WO2001032191A2

WO2001032191A2 - Phytoestrogenic pharmaceutical preparations

Info

Publication number: WO2001032191A2
Application number: PCT/IL2000/000700
Authority: WO
Inventors: Jacob Vaya; Snait Tamir
Original assignee: Gavish - Galilee Bio Applications Ltd.
Priority date: 1999-11-01
Filing date: 2000-11-01
Publication date: 2001-05-10
Also published as: IL132666A; AU1172101A; IL132666A0; WO2001032191A3

Abstract

Phytoestrogenic pharmaceutical compositions having estrogen-like activity, comprising an active agent selected from licorice root extract, an isoflavanoid isolated from licorice root, particularly glabridin, hispaglabridin A, hispaglabridin B and glabrene, and any mixture thereof, and optionally further comprising a pharmaceutically acceptable carrier, excipient or diluent.The phytoestrogenic compositions of the invention are particularly suitable for the treatment and/or prevention of post-menopausal osteoporosis. and for hormone replacement post-menopausal therapy.

Description

Phytoestrogenic Pharmaceutical Preparations

Field of the Invention

The present invention relates to the phytoestrogenic pharmaceutical compositions having estrogen-like activity comprising isoflavans and isoflavenes, isolated from licorice roots, particularly glabridin and glabrene. The invention also relates to phytoestrogenic compositions having estrogen-like activity comprising licorice root extract. The compositions of the invention may be used in hormone replacement therapy and in the prevention and treatment of osteoporosis.

Background of the Invention

Estrogen: Steroid hormones exhibit a broad range of physiological activities. Estrogen is active in the development of the mammary gland and the uterus, in maintaining pregnancy and bone density and in protecting from cardiovascular diseases [Korach, K.S., Science 266(5190):1524-7 (1994); Iafrati, M.D., et al., Nat. Med. 3(5):545-8 (1997); Seed, M., Atherosclerosis 90(1): 1-7 (1991)]. However, estrogen can also stimulate malignant growths and thus contribute to the development of estrogen-dependent tumors, such as breast cancer [(Phillips, D.M. et al., Am Fam Physician 53(2):657-65 (1996)]. Estrogen acts by binding to an estrogen receptor (ER), forming a complex that undergoes dimerization and binds to specific adaptor proteins that allow contact with the target gene control region [Jordan, V.C., Sci Am 279(4):60-7 (1998)]. In addition to ER-α, a second receptor, ER-β, has been discovered with different structure and tissue distribution. The biological effect of an ER-ligand in a specific tissue is determined by the expression of ER-α and ER-β in that tissue [Paech, K., et al, Science 277(5331):1508-10 (1997)]. Furthermore, some estrogens function as antagonists in some tissues and as agonists in others. For example, tamoxifen, which is an anti-estrogen or pure antagonist in breast tissue used to treat breast cancer, acts as an estrogen agonist in bone [Phillips et al. (1996) ibid,.]. The newest selective estrogen receptor modulator (SERM), raloxifene, functions similarly to tamoxifen in the bone, breast, and cardiovascular system, but exhibits minimal antagonist activity in the uterus [Yang, N.N., et al, Endocrinology 137(5):2075-84 (1996)]. The two key events that control the tissue selectivity of an estrogen are the receptor shape and the interaction with adaptor proteins [Horwitz, K.B., et al, Mol Endocrinol 10(10): 1167-77 (1996); Smith, C.L., et al., Mol Endocrinol ll(6):657-66 (1997)]. Estrogen is also beneficial in reducing the risk of cardiovascular disease (lafrati et al., ibid.; Seed, ibid.]. The incidence of heart diseases among pre-menopausal women is low compared with males, whereas among post-menopausal women incidence approaches that of males. Administration of estrogen to post-menopausal women decreases the incidence of heart diseases [Stampfer, M.J. and G.A. Colditz, Prev Med 20(l):47-63 (1991)]. This protective effect of estrogen may partially result from its effect on decreasing the ratio between LDL and HDL [Shewmon, D.A., et al., Arterioscler Thromb 14(10):1586-93 (1994)].

Phytoestrogens: Phytoestrogens are naturally occurring substances, derived from plants. They are part of the human diet, and have estrogen-like activity [Tham D.M., et al, J Clin Endocrinol Metab 83(7):2223-35 (1998); Setchell, K.D. and A. Cassidy, J Nutr 129(3):758S-767S (1999); Zava et al., P.S.E.B.M. 217:369:378 (1998)]. Phytoestrogens include the subclasses lignans, isoflavonoids and coumestans, widely distributed in oilseeds (flax, cereals), vegetables and soybeans. The major food active isoflavonoids are genistein (IV) and daidzein (V) which, can be detected in human plasma. Several lines of evidence in the literature show a correlation between diet and bone diseases, coronary heart disease and major cancer activity [Tham et al., ibid.; Setchell et al., ibid.; Zava, D.T., et al, Environ Health Perspect 105(Suppl 3):637-45 (1997); Knight D.C., et al, Obstet Gynecol 87:897-904 (1996); Lamartiniere. et al, Am J Clin Nutr 68(6 Suppl):1400S-1405S (1998)]. Breast cancer is the most common malignancy among women in Western society and it is the leading cause of death among American women of 40-55 years of age. Epidemiological evidence indicates that soy intake (rich in isoflavonoids) is associated with lower breast cancer risk in women [Lee H.P., et al, Lancet 337:1197-1200 (1991); Fournier B.F., et al, Cancer Epidemiology, Biomarkers and Prevention 7:1055-1065 (1998)]. Japanese women with a low risk of breast cancer, showed high isoflavonoid excretion. Women with diet that was rich with isoflavonoids showed a low incidence of breast cancer [Adlercreutz, C.H., et al, J Nutr, 125(3 Suppl):757S-770S (1995); Adlercreutz, H. et al Lancet 342(8881):1209-10 (1993).

