US 2012O1641.22A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2012/0164.122 A1 Brinton et al. (43) Pub. Date: Jun. 28, 2012

(54) PHYTOESTROGENIC FORMULATIONS FOR A6IP 9/10 (2006.01) ALLEVATION OR PREVENTION OF A6IP35/00 (2006.01) NEURODEGENERATIVE DISEASES A6IP3L/00 (2006.01) A6IP3/02 (2006.01) (76) Inventors: Roberta Diaz, Brinton, Rancho A6II 45/00 (2006.01) Palos Verdes, CA (US); Liqin A6IP 25/28 (2006.01) Zhao, Los Angeles, CA (US) (52) U.S. Cl...... 424/94.1: 514/456 (21) Appl. No.: 13/362,825 (57) ABSTRACT (22) Filed: Jan. 31, 2012 Select phytoestrogen pharmaceutical compositions and methods of use for promoting neurological heath and preven Related U.S. Application Data tion of age-related neurodegeneration, Such as AD, have been - - - developed. These select phytoestrogen formulations are com (62) Division of application No. 1 1/777,951, filed on Jul. posed of a number of plant-derived estrogenic molecules 13, 2007. and/or their structural analogues and exhibit binding prefer (60) Provisional application No. 60/819,849, filed on Aug. ence to ERB over ERC. and agonist activity in the brain. These ERB-selective phytoestrogen formulations cross the blood 2, 2006, provisional application No. 60/889,920, filed brain-barrier and promote estrogen-associated neurotro on Feb. 14, 2007, provisional application No. 60/943, phism and neuroprotections mechanisms in the brain, without 190, filed on Jun. 11, 2007. activating proliferative mechanisms in the reproductive tis O O Sues and are therefore devoid of other estrogen-associated Publication Classification problematic aspects. These are administered enterally, trans (51) Int. Cl. dermally, transmucosally, intranasally or parenterally, in a A6 IK3I/353 (2006.01) dosage effective to prevent or alleviate neuronal damage, A6IP 5/30 (2006.01) effect neuronal regeneration or Sustain viability, increase A6IP 25/00 (2006.01) expression of anti-apoptotic proteins, and/or decrease indica A6IP39/06 (2006.01) tors of Alzheimer's Disease.

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PHYTOESTROGENC FORMULATIONS FOR negative results of the Women's Health Initiative Memory ALLEVATION OR PREVENTION OF Study (“WHIMS) trial was the advanced age, more than ten NEURODEGENERATIVE DISEASES years following menopause, at which ET/HT was initiated in women. Data from both basic Science analyses and clinical CROSS-REFERENCE TO RELATED studies indicate a “healthy cell bias” of estrogen action in the APPLICATIONS neurons/brains, Suggesting that ET/HT acts as an effective 0001. This application claims priority to U.S. Ser. No. preventative therapeutic strategy for age-related cognitive 60/819,849 mailed by express mail label no. ER 45595.9795 decline and neurodegenerative disorders, such as Alzheimer's US on Aug. 1, 2006: U.S. Ser. No. 60/889,920 filed Feb. 14, disease (AD), while it is not an effective treatment strategy. 2007, and U.S. Ser. No. 60/943,190 filed Jun. 11, 2007. The 0006. The current widely prescribed ET, conjugated disclosure(s) in the application(s) listed above are herein equine estrogens (“CEE), is a highly complex ET with over incorporated by reference. 200 different components. Whether CEE provides the opti mal therapeutic efficacy has been questioned. Another key STATEMENT REGARDING FEDERALLY issue challenging HT is the optimal composition. The proges FUNDED RESEARCH tin and its timing of administration in conjunction with ET, remains to be determined. Moreover, while ET/HT has long 0002 This work was supported by National Institute of been used in postmenopausal women to delay or reverse some Mental Health Intramural Research Program (P.J.S.) and of the problems associated with menopause, epidemiological Grant MH67159 (R.D.B.), National Institute of Aging Grants and clinical studies have uncovered potential long-term risks AG06647 (J.H.M.), AG16765 (J.H.M., A.C.G.), and related to this therapy. The recently revealed risks associated AG14751 and AG026572 (R.D.B.), and the Kenneth T. and with ET/HT have greatly increased interest in the develop Eileen L. Norris Foundation (R.D.B.). ment of estrogen alternatives that promote beneficial effects of estrogen in brain, bone and the cardiovascular system, BACKGROUND OF THE INVENTION while not eliciting deleterious effects in other organs, particu 0003. The demographics suggest that we face a devastat larly in breast and uterine tissues. ing increase in the prevalence of AD, reinforcing the imme 0007 Two nuclear receptors forestrogen (ERs), ERC. and diate need for basic and translational neuroscience to develop ERB, have been identified. In the central nervous system, both safe and efficacious ET and HT regimens for the brain. Of ERC. and ERB are expressed in the hippocampus and cortex of those affected with AD, 68% are female and 32% are male rodent and human brains. Previous studies have demonstrated (Brookmeyer et al., 1998 Am J Public Health 88:13372). that both ERC. and ERB can equivalently promote neuronal Because women have a longer life expectancy than men, the Survival by activating estrogen mechanisms of action in rat absolute number of women with AD exceeds that of men. hippocampal neurons. Increasing evidence indicates that However, a double danger exists for women. Results of a ERB is a key requirement for activation of mechanisms that meta-analysis of seven sex-specific studies concluded that underlie estrogen-inducible neuronal morphological plastic women are 1.5 times more likely to develop AD than age ity, brain development, and cognition. ERO, on the other matched men (Gao et al., 1998 Arch Gen Psychiatry 55:809), hand, is more predominant in mediating the sexual character which was supported by the Cache County analysis that istics of estrogen effects in the reproductive organs such as showed a clear female gender increase in the incidence of AD breast and uterus. Taken together, these data establisha poten (Zandi et al., 2002 JAMA 288:21239). tial therapeutic application for ERB as a pharmacological 0004 At the turn of the new millennium in the United target to promote memory function and neuronal defense States, there were nearly 42 million women over the age of 50 mechanisms against age-related neurodegeneration Such as years and, of these, more than 31 million women were over Alzheimer's disease (AD), while avoiding activating unto the age of 55 years (North American Menopause Society, wardestrogenic proliferative effects in the breast and uterus, 2004). Worldwide, there are currently more than 470 million although this might be at the cost of lower efficacy due to the womenaged 50 years or older, and 30% of those are projected lack of activation of ERB in the brain. Other potential thera to live into their 80s (North American Menopause Society, peutic advantages associated with ERB include regulation of 2004). These women can anticipate spending one-third to estrogen vasculoprotective action and development of inter one-half of their lifetime in the menopausal state. Reports on ventions targeting diseases Such as depression, colon cancer, prevalence of AD vary, but of the 18 million American women prostate cancer, obesity, leukemia, and infertility. However, a in their mid to late 70s, as many as 5 million may suffer from potential disadvantage of an ERB-selective ligand is the lack AD, and this figure increases dramatically at older ages of activation of ERC. in bone, as ERC. has been demonstrated (Brookmeyer et al., 1998). The projected exponential to mediate estrogen regulation of bone density. increase in the prevalence of AD, along with the anticipated 0008 Although there is still controversy regarding the impact on families and Society, highlights the imperative for differential roles of two estrogen receptor (“ER”) subtypes, developing strategies to prevent or delay the onset of AD ERC. and/or ERB, in mediating estrogen actions in the brain Sooner rather than later. and/or neurons, it has been widely demonstrated that ERB 0005. The profound disparities between the largely posi plays a key role in regulating brain development, neurogen tive basic Science findings of gonadal steroidal action in brain esis and estrogen-induced improved neuronal plasticity and and the adverse outcomes of recent estrogen or hormone survival. In addition, as compared with ERO, ERB is less therapy (“ET/HT”) clinical trials in women who are either effective in mediating the sexual characteristics of estrogen aged postmenopausal or postmenopausal with Alzheimer's action in reproductive tissues, avoiding activating untoward disease (AD), has led to an intense reassessment of gonadal estrogenic proliferative effects in the breast and uterus. hormone action and the model systems used in basic and Therefore, ERB represents a potentially safer therapeutic tar clinical science. One key factor that could contribute to the get for promoting memory function and neuroprotection. US 2012/0164122 A1 Jun. 28, 2012

