AU2011236080A1 - Phytoestrogenic formulations and uses thereof - Google Patents

Abstract

Select phytoestrogen pharmaceutical compositions and methods of use for promoting neurological health and prevention of age-related neurodegeneration, such as AD, have been developed. These select phytoestrogen formulations are composed of a number of plant-derived estrogenic molecules and/or their structural analogues and exhibit binding preference to ERB over ERa and agonist activity in the brain. These ERB-selective phytoestrogen formulations cross the blood-brain-barrier and promote estrogen-associated neurotrophism and neuroprotection mechanisms in the brain, without activating proliferative mechanisms in the reproductive tissues and are therefore devoid of other estrogen-associated problematic aspects. The select phytoestrogen formulations are therapeutically useful to both women and men for sustaining neurological health and preventing age-related cognitive decline and neurodegenerative disorders, such as AD. These are administered enterally, transdermaly, transmucosally, intranasally or parenterally, in a dosage effective to prevent or alleviate neuronal damage, effect neuronal regeneration or sustain viability, increase expression of anti-apoptotic proteins, and/or decrease indicators of Alzheimer's Disease. The formulations preferably contain combinations of compounds, and can be formulated for daily, sustained, delayed or weekly/monthly administration. In a preferred embodiment, these are administered to women who are in menopause or post menopausal, most preferably early in menopausal.

Description

1 AUSTRALIA Patents Act 1990 UNIVERSITY OF SOUTHERN CALIFORNIA COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Phytoestrogenic formulations and uses thereof The following statement is a full description of this invention including the best method of performing it known to us:- PHYTOESTROGENIC FORMULATIONS AND USES THEREOF Background of the Invention This work was supported by National Institute of Mental Health 5 Intramural Research Program (P.J.S.) and Grant MH67159 (R.D.B.), National Institute of Aging Grants AG06647 (J.H.M.), AG16765 (J.H.M., A.C.G.), and AG14751 and AG026572 (R.D.B.), and the Kenneth T. and Eileen L. Norris Foundation (R.D.B.). This application claims priority to U.S.S.N. 60/819,849 mailed by 10 express mail label number ER455959795US on August 1, 2006; U.S.S.N. 60/889,920 filed February 14, 2007, and U.S.S.N. 60/943,190 filed June 11, 2007. The demographics suggest that we face a devastating increase in the prevalence of AD, reinforcing the immediate need for basic and translational 15 neuroscience to develop safe and efficacious ET and HT regimens for the brain. Of those affected with AD, 68% are female and 32% are male (Brookmeyer et al., 1998 Am J Public Health 88:13372). Because women have a longer life expectancy than men, the absolute number of women with AD exceeds that of men. However, a double danger exists for women. 20 Results of a meta-analysis of seven sex-specific studies concluded that women are 1.5 times more likely to develop AD than age-matched men (Gao et al., 1998 Arch Gen Psychiatry 55:809), which was supported by the Cache County analysis that showed a clear female gender increase in the incidence of AD (Zandi et al., 2002 JAMA 288:21239). 25 At the turn of the new millennium in the United States, there were nearly 42 million women over the age of 50 years and, of these, more than 31 million women were over the age of 55 years (North American Menopause Society, 2004). Worldwide, there are currently more than 470 million women aged 50 years or older, and 30% of those are projected to live into their 80s 30 (North American Menopause Society, 2004). These women can anticipate spending one-third to one-half of their lifetime in the menopausal state. Reports on prevalence of AD vary, but of the 18 million American women in

