serum.

Example 6

MCF-7 mammary cancer cells were stimulated by genistein (4 μM) and test cultures were

additionally supplemented with lycopene or phytoene or a combination of both, at either

physiological concentrations (Fig. 6 panel A) or at about one order of magnitude greater than the physiological concentrations (Fig. 6 panel B). The results demonstrate that high,

non-physiological concentrations of the individual carotenoids were effective in

significantly inhibiting phyto estrogen-induced cell proliferation, and the combination of the

two was even more effective(Fig. 6 panel B). At lower physiological concentrations,

lycopene was only marginally effective in inhibiting phyto estrogen-induced cell

proliferation, and phytoene was not effective. The combination of lycopene and phytoene

however, at physiological concentrations, was effective in significantly inhibiting

phyto estrogen-induced cell proliferation.

While embodiments of the invention have been described by way of illustration, it will be

apparent that the invention may be carried out with many modifications, variations and adaptations, without departing from its spirit or exceeding the scope of the claims.

Claims

1. A method of preventing adverse effects which may result from the administration

of at least one hormone to a subject without detectable cancer comprising administering to said subject at least one carotenoid in an amount from about 2 to

about 10 mg per day.

2. The method of claim 1 in which the carotenoid is administered in an amount from

about 2 to about 6 mg per day.

3. The method of claim 1 in which the carotenoid is administered in an amount of

about 2 mg per day.

4. A method of preventing adverse effects which may result from the adrninistration

of at least one hormone to a subject without detectable cancer comprising administering to said subject at least one carotenoid which does not exhibit substantial provitamin A activity in an amount of about 2 mg per day.

5. A method of preventing adverse effects which may result from the administration

of at least one hormone to a subject without detectable cancer comprising

administering to said subject at least one carotenoid selected from the group

consisting of alpha-carotene, beta-carotene and cryptoxanthin in an amount of

about 2 mg per day.

6. The method of any of claims 1, 4 or 5 wherein said carotenoid is in an amount

sufficient to cause an effective serum concentration of said carotenoid of up to

about 1.5 μM.

7. The method of any of claims 1, 4 or 5 wherein said hormone comprises phytoestrogen, nonsteroidal estrogen, or a mixture thereof.

8. The method of any of claims 1, 4 or 5 wherein said hormone comprises steroidal

estrogen, progestin, or a mixture thereof.

9. The method of claim 1 or 4 wherein said carotenoid is lycopene.

10. The method of claim 1 or 4 wherein said carotenoid is selected from the group consisting of lycopene, zeta-carotene, phytoene, phytofluene, lutein, zeaxanthin,

and astaxantine.

11. The method of any of claims 1 - 4 wherein said carotenoid comprises a mixture of

lycopene and phytoene.

12. The method of any of claims 1 - 4 wherein said carotenoid comprises a mixture of

lycopene and phytofluene.

13. The method of any of claims 1 - 4 wherein said carotenoid comprises a mixture of

lycopene, phytoene and phytofluene.

14. The method of any of claims 1, 4 or 5 wherein said carotenoid is co-administered

with said hormone in a composition separate from said hormone.

15. The method of any of claims 1, 4 or 5 wherein said carotenoid is co-administered

with said hormone in a composition further comprising said hormone.

16. A method of preventing adverse effects which may result from the administration of at least one hormone selected from group consisting of estrogen, estradiol, estrone, medroxyprogesterone, norethindrone, norethisterone, norgestrel,

progestin, and progesterone to a subject without detectable cancer comprising administering to said subject at least one carotenoid which does not exhibit

substantial provitamin A activity in an amount from about 2 to about 10 mg per

day without inhibiting the beneficial activity of said hormone.

17. A method of preventing adverse effects which may result from the administration

of at least one hormone selected from the group consisting of estrogen, estradiol, estrone, medroxyprogesterone, norethindrone, norethisterone, norgestrel,

progestin, and progesterone to a subject without detectable cancer comprising

administering to said subject at least one carotenoid which does not exhibit

substantial provitamin A activity in an amount from about 2 to about 6 mg per day

without inhibiting the beneficial activity of said hormone.

18. A method of preventing adverse effects which may result from the administration

of at least one hormone selected from the group consisting of estrogen, estradiol,

estrone, medroxyprogesterone, norethindrone, norethisterone, norgestrel, progestin, and progesterone to a subject without detectable cancer comprising

administering to said subject at least one carotenoid which does not exhibit

substantial provitamin A activity in an amount of about 2 mg per day without inhibiting the beneficial activity of said hormone.

19. A method of preventing adverse effects which may result from the administration of at least one hormone selected from the group consisting of estrogen, estradiol,

estrone, medroxyprogesterone, norethindrone, norethisterone, norgestrel,

progestin, and progesterone to a subject without detectable cancer comprising administering to said subject at least one carotenoid selected from the group

consisting of alpha-carotene and cryptoxanthin in an amount of about 2 mg per day without inhibiting the beneficial activity said hormone.

20. The method of any of claims 16 - 18 wherein said carotenoid is selected from the

group consisting of lycopene, zeta-carotene, phytoene, phytofluene, lutein,

zeaxanthin, and astaxantine.

21. The method of any of claims 16 - 18 wherein said carotenoid comprises a mixture

of lycopene and phytoene.

22. The method of any of claims 16 - 18 wherein said carotenoid comprises a mixture

of lycopene and phytofluene.