Osteoporosis is characterized by a reduction in bone mineral density to the extent that fractures occur after minimal trauma. It is a major health problem in Western countries among elderly women. It affects more than 25 million women, causing some 250,000 hip fractures yearly [Melton, L.J., 3rd, et al, J Bone Miner Res 12(1): 16-23 (1997)] with mortality in the first year after hip fractures reached 20% [Cooper, C, et al, Amer J Epidem 137, 1001-5 (1993)]. The phytoestrogen, genistein, is reported to prevent cancellous bone loss, and to maintain or to increase bone density in post-menopausal women [Valente, M., et al, Calcif Tissue Int 54(5):377-80 (1994)]. Pharmaceutical and dietary compositions for the treatment of osteoporosis, comprising such phytoestrogens, in combination with many other ingredients including licorice root extract, are described for example, in US Patent No. 5,424,331.

Licorice root which consist of the dried unpeeled root and stolons of glycyrrhiza glabra are widely used as flavoring and sweating agents, as well as demulcents and expectorants in western countries. Furthermore, licorice root extracts has been used in medicine for more than 3000 years, and a wide range of therapeutic uses have been ascribed to it. Several pharmacological activities such as antibacterial and antiviral, anti-inflammatory, anti-allergic and antihepatotoxic effects have been attributed to licorice constituents glycyrrhizin and its aglicone glycyrrhizic acid. Licorice root also contains isoflavans and isoflavenes, subclasses of the flavonoid compounds, containing ring A fused to ring C connected to ring B through carbon 3. Several functional groups may be attached to this basic skeleton mainly hydroxyl groups. In the isoflavans subclass (Fig. 1), the heterocyclic ring C does not contain double bond between carbons 2 and 3 neither carbonyl group attached to carbon 4. This structure does not allow conjugation of the double bonds between ring A and B. Several isoflavans and isoflavene isolated by the inventors from licorice root, which were known to exhibit anti-oxidative activity [Vaya, J., et al, Free Radic Biol Med 23(2):302-13 (1997)], have recently been shown by the inventors to possess phytoestrogenic activities [Tamir S. et al, Cancer Res. 60:5704-9 (2000)]. Glabridin is the major constituent (11%) of the alcoholic extract of licorice root and was shown to have the highest antioxidative activity [Belinky PA, et al. Atherosclerosis 137:49-61 (1998)). Glabrene is another licorice constituent of an isoflavene subclass of the flavonoid family, has two hydroxyl groups at the 2' and 7 positions, a 2,2-dimeth l-γ-pyran ring fused to B ring, and a double bond between carbons 3 and 4 in C ring, which confers maximal conjugation of the double bonds on the molecule (Fig. 1). The structure and lipophilicity of the isolated glabridin and glabrene differ considerably from those of the known isoflavonoids, such as genistein and daidzein, the major phytoestrogens of the soy beans. These differences affect both their physical and chemical behavior, such as their three dimensional structure and their ability to stabilize free radicals. The flavanoids of the present invention are less polar compounds (similar to estradiol) than known isoflavonoids. These structural lipophilic similarity between glabridin and estradiol (Fig. 1), makes them possible candidates to function as estrogen-like substances.

In search for substances that could replace estrogen in different treatment protocols, over the last few years the inventors studied the phytoestrogenic properties of glabridin and glabrene and other isoflavans isolated from licorice root, by comparing their ability to bind to the human estrogen receptor (ER¹) and their effect on estrogen-responsive human breast cancer cells, over a broad range of concentrations. In vivo studies included their effects on rat uterus wet weight and on the induction of the immediate early "estrogen-induced protein" creatine kinase (KC) in rat skeletal and cardiovascular tissues as well as uterus. The results indicate that glabridin, the major isoflavan, and glabrene a minor component in licorice extract, bind to the human estrogen receptor and exhibit varying degrees of estrogen receptor agonism in vitro and in vivo. Their agonistic effects are not the same in different tissues, presenting tissue specificity. This suggests that isoflavans and isoflavenes may serve as natural estrogen agonists in preventing the symptoms and diseases associated with estrogen deficiency.

It is thus an object of the invention to provide phytoestrogenic pharmaceutical compositions which would have estrogen-like activity, but would have advantages over estradiol, especially substances that would not lead to increased cell proliferation and induction of estrogen-responsive tumors, particularly breast cancer.

It is another object of the invention to provide phytoestrogenic pharmaceutical compositions for post-menopausal hormone replacement therapy.

A yet further object of the invention is to provide phytoestrogenic pharmaceutical compositions for the treatment and/or prevention of post-menopausal osteoporosis.

These and other objects of the invention will be detailed as the description proceeds. Summary of the Invention

The invention relates to phytoestrogenic pharmaceutical compositions having estrogen-like activity, comprising an active agent selected from hcorice root extract, an isoflavanoid isolated from hcorice root and any mixture thereof, and optionally further comprising a pharmaceutically acceptable carrier, excipient or diluent.

In a particular embodiment, the said isoflavanoid is selected from isoflavans and isoflavenes, more particularly said isoflavan is selected from glabridin, hispaglabridin A and hispaglabridin B and said isoflavene is glabrene.