However, this safety may be at the cost of lower efficacy, due on this analysis, a presumption can be made that the ineffec to the lack of activation of Ma in the brain. Other potential tiveness of administering a mixture of phytoestrogens (i.e. a advantages for ERB-target therapeutics arise from its regula soy protein Supplement) may partly come from the antago tion of estrogen's cardioprotective effects. ERB-selective nizing actions among different phytoestrogens, which may be ligands may also provide effective therapeutics for preventing ERC. selective or ERB selective. These findings indicate that or treating inflammation, depression, anxiety, colon cancer, although both ERC. and ERB contribute to estrogen promo prostate cancer, obesity, leukaemia, and infertility. tion of neuronal survival, simultaneous activation of both ER 0009. In searching for an effective ERB-selective estrogen subtypes, ERC. and ERB, in the same context may diminish alternative replacement therapy for promoting neurological the efficacy. In addition, the different ratio and distinct func function and preventing age-related neurodegeneration, Such tion of homodimer and heterodimer induced by co-adminis as AD, in postmenopausal women, it is of particularinterest to tration of an ERC.-selective agonist and an ERB-selective identify and develop naturally occurring molecules or ana agonist may also account for the reduced efficacy exerted by logues that potentially have a less toxic profile for long-term the combination of both agonists. administration. It is known that several plant-derived estro 0013 Development of an ERB-selective phytoestrogen genic molecules (referred to as “phytoestrogens') bind to formulation could maximize the therapeutic benefits associ ERC. and to ERB subtypes, and some of these molecules ated with activation of ERB in the brain while minimizing the possess moderate binding selectivity for ERB and exert estro adverse effects associated with the activation of ERC. in repro genic effects in multiple tissues. ductive tissues. Moreover, selective targeting of ERB poten 0010. The therapeutic efficacy of phytoestrogens in the tially reduces antagonistic actions that may occur in a com brain remains controversial. On the one hand, when admin plex Soy-derived preparation. This naturally occurring ideal istered singly, phytoestrogens appeared to be moderately neu formulation would have tremendous therapeutic value in pro roprotective. On the other hand, a recent clinical trial revealed moting neurological function and preventing AD in a popu that a soy protein Supplement that contains a mixture of lation at risk for losing neurological capacity and losing phytoestrogens did not show improved cognitive function in memory function, i.e., postmenopausal women. To date, no postmenopausal women, when treatment was initiated at the Such phytoestrogen formulation exists. Thus, there is a need age of 60 years or older. The clinical trial of phytoestrogens to discover and develop a novel select phytoestrogen formu reported that a soy protein Supplement containing a complex lation, generally, and particularly, a formulation that func formulation of isoflavones did not improve cognitive function tions in the brain. in postmenopausal women when treated at the age of 60 years 0014. It is therefore an object of the present invention to or older, Kreijkamp-Kaspers, et al. JAMA 2004, 292, 65-74, provide an ERB-selective phytoestrogen formulation maxi also indicating that when started 10 or more years following mizing the therapeutic benefits associated with activation of menopause in postmenopausal women when age-related neu ERB in the brain while minimizing the adverse effects asso ronal reorganization has taken place, ET/HT has no benefit on ciated with the activation of ERC. in reproductive tissues. neural function. Age and hormonal “history' may be impor 0015. It is a further object of the invention to provide such tant factors that were responsible for these negative results, as a composition wherein the active ingredients are isolated was the case for the WHIMS trials. from natural Substances. 0011. Another issue that can substantially impact the effi cacy of a mixture of phytoestrogens action in the brain is the SUMMARY OF THE INVENTION formulation of phytoestrogens, since when administered alone, a number of phytoestrogens were protective to neurons 0016 Select phytoestrogen pharmaceutical compositions from neurodegenerative insults. Zhao, et al. Exp. Biol. Med. and methods of use for promoting neurological health and 2002, 227, 509-519. Soy extracts or soy protein supplements prevention of age-related neurodegeneration, Such as AD, generally contain multiple phytoestrogenic molecules, some have been developed. These select phytoestrogen formula of which may be ERC.-selective agonists, while others may be tions are composed of a number of plant-derived estrogenic ERB-selective agonists, and others may be ineffective in acti molecules and/or their structural analogues and exhibit bind vating either ERC. or ERB but may function as inhibitors of ing preference to ERB over ERC. and agonist activity in the ER binding of those ERC. and/or ERB phytoestrogenic ago brain. These ERB-selective phytoestrogen formulations cross nists. The ineffectiveness of a complex formulation of phy the blood-brain-barrier and promote estrogen-associated toestrogens in promoting beneficial effects of estrogen in neurotrophism and neuroprotection mechanisms in the brain, brain, such as a Soy-derived preparation, may also arise from without activating proliferative mechanisms in the reproduc antagonizing actions among the different phytoestrogens, in tive tissues and are therefore devoid of other estrogen-asso addition to the possible ER antagonism, likely from the acti ciated problematic aspects. The select phytoestrogen formu vation of both ERC. and ERR in the same context. Co-admin lations are therapeutically useful to both women and men for istration of an ERC-selective agonist and an ERB-selective Sustaining neurological health and preventing age-related agonist is less effective than treatment with either agonist cognitive decline and neurodegenerative disorders, such as alone in various neuroprotective measurements. AD. 0012 ERC. and ERB have a yin/yang relationship in many 0017. These are administered enterally, transdermally, contexts where one receptor may antagonize the actions of the transmucosally, intranasally or parenterally, in a dosage other. Weihua, et al. FEESLett. 2003, 546, 17-24: Gustafsson, effective to prevent oralleviate neuronal damage, effect neu J. A. Trends Pharmacol. Sci. 2003, 24, 479-485. Studies ronal regeneration or Sustain viability, increase expression of confirmed this observation, showing that coadministration of anti-apoptotic proteins, and/or decrease indicators of Alzhe ERC.-selective agonist PPT and ERB-selective agonist DPN imer's Disease. The formulations preferably contain combi was less efficacious than either PPT or DPN alone in protect nations of compounds, and can be formulated for daily, Sus ing hippocampal neurons against excitotoxic insults. Based tained, delayed or weekly/monthly administration. In a US 2012/0164122 A1 Jun. 28, 2012

