WO2008004223A2 - Compounds and methods for the treatment of menopause and bone related disorders - Google Patents

Abstract

There is provided a method for treating a menopausal symptom, which may include administering an effective amount of a Femarelle (DT56a) compound to a female suffering from a menopausal symptom. Also provided is a method for treating a bone density disorder, which may include administering a Femarelle (DT56a) compound to a subject suffering form bone density disorder. Bone density disorder may include osteopenia, osteoporosis, age-related bone disorder or any combination thereof. Also provided is a method for of inhibiting estrogen binding to estrogen receptors (ER) in a uterus, the method comprising administering an effective amount of a Femarelle (DT56a) to a female. Also provided is a method for inhibiting estrogen binding to estrogen receptors (ER) in a breast tissue, the method may include administering an effective amount of a Femarelle (DT56a) to a female.

Description

COMPOUNDS AND METHODS FOR THE TREATMENT OF

MENOPAUSE AND BONE RELATED DISORDERS

BACKGROUND

[001] Maintaining human health and restoring it, is one of the most significant problems health care providers deal with every day. When entering middle age (usually age 35-70 years for human females) and even before or after, certain conditions occur or evolve, which may affect health and decrease the

quality of life. Menopausal symptoms and osteoporosis are two common conditions often related to middle age.

[002] Menopause is a stage of a female, usually associated with the

reproductive cycle, traditionally characterized by the ovaries decreasing or ceasing production of estrogen (and/or other hormones), which may relate to the reproductive system and other functions. Further information and description on the causes and effects of menopause can be found in (herein incorporated by reference in their entirety):

http://www.niapublications.org/agepages/menopause.asp;

http://www.mayoclinic.com/print/menopause/DS00119/DSECTION=all&METH OD=print; http://www.wrongdiagnosis.com/rn/menopause/causes.htm;

http://www.safemenopausesolutions.com/causesmenopause.html; http://www.earlymenopause.com/causes.htm; and http://www.4woman.gov/faq/menopaus.htm. [003] As the body adapts to the changing levels of hormones, vasomotor symptoms (which may include alteration of the diameter of the vessels, especially the blood vessels) appear. Such vasomotor symptoms can include or be related to hot flashes and increased heart beats (also known as palpitations). Psychological symptoms may also appear, such as increased depression, anxiety, irritability, mood swings and lack of concentration. Atrophic symptoms (which may include symptoms related to degeneration of

the body), such as vaginal dryness and urgency of urination, may appear. The decrease in estrogen levels may also lead to a loss in bone density, since estrogen relates to building and maintaining bone density. In some cases, this loss in bone density may lead to osteoporosis, which will be further discussed hereinafter. Further information and description on the causes and effects of estrogen can be found in (herein incorporated by reference in their entirety):

http://www.labtestsonline.org/understanding/analytes/estrogen/test.html; http://www.nlm. nih.gov/medlineplus/news/fullstory_29361. html; http://www.medem.com/medlb/article_detaillb.cfm?articleJD=ZZZJJKPNWLC &sub_cat=502;

http://www.nytimes.com/specials/women/warchive/970304_1671.html; http://www.bio.cmu.edu/courses/BiochemMols/ER/ERIntro.html;

http://wwwlabtestsonline.org/understanding/analytes/estrogen/multiprint.html; and

http://wwwlabtestsonline.org/understanding/analytes/estrogen/test.html.

[004] Menopausal symptoms might appear before middle age, for example in premature menopause, which is usually considered as menopause occuring under the age of 35 for human females (hereinafter referred to as "women"). Premature menopause can be due to genetics and/or malnourishment (which can be the outcome of a disease like anorexia and/or simply a lack of essential food elements in diet). Premature menopause, may include

additional disorders such as autoimmune disorders, thyroid disease or other disease(s) of the endocrinology system such as diabetes. Menopausal symptoms can be surgically induced for example, if the woman has had a previous hysterectomy or by various procedures such as chemotherapy or exposure to ionizing radiation. Menopause symptoms can also appear due to other medical conditions, for example, when a person suffers from a certain disease which does not allow her to digest or absorb vital ingredient(s) derived from nutrition or if the person had suffered from cancer or another serious illness. Further information and description on the causes and effects

of menopause can be found in (herein incorporated by reference in their entirety):http://www.menopause- online.com, http://nccam.nih.gov/health/menopauseandcam/; and

http://womenshealth.gov/faq/menopaus.htm.

[005] Osteoporosis is a disease of the bones often characterized by bone mineral density (BMD) being reduced and/or bone architecture being disrupted (such bone matter loosing its critical mass and/or its normal structure, therefore potentially becoming less capable and tending to fracture

easily). Due to different hormone component(s), female(s) reputedly suffer from osteoprosis more than males. Further information and description on the

causes and effects of osteoporosis can be found in (herein incorporated by reference in their entirety):

http://www.wheelessonline.com/ortho/search?search=Osteoporosis&go.x=7& go.y=10; http://www.nof.org/osteoporosis.html; http://www.osteo.org/default.asp; http://www.nih.gov/news/WordonHealth/dec2003/osteo.htm; http://nihseniorhealth.gov/osteoporosis/toc.html; http://www.niams.nih.gov/bone/index.htm; http://www.doctorsforaduits.com/topics/dfa_oste.htm; and http://www.medes.fr/Eristo/Osteoporosis/BoneRemodeling.html.

[006] While treatments are becoming available, prevention is still the most important way to reduce fractures related to osteoporosis and/or reduce menopausal symptoms to improve quality of life. Numerous products have been disclosed or described which advertise their use or capability of slowing or inhibiting either or both the process of osteoporosis and/or menopausal symptoms.

[007] Since estrogen is one of the key factors related to menopause and may be related to osteoporosis, understanding its mechanism is advantageous. The mechanism of estrogen action is believed to include a binding to an estrogen receptor (ER) in a responsive cell (a cell which is adapted to contact with an estrogen receptor). The estrogen-ER complex, or at least a portion thereof, is then trans-located into the nucleus, where it expedtedly binds to the DNA (deoxyribonucleic acid) and acts to modulate the rate of transcription of specific genes. Two ERs have been currently identified, ERa and ERβ, which differ in their structure, tissue distribution and their biological effects. Further information and description on the causes and effects of estrogen receptor can be found in (herein incorporated by reference in their entirety):

http://www.bio. emu. edu/Courses/BiochemMols/ER/ERIntro. html; http://www.bio. emu. edu/Courses/BiochemMols/ER/ERIntro.html#overview; http://www.bio. emu. edu/Courses/BiochemMols/ER/ERIntro.htm^Erchime;

B. Hanstein, et ai, "p300 is a component of an estrogen receptor coactivator complex", Proceedings of the National Academy of Science, PNAS, Vol. 93, Issue 21 , 11540-11545, October 15, 1996;

AK. Shiau.D.Barstad, PM. Loria.LCheng.PJ. Kushner.DA. Agard, and GL. Greene "The Structural Basis of Estrogen Receptor/Coactivator Recognition and the Antagonism of This Interaction by Tamoxifen", Cell, Vol. 95, 927-937, December 23, 1998; http://www.pnas.Org/cgi/content/abstract/81/2/429; and http://www.pnas.Org/cgi/content/abstract/74/9/3681.

[008] Hormone Replacement Therapy (HRT), is commonly used in relation to

menopause and osteoporosis. HRT attempts to provide a suplement for various hormones such as estrogens, progesterone or progestins, and sometimes testosterone. HRT can have non-desired side effects, such as depression, headaches, breast sensitivity, skin irritation, weight gain, menstrual bleeding and others, additionally, there appears to be an increased risk for breast cancer, stroke, and/or cardiovascular disease. These severe side effects led to extensive research aimed at finding compounds having beneficial estrogenic effects on selected sites, such as bones and cardiovascular system, while minimizing harmful side effects. Further information and description on the causes and effects of HRT can be found in (herein incorporated by reference in their entirety):

http://www.osteoporosis.ca; http://training.seer. cancer. gov/module_anatomy/unit6_3_endo_glnds. html; http://www.nlm. nih.gov/medlineplus/news/fullstory_29814. html; http://www.nlm. nih.gov/medlineplus/news/fullstory_29967. html;

http://www.nlm. nih.gov/medlineplus/alphanews_h.html#HormoneReplacement Therapy; http://www.nlm. nih.gov/medlineplus/news/fullstory_29293. html; and http://www.mayoclinic.com/health/cancer-treatment/CA00039.

[009] There are various other medical and pharmaceutical treatments, which have beem used in relation to menopause and osteoporosis. Biophosphonates are a drug family, which may be used to treat osteoporosis. Biophosphonates are assumed to bind permanently to the surfaces of bones and to inhibit osteoclasts (bone-destroying cells). Further information and description on the causes and effects of osteoclasts can be found in (herein incorporated by reference in their entirety):

http://www.wheelessonline.com/ortho/osteoclasts; http://www.medterms.com/script/main/art.asp?articlekey=11794; http://users.rcn.eom/jkimball.ma.ultranet/BiologyPages/B/Bone.html; http://www.fda. gov/fdac/features/796_bone. html; and http://www.gentaur.com/new_page_77.htm.

[0010] Biophosphonates alledgedly allow osteoblasts (bone-building cells) to work more effectively. Biophosphonates may also have non-desired side effects. Further information and description on the causes and effects of biophosphonates can be found in (herein incorporated by reference in their entirety):

http://www.emedicinehealth.com/articles/45766-8.asp; http://www.endocrineweb.com/osteoporosis/fosamax.html; http://www.truestarhealth.com/Notes/1306002.html; http://www.manxbonezone.com/treatments.htm; http://www.expertcIick.com/NewsReleaseWire/default.cfm ?Action=ReteaseDe tail&ID=7388; and http://www.malecentrum.sk/kniha.php?!D=170&kat=19.

[0011] Another method which alledgedly relates to the treatment of osteoprosis and menopause is the usage of Selective Estrogen Receptor

Modulators (SERMs). SERMs are principally non-hormonal compounds, which can act like an estrogen hormone in some parts of the body, such as the bones and heart, but do not affect other parts of the body, such as the

uterus and breast(s). Some SERMs, such as Raloxifene, while reported to be helpful in treating osteoporosis, worsen menopausal symptoms. For example, Raloxifene may increase hot flashes. Moreover the use of SERMs may increase the risk of blood clots, which may lead to a stroke. Further information and description on the causes and effects of SERMs can be found in (herein incorporated by reference in their entirety):

http://www.breastcancer.org incorporated herein by reference.

[0012] Another method for treating osteoprosis is by using parathyroid

hormone (PTH) its analogue, teriparatide, and the like, which are known as "bone formation agents". These bone formation agents are believed to affect the process by which a bone is constantly renewed, as by activating osteoblasts (bone-building cells). Further information and description on the causes and effects of osteoblasts can be found in (herein incorporated by reference in their entirety):

http://www.wheelessonline.com/ortho/search?search=Osteoblasts&go.x=9&g o.y=8; http://medical-dictionary.thefreedictionary.com/osteoblasts; http://www.nature.com/glossary/clinicalpractice/defDetails. do?uid=ncp_1283; http://www.medes.fr/Eristo/Osteoporosis/BoneRemodeling.html;

http://education.vetmed.vt.edu/Curriculum/VM8054/Labs/Lab8/Examples/exos blst.htm; http://users.rcn.eom/jkimball.ma.ultranet/BiologyPages/B/Bone.htm;! http://www-medlib.med.utah.edU/WebPath/BONEHTML/BONE012.html: and http://encyclopedia.thefreedictionary.com/osteoblasts.

[0013] Also used in relation to menopause are phytoestrogens. Phytoestrogens are plant compounds having estrogen-like biological activity, which are considered to be a natural alternative to the use of hormone replacement therapy (HRT) for the attributed relief of experiencing mild menopausal symptoms. Phytoestrogens, found in a wide variety of edible plants, may display both estrogenic and antiestrogenic effects. Reputedly, Japanese women are said to experience a lower frequency of hot flashes or flushes at or in menopause than Western women, and this has been partly attributed to their high phytoestrogen consumption. The biological actions of these compounds are extremely complex. Their ultimate actions, reactions and effects are determined by many factors, including the relative levels of estrogen receptors rc. and P. Further information and description on the causes

and effects of phytoestrogens can be found in (herein incorporated by reference in their entirety): Journal of the Society for Gynecologic Investigation, volume 9, number 4, July 2002, pp.238-242, "A Pilot Study of the Effects of Phyto Estrogen Supplementation on Postmenopausal Endometrium". [0014] There is a need for more efficient phytoestrogenic and other compounds and/or compositions containing them that may be used for the treatment of menopausal symptoms and/or osteoporosis having reduced or no side effects.