The phytoestrogenic compositions of the invention are particularly suitable for the treatment and or prevention of post-menopausal osteoporosis. Compositions for the treatment of osteoporosis preferably comprise as the active agent hcorice root extract and/or an isoflavanoid isolated from licorice root, preferably an isoflavanoid is selected from isoflavans and isoflavenes, and particularly glabridin, hispaglabridin A and hispaglabridin B, and glaberene as isoflavene.

The phytoestrogenic composition of the invention are also intended as hormone replacement post-menopausal therapy. Such compositions preferably comprise as the active agent hcorice root extract and/or an isoflavanoid isolated from licorice root, preferably an isoflavanoid is selected from isoflavans and isoflavenes, and particularly glabridin, hispaglabridin A and hispaglabridin B, and glaberene as isoflavene.

In a further embodiment the invention relates to a method of treating and/or preventing post-menopausal osteoporosis comprising administering to a patient in need of such treatment a pharmaceutically effective amount of an active agent selected from hcorice root extract, an isoflavanoid isolated from hcorice root and any mixture thereof, or of a pharmaceutical composition of the invention. Thus, the active agent may be hcorice root extract and/or an isoflavanoid isolated from hcorice root, preferably an isoflavanoid is selected from isoflavans • and isoflavenes, and particularly glabridin, hispaglabridin A and hispaglabridin B, and glaberene as isoflavene, and any mixture thereof.

Still further, the invention relates to a method of providing post-menopausal hormone replacement therapy to a patient in need of such treatment comprising administering to said patient a therapeutically effective amount of an isoflavanoid isolated from hcorice root, or of a pharmaceutical composition of the invention containing the same. The active agent is preferably an isoflavanoid is selected from isoflavans and isoflavenes, and particularly glabridin, hispaglabridin A and hispaglabridin B, and glaberene as isoflavene, and any mixture thereof.

AH the above and other characteristics and advantages of the invention will be further understood from the following illustrative and non-limiting examples of preferred embodiments thereof.

Description of the Figures

The present invention will be more clearly understood from the detailed description of the preferred embodiments and from the attached drawings in which:

Figure 1 The structure of the isoflavan glabridin, Isoflavene glabrene and the estrogen (estradiol)

Figure 2 Competition of glabridin and glabrene for the human estrogen receptor with [³H]labeled 17 β-estradiol in T-47D cells Cells were incubated with [³H] 17 β-estradiol and increasing concentrations of glabridin, glabrene or 0.1% of ethanol as a control. Radioactivity of the cells' nuclei was counted and plotted as percentage of control. Values are means±SD of >3 experiments. Figure 3A and 3B The effect of glabridin, glabrene, hispaglabridin A and hispaglabridin B on the growth of estrogen-responsive breast cancer cells

T47D (ER+) cells were incubated with increasing concentrations of tested compound for ~7 days. Proliferation was estimated using XTT cell proliferation reagent. Results are presented as percentage of control (0.1% of ethanol) (means±SD, n>3).

Figures 4A and 4B The Effect of glabridin on anchorage-independent growth

MCF-7 cells were plated onto soft agar plates in the presence of increasing concentrations of glabridin, with and without InM estradiol. Colony formation was observed after 3 weeks. A: Increasing concentrations of glabridin 1, 10 and 25μM. B: Increasing concentrations of glabridin 1, 10 and 25μM in the presence of InM estradiol.

Figure 5 Glabrene activates ER mediated transcription

Hela cells were transiently transfected as described in Methods and Materials. Cells were incubated with increasing concentrations of glabrene or 0.1% ethanol as control. After two days cells lysate were tested for CAT activity. Results are presented as relative CAT activity (experimental control). Values are means±SD of >3 experiments.

Figure 6 Illustrates the procedure for separation of phytoestrogens from licorice root.

Detailed Description of the Invention

The present invention is based on the finding that certain isoflavans and isoflavenes, isolated from hcorice extract, and particularly glabridin and glabrene possess very potent estrogen-hke activity, whilst having a very weak cell proliferative activity. As such they can be used in replacement post-menopausal hormone therapy. The invention thus relates to phytoestrogenic pharmaceutical compositions comprising as active agent at least one isoflavamoid isolated from hcorice root. Preferred such active agent are isoflavans, particularly glabridin and certain glabridin derivatives, and isoflavenes, particularly glabrene, found in hcorice, which are phytochemicals with several known biological activities and a chemical structure and lipophilicity which resemble those of estradiol. The structure of estradiol, glabridin, glabrene and the glabridin derivatives hispaglabridin A and hispaglabridin B is illustrated in Fig. 1.

The inventors have investigated the estrogenic and estrogen-dependent proliferation-inducing activity of glabridin and certain glabridin derivatives, and of glabrene. As will be shown in the following Examples, these compounds bind to the human estrogen receptor (Example 1.1), enhance the proliferation of estrogen-dependent human breast cancer cells (MCF7 and T47D) in vitro at concentrations as low as lμM, achieving proliferative effects similar to those of InM estradiol and lμM genistein, a known phytoestrogen from soy, and activate the expression of endogenous estrogen-regulated genes. Both glabridin and glabrene markedly inhibited the growth of human breast cancer cells at approximately 25μM (Example 1.2).