23. The method of any of claims 16 -18 wherein said carotenoid comprises a mixture of lycopene, phytoene and phytofluene.

24. The method of any of claims 16 - 19 wherein said carotenoid is co-administered

with said hormone in a composition separate from said hormone.

25. The method of any of claims 16 - 19 wherein said carotenoid is co-administered

with said hormone in a composition further comprising said hormone.

26. A unit dosage form suitable for adrninistration to a human comprising a physiologically effective amount of at least one hormone selected from the group consisting of steroidal estrogen, estradiol, estrone, medroxyprogesterone,

norethindrone, norethisterone, progestin, norgestrel, and progesterone, and at least

one carotenoid wherein said carotenoid is in an amount effective to prevent the

adverse effects which may result from the administration of said hormone.

27. A unit dosage form suitable for adrninistration to a human comprising a

physiologically effective amount of at least one hormone selected from the group

consisting of steroidal estrogen, estradiol, estrone, medroxyprogesterone,

norethindrone, norethisterone, progestin, norgestrel and progesterone, and at least

one carotenoid, wherein said carotenoid is in an amount effective to prevent the adverse effects which may result from the administration of said hormone without

inhibiting the beneficial activity of said hormone.

28. A unit dosage form to any of clams 26 and 27 wherein said carotenoid is in an

amount from about 2 to about 6 mg.

29. A unit dosage form suitable for once daily administration to a human comprising a

physiologically effective amount of at least one hormone selected from the group consisting of steroidal estrogen, estradiol, estrone, medroxyprogesterone, norethindrone, norethisterone, progestin, norgestrel, and progesterone, and at least

one carotenoid wherein said carotenoid is in an amount effective to prevent the

adverse effects which may result from the administration of said hormone.

30. A unit dosage form suitable for once daily administration to a human comprising a

physiologically effective amount of at least one hormone selected from the group consisting of steroidal estrogen, estradiol, estrone, medroxyprogesterone,

norethindrone, norethisterone, progestin, norgestrel and progesterone, and at least one carotenoid, wherein said carotenoid is in an amount effective to prevent the adverse effects which may result from the administration of said hormone without

inhibiting the beneficial activity of said hormone.

31. A unit dosage form suitable for once daily administration to a human comprising a

consisting of steroidal estrogen, estradiol, estrone, medroxyprogesterone,

\ norethindrone, norethisterone, progestin, norgestrel, and progesterone, and at least

one carotenoid wherein said carotenoid is in an amount from about 2 to about 10

mg.

32. A unit dosage form suitable for once daily adrninistration to a human comprising a

one carotenoid wherein said carotenoid is in an amount from about 2 to about 6 mg.

33. A unit dosage form suitable for once daily administration to a human comprising a

one carotenoid wherein said carotenoid is in an amount of about 2 mg.

34. A unit dosage form according to any of claims 26 - 33 wherein said carotenoid is selected from the group consisting of lycopene, alpha-carotene, beta-carotene,

zeta-carotene, phytoene, phytofluene, lutein, zeaxanthin, cryptoxanthin and

astaxantine.

35. A unit dosage form according to any of claims 26 - 33 wherein said carotenoid

comprises a mixture of lycopene and phytofluene.

36. A unit dosage form according to any of claims 26 - 33 wherein said carotenoid

comprises a mixture of lycopene and phytoene.

37. A unit dosage form according to any of claims 26 - 33 wherein said carotenoid comprises a mixture of lycopene, phytoene and phytofluene.

38. A unit dosage form according to claim 26, 27, 32 or 33 wherein said carotenoid is

in an amount sufficient to cause effective serum concentrations of said carotenoid

of up to about 1.5 μM.

39. A unit dosage form suitable for once daily administration to a human comprising a physiologically effective amount of at least one phytoestrogen and at least one carotenoid selected from the group consisting of zeta-carotene, phytoene,

phytofluene, lutein, zeaxanthin, and astaxantine, wherein said carotenoid is in an

amount of about 2 mg.

40. A unit dosage form suitable for once daily administration to a human comprising a physiologically effective amount of at least one phytoestrogen and at least one

carotenoid selected from the group consisting of zeta-carotene, phytoene,

amount sufficient to cause effective serum concentrations of said carotenoid of up

to about 1.5 μM.

41. A unit dosage form suitable for once daily administration to a human comprising a

physiologically effective amount of at least one phytoestrogen and a mixture of

lycopene and phytoene, wherein said mixture is in an amount of about 2 mg.

42. A unit dosage form suitable for once daily administration to a human comprising a

physiologically effective amount of at least one phytoestrogen and a mixture of lycopene and phytofluene, wherein said mixture is in an amount of about 2 mg.

43. A unit dosage form suitable for once daily administration to a human comprising i physiologically effective amount of at least one phytoestrogen and a mixture of lycopen<

phytoene and phytofluene, wherein said mixture is in an amount of about 2 mg.

44. A unit dosage form suitable for once daily administration to a human comprising a physiologically effective amount of at least one phytoestrogen and at least one

carotenoid selected from the group consisting of alpha-carotene and cryptoxanthin, wherein said carotenoid is in an amount of about 2 mg.

45. The unit dosage form of claim 44 wherein said carotenoid is in an amount sufficient

to cause effective serum concentrations of said carotenoid of up to about 1.5 μM.