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their mid to late 70s, as many as 5 million may suffer from AD, and this figure increases dramatically at older ages (Brookmeyer et al., 1998). The projected exponential increase in the prevalence of AD, along with the anticipated impact on families and society, highlights the imperative for 5 developing strategies to prevent or delay the onset of AD sooner rather than later. The profound disparities between the largely positive basic science findings of gonadal steroidal action in brain and the adverse outcomes of recent estrogen or hormone therapy ("ET/HT") clinical trials in women who 10 are either aged postmenopausal or postmenopausal with Alzheimer's disease (AD), has led to an intense reassessment of gonadal hormone action and the model systems used in basic and clinical science. One key factor that could contribute to the negative results of the Women's Health Initiative Memory Study ("WHIMS") trial was the advanced age, more than ten years following 15 menopause, at which ET/HT was initiated in women. Data from both basic science analyses and clinical studies indicate a "healthy cell bias" of estrogen action in the neurons / brains, suggesting that ET/HT acts as an effective preventative therapeutic strategy for age-related cognitive decline and neurodegenerative disorders, such as Alzheimer's disease ("AD"), while it is 20 not an effective treatment strategy. The current widely prescribed ET, conjugated equine estrogens ("CEE"), is a highly complex ET with over 200 different components. Whether CEE provides the optimal therapeutic efficacy has been questioned. Another key issue challenging HT is the optimal composition. The progestin and its timing of administration in 25 conjunction with ET, remains to be determined. Moreover, while ET/HT has long been used in postmenopausal women to delay or reverse some of the problems associated with menopause, epidemiological and clinical studies have uncovered potential long-term risks related to this therapy. The recently revealed risks associated with ET/HT have greatly increased interest 30 in the development of estrogen alternatives that promote beneficial effects of estrogen in brain, bone and the cardiovascular system, while not eliciting deleterious effects in other organs, particularly in breast and uterine tissues. 2 Two nuclear receptors for estrogen (ERs), ERa and ER, have been identified. In the central nervous system, both ERa and ER3 are expressed in the hippocampus and cortex of rodent and human brains. Previous studies have demonstrated that both ERa and ERp can equivalently promote 5 neuronal survival by activating estrogen mechanisms of action in rat hippocampal neurons. Increasing evidence indicates that ERD is a key requirement for activation of mechanisms that underlie estrogen-inducible neuronal morphological plasticity, brain development, and cognition. ERa, on the other hand, is more predominant in mediating the sexual 10 characteristics of estrogen effects in the reproductive organs such as breast and uterus. Taken together, these data establish a potential therapeutic application for ERD as a pharmacological target to promote memory function and neuronal defense mechanisms against age-related neurodegeneration such as Alzheimer's disease (AD), while avoiding activating untoward 15 estrogenic proliferative effects in the breast and uterus, although this might be at the cost of lower efficacy due to the lack of activation of ERs in the brain. Other potential therapeutic advantages associated with ERP include regulation of estrogen vasculoprotective action and development of interventions targeting diseases such as depression, colon cancer, prostate 20 cancer, obesity, leukemia, and infertility. However, a potential disadvantage of an ERP-selective ligand is the lack of activation of ERa in bone, as ERa has been demonstrated to mediate estrogen regulation of bone density. Although there is still controversy regarding the differential roles of two estrogen receptor ("ER") subtypes, ERa and/or ERp, in mediating 25 estrogen actions in the brain and/or neurons, it has been widely demonstrated that ERp plays a key role in regulating brain development, neurogenesis and estrogen-induced improved neuronal plasticity and survival. In addition, as compared with ERa, ERp is less effective in mediating the sexual characteristics of estrogen action in reproductive tissues, avoiding activating 30 untoward estrogenic proliferative effects in the breast and uterus. Therefore, ERp represents a potentially safer therapeutic target for promoting memory function and neuroprotection. However, this safety may be at the cost of 3 lower efficacy, due to the lack of activation of ERa in the brain. Other potential advantages for ERp-target therapeutics arise from its regulation of estrogen's cardioprotective effects. ERP-selective ligands may also provide effective therapeutics for preventing or treating inflammation, depression, 5 anxiety, colon cancer, prostate cancer, obesity, leukaemia, and infertility. In searching for an effective ERp-selective estrogen alternative replacement therapy for promoting neurological function and preventing age related neurodegeneration, such as AD, in postmenopausal women, it is of particular interest to identify and develop naturally occurring molecules or 10 analogues that potentially have a less toxic profile for long-term administration. It is known that several plant-derived estrogenic molecules (referred to as "phytoestrogens") bind to ERa and to ERs subtypes, and some of these molecules possess moderate binding selectivity for ERp and exert estrogenic effects in multiple tissues. 15 The therapeutic efficacy of phytoestrogens in the brain remains controversial. On the one hand, when administered singly, phytoestrogens appeared to be moderately neuroprotective. On the other hand, a recent clinical trial revealed that a soy protein supplement that contains a mixture of phytoestrogens did not show improved cognitive function in postmenopausal 20 women, when treatment was initiated at the age of 60 years or older. The clinical trial of phytoestrogens reported that a soy protein supplement containing a complex formulation of isoflavones did not improve cognitive function in postmenopausal women when treated at the age of 60 years or older, Kreijkamp-Kaspers, et al. JAMA 2004, 292, 65-74, also indicating that 25 when started 10 or more years following menopause in postmenopausal women when age-related neuronal reorganization has taken place, ET/HT has no benefit on neural function. Age and hormonal "history" may be important factors that were responsible for these negative results, as was the case for the WHIMS trials. 30 Another issue that can substantially impact the efficacy of a mixture of phytoestrogens action in the brain is the formulation of phytoestrogens, since when administered alone, a number of phytoestrogens were protective 4 to neurons from neurodegenerative insults. Zhao, et al. Exp. Biol. Med 2002, 227, 509-519. Soy extracts or soy protein supplements generally contain multiple phytoestrogenic molecules, some of which may be ERa-selective agonists, while others may be ERP-selective agonists, and others may be 5 ineffective in activating either ERa or ERp but may function as inhibitors of ER binding of those ERa and/or ERp phytoestrogenic agonists. The ineffectiveness of a complex formulation of phytoestrogens in promoting beneficial effects of estrogen in brain, such as a soy-derived preparation, may also arise from antagonizing actions among the different phytoestrogens, in 10 addition to the possible ER antagonism, likely from the activation of both ERa and ERp in the same context. Co-administration of an ERa-selective agonist and an ERp-selective agonist is less effective than treatment with either agonist alone in various neuroprotective measurements. ERa and ERO have a yin/yang relationship in many contexts where 15 one receptor may antagonize the actions of the other. Weihua, et al. FEBS Left. 2003, 546, 17-24; Gustafsson, J. A. Trends Pharmacol. Sci. 2003, 24, 479-485. Studies confirmed this observation, showing that coadministration of ERt-selective agonist PPT and ER-selective agonist DPN was less efficacious than either PPT or DPN alone in protecting hippocampal neurons 20 against excitotoxic insults. Based on this analysis, a presumption can be made that the ineffectiveness of administering a mixture of phytoestrogens (i.e. a soy protein supplement) may partly come from the antagonizing actions among different phytoestrogens, which may be ERa selective or ERp selective. These findings indicate that although both ERa and ERp 25 contribute to estrogen promotion of neuronal survival, simultaneous activation of both ER subtypes, ERa and ERp, in the same context may diminish the efficacy. In addition, the different ratio and distinct function of homodimer and heterodimer induced by co-administration of an ERa selective agonist and an ERp-selective agonist may also account for the 30 reduced efficacy exerted by the combination of both agonists. Development of an ERp-selective phytoestrogen formulation could maximize the therapeutic benefits associated with activation of ERp in the 5 brain while minimizing the adverse effects associated with the activation of ERa in reproductive tissues. Moreover, selective targeting of ERp potentially reduces antagonistic actions that may occur in a complex soy derived preparation. This naturally occurring ideal formulation would have 5 tremendous therapeutic value in promoting neurological function and preventing AD in a population at risk for losing neurological capacity and losing memory function, i.e., postmenopausal women. To date, no such phytoestrogen formulation exists. Thus, there is a need to discover and develop a novel select phytoestrogen formulation, generally, and particularly, 10 a formulation that functions in the brain. It is therefore an object of the present invention to provide an ERP selective phytoestrogen formulation maximizing the therapeutic benefits associated with activation of ERP in the brain while minimizing the adverse effects associated with the activation of ERa in reproductive tissues. 15 It is a further object of the invention to provide such a composition wherein the active ingredients are isolated from natural substances. Summary of the Invention Select phytoestrogen pharmaceutical compositions and methods of use for promoting neurological health and prevention of age-related 20 neurodegeneration, such as AD, have been developed. These select phytoestrogen formulations are composed of a number of plant-derived estrogenic molecules andlor their structural analogues and exhibit binding preference to ERp over ERa and agonist activity in the brain. These ERp selective phytoestrogen formulations cross the blood-brain-barrier and 25 promote estrogen-associated neurotrophism and neuroprotection mechanisms in the brain, without activating proliferative mechanisms in the reproductive tissues and are therefore devoid of other estrogen-associated problematic aspects. The select phytoestrogen formulations are therapeutically useful to both women and men for sustaining neurological health and preventing age 30 related cognitive decline and neurodegenerative disorders, such as AD. These are administered enterally, transdermally, transmucosally, intranasally or parenterally, in a dosage effective to prevent or alleviate 6 neuronal damage, effect neuronal regeneration or sustain viability, increase expression of anti-apoptotic proteins, and/or decrease indicators of Alzheimer's Disease. The formulations preferably contain combinations of compounds, and can be formulated for daily, sustained, delayed or 5 weekly/monthly administration. In a preferred embodiment, these are administered to women who are in menopause or post menopausal, most preferably early in menopausal. Brief Description of the Drawings Figure 1 shows the chemical Structures of 17p-estradiol and the 10 phytoSERMs genistein, daidzein, equol, and IBS003569. Figures 2A and 2B show the competition binding curves for ERot and ERP (molar concentration versus fluorescence polarization (mP)) of G, D, E, I or combinations: G+D, CrD+E, or G+D+E+I. Figure 3 is a graph showing the neuroprotective efficacy of four 15 ERD-selective phytoestrogenic molecules when administered alone at concentrations that elicited the maximal neuroprotective effects as revealed from the dose-response analyses (100 nM for all four molecules Genistein (G), Daidzein (D), Equol (E) and IBSO03569 (I)), or co-administered: G+D, G+D+E, or G+D+E+I, against supraphysiological glutamate (100 PM) 20 induced neurotoxicity in primary hippocampal neurons by measurement of calcein AM staining. Figures 4A and 4B are graphs showing the effect of four ERp selective phytoestrogenic molecules when co-administered (100 nM for all four molecules) as G+D, G+D+E, or G+D+E+I, on the expression of the 25 anti-apoptotic proteins, Bcl-2 and Bcl-xL, in primary hippocampal neurons. Figure 5 is a graph illustrating the effect of four ERP-selective phytoestrogenic molecules when co-administered (100 nM for all four molecules), G+D, G+D+E, or G+D+E+I, on the expression of the anti-p amyloid protein, insulin-degrading enzyme ("IDE"), in primary hippocampal 30 neurons. Figure 6 is a graph illustrating the effect of four ERp-selective phytoestrogenic molecules when co-administered (100 nM for all four 7 molecules): G+D, G+D+E, or G+D+E+I, on the expression of the spine marker, spinophilin, in primary hippocampal neurons. Figures 7A-7D are graphs shows the neuroprotective efficacy of G, D, E, and I, alone and in combination: G+D, G+D+E, or G+D+E+I, against 5 (7A) glutamate- and (7B) P-amyloidI-42-induced neurotoxicity in rat primary hippocampal neurons, controls live/dead cells (7C); dead cells (7D). Figures 8A-8C are graphs showing the effects of G, D, E, and I, alone and in combination: G+D, G+D+E, and G+D+E+I, on insulin degrading enzyme (IDE) expression on neprilysin (NEP) expression in 10 hippocampal tissues derived from adult ovariectomized rats. Figures 9A-9E are graphs showing the effects of G, G+D+E, and G-+D+E+I on forebrain mitochondrial cytochrome c oxidase (COX) activity in adult ovariectomized rats. Figures 10A-10E are graphs showing the effects of G, G+D+E, and 15 G+D+E+I on percent increase forebrain mitochondrial respiratory activity in adult ovariectomized rats. Figures 1 1A-11 C are schematics showing estrogen mechanisms of action that lead to neurotrophic and neuroprotective outcomes. 