In vivo, it has been found that feeding 25mg/day of a hcorice root extract to ovariectomized rats had a stronger effect on specific activity of the "estrogen-induced protein" creatine kinase in skeletal tissues than lOmg/day estradiol (Example 2.1). Glabridin and glabrene showed the same effect as estradiol on CK activity in the uterus, but had an increased effect in cardiovascular tissues and skeletal tissues (Examples 2.2 to 2.4). These results suggest a possible use of hcorice extract, as well as glabridin and glabrene as estrogen agonists, in the prevention of estrogen-responsive bone and cardiovascular diseases. Prehminary data from structure -activity studies suggest that the position of the hydroxyl group at B ring in an isoflavan derivative may have a significant role in binding to the human estrogen receptor and in proliferation-inducing activity [Belinky PA, et al, Free Radic Biol Med 24(9): 1419-29 (1998)].

The cardiovascular effects of glabridin were studied in vitro on human endothelial cells and smooth muscle cells. Example 1.5 shows that glabridin and estradiol modulate DNA synthesis in human smooth muscle cells in a parallel fashion. Their effects in SMCs are bi-modal, inducing stimulation at low concentrations and inhibiting at high concentrations. In contrast, both estradiol and glabridin positively influence DNA synthesis by cells of endothelial origin. These results suggest a beneficial role for glabridin, as estrogen agonist, in the prevention of atherosclerosis.

Thus, hcorice root extract, glabridin and glabrene as well as certain glabridin derivatives such as hispaglabridin A and hispaglabridin B, may be used in the treatment or prevention of various hormone-dependent conditions, as hormone replacement therapeutic agents for menopausal and post-menopausal symptoms. Particularly preferred licorice extract is the acetone extract exemplified in the present invention, but alcohohc extracts can also be used.

The phytoestrogenic pharmaceutical composition having estrogen-like activity thus comprise as an active ingredient an active agent selected from licorice root extract, an isoflavanoid and isoflavene isolated from licorice root and any mixture thereof, and optionally further comprising a pharmaceutically acceptable carrier, excipient or diluent. Preferred active agents are hcorice root extract as herein described and glabridin, hispaglabridin A, hispaglabridin B and glabrene. The compositions of the invention are particularly useful in the treatment and/or prevention of osteoporosis; particularly post-menopausal osteoporosis, and in post-menopausal hormone replacement therapy.

The invention also relates to a method of treating and/or preventing post-menopausal osteoporosis comprising administering to a patient in need of such treatment a pharmaceutically effective amount of an active agent selected from hcorice root extract, an isoflavanoid isolated from licorice root and any mixture thereof, or of a pharmaceutical composition of the invention. Preferred active agents to be administered are licorice root extract, particularly the extract exemplified in the application, and glabridin and glabrene.

The invention is further directed at a method of providing post-menopausal hormone replacement therapy to a patient in need of such treatment, comprising administering to said patient a therapeutically effective amount of an isoflavanoid isolated from hcorice root, or of a pharmaceutical composition of the invention. Preferred isoflavanoids and isoflavene are glabridin and glabrene.

The preparation of pharmaceutical compositions is well known in the art of pharmacy, see e.g., US Patents 5,736,519, 5,733,877, 5,554,378, 5,439,688, 5,418,219, 5,354,900, 5,298,246, 5,164,372, 4,900,549, 4,755,383, 4,639,435, 4,457,917, and 4,064,236, and Remington's Pharmacopea, all incorporated herein by reference. The active agents in accordance with the present invention are preferably mixed with an excipient, carrier, diluent, and optionally, a preservative or the like, and any other pharmaceutically acceptable vehicles as known in the art, see e.g., the above US patents. Examples of excipients include, glucose, mannitol, inositol, sucrose, lactose, fructose, starch, corn starch, microcrystalline cellulose, hydroxypropyl- cellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone and the like. Optionally, a thickener may be added, such as a natural gum, a cellulose derivative, an acrylic or vinyl polymer, OF the like.

The pharmaceutical composition is provided in solid, hquid or semi-solid form. A solid preparation may be prepared by blending the above components to provide a powdery composition. The hquid preparation is provided preferably as aqueous solution, aqueous suspension, oil suspension or microcapsule composition. A semi-sohd composition is provided preferably as hydrous or oily gel or ointment. About 1% to about 50% w/v %, preferably about 5% to 20% w/v % of the active principal are provided in the composition.

A solid composition may be prepared by mixing an excipient with a solution of the active agent, gradually adding a small quantity of water, and kneading the mixture. After drying, the mixture is pulverized. A liquid composition may be prepared by dissolving, suspending or emulsifying the peptide of the invention in water, a buffer solution or the like. An oil suspension may be prepared by suspending or emulsifying the peptide of the invention or protein in an oleaginous base, such as sesame oil, olive oil, corn oil, soybean oil, cottonseed oil, peanut oil, lanohn, petroleum jeUy, paraffin, Isopar, silicone oil, fatty acids of 6 to 30 carbon atoms or the corresponding glycerol or alcohol esters. Buffers inlcude Sorensen buffer (Ergeb. Physiol., 12, 393 1912), Clark-Lubs buffer (J. Bact., 2, (1), 109 and 191, 1917), Macllvaine buffer (J. Biol. Chem., 49, 183, 1921), Michaehs buffer (Die Wasserstoffinonenkonzentration, p. 186, 1914), and Kolthoff buffer (Biochem. Z., 179, 410, 1926).