preferred embodiment, these are administered to women who PKCS, (7) activating Src kinase, (8) activating the MEK/ are in menopause or post menopausal, most preferably early ERK 1/2 pathway, (9) ERK translocates to the nucleus, (10) in menopausal. activating and phosphorylating CREB, (11) enhancing tran Scription of antiapoptotic genes Bcl-2 and Bcl-X1, which BRIEF DESCRIPTION OF THE DRAWINGS enhance mitochondrial vitality, and spinophilin, which 0018 FIG. 1 shows the chemical Structures of 173-estra encourages synaptic growth, (12) simultaneously, estrogen diol and the phytoSERMs genistein, daidzein, equol, and activation of PI3K leads to activation of Akt, which phospho IBSOO3569. rylates and inhibits the proapoptotic protein BAD. 11B, 0019 FIGS. 2A and 2B show the competition binding Estrogen-induced neuroprotective mechanisms converge on curves for ERC. and ERB (molar concentration versus fluo mitochondria. Estrogen-activated cellular signaling cascade rescence polarization (mP)) of G, D, E, I or combinations: promotes enhanced mitochondrial function, leading to G+D, G+D+E, or G+D+E+I. increased calcium load tolerance, enhanced electron trans 0020 FIG. 3 is a graph showing the neuroprotective effi port chain efficiency, and promotion of antioxidant defense cacy of four ERB-selective phytoestrogenic molecules when mechanisms. These actions are mediated by the regulation of administered alone at concentrations that elicited the maxi both nuclear and mitochondrial encoded genes initiated by mal neuroprotective effects as revealed from the dose-re the activation of second-messenger signaling cascades. 11C, sponse analyses (100 nM for all four molecules Genistein Conceptual schematic of NeuroSERM design and therapeu (G), Daidzein (D), Equol (E) and IBSO03569 (I)), or co tic use. Consistent with the healthy cell bias of estrogen administered: G+D, G+D+E, or G+D+E+I, against Supra benefit hypothesis, selective molecules would be adminis physiological glutamate (100LM)-induced neurotoxicity pri tered before neurodegenerative insult while neurons are still mary hippocampal neurons by measurement of calcein AM healthy. NeuroSERM exposure would lead to enhanced neu staining. ral Survival mechanisms, represented as mitochondria with 0021 FIGS. 4A and 4B are graphs showing the effect of Bcl-2 additions, that promote neural defense against neuro four ERB-selective phytoestrogenic molecules when co-ad degenerative insults associated with age-associated diseases ministered (100 nM) for all four molecules) as G+D, G+D+E, such as Alzheimer's and Parkinson's. Designer NeuroSERM or G+D+E+I, on the expression of the anti-apoptotic proteins, molecules target the membrane site of estrogen action, Bcl-2 and Bcl-XL, in primary hippocampal neurons. whereas PhytoSERM molecules preferentially target estro 0022 FIG. 5 is a graph illustrating the effect of four ERB gen receptor?. Abbreviations: AMPAR, AMPA receptor; C, selective phytoestrogenic molecules when co-administered cytochrome oxidase; Fo, F, ATPase subunits; LTD, long (100 nM for all four molecules), G+D, G+D+E, or G+D+E+I, term depression; LTP. long-term potentiation; NAD, nicoti on the expression of the anti-B-amyloid protein, insulin-de namide adenine dinucleotide; NADH, nicotinamide adenine grading enzyme ("IDE'), in primary hippocampal neurons. dinucleotide; VDCC, Voltage-dependent calcium channel. 0023 FIG. 6 is a graph illustrating the effect of four ERB selective phytoestrogenic molecules when co-administered DETAILED DESCRIPTION OF THE INVENTION (100 nM for all four molecules): G+D, G+D+E, or G+D+E+I, I. Definitions on the expression of the spine marker, spinophilin, in primary hippocampal neurons. 0029. “Estrogen Receptor, as used herein, refers to any 0024 FIGS. 7A-7D are graphs shows the neuroprotective protein in the nuclear receptor gene family that binds estro efficacy of G, D, E, and I, alone and in combination: G+D. gen, including, but not limited to, any isoforms and variants G+D+E, or G+D+E+I, against (7A) glutamate- and (7B) thereof. Human estrogen receptors include the alpha- and B-amyloid1-42-induced neurotoxicity in rat primary hippoc beta-isoforms (referred to herein as “ERC and “ERB'). ampal neurons, controls live/dead cells (7C); dead cells (7D). 0030 “Estrogen Receptor Modulator, as used herein, 0025 FIGS. 8A-8C are graphs showing the effects of G, refers to a compound that can act as an estrogen receptor D, E, and I, alone and in combination: G+D, G+D+E, and agonist or antagonist of an estrogen receptor or estrogen G+D+E+I, on insulin-degrading enzyme (IDE) expression on receptor isoform having an ICso or ECso with respect to ERC. neprilysin (NEP) expression in hippocampal tissues derived ERB and/or other estrogen receptor isoforms of no more than from adult ovariectomized rats. about 50 uMas determined using the ERC, and/or ERB trans 0026 FIGS. 9A-9E are graphs showing the effects of G, activation assay described herein. More typically, estrogen G+D+E, and G+D+E+I on forebrain mitochondrial cyto receptor modulators have ICso or ECso values (as agonists or chrome c oxidase (COX) activity in adult ovariectomized rats. antagonists) of not more than about 10 uM. Representative 0027 FIGS. 10A-10E are graphs showing the effects of G, compounds are predicted to exhibit agonist or antagonist G+D+E, and G+D+E+I on percent increase forebrain mito activity via an estrogen receptor. Compounds preferably chondrial respiratory activity in adult ovariectomized rats. exhibit an antagonist or agonist ICs or ECs with respect to 0028 FIGS. 11A-11C are schematics showing estrogen ERC. and/or ERB of about 10 uM, more preferably, about 500 mechanisms of action that lead to neurotrophic and neuro nM, even more preferably about 1 nM, and most preferably, protective outcomes. 11A, 17-B-Estradiol (E2) acting via a about 500 pM, as measured in the ERC. and/or ERB transac membrane-associated site (mER) activates a cascade required tivation assays. “ICso is that concentration of compound for multiple responses that lead to enhanced neural plasticity, which reduces or inhibits the activity of a target (e.g., ERC. or morphogenesis, neurogenesis, and neural Survival. The sig ERB) to half-maximal level. “ECs” is that concentration of naling sequence induced by E2 at the membrane site is as compound which provides half-maximum effect. follows: (1) E2 binding to mER, (2) E2-mER complexes with 0031) “Selective Estrogen Receptor Modulator” (or p85 to activate PI3K, (3) activating calcium-independent “SERM), as used herein, refers to a compound that exhibits PKC, (4) phosphorylating the L-type calcium channel, (5) activity as an agonist or antagonist of an estrogen receptor inducing calcium influx, (6) activating calcium-dependent (e.g., ERO, ERB or other estrogen receptor isoform) in a US 2012/0164122 A1 Jun. 28, 2012 tissue-dependent or receptor dependent manner. Thus, as will bromides, and iodides; dialkylsulfates like dimethyl, diethyl, be apparent to those of skill in the biochemistry, molecular dibutyl, and diamylsulfates, long chainhalides such as decyl. biology and endocrinology arts, compounds that function as lauryl, myristyl and Stearyl chlorides, bromides and iodides, SERMs can act as estrogen receptoragonists in Some tissues, aralkyl halides like benzyl and phenethyl bromides, and oth e.g., bone, brain, and/or cardiovascular, and as antagonists in ers. Wafer or oil-soluble or dispersible products are thereby other tissue types, e.g., the breast and/or uterine tissue. obtained. 0032 “Phytoestrogen refers to a naturally occurring 0042 Examples of acids which may be employed to form compound of plants, such as soybeans, or plant products. Such pharmaceutically acceptable acid addition salts include Such as whole grain cereals, that acts like estrogen or binds to an inorganic acids as hydrochloric acid, Sulfuric acid, and phos estrogen receptor. phoric acid, and organic acids such as oxalic acid, maleic 0033. As used herein, the term “NeuroSERM' refers to acid, Succinic acid and citric acid. Basic addition salts can be compounds that target the membrane site of estrogen action. prepared in situ during the final isolation and purification of 0034. As used herein, the term “PhytoSERM' refers to the compounds, or separately by reacting carboxylic acid natural source compounds that preferentially target estrogen moieties with a suitable base such as the hydroxide, carbonate receptor beta. orbicarbonate of a pharmaceutically acceptable metal cation 0035. As used herein, the term “analogue' refers to a or with ammonia, oran organic primary, secondary or tertiary chemical compound with a structure similar to that of another amine. Pharmaceutically acceptable salts include, but are not (reference compound) but differing from it in respect to a limited to, cations based on the alkali and alkaline earth particular component, functional group, atom, etc. metals, such as Sodium, lithium, potassium, calcium, magne 0036. As used herein, the term "derivative' refers to com sium, and aluminum salts, as well as non-toxic ammonium, pounds which are formed from a parent compound by chemi quaternary ammonium, and mine cations, including, but not cal reaction(s). limited to ammonium, tetramethylammonium, tetraethylam monium, methylamine, dimethylamine, trimethylamine, tri II. Compositions ethylamine, ethylamine, and the like. Other representative 0037 Compositions containing one or more phytoestro organic amines useful for the formation of base addition salts gens are described herein. A number of phytoestrogens have include diethylamine, ethylenediamine, ethanolamine, been isolated and identified and additional analogs created, diethanolamine, and piperazine. all of which have estrogen receptor binding selectivity. In one 0043. Appropriate carriers can be added that assist the embodiment, of the composition contains two or more plant compounds to cross the blood-brain-barrier. derived estrogenic molecules and/or structural analogues, 0044 B. Additional Active Agents which possess ERB-binding selectivity and exhibit neuropro 0045 While the compounds can be administered as the tective activity when administered individually. These com sole active pharmaceutical agent, they can also be used in positions are useful for preventing estrogen-deficiency asso combination with one or more other compound as described ciated symptoms and disorders, particularly age-related herein, and/or in combination with other agents used in the cognitive decline and neurodegenerative diseases, such as treatment and/or prevention of estrogen receptor-mediated Alzheimer's disease (AD'). disorders. Alternatively, the compounds can be administered 0038 A. PhytoSERMs sequentially with one or more Such agents to provide Sus 0039. The compositions described herein contain one or tained therapeutic and prophylactic effects. Suitable agents more phytoestrogens or natural source selective estrogen include, but are not limited to, other SERMs as well as tradi receptor modulators (SERMs) exhibiting a binding prefer tional estrogen agonists and antagonists. ence for ERB. PhytoSERMs can be identified as described in 0046 Representative agents useful in combination with Example 1. Suitable phytoSERMs include, but are not limited the compounds for the treatment of estrogen receptor-medi to, genistein, daidzein, equol. IBSO03569 and combinations ated disorders include, for example, tamoxifen, 4-hydrox thereof. The structures of genistein, daidzein, equol, and ytarnoxifen, raloxifene, toremifene, droloxifene, TAT-59, IBSO03569 are shown in FIG.1. Others are listed in Table 1. idoxifene, RU 58,688, EM 139, ICI 164,384, ICI 182,780, Preferred compounds cross the blood brain barrier. clomiphene, MER-25, DES, nafoxidene, CP-336,156, 0040. As demonstrated by Example 2, combinations of GW5638, LY 139481, LY353581, Zuclomiphene, enclomi two or more PhytoSERMS are more effective than adminis phene, ethamoxytriphetol, delmadinone acetate, bisphospho tration of one PhytoSERM. nate. Other agents that can be combined with one or more of 0041. The compounds can be used in the form of salts the compounds include aromatase inhibitors such as, but not derived from inorganic or organic acids. These salts include, limited to, 4-hydroxymdrostenedione, plomestane, exemes but are not limited to, the following: acetate, adipate, alginate, tane, aminogluethimide, rogletimide, fadrozole, Vorozole, citrate, aspart ate, benzoate, benzenesulfonate, bisulfate, letrozole, and anastroZole. butyrate, camphorate, camphorsulfonate, digluconate, cyclo 0047 Still other agents useful in combination with the pentanepro-pionate, dodecylsulfate, ethanesulfonate, gluco compounds described herein include, but are not limited to heptanoate, glycerophosphate, hemi-sulfate, heptanoate, antineoplastic agents, such as alkylating agents, antibiotics, hexamate, fumarate, hydrochloride, hydrobromide, hydroio hormonal antineoplastics and antimetablites. An example dide, 2-hydroxyethanesulfonate, lactate, maleate, methane includes the compounds used to treat or prevent osteoporosis. Sulfonate, nicotinate, 2-napthalenesulfanate, oxalate, pamo Other ingredients include vitamins, nutritional Supplements, ate, pectinate, Sulfate, 3-phenylpropionate, picrate, pivalate, anti-oxidant agents, coenzymes, etc. propionate. Succinate, tartrate, thiocyanate, p-toluene 0048. The additional active agents may generally be Sulfonate and undecanoate. Also, any basic nitrogen-contain employed in therapeutic amounts as indicated in the PHYSI ing groups can be quaternized with agents such as lower alkyl CIANS DESK REFERENCE (PDR) 53rd Edition (2003), or halides, such as methyl, ethyl, propyl, and butyl chloride, Such therapeutically useful amounts as would be known to US 2012/0164122 A1 Jun. 28, 2012