1 IA, 17-P Estradiol (E2) acting via a membrane-associated site (mER) activates a 20 cascade required for multiple responses that lead to enhanced neural plasticity, morphogenesis, neurogenesis, and neural survival. The signaling sequence induced by E2 at the membrane site is as follows: (1) E2 binding to mER, (2) E2-mER complexes with p85 to activate P13K, (3) activating calcium-independent PKC, (4) phosphorylating the L-type calcium channel, 25 (5) inducing calcium influx, (6) activating calcium-dependent PKCs, (7) activating Src kinase, (8) activating the MEK/ERK1/2 pathway, (9) ERK translocates to the nucleus, (10) activating and phosphorylating CREB, (11) enhancing transcription of antiapoptotic genes Bcl-2 and Bcl-xl, which enhance mitochondrial vitality, and spinophilin, which encourages synaptic 30 growth, (12) simultaneously, estrogen activation of P13K leads to activation of Akt, which phosphorylates and inhibits the proapoptotic protein BAD. I IB, Estrogen-induced neuroprotective mechanisms converge on 8 mitochondria. Estrogen-activated cellular signaling cascade promotes enhanced mitochondrial function, leading to increased calcium load tolerance, enhanced electron transport chain efficiency, and promotion of antioxidant defense mechanisms. These actions are mediated by the 5 regulation of both nuclear and mitochondrial encoded genes initiated by the activation of second-messenger signaling cascades. 11 C, Conceptual schematic of NeuroSERM design and therapeutic use. Consistent with the healthy cell bias of estrogen benefit hypothesis, selective molecules would be administered before neurodegenerative insult while neurons are still healthy. 10 NeuroSERM exposure would lead to enhanced neural survival mechanisms, represented as mitochondria with Bcl-2 additions, that promote neural defense against neurodegenerative insults associated with age-associated diseases such as Alzheimer's and Parkinson's. Designer NeuroSERM molecules target the membrane site of estrogen action, whereas PhytoSERM 15 molecules preferentially target estrogen receptors. Abbreviations: AMPAR, AMPA receptor; C, cytochrome oxidase; FO, FI, ATPase subunits; LTD, long-term depression; LTP, long-term potentiation; NAD, nicotinamide adenine dinucleotide; NADH, nicotinamide adenine dinucleotide; VDCC, voltage-dependent calcium channel. 20 Detailed Description of the Invention I. Definitions "Estrogen Receptor", as used herein, refers to any protein in the nuclear receptor gene family that binds estrogen, including, but not limited to, any isoforms and variants thereof. Human estrogen receptors include the 25 alpha- and beta-isoforms (referred to herein as "ERa" and "ERp"). "Estrogen Receptor Modulator", as used herein, refers to a compound that can act as an estrogen receptor agonist or antagonist of an estrogen receptor or estrogen receptor isoform having an IC 50 or EC5 0 with respect to ERa, ERp and/or other estrogen receptor isoforms of no more than about 50 30 pM as determined using the ERa, and/or ERp transactivation assay described herein. More typically, estrogen receptor modulators have ICso or ECso values (as agonists or antagonists) of not more than about 10 JpM. 9 Representative compounds are predicted to exhibit agonist or antagonist activity via an estrogen receptor. Compounds preferably exhibit an antagonist or agonist IC 50 or EC 5 o with respect to ERL and/or ERD of about 10 pM, more preferably, about 500 nM, even more preferably about I nM, 5 and most preferably, about 500 pM, as measured in the ERa and/or ERp transactivation assays. "ICso" is that concentration of compound which reduces or inhibits the activity of a target (e.g., ERa or ERp) to half-maximal level. "EC 5 o" is that concentration of compound which provides half maximum effect. 10 "Selective Estrogen Receptor Modulator" (or "SERM"), as used herein, refers to a compound that exhibits activity as an agonist or antagonist of an estrogen receptor (e.g, ERa, ERp or other estrogen receptor isoform) in a tissue-dependent or receptor dependent manner. Thus, as will be apparent to those of skill in the biochemistry, molecular biology and 15 endocrinology arts, compounds that function as SERMs can act as estrogen receptor agonists in some tissues, e.g., bone, brain, and/or cardiovascular, and as antagonists in other tissue types, e.g., the breast and/or uterine tissue. "Phytoestrogen" refers to a naturally occurring compound of plants, such as soybeans, or plant products, such as whole grain cereals, that acts 20 like estrogen or binds to an estrogen receptor. As used herein, the term "NeuroSERM" refers to compounds that target the membrane site of estrogen action. As used herein, the term "PhytoSERM" refers to natural source compounds that preferentially target estrogen receptor beta. 25 As used herein, the term "analogue" refers to a chemical compound with a structure similar to that of another (reference compound) but differing from it in respect to a particular component, functional group, atom, etc. As used herein, the term "derivative" refers to compounds which are formed from a parent compound by chemical reaction(s). 30 II. Compositions Compositions containing one or more phytoestrogens are described herein. A number of phytoestrogens have been isolated and identified and 10 additional analogs created, all of which have estrogen receptor binding selectivity, In one embodiment, of the composition contains two or more plant-derived estrogenic molecules and/or structural analogues, which possess ERP-binding selectivity and exhibit neuroprotective activity when 5 administered individually. These compositions are useful for preventing estrogen-deficiency associated symptoms and disorders, particularly age related cognitive decline and neurodegenerative diseases, such as Alzheimer's disease ("AD"). A. PhytoSERMs 10 The compositions described herein contain one or more phytoestrogens or natural source selective estrogen receptor modulators (SERMs) exhibiting a binding preference for ERs. PhytoSERMs can be identified as described in Example 1. Suitable phytoSERMs include, but are not limited to, genistein, daidzein, equol, IBS003569 and combinations 15 thereof. The structures of genistein, daidzein, equol, and IBSO03569 are shown in Figure 1. Others are listed in Table 1. Preferred compounds cross the blood brain barrier. As demonstrated by Example 2, combinations of two or more PhytoSERMS are more effective than administration of one PhytoSERM. 20 The compounds can be used in the form of salts derived from inorganic or organic acids. These salts include, but are not limited to, the following: acetate, adipate, alginate, citrate, aspart ate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, cyclopentanepro-pionate, dodecylsulfate, ethanesulfonate, 25 glucoheptanoate, glycerophosphate, hemi-sulfate, heptanoate, hexamate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2 hydroxyethanesulfonate, lactate, maleate, methanesulfonate, nicotinate, 2 napthalenesulfanate, oxalate, parnoate, pectinate, sulfate, 3 phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, 30 thiocyanate, p-toluenesulfonate and undecanoate. Also, any basic nitrogen containing groups can be quaternized with agents such as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides, and 11 iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl, and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides, and others. Wafer or oil-soluble or dispersible products are thereby obtained. 5 Examples of acids which may be employed to form pharmaceutically acceptable acid addition salts include such inorganic acids as hydrochloric acid, sulfuric acid, and phosphoric acid, and organic acids such as oxalic acid, maleic acid, succinic acid and citric acid. Basic addition salts can be prepared in situ during the final isolation and purification of the compounds, 10 or separately by reacting carboxylic acid moieties with a suitable base such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation or with ammonia, or an organic primary, secondary or tertiary amine. Pharmaceutically acceptable salts include, but are not limited to, cations based on the alkali and alkaline earth metals, such as sodium, lithium, 15 potassium, calcium, magnesium, and aluminum salts, as well as non-toxic ammonium, quaternary ammonium, and mine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Other representative organic amines useful for the formation of 20 base addition salts include diethylamine, ethylenediamine, ethanolamine, diethanolamine, and piperazine. Appropriate carriers can be added that assist the compounds to cross the blood-brain-barrier. B. Additional Active Agents 25 While the compounds can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more other compound as described herein, and/or in combination with other agents used in the treatment and/or prevention of estrogen receptor-mediated disorders. Alternatively, the compounds can be administered sequentially 30 with one or more such agents to provide sustained therapeutic and prophylactic effects. Suitable agents include, but are not limited to, other SERMs as well as traditional estrogen agonists and antagonists. 12 Representative agents useful in combination with the compounds for the treatment of estrogen receptor-mediated disorders include, for example, tamoxifen, 4-hydroxytamoxifen, raloxifene, toremifene, droloxifene, TAT 59, idoxifene, RU 58,688, EM 139, ICI 164,384, ICI 182,780, clomiphene, 5 MER-25, DES, nafoxidene, CP-336,156, GW5638, LY 139481, LY353581, zuelomiphene, enclomiphene, ethamoxytriphetol, delmadinone acetate, bisphosphonate. Other agents that can be combined with one or more of the compounds include aromatase inhibitors such as, but not 1 imited to, 4 hydroxymdrostenedione, plomestane, exemestane, aminogluethimide, 10 rogletimide, fadrozole, vorozole, letrozole, and anastrozole . Still other agents useful in combination with the compounds described herein include, but are not limited to antineoplastic agents, such as alkylating agents, antibiotics, hormonal antineoplastics and antimetablites. An example includes the compounds used to treat or prevent osteoporosis. 15 Other ingredients include vitamins, nutritional supplements, anti-oxidant agents, coenzymes, etc. The additional active agents may generally be employed in therapeutic amounts as indicated in the PHYSICIANS' DESK REFERENCE (PDR) 53rd Edition (2003), or such therapeutically useful amounts as would 20 be known to one of ordinary skill in the art. The compounds and the other therapeutically active agents can be administered at the recommended maximum clinical dosage or at lower doses. Dosage levels of the active compounds in the compositions may be varied to obtain a desired therapeutic response depending on the route of administration, severity of the disease 25 and the response of the patient. The combination can be administered as separate compositions or as a single dosage form containing both agents. When administered as a combination, the therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single 30 composition. 13 C. Pharmaceutical Compositions The compounds can be administered enterally, transdermally, transmucisally, intranasally or parenterally. Excipients for oral formulation are known to those skilled in the art, as discussed briefly below, and can be 5 used to provide immediate, sustained, delayed, or pulsed release. The compounds can also be administered via a transdermal patch, a depo, vaginally or rectally using a topical carrier such as a gel, lotion, ointment, liposomal formulation, suspension, foam, spray or supposity, via the pulmonary or nasal route, buccally or sublingual via the mucosal membranes 10 of the mouth. The appropriate excipients for all of these formulations are known. The compounds may be dissolved or suspended in saline, sterile water or phosphate buffered saline, or a suitable oil for injection iv, im, subcu, or ip. Suitable pharmaceutically acceptable excipients include processing 15 agents and drug delivery modifiers and enhancers, such as, for example, calcium phosphate, magnesium stearate, tale, monosaccharides, disaccharides, starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose, dextrose, hydroxypropyl-.beta.-cyclodextrin, polyvinylpyrrollidone, low melting waxes, and ion exchange resins, as well 20 as combinations of any two or more thereof. Other suitable pharmaceutically acceptable excipients are described in Remington's Pharmaceutical Sciences, Mack Pub. Co., New Jersey (1991). Pharmaceutical compositions containing estrogen receptor modulating compounds may be in any form suitable for the intended method 25 of administration, including, for example, a solution, a suspension, or an emulsion. Liquid carriers are typically used in preparing solutions, suspensions, and emulsions. Liquid carriers contemplated for use include, for example, water, saline, pharmaceutically acceptable organic solvent(s), pharmaceutically acceptable oils or fats, as well as mixtures of two or more 30 thereof. The liquid carrier may contain other suitable pharmaceutically acceptable additives such as solubilizers, emulsifiers, nutrients, buffers, preservatives, suspending agents, thickening agents, viscosity regulators, or 14 stabilizers. Suitable organic solvents include, for example, monohydric alcohols, such as ethanol, and polyhydric alcohols, such as glycols. Suitable oils include, for example, soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil. For parenteral administration, the carrier can also be an oily 5 ester such as ethyl oleate, isopropyl myristate. Compositions may also be in the form of microparticles, microcapsules, liposomal encapsulates, as well as combinations of any two or more thereof. The compounds may be administered orally, parenterally, sublingually, by inhalation spray, rectally, vaginally, or topically in dosage 10 unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired. Topical administration may also involve the use of transdermal administration such as transdermal patches or ionophoresis devices. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, 15 intrasternal injection, or infusion techniques. Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension 20 in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-propanediol. Among the acceptable vehicles and solvents that may be employed are water; Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be 25 employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can be useful in the preparation of injectables. Suppositories for rectal or vaginal administration of the drug can be prepared by mixing the drug with a suitable nonirritating excipient such as cocoa butter and polyethylene glycols that are solid at ordinary temperatures 30 but liquid at the rectal temperature and will therefore melt in the rectum and release the drug. 15 Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound may be admixed with at least one inert diluent such as sucrose lactose or starch. Such dosage forms may also comprise, as is normal 5 practice, additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings. Liquid dosage forms for oral administration may include 10 pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water. Such compositions may also comprise adjuvants, such as wetting agents, emulsifing and suspending agents, cyclodextrins, and sweetening, flavoring, and perfuming agents. 