IL132666A

IL132666A - Phytoestrogenic pharmaceutical preparations

Info

Publication number: IL132666A
Application number: IL132666A
Authority: IL
Original assignee: Migal Galilee Technological Ct
Priority date: 1999-11-01
Filing date: 1999-11-01
Publication date: 2007-07-24
Also published as: WO2001032191A2; AU1172101A; IL132666A0; WO2001032191A3

Description

PHYTOESTROGENIC PHARMACEUTICAL PREPARATIONS Field of the Invention The present invention relates to phytoestrogen^ pharmaceutical compositions having estrogen-like activity, containing isoflavans and like substances and to their various uses.

Background of the Invention The importance of estrogens in homeostatic regulation of many cellular and biochemical events is well illustrated by the pathophysiologic changes that occur with estrogen deficiency (Iafrati et al 1997; Korach et al 1994). Many of the major diseases of Western population are hormone-dependent, namely cardiovascular diseases, cancer, menopausal symptoms and post-menopausal osteoporosis. Estrogen has also deleterious effects, it can support growth in estrogen responsive target tissues including the breast and uterus. One peculiarity of estrogenic regulation is that it is affected by a wide variety of non-steroidal compounds in addition to estradiol, the natural ligand. Some of these compounds, including tamoxifen, have varying effects as agonists or antagonists, depending on the particular organ system or gene examined (Harris et al 1992; Phillips 1996). Thus, there is a search for natural compounds, which would mimic the effects of estrogen, and would be devoid of adverse effects.

Phytoestrogens are naturally occurring substances, derived from plants which are part of the human diet, and have a structure and activity similar to those of estradiol (4). Steroid-like activity of a compound may result from different mechanisms of action: a) altering the level of the endogenous steroid by affecting its synthesis, metabolism, distribution, or clearance; b) affecting the receptor function and thus altering tissue responsiveness to the steroid; c) interacting directly with the steroid receptor as an agonist. Phytoestrogens are capable of such activity, including subclasses such as lignans, isoflavonoids and coumestans, and they are widely distributed in oilseeds (flax, cereals), vegetables and soybeans. The main mammalian lignans are enterolactone and enterodiol, and the major active isoflavonoids are genistein and daidzein, both detected in human plasma. Most of the isoflavonoids occurring in plants are in bound form, as glycosides, and in this form they are biologically inactive. To retain the activity, the ether bond has first to be cleaved, a process which is likely to occur in the gut flora.

Isoflavans, like lignans, were found to have weak estrogenic activity, ranging from 500-15,000 times less than the estradiol. Accumulating evidence from molecular and cellular biology experiments, animal studies, and to a limited extent, human clinical trials, suggests that phytoestrogens may potentially confer health benefits related to cardiovascular diseases, cancer, osteoporosis, and menopausal symptoms. These potential benefits are consistent with the epidemiological evidence that rates of heart disease, various cancers, osteoporotic fractures, and menopausal symptoms are more lower amongst populations that consume plant-based diets, particularly cultures with diets high in soy products (Tham et al 1998; Zava et al 1997a; Cassidy et al 1993, Fournier et al 1998; Herman et al 1995; Knight et al 1996).

Isoflavans are subclass of the flavonoid compounds, contain ring A fused to ring C connected to ring B through carbon 3 (Fig. 1). Several functional groups may be attached to this basic skeleton mainly hydroxyl groups. In the isoflavan sub-class, the heterocychc ring C does not contain double bond between carbon 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. Their structure and lipophilicity resemble those of estradiol (Fig. 1), which makes them possible candidates to function as estrogen- like, in contrast to most known flavonoids. Several isoflavans which exhibited anti-oxidative activity were isolated by the inventors from licorice roots (Fig. 2) (Vaya et al 1997). Glabridin is the major constituent (11%) of the alcoholic extract and had the highest antioxidative activity (Belinki et al, 1997).

Application of glabridin in cosmetic compositions is disclosed by the prior art. For example, JP 09227341, describes the use of glabridin compositions for the treatment of androgen related conditions, particularly male alopecia, acne, prostatic hypertrophy and the like, and for cosmetic conditions such as pimples. However, application of isoflavans, such as glabridin, for the treatment and prevention of menopausal symptoms, post-menopausal osteoporosis and cardiovascular diseases is not suggested by the prior art. 2a Summary of the Invention The present invention relates to phytoestrogenic pharmaceutical compositions comprising as active ingredient at least one isoflavan having estrogen-like activity.