one of ordinary skill in the art. The compounds and the other may also involve the use of transdermal administration Such therapeutically active agents can be administered at the rec as transdermal patches or ionophoresis devices. The term ommended maximum clinical dosage or at lower doses. Dos parenteral as used herein includes Subcutaneous injections, age levels of the active compounds in the compositions may intravenous, intramuscular, intrasternal injection, or infusion be varied to obtain a desired therapeutic response depending techniques. on the route of administration, severity of the disease and the 0054 Injectable preparations, for example, sterile inject response of the patient. The combination can be administered able aqueous or oleaginous Suspensions may be formulated as separate compositions or as a single dosage form contain according to the known art using Suitable dispersing or wet ing both agents. When administered as a combination, the ting agents and Suspending agents. The sterile injectable therapeutic agents can be formulated as separate composi preparation may also be a sterile injectable solution or Sus tions that are given at the same time or different times, or the pension in a nontoxic parenterally acceptable diluent or Sol therapeutic agents can be given as a single composition. vent, for example, as a solution in 1,3-propanediol. Among 0049 C. Pharmaceutical Compositions the acceptable vehicles and solvents that may be employed 0050. The compounds can be administered enterally, are water, Ringer's solution, and isotonic sodium chloride transdermally, transmucisally, intranasally or parenterally. Solution. In addition, Sterile, fixed oils are conventionally Excipients for oral formulation are known to those skilled in employed as a solvent or Suspending medium. For this pur the art, as discussed briefly below, and can be used to provide pose, any bland fixed oil may be employed including Syn immediate, Sustained, delayed, or pulsed release. The com thetic mono- or diglycerides. In addition, fatty acids Such as pounds can also be administered via a transdermal patch, a oleic acid can be useful in the preparation of injectables. depo, vaginally or rectally using a topical carrier Such as agel, 0055 Suppositories for rectal or vaginal administration of lotion, ointment, liposomal formulation, Suspension, foam, the drug can be prepared by mixing the drug with a suitable spray or suppository, via the pulmonary or nasal route, buc nonirritating excipient such as cocoa butter and polyethylene cally or Sublingual via the mucosal membranes of the mouth. glycols that are solidat ordinary temperatures but liquid at the The appropriate excipients for all of these formulations are rectal temperature and will therefore melt in the rectum and known. The compounds may be dissolved or Suspended in release the drug. saline, sterile water orphosphate buffered saline, or a suitable 0056 Solid dosage forms for oral administration may oil for injection iv, im, Subcu, or ip. include capsules, tablets, pills, powders, and granules. In Such 0051 Suitable pharmaceutically acceptable excipients Solid dosage forms, the active compound may be admixed include processing agents and drug delivery modifiers and with at least one inert diluent such as Sucrose lactose or starch. enhancers, such as, for example, calcium phosphate, magne Such dosage forms may also comprise, as is normal practice, sium Stearate, talc, monosaccharides, disaccharides, starch, additional Substances other than inert diluents, e.g., lubricat gelatin, cellulose, methyl cellulose, sodium carboxymethyl ing agents such as magnesium Stearate. In the case of cap cellulose, dextrose, hydroxypropyl-..beta.-cyclodextrin, poly Sules, tablets, and pills, the dosage forms may also comprise vinylpyrrollidone, low melting waxes, and ion exchange res buffering agents. Tablets and pills can additionally be pre ins, as well as combinations of any two or more thereof. Other pared with enteric coatings. Suitable pharmaceutically acceptable excipients are 0057 Liquid dosage forms for oral administration may described in Remington's Pharmaceutical Sciences, Mack include pharmaceutically acceptable emulsions, Solutions, Pub.Co., New Jersey (1991). Suspensions, syrups, and elixirs containing inert diluents 0052 Pharmaceutical compositions containing estrogen commonly used in the art, Such as water. Such compositions receptor modulating compounds may be in any form Suitable may also comprise adjuvants, such as wetting agents, emul for the intended method of administration, including, for sifing and Suspending agents, cyclodextrins, and Sweetening, example, a solution, a suspension, or an emulsion. Liquid flavoring, and perfuming agents. carriers are typically used in preparing Solutions, Suspen 0058. The compounds can also be administered in the sions, and emulsions. Liquid carriers contemplated for use form of lipsomes. AS is known in the art, liposomes are include, for example, water, saline, pharmaceutically accept generally derived from phospholipids or other lipid sub able organic solvent(s), pharmaceutically acceptable oils or stances. Liposomes are formed by mono- or multilamellar fats, as well as mixtures of two or more thereof. The liquid hydrated liquid crystals that are dispersed in an aqueous carrier may contain other Suitable pharmaceutically accept medium. Any non-toxic, physiologically acceptable and able additives such as solubilizers, emulsifiers, nutrients, metabolizable lipid capable of forming liposomes can be buffers, preservatives, Suspending agents, thickening agents, used. The present compositions in liposome form can con Viscosity regulators, or stabilizers. Suitable organic solvents tain, in addition to a compound, stabilizers, preservatives, include, for example, monohydric alcohols, such as ethanol, excipients. The preferred lipids are the phospholipids and and polyhydric alcohols, such as glycols. Suitable oils phosphatidylcholines (lecithins), both natural and synthetic. include, for example, soybean oil, coconut oil, olive oil, saf Methods to form liposomes are known in the art (Prescott flower oil, cottonseed oil. For parenteral administration, the 1976). carrier can also be an oily ester Such as ethyl oleate, isopropyl 0059 Transdermal patches are well known for delivery of myristate. Compositions may also be in the form of micro nicotine, nitroglycerin and birth control. These can be utilized particles, microcapsules, liposomal encapsulates, as well as with these formulations as well. Depos that are implanted combinations of any two or more thereof. under the skin orip can also be used, similarly to the manner 0053. The compounds may be administered orally, of delivering birth control. parenterally, Sublingually, by inhalation spray, rectally, vagi nally, or topically in dosage unit formulations containing III. Methods of Administration conventional nontoxic pharmaceutically acceptable carriers, 0060 Compounds can be administered in a variety of adjuvants, and vehicles as desired. Topical administration ways including enteral, parenteral, pulmonary, nasal, US 2012/0164122 A1 Jun. 28, 2012