[0015] The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification and a study of the figures

SUMMARY

[0016] The following embodiments and aspects thereof are described and illustrated in conjunction with systems, tools and methods which are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other advantages or improvements.

[0017] There is provided, in an embodiment, a method for treating a menopausal symptom, which may include administering an effective amount of a Femarelle (DT56a) compound to a female suffering from a menopausal symptom.

[0018] Treating may include reducing, preventing or delaying the occurrence or reoccurrence of a menopausal symptom. Treating may also include improving the quality of life of a female. [0019] The menopausal symptom may include a vasomotor symptom, a psychological symptom, a physiological symptom or any combination thereof.

[0020] The vasomotor symptom may include hot flashes, night sweats, sleep disturbances, headaches, palpitations or any combination thereof.

[0021] The psychological symptom may include increased depression, anxiety, irritability, mood swings, lack of concentration, reduced sexual desire/arousal, fatigue or any combination thereof.

[0022] The physiological symptom may include, arthralgia and myalgia, vaginal dryness, dyspareunia, Vaginal itching/burning, urinary frequency, dysuria urgency or any combination thereof.

[0023] The method, as disclosed herein, may include treating a menopausal symptom essentially without inducing an estrogenic effect on the reproductive tract.

[0024] The effective amount of the Femarelle (DT56a) compound may include about 500-1000 mg/day. The effective amount of the Femarelle (DT56a) compound may include about 644 mg/day. The effective amount of the Femarelle (DT56a) compound may include about 200-500 mg/day. The effective amount of the Femarelle (DT56a) compound may include about 322 mg/day.

[0025] Also provided is a method for treating a bone density disorder, which may include administering a Femarelle (DT56a) compound to a subject suffering form bone density disorder. Bone density disorder may include osteopenia, osteoporosis, age-related bone disorder or any combination thereof. Osteoporosis may include postmenopausal osteoporosis or osteopenia. Treating the bone density disorder may include increasing the bone mineral density (BMD). Increasing the bone mineral density (BMD) may include increasing the BMD by between 1-5%. Treating a bone density disorder may include inhibiting bone loss. Treating a bone density disorder may include increasing bone formation. Treating a bone density disorder may include stimulating osteoblast formation. The method may include treating a bone density disorder essentially without inducing an estrogenic effect on the reproductive tract. The effective amount of the Femarelle (DT56a) compound may include about 500-1000 mg/day. The effective amount of the Femarelle (DT56a) compound may include about 644 mg/day. The effective amount of the Femarelle (DT56a) compound may include about 200-500 mg/day.

[0026] Also provided is a method for enhancing regulation of proliferation of the bone cells, the method comprising contacting a Femarelle (DT56a) compound with bone tissue.

[0027] Moreover, there is provided a method for enhancing regulation of differentiation of bone cells, the method comprising contacting a Femarelle (DT56a) compound with bone cells.

[0028] Also provided is a method for stimulating osteoblast formation, which may include administering a Femarelle (DT56a) compound to a subject suffering from a bone density disorder.

[0029] Also provided is a method for enhancing regulation of differentiation of bone cells, the method comprising contacting a Femarelle (DT56a) compound with bone tissue. [0030] The Femarelle (DT56a) compound may enhance the production of mRNA, which may induce the production of an estrogen receptor (ER) in bone cells. ER may include ERβ. ER may include ERa. The mRNA may induce the production of ERa and ERβ on bone cells, wherein the production of ERβ may be higher than the production of ERa.

[0031] The Femarelle (DT56a) compound may further enhance regulation of proliferation of the bone cells. The bone cells as referred to herein may include osteoblasts.

[0032] Also provided is a method for increasing the intracellular levels of [Ca2+], the method comprises administering a Femarelle compound to a subject suffering from a bone density disorder.

[0033] Also provided is a method for stimulating bone cell formation, the method comprising administering a Femarelle (DT56a) compound to a subject suffering from a bone density disorder. The bone cells may include osteoblasts. The Femarelle compound may be adapted to enhance the production of mRNA. The enhancement of the production of mRNA may induce the production of estrogen receptors (ER) in bone cells.

[0034] Also provided is a method for stimulating osteoblast formation in a cell culture, the method comprises contacting a Femarelle (DT56a) compound with an osteoblast cell culture.

[0035] Also provided is a method for increasing the intracellular levels of [Ca²⁺] in a cell culture, the method comprises contacting a Femarelle compound with an osteoblast cell culture. [0036] Also provided is a method for enhancing regulation of differentiation of bone cells, the method comprising contacting a Femarelle (DT56a) compound with bone cells. The bone cells may include osteoblasts.

[0037] Also provided is a method for regulation of differentiation of bone cells, the method comprising contacting a Femarelle (DT56a) compound with bone tissue. The bone cells may include osteoblasts.

[0038] Also provided is a method for regulation of proliferation of bone cells, the method comprising contacting a Femarelle (DT56a) compound with bone tissue. The bone cells may include osteoblasts.

[0039] Also provided is a method for of inhibiting estrogen binding to estrogen receptors (ER) in a uterus, the method comprising administering an effective amount of a Femarelle (DT56a) to a female. The Femarelle (DT56a) may have an antagonistic effect on estrogen receptors (ER) in a uterus. The effective amount of the Femarelle (DT56a) compound may include about 500- 4000 mg/day. The effective amount of the Femarelle (DT56a) compound may include about 1300 mg/day. The effective amount of the Femarelle (DT56a) compound comprises about 644 mg/day.

[0040] Also provided is a method for inhibiting estrogen binding to estrogen receptors (ER) in a breast tissue, the method may include administering an effective amount of a Femarelle (DT56a) to a female. The effective amount of the Femarelle (DT56a) compound may include about 200-4000 mg/day. The effective amount of the Femarelle (DT56a) compound may include about 644 mg/day. The effective amount of the Femarelle (DT56a) compound comprises about 322 mg/day. [0041] In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the figures and by study of the following detailed description.

DETAILED DESCRIPTION

[0042] While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and sub-combinations thereof, it is therefore intended that the following appended claims and claims hereafter introduced be interpreted to include ail such modifications, permutations, additions and sub- combinations as are within their true spirit and scope.

[0043] In the following description, various aspects of the invention will be described. For the purpose of explanation, specific configurations and details are set forth in order to provide a thorough understanding of the invention. However, it will also be apparent to one skilled in the art that the invention may be practiced without specific details being presented herein. Furthermore, well-known features may be omitted or simplified in order not to obscure the disclosure.

1. Femarelle

[0044] Femarelle (interchangeably referred to herein also as DT56a and Tofupill) may refer to one or more compounds which may be produced from soybean. Femarelle may include one or more phytoestrogen ingredients. Femarelle may include one or more isoflavones which is a main subclass of phytoestrogen. [0045] Femarelle is associated with soybean in a refining, extracting, purifying or other process that may include one or more of any of the following processes: enzymatic reaction, extraction, distillation, hydrolyzing, separation, cleaning, purification, isolation, heating, cooling, freezing, crystallization, vaporizing or any other suitable process known in the art.

[0046] Various terms and acronyms may be used herein. The term "BMD" as used herein refers to bone mineral density. In another embodiment, the term "FSH" as used herein refers to follicle-stimulated hormone. The term "CK" as used herein refers to creatine kinase. The term "MCF-7" estrogen as used herein refers to estrogen-dependent breast cancer cell line. The term "ER" as used herein refers to an estrogen receptor. The term "HRT" as used herein refers to hormone replacement therapy. The term "SERM" as used herein refers to a selective estrogen-receptor modulator. The term "E2" as used herein refers to estradiol, a type of estrogen. The term "hObs" as used herein refers to human osteoblasts.

2. The Effect of Femarelie on menopausal symptoms

[0047] There is herein provided a method for treating a menopausal symptom which may include administering an effective amount of a Femarelle compound to a female suffering from a menopausal symptom(s).

[0048] There is further provided herein a method for reducing a menopausal symptom which may include administering an effective amount of a Femarelle compound to a female suffering from a menopausal symptom(s).

[0049] There is also provided a method for treating a symptom of hormonal variation in a female which may include administering an effective amount of a Femarelle compound to the female suffering from a hormonal variation related condition.

[0050] There is also provided a method for improving the quality of life of a female which may include administering an effective amount of a Femarelle compound to the female suffering from a hormonal variation related condition, such as menopause.

[0051] There is provided a method for preventing or delaying the occurrence or reoccurrence of a menopausal symptom which may include administering an effective amount of a Femarelle compound to the female.

[0052] Menopausal symptoms may include, for example but are not limited to, vasomotor symptoms such as hot flashes, flushes and palpitations (awareness of the beating of the heart), psychological symptoms such as increased depression, anxiety, irritability, mood swings and/or lack of concentration, headache, insomnia, arthralgia, mylagia, atrophic symptoms such as vaginal dryness and urgency of urination, increasingly scanty and erratic menstrual periods or any combination thereof. Menopausal symptom(s) may be measured according to an instrument, test or measure relevant for women in their menopausal transition, such as the Kupperman index.

[0053] The term "treating" as used herein may include preventing, reducing, suppressing and/or inhibiting. The terms "reducing", "suppressing" and "inhibiting" may have their commonly understood meaning(s) including lessening or decreasing. "Progression" as referred to herein, includes increasing in scope or severity, advancing, growing or becoming worse — for example, as related to a symptom. "Recurrence" as referred to herein, may include the return of a disease or a symptom after a remission or after reduction of the symptom.

[0054] The female may be pre, post or menopausal. The menopause may be drug induced, surgically induced or naturally or otherwise occurring.

[0055] A Femarelle compound may be present in a pharmaceutical composition. The pharmaceutical composition may include a pharmaceutically acceptable carrier or excipient.

[0056] The term "Femarelle compound" may refer to one or more compounds present in- Femarelle, its analog, derivative, isomer, metabolite, degradation product, pharmaceutically acceptable salt, pharmaceutical product, hydrate or any combination thereof.

[0057] A Femarelle compound, mixture, mixture of natural materials, composition or a pharmaceutical composition containing Femarelle compound may be administered to a subject by any method known to a person of skill in the art, for example, orally, subcutaneously, ocularly, transcutaneous^, intravenously, intramuscularly, intraperitoneal^, nasal inhalation, implantation, intra-arterially, intralesionaliy, by application to mucous membranes, by transdermal patch or by any other suitable way. The Femarelle compound or a pharmaceutical composition containing the Femarelle compound may be topically administered to one or more of a subject's body surfaces, and/or may be formulated in any form suitable for topical administration, for example, gels, ointments, creams, pastes, lotions, drops and the like. [0058] The term "pharmaceutical composition" as used herein, may include a "therapeutically effective amount" of the Femarelle compound together with a pharmaceutically acceptable carrier or excipient. The term "therapeutically effective amount" as used herein may refer to that amount which provides a therapeutic effect for a given condition and administration regimen.

[0059] The term "administering" as used herein may refer to bringing into contact a subject and a Femarelie compound. Administration may be accomplished in vitro (in an artificial environment) or in vivo (in a living organism), for example, in cells or tissues of living organisms, for example humans.

[0060] The pharmaceutical compositions may be formulated for oral administration, for example, as a solid or liquid preparation. Suitable solid oral formulations may include tablets, capsules, caplets, pills, granules, pellets, powders, and the like. Suitable liquid oral formulations may include, solutions, suspensions, dispersions, emulsions, oils and the like.

[0061] The term "pharmacological dose" as used herein may refer to approximately 644 mg/day, and may sometimes may be used interchangeably with the term "standard dose", as opposed to a "low dose" which is approximately 344 mg/day.

[0062] A Femarelle compound may be delivered in a vesicle, for example but not limited to, a liposome (as described in Langer, Science 249: see especially pages 1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, N.Y., see especially pages 353-365 (1989); all incorporated in their entirety herein by reference).

[0063] The term "pharmaceutically acceptable carrier(s) or excipient(s)" as used herein may be well known to those of skill in the art, for example, a solid carrier or excipient for solid formulations, a liquid carrier or excipient for liquid formulations, a diluent, or any combination thereof. Solid carriers, excipients and diluents may include but are not limited to, according to some embodiments, a gum, a starch (for example, corn starch, pregeletanized starch), a sugar (for example, lactose, mannitol, sucrose, dextrose), a cellulosic material (for example, microcrystalline cellulose), an acrylate (for example, polymethylacrylate), calcium carbonate, magnesium oxide, talc, or any mixture thereof. Liquid pharmaceutically acceptable carriers may include but are not limited to, according to some embodiments, aqueous (for example, water, alcohol / water solutions) or non-aqueous (for example, propylene glycol, polyethylene glycol) solutions, suspensions, emulsions or oils (for example, peanut oil, soybean oil, mineral oil, olive oil, sunflower oil, and fish-liver oil).