The active agents and compositions of the invention may be used as pharmaceuticals as weU as dietary additives. Preferably, the active agents and compositions of the invention are administered orally, although other modes of administration, e.g. intravenous, intramuscular or subcutaneous administration may be possible. The dosage of the active agent may depend upon the condition to be treated, the patient's age, bodyweight, and the route of administration, and will be determined by the attending physician. The patient is usuaUy a woman at menopausal or post-menopausal age, who suffer estrogen deficiency, classically between 48 to 60 years of age. Patients may suffer from osteoporosis also before menopause is manifested, and the active agents and compositions of the invention may be used also for the treatment of such pre-menopausal patients. Doses may range from about 0.04mg/kg body weight to 2mg/Kg, preferably from 0.4mg/Kg to 1.2mg/Kg of active material. A therapeutically effective amount is an amount effective to improve and/or prevent the disease. For example, when treating osteoporosis, an effective amount will be sufficient to preserve bone mass of the treated patient, and prevent bone fractures.

The compositions of the invention may comprise pharmaceutically compatible biodegradable polymers affording sustained-release of the active principal. Various polymers may be employed, as described e.g., in USP 5,439,688. The compositions of the invention may also be in the form of a fat emulsion. The preparation of such pharmaceutical compositions is described, e.g., in US 5,733,877.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The invention will be illustrated on hand of the following Examples, which are illustrative only and do not limit the invention thereto. Examples

Materials and Methods

Chemicals and Reagents: 17β-Estradiol was purchased from Sigma, and

[³H]-17β-estradiol for competition assay from NEN™ Life Science Products

(Boston, MA). Leibovitz L-15, fetal calf serum (FCS), RPMI-1640, Tripsin

EDTA, L-Glutamine, Hepes buffer, penicillin-streptomycin, sodium pyruvate solutions and XTT Reagent Cell Proliferation Kit were all purchased from Biological Industries (Beth Haemek, Israel).

Isolation of phytoestrogens from licorice: Glabridin, hispaglabridin A, hispaglabridin B and glabrene were isolated from the acetone extract of the roots of Glycyrrhiza glabra, as previously described [Vaya et al, 1997, ibid., fully incorporated by reference]. The procedure is illustrated in Fig. 6.

Human Breast Cancer Cells: Different lines of human breast cancer cells (T-47D, MCF-7) were purchased from the American Type Culture Collection (ATCC). The cells were grown in DMEM, supplemented with 2μg/ml insulin, ImM sodium pyruvate, ImM nonessential amino acids, 4mM glutamine, 10% fetal calf serum, and antibiotics (penicilhn/streptomycin). One week before experiments, cells were transferred to phenol red-free medium, supplemented with 5% charcoal-stripped fetal calf serum (C-SFCS).

Cell proliferation: Cells were seeded into 96-well tissue culture plates (5000 cells/well) in 5% C-SFCS RPMI phenol red-free medium (T-47D cells) or 5% C-SFCS Leibovitz L-15 medium (MDA-MB-468 cells) and incubated at 37°C for 48h. The medium was then removed and fresh media with test compounds were added (control contained 0.1% ethanol). The medium was changed every three days. To evaluate relative cell concentration, XTT reagent was used. Absorbance was measured at 450nm, using a Spectra II spectrophotometer (SLT-Labinstrument, Austria). Transient gene expression assay in Hela cells: The human ERα expression plasmid pSG5-hERGO (kindly provided by Prof. Kaye, The Waizamann Institute, Rehovot, Israel, and with the approval of Prof. Gustafsson) was used. Hela human cervical carcinoma cells (kindly provided by Prof. Kaye) were cultured in phenol red free DMEM supplemented with 10% charcoal-stripped FCS.

Cells were seeded into 6cm Petri dishes at 4xl0⁵ cells per dish and calcium phosphate transfection was performed according to Gorman et al. [Gorman GM, In: Glover DM (ed), DNA Cloning, IRL Press, Washington Dc, USA, Vol. 3, 143-190 (1985)]. Cells were co-transfected with 2.4μg PAM117, 2μg HEGO (The human ERα expression plasmid pSG5-hERG), and 2μg luciferase expression plasmid (all plasmids were kindly provided by Prof. Kaye) as an internal reference for transfection efficiency. Four hours after transfection cells were supplemented with fresh medium containing test compounds. 48 h later cells were harvested into 1 ml extraction buffer (0.1M KPO , ImM DTT), centrifuged for 5 min at 4°C, 14,000xrpm and were lysed by three freeze-thaw cycles. Cell extracts were used for luciferase and CAT assays.

Luciferase assay: Enhanced chemiluminescence (ECL) kit, containing luciferin and ATP, was purchased from Amersham Life Science International pic. (Buckinghamshire, England). Luciferase activity was monitored using luminometer (Lumac 13M biocounter M 2010A, Ilpen).

CAT assay: CAT activity was estimated using butyril coenzyme A and ¹⁴C-chloramphenicol. The reaction products were separated by a phase extraction technique according to Seed and Sheen 1988. Radioactive spots were excised and counted by hquid scintillation and CAT activity was expressed as fold increase over control. Colony formation in soft agar: MCF-7 cells were plated on to soft agar plates in the presence of various concentrations of the test compounds for 3 weeks and assayed for colony formation. Cells (10³) were first suspended in 0.15ml medium (MEM supplemented with 2μg/ml insulin and 5% C-SFCS) containing 0.3% agar. The mixture was added over a layer of 0.5% agar in MEM on a 24-well plate. Plates were fed weekly, and after three weeks were stained with vital stain 2-(p-isodiphenyl)-3-(p-nitrophenyl)-5-phenyl- tertazohum chloride hydrate. Colonies larger than 0.15mm diameter were scored.