15 The compounds can also be administered in the form of lipsomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multilamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non toxic, physiologically acceptable and metabolizable lipid capable of forming 20 liposomes can be used. The present compositions in liposome form can contain, in addition to a compound, stabilizers, preservatives, excipients. The preferred lipids are the phospholipids and phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art (Prescott 1976). 25 Transdermal patches are well known for delivery of nicotine, nitroglycerin and birth control. These can be utilized with these formulations as well. Depos that are implanted under the skin or ip can also be used, similarly to the manner of delivering birth control. III. Methods of Administration 30 Compounds can be administered in a variety of ways including enteral, parenteral, pulmonary, nasal, mucosal and other topical or local routes of administration. For example, suitable modes of administration 16 include oral, subcutaneous, transdermal, transmucosal, iontophotetic, intravenous, intramuscular, intraperitoneal, intranasal, subdural, rectal, vaginal and inhalation. An effective amount of the compound or composition is administered 5 to treat and/or prevent an estrogen receptor-mediated disorder in a human or animal subject. Modulation of estrogen receptor activity results in a detectable suppression or up-regulation of estrogen receptor activity either as compared to a control or as compared to expected estrogen receptor activity. Effective amounts of the compounds generally include any amount sufficient 10 to detectably modulate estrogen receptor activity by any of the assays described herein, by other activity assays known to those having ordinary skill in the art, or by detecting prevention and/or alleviation of symptoms in a subject afflicted with an estrogen receptor-mediated disorder. The effective amount will also be determined based on when the 15 compounds are administered. Estrogen/hormone therapy (ET/HT) has been associated with the reduced risk of developing AD when treated at the menopausal transition in women Brinton, R. D. Impact of estrogen therapy on Alzheimer's disease: a fork in the road? CNS Drugs 2004, 18, 405-422. For example, results of the Cache County Study indicate that women who 20 receive ET/HT at the time of menopause and continue for 10 years have a 3 fold lower risk of developing AD, Zandi, et al. JAMA 2002, 288, 2123-2129, whereas the recent data from the Women's Health Initiative Memory Study indicate that women who begin the therapy late in menopause have a greater risk of developing AD , Espeland, et al. Women's Health Initiative Memory 25 Study. JAMA 2004, 291, 2959-2968; Shumaker, et al., JAMA 2004, 291, 2947-2958. These clinical observations are consistent with basic science analyses of estrogen-inducible molecular mechanisms in the brain, indicating a healthy cell bias of estrogen action. Estrogen receptor-mediated disorders that may be treated include any 30 biological or medical disorder in which estrogen receptor activity is implicated or in which the inhibition of estrogen receptor potentiates or retards signaling through a pathway that is characteristically defective in the 17 disease to be treated. The condition or disorder may either be caused or characterized by abnormal estrogen receptor activity. Representative estrogen receptor-mediated disorders include, for example, osteoporosis, atherosclerosis, estrogen-mediated cancers (e.g., breast and endometrial 5 cancer), Turner's syndrome, benign prostate hyperplasia (i.e., prostate enlargement), prostate cancer, elevated cholesterol, restenosis, endometriosis, uterine fribroid disease, hot flashes, and skin and/or vagina atrophy. Other estrogen receptor-mediated conditions that may be treated include neurological diseases and disorders including memory loss and 10 dementia, and neurodegenerative disease, including Alzheimer's disease. In addition to the potential beneficial effects of estrogen on episodic memory, some evidence suggests that HT reduced the risks of both dementia (including AD) and mild cognitive impairment (MCI). MCI is a condition thought to represent a transitional state between normal cognition and 15 dementia in some individuals, with a 12% conversion rate from MCI to dementia each year. Observational studies repeatedly document that women taking HT enjoy an 30% reduced risk for dementia compared with women not taking HT [odds ratio range, 0.306 (Yaffe et al.,1998 JAMA 279:688; Hogervorst et al., 2003 Cochrane Database Syst Rev CD003122)]. Thus, 20 observational studies suggest that declining reproductive function could be a modifiable risk factor for dementia or that HT/ET could serve a protective role against some of the risks for developing dementia. Several recent observational studies have identified that the stage of reproductive aging at which HT/ET is started modifies the risk of dementia. 25 In these studies, women who take HT/ET during the late menopause transition or early postmenopause have a lower risk of dementia than those starting HT/ET later (Zandi et al., 2002 JAMA 288:21239; Henderson et al., J Neurol Neurosurg Psychiatry 76:103 2005). Thus, the timing of starting HT/ET relative to the menopause has been proposed to be one factor 30 explaining the otherwise discordant observations between the observational studies and the RCTs (Resnick and Henderson, 2002 JAMA 288:21702; 18 Manson et al., 2006 Menopause 13:139). Recent preclinical studies reviewed below highlight the importance of timing of ET in this report. Successful treatment of a subject may result in the prevention, inducement of a reduction in, or alleviation of symptoms in a subject 5 afflicted with an estrogen receptor-mediated medical or biological disorder. Thus, for example, treatment can result in a reduction in breast or endometrial tumors and/or various clinical markers associated with such cancers. Treatment of Alzheimer's disease can result in a reduction in rate of disease progression, detected, for example, by measuring a reduction in the 10 rate of increase of dementia. Historically, there has been a presumption that declining reproductive function plays no role in the onset of mood disorders that occur during midlife in women. The symptoms of depression during the menopause transition also were assumed to be transient and of such minor severity that 15 they were dismissed to be of little clinical consequence. Recent studies, however, suggest that these presumptions are incorrect. First, several community-based longitudinal studies have reported the relative independence of depressions during the menopause transition and hot flushes: both occur at this stage of life, but depression is not simply caused 20 by hot flushes (Avis et al., 2001 Soc Sci Med 52:345). Second, recent longitudinal studies that followed women with no past history of depression demonstrated an increased risk of first-onset depressions during the late menopause transition (Schmidt et al., 2004 Am J Psychiatry 161:22384; Cohen et al., 2006 Arch Gen Psychiatry 63:385; Freeman et al., 2006 Arch 25 Gen Psychiatry 61:62). Finally,both major and minor depressions are clinically significant to women at midlife, because both are associated with an increased risk for several other medical conditions (Wassertheil-Smoller et al., 2004 Arch Intern Med 164:289) relevant to the health of women at midlife (e.g., cardiovascular disease, dementia, and the metabolic syndrome). 30 The majority of women do not develop depression during the menopause transition, and, therefore, reproductive aging is not uniformly associated with either depressive symptoms or the syndrome of depression. 19 Nonetheless, despite numerous studies concluding that the menopause is not associated with an increased risk for developing depression in women, several other longitudinal, community-based studies reported an association between the menopause transition and an increased risk for depression S (Schmidt, 2005 Am J Med 118:54). Indeed, five recent longitudinal studies all have documented an increased risk for depression during the menopause transition, with odds ratios ranging from 1.8 to 2.9 compared with the premenopause (Bromberger et al., 2001 Am J Public Health 91:14352; Freeman et al., 2004 Arch Gen Psychiatry 61:62, 2006 Arch Gen Psychiatry 10 63:375; Schmidt et al., 2004 Am J Psychiatry 161:22384; Cohen et al., 2006 Arch Gen Psychiatry 63:385). These data suggest that events surrounding the final menstrual period may predispose some women to develop clinically significant depressive illness. Although several factors could precipitate depression in these women, endocrine events are suggested by the stage of 15 the menopause transition (i.e., late) during which depressions appeared. The late transition is characterized by more prolonged hypogonadism than the early perimenopause, during which estradiol secretion may be increased. Thus, the timing of appearance of the depressions observed suggest an endocrine mechanism related to the perimenopause (estradiol withdrawal 20 and/or recent-onset of prolonged hypogonadism) in the pathophysiology of perimenopausal depression. Efforts to investigate the potential role of declining ovarian hormone secretion in the onset of depression have examined the effects on mood of administering HT[ET in women with perimenopausal and postmenopausal 25 depression. The antidepressant efficacy of estradiol has been examined in three relatively recent RCTs of women meeting standardized diagnostic criteria for major and minor depression, who were randomly assigned to enter double-blind, placebo-controlled trials (Schmidt et al., 2000; Soares et al., 2001 Arch Gen Psychiatry 58:529 ; Morrison et al., 2004 Biol Psychiatry 30 55:406). In perimenopausal women, short-term administration (3 weeks) of estradiol significantly decreased depression scores compared with both baseline and placebo conditions. In one study, a full or partial therapeutic 20 response was seen in 80% of perimenopausal women on estradiol compared with 22% of those on placebo (Schmidt et aL, 2000). The efficacy of ET in perimenopausal depression is consistent with the observed effect size (0.69) in a recent meta-analysis of studies examining the effects of estrogen on 5 mood (Zweifel and O'Brien, 1997 Psychoneuroendocrinology 22:189). The therapeutic response to estradiol was observed in both major and minor depression as well as in women with and without hot flushes. Thus, the efficacy of ET in perimenopausal depression is not solely a product of its ability to reduce the distress of hot flushes. In contrast to these studies in 10 perimenopausal depression, the administration of estradiol under similar conditions failed to improve mood in depressed women who were 5 years postmenopause (Morrison et al., 2004). Thus, the effects of estradiol on depression may be limited to perimenopausal women. Additionally, as with the potential effects of estrogen on the course of dementia, the stage of 15 reproductive aging at which women present and/or commence ET might modify the observed outcomes. In summary, the majority of women do not develop depression during or after the menopause transition. Nevertheless, recent prospective studies monitoring both reproductive status and mood have documented that, for 20 some women, perimenopause-related events increase the risk for the onset of depression. The role of ovarian function in these episodes of depression is suggested by both the timing of their onset relative to the last menstrual period and the antidepressant efficacy of short-term ET. The amount of active ingredient that may be combined with the 25 carrier materials to produce a single dosage form will vary depending upon the estrogen-mediated disease, the host treated and the particular mode of administration. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general 30 health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity of the particular disease undergoing therapy. The prophylactically or therapeutically effective amount 21 for a given situation can be readily determined by routine experimentation and is within the skill and judgment of the ordinary clinician. For exemplary purposes, a prophylactically or therapeutically effective dose will generally be from about 0.01 mg/kg/day to about 100 5 mg/kg/day, preferably from about 0.1 mg/kg/day to about 2 0 mg/kg/day, and most preferably from about I mg/kg/day to about 10 mg/kg/day of a estrogen receptor modulating compound , which may be administered in one or multiple doses. IV. Kits 10 Kits may be provided which contain the formulation to be administered. The formulation may be administered once a day or more than once a day. The formulation can be administered enterally, parenterally, or topically. The kits typically contain the active agent(s) to be administered, excipients and carriers, and instructions for administration of the 15 formulation. The kits may also contain equipment/devices used to administer the formulation, such as syringes. The present invention will be further understood by reference to the following non-limiting examples. 