mucosal and other topical or local routes of administration. 688; Hogervorst et al., 2003 Cochrane Database Syst Rev For example, Suitable modes of administration include oral, CD003122). Thus, observational studies suggest that declin Subcutaneous, transdermal, transmucosal, iontophoretic, ing reproductive function could be a modifiable risk factor for intravenous, intramuscular, intraperitoneal, intranasal, Sub dementia or that HT/ET could serve a protective role against dural, rectal, vaginal and inhalation. Some of the risks for developing dementia. 0061 An effective amount of the compound or composi 0065. Several recent observational studies have identified tion is administered to treat and/or prevent an estrogen recep that the stage of reproductive aging at which HT/ET is started tor-mediated disorder in a human or animal Subject. Modu modifies the risk of dementia. In these studies, women who lation of estrogen receptor activity results in a detectable take HT/ET during the late menopause transition or early Suppression or up-regulation of estrogen receptor activity postmenopause have a lower risk of dementia than those either as compared to a control or as compared to expected starting HT/ET later (Zandi et al., 2002 JAMA 288:21239; estrogen receptor activity. Effective amounts of the com Henderson et al., J Neurol Neurosurg Psychiatry 76:103 pounds generally include any amount Sufficient to detectably 2005). Thus, the timing of starting HT/ET relative to the modulate estrogen receptor activity by any of the assays menopause has been proposed to be one factor explaining the described herein, by other activity assays known to those otherwise discordant observations between the observational having ordinary skill in the art, or by detecting prevention studies and the RCTs (Resnick and Henderson, 2002 JAMA and/or alleviation of symptoms in a subject afflicted with an 288:21702: Manson et al., 2006 Menopause 13:139). Recent estrogen receptor-mediated disorder. preclinical studies reviewed below highlight the importance 0062. The effective amount will also be determined based of timing of ET in this report. on when the compounds are administered. Estrogen/hormone 0.066 Successful treatment of a subject may result in the therapy (ET/HT) has been associated with the reduced risk of prevention, inducement of a reduction in, or alleviation of developing AD when treated at the menopausal transition in symptoms in a subject afflicted with an estrogen receptor women Brinton, R. D. Impact of estrogen therapy on Alzhe mediated medical or biological disorder. Thus, for example, imer's disease: a fork in the road'? CNS Drugs 2004, 18, treatment can result in a reduction in breast or endometrial 405-422. For example, results of the Cache County Study tumors and/or various clinical markers associated with Such indicate that women who receive ET/HT at the time of meno cancers. Treatment of Alzheimer's disease can result in a pause and continue for 10 years have a 3-fold lower risk of reduction in rate of disease progression, detected, for developing AD, Zandi, et al. JAMA 2002, 288, 2123-2129, example, by measuring a reduction in the rate of increase of whereas the recent data from the Women's Health Initiative dementia. Memory Study indicate that women who begin the therapy 0067. Historically, there has been a presumption that late in menopause have a greater risk of developing AD, declining reproductive function plays no role in the onset of Espeland, et al. Women's Health Initiative Memory Study. mood disorders that occur during midlife in women. The JAMA 2004, 291, 2959-2968; Shumaker, et al., JAMA 2004, symptoms of depression during the menopause transition also 291, 2947-2958. These clinical observations are consistent were assumed to be transient and of Such minor severity that with basic science analyses of estrogen-inducible molecular they were dismissed to be of little clinical consequence. mechanisms in the brain, indicating a healthy cell bias of Recent studies, however, Suggest that these presumptions are estrogen action. incorrect. First, several community-based longitudinal stud 0063 Estrogen receptor-mediated disorders that may be ies have reported the relative independence of depressions treated include any biological or medical disorder in which during the menopause transition and hot flushes: both occurat estrogen receptor activity is implicated or in which the inhi this stage of life, but depression is not simply caused by hot bition of estrogen receptor potentiates or retards signaling flushes (Avis et al., 2001 Soc SciMedS2:345). Second, recent through a pathway that is characteristically defective in the longitudinal studies that followed women with no past history disease to be treated. The condition or disorder may either be of depression demonstrated an increased risk of first-onset caused or characterized by abnormal estrogen receptor activ depressions during the late menopause transition (Schmidt et ity. Representative estrogen receptor-mediated disorders al., 2004 Am J Psychiatry 161:22384; Cohen et al., 2006 Arch include, for example, osteoporosis, atherosclerosis, estrogen Gen Psychiatry 63:385; Freeman et at, 2006 Arch Gen Psy mediated cancers (e.g., breast and endometrial cancer), Turn chiatry 61:62). Finally, both major and minor depressions are er's syndrome, benign prostate hyperplasia (i.e., prostate clinically significant to women at midlife, because both are enlargement), prostate cancer, elevated cholesterol, resteno associated with an increased risk for several other medical sis, endometriosis, uterine fribroid disease, hot flashes, and conditions (Wassertheil-Smoller et al., 2004 Arch Intern Med skin and/or vagina atrophy. Otherestrogen receptor-mediated 164:289) relevant to the health of women at midlife (e.g., conditions that may be treated include neurological diseases cardiovascular disease, dementia, and the metabolic Syn and disorders including memory loss and dementia, and neu drome). rodegenerative disease, including Alzheimer's disease. 0068. The majority of women do not develop depression 0064. In addition to the potential beneficial effects of during the menopause transition, and, therefore, reproductive estrogen on episodic memory, some evidence Suggests that aging is not uniformly associated with either depressive HT reduced the risks of both dementia (including AD) and symptoms or the syndrome of depression. Nonetheless, mild cognitive impairment (MCI). MCI is a condition thought despite numerous studies concluding that the menopause is to represent a transitional state between normal cognition and not associated with an increased risk for developing depres dementia in some individuals, with a 12% conversion rate sion in women, several other longitudinal, community-based from MCI to dementia each year. Observational studies studies reported an association between the menopause tran repeatedly document that women taking HT enjoy an 30% sition and an increased risk for depression (Schmidt, 2005 reduced risk for dementia compared with women not taking Am J Med 118:54). Indeed, five recent longitudinal studies all HT odds ratio range, 0.306 (Yaffe et al., 1998 JAMA 279: have documented an increased risk for depression during the US 2012/0164122 A1 Jun. 28, 2012