Estrogen receptor binding assay: Test compounds were prepared in 100% ethanol and the stock solutions were diluted in 1% C-SFBS in RPMI-1640 medium. Control tubes contained 0.4% ethanol (0.1% final concentration in incubation). Triphcate 50μl ahquots of test compounds were added to 50μl [³H] E2 (100 Ci/mmol) diluted in 1% C-SFBS to a concentration of 0.4nM. The test tubes were equilibrated at 37°C while the cells were prepared. T47D cells were fed with 1% C-SFBS in RPMI-1640 (containing 0.2ng/ml insulin), without phenol red, at least two days prior to assay. Cells were removed with trypsin-EDTA and diluted in 1% CT RPMI-1640 to 3xl0⁶ cells/ml. One hundred μl diluted cells and [³ H]-estradiol were added to the test compounds. The tubes were mixed gently and incubated at 37°C for lh. After incubation the cells were sedimented by centrifugation at 3000xRPM for 5min at 4°C. After removal of the supernatant, the cells were washed once with ice-cold TPSG (0.2% Triton X-100 + PBS containing 0.1M sucrose and 10% glycerol). Tubes were vigorously vortexed in fresh TPSG and incubated for 5 min. Intact nuclei were sedimented by centrifugation, 3000xrpm for 5min at 4°C. The supernatant was aspirated and [³H] -estradiol remaining in the nuclei was measured by a beta counter Zava et al, 1997, ibid.]. Results are presented as percent of [³H] -estradiol binding to ER in the nucleus in the absence (control=100%) or presence of increasing concentrations of test compounds. In Vivo Experiments: Twenty-five day-old Wistar-derived female rats at a weight of about 60g were housed in metal cages in groups of five per cage and maintained on a 14h light/ 1 Oh- dark cycle, at 23°C. Access to food and tap water was ad libitum.

The animals were injected with 0.5ml PBS containing test compounds dissolved in ethanol, or ethanol as a control. Final concentration of ethanol in PBS was 1%. After 24h, the animals were killed and the uterus removed through a midline incision. The wet uterine weight was determined. In addition, aorta, left ventricle, diaphysis and the epiphysis of the femur were removed and all organs were frozen at -20°C for later analysis of CK activity.

Creatine kinase activity: Frozen organs were collected in cold isotonic extraction buffer (0.25M-sucrose, 0.05M-tris, 0.4mM-EDTA, 2.5mM-DTT, 5mM-sodium acetate) and homogenized in a Polytron homogenizer (Kinematica A.G.). Homogenates were centrifuged at 14,000x for 5min at 4°C. The supernatant was tested for CK activity in a Kontron model 922 Uvicon spectrophotometer at 340nm, using a coupled assay for ATP, as described by Somjen et al [ibid.]. Protein was determined by Coomassie brilliant blue.

Statistical analysis: Statistical significance was determined by the ANOVA test.

Results

1. In Vitro Experiments

1.1 Glabridin and glabrene bind to the human estrogen receptor

To appreciate how the molecular structure of glabridin and glabrene related to their estrogenic actions, their effect was compared with those of estradiol. The concentrations of these flavanoids required to inhibit the binding of a single sub-saturating concentration (O.lnM) of radiolabled estradiol to estrogen receptor (ER) in intact human breast cancer cells are shown in Fig. 2. Competition binding studies were performed by using T47D cells, which are known to contain estrogen receptor. As shown in Fig. 3, the binding affinities of glabridin and glabrene for ER were about 10³, 10⁴ lower, than that of estradiol, respectively. The IC50 for glabridin was approximately 5μM, of glabrene lμM, indicating that they are relatively weak ligands for the receptor. Nevertheless, these concentrations are similar to other known phytoestrogens such as genestein [Zava, D.T., et al, Nutr Cancer 27(l):31-40 (1997b); Wang, C. and Kurzer, M.S., Nutr Cancer 28(3):236-47 (1997] and are about 10³ lower than that of estradiol.

1.2 Effects of glabridin and glabrene on proliferation of breast cancer cells

The effects of increasing concentrations of glabridin and glabrene on cell growth were compared with those of estradiol and are shown in Fig 3. Maximal stimulation of cell proliferation occurred with about lOOpM estradiol, lOμM glabridin and lOμM glabrene. They both markedly inhibited the growth of human breast cancer cells at approximately 25μM.

1.3 Effect of glabridin on anchorage-independent growth of MCF7 cells

When the cells are grown in suspension in 0.3% agar in complete medium they formed large colonies in the presence of lOμM glabridin (Fig. 4, Table 1) or lOnM estradiol. In contrast to its promotion of colony formation at lower concentrations, glabridin inhibited anchorage independent growth at concentrations of 25μM. Table 1 details the effect of increasing concentrations of glabridin on anchorage-independent growth of MCF7 cells. MCF-7 cells were plated onto soft agar plates in the presence of increasing concentrations of glabridin, with and without InM estradiol. Colonies larger than 0.15mm were counted after 3 weeks. Table 1

Effect of increasing concentrations of glabridin on anchorage-independent growth of MCF7 cells

When glabridin was tested in the presence of lOnM estradiol, it had no effect on the anchorage independent growth-promoting effects of estradiol. The pronounced growth-inhibiting action of glabridin at concentrations of >25μM reached control levels and was not modified by estradiol (Fig.4 and Table 1).