20 Examples Example 1: Identification of PhytoSERMS ERs has been associated with estrogen-induced promotion of memory function and neuronal survival. Based on the optimized complex structure of human ERp LBD bound with genistein, computer-aided 25 structure-based virtual screening against a natural source chemical database was conducted to determine the occurrence of plant-based ERO-selective ligands. Twelve representative hits derived from database screening were assessed for their binding profiles to both ERs, three of which displayed over I 00-fold binding selectivity to ER over ERa. 22 Materials and Methods Identification of Molecules Identification of Compounds in Database All computational work was performed on a SGI Octane workstation 5 equipped with the IRIX 6.5 operating system (Silicon Graphic Inc.). First, the 3D crystallographic structure of human ERp LBD complexed with genistein was downloaded from the Protein Data Bank (PDB ID: 1QKM). The complex structure was fixed and energy minimized with the Accelrys molecular modeling software package InsightI 2000 (Accelrys Inc.). An in 10 house 2D natural source chemical collection containing approximately 25 000 plant-based natural molecules or derivatives was converted to a 3D multiconformational database with the Accelrys modeling software package Catalyst 9.8 (Accelrys Inc.). The receptor-docking site was defined based on the binding position 15 of genistein in the receptor and specified as all atoms within 10 A of the center carbon of genistein. GOLD 2.0 (Genetic Optimization for Ligand Docking), an automated ligand docking program distributed by CCDC (Cambridge Crystallographic Data Center), was applied to calculate and rank the molecules based on their complementarities with the receptor binding 20 site, on both geometrical and chemical features. Prior to the database screening, initial validation using genistein as the test ligand was conducted. The aim of the validation test was to evaluate the effectiveness of the algorithm of the docking program in identifying the experimentally observed binding mode of the ligand in the receptor, to 25 determine whether the program is applicable to the specific target system in this study. In addition, the validation test was used to determine the optimal parameter settings for the later database screening. Twenty docking runs were carried out on the test complex, using the fastest default generic algorithm parameters optimized for virtual library screening, and the 30 GoldScore fitness function was applied. The validation test demonstrated that, based on the specified parameter settings, GOLD was effective in capturing the contributive hydrogen bond donor (NDI in His 475) crucial to 23 the binding and reproducing the nearly coincident solution in terms of both the binding orientation and conformation of genistein as observed in the experimental measurement (see Figure 1). The root-mean-square (RMS) deviations were computed between the observed experimental position and 5 the GOLD solutions, with RMSD 0.3299 and 0.4483 compared to top-ranked and worst solutions, respectively. The average RMSD of all solutions was 0.3566, which is regarded as a good prediction based on the subjective classifications defined by the program developer (refer to the program manual), suggesting that this program is reliable and applicable to the 10 database screening toward ERP. Using the parameter settings determined in the validation test, the 3D natural source chemical database was input and docked into the prepared ERP binding site in a flexible docking mainer (full ligand and partial protein) and scored based on the GoldScore fitness function. Five hundred 15 resultant top-scoring molecules were filtered via visual screening in the context of the receptor in InsightIl. Based on visual analysis, 100 molecules underwent further analysis by Affinity, a more complex and predictive ligand docking program to refine the binding modes predicted by GOLD. The criteria used for the selection of candidate molecules for investigation 20 included the following (a) formation of hydrogen bond with donor atom NDl in His 475; (b) hydrophobic and hydrophilic balance appearing in the structure (e.g., the molecule should potentially have two relatively hydrophilic sides and a hydrophobic center to enhance both the steric and electrostatic complementarity with the receptor); (c) bound pose of the 25 molecule in the receptor; and d) structural diversity. Finally, molecules that met the above criteria were computationally predicted for their drug-likeness (Lipinski's Rule of Five) and blood-brain barrier (BBB) penetration properties. The ligand binding domains of the human ERot and ER$ are 30 approximately 60% homologous. Structural modeling and mutational analyses indicate that two variant amino acid residues along the ligand binding pocket, Leu 384 and Met 421 in ERt, which are replaced with Met 24 336 and Ile 373, respectively, in ERp, are the key molecular constituents underlying discriminative binding of selective ligands to either receptor subtypes. Sun, ct al. Mol. Endocrinol. 2003, 17, 247-258. This slight structural variance serves as the foundation for both design and discovery of 5 ER specific ligands. The similarities in the chemical features of both pairs of residues presents a substantial challenge to discover a selective ligand based on this difference. Of the known natural source ERp-selective ligands, genistein remains the most selective. However, an increasing number of synthetic compounds are emerging showing greater selectivity than genistein 10 for ERp, as evidenced by the compound DPN developed in Katzellenebogen's laboratory. Computer-aided structure-based virtual database screening provides an efficient approach to rationally highlight a small group of lead candidates from a large number of compounds for investigation at the bench. 15 Determination of Binding Affinity and Selectivity The binding affinity and selectivity of candidate molecules yielded from database screening were determined by a fluorescent polarization competitive binding assay using purified baculovirus-expressed human ERp or ERa and a fluorescent estrogen ligand EL Red (PanVera Corp.). Test 20 molecules were serially diluted to a 2x concentration in assay buffer (200 pM to 200 pM). Fifty microliters of preincubated 2x complex of ERP (30 nM) or ERp (60 nM) and EL Red (2 nM) was added to each well in a 96 well Non-binding Surface black microplate (Corning Life Sciences) for a final volume of 100 [L. Negative controls containing ER and EL Red 25 (equivalent to 0% inhibition) and positive controls containing only free EL Red (equivalent to 100% inhibition) were included. After a 2-h incubation period at room temperature, the polarization values were measured using a Tecan GENios Pro reader at 535 nm/590 nm excitation/emission and plotted against the logarithm of the test molecule concentration. IC 5 o values 30 (concentration of test molecule that displaces half of the EL Red from ER) were determined from the plot using a nonlinear least-squares analysis. 25 Results 31 molecules that can form a hydrogen bond with ND1 in His 475 were selected and grouped into three categories based upon the chemical features that favored both the van der Waals (VDW) contact (the number of 5 the rings in the structure) and electrostatic interactions (the number of the hydrogen bonds) with the receptor. 10 molecules that have strong VDW interactions with the receptor, but without contributive hydrogen bonding, were grouped in Category IV. These molecules contain three or four five- or six-membered rings in their structures that could promote the hydrophobic 10 interactions with the center of the receptor binding site as observed in endogenous estrogen 17p-estradiol that consists of four rings in its structure and binds to the estrogen receptor with a high affinity. Table 1 summarizes the ICso binding results of test molecules to both ERa and ERO as well as the binding selectivity of representative molecules 15 selected from four categories. 26 Table 1. Binding Afinity (1z) and Selectivity of Representative Molecules for Eeroga Receptor a and # CoMpd Structure _ , SeIetivity Pageste NC* NC geisteii 4.66 98.7 47.2 jiM nM 175.. 18.6 4.07 nM nM 2 NC 0,68 > 100 3 12 Z5 0.4 fig nM 4 N NC NC 2,A >100 6 NC NC 7 85.7 4&0 1,99 8 NC 4A6 > 100 9 NC NC 10 NC NC 11 2.32 NC 0.01 12 NC NC *NC: Nonconvergence within the dose range, predicting that either the molecule does not bind to the receptor or that the binding ninity is very low, with an IC 0 great than I mK As expected, the negative control steroid, progesterone, does not bind to either ER. As a positive natural source estrogen control, genistein was 5 found to bind to ERp with a 47.2-fold greater binding selectivity over ERa, 27 but at an affinity one-fourth of 170-estradiol. Among 12 molecules tested, five molecules, 1, 2, 5, 7, and 8, showed binding selectivity to ERp over ERa, 3 of which, 2, 5, and 8, displayed the selectivity over 100-fold. Preliminary structure and binding activity relationship analyses revealed that 5 both the central hydrophobic skeletal structure and the connected two polar 'arms' contribute to the binding affinity of ligands to both ERs. The enhanced VDW contact derives mainly from the central hydrophobic feature of the molecule. For example, the number of rings increases the binding affinity of molecules to the receptor, as indicated by the VDW value of 17$ 10 estradiol (-67.98) versus that of genistein (-60.75) and molecule 9 (-58.04), which are well correlated with their order-different binding affinities. Meanwhile, the hydrogen bonds derived from the two polar "arms" of the molecule are essential for the binding as well. The lack of one "arm" of the hydrogen bond, as represented by molecule 4 and 6, or two 'arms', as 15 represented by 10 and 12, even though the latter two molecules can elicit strong VDW interactions with the receptor, with the VDW value of -72.58 and -69.19, respectively, leads to either very weak or no binding. With respect to the binding selectivity, as demonstrated in the modeling complex structures of a synthetic ERp-selective agonist, PPT, Stauffer, et al.. J. Med. 20 Chem. 2000, 43,4934-4947 and a synthetic ERp-selective agonist, DPN, Meyers, et al., J. Med Chem. 2001, 44, 42304251, with both ERs, Zhao, et al. 2004 Abstract Book; The Keystone Symposia: Nuclear Receptors: Steroid Sisters, Keystone, CO; February 2004, relatively larger molecular size favors the binding selectivity for ER over ERa, as represented by molecule 3 and 25 11. These analyses shed light on the future search and design of more active and selective ER subtype-selective ligands. Further, 3 out of 12 representative molecules yielded from database searching displayed over 100-fold selectivity toward ERP over ERa, demonstrating the effectiveness 30 of this computer-aided virtual screening approach applied in the present study in the discovery of potential molecules that preferentially interact with ERs. 28 Example 2: Preclinical Identification of ERP-Selective PhytoSERM Combinations for Prevention of Neurodegeneration The impact of ERb-selective PhytoSERMs when administered singly 5. or in combination on neuronal survival and molecular/functional markers associated with prevention of neurodegeneration and Alzheimer's disease (AD) was investigated. Materials and Methods 17B-Estradiol was purchased from Steraloids (Newport, RI). 10 Genistein, daidzein and equol were purchased from Indofine Chemical (Hillsborough, NJ). IBS003569 was purchased from InterBioScreen (Moscow, Russia). The structures of these compounds are shown in Figure 1. In Vitro Treatments: Test compounds (or combinations) were first 15 dissolved in analytically pure DMSO (10 mM) and diluted in Neurobasal medium to the working concentrations right before treatments. In Vivo Treatments: Test compounds (or combinations) were first dissolved in analytically pure DMSO and diluted in corn oil (50 ml of DMSO in 950 ml of corn oil) to the working concentrations at 100 mg/ml for 20 175-estradiol and 10 mg/ml for phytoSERMs. In vitro Assays ERa Binding Assays ERa receptor (about.0.2 mg/ml, Affinity Bioreagents) is diluted to about 2 x 103 mg/ml in phosphate-buffered saline ("PBS") at a pH of 7.4. 25 Fifty microliters of the EPa -PBS solution is then added to each of the wells of a flashplate. The plates are sealed and stored in the dark at 4*C for 16-18 hours. The buffered receptor solution is removed just prior to use, and the plates are washed 3 times with 200 microliters per well of PBS. The washing is typically performed using a slow dispense of reagent into the wells to 30 avoid stripping the receptor from the well surface. For library screening, 150 microliters of I iM 3 H-estradiol (New England Nuclear, Boston, Mass.) in 20 mM Tris-HCl, 1 mM EDTA, 10% 29 glycerol, 6 mM monothioglycerol, 5 mM KCI, pH 7.8 is mixed with 50 microliters of the test compound (in same buffer) in a 96 well mictrotiter plate, resulting in a final estradiol concentration of 0.6 nM. In addition, several dilutions of estradiol, centered on the IC 50 of 1-2 nM, are also added 5 to individual wells to generate a standard curve. The plates are gently shaken to mix the reagents. A total of 150 microliters from each of the wells is added to the corresponding wells of the pre-coated ER% plates. The plates are sealed and the components in the wells are incubated either at room temperature for 4 hours or at 4"C overnight. The receptor bound ligand is 10 read directly after incubation using a scintillation counter. The amount of receptor bound ligand is determined directly, i.e., without separation of bound from free ligand. If estimates of both bound and free ligand are required, the supernatant is removed from the wells, liquid scintillant is added, and the wells are counted separately in a liquid scintillation counter. 15 ERP Binding Assays ER5 receptor (.about.0.2 mg/ml, Affinity Bioreagents) is diluted to about 2 x10 3 mg/ml in phosphate-buffered saline ("PBS") at a pH of 7.4. Fifty microliters of the ERg -PBS solution is then added to each the wells of a flashplate. The plates are sealed and are stored in the dark at 4 0 C for 16-18 20 hours. The buffered receptor solution is removed just prior to use, and the plates are washed 3 times with 200 microliters per well of PBS. The washing is typically performed using a slow dispense of reagent into the wells to avoid stripping the receptor from the well surface. For library screening, 150 microliters of 1 nM 3 H-estradiol (New 25 England Nuclear, Boston, Mass.) in 20 mM Tris-HCl, 1 mM EDTA, 10% glycerol, 6 mM monothioglycerol, 5 mM KCI, pH 7.8 was mixed with 50 microliters af the test compound (in same buffer) in a 96 well microtiter plate, resulting in a final estradiol concentration of 0.6 nM. In addition, several dilutions of estradiol, centered on the IC 50 of 1-2 nM is also added to 30 individual wells to generate a standard curve. The plates are then gently shaken to mix the reagents. A total of 150 microliters from each of the wells is added to the corresponding wells of the pre-coated ERA plates. The plates 30 are sealed and the components in the wells are incubated at room temperature either for 4 hours or at 4*C overnight. The receptor bound ligand is read directly after incubation using a scintillation counter. The amount of receptor bound ligand is determined directly, i.e., without separation of 5 bound from free ligand. If estimates of both bound and free ligand are required, the supernatant is removed from the wells, liquid scintillant is added, and the wells are counted separately in a liquid scintillation counter. ERa/ERf Transactivation Assays Construction of Transfected CHO Cells 10 Transfected CHO cells were derived from CHO KI cells obtained from the American Type Culture Collection ("ATCC", Rockville, Md.). The transfected cells were modified to contain the following four plasmid vectors: (1) pKCRE with DNA for the human estrogen receptor, (2) pAG-60 neo with DNA for the protein leading to neomycin resistance, (3) pRO-LUC 15 with DNA for the rat oxytocin promoter and for firefly luciferase protein, and (4) pDR2 with DNA for the protein leading to hygromycine resistance. All transformations with these genetically modified CHO cells are performed under rec-VMT containment according to the guidelines of the COGEM (Commissie Genetische Modificatie). Screening is performed either in the 20 absence of estradiol (estrogenicity) or in the presence of estradiol (anti estrogenicity). Assays to Assess Neuronal Function Neuronal culture preparation Primary cultures of hippocampal neurons were obtained from 25 Embryonic Day 18 (El 8d) rat fetuses. Briefly, after dissected from the brains of the rat fetuses, the hippocampi were treated with 0.02% trypsin in Hank's balanced salt solution (137 mMNaCl, 5.4 mM KCI, 0.4 mM KH 2 PO4, 0.34 mM Na 2