[0064] The pharmaceutical composition may be delivered in a controlled (sustained) release system, for example but not limited to controlled release systems described in the review^' by Langer (Science 249: see especially pages 1527-1533 (1990) which is herein incorporated by reference in its entirety.

[0065] Femarelle may not affect estrogen type MCF-7 which is an estrogen- dependent breast cancer cell-line. Also, creatine kinase (CK), a marker of estrogenic activity may be used to determine the effect of Femarelle on skeletal tissues.

[0066] Femarelle may relieve vasomotor symptoms in females. In a study, Femarelle was found to relieve vasomotor symptoms in proximally 75% of the patients. Patients receiving pharmacological dose had a significant increase in BMD. Patients receiving a pharmacological dose had a statistically significant increase in BMD. Side effects which are commonly associated with various treatments, such as endometrial thickening and changes in serum E2 and in FSH hormone levels where not detected. Patients receiving pharmacological dose experienced no change in the endometrial thickness, serum E2 and in the FSH. Additionally, subjects receiving a pharmacological dose of Femarelle did not generate any noticable effect in the MCF-7 cell line.

[0067] It is well accepted that hormonal treatment by administration of E2 tends to desirably stimulate skeletal tissues but undesirably increases CK activity in the uterus. Femarelle stimulated skeletal tissues similarly to E2, but without increased CK activity in the uterus.

[0068] Raloxifene blocks the stimulation of CK induced by either Femarelle or E2 which indicates that Femarelle and E2 may have a common mechanism of action which includes binding to estrogen receptors (ER). Femarelle selectively activates ER in brain and bone tissues, yet does not have any effect on the breast and uterus.

[0069] Femarelle selectively activates estrogen receptors, for example by stimulating ER in the brain. Such stimulation can relive, inhibit or delay menopausal symptoms; stimulation of ER in the bones (including teeth) may result in an elevation in BMD both accomplished without noticeably changing FSH and E2 hormonal levels and without affecting ER in the uterus.

[0070] Example 1 shows multiple experiments on Femarelle exhibiting a reduction in menopausal symptoms without noticeable undesirable estrogenic effect. More details relating to Example 1 and the following Fig. 1-Fig. 5 (inclusive) may be found in I. Yoles, B. Chayen. Department of Obstetrics & Gynecology "selective estrogen receptor modulation by DT56a (Tofupill) a unique phyto-estrogen for menopausal treatment" which is incorporated herein by reference in its entirety.

Example Ka): In vivo trial of estrogenic effect of Femarelle

[0071] Femarelle DT56a had no measurable or noticeable estrogenic effect on estrogen dependent MCF-7 cells, in comparison to estrogen (E2) as shown in Fig.1. More specifically, the percentage of estrogen dependent MCF-7 cell growth was measured in relation to set concentrations (Cone.) of Femarelle (referenced as DT56a). As seen in Fig. 1 , the curve of the percentage of estrogen dependent MCF-7 cell growth versus the amount of Femarelle is shown at 100 and the curve of estrogen dependent MCF-7 cell growth versus the amount of E2 is shown at 120. The various points on curve 100 show specific cell growth when exposed respectively to Femarelle dosages of 0 (zero or none at 100a), a pharmacological dose (at 100b), four times a pharmacological dose (at 100c), and so on, up to 66,000 times a pharmacological dose shown at 100i. Similarly, the two points on curve 120 show specific cell growth when exposed respectively to E2 dosages of 0 (zero or none at 120a) and a pharmacological dose (at 120b). For example, in reference to Fig.1 , no increase in cancer ceil growth is detected at a pharmacological dosage of Femarelle. Only a minor increase in cancer cell growth is induced at high dosages of Femarelle (over 2000 times the pharmacological dosage). There is no increase in cell growth at lower dosages of Femarelle. In contrast, a large increase in cancer cell growth is induced at a pharmacological dosage of E2.

[0072] . E2 was added at pharmacological dose and Femarelle was added at incremental doses starting at the calculated pharmacological dose (0.3mg/ml) and ending at 66,000 times of the pharmacological dose.

Example Kb): In vitro trial of skeletal stimulation by Femarelle

[0073] Immature female rats were used intact or after ovariectomy. Rats (n=5 per group) were fed daily for 4 days with 650 mg Femarelle (calculated pharmacological dose) or E2 (5 mg). Creatine kinase (CK) specific activity was measured in order to assess the activation estrogen receptor. CK activity was previously described as a response marker for estrogenic activity. Long- term effect (2 months of daily ingestions) was analyzed by histological preparations. When administered in multiple oral doses, Femarelle stimulated skeletal tissues similarly to E2, but whereas E2 significantly increased CK specific activity in the uterus, Femarelle did not. The selective estrogen receptor modulator (SERM) Raloxifene blocked the stimulation of CK by either Femarelle or E2 in all tissues tested.

[0074] Femarelle stimulated skeletal tissues, without affecting the uterus, as shown in Fig.2 which includes Fig.2a, Fig.2b, Fig.2c, Fig.2d. Fig.2a exhibits a comparison between a control, Femarelle (referenced herein as DT56a) and estrogen in bone epiphysis (the end part of the bone). Fig.2b exhibits a comparison between a control, Femarelle (referenced herein as DT56a) and estrogen in a uterus. Fig.2a and Fig.2b show that when administered in multiple oral doses, Femarelle stimulated skeletal tissues similarly to estrogen, but whereas estrogen significantly increased CK specific activity in the uterus, Femarelle did not. Fig. 2c exhibits a comparison between a control, estrogen, Femarelle, Raloxifene, estrogen combined with Raloxifene and Femarelle combined with Raloxifene, in immature female rats. Fig. 2d exhibits a comparison between a control, estrogen, Femarelle, Raloxifene, estrogen combined with Raloxifene and Femarelle combined with Raloxifene, in ovariectomized (surgically remove an ovary) female rats. Fig.2c and Fig.2d show that the selective estrogen receptor modulator (SERM) Raloxifene blocked the simulation of CK by either Femarelle or E2 in all tissues tested.

[0075] Fig.3 shows pictures of the results from the lab trial ("in-vitro") of stimulated skeletal tissue samples form an ovariectomized female rats. The inhibition by Raloxifene may indicate a possible common receptor or receptors for E2 and Femarelle. The effect of Femarelle on bones was thus shown both by the biochemical assays and the histological preparations.

Example Kc): Clinical trial of skeletal stimulation by Femarelle

[0076] Ninety eight healthy postmenopausal women were randomly allocated to receive either pharmacological dose of Femarelle (644 mg/day) or a low dose of Femarelle (322 mg/day). A detailed medical history, physical examination, pelvic ultrasound and blood tests were performed. The physician completed a detailed Kupperman index and quality of life questionnaire for each patient. These measures were repeated every 3 months throughout the 12 months of treatment. Bone mass density (BMD) was assessed by dual- energy x-ray absorptiometry of the lumbar spine and femoral neck prior to the study and following 12 months of treatment.

[0077] Femarelle notably reduced menopausal symptoms without any undesirable estrogenic effect as shown in Fig.4 which shows the results of the clinical trial on postmenopausal women. Fig.4a exhibits the Femarelle effect on menopausal symptoms as a function of time. According to the graphs, the relief of at least one symptom was achieved in 81 % of the patients and was sustained in 76% following 12 months of treatment. For all 15 variables assessed all improvement achieved after 12 weeks of treatment remained consistent throughout the 12 months. Fig.4b shows a statistical elevation in BMD in both the spine and the femoral neck. According to Fig.4b, in the pharmacological dose study group there was a statistically significant elevation of 3.6% in the spine compared to a 0.6% reduction in the low dose group. The effect in the femoral neck was 2.6%.

[0078] Fig.5 shows the results of the clinical trial of postmenopausal women regarding the endometrial thickness, FSH levels and the E2 levels. Fig.5 includes three graphs Fig.5a, Fig.5b and Fig.5c which show no change in the endometrial thickness (Fig.5a), no change in the FSH levels (Fig.5b), no change in the E2 levels (Fig.5c) after 12 weeks and 12 months of Femarelle treatment in both the pharmacological and low dose groups. The effect of Femarelle remained constant during 12 months of treatment. Femarelle increased BMD without altering serum sex hormone levels or endometrial thickness. [0079] Example 2 shows a comparison between the efficacy of Femarelle vs. placebo. More details relating to Example 2 and the following Fig.6-Fig.9 (inclusive) may be found in I.Yoles, B.Chayen, B.Kaplan, DS Seidman, "phytoestrogen time-tested solution for menopausal symptoms revisited", which is incorporated herein by reference in its entirety.

Example 2(a): Experimental group

[0080] One hundred and two healthy postmenopausal ^' patients were randomized to receive daily tablets of either Femarelle or placebo. Detailed medical history, physical examination, pelvic ultrasound, hormonal and lipid blood test were taken. A detailed questionnaire regarding patient's complaints was completed at the time of enrolment and after 12 weeks.

Example 2(b): Detailed enrollment

[0081] A statistically significant improvement in menopause symptoms was observed in 12 out of 15 variables examined after treatment with Femarelle. Hot flushes decreased in 76% of the patients treated by Femarelle vs. 19% by placebo, palpitations 77% vs. 17%, sleeping disturbances 69% vs.16% and nervousness 56% vs. 14% in the placebo group (p<0.001). The percentage of patients reported a relief is shown in Fig. 6 and Fig.7. Fig.6 shows the percent of patients reported relief, in at least one symptom when administering different amounts of Femareile, more specifically: a placebo dose (no Femarelle existing in this dose), a low dose of Femarelle and a high dose of Femarelle. According to Fig.6 there was no significant change in the percentage of relief between the low dose and the high dose. Fig.7 shows the percent of patients relieved from main complaints such as hot flashes, insomnia, palpitations and libido when administering different amounts of Femarelle: a placebo dose (no Femarelle existing in this dose), a low dose and a high dose. According to the Fig 7, high dose showed better results in the relief of hot flashes, insomnia and palpitations, whereas low dose provide better results in the relief of libido Fig.8 shows the difference in the endometrial thickness at pre treatment and post treatment in a placebo and in a phytoestrogen (referenced herein as PE). Fig.9 shows the difference in the FSH levels at pre treatment and post treatment in a placebo and in a phytoestrogen (referenced herein as PE). According to Fig.9 no changes in the FSH of pre treatment and post treatment were detected.

Example 2(cV Preparations

[0082] The preparation when compared with a placebo significantly reduced menopausal symptoms without altering the serum sex hormone levels or endometrial thickness. A preparation containing high concentrations of active Femarelle may achieve a significant relief of menopausal symptoms.

[0083] Example 3 shows a clinical trial for the relieve of main menopausal symptoms. More details relating to Example 3 and the following Fig.10-Fig.15 (inclusive) may be found in I Yoles, Y.Yogev, Y.Frenkel, M.Hirsch, R.Nahum, B.Kaplan, "efficacy and safety of standards versus low-dose Femarelle (DT56a) for the treatment of menopausal symptoms", Abstract from clinical and experimental obstetrics&gynecology, Vol. XXXI₁ n. 2, 2004, which is incorporated herein by reference in its entirety

Example 3(a): Experimental group [0084] Eighty healthy postmenopausal women were randomly allocated to receive either the standard dose (SD) or low dose (LD) of Femarelle (644 mg/day vs. 344 mg/day). A detailed medical history was taken on enrollment, followed by a physical examination, pelvic ultrasound, and sex hormone and lipid profiles. A detailed Kupperman index for each patient. These measures were repeated every 3 months during 12 months of treatment. Eligibility for participating was limited to patients who had an intact uterus, no menses within the last six months, and blood hormone levels indicating menopause: follicle-stimulating hormone (FSH) >30 mlU/mL and estradiol (E₂) <150 pmol/L. Women with any pre-existing chronic illness, endometrial polyps or hyperplasia, and moderate or severe hypertension were excluded, as were habitual users of drugs or alcohol. The study was approved by the local ethic committee of the Rabin Medical Center, and all patients signed a written informed consent form, after receiving a comprehensive explanation of the study.