1.4 Effect of glabrene on colony formation ofMCF7 cells

The effects of increasing concentrations of glabrene on colony formation were found to be similar to its effects on cell proliferation, i.e. the effect was bi-phasic (Table 2). Maximal colony stimulation by glabrene at lμM was equal to that of estradiol at lOnM. In contrast to its colony formation-promoting effects at lower concentrations, glabrene inhibited colony formation at higher concentrations. Results are given in Table 2, which shows the effect of increasing concentrations of glabrene on colony formation. MCF-7 cells were plated in the presence of increasing concentrations of glabrene. Colonies larger than 0.15mm were counted after 3 weeks. Table 2

Effect of increasing concentrations of glabrene on colony formation

1.5 Effects of Glabridin on DNA synthesis in human vascular cells and endothelial cells

Animal and human studies indicate that estrogens are protective against coronary atherosclerosis [lafrati et al, ibid.]. Because endothelial repair and vascular smooth muscle cells (VSMC) proliferation have well defined pathophysiological roles in vascular injury and atherogenesis, the potential modulation of such processes by estrogen is of obvious interest. The present experiments were undertaken to explore the effects of glabridin on DNA synthesis in human endothelial cells and VSMCs. The results in Table 3 show that glabridin as estradiol induced dose-dependent increase of ³H-thymidine incorporation into DNA of endothelial cells and had the same bi-phasic effect on the smooth muscles cells. The inhibition of SMC proliferation and the induction of endothelial cells proliferation by estradiol and glabridin, which acts as agonist, is beneficial for the prevention of atherosclerosis. In the experiment summarized in Table 3, E304 cells (human endothehal cells) and E/C cells (human primary arterial smooth muscle cells) were exposed to increasing concentrations of glabridin. DNA synthesis was tested using ³H-thymidine incorporation. Results are presented as increase fold of control. Table 3

Estrogenic activity of glabridin in vascular cells

1.6 Effects of glabrene on ER-mediated transcription

The ability of glabrene to bind to the human ER raised the possibility that it might function as an agonist or antagonist. In the presence of an agonist, the ER initiates transcriptional activation by binding to specific ER elements in the promoters of target genes [Behrens J., et al, Proc Natl Acad Sci USA 88:11495-11499 (1991)]. The action of glabrene was tested first by using pSG5-hERGO, a reporter gene that contains a single copy of an ER element upstream of the CAT promoter. In Hela cells, glabrene produced dose-dependent transcriptional activation with half-maximal induction at lμM (Fig. 5), corresponding to the concentration required for inhibition of estradiol binding. Glabrene produced a maximal level of induction that was similar to that achieved by lOnM estradiol.

2. In Vivo Experiments

2.1 Animals fed with licorice extract

Licorice extract was tested in vivo in relation to atherosclerosis and found to inhibit the formation of lesions in E° mice [Fuhrman B., et al, Am J Clin Nutr 66:267-275 (1997)]. The present study tested the estrogen-like effects of hcorice extract in vivo. Prepubertal female rats fed with estradiol (0.5μg/day) or hcorice extract (25μg/day) for 2 weeks showed a significant increase in CK activity in the left ventricle, the pituitary and the diaphyseal bone (Table 4). Creatine kinase activity is known to be induced by estrogens in vivo and in vitro [Somjen D., et al Hypertension 32:39-45 (1998)]. The results show that estradiol, at 0.5μg/day/rat, stimulated CK activity to the same level as licorice extract at 25μg/rat/day. Estradiol had a weaker stimulatory effect on CK activity in the left ventricle, and the diaphysis, possibly due to tissue specificity. In the experiment described in Table 4, rats were fed with 0.5μg estradiol or 25μg/day hcorice for two weeks. CK was extracted and assayed as described in Materials and Methods. Results are presented as increase fold of enzyme activity (experimental/control). P<0.05 in all cases compared to control.

Table 4

The effect of Licorice extract on induction of creatine kinase activity in various immature female rat tissues

IvJ

2.2 Effects of glabridin Injections on immature female rats

Injection into prepubertal female rats of estradiol (5μg /rat) or glabridin (2.5, 25, 200, and 250μg/rat) resulted 24h later in a significance increase of uterus weight (Table 5), and in a significant increase of the specific activity of the "estrogen induced protein" - creatine kinase B (CK), in rat uterus, epiphyseal cartilage, diaphyseal bone and cardiovascular tissues (Table 6). In several tissues (bone, cartilage, uterine and cardiovascular tissues) the effects of estrogen, including growth modulation, are hnked to the induction of CK activity, and this has been used as general genomic response marker for steroids. CK is involved in cellular energy buffering and is closely related to changes in cell replication rate in various cell types [Malnick, S.D., et al. Endocrinology 113(5):1907-9 (1983); Somjen D., et al. Proc Natl Acad Sci USA 86(9):3361-5 (1989)]. The results show that estradiol at 5 μg/rat stimulated CK activity to the same level as glabridin at 2.5 μg/rat in the diaphysis and uterus. Glabridin had a weaker affect on the stimulation of the CK activity in left ventricle than estradiol, w^τhich may be due to tissue specificity. In the experiments described in Table 5, rats were killed 24h after injection with 5μg estradiol or 200μg glabridin and wet uterine weight was determined. Results are presented as wet uterus weight+SD.

Table 5

Glabridin stimulates uterus growth in female rats

In the experiments described in Table 6, rats were killed 24h after injection with 5μg estradiol or 2.5, 25 or 250μg glabridin animal. CK was extracted and assayed as described in Materials and Methods. Results are presented as means+SD of CK specific activity (exp/control).