HPO

4 .7H20, 10 mM glucose, and 10 mM HEPES) for 5 min at 37*C and dissociated by repeated passage through a series of fire-polished 30 constricted Pasteur pipettes. Between 2 x 104 and 4 x 10 4 cells were plated onto poly-D-lysine (10 g/ml) -coated 22 mm coverslips in covered 35 mm petri dishes for morphological analysis, and 1 x 105 cells/ml were plated 31 onto poly-D-lysine-coated 24-well, 96-well culture plates or 3-5 x 105 cells/ml onto 0.1% polyethylenimine-coated 60mm petri dishes for biochemical analyses. Nerve cells were grown in phenol-red free Neurobasal medium (NBM, Invitrogen Corporation, Carlsbad, CA) supplemented with 5 B27, 5 U/ml penicillin, 5 pg/ml streptomycin, 0.5 mM glutamine and 25 pM glutamate at 3 7*C in a humidified 10% CO 2 atmosphere at 37*C for the first 3 days and NBM without glutamate afterwards. Cultures grown in serum free Neurobasal medium yield approximately 99.5% neurons and 0.5% glial cells. 10 Neuroprotection measurements Glutamate exposure Primary hippocampal neurons were pretreated with compounds for 48 hr followed by exposure to 100 gM glutamate for 5 min at room temperature in HEPES buffer containing 100 mM NaCl, 2.0 mM KCI, 2.5 15 mM CaCl 2 , 1.0 mM MgSO4, 1.0 mM NaH 2 PO4, 4.2 mnM NaHCO3, 10-0 mM glucose and 12.5 mnM T-LEPES. Immediately following glutamate exposure, cultures were washed once with HEPES buffer and replaced with fresh Neurobasal medium containing the test compounds. Cultures were returned to the culture incubator and allowed to incubate for 24 hr prior to 20 cell viability measurements on the following day. Western immunoblotting CREB phosphorylation Nuclear lysates were prepared as following: Briefly, hippocampal neurons grown on poly-D-lysine coated culture dishes were treated with 25 compounds for appropriate periods, washed with cold PBS once and scraped into I ml PBS. Cells were then centrifuged at 5,000 rpm for 5 min, and the pellet was dissolved in Cytoplasm Extraction buffer (10 mM HEPES, 1 mM EDTA, 60 mM KCl, 0.075% Igepal and protease and phosphatase inhibitor cocktail) and suspended by passage through a 200 .1 pipette tip. After 30-45 30 RPM of incubation at 4*C, the samples were centrifuged at 5,000 rpm for 5 min to generate the cytoplasmic extract in the supernatant. The supernatant cytoplasmic extract was removed, and Nuclear Extraction buffer (20mM Tris 32 HCI, 1.5mM MgC1 2 , 420mM NaCl, 0.2mM EDTA, 25% glycerol, 0.5% Igepal and protease and phosphatase inhibitor cocktail) was added to the pellet followed by 5M NaCl to break the nuclear membrane. Following 30 45 RPM of incubation at 4*C, the samples were centrifuged at 12,000 rpm 5 for 10 min to generate a supernatant containing the nuclear extract. Protein concentration was determined by the BCA method. An appropriate volume of 2X sample buffer was added to the protein samples, and samples were boiled at 95"C for 5 min. Samples (25 pg of proteins per well) were loaded on a 10% SDSPAGE gel and resolved by standard 10 electrophoresis at 90V. Proteins were then electrophoretically transferred to Immobilon-P PVDF membranes overnight at 32 V at 4*C. Membranes were blocked for 1 hr at room temperature in 10% non-fat dried milk in PBS containing 0.0 5% Tween 20 (PBS-T), incubated with appropriate primary antibodies against phospho-CREB (pSER1 33 , mouse monoclonal, 1:2000; 15 Cell Signaling Technology, Beverly, MA), CREB (rabbit polyclonal, 1:1000; Cell Signaling Technology, Beverly, MA), spinophilin (rabbit polyclonal, 1:1000; Upstate Biotecholagy, Lake Placid, NY), actin (mouse monoclonal, 1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or histone H1 (mouse monoclonol, 1:250; Santa Cruz Biotechology, Inc., Santa Cruz, CA) 20 at temperatures and times specified by the antibody providers. All primary antibodies were dissolved in PBS-T with 1 % horse serum (for mouse monoclonal antibody) or goat serum (for rabbit polyclonol). After washing in PBS-T, the membranes were incubated with horseradish peroxidase conjugated anti-mouse IgG (1:5000; Vector Laboratories, Inc., Burlingame, 25 CA) in PBS-T with I % horse serum or anti-rabbit IgG (1:5000; Vector Laboratories, Inc., Burlingame, CA) in PBS-T with 1% goat serum for 1 hr. Immunoreactive bands were visualized by TMI3 detection kit (Vector Laboratories. Inc., Burlingame, CA) and quantified using Un-Scan-It gel image software (Silk Scientific, Inc., Orem, UT). Following transfer, gels 30 were stained with Coomassie blue (Bio-rad Laboratories, Hercules, CA) to ensure equal protein loading. 33 Bcl-2 and Bcl-xl expression Primary hippocampal neurons were pretreated with compounds for 48 hr before the cells were lysed by incubation in ice-cold lysis buffer containing: 0.005% SDS, 0.1% Igepal, 0.2 mM sodium orthovanadate, 0.2 5 mM phenylmethylsulfonylfluoride and protease inhibitor mixture in PBS at 4*C for 45 min. Cell lysates were centrifuged at 10,000 rpm at 4"C for 10 min, and the concentration of protein in the supernatant was determined using the BCA Protein Assay (Pierce Biotechnslogy, Inc., Rockford, IL). 25 pg of total protein were diluted in 15 p1 2X SDS containing sample buffer 10 and the final volume was made 30 pl with water. After denaturalization on a hot plate at 95-100*C for 5 min, 25 pl of the mixture were loaded per lane on 10% SDS-polyacrylamide mini-gels followed by electrophoresis at 90V. The proteins were then electro-transferred to polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA) from the gels. Nonspecific 15 binding sites were blocked with 5% nonfat dry milk in PBS containing 0.05% TweenTM-20 (PBS-TweenTm). Membranes were incubated with the primary monoclonal antibody against Bcl-2 (Zymed Laboratories, Inc., S. San Francisco, CA) diluted 1:250 in PBS-Tweenm with 1% horse serum (Vector Laboratories, Inc., Burlingame, CA) overnight at 4 "C, then 20 incubated with the secondary horseradish peroxidase (HRP)-conjugated horse anti-mouse IgG (Vector Laboratories, Inc, Burlingame, CA) diluted 1:5,000 in PBS-TweenTM with 1% horse serum for 2 hr at room temperature, and Bcl-2 proteins were visualized by developing the membranes with TMB substrate for peroxidase (Vector Laboratories, Inc., Burlingame, CA). D 25 Actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) level was determined to ensure equal protein loading, and high-range Precision Protein Standards (Bio-Rad Laboratories, Hercules, CA) was used to determine protein sizes. Relative intensities of bands were quantified by optical density analysis using an image digitizing software Un-Scan-Tt version 5.1 (Silk 30 Scientific, Inc., Orem, UT). 34 Statistics Statistically significant differences between groups were determined by a one way analysis of variance (ANOVA) followed by a Newman-Keuls post hoc analysis. 5 In vivo Assays Immature Rat Uterotrophic Bioassay for Estrogenicity Anti-Estrogenicity Antiestrogenic activity was determined by the ability of a test compound to suppress the increase in uterine wet weight resulting from the administration of 0.2 gg 17-p-estradiol ("E 2 ") per day. Any statistically 10 significant decreases in uterine weight in a particular dose group as compared with the E 2 control group are indicative of anti-estrogenicity . One hundred forty (140) female pups (19 day s old) in the 35-50 g body weight range are selected for the study. On day 19 of age, when the pups weigh approximately 35-50 g, they are body weight-order randomized 15 into treatment pups. Observations for mortality, morbidity, availability of food and water, general appearance and signs of toxicity are made twice daily. Pups not used in the study are euthanized along with the foster dams. Initial body weights are taken just prior to the start of treatment at day 19 of age. The final body weights are taken at necropsy on day 22 of age. 20 Treatment commences on day 19 of age and continues until day 20 and 21 of age. Each animal is given three subcutaneous ("sc") injections daily for 3 consecutive days. Three rats in each of the control and mid- to high-level dose test groups are anesthetized with a ketamine/xylazine mixture. Their blood is collected by exsanguination using a 22 gauge needle 25 and 5 ml syringe flushed with 10 USP with sodium heparin/ml through the descending vena cava; and then transferred into a 5 ml green top plasma tube (sodium heparin (freeze-dried), 72 USP units). Plasma samples are collected by centrifugation, frozen at -70*C, and analyzed using mass spectrographic to determine the presence and amount of test compound in the serum . Blood 30 chemistry is also analyzed to determine other blood parameters. The uteri from the rats are excised and weighed. The remaining rats are sacrificed by 35 asphyxiation under CO 2 . The uteri from these rats are excised, nicked, blotted to remove fluid, and weighed to the nearest 0.1 mg. In order to determine whether the test compound significantly affected final body weight, a parametric one-way analysis of variance 5 (ANOVA) is performed (SIGMASTAT version 2.0, available commercially from Jandel Scientific, San Rafael, Calif.). Estrogen agonist and antagonist activity is assessed comparing uterine wet weights across treatment groups using a parametric ANOVA on loglo transformed data. The data are transformed to meet assumptions of normality and homogeneity of variance 10 of the parametric AWQVA. The F value is determined and a Student Newman-Kuel s multiple range test is performed to determine the presence of significant differences among the treatment groups. The test compound is determined to act as a mixed estrogen agonist/antagonist if the test compound does not completely inhibit the I 7.--estradiol stimulated 15 uterotrophic response. The use of animals was approved by the Institutional Animal Care and Use Committee at the University of Southern California (Protocol Number: 10780). Embryonic day 18 Sprague-Dawley rat (Harlan, Indianapolis, IN) fetuses were used to obtain primary hippocampal neuronal 20 cultures for in vitro experiments. Young adult (14 to 16-week-old, weighing from 270-290 g) female ovariectomized Sprague-Dawley rats (Harlan) were used for in vivo experiments. In vitro neuroprotection and associated mechanistic studies were conducted in primary hippocampal neurons obtained from embryonic day 18 25 rat fetuses. Adult female ovariectomized rats were used to relate the in vitro findings to in vivo environment, along with the assessment of the impact of PhytoSERMs on brain mitochondrial functions and uterine weight. During a 2-week surgery recovery following ovariectomy, but before treatment, rats were placed on a phytoSERM-reduced diet, TD.96155 30 (Harlan Teklad). Rats were given, once daily, 2 subcutaneous injections of vehicle (control), 17 P -estradiol (70 pg/kg BW), genistein (6 mg/kg BW), or 36 phytoSERM combinations (6 mg/kg BW). Dosages used here are commensurate with those used in humans. Following the second injection, animals fasted for 24 hours prior to sacrifice and brain dissection. Hippocampal and cortical tissues were 5 collected from one hemisphere and stored for biochemical analyses. The remaining brain tissues minus cerebellum, pineal gland, and brainstem were utilized for mitochondrial isolations, followed immediately by mitochondrial respiratory activity measurements. The rest of mitochondrial samples were stored for cytochrome c oxidase activity measurements. Uteri were excised, 10 trimmed of fat and connective tissue, and both a wet weight and a dry weight were recorded. Results The PhytoSERMs tested are shown in Figure 1. Selective Binding for both ERP and ERa 15 Figure 2 presents the competition binding curves of four known ER ligands for both ERp and ERt. The IC 50 determined for these ligands from the binding curves are consistent with the previously reported values using alternative methods such as radioligand assay, demonstrating the reliability of this assay in determining the binding profiles of small molecules to both 20 ERs. Figures 2A and 2B show the competition binding curves for ERa and ERs. Data were generated with a fluorescence polarization-based competitive binding assay using full-length human ERa and ER p, and plotted against the logarithm of serially diluted concentrations of the test 25 compounds (or combinations). Progesterone served as a negative control. 17 s -Estradiol served as a positive control. Combined formulations were composed of equivalent molar of individual phytoSERMs included. G: genistein; D: daidzein; E: equol; I: IBS003569. 17p-estradiol has no binding preference to ERa or to ERs. The concentration of a test molecule 30 resulting in the half-maximum shift in polarization value equals its ICSo. Non-convergence within the dose range, predicts that either the molecule does not bind to the receptor or that the binding affinity is very low. 37 Table 2 shows the binding data for ERcc and ERP. Table 2. Binding data for ERa and ERp CompouNda ERA ER Seeetvty ICSO RBA RO ICa RBA R M% (%t WA) (%/ Propsterone Nn-Binig N-Bi 17..Estdio 0.0253 100.0 0.9791 0.0325 100.0 0.9611 0.78 Genistein 4.735 0.5343 09811 0.0789 41.12 0.998 60.0 DAlzAin 26.65 0.0949 0.7876 1.738 1.867 03883 14.27 Equal 5.876 0.4306 03948 0.5825 5.571 0986 10.09 IBS003569 1695 0:0015 0.9917 7.819 0.415 0.9959 >100 G*D 9.896 02557 09865 0.1574 20.62 0.9970 62.87 G+D+E 15.71 0.1610 09925 0.1902 17.06 0.9969 52.60 G+0D+E+I 15.85 03596 09932 0.2615 12.41 0.9891 60.61 ARBA (%) refers to the relative binding affinity of the test compound (combination) that is 5 expressed as the percent of the binding affinity of 17 P -estradiol (RBA = 100%).