Example 3(b): Detailed enrollment

[0085] A complete medical and gynecological history was obtained for each patient, and the physician completed an assessment questionnaire of the frequency-severity of menopausal symptoms. All participants underwent a physical examination, gynecological and breast examination, and transvaginal ultrasonography. Laboratory tests included hormonal and lipid profiles, tests for liver and kidney function and mammography.

[0086] For the trial, externally identical SD (standard dose) and LD (low dose) capsules were placed in identical white bottles with no identifying marks apart from a serial number. The bottles were placed in front of the participants, and they were asked to choose one at random. The contents were unknown to the research team. Patients were randomized to receive the SD or LD of Femarelle, 644 mg/day vs. 344 mg/day for 12 months. All tests and assessments done at the time of enrollment were repeated after 3, 6, and 12 months, Mammogram was performed at enrollment and after 12 months. Compliance and side effects were assessed every 3 months. The Kupperman index was analyzed at the time of enrollment, after 12 weeks and 12 months.

Example 3(c): tools for Statistical analysis

[0087] Data were analyzed by a matched paired t-test. A p-value less than 0.05 was considered statistically significant.

Example 3(d): Reduction in the Kupperman index

[0088] In both groups, treatment resulted in a significant reduction in the Kupperman index: In the SD group 16±7.2 at enrollment and 9±6 and 8.7±7 at 12 weeks and 12 months respectively (p< 0.01). Similarly, in the LD group there was a significant decrease from enrollment (17±8.4) to 12 weeks (10±5.7) and to 12 months (8.6±7.3) (p < 0.01). Following 12 weeks and 12 months of treatment, there was no statistical difference in the decline in the Kupperman index between the SD and LD groups as shown in Fig.10. referenced from "efficacy and safety of standards versus low-dose Femarelle (DT56a) for the treatment of menopausal symptoms" The majority of patients reported relief in main menopausal symptoms in both groups; vasomotor: 76- 78% in the SD and LD respectively, headaches: 68-60%, Insomnia: 59-52% and arthralgia and myalgia: 70-70% of patients reported relief. This

23 improvement was maintained following 12 months of treatment as shown in Fig.11. Relief of at least one symptom was achieved in 81 % of the patients and sustained in 76% after 12 months, in both groups.

[0089] There was no statistically significant change in endometrial thickness as shown in Fig.12, FSH as shown in Fig.13, E₂ as shown in Fig.14 or TSH levels as shown in Fig.15 from enrollment to the end of the study in both groups.

3. The effect of Femarefte on bone density

[0090] In one embodiment, there is provided a method for treating a bone density disorder, the method may include administering a Femarelle compound to a subject suffering form bone density disorder.

[0091] In another embodiment, there is provided a method for increasing the bone mineral density (BMD) in a subject in a need thereof, the method may include administering a Femarelle compound to a subject.

[0092] In another embodiment, there is provided a method for inhibiting the bone loss in a subject, the method may include administering a Femarelle compound to a subject suffering from a bone loss.

[0093] In one embodiment, there is provided a method for treating a bone density disorder without inducing an estrogenic effect on the reproductive tract, for example, the uterus, the method may include administering a Femarelle compound to a subject suffering form bone density disorder.

[0094] In another embodiment, there is provided a method for increasing the BMD in a subject in a need thereof without inducing an estrogenic effect on the reproductive tract, for example, the uterus, the method may include administering a Femarelle compound to a subject.

[0095] In another embodiment, there is provided a method for inhibiting the bone loss in a subject, without inducing an estrogenic effect on the reproductive tract, for example, the uterus, the method may include administering a FemareUe compound to a subject suffering from a bone loss.

[0096] In one embodiment, the term "bone density disorder" may include any disease or condition that may relate to a temporary or permanent reduction of bone mineral density. In another embodiment, the bone density disorder may relate to osteoporosis, genetic bone disorder, age-related bone disorder or any other bone related condition, the term "Bone disorder" refers a disease which detrimentally affect the function and the structure of a skeletal system. In another embodiment, the may be localized, for example occurring in a part of a bone. In another embodiment the term "Bone density disorder" refers a disease of bone in which bone mineral density (BMD) is reduced and bone microarchitecture is disrupted, this kind of affected bones are susceptible to fracture since they are weak due to lack of vital substances. Osteoporosis is defined as a bone mineral density disorder (2.5 standard deviations below peak bone mass for 20-year-old person standard), the bone density disorder may be caused by an injury or a trauma.

[0097] The term "bone loss" may refer to a reduction of BMD or to any process that may result in a reduction in BMD.

[01] Osteoporosis may relate to postmenopausal osteoporosis. In another embodiment, osteoporosis may be type 1 or type 2 osteoporosis. [0098] The term "postmenooausal osteoporosis" as used herein, may refer to a form of osteoporosis that commonly occurs after menopause and may include any one of the following low bone mass, bone fragility, and susceptibility to fractures Osteoporosis may be assessed as bone mineral density (BMD) The T-score is a representation of patient's BMD expressed as number of standard deviations below or above mean BMD of the young adult. T-score values below 2 5 SD may be defined as characteristic of osteoporosis Values between -1 SD and -2 5 SD may be defined as osteopenia (precuisor condition to osteoporosis)

(http7/en wikipedia org/wiki/Postmenopausal osteoporosis, incorporated herein by reference)

[0099] The increase in BfMD resulted from any one of the methods disclosed herein, may be between 0 5-15% In another embodiment, the increase in BMD, resulted from any one of the methods disclosed herein, may be between 1-10% In anothei embodiment, the increase in BMD may be between 2-7% In another embodiment, the increase in BMD, resulted from any one of the methods disclosed herein, may be between 2-5%. In another embodiment, the increase in BMD, resulted from any one of the methods disclosed herein, may be more than 2%

[00100] The subject may be a female In another embodiment, the female may be postmenopausal In another embodiment, the menopause is drug induced, surgically induced, or naturally-occurring In another embodiment, the menopause is hormone related [00101] A treatment with a daily standard dose of Femarelle may result in an increase in BMD. A reduced dose may result in a decrease in BMD.

[02] Femarelle may stimulate skeletal tissues similarly to E2, but whereas E2 increases CK specific activity in the uterus, Femarelle does not. An increase was found in the trabecular bone volume and cortical wifemarelleh compared to the placebo-fed group according to experiments with ovariectomized rats, wh'ch were fed for two months with Femarelle in the equivalent of the standard recommended dose.

[00102] For example, a treatment of menopausal women with 644 mg/day of Femarelle for 12 months resulted in a substantial elevation in the BMD of the spine and the hip, compared to treatment with a low-dose regime.

[00103] A treatment v- ith a standard dose of Femarelle may result in a reduction in a menooausal symptom, which may be measured, for example, by the Kupperman index A tteatment with a low dose of Femarelle may result in a reduction in menopausal symptom, which may be measured, for example, by the Kupperman index A low dose of Femarelle may be a sufficient treatment for relieving menopausal symptoms. However this dosage does not provide protection against the development of osteoporosis since it resulted in a decrease in BMD following 12 months of treatment. The low dose of Femarelle may be use for treatment for menopausal symptoms. The standard dose of Femarelle may be used for the combined treatment of a menopausal symptom and osteoporosis [00104] There is provided a method for treating a bone density disorder and a menopausal symptom, the method may include administering a Femarelle compound to a subject suffering form bone density disorder.

[00105] Example 4 shows an experiment which examines the treatment of Femarelle on bone loss in postmenopausal women. More details relating to Example 4 and Fig.16 may be found in I.Yoles, Y.Yogev, Y.Frenkel, M.Hirsch, R.Nahum, B.Kaplan, "tofupil! (DT56a) for the treatment of postmenopausal Bone loss' a New Phyto-Selective Estrogen Receptor Modulator-like Substance", which is incorporatec! herein by reference in its entirety.

Example 4(a)" experimental group

[00106] A control group necessarily contained women who had recently become menopausal, like the study group. Therefore, their climacteric symptoms, too, were prominent and we had to offer them some type of treatment to ensure compliance. As a result, the low dose with the high dose of Femarelle was examined. Since BMD decreases rapidly at early menopause, evaluating the di ug's dose-effect was efficient.

[00107] Examining the effects of two dosages of Femarelle on postmenopausal women seeking hormone replacement therapy at women's health care centers in Israel Inclusion criteria were healthy women with an intact uterus who had no menses within the previous 6 months and who had blood hormone levels indicating menopause: follicle-stimulating hormone (FSH) >30 mlU/mL, estradiol (E₂) <150 pmol/L. Exclusion criteria were any pre-existing illness, habitual use of drugs or alcohol, presence of endometrial polyps or hyperplasia, and moderate or severe hypertension. The experiment was approved by the Human Investigation Review Board of the Rabin Medical Center, and all patients signed an informed consent form after receiving a comprehensive explanation of the study.

[00108] Of the 98 women who met the inclusion criteria and agreed to participate in the study, 82 (83%) completed all 12 months of treatment, 39 of them in the study group and 43 in the control group, the other 16 dropped out for personal reasons. No side effects or adverse reactions were reported by any of the participants. Baseline characteristics were similar in both study and control group as shown in table 1 referenced from "efficacy and safety of standards versus low-dose Femarelle (DT56a) for the treatment of menopausal symptoms".

Table 1 :Demoqraphic data of the study and control groups.

Study group Control

Age (years) 55.51+5.4 55.83 + 5.1

Time since 5.97+5.8 ' 5.73+4.48 menopause

(years)

Height (cm) 160.9+6 159.43+6.5

Weight (kg) 66.16+9.2 66.82+11.1

Body mass 25.50+3 26.31+4.3 index (kg/m²)

Smokers 3 (8.1) 6 (13.3) number (%) All values except smokers are mean+SD

Example 4(b): Detailed enrolment and procedures

[00109] A complete medical and gynecological history was obtained for each patient, and the attending physician completed an assessment questionnaire detailing the patient's menopausal symptoms. All participants underwent a physical examination (blood pressure, height and weight), a gynecological and breast examination, and transvaginal ultrasonography. Laboratory tests included hormonal and lipid profiles and tests of liver and kidney function. A baseline mammography was done as well, in addition to dual-energy x-ray absorptiometry (DEXA) of the lumbar spine and femoral neck.

[00110] For the trial, externally identical study (high dose) and control

(low-dose) capsules were placed in identical white bottles with no identifying marks apart from a serial number. The bottles were placed in front of the participants, and they were asked to choose one at random. The contents were unknown to the research team. Patients were asked to take two capsules a day, for a total of 644 mg/day of Femarelle for the study group and of 344 mg/day Femarelle for the control group. The control capsules were supplemented with 250-rng calcium phosphate and 8-mg natural vitamin E. The duration of the trial was 12 months.

[00111] Compliance and side effects were assessed every three months, and all tests and assessments done at the time of enrollment were repeated after 3, 6, and 12 months. After 9 months, each patient was interviewed DEXA of the lumbar spine and femoral neck was repeated after

12 months in the same institute us'ng the same machine

Example 4(c) Statistical analysis

[00112] Data was statistical!/ analyzed by paired sample t-test and by

two-sample t-test A p v?lue below 0 05 was considered statistically

significant

Example 4(d) Examining bon ³ mineral density (BMP) after treatment

[00113] On DEXA scanning after 12 months, BMD in the lumbar spine

increased from baseline in 'he study group by an average of 3.6% and

decreased in the control group bv an average of 0 6% as shown in Fig.16.

The improvement in the study group was statistically significant (p=0.039) and

there was a difference between the study group and the control group

(p=0 037) BMD in the femoral neck increased by 2 0% in the study group and

decreased by 0 6% in the control group as shown in Fig 16 Moreover, Fig. 16

shows no changes in BMD following 12 months of treatment. In the study

group, there was a statistically significant elevation of 3 6% in the spine

compared to 0 6% reduction n the control group (p=0 037) The net effect in

the femoral neck was 2 6% (NS) 'n the femoral neck, there was no statistical

difference in the study group or between the two groups Laboratory testing at

12 months yielded no ti eatment-related changes in serum FSH or E₂ or in

endometrial thickness in either the treatment or control group as shown in

Table 2

Table 2 Measures of endometnal thickness, FSH and E?, pre and post treatment Study group Control

P re Post Pre Post

Endometrium (mm) 3.2 2.8 3.7 3.4

Mean FSH (mlU/mL) 73.7 66.9 79.4 73.3

Mean E₂ (μmol/L) 105 92 110 105

[00114] The results mentioned in the experimental details, show that

Femarelle selectively affects bone formation in postmenopausal women, without affecting the endometrium or sex hormone levels. Femarelle is a novel phyto-SERM suitable candidate for the pharmacological treatment of postmenopausal osteoporosis

3a Stimulation of bone formation

[00115] There is provided a method for treating bone loss, the method may include administering a Femarelle compound to a subject suffering from bone loss, wherein the Femarelle compound stimulates the formation of bone and thus treats bone loss, in another embodiment, a Femarelle compound may prevent bone loss, which is caused by estrogen deficiency. One of the most common findings in skeletal radiology is increased radiolucency of bone named osteopenia.