Table 6

Glabridin induction of creatine kinase activity in various female rat tissues

2.3 Effects of glabridin feeding on ovariectomized female rats

Feeding female rats, which were ovariectomized, with 25μg/day glabridin or lOμg/day estradiol for 7 days resulted in an increase of CK activity, in all estrogen responsive tissues tested (Table 7). Feeding glabridin had the same effect as estradiol on the uterus, epiphysis and pituitary but a stronger effect on the stimulation of the CK activity in the diaphysis and aorta. The results also show that orally giving glabridin had a weaker effect on the cardiovascular tissues than injections of glabridin. In the experiments described in Table 7, ovariectomized rats were killed after feeding during 7 days with lOμg/animal estradiol or 25μg/animal glabridin. CK was extracted and assayed as described in Materials and Methods. Results are presented as increase fold of enzyme activity over control. Table 7

The effect of feeding glabridin on the induction of creatine kinase activity in various ovx-female rat tissues

2.4 Effects of glabrene on immature female rats

Injection of estradiol (5μg) or glabrene (2.5, 25, 250μg) into prepubertal female rats resulted in a significance increase in CK activity in the left ventricle, uterus, pituitary and diaphyseal bone measured after 24h (Table 8). CK activity is known to be induced by estrogens in vivo and in vitro [Somjen et al. (1998) ibid.]. The results show that estradiol, at 5μg/rat, stimulated CK activity to the same level as glabrene at 2.5μg/rat in the uterus and diaphysis. and at 25μg/rat in the aorta and epiphysis. Glabrene had a weaker effect on the stimulation of CK activity in the pituitary (1.24±0.06 E/C) than estradiol (2.00+0.14 E/C), which may be due to tissue specificity. In the experiments described in Table 8, rats were killed 24h after injection with 5μg estradiol or 2.5, 25, 250μg glabrene. CK activity was assayed as described in Materials and Methods. Results are presented as increase fold of enzyme activity (experimental/control). Table 8

Glabrene induction of creatine kinase activity in various female rat tissues

* One-way ANOVA (p < 0.05) ** One-way ANOVA (p < 0.01)

Claims

Claims:

1. A phytoestrogenic pharmaceutical composition having estrogen-like activity, comprising an active agent selected from licorice root extract, an isoflavanoid isolated from hcorice root and any mixture thereof, and optionally further comprising a pharmaceutically acceptable carrier, excipient or diluent.

2. The phytoestrogenic composition of claim 1, wherein said isoflavanoid is selected from isoflavans and isoflavenes.

3. The phytoestrogenic composition of claim 2, wherein said isoflavan is selected from glabridin, hispaglabridin A and hispaglabridin B.

4. The phytoestrogenic composition of claim 2 or claim 3, wherein said isoflavene is glabrene.

5. The phytoestrogenic composition of claim 1, for the treatment and/or prevention of post-menopausal osteoporosis.

6. The phytoestrogenic composition of claim 5, wherein said active ingredient is a hcorice root extract.

7. The phytoestrogenic composition of claim 5, wherein said active ingredient is an isoflavanoid isolated from hcorice root.

8. The phytoestrogenic composition of claim 7, wherein said isoflavanoid is selected from isoflavans and isoflavenes.

9. The phytoestrogenic composition of claim 8, wherein said isoflavan is selected from glabridin, hispaglabridin A and hispaglabridin B.

10. The phytoestrogenic composition of claim 8, wherein said isoflavene is glabrene.

11. The phytoestrogenic composition of claim 1, for hormone replacement post-menopausal therapy.

12. The phytoestrogenic composition of claim 11, wherein said active ingredient is a hcorice root extract.

13. The phytoestrogenic composition of claim 11. wherein said active ingredient is an isoflavanoid isolated from hcorice root.

14. The phytoestrogenic composition of claim 13, wherein said isoflavanoid is selected from isoflavans and isoflavenes.

15. The phytoestrogenic composition of claim 14, wherein said isoflavan is selected from glabridin, hispaglabridin A and hispaglabridin B.

16. The phytoestrogenic composition of claim 14, wherein said isoflavene is glabrene.

17. A method of treating and/or preventing post-menopausal osteoporosis comprising administering to a patient in need of such treatment a pharmaceutically effective amount of an active agent selected from licorice root extract, an isoflavanoid isolated from licorice root and any mixture thereof, or of a pharmaceutical composition comprising said active agent and optionally further comprising a pharmaceutically acceptable carrier, excipient or diluent.

18. The method of claim 17, wherein said active agent is hcorice root extract.

19. The method of claim 17, wherein said isoflavanoid is selected from isoflavans and isoflavenes.

20. The method of claim 19, wherein said isoflavan is selected from glabridin, hispaglabridin A and hispaglabridin B and any mixture thereof.

21. The method of claim 19, wherein said isoflavene is glabrene.

22. A method of providing post-menopausal hormone replacement therapy to a patient in need of such treatment comprising administering to said patient a therapeutically effective amount of an isoflavanoid isolated from licorice root, or of a pharmaceutical composition comprising said active agent and optionally further comprising a pharmaceutically acceptable carrier, excipient or diluent.

23. The method of claim 22, wherein said isoflavanoid is selected from isoflavans, isoflavenes and any mixture thereof.

24. The method of claim 23, wherein said isoflavan is selected from glabridin, hispaglabridin A and hispaglabridin B and any mixture thereof.

25. The method of claim 23, wherein said isoflavene is glabrene.