The invention also relates to phytoestrogenic pharmaceutical compositions comprising as active ingredient a non-polar fraction of a licorice extract having estrogen-like activity.

The isoflavan can be is glabridin, 4'-0-methylglabridin, 2'-0-methyl-glabridin or 2',4'-0-methylglabridin.

Description of the Figures Figure 1 The structure of the isoflavan glabridin and the estrogen (estradiol).

Figure 2 The structure of the isolated isoflavans from Licorice roots and the synthesized glabridin derivatives.

Figure 3 Competition of glabridin and its derivatives for binding of [3H] labeled 17U-estradiol to T47D cells Cells were incubated with [3H]17p-estradiol and increasing concentrations of glabridin, its derivatives 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 4 The effect of glabridin and its derivatives on the growth of estrogenic 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). 3 Figure 5 The Effect of glabridin on anchorage-independent growth of MCF7 cells MCF-7 cells were plated onto soft agar plates (containing 0.3% agar) in the presence of increasing concentrations of glabridin with and without ΙΟηΜ estradiol. After 3 weeks colony formation was observed.

A: Increasing concentration of glabridin. Magnification x 5.

B: Glabridin+estradiol. Magnification x 5.

Detailed Description of the Invention The present invention is based on the finding that various isoflavans possess estrogen-like activity.

The invention relates to phytoestrogenic pharmaceutical compositions comprising as active ingredient at least one isoflavan having estrogen-like activity, optionally also comprising pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

The invention also relates to phytoestrogenic pharmaceutical composition comprising as active ingredient a non-polar fraction of a licorice extract having estrogen-like activity, optionally also comprising pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

Preferred isoflavans are glabridin, 2'-0-methylglabridin, 4'-O-methyl-glabridin and 2',4'-0-methylglabridin.

The preferred active agent in the compositions of the invention is glabridin, found in licorice, which is a phytochemical with several known biological activities and a chemical structure and lipophihcity which resemble those of estradiol (Fig. 1). As will be shown in the following Examples, the inventors have investigated the estrogenic and proliferation-inducing activity of 4 glabridin and glabridin derivatives, and have demonstrated that glabridin binds to the human estrogen receptor, enhances the proliferation of estrogen-dependent human breast cancer cells (MCF7 and T47D) in vitro at concentration of ΙμΜ, achieving proliferative effects similar to those of InM estradiol and ΙμΜ genistein, a known phytoestrogen from soy. In vivo, it has been shown that glabridin affects the uterus wet weight and stimulates the specific activity of the "estrogen-induced protein" creatine kinase B in rat skeletal tissues, cardiovascular tissues and uterus.

The estrogenic activity of several other isoflavans isolated from licorice (Fig. 2) was studied in order to shed some light on the relevance of the chemical structure of the molecules to the estrogenic activity. Preliminary data suggest that the position of the hydroxyl group at B ring in an isoflavan derivative has a significant role in binding to the human estrogen receptor and in proliferation-inducing activity.

The cardiovascular effects of glabridin were studied in vitro on human endothelial cells and smooth muscle cells. Preliminary results show 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, the compositions of the invention may be used in the treatment or prevention of various hormone-dependent condition. The compositions are particularly useful in the treatment and prevention of cardiovascular diseases, cancer, menopausal symptoms and post-menopausal osteoporosis.

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

Examples Procedures: Details of procedures are given in the description of the Figures and in the Results section.

Results Glabridin and its derivatives bind to the human estrogen receptor To appreciate how the molecular structure of glabridin and its derivatives 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. 3. 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, 2'-0-methylglabridin and 4'-0-methylglabridin for ER were about 103, 104 and 10G lower, than that of estradiol, respectively. 2,4-O-methylglabridin did not bind to the human ER. The IC50 for glabridin was approximately 5μΜ, indicating that it is relatively weak ligand for the receptor. Nevertheless, this concentration is similar to other known phytoestrogen such as genestein (Zava et al 1997b, Wang et al 1997) and is about 103 lower than that of estradiol.