menopause transition, with odds ratios ranging from 1.8 to tion. It will be understood, however, that the specific dose 2.9 compared with the premenopause (Bromberger et al., level for any particular patient will depend upon a variety of 2001 Am J Public Health91:14352: Freeman et al., 2004 Arch factors including the activity of the specific compound Gen Psychiatry 61:62, 2006 Arch Gen Psychiatry 63:375; employed, the age, body weight, general health, sex, diet, Schmidt et al., 2004 Am J Psychiatry 161:22384; Cohen et al., time of administration, route of administration, rate of excre 2006 Arch Gen Psychiatry 63:385). These data suggest that tion, drug combination, and the severity of the particular events Surrounding the final menstrual period may predispose disease undergoing therapy. The prophylactically or thera Some women to develop clinically significant depressive ill peutically effective amount for a given situation can be ness. Although several factors could precipitate depression in readily determined by routine experimentation and is within these women, endocrine events are Suggested by the stage of the skill and judgment of the ordinary clinician. the menopause transition (i.e., late) during which depressions 0072 For exemplary purposes, a prophylactically or appeared. The late transition is characterized by more pro therapeutically effective dose will generally be from about longed hypogonadism than the early perimenopause, during 0.01 mg/kg/day to about 100 mg/kg/day, preferably from which estradiol Secretion may be increased. Thus, the timing about 0.1 mg/kg/day to about 20 mg/kg/day, and most pref of appearance of the depressions observed suggest an endo erably from about 1 mg/kg/day to about 10 mg/kg/day of a crine mechanism related to the perimenopause (estradiol estrogen receptor modulating compound, which may be withdrawal and/or recent-onset of prolonged hypogonadism) administered in one or multiple doses. in the pathophysiology of perimenopausal depression. 0069 Efforts to investigate the potential role of declining IV. Kits ovarian hormone secretion in the onset of depression have 0073 Kits may be provided which contain the formulation examined the effects on mood of administering HT/ET in to be administered. The formulation may be administered women with perimenopausal and postmenopausal depres once a day or more than once a day. The formulation can be Sion. The antidepressant efficacy of estradiol has been exam administered enterally, parenterally, or topically. The kits ined in three relatively recent RCTs of women meeting stan typically contain the active agent(s) to be administered, dardized diagnostic criteria for major and minor depression, excipients and carriers, and instructions for administration of who were randomly assigned to enter double-blind, placebo the formulation. The kits may also contain equipment/devices controlled trials (Schmidt et al., 2000; Soares et al., 2001 Arch used to administer the formulation, such as Syringes. Gen Psychiatry 58:529; Morrisonet al., 2004 Biol. Psychiatry 0074 The present invention will be further understood by 55:406). In perimenopausal women, short-term administra reference to the following non-limiting examples. tion (3 weeks) of estradiol significantly decreased depression scores compared with both baseline and placebo conditions. EXAMPLES In one study, a full or partial therapeutic response was seen in 80% of perimenopausal women on estradiol compared with Example 1 22% of those on placebo (Schmidt et al., 2000). The efficacy of ET in perimenopausal depression is consistent with the Identification of PhytoSERMS observed effect size (0.69) in a recent meta-analysis of studies 0075 ERB has been associated with estrogen-induced examining the effects of estrogen on mood (Zweifel and promotion of memory function and neuronal Survival. Based O'Brien, 1997 Psychoneuroendocrinology 22:189). The on the optimized complex structure of human ERB LBD therapeutic response to estradiol was observed in both major bound with genistein, computer-aided structure-based virtual and minor depression as well as in women with and without screening against a natural source chemical database was hot flushes. Thus, the efficacy of ET in perimenopausal conducted to determine the occurrence of plant-based ERB depression is not solely a product of its ability to reduce the selective ligands. Twelve representative hits derived from distress of hot flushes. In contrast to these studies in peri database screening were assessed for their binding profiles to menopausal depression, the administration of estradiol under both ERs, three of which displayed over 100-fold binding similar conditions failed to improve mood in depressed selectivity to ERB over ERC. women who were 5 years postmenopause (Morrison et al., 0076 Materials and Methods 2004). Thus, the effects of estradiol on depression may be 0077. Identification of Molecules limited to perimenopausal women. Additionally, as with the 0078 Identification of Compounds in Database potential effects of estrogen on the course of dementia, the 0079 All computational work was performed on a SOT stage of reproductive aging at which women present and/or Octane workstation equipped with the IRIX 6.5 operating commence ET might modify the observed outcomes. system (Silicon Graphic Inc.). First, the 3D crystallographic 0070. In summary, the majority of women do not develop structure of human ERB LBD complexed with genistein was depression during or after the menopause transition. Never downloaded from the Protein Data Bank (PDB ID: 1GKM). theless, recent prospective studies monitoring both reproduc The complex structure was fixed and energy minimized with tive status and mood have documented that, for some women, the Accelrys molecular modeling software package Insight I perimenopause-related events increase the risk for the onset 2000 (Accelrys Inc.). An in-house 2D natural source chemi of depression. The role of ovarian function in these episodes cal collection containing approximately 25 000 plant-based of depression is Suggested by both the timing of their onset natural molecules or derivatives was converted to a 3D mul relative to the last menstrual period and the antidepressant ticonformational database with the Accelry's modeling soft efficacy of short-term ET. ware package Catalyst 9.8 (Accelrys Inc.). 0071. The amount of active ingredient that may be com 0080. The receptor-docking site was defined based on the bined with the carrier materials to produce a single dosage binding position of genistein in the receptor and specified as form will vary depending upon the estrogen-mediated dis all atoms within 10 A of the centercarbon of genistein. GOLD ease, the host treated and the particular mode of administra 2.0 (Genetic Optimization for Ligand Docking), an auto US 2012/0164122 A1 Jun. 28, 2012 mated ligand docking program distributed by CCDC (Cam underlying discriminative binding of selective ligands to bridge Crystallographic Data Center), was applied to calcu either receptor subtypes. Sun, et al. Mol. Endocrinol. 2003, late and rank the molecules based on their complementarities with the receptor binding site, on both geometrical and 17, 247-258. This slight structural variance serves as the chemical features. foundation for both design and discovery of ER specific 0081 Prior to the database screening, initial validation ligands. The similarities in the chemical features of both pairs using genistein as the test ligand was conducted. The aim of of residues presents a Substantial challenge to discover a the validation test was to evaluate the effectiveness of the selective ligand based on this difference. Of the known natu algorithm of the docking program in identifying the experi ral source ERB-selective ligands, genistein remains the most mentally observed binding mode of the ligand in the receptor, selective. However, an increasing number of synthetic com to determine whether the program is applicable to the specific pounds are emerging showing greater selectivity than target system in this study. In addition, the validation test was genistein for ERB, as evidenced by the compound DPN devel used to determine the optimal parameter settings for the later oped in Katzellenebogen's laboratory. Computer-aided struc database screening. Twenty docking runs were carried out on ture-based virtual database screening provides an efficient the test complex, using the fastest default generic algorithm approach to rationally highlight a small group of lead candi parameters optimized for virtual library Screening, and the dates from a large number of compounds for investigation at GoldScore fitness function was applied. The validation test the bench. demonstrated that, based on the specified parameter settings, GOLD was effective in capturing the contributive hydrogen Determination of Binding Affinity and Selectivity bond donor (ND1 in His 475) crucial to the binding and reproducing the nearly coincident Solution in terms of both I0084. The binding affinity and selectivity of candidate the binding orientation and conformation of genistein as molecules yielded from database screening were determined observed in the experimental measurement (see FIG. 1). The by a fluorescent polarization competitive binding assay using root-mean-square (RMS) deviations were computed between purified baculovirus-expressed human ERB or ERC. and a the observed experimental position and the GOLD solutions, fluorescent estrogen ligand EL Red (Pan Vera Corp.). Test with RMSD 0.3299 and 0.4483 compared to top-ranked and molecules were serially diluted to a 2x concentration in assay worst solutions, respectively. The average RMSD of all solu buffer (200 uM to 200 uM). Fifty microliters of preincubated tions was 03566, which is regarded as a good prediction based 2x complex of ERB (30 nM) or ERB (60 nM) and EL Red (2 on the Subjective classifications defined by the program nM) was added to each well in a 96-well Non-binding Surface developer (refer to the program manual), Suggesting that this black microplate (Corning Life Sciences) for a final volume program is reliable and applicable to the database screening of 100 uL. Negative controls containing ER and EL Red toward ERB. (equivalent to 0% inhibition) and positive controls containing 0082. Using the parameter settings determined in the vali only free EL Red (equivalent to 100% inhibition) were dation test, the 3D natural source chemical database was input included. After a 2-h incubation period at room temperature, and docked into the prepared ERB binding site in a flexible the polarization values were measured using a Tecan GENios docking manner (full ligand and partial protein) and scored Pro reader at 535 nm/590 nm excitation/emission and plotted based on the GoldScore fitness function. Five hundred result against the logarithm of the test molecule concentration. ICso ant top-scoring molecules were filtered via visual screening values (concentration of test molecule that displaces half of in the context of the receptor in Insight|I. Based on visual the EL Red from ER) were determined from the plot using a analysis, 100 molecules underwent further analysis by Affin nonlinear least-squares analysis. ity, a more complex and predictive ligand docking program to 0085. Results refine the binding modes predicted by GOLD. The criteria I0086 31 molecules that can form a hydrogen bond with used for the selection of candidate molecules for investigation ND1 in His 475 were selected and grouped into three catego included the following (a) formation of hydrogen bond with ries based upon the chemical features that favored both the donoratom ND1 in His 475; (b) hydrophobic and hydrophilic van der Waals (VDW) contact (the number of the rings in the balance appearing in the structure (e.g., the molecule should structure) and electrostatic interactions (the number of the potentially have two relatively hydrophilic sides and a hydro hydrogen bonds) with the receptor. 10 molecules that have phobic center to enhance both the steric and electrostatic strong VDW interactions with the receptor, but without con complementarity with the receptor); (c) bound pose of the tributive hydrogen bonding, were grouped in Category IV. molecule in the receptor; and d) structural diversity. Finally, These molecules contain three or four five- or six-membered molecules that met the above criteria were computationally rings in their structures that could promote the hydrophobic predicted for their drug-likeness (Lipinski's Rule of Five) and interactions with the center of the receptor binding site as blood-brain barrier (BBB) penetration properties. observed in endogenous estrogen 17B-estradiol that consists 0083. The ligand binding domains of the human ERC. and of four rings in its structure and binds to the estrogen receptor ERB are approximately 60% homologous. Structural model with a high affinity. ing and mutational analyses indicate that two variant amino I0087 Table 1 summarizes the ICso binding results of test acid residues along the ligand binding pocket, Leu 384 and molecules to both ERC. and ERB as well as the binding selec Met 421 in ERC, which are replaced with Met 336 and Ile tivity of representative molecules selected from four catego 373, respectively, in ERB, are the key molecular constituents ries. US 2012/0164122 A1 Jun. 28, 2012