[00116] There is also provided a method for restoring bone loss, the method may include administering a Femarelle compound to a subject suffering from bone loss, wherein the Femarelle compound stimulates the formation of bone and thus restore bone loss.

[00117] Osteopenia induced by overcosemized (OVX) in rats has been widely used as a model for studying postmenopausal osteoporosis. Treatment with E2 is known to reverse ^τhe osteoporotic changes caused by OVX. Two months after OVX, the vehicle-treated rats were profoundly osteopenic, but most of the bone loss was restored by treatment with E2 as well as by Femarelle. This positive effect was found in all the tested variables, the trabecular bone volume cortical thickness and growth plate width. In another embodiment, said positive effects of treatment on the growth plate width of the OVX rats and the cortical thickness in the non-OVX rats were better in the Femarelle treated rats than it the E2-treated rats. Histological appearance of the OVX rats treated with Femarelle was similar to the non-OVX rats.

[00118] Significant elevation in creatine kinase (CK) activity, as a marker of estrogen receptor activation was found in skeletal tissues of both OVX and non-OVX rats. However, Femarelle did not activate estrogen receptors in the uterus. In another embodiment, CK activation in the skeletal tissues following Femarelle treatment supported the results found in the histomorphometric measurements. [00119] Example 5 shews experimental details, substantiate findings from clinical studies showing that Femarelle prevents postmenopausal osteoporosis, and experimental details showing that short term treatment with Femarelle, as with E2, increases the specific activity of CK in skeletal tissues. More details relating to Example 5 and the following Fig.17-Fig.19 (inclusive) may be found in D.Somjen, S.Katzburg, E.Livne, I.Yoles, "DT56a (Femarelle) stimulates bone formation in female rats", BJOG : an International Journal of Obstetrics and Gynaecology, July 2005, which is incorporated herein by reference in its entirety.

Example 5 (a): experimental group

[00120] Thirty Wistar-de rived, locally bred female rats, aged 25 days and weighing about 60 g at the start of the experiment, were maintained on a 14: 10-hour light/dark schedule at 23 degree Celsius, and provided with food pellets and water ad libitum.

Example 5 (b): Detailed enrollment and procedures

[00121] Two equal groups were formed, in one of which the rats underwent bilateral OVX and in the other they were left intact (non-OVX). All rats were handled according to the NIH guidelines and the regulations and studies were approved by the Committee on Experimental Animals, TeI Aviv- Sourasky Medical Center.Rats in the non-OVX group were injected five days per week with 5 Ag E2 (n ^Λλ 5), 650 Ag Femarelle or with vehicle (n % 5 on- OVX control). Starting two weeks after surgery, OVX rats were injected five days per week with 10 Ag E2 (n ^ΛA 5), 1300 Ag Femarelle (n % 5 ) or with vehicle (n % 5 OVX control). The injected doses were calculated according to the animal weight and the dose response curves, previously shown to be the effective dose.

[00122] After eight weeks of treatment, and 24 hours after the last injection, the rats were sacrificed by cervical dislocation and organs were removed for histomorphometry and biochemical tests. Samples of whole tibiae from each treatment group of the OVX and the non-OVX rats were fixed for 48 hours in neutral buffered 4% formaldehyde in 0.1 M sodium phosphate buffer pH 7.4, decalcified (two to three weeks, room temperature) in 10% ethylene diamine teirascetic acid (EDTA), dehydrated in graded alcohol and embedded in paraffin. Sections (6 μm thick) were stained with haematoxylin and eosin for general morphology.

[00123] Trabecular bone from each treatment group was measured in the proximal tibial metaphysis at 50 Am below the growth plate. Measurements were performed in multiple randomized frames. The height of the growth plate, the width of primary trabeculae (spicules) underneath the growth plate and the arrangement of cells in the growth plate were also determined. For analysis of cortical bone, measurements were taken at 50- Am intervals on three seasons of the tibial midshaft of each rat. For histomorphometry, we used a transmitted light microscope (Zeiss) linked to a CCD video camera and a computerized histomorphometric system (Galai, Migdal Ha-Emek, Israel). An ocular micrometer, magnification x40 and x100, was used for measurement. The measured parameters included trabecular bone volume (TBV) which is expressed as a percentage of total bone volume, and obtained by dividing the trabecular area by the tissue area), trabecular width, cortical thickness and growth plate width. [00124] Rat bones were collected in cold isotonic extraction buffer,6 homogenized for a few seconds using a Polytron homogenizer and enzyme extracts obtained by centrifugation of homogenates at 14000 _ g for 5 minutes at 4°C in an Eppendorf microcentrifuge. CK activity was measured in a Kontron Model 922 Uvicon Soectrophotometer using a Sigma coupled assay kit (UV-47). Protein was assayed by Coomassie brilliant blue dye binding. Results are means of n ^ΛA 5 and are expressed as percent stimulation of the specific activities of CK in hormone-treated and control animals.

Example 5(c): statistical analysis

[00125] The significance of differences between mean values obtained from the different experimental and control groups was evaluated by ANOVA. A value of P < 0.05 was considered significant.

Example 5(d): The effects cf E2 and Femarelle on bone histomorphological parameters

[00126] Bone histomoφhological parameters are shown in Table 3.

Despite the small sizes of the groups, the results are statistically significant because the variations between animals in each group are very small. Vehicle-treated OVX rats (controls) were markedly osteopenic relative to control (vehicle-treated) non-OVX rats, having lost 31 % of the TBV (P < 0.01) and 21 % of the cortical thic kness (P < 0.001), Figs 17 and Fig.18. OVX resulted in a 21 % reduction in the cortical thickness. Treatment with E2 completely restored this reduction (P < 0.001). Treatment of rats with Femarelle increased the conical thickness in the non-OVX group by 32% (P < 0.001) and by 38%ιn the OVX group (P < 0 005). OVX rats treated with Femarelle had a thicker co'iex than the non-OVX control group 458 [] Am

(Figs 17a and 17b) OVX caused a reduction in growth plate width (34%, P <

0.05). In the bones of non-OVX rats, the growth plate width was significantly

increased by treatment with E2 (P < 0.01) but was not affected by treatment

with Femarelle (Fig. 17b)

Table 3

Treatment Percentage of Trabectilar Cortical Growth plats; trabecular bone volume (% thickness (μm) thickness width (μm) TBV) (μrn)

Nmi-OVX

Comrol 62 [1.51 236 [1.8] 458 [13] 177 [6) E2 63[2] 24.2[ 1 . 1 ] 423 [15] 217 [2O] DT56a 59 [3] 27. 8 [1 .3] 673 [59] 171 [9]

OVX

Control 43 [2] 30.7 [1 .1] 363 [15] 117 [1 ] E; 57 [4] 26.5 [1.1 ] 472 [24] 157 [4] DT56a 53 [3] 26.1 [1.1] 502 [16] 176[5]

[00127] In OVX rats, Femarelle treatment completely prevented the

OVX-induced reduction of the growth plate width, restoring the growth plate to

the same width as that in non-OVX controls (P < 0.01), Fig. 19. Moreover, in

the OVX rats the growth plates of Femarelle-treated rats were more mature in

appearance that those of E2-treated rats, containing relatively more

chondroblastic and hypertrophic cells and fewer proliferative cells.

[00128] Treatment with E2 restored bone loss in the OVX rats by 32%

compared with the control OVX rats (P < 0.01). Treatment of OVX rats with Femarelle increased TBV, restoring the bone loss by 23% (P < 0.01). There was statistically significant difference between E2 and Femarelle treatment in restoring the TBV, Fig. 17c.

[00129] CK-specific activity was measured in epiphysis (Ep), diaphysis

(Di) and the uterus (Ut) following the different treatments. An increase in CK activity was found following E2 or Femarelle treatment in both Ep and Di. However, while E2 produced significant CK activation in the uterus, Femarelle had no stimulatory effect in that site (Fig. 19). These results were found in both OVX and non- OVX rats. The basal activity of CK for non-OVX rats was in Ep 0.58 0 .10 Amol/minute/mg protein, in Di 0.68 0 .08 Amol/minute/mg protein and in Ut 1.47 mo!/ minute/mg protein. For OVX, in Ep 0.43 0 .08 Amol/minute/mg protein, in Di 0.66 0 .09 Amol/minute/mg protein and in Ut 1.34 0 .15 Amol/minute/mg protein.

4. Effect of Femareile on osteoblasts

[00130] Femarelle may be used as a bone forming agent. In another embodiment, Femarelle may be used as an anti-resorptive agent. In another embodiment, Femarelle may be used as a bone forming agent and an anti- resorptive agent.

[00131] There is provided a method for bone forming, the method may include administering a Femarelle compound to a subject suffering from a bone density disorder.

[00132] There is provided a method for bone forming and resorption inhibition, the method may include administering a Femarelle compound to a subject suffering from a bone density disorder. [00133] There ^;s provided a method for stimulating osteoblast formation, the method may include administering a Femarelle compound to a subject suffering from a bone density disorder.

[00134] There is provided a method for stimulating osteoblast formation in a female, the method may include administering a Femarelle compound to a subject suffering from a bone density disorder.

[00135] There <s provided a method for stimulating osteoblast formation in a female, the me' hod ma^ include administering a dose of Femarelle to a subject suffering from a bone density disorder. In another embodiment, the female may be pre- and post- menopausal.

[00136] There is provided a method for stimulating osteoblast formation in a cell culture, the method may include contacting a Femarelle compound with an osteoblast cell culture

[00137] There is provided a method for stimulating osteoblast formation in a cell culture, the method may include contacting a dose of Femarelle compound with an osteoblast cell culture.

[00138] There is provided a method for increasing the intracellular levels of [Ca2+], the method may include administering a Femarelle compound to a subject suffering from a bone density disorder.

[00139] There is provided a method for increasing the intracellular levels of [Ca²⁺] in a female, the method may include administering a Femarelle compound to a subject suffering from a bone density disorder.

[00140] [00141] There is provided a method for increasing the intracellular levels of [Ca2+] in a female, the method may include administering a dose of Femarelle to a subject suffering from a bone density disorder. In another embodiment, the female may be pre and post menopausal.

[00142] There is provided a method for increasing the intracellular levels of [Ca²⁺] in a cell culture, the method may include contacting a Femarelle compound with an osteoblast cell culture.

[00143] The effects Femarelle may be via putative membranal ER, by increasing intracellular cytosoϋc free calcium concentration [Ca²⁺]. This may be due to the increase in [Ca²+] in both age groups was mediated by two mechanisms: 1) [Ca²⁺] influx through voltage-gated [Ca²⁺] channels and 2) [Ca²⁺] mobilization from the endoplasmic reticulum, which involved a Pertussis toxin-sensitive G-protein.

[03] There is provided a method of treating a bone density disorder, the method may include administering a Femarelle compound to a subject suffering form a bone density disorder.

[00144] There is provided a method for enhancing regulation of proliferation of the bone cells, the method may include contacting at least one Femarelle compound with bone cells.

[00145] There Femareϋe compound may further enhance regulation of differentiation of the bone cells. [00146] There is provided a method for enhancing regulation of differentiation of the bone ceils, the method comprising contacting at least one compound present in Femarelle with bone cells.

[00147] There is provided a method for enhancing regulation of proliferation of the bone cells, the method comprising contacting at least one compound present in Femarelle with bone tissue.

[00148] There is pro 'ided a method for enhancing regulation of differentiation of the oone cells, the method comprising contacting at least one compound present in Femarelle with bone tissue.

[00149] The Famarelie compound may enhance the production of mRNA, which may induce the production of an estrogen receptor (ER) in bone cells. In addition, Femarelle may bind to ER(s). Femarelle may selectively bind to the Ers. ER may include ER alpha, ER beta or a combination thereof. Bone cells may include osteoblasts.

[00150] A Femarelle compound's affinity to ERβ may be higher than that of ERa. The mRNA may induces the production of ERa and ERβ on bone cells, wherein the production of ERβ may be higher than the production of ERa.