Effects of glabridin and its derivatives on proliferation of breast cancer cells The effects of increasing concentrations of glabridin and its derivatives on cell growth were compared with those of estradiol and are shown in Fig 4. Maximal stimulation of cell proliferation occurred with about ΙΟΟρΜ estradiol (data not shown), ΙΟμΜ glabridin. 2'-0-methylglabridin, μΜ 4'-0-methylglabridin, 2,4-O-methylglabridin, HispA and HispB had weak effect on cell proliferation. Glabridin at 25μΜ markedly inhibited growth whereas 2'-0-methylglabridin, 4'-0-methylglabridin, 2,4-O-methylglabridin, HispA and HispB inhibited the growth of the human breast cancer cells at approximately ΙΟΟμΜ. 6 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 lOnM estradiol or ΙΟμΜ glabridin. The effects of increasing concentrations of glabridin on colony formation were found to be similar to its effects on cell proliferation, i.e. the effect was bi-phasic. Glabridin alone stimulated colony formation over 1μΜ-10μΜ (Fig. 5A). Maximal colony stimulation by glabridin at ΙΟμΜ was equal to that of estradiol at lOnM (data not shown). In contrast to its colony formation-promoting effects at lower concentrations, glabridin inhibited anchorage-independent growth at concentrations >15μΜ. When glabridin was tested in the presence of lOnM estradiol, it had no effect on the anchorage-independent growth promoting effects of estradiol over the concentration range from lOnM to ΙΟμΜ (Fig. 5B). The growth-inhibitory actions of glabridin over the 15μΜ to 50μΜ concentration range were not modified by estradiol, i.e. glabridin affects the number and size of colonies.

Effects of glabridin in vivo on immature female rats Injection into prepubertal female rats of estradiol ^g/rat) or glabridin (20(^g/rat) resulted in a significance increase of uterus weight after 24 hours (Table 1), and in a significance 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 2). In several tissues (bone, cartilage, uterine and cardiovascular tissues) the effects of estrogen, including growth modulation, are linked 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 et al 1983, Somjen et al 1989). 7 Table 1 Glabridin stimulates uterus growth in female rats Rats were sacrificed 24h after injection with 5μg estradiol or 20(^ glabridin and wet uterine weight was determined. Results are presented as wet uterus weight ±SD.

Table 2 Glabridin induction of creatine kinase activity in various female rat tissues 8 Rats were sacrificed 24h after injection with 5 μ£ estradiol or 200μ¾ glabridin. CK was extracted and assayed. Results are presented as means +SD of CK specific activity (μιηοΐ/mm/mg protein).

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 (Iafrati et al 1997). 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.

Table 3 Estrogenic activity of glabridin in vascular cells 9 E304 cells (human endothelial 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.

References Belinky PA, et al. Atherosclerosis 137(1):49-61 (1998).

Belinky PA, et al. Free Radic Biol Med 24(9): 1419-29 (1998).

Cassidy A, et al. FASEB J. 7(3 Pt II):5000 (1993).

Fournier BF, et al. Cancer Epidemiology, Biomarkers and Prevention 7:1055-1065 (1998).

Herman C, et al. J. Nutr. 125:757S-770S (1995).

Iafrati MD, et al. Nat. Med. 3(5):545-8 (1997).

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Claims

9865/99 132666/3

1. Phytoestrogenic pharmaceutical compositions comprising as active ingredient at least one isoflavan having estrogen-like activity, optionally also comprising a pharmaceutically acceptable carrier, adjuvant, excipient or diluent.

2. A pharmaceutical composition according to claim 1, wherein said isoflavan is glabridin.

3. A pharmaceutical composition according to claim 1, wherein said isoflavan is 4'-0-methylglabridin.

4. A pharmaceutical composition according to claim 1, wherein said isoflavan is 2'-0-methylglabridin.

5. A pharmaceutical composition according to claim 1, wherein said isoflavan is 2',4'-0-methylglabridin.

6. A pharmaceutical composition according to any one of the preceding claims, for the treatment and prevention of menopausal symptoms.

7. A pharmaceutical composition according to any one of claims 1 to 5, for the treatment and prevention of post-menopausal osteoporosis.

8. A pharmaceutical composition according to any one of claims 1 to 5, for the treatment and prevention of cardiovascular diseases.

9. Use of an isoflavan having estrogen-like activity in the preparation of a phytoestrogenic pharmaceutical composition.

10. The use according to claim 9, wherein said isoflavan is glabridin. 1 1 132666/1

11. The use according to claim 9, wherein said isoflavan is any one of 4'-0-methylglabridin, 2'-0-methylglabridin and 2',4'-0-methylglabridin.

12. The use of any one of claims 9 to 11, wherein said pharmaceutical composition is intended for the treatment and prevention of menopausal symptoms.

13. The use of any one of claims 9 to 11, wherein said composition is intended for the treatment and prevention of post-menopausal osteoporosis. 14. The use of any one of claims 9 to 11, wherein said composition is intended for the treatment and prevention of cardiovascular diseases.

14. A pharmaceutical composition according to any one of claims 1 to 8, substantially as herein described and exemplified. 12