TABLE 1. Binding Affinity (ICs) and Selectivity of Representative Molecules for Estrogen Receptor C. and B

ICso Selectivity

Compd ERC ERB (ERC/ERB)

Progesteron NC: NC

genistein 4.68 IM 98.7 nM 47.2

HO

75.7 M 18.6 nM 4.07

NC 0.68 M >100

120 nM 250 nM O.48

NC NC US 2012/0164122 A1 Jun. 28, 2012 10

TABLE 1-continued

Binding Affinity (ICs) and Selectivity of Representative Molecules for Estrogen Receptor C. and B

ICso Selectivity

Compd Structure ERC ERB (ERC/ERB)

5 NC 2.80 IM >100 O

HO cCO NC NC O o1

HO Or(3)

85.7 M 43.0 M 1.99

OH O O O S. HO

O NC 4.48 M >100 (2)

O O OS

OH

O NC NC

OroOH 10 NC NC O -O- O US 2012/0164122 A1 Jun. 28, 2012 11

TABLE 1-continued Binding Affinity (ICs) and Selectivity of Representative Molecules for Estrogen Receptor C. and ICso Selectivity Compd Structure ERC ERB (ERC/ERB) 11 2.32 IM NC <0.01

\ N O O O O

12 NC NC

() *NC: Nonconvergence within the dose range, predicting that either the molecule does not bind to the receptor or that the binding affinity is very low, with an IC50 greater than 1 mM. (2) indicates text missing or illegible when filed

0088 As expected, the negative control steroid, progest Steroid Sisters, Keystone, Colo.; February 2004, relatively erone, does not bind to either ER. As a positive natural source larger molecular size favors the binding selectivity for ERB estrogen control, genistein was found to bind to ERB with a over ERC, as represented by molecule 3 and 11. 47.2-fold greater binding selectivity over ERC, but at an I0089. These analyses shed light on the future search and affinity one-fourth of 17 B-estradiol. Among 12 molecules design of more active and selective ER subtype-selective tested, five molecules, 1, 2, 5, 7, and 8, showed binding ligands. Further, 3 out of 12 representative molecules yielded selectivity to ERB over ERC, 3 of which, 2.5, and8, displayed from database searching displayed over 100-fold selectivity the selectivity over 100-fold. Preliminary structure and bind toward ERB over ERO, demonstrating the effectiveness of ing activity relationship analyses revealed that both the cen this computer-aided virtual screening approach applied in the tral hydrophobic skeletal structure and the connected two present study in the discovery of potential molecules that polar arms contribute to the binding affinity of ligands to preferentially interact with ERB. both ERs. The enhanced VDW contact derives mainly from the central hydrophobic feature of the molecule. For example, Example 2 the number of rings increases the binding affinity of mol ecules to the receptor, as indicated by the VDW value of Preclinical Identification of ERB-Selective Phy 17 B-estradiol (-67.98) versus that of genistein (-60.75) and toSERM Combinations for Prevention of Neurode molecule 9 (-58.04), which are well correlated with their generation order-different binding affinities. Meanwhile, the hydrogen bonds derived from the two polar “arms of the molecule are (0090. The impact of ERb-selective PhytoSERMs when essential for the binding as well. The lack of one "arm' of the administered singly or in combination on neuronal Survival hydrogen bond, as represented by molecule 4 and 6, or two and molecular/functional markers associated with prevention arms, as represented by 10 and 12, even though the latter of neurodegeneration and Alzheimer's disease (AD) was two molecules can elicit strong VDW interactions with the investigated. receptor, with the VDW value of -72.58 and -69.19, respec 0091. Materials and Methods tively, leads to either very weak or no binding. With respect to 0092] 17 B-Estradiol was purchased from Steraloids (New the binding selectivity, as demonstrated in the modeling com port, R.I.). Genistein, daidzein and equol were purchased plex structures of a synthetic ERB-selective agonist, PPT. from Indofine Chemical (Hillsborough, N.J.). IBSO03569 Stauffer, et al. J. Med. Chem. 2000, 43, 4934-4947 and a was purchased from InterBioScreen (Moscow, Russia). The synthetic ERB-selective agonist, DPN, Meyers, et al., J. Med. structures of these compounds are shown in FIG. 1. Chem. 2001, 44, 4230-4251, with both ERs, Zhao, et al. 2004 0093. In Vitro Treatments: Test compounds (or combina Abstract Book: The Keystone Symposia: Nuclear Receptors: tions) were first dissolved in analytically pure DMSO (10 US 2012/0164122 A1 Jun. 28, 2012