[00151] The Femarelle may further enhance the regulation of proliferation of the bone cells

[00152] The bone cells may include osteoblasts. [00153] The term "stimulating", "stimulation" or "stimulated" may refer to increasing the number, increasing the activity or both.

[00154] The term "formation' may refer to proliferation, multiplication, generation or any other mearing known in the art. In another embodiment, the term "osteoblast cell culture ' may refer to sex-specifically female derived cultured osteoblasts

[00155] Femareϋe may affect human bone derived cultured osteoblasts.

Femarelle may stimulate CK and DNA synthesis in both pre and post menopausal female bone cells. Femarelle may stimulate CK and DNA synthesis in both pre- and post- menopausal female bone cells with maximal effect at 100ng/ml fo^r both age groups. In addition, Femarelle stimulated ALP in cells from both pra- and πost- menopausal women with maximal effect at lower dose of 50nπ/ml, w,th higher response of pre-menopausal cells. Raloxifene (RaI) inhibited all Femarelle- stimulated changes in cells derived from both age groups. Femarelle, when given together with E2, completely antagonized E₂- stimulates effects demonstrating its nature as a phyto-SERM. Femarelle also, dose dependently, stimulated the intracellular levels of [Ca²⁺] with maximal effect at 10mg/ml.

[00156] Femareile may stimulate sex-specifically female derived cultured osteoblasts, indicating its unique nature compared to the compounds currently used for postmenopausal osteoporosis by being bone- forming and not only an anti-resorptive agent. [00157] Femarelle may activate osteoblasts in vitro, in all the tested parameters in a sex specific manner, namely it stimulated only female and not male-derived bone calls in both pre and postmenopausal age groups.

[00158] Femarelle may have an estrogen-like activity at both the nuclear and the membrane level? in female human osteoblasts. In another embodiment, Femarelle therefore may interact with membrane binding sites, penetrate the cells, and reach the nuclear receptors by an as yet uncharacterized mechanism and work at both levels.

[00159] Femareϋe may stimulate cultured osteoblasts, suggesting another mechanism of action of being not only anti-resorptive agent, but also a bone forming agent

[00160] Example 6 shows multiple experiments on female bone cell for the evaluating stimuiation of bone cell. More details relating to Example 6 and Fig.20-Fig.26 (inclusive) may be found in D.Somjen, S.Katzburg, M.Lieberherr, D.Hendel, I.Yoles, referenced from "DT56a stimulates gender- specific human cultured bone cells in vitro", Journal of Steroid Biochemistry&Molecular Biology, 15 March 2005, which is incorporated herein by reference in its entirety.

[00161] Femareile like E2 did not stimulated male derived bone cells, which respond only to DHT, demonstrating that like E2, Femarelle is sex- specific in its beneficial effects on bone cells in culture. Femarelle's activity was blocked by the SERM raloxifene (RaI), identical with Ral's effects on E2, and also displayed SERM-like activity in blocking E2's effect.

Example 6(a): experiment group [00162] Human female bone cells from pre- and post- menopausal women were prepared from bone explants, by a non-enzymatic method as described previously Samples of the trabecular surface of the iliac crest or long bones were cut into 1mm³ pieces and repeatedly washed with phosphate buffered saline to remove blood components.

Example 6(b): Detailed enrollment

Cell Cultures

[00163] The erolants were incubated in DMEM medium without calcium (to avoid fibroblastic growth) containing 10% fetal calf serum (FCS) and antibiotics. First passage eel's were seeded at a density of 3x10⁵ cells/35mm tissue culture dish, m phenni red- free DMEM with 10% charcoal stripped FCS, and incubated at 37⁰C <n 5% CO₂.

Creatine kinase extraction ard assay in human osteoblasts

[00164] Cells were treated for 24 h with the various agents as specified, scraped off and homogenized by freezing and thawing three times in an extraction buffer. Supemarant extracts were obtained by centrifugation of homogenates at 14000x g for 5 min at 4⁰C in an Eppendorf micro-centrifuge. Creatine kinase activity (CK) was assayed by a coupled spectrophotometry assay. Protein was determined by Coomasie blue dye binding, using bovine serum albumin (BSA) as the standard.

Alkaline phosphatase extraction and assay in human osteoblasts

[00165] After ti eatment with the different agents, cells were collected and homogenized by freezing and thawing three times in cold PBS and the soluble ALP was obtained by centrifugation at 14000xg. Enzyme activity was assayed in an ELlSA reader, measuring the hydrolysis of p-nitrophenyl phosphate at 37⁰C in a buffer containing 2mM MgCI₂ and 10OmM 2-amino 2- methyl isopropanol at pH 10 3 The reaction was stopped with 1 M NaOH and the extracts were analyzed at 410nm. Enzyme specific activity was determined as OD x 4.3 per mg protein.

DNA synthesis in human osteoblasts

[00166] Cells were grown until sub confluence and then treated with various hormones or agents as indicated. Twenty-two hours following the exposure to these agents, !³H] thymidine was added for two hours. Cells were then treated with 10% ice-cold trichloroacetic acid (TCA) for 5 min and washed twice with 5% TCA and then with cold ethanol. The cellular layer was dissolved in 0.3ml of 0.3N NsOH, samples were aspirated and [³H] thymidine incorporation into DMA was determined.

Intracellular Calcium concentration

[00167] Cells grown on cover slips were washed with Hanks' Hepes, pH

7.4 buffer, and loaded withi μM Fura-2/AM for 40 min in the same buffer at room temperature. The glass cover slips carrying the cells were inserted into a cuvette containing Hanks' Hepes, pH 7.4 buffer. The cuvette was placed in a thermo stated (37°C) Hitachi F-2000 spectrofluorimeter. Femarelle, E2 or vehicle alone were ar-dei d'rectly to the cuvette under continuous stirring. The Fura-2/AM fluorescence response to intracellular calcium concentration ([Ca²⁺Ji) was calibrated from the ratio of 340- 380 nm fluorescence values after subtraction of the background levels. The values for R_max and R_mj_n were calculated from measurements using25 μM digitonin and 4mM EGTA and enough Tris base to raise the pH to 8.3 or higher.

Example 6(c): tools for Statistical analysis

[00168] Differences bet-ween the mean values of experimental and control groups were evaluated by analysis of variance (ANOVA). P values less than 0.05 were considered significant.

Example 6(d): The effects of Femarelle on CK specific activity in human female bone :el!s

[00169] To determine their responsiveness to Femarelle compared to

E2 human bone ceϋs were incubated with 0.3- 300 nM E2, or 1-250ng/ml Femarelle. Femarelle, similar to E₂ treated cells, showed an increase in CK specific activity, in both pre- and post-menopausal cells as shown in Fig. 20. The increase in the E2 treated cells was greater in the pre- menopausal compared to postmenopausal women.

Example 6(e): The effects of Femarelle on DNA synthesis in human female bone cells

[00170] To determine their responsiveness to Femarelle compared to

E2, human female bone ceils were incubated with 0.3- 300 nM E2, or 1- 250ng/ml Femarelle. A si2mιlar increase in thymidine incorporation was found in both Femarelle and E groups, in both pre and post menopausal cells as shown in Fig. 21.

Example 6(f): The effects of Femarelle on ALP specific activity in human female bone cells [00171] To determine heir responsiveness to Femarelle compared E2, human female bone cells were incubated with 0.3- 300 nM E2, or 1-200ng/ml Femarelle. A similar increase in thymidine incorporation was found in both Femarelle and E2 groups as °-hown in Fig 22. In both treatments the maximal increase was higher in the pre menopausal cells compared to post menopausal cells.

Example 6(g): The effects of Femarelle on CK specific activity, DNA synthesis and ALP s oecific activity in human male bone cells

[00172] To deieπniπe their responsiveness to Femarelle compared to E₂ and to dihydrotesto^erone (DHT), human male bone cells were incubated with 0.3- 300 nM E₂, with 1-200ng/ml Femarelle or with 30-300OnM DHT. An increase in all parameters, were found only with DHT at optimum of 30OnM for CK and of 300OnM for ALP and DNA synthesis as shown in Fig. 23.

Example 6(h): The effects of Raloxifene or Femarelle on CK specific activity, DNA synthesis and on ALP specific activity stimulated by E? in human female bone cells

[00173] When human female bone cells were incubated with both

Femarelle and raloxifene oι^~ E₂, Femarelle activity in both age groups was blocked by Raloxifene E₂'s activity, was also blocked by Femarelle as shown in Fig. 24, as measured Lv CK specific activity (24a), DNA synthesis (24b) and ALP (24c) in both pre and pest menopausal age groups.

Example 6(i): The effects of Femarelle on intracellular [Ca²⁺I concentration in human female bone cells [00174] To determine their responsiveness to Femarelie compared to E2, human female bone cells from post- menopausal origin, were incubated with 0.01- 0.10 nM E2, or with 0.01-1 Ong/ml Femarelie. An increase in intracellular [Ca2+]i concentration was found with both hormones (Figs. 25- 26) with higher increase wϋh Femarelle and with higher sensitivity to this treatment as determined by the E2 increased intracellular [Ca2+] concentration at 0.01 nM by about 120% and Femarelie at 10ng/ml by about 240% .

5. Effect of Femsro'ie on the formation of vitamin D

[00175] The effct of Femarelie on the production of vitamine D was evaluated in a study "Less-calcemic vitamin D analog enhance creatine kinase activity and modulate responsiveness to DT56a in human bone cells in culture", which is incorporated herein by reference in its entirety.

[00176] Vitamin D receptors are widely expressed in the skeletal system. Vitamin D and its metabolites and analogs exert a variety of biological activities such as regulations of cellular proliferation and differentiation, cell calcium transients and energy metabolism. Energy metabolism is exerted through the control of the brain type isozyme of creatine kinase specific activity (CK), which serves to provide a readily available reservoir for ATP generation under increased workload.

[00177] Pre-treatment with the less calcemic analog of vitamin D JKF

1624F₂-2 (JKF) up-rsgulated the responsiveness to estradiol-17β and other estrogenic compounds via modulation of the expression of mRNA for ERs. [00178] Mutual modulation of the vitamin D system and Femarelle in human cultured female bone cells (hObs), by comparing the effects of Femarelle to those of E₂ on the induction of CK specific activity in hObs.

[00179] Femarelie as well as E₂ stimulates CK in pre and postmenopausal cells. In another embodiment, pre-treatment of the cells with JKF resulted in increased CK activity response to E₂ but not to Femarelle. In another embodiment, treatment of the cells with Femarelle similar to E₂ resulted in the increase of 1α25 vitamin D hydroxylase mRNA expression, furthermore enzyme activity resulted in increased production of 1 ,25(OH)₂D₃ in these cells.

[00180] Vitamin D analogs and Femarelle modulate each others activity on CK response in human bone cells as shown in Fig. 27 and Fig. 28 both referenced from "Less-calcemic vitamin D analog enhance creatine kinase specific activity and modulate responsiveness to DT56a in human bone cells in culture". Femareile may modulate the response to estrogen by direct modulation of ERs mRNA expression and by indirect modulation via increasing vitamin D in bone cells leading to modulation of responsiveness to estrogens by this system as well as shown in Fig.29 also referenced from "Less-calcemic vitamin D analog enhance creatine kinase specific activity and modulate responsiveness to DT56a in human bone cells in culture".

6. Estrogenic effect of Femsrelle

[00181] The term "estrogenic effect", as referred to herein, may be directed to an effect on uterine weight, vaginal epithelium, serotonin-induced contractile activity or any other estrogenic effect known in the art or any combination thereof Estrogenic effect on uterine weight may include an increase of uterine weight

[00182]

[00183] FemarHie may have a reduced estrogenic effect on the female reproductive tract in another embodiment, Femarelle may have a reduced estrogenic effect on uterine weight. In another embodiment, Femarelle may have a reduced strogenic effect on vaginal epithelium. In another embodiment, Fema'olle ma^ have a reduced estrogenic effect on serotonin- induced contractile a'-πvnv

[00184] The erm "reduced estrogenic effect" may refer to any estrogenic effect which is

than 20%. According to other embodiments, the term "reduced estrogenic effect" may refer to any estrogenic effect which is lower than 10%.

[00185] Femarplle may display a lower estrogenic effect than other commonly used hoimone replacement therapy, such as conjugated equine estrogens, for postmenopausal women Femarelle may diminish hot flashes in postmenopausal women Femarelle may act as selective estrogen receptor modulators (SERM) which tor example but not limited to, may diminish hot flashes or any other oostmenopausal symptom, without exerting any effect on the female reproductive tract, such as the uterus or vaginal epithelium. In one embodiment, phyto-estrogens which may be contained in Femarelle may act as SERM.