BR

2 refers to goodness of fit of nonlinear regression between the binding curve and the data. Between 0.0 and 1.0, higher values indicate that the curve fits the data better. A fit with a R2 at 1.0 indicates that all points lie exactly on the curve with no scatter. Neuroprotective Effect 10 Table 3 and Figure 3 show the dose-dependent neuroprotective effects of four ERp-selective phytoestrogenic molecules against supraphysiological glutamate (100 gM)-induced neurotoxicity in primary hippocampal neurons by measurement of LDH release. * P < 0.01 compared to vehicle alone-treated cultures; * P <0.05 and ** P <0.01 15 compared to glutamate alone-treated cultures. 38 Table 3. Dose-dependent effects of individual phytoSERMs against glutamate-induced neurotoxicity in primary hippocampal neurons by LDH measurementsA TreatmentGenistein LDH Release (% of Control) Control 100.00± 3.09 Glutamate alone 410.99 t 8.27 " 1 nM 361.03 7.71 ** 10 nM 350.02 8.21 ** 100 nM 347.24 16.96 ** 1 p.M 356.79 ± 11.15 ** 10 M 377.84 ± 8.45 ** 5 TreatmentDaidzein LDH Release (% of Control) Control 100.00 ± 4.28 Glutamate alone 378.26 ± 11.95 4 I nM 338.39 ± 16.49 10 nM 333.98 ± 9.10 * 100 nM 301.42 ± 7.70 ** 1 pM 318.49 ±15.92 ** 10 pM 325.41 ± 26.12 * TreatmentEquol LDH Release (% of Control) Control 100.00 ± 14.95 Glutamate alone 460.27 ± 12.20 " I nM 453.50 ± 23.37 10 nM 403.78 ± 17.02 * 100 nM 331.59 ± 9.67 ** 1 PM 381.80 ± 12.01 ** 10 M 390.21 ± 9.40 ** TreatmentIBSO03569 LDH Release (% of Control) Control 100.00 ± 2.05 Glutamate alone 281.17 ± 6.77 * I nM 262.41 ± 10.60 10 nM 270.86 ± 12.94 100 nM 220.56 ± 6.80 ** 1 M 246.30 ± 7.70 ** 10 PM 307.53 ±2.62 39 APrimary hippocampal neurons grown for 7 DIV were pretreated with the test phytoSERMs at serially diluted concentrations for 48 h, followed by a 5-min exposure to 100 mM glutamate. The amount of LDH released into the culture media was measured 24 h later. BData are derived from a single experiment and are representative of at lease three 5 independent experiments. Results are presented as the percent of LDH release from vehicletreated control cultures and expressed as means * S.E.M., n 6. #P <0.01 compared to vehicle-treated control cultures, *P <0.05 and **P <0.01 compared to glutamate alone treated cultures; VP <0.05 and 1P <0.01 compared to cultures treated with 10 nM phytoSERMs; PP <0.05 and 9P <0.01 compared to cultures treated with I pM 10 phytoSERMs; fP <0.05 and ffP < 0.01 compared to cultures treated with 10 mM phytoSERMs. Figure 3 shows the neuroprotective efficacy of four ERp-selective phytoestrogenic molecules when administered alone at concentrations that 15 elicited the maximal neuroprotective effects as revealed from the dose response analyses (100 nM for allfour molecules), or co-administered, against supraphysiological glutamate (100 pM)-induced neurotoxicity in primary hippocampal neurons by measurement of calcein AM staining. Results are presented in terms of neuroprotective efficacy 20 NE = (Vteatment - Vgiunate) / (Vcoonoi - Vgautwae) * 100%, (1) where Vtieatment is the individual value from phytoestrogen-treated cultures, Vsumate is a mean value from glutamate alone-treated cultures, and Veot,, is a mean value from vehicle-treated control cultures. * P <0.01 compared to 25 vehicle alone-treated cultures; * P <0.05 and **P <0.01 compared to glutamate alone-treated cultures. Data presented in Figure 3 and Table 3 demonstrate that although the four ERp--selective phytoestrogenic molecules, when administered individually, are concentration-dependent and are protective against 30 excitotoxic glutamate-induced neurotoxicity in primary neurons, these effects are moderate and arise from the weaker binding to the estrogen receptor compared to the endogenous estrogen 17p-estradiol (E2). Figure 3 demonstrates that co-administration of 3 or 4 of these phytoestrogens afforded much greater neuroprotective efficacy compared to administration 35 of single phytoestrogens or a combination of 2 phytoestrogens. 40 Expression of Anti-Apoptotic Proteins Bcl-2 and Bcl-xL These outcomes are paralleled by the results derived from the western analyses of the expression of anti-apoptotic proteins, Bcl-2 and Bcl-xL, in primary neurons. Figures 4A-4B shows the effects on Bel-2 and Bcl-XL 5 expression in rat primary hippocampal neurons and hippocampal tissues derived from adult ovariectomized rats. Primary hippocampal neurons grown for 7 divisions were treated with the test compounds (or combinations) for 48 hr followed by Western blot analyses. Adult ovariectomized rats were given, once daily, 2 subcutaneous injections of the 10 test compounds (or combinations). Rats were sacrificed 24 h later following the 2nd injection. Hippocampal tissues were homogenized followed by Western blot analyses. Combined formulations were composed of equivalent molar in (A) and equivalent weight in (B) of individual phytoSERMs including G: genistein; D: daidzein; E: equol; and I: IBS003569. 15 Incubation of neurons with a combination of four phytoestrogens for 48 hours induces a significantly increased expression of both proteins comparable to those induced by E2. This is illustrated in Figure 4, which shows the effect of four ERP-selective phytoestrogenic molecules when co administered (100 nM for all four molecules) on the expression of the anti 20 apoptotic proteins, Bcl-2 and Bcl-xL, in primary hippocampal neurons. **P <0.01 compared to vehicle alone-treated cultures. By comparison, a combination of two phytoestrogens is not sufficient to induce a significant increase in the expression of both proteins, as also illustrated in Figures 4A and 4B. 25 Up-regulation of the Bel-2 family anti-apoptotic proteins have been associated with the neuroprotective mechanism elicited by E2. These data indicate that a combined used of multiple ERp-selective phytoestrogens is effective to activate the neuroprotective mechanism leading to improved neuronal survival against neurodegenerative insults. Estrogen receptor 30 interaction with p851PI3K also enhances pAkt, which phosphorylates the proapoptotic protein Bcl-2-associated death protein (BAD) to prevent heterodimerization with, and inactivation of, Bcl-2 . In cortical neurons, 41 estradiol induced pAkt translocation to the nucleus. Recent analyses indicate that estradiol, via the P13K signaling pathway, activates both the Akt and the ERK1/2 cascades in the same population of cortical and hippocampal neurons. Simultaneous activation of two pathways that prevent mitochondria 5 from activating cell-death cascades is likely to promote neuron survival. Increased Expression of the anti-p-amyloid proteinIDE Figure 5 illustrates the effect of four ERD-selective phytoestrogenic molecules when co-administered (100 nM for all four molecules) on the expression of the anti-p-amyloid protein, insulin-degrading enzyme ("IDE") 10 in primary hippocampal neurons. **P < 0.01 compared to vehicle alone treated cultures. Figure 5 demonstrates the effects of these various combinations of phytoestrogens along with E2 on the expression of the anti 0-amyloid (anti-Ap) protein, insulin-degrading enzyme (IDE) in primary neurons. Data showed that all three combinations composed of two, three or 15 four phytoestrogens significantly increases IDE expression in neurons. Among them, a combination of three phytoestrogens induced the greatest neuronal response, with an efficacy greater than E2 as well as a combination of two phytoestrogens. It is clear that one neuropathological hallmark of AD is a significant 20 deposition of extracellular AP peptide, as referred to As plaque. Impaired AD clearance and/or degradation has been demonstrated to contribute in part to As plaque formation in AD brain. Besides degrading insulin and several regulatory peptides, IDE, a metalloprotease enzyme, has been demonstrated to play a key role in degrading As peptide monomer in the brain. Choronic 25 upregulation of IDE represents a novel efficacious therapeutic approach to lowering the steady-state As level in the brain and eventually preventing the occurrence of Alzheimer-type pathology. Therefore, these data indicate that coadministration of multiple ERD-selective phytoestrogens have the potential to activate the anti-Ap mechanism, and as a result, maintain the brain in a 30 long-term healthy status. 42 Upregulation of Spinophilin Figure 6 illustrates the effect of four ERs.-selective phytoestrogenic molecules when co-administered (100 nM for all four molecules) on the expression of the spine marker, spinophilin, in primary hippocampal 5 neurons. **P <0.01 compared to vehicle alone-treated cultures. Spinophilin, a protein that is enriched in the heads of neuronal dendritic spines, has been demonstrated to play a significant role in modulating both dendritic morphology and glutamatergic synaptic activity. Upregulation of spinophilin has been correlated with estrogen regulation of neuronal synaptic 10 plasticity. Therefore, theses results indicate that these phytoestrogen combinations are effective to promote neurotrophism, thereby sustaining the brain staying in a synaptically active status, and prevent cognitive decline and memory loss. Neuroprotection against Glutamate 15 Figures 7A-7D shows the neuroprotective efficacy of the compounds against glutamate (Figure 7A) and amyloid 1 -4 2 -induced neurotoxicity in rat primary hippocampal neurons. Primary hippocampal neurons grown for 7 divisions were pretreated with the test compounds (or combinations) for 48 h, followed by a 5-min exposure to 100 mM glutamate. Neurons were 20 incubated for an additional 24 h prior to neuronal viability analyses by calcein AM staining. Following pretreatment with the compounds (or combinations) for 48 hr, neurons were exposed to 3 mM p-amyloidt.42 for 2 d. Neuronal viability was analyzed by fluorometric measurements of activities of the LDH and dead-cell protease released in the culture media, 25 and the live-cell protease exclusively entering intact viable neurons. Results are presented as neuroprotective efficacy (NE), which is defined as the percentage of neurotoxin-induced toxicity prevented by the test compounds (or combinations) and quantitated by the equation: NE = (Vcatment Vneuwtoxta) / (Veontroi - Vnewrotox-m) * 100% 30 where Vteatment is the individual value from the test compounds (or combinations)-treated cultures, Vmotxin is a mean value from glutamate or P-amyloid 14 2 alone-treated cultures, and Veentci is a mean value from 43 vehicle-treated control cultures. "P < 0.01 compared to vehicle-treated control cultures; *P <0.05 and **P <0.01 compared to glutamate or p amyloid1 4 2 alone treated cultures; 8 P <0.05 compared to E2-treated cultures; CP <0.05 compared to genistein-treated cultures; "'P <0.05 and "P <0.01 5 compared to combination (G+D)-treated cultures; "P <0.05 compared to combination (G+D+E+I)-treated cultures. Combined formulations were composed of equivalent molar of individual phytoSERMs included. G: genistein; D: daidzein; E: equol; I: IBS003569. Effect on IDE/NEP Exporession 10 Figures 8A-8C show the effects on insulin-degrading enzyme (IDE)/neprilysin (NEP) expression in (A) rat primary hippocampal neurons and (B) hippocampal tissues derived from adult ovariectomized rats. (A) Primary hippocampal neurons grown for 7 DIV were treated with the test compounds (or combinations) for 48 hr followed by Western blot analyses. 15 (B) Adult ovariectomized rats were given, once daily, 2 subcutaneous injections of the test compounds (or combinations). Rats were sacrificed 24 h later following the second injection. Hippocampal tissues were homogenized followed by western blot analyses. Results are presented as the fold increase in protein expression and expressed as the percent of control, n 2 4. *P < 20 0.05 and **P <0.01 compared to vehicle-treated control cultures or animals. 'P <0.05 and "P <0.01 compared to E2-treated cultures; "'P <0.01 compared to combination (G+D) or genisteintreated cultures; *P <0.05 compared to combination (G+D+E+l)-treated cultures. Combined formulations were composed of equivalent molar in (A) and equivalent 25 weight in (B) of individual phytoSERMs including G: genistein; D: daidzein; E: equol; and I: IBSO03569. Effect on Forebrain Mitochondria Figures 9A-9E show the effects on forebrain mitochondrial cytochrome c oxidase (COX) activity in adult ovariectomized rats. Rats 30 were given, once daily, 2 subcutaneous injections of the test compounds (or combinations). Rats were sacrificed 24 h later following the 2nd injection. Forebrain mitochondria were isolated followed by a spectrophotometric 44 measurement of COX activity using an immunocapture method. Colorimetric absorbance at 550 nm was recorded every 5 min for 115 min. COX activity is presented as the initial rate of oxidation of reduced cytochrome c, and determined by calculating the initial slope between two time points (<20 5 min) within the linear region. (Upper Panel) Time-lapse change in absorbance; (Lower Panel) % increase in mitochondrial COX activity, n 4; *P < 0.05 and **P <0.01 compared to vehicle-treated control animals; VP < 0.05 compared to genistein-treated animals. Combined formulations were composed of equivalent weight of individual phytoSERMs including E2: 10 17bestradiol; G: genistein; D: daidzein; E: equol; I: 1BS003569. Figures 1OA-OE show the effects on forebrain mitochondrial respiratory activity in adult ovariectomized rats. Rats were treated as above. Forebrain mitochondria were isolated followed immediately by a polygraphical measurement of respiratory activity using an oxygen electrode. 15 Following a basal recording, mitochondrial state 4 respiration was measured following the addition of substrates, malate/glutamate. State 3 respiration was measured following the addition of ADP. Respiratory control ratio (RCR) was calculated as the ratio between the rate of oxygen uptake at state 3 and the rate of oxygen uptake at state 4. (Figures 12A-12D) Time-lapse 20 oxygen uptake; (Figure 12E) % increase in mitochondrial respiratory activity, n 4; *P < 0.05 and **P <0.01 compared to vehicle-treated control animals; VP < 0.05 compared to genistein-treated animals. Combined formulations were composed of equivalent weight of individual phytoSERMs including E2: 17b-estradiol; G: genistein; D: daidzein; E: 25 equol; and I: IBSO03569; Mito: mitochondria; Mal/Glut: malate/glutamate. Effect on Uterine Weight Table 4 shows the effects on uterine weight in adult ovariectomized rats. Changes in uterine weight in response to estrogenic stimulation can be used to evaluate the estrogenic characteristics of test compounds on uterine 30 tissues. In one example, described below, immature female rats having low endogenous levels of estrogen are dosed with test compound (subcutaneously) daily for 3 days. Compounds are formulated as appropriate 45 for subcutaneous injection. As a control, 17-P estradiol is administered alone to one dose group. Vehicle control dose groups are also included in the study. Twenty-four hours after the last treatment, the animals are necropsied, and their uteri excised, nicked, blotted and weighed. Any statistically 5 significant increases in uterine weight in a particular dose group as compared to the vehicle control group demonstrate evidence of estrogenicity. Table 4. Effects on uterine weight in adult ovariectomized ratsA Treatment Uterine Weight Wet Weight (mg) lumase (%)f Dry Weight (mg) Increase (%) Control 127.62 ±10.75 0.00 : 8.42 26.42 ± 2.45 0.00 ±9.27 vehicle) 17-Estradiol 281.06t 32.00 *' 120.23±25.07 **P 46.70 14.13 ** 76.74 ± 15.63 **D (70 Pg&g BW) Genistein 144.11± 10.18 12.92±7.97 28.14 ±2.04 6.49±7.71 (6 mg/kg BW) G+D+E 119.84±1.19 -6.10 t 0.93 23.71 ±0.04 -10.26 ±0.13 (6 mg/kg BW)c G+DoE+I 146.99± 18.45 15.17 t 14.46 28.73 ± 3.67 8.73 ± 13.90 (6 m~g DVf AAdult ovariectomized rats were given, daily once, 2 subcutaneous injections of the test 10 compounds (or combinations) (n > 4 for each group). Rats were sacrificed 24 h later following the 2nd injection. Uteri were immediately excised and a wet weight was recorded. Uteri were then air dried for I hour followed by at 70 *C overnight, and the dry weight was recorded. 15 BIncrease in uterine weight compared with vehicle-treated control animals and expressed as the percent of control (set as 0). cCombined formulations were composed of equivalent weight of individual phytoSERMs included for a total amount of 6 mg/kg BW given to animals. G: genistein; D: daidzein; B: equol; I: IBS003569. 20 ***P < 0.01 compared to any other treatment groups. Summary Both in vitro and in vivo analyses demonstrated that combined use of select test phytoSERMs provided significantly increased efficacy in sustaining neuronal survival when challenged with neurotoxins, promoting 25 expression of proteins as key players in neuroprotection and metabolism/clearance of p-amyloid in neurons/brain, and enhancing brain mitochondrial functions. In particular, combined use of genistein, daidzein and equol at an equivalent weight afforded the maximal efficacy comparable 46 or greater than 17b-estradiol in neuronal/brain assays. In contrast, such a combination showed no impact on uterine weight, which however was markedly increased by 17b-estradiol. The present study indicates that combined use of select ERp-selective 5 PhytoSERMs can be more therapeutically effective than single administrations and alternative combined formulations. In particular, the present study suggests the potential of the combination of genistein, daidzein and equol, at an equivalent weight, for prevention of neurodegeneration and AD, along with management of climacteric symptoms in postmenopausal 10 women. Figures 11A-I IC are schematics showing estrogen mechanisms of action that lead to neurotrophic and neuroprotective outcomes. 17-$ Estradiol (E2) acting via a membrane-associated site (mER) activates a cascade required for multiple responses that lead to enhanced neural 15 plasticity, morphogenesis, neurogenesis, and neural survival. The signaling sequence induced by E2 at the membrane site is as follows: (1) E2 binding to mER, (2) E2-mER complexes with p85 to activate P13K, (3) activating calcium-independent PKC, (4) phosphorylating the L-type calcium channel, (5) inducing calcium influx, (6) activating calcium-dependent PKCs, (7) 20 activating Src kinase, (8) activating the MEK/ERK1/2 pathway, (9) ERK translocates to the nucleus, (10) activating and phosphorylating CREB, (11) enhancing transcription of antiapoptotic genes Bcl-2 and Bcl-xl, which enhance mitochondrial vitality, and spinophilin, which encourages synaptic growth, (12) simultaneously, estrogen activation of P13K leads to activation 25 of Akt, which phosphorylates and inhibits the proapoptotic protein BAD. Estrogen-induced neuroprotective mechanisms converge on mitochondria. Estrogen-activated cellular signaling cascade promotes enhanced mitochondrial function, leading to increased calcium load tolerance, enhanced electron transport chain efficiency, and promotion of 30 antioxidant defense mechanisms. These actions are mediated by the regulation of both nuclear and mitochondrial encoded genes initiated by the activation of second-messenger signaling cascades. 47 These mechanisms and the data herein demonstrate that, consistent with the healthy cell bias of estrogen benefit hypothesis, selective molecules can be administered before neurodegenerative insult while neurons are still healthy and that phytoSERM exposure will lead to enhanced neural survival 5 mechanisms, represented as mitochondria with Bcl-2 additions, that promote neural defense against neurodegenerative insults associated with age associated diseases such as Alzheimer's and Parkinson's. These studies exemplify the therapeutic promise of select ERD selective phytoestrogens when used in combination for sustaining memory 10 function and preventing age-related neurodegenerative insults and AD. These ERD-selective phytoestrogen formulations, which optimize activation of ERP3 while minimizing or avoiding activating ERot, should serve as an effective estrogen alternative replacement therapy for sustaining neurological health, function and prevention of AD without induction of proliferative 15 responses in the reproductive tissues as seen with the current ET/HT. Moreover, in light of the most recent data indicating that activation of ERs significantly reduces both ApoE mRNA and protein expression in neurons, ERp-selective phtoestrogen formulations may serve as a particular viable strategy for reducing a major risk factor of AD in ApoE4 carriers. 48