[00186] The different estrogenic potency of phyto-estrogens in relation to estrogens may be evaluated For Example, flavonoids are 10³ to 10⁴ -fold less potent agonists than 17 beta-estradiol, and micromolar concentrations are required to generate estrogenic activity. In one embodiment, the estrogenic actions of Femarelle are approved at low doses.

[00187] Femarelle may not display either uterotropic effect or uterine smooth muscle sensitization to serotonin. Phyto-estrogens in Femarelle may lack a clear peripheral estrogenic actions on female reproductive tract. Phyto- estrogens contained in Femarelle may control hot flashes in postmenopausal women.

[00188] Etrogeπic actions of Femarelle may appear at low doses and may be used for treating postmenopausal women.

[00189] Example 7 shows experiment of estrogenic effects of Femarelle.

[00190] More details relating to Example 7 and the following Fig.30-

Fig.35 (inclusive) may be found in, "tofupill lacks peripheral estrogen-like actions in the rat reproductive tract", Reproductive Toxicology 20 (2005) 261- 266, which is incorporated herein by reference in its entirety.

Example 7(a): experiment group

[00191] An adult female bilaterally ovariectomized rat, 200 — 25Og body weight, 10-12 weeks old, bred in the center for laboratory animals production and maintenance, with free access to food and water, and housed in groups of 4-5 per cage under a 12 : 12 h light. The rats were bilaterally ovariectomized under xylacine (20 mg/kg body weight) and ketamine (45 mg/kg body weight) anesthesia.

Example 7(b): Detailed enrollment [00192] After 3 recover*, period of 2 weeks, rats were randomly assigned to one of the following groups of 14-day treatment

Group Treatment Dose n

I Conjugated equine 10.4 μg/kg 5 estrogens, low dose

Il Femarelie, Ow dose 3 4mg/kg 5

III Vehicle, low dose 1 OmL 6

IV Conjugatec equine 31.2 μg/kg 6 estroαens iiαh dost;

V Femarelle, high αos<-' 10.2 mglkg 6

Vl Vehicle, high dose 1 5mL 5

VII 17D-Estradιoi 40.0 μglkg 4

[00193] Low dose was calculated on the body weight basis as three times the dose recommended to postmenopausal women, whereas high dose was calculated as three times the low dose. Conjugated equine estrogens (CEE) used were the commercial presentation of commonly used estrogen replacement therapy in postmenopausal women and served as positive control. Drugs were orepared from the commercial presentation of conjugated equine estrogens or the food supplement, Femarelie.

[00194] The duration of treatment was daily oral doses, which were administered during 14 days in pre-menopausal women, soy protein given daily during 30 days (one menstrual cycle) was sufficient to exert effects on the menstrual cycle Rat estrous cycle lasts 4 days, thus, the treatment schedule used in the present study was equivalent to more than three estrous cycles. Soybean estrogens din' not demonstrate estrogenic activity on uterine weight of rats in a 2 month treatment schedule. Similarly, soybean estrogens administered durinq 3 years did not exert estrogenic actions on vaginal epithelium of ovariectomized macaques.

Example 7(c): examining esti ocienic effect on Uterine weight

[00195] Before performing contractile activity assays, the dissected uterus was placed on a filter paper to eliminate liquid excess and to record wet weight. At the end of contractile assays, tested segments and remainder of the uterine tissue from each rat was dried in a laboratory oven and dry weight was recorder!

[00196] Wet weight of uterus from rats treated with either low or high- dose CEE was higher than that from Femarelle-treated or non-treated ovariectomized controls as shown in Fig. 30.OnIy uterus from the CEE- treated rats showed the classical hyperemic appearance due to estrogenic stimulation. Similar values of dry weight were recorded for all of the low-dose treatments. On the contrary dry weight of high-dose CEE-treated rats was higher than those of either high-dose Femarelle-treated rats or non-treated ovariectomized controls as shown in Fig. 31. the uterine weight (wet and dry) from Femarelle-treated rats was similar to that of the ovariectomized controls (Figs. 30 and Fig.31}

Example 7(d): examining estrogenic effect on Vaginal epithelium

[00197] Daily vaginal smears were taken to observe the estrogenic effect on vaginal epithelium Vaginal smears were observed fresh and were subsequently fixed m absolute ethanol and stained with hematoxilin-eosin. In ovariectomized rats the vaginal smear showed an atrophic pattern consisting of leukocytes, mucus and scarce nucleated epithelial cells. When estrogens were administered to ovariectomized rats, the vaginal smear consisted mainly of exfoliated cornified cells.

[00198] Vaginal epitheiium from rats treated with low doses of either CEE or Femarelle was similar to that of non-treated ovariectomized controls. In the case of high-dose "reatments, only CEE-induced keratinization of vaginal epithelium as evdenred by abundance of cornified cells and presence of some epithelial nuc'eateri cells as shown in Fig. 32.Vaginal epithelium of Femarelle -treated 3:s aε shown in Fig. 33. On the other hand, only leukocytes were observed in vaginal epithelium of control rats as shown in Fig. 34.

Example 7(e): Examining Uterine smooth muscle sensitivity to serotonin

[00199] Rings approximately 5-7 mm long were dissected from cervical segment of each uterine horn a day after the last day of treatment. Uterine cervical segments were placed longitudinally in a 5 mL organ bath containing Krebs-Ringer-bicarbπnate (KRB) solution. Each uterine segment was placed under optimum resting force of 1 g and allowed to equilibrate for 1 h before exposure to drugs, ->i!.ing th's equilibration period tissues were washed with fresh KRB every 10 min After equilibration, uterine rings were bathed in a depolarizing solution (60 ml ^r\ KC1), prepared by equimolar substitution of NaCI for KCI. This solution oroduces uterine smooth muscle contractile response that was considered a maximal standard response. Contractile responses were recorded isometrically with a FT03 Grass tension transducer connected to a GIVISS mode! 7B polygraph. After two consecutive similar responses to 60 mM KC 1 , uterine segments were exposed to different concentrations of serotonin in a non-cumulative manner (10-^δ to 10-⁴ M). Each serotonin concentre ι-on remaned in the tissue bath for 10 min.

Example 7 (f) Contractile aαvitv assays

[00200] Uterus from rπ'r. treated with either conjugated estrogens or

Femarelle, or in vehicle-tre^'ed ovariectomized controls (data not shown), serotonin did not ιnri"ce conπ actile activity. Only rats treated with 17-estradiol displayed a clear se. -ϊtonin-imiuced contractile response as shown in Fig. 35.

7. Effects of Fen^roH^ on vascular tissues by Stimulating Creatine Kinase

[00201] Femareϋe ma- obtain beneficial effects not only in skeletal tissues without affertipg ih^p "terus, but also in vascular tissues. Femarelle may have similar be^πεfιcιal en^'ects on vascular tissues like estradiol-17β (E2).

[00202] Fema'ells stirr.t .Jates bone, cartilage and uterus from immature or ovariectomized female rats By measuring the changes in the specific activity of the BB isozyme of creatine kinase (CK), such bone stimulation can be confirmed

[00203] When adminisiered in multiple oral doses, Femarelle may stimulate skeletal Kcues similarly to E2 The selective estrogen receptor modulator raloxifene may b!-->ck the stimulation of creatin kinase by either Femarelle or by E2 in >ssues

[00204] Femai e'le may effect vascular tissues. In another embodiment,

Femarelle may effects the ao.ia (Ao) ana the left ventricle of the heart (Lv). In another embodiment, a single or multiple administration of Femarelle may result in the same vascular eϋ^'ects as E2.

[00205] Creatine kinase (CK) specific activity, which is a general estrogenic marker and is also correlated with bone formation which may be stimulated by Femarelle CK specific activity in vascular tissues may be induced by Femarelle, when administrating Femarelle at high dosages by a single injection. In another embodiment, CK specific activity in vascular tissues may be induced by -emarelle, when administrating Femarelle at the same range as thst ^■ i ¹Z 2

[00206] The pa'tern of nhibition by raloxifene of the stimulation of CK by both E2 and Femai eϋe in ^oth vascular and skeletal tissues points to a common mechanism of action by these compounds, probably by utilizing the same ERs.

[00207] Example 8^' shows in vivo experiment for determining estrogenic properties of femarelle on vascular tissues. More details relating to Fig.36- Fig.40 (inclusive) may be found in D.Somjen.l.Yoles, "DT56a (Tofupill/Femarelle) se'ectiveiy stimulates creatine kinase specific activity in skeletal tissues of rεts but ^pot in the uterus", Journal od Steriod Biochemistry & Molecular Biology 86, 27 February 2003, which is incorporated herein by reference in its entirety

Example 8(a): experiment gro^pp

[00208] Immature female Wistar rats were used intact at the age of 25 days or after ovariectomy performed at the age of 25 days. Treatments were administered starting 2 weeks after surgery, at the age of 39 days. Rats were housed in air-conditioned quarters with light from 05 00 to 19 00 hrs and were exposed to food and water ad libidum Experiments were carried out according to the rεgu'atnns α the Committee on Experimental Animals of the Tel-Aviv Sourasky M-'d'cal Center

Example 8(b) Detailed enro¹1 nent

[00209] Chaππ c in ttr specific activity cf CK in aorta (Ao) and left ventricle of the heai ¹ ' j¹) ino ed by a single intraperitoneal injection (i.p.) of E2 or DT56a, were aε ^a o i in o ^nectomized female rats, 2 weeks after surgery, or in 25-day-old i \ \> ^~ό\\re male rats (n=5 per group for each experiment, repeated 2 or 3 times) Matted control rats (_n=5) were injected with 0.05% ethanol in phosphat^-oi-ffer;^' saline (PBS) E2 was injected at 5 ug for immature and 30 ι, g ^ror \'arιectomιzed female rats and animals were sacrificed 24 h aftei rjection in multiple daily applications, rats were fed daily directly into the stoi .iarh foi ^Λ davs at the indicated doses Organs examined were removed and siorod at -?0 ⁰C until processed for enzymic assay

[00210] Differe nces b ^> 'een the mean values obtained from the experimental and th^p "ontro! groups were evaluated by analysis of variance (ANOVA)

Example 8(d) Sti^mulation 3f creatine kinase specific activity, by a single injection of F-maielle n estradiol

[0021 1] Immature femai^p rats, weighing about 6Og, were injected with

E2 (5 μg/rat) or ιnc^rα?3ing concentrations of Femarelle (ranging from 64μg/rat to 3200 μg/rat) These concentrations are equivalent, respectively, to 0.1 to 5 times the human pharmacological dosage, calculated on the basis of weight. Femarelle and E2 y,-^» o tested for their effects on the specific activity of CK in rat tissues [aorta (Ao), left ventricle of the heart (Lv) as shown in Fig. 36. Stimulation of CK Oy Ferrvrelle in all organs was maximal at 1300 μg Femarelle, with a rignificam increase relative to vehicle-treated controls evident by 130 μg F^emarelle E2 stimulated CK to lower extent at the specific dose compared to th-^> optimal dose of Femarelle.

Example 8(e) Inh'bn > -M by '>;'oxιfene of the creatine kinase specific activity, stimulated by Femai- 'Ie or by estradiol in immature female rats.

[00212] Immar .' ^p fem÷'e rats were injected with Femarelle at the optimal dosage (13 0 ug) oi Λ/ιth E2 (5 μg), alone or together with 0.5 mg raloxifene (RaI) Ra' vhich v< 13 stimulatory in the vascular tissues, completely blocked the sπmula T/ acth/'rv of both compounds in Ao. In Lv RaI blocked Femarelle by about 70% but did not block at all E2 stimulated CK as shown in Fig. 37.

Example 8(f) Inhibi^tion by raloxifene of the creatine kinase specific activity, stimulated by Fenweϋe or e^radiol injection in ovariectomized female rats

[00213] Ovariectomized ^'emale rats were injected with Femarelle at the equivalent optimal ^oncentr-'ion for adult rats (3200 μg, which is 2.5 times higher than for immnrjre rat- -nnd this is similar dose per weight), or with E2 (10 μg), alone or toπether

ιih 0 5 mg of raloxifene (RaI) Femarelle, E2 and RaI stimulated enzyme activity similarly in both Ao and in Lv as shown in Fig. 38 referenced from '"3T56a (Tofupill/Femarelle) selectively stimulates creatine kinase specific activity in skeletal tissues of rats but not in the uterus". RaI, which was stimulatory in the vascular tissues, completely blocked the stimulatory activity of both compounds in Ao as well as in LV as shown in Fig.38.

Example 8(g): Stimulation of creatine kinase specific activity by repeated oral administration of Femarelle or estradiol in immature and in ovariectomized female rats.

[00214] Daily ingestion of Femareϋe (650μg in immature and 1600μg for ovx, both are haif if the ->otimal concentration injected), or E2 (5μg for immature and 10μg for ovx) for 4 days stimulated similarly CK specific activity in Ao of immature as well a? in ovx rats. In Lv from both animals Femarelle stimulated CK to about two third of the stimulation by E2 itself as shown in Fig.39 and Fig.4O

8. Chemical structure of cfci^zein

[00215] DaidzeO may be incorporated in foods and in dietary supplements. In ano'her embodiment, daidzein may be provided by isolation used in the conven^tional industrial processing incorporating fractionation and chemical extraction These methods may include aggressive extracting processes, which may change the original structure of the daidzein. The changes in the natural structure consequently may alter the original function of the material. This change may lead to a reduced potency of the natural compound and might lead to dangerous physical consequences.

[00216] In order to take advantage of the positive characteristics of the daidzein, it is crucial to preserve its chemical structure during the industrial processing or alteπ^"> nsiy crea^fe a material that comprises the basic elements found in the natural ^rnidzein When exposed to the physiological environment in the human bodv this rr iieπal will potentiate the daidzein, and create availability to the bo iy in tl "³ same structure as found in the soybean. The contribution of the laidzein has been revealed through numerous clinical studies. This has shed light on the importance of the biochemical properties concerning this matiMal in sry foods and in the different dietary supplements derived from the so^{v κ}ean in ^rder to preserve its beneficial properties.

[00217] Keep¹ "' ^J l >e ci ^'^"nical structure of the daidzein intact following its processing intc ^ ^food s¹ inclement is significant In another embodiment, biochemical pioperVs whic'i followed the reaction during the immersion of the soybean, is pιe^^~τved in 'he material Femarelle. In another embodiment, the potentiation th?; characterizes the soybeans (9 27-fold potentiation) is expressed more s<gr firantly Ή the Femarelle, a 23-fold increase. In another embodiment, the composition of the daidzein derived from the potentiation is identical to its compo^sition found in the original soybean.

[00218] In the ^Pharmaceutical world where the potentiation is expressed in the effective chf mical e^gression of the active materials, the material Femarelle succeeds >n expi ^ssing the daidzein in values of 55.45-fold the active material that ^■ - found iι > the originating soybean

[00219] Examt ¹^ 9 sh'-^'s the effect of immersion in H₂O on ground soybean and

-MIe Mf details relating to Example 9 and the following Fig.41-Fig.42 (inclusive) may he found in Dr M Cook^'The potentiating effect of water immersion on daidzem content in soybean, compared to DT56a-A novel phytoestrogen Ompound", Soy &Health 2002-clinical Evidence Dietetic Application which is incorporated herein by reference in its entirety.

Example 9(aV expein nental g roup

[00220] Two i Materials soy and Femarelle, were divided into two categories and were ^eparat^r-i-/ analyzed.

Example 9(b): Detai l -d procedures

[00221] Each '¹TpOUn". ^w/as analyzed in two forms; its original dry form and after immersioi n water for 8 hrs Each category contained 100 gr. of material. The imme^sed form vas grounded in a blender and extracted with 600 ml of acetonitnie. The mixtures were stirred gently for 24 hr. at room temperature (23 C^0-: following which they were filtered through a Gelman type A/E size 13 mm olass fib^pr filter; 100 microliters of filtrates after short centrifugation were applied for HPLC analysis.

[00222] High . erformance liquid chromatography of the acetonitrile extracts was carried out u?>ng C₁₃ [A LiChroCART (4.6 x 250 mm, 5μm)] Purospher STAR cohmn av-, ihe stationary phase A flow rate of 0.8 ml/min was used for the coH'mn, which was maintained at a pressure of 170 bar. The operating condition Λ'as at ioom temperature. Elution was carried out by a reversed-phase method with a linear gradient system of acetonitrile, concentrations from IO to 100% in 30 min. followed by an isocratic hold period of at least 40 min. The samples were injected, volume of 100 μl., elutes were detected at 256 nπi [00223] All the samples vere collected by fraction collector. Fraction 14-

16-retention time (dsidzein) were further fractionated (rescreen) in the same condition on HPLC ^■'■^;-■ order ÷o demonstrate overlapping equivalence retention time peaks from diffe^rent samoles).

[00224] The do'c'zein vπs identified by comparison of its retention times to authentic daidzem (Sigma Chemical Co., St. Louis, MO). Concentration of the daidzein was emulated from standard curves of the area responses to the authentic daid∑eπ- .

Example 9(c): resuN ^f the e^xperiment

[00225] The amount of -,3idzein in the dry soy and following immersion were 0.22 and 2.04.

a ten-fold increase. However, the results of the content of daidr^n in d>^" - and immersed Femarelle, were 0.53 and 12.2 (mg/100g, dry basis' ^resρeci'"eiy, a twenty-three -fold increase. Furthermore, the diadzein voltage; measui ^ments in dry soy and after immersion were 2 and 10 mV respectively, a five-fold increase. On the other hand, the results of the content of di3dz-Mn in drv and immersed Femarelle, were 6 and 56 mV respectively, a nine-iHd increase.

[00226] The results are ihown in Table 4 and Fig. 41 and Fig.42. Table 4 illustrates the effer of immersion in H₂O on ground soybean and Femarelle, on apparent free darlzein consent in dry form vs. immersed form.

Table 4 (1)1.1.Soyfood rnπ/IOOg, dry basis Height (mV)

Soy (in dry form) 0 2 2.0

Soy (immersed) 2 0 10.0

in dry form) 0.53 6.0 imers eφ 12 2 56.0

[00227] Fig. 4 i -hows O-fidzein levels (mg/100g, dry basis) determined by HPLC after extras .ion with acetonitrile. In order to demonstrate overlapping equivalence

time peaks from different samples, fraction 14-16 retention time (daid - sin), w* further fractioned (re-screened) in the same condition on HPLC a- shown in Figure 41.

[00228] Figure ^J2 shoe's re-screened fraction 14-16 retention time in same condition on ^PLC. From Figures 41-42 above it is clear that the highest amount of iaidzein was found in the immersed Femarelle. The original glucosides embeci'i ϊd in the soybeans and femarelle, were hydrolyzed during ¹J i^ soakinπ

Claims

CLAIMSWhat we clasm is

1. A method fe ireatin ^• a menopausal symptom, the method comprising a niniste_' tnj an effective amount of a Femareile (DT56a) ccrrvound v s remale suffering from a menopausal symptom

2. The method ^' claim ¹ wherein treating comprises reducing, preventing o< deiayirq the occurrence or reoccurrence of a menopausal ^o^mptom

3. The methoi • claim " wherein treating comprises improving the quality of ^{h '}e of a ^male

4 The method ^f clan 1 wherein the menopausal symptom comprises a '

svmptom, a psychological symptom, or any combination thereof

5. The method Df claim 4, wherein the vasomotor symptom comprises br[ flashes, night sweats, sleep disturbances, headaches ^ alpitation^ or any combination thereof.

6. The method >f claim 4 wherein the psychological symptom comprises rrreaseσ depression, anxiety, irritability, mood swings, lack ' f concentration, reduced sexual desire/arousal, fatigue or an HDmbinDt'on thereof

7. The method of claim 4, wherein the physiological symptom comprises, arthralgia and myalgia, vaginal dryness, dyspareunia, Vaginal etching/burning, urinary frequency, dysuria urgency or any combination thereof.

8. The method of claim 1 , comprises treating a menopausal symptom essentially wiihout inducing an estrogenic effect on the reproductive >■ ict.

9. The method " f claim 1 , wherein the effective amount of the Femarelle (t?T56a) compound comprises about 500-1000 mg/day.

10. The method vf claim 1 , wherein the effective amount of the Femarelle (D^T56a) compound comprises about 644 mg/day.

11. The method ^■ f claim 1 , wherein the effective amount of the Femareile (DT56a) compound comprises about 200-500 mg/day.

12. The method f claim i , wherein the effective amount of the Femarelle (D^~56a) compound comprises about 322 mg/day.

13. A method for treating post menopause bone density disorder, the method comprising administering an effective amount of a Femarelle (DT56a) compound to a subject suffering form bone density disorder.

14. The method ->f claim 13, wherein the bone density disorder comprises osteopenia osteoporosis, age-related bone disorder or any combination thereof.

15. The method of claim 13, wherein osteoporosis comprises postmenopausal osteoporosis or osteopenia.

16. The method of claim 13, wherein treating the bone density disorder comprises increasing the bone mineral density (BMD).

17. The method of claim 16, wherein increasing the bone mineral density (BMD^X compπr-es increasing the BMD by between 1-5%.

18. The method of claim 13. wherein treating a bone density disorder corrurises inhibiting bone loss.

19. The method of claim 13, wherein treating a bone density disorder com^prises in' -easing bone formation.

20. The method 3f claim 13, wherein treating a bone density disorder comprises stimulating osteoblast formation.

21. The method Df claim 13, comprises treating a bone density disorder essentially without inducing an estrogenic effect on the reproductive Vict.

22. The method ^f claim 13, wherein the effective amount of the Femarelle (^¹ T 56a) compound comprises about 500-1000 mg/day.

23. The method ^<-f claim ¹ J, wherein the effective amount of the Femarelle (DT56a) coπviound comprises about 644 mg/day.

24. The method of claim 13, wherein the effective amount of the Femarelle < DT56a) compound comprises about 200-500 mg/day.

25.A method KX increasing the intracellular levels of [Ca2+], the method comi ' ises

a Femarelle compound to a subject suffix i ' Q from 3 υone density disorder.

26.A method ^f v sπmi.iatmg bone cell formation, the method comprising at mimstei 'ng a Femarelle (DT56a) compound to a subject suffemg from a bone density disorder

27. The method of claim 26, wherein the bone cells comprise osteoblasts

28. The method ^~f claim 26 wherein the Femarelle compound is adapted to emance t^° oroduction of mRNA.

29. The method if claim 28, wherein the enhancement of the production oi mRNΛ induces the pioduction of estrogen receptors (E^{p i} in bon^ ceils

30. A method foi 3tιmulat no osteoblast formation in a cell culture, the method comprr.es contacting a Femarelle (DT56a) compound with an osteoblast cell culture.

31. A method for ncreasing the intracellular levels of [Ca²⁺] in a cell culture, the method comprises contacting a Femarelle compound vyi'ⁱ-> an osteoblast cell culture.

32.A method foi enhancing regulation of differentiation of bone cells, the IΓK- 'iod comυπsing contacting a Femarelle (DT56a) compound w.v bone ce'ii

33. The method jf claim 32, wherein the bone cells comprise osteoblasts

34.A method fc enhancing regulation of differentiation of bone cells, the m^-hod comprising contacting a Femarelle (DT56a) compound wfh bone tissue

35. The method of claim ?Λ_} wherein the bone cells comprise osteoblasts

36. A method fo^r enhance ιq "βgulation of proliferation of bone cells, the method comprising contacting a Femarelle (DT56a) compound with bone tπsue

37. The method of claim 36, wherein the bone cells comprise osteoblasts

38. A method o^f inhibiting estrogen binding to estrogen receptors (ER) in a uterus, the method comprising administering an effective amount of a Femarelle (DT56a) to a female.

39. The method nf claim 38, wherein the Femarelle (DT56a) has an antagonistic etfect on estrogen receptors (ER) in a uterus.

40. The method ^fΛf claim 38, wherein the effective amount of the Femarelle (DT56a) compound comprises about 500-4000 mg/day.

41. The method if claim 33 wherein the effective amount of the Femarelle (D^"' 55a) corroound comprises about 1300 mg/day.

42. The method -f claim ^8, wherein the effective amount of the Femarelle (Dl 56a) comoound comprises about 644 mg/day.

43. A method of inhibiting estrogen binding to estrogen receptors (ER) in a breast tissue the method comprising administering an effective amount of a Femarelle (DT56a) to a female.

44. The method of claim 43 wherein the effective amount of the Femarelle ;DT56a) compound comprises about 200-4000 mg/day.

45. The method ^r>f claim Η wherein the effective amount of the Femarelle (DT 56a) comoound comprises about 644 mg/day.

46. The method of claim £3, wherein the effective amount of the Femarelle (D^T56a) conoound comprises about 322 mg/day.