Claims (14)

1. A formulation for administration to an individual in need of treatment or prevention of neurological disorders or aging comprising two or more naturally occurring compounds that selectively preferentially bind to estrogen receptor p, which cross the blood brain barrier.

2. The formulation of claim 1, wherein the two or more naturally occurring compounds are selected from the group consisting of genistein, daidzein, equol, IBS003569, and combinations thereof.

3. The formulation of claim 1, further comprising one or more excipients or carriers.

4. The formulation of claim 1, further comprising one or more additional active agents selected from the group consisting of other selective estrogen receptor modulators, estrogen agonists, estrogen antagonists, antineoplastic agents, alkylating agents, antibiotics, hormones, antimetablites, anti-osteoporosis drugs, vitamins, nutritional supplements, antioxidants and coenzymes.

5. The formulation of claim 1 formulated for enteral, parenteral, or topical administration.

6. A method for alleviating or preventing one or more symptoms associated with estrogen deficiency comprising administering to the patient an effective amount of the formulation of any of claims 1-5.

7. The method of Claim 6, wherein the patient is a postmenopausal women.

8. The method of Claim 6, wherein the estrogen-deficiency associated risks are a neurological disease or disorder.

9. The method of Claim 8, wherein the neurological disease is a neurodegenerative disease.

10. The method of Claim 9, wherein the neurodegenerative disease is cognitive deficit or memory loss.

11. The method of Claim 9, wherein the neurodegenerative disease is Alzheimer's disease. 49

12. The method of claim 6 further to treat or prevent hot flashes comprising administering to a subject in need thereof an effective amount of the formulation of claim 1.

13. The method of claim 6 to treat or prevent cognitive decline associated with estrogen deficiency comprising administering to a subject in need thereof an effective amount of the formulation of claim 1.

14. The method of claim 6 to treat or prevent osteoporosis associated with estrogen deficiency comprising administering to a subject in need thereof an effective amount of the formulation of claim 1. 50