mM) and diluted in Neurobasal medium to the working con incubation using a scintillation counter. The amount of recep centrations right before treatments. tor bound ligand is determined directly, i.e., without separa 0094. In Vivo Treatments: Test compounds (or combina tion of bound from free ligand. Ifestimates of both bound and tions) were first dissolved in analytically pure DMSO and free ligand are required, the Supernatant is removed from the diluted in corn oil (50 ml of DMSO in 950 ml of corn oil) to wells, liquid scintillant is added, and the wells are counted the working concentrations at 100 mg/ml for 17 B-estradiol separately in a liquid Scintillation counter. and 10 mg/ml for phytoSERMs. 0102 ERC/ERB Transactivation Assays 0095. In vitro Assays (0103 Construction of Transfected CHO Cells 0096 ERC. Binding Assays 01.04 Transfected CHO cells were derived from CHO KI 0097 ERC. receptor (about 0.2 mg/ml, Affinity cells obtained from the American Type Culture Collection Bioreagents) is diluted to about 2x10 mg/ml in phosphate (“ATCC, Rockville, Md.): The transfected cells were modi buffered saline (“PBS) at a pH of 7.4. Fifty microliters of the fied to contain the following four plasmid vectors: (1) pKCRE EPO-PBS solution is then added to each of the wells of a with DNA for the human estrogen receptor, (2) p.AG-60-neo flashplate. The plates are sealed and stored in the dark at 4°C. with DNA for the protein leading to neomycin resistance, (3) for 16-18 hours. The buffered receptor solution is removed pRO-LUC with DNA for the rat oxytocin promoter and for just prior to use, and the plates are washed 3 times with 200 firefly luciferase protein, and (4) plDR with DNA for the microliters per well of PBS. The washing is typically per protein leading to hygromycine resistance. All transforma formed using a slow dispense of reagent into the wells to tions with these genetically modified CHO cells are per avoid stripping the receptor from the well Surface. formed under rec-VMT containment according to the guide 0098. For library screening, 150 microliters of 1 nM lines of the COGEM (Commissie Genetische Modificatie). H-estradiol (New England Nuclear, Boston, Mass.) in 20 Screening is performed either in the absence of estradiol mM Tris-HCl, 1 mM EDTA, 10% glycerol, 6 mM mono (estrogenicity) or in the presence of estradiol (anti-estroge thioglycerol, 5 mMKC1, pH 7.8 is mixed with 50 microliters nicity). of the test compound (in same buffer) in a 96 well microtiter 0105 Assays to Assess Neuronal Function plate, resulting in a final estradiol concentration of 0.6 nM. In 0106 Neuronal Culture Preparation addition, several dilutions of estradiol, centered on the ICs of 0107 Primary cultures of hippocampal neurons were 1-2 nM, are also added to individual wells to generate a obtained from Embryonic Day 18 (E18d) rat fetuses. Briefly, standard curve. The plates are gently shaken to mix the after dissected from the brains of the rat fetuses, the hippoc reagents. A total of 150 microliters from each of the wells is ampi were treated with 0.02% trypsin in Hank's balanced salt added to the corresponding wells of the pre-coated ERC. solution (137 mMNaCl, 5.4 mMKC1, 0.4 mMKHPO, 0.34 plates. The plates are sealed and the components in the wells mM NaHPO.7HO, 10 mM glucose, and 10 mM HEPES) are incubated either at room temperature for 4 hours or at 4 for 5 min at 37° C. and dissociated by repeated passage C. overnight. The receptor bound ligand is read directly after through a series of fire-polished constricted Pasteur pipettes. incubation using a scintillation counter. The amount of recep Between 2x10 and 4x10" cells were plated onto poly-D- tor bound ligand is determined directly, i.e., without separa lysine (10 g/ml)-coated 22 mm coverslips in covered 35 mm tion of bound from free ligand. If estimates of both bound and petri dishes for morphological analysis, and 1x10 cells/ml free ligand are required, the Supernatant is removed from the were plated onto poly-D-lysine-coated 24-well, 96-well cul wells, liquid scintillant is added, and the wells are counted ture plates or 3-5x10 cells/ml onto 0.1% polyethylenimine separately in a liquid Scintillation counter. coated 60 mm petri dishes for biochemical analyses. Nerve 0099 ERB Binding Assays cells were grown in phenol-red free Neurobasal medium 0100 ERB receptor (about.0.2 mg/ml, Affinity (NBM, Invitrogen Corporation, Carlsbad, Calif.) supple Bioreagents) is diluted to about 2x10 mg/ml in phosphate mented with B27. 5 U/ml penicillin, 5 g/ml streptomycin, buffered saline (“PBS) at a pH of 7.4. Fifty microliters of the 0.5 mM glutamine and 25 Mglutamate at 37°C. in a humidi ERB-PBS solution is then added to each the wells of a flash fied 10% CO, atmosphere at 37° C. for the first 3 days and plate. The plates are sealed and are stored in the dark at 4°C. NBM without glutamate afterwards. Cultures grown in for 16-18 hours. The buffered receptor solution is removed serum-free Neurobasal medium yield approximately 99.5% just prior to use, and the plates are washed 3 times with 200 neurons and 0.5% glial cells. microliters per well of PBS. The washing is typically per 0108) Neuroprotection Measurements formed using a slow dispense of reagent into the wells to 0109 Glutamate Exposure avoid stripping the receptor from the well Surface. 0110 Primary hippocampal neurons were pretreated with 0101 For library screening, 150 microliters of 1 nM compounds for 48 hr followed by exposure to 100 glutamate H-estradiol (New England Nuclear, Boston, Mass.) in 20 for 5 min at room temperature in HEPES buffer containing mM Tris-HCl, 1 mM EDTA, 10% glycerol, 6 mM mono 100 mMNaCl, 2.0 mMKC1, 2.5 mM CaCl, 1.0 mMMgSO, thioglycerol, 5 mM KC1, pH 7.8 was mixed with 50 microli 1.0 mM NaH2PO, 4.2 mM NaHCOs, 10.0 mM glucose and ters of the test compound (in same buffer) in a 96 well micro 12.5 mMT-LEPES. Immediately following glutamate expo titer plate, resulting in a final estradiol concentration of 0.6 sure, cultures were washed once with HEPES buffer and nM. In addition, several dilutions of estradiol, centered on the replaced with fresh Neurobasal medium containing the test ICs of 1-2 nM is also added to individual wells to generate a compounds. Cultures were returned to the culture incubator standard curve. The plates are then gently shaken to mix the and allowed to incubate for 24 hr prior to cell viability mea reagents. A total of 150 microliters from each of the wells is Surements on the following day. added to the corresponding wells of the pre-coated ERB 0111 Western Immunoblotting plates. The plates are sealed and the components in the wells (O112 CREB Phosphorylation are incubated at room temperature either for 4 hours or at 4 0113 Nuclear lysates were prepared as following: Briefly, C. overnight. The receptor bound ligand is read directly after hippocampal neurons grown on poly-D-lysine coated culture US 2012/0164122 A1 Jun. 28, 2012

dishes were treated with compounds for appropriate periods, electro-transferred to polyvinylidene difluoride membranes washed with cold PBS once and scraped into 1 ml PES. Cells (Millipore Corp., Bedford, Mass.) from the gels, Nonspecific were then centrifuged at 5,000 rpm for 5 min, and the pellet binding sites were blocked with 5% nonfat dry milk in PBS was dissolved in Cytoplasm Extraction buffer (10 mM containing 0.05% TweenTM-20 (PBS-TweenTM). Membranes HEPES, 1 mM EDTA, 60 mM KC1, 0.075% gepal and pro were incubated with the primary monoclonal antibody tease and phosphatase inhibitor cocktail) and Suspended by against Bcl-2 (Zymed Laboratories, Inc., S. San Francisco, passage through a 200 ul pipette tip. After 30-45 RPM of Calif.) diluted 1:250 in PBS-Tween with 1% horse serum incubation at 4°C., the samples were centrifuged at 5,000 (Vector Laboratories, Inc., Burlingame, Calif.) overnight at rpm for 5 min to generate the cytoplasmic extract in the 4°C., then incubated with the secondary horseradish peroxi Supernatant. The Supernatant cytoplasmic extract was dase (HRP)-conjugated horse anti-mouse IgG (Vector Labo removed, and Nuclear Extraction buffer (20 mM Tris HCl, 1.5 mM MgCl, 420 mM. NaCl, 0.2 mM EDTA, 25% glycerol, ratories, Inc, Burlingame, Calif.) diluted 1:5,000 in PBS 0.5% Igepal and protease and phosphatase inhibitor cocktail) TweenTM with 1% horse serum for 2 hr at room temperature, was added to the pellet followed by 5 M NaCl to break the and Bcl-2 proteins were visualized by developing the mem nuclear membrane. Following 30-45 RPM of incubation at 4 branes with TAB substrate for peroxidase (Vector Laborato C., the samples were centrifuged at 12,000 rpm for 10 minto ries, Inc., Burlingame, Calif.). B-Actin (Santa Cruz Biotech generate a Supernatant containing the nuclear extract. nology, Inc., Santa Cruz, Calif.) level was determined to 0114 Protein concentration was determined by the BCA ensure equal protein loading, and high-range Precision Pro method. An appropriate volume of 2x sample buffer was tein Standards (Bio-Rad Laboratories, Hercules, Calif.) was added to the protein samples, and samples were boiled at 95° used to determine protein sizes. Relative intensities of bands C. for 5 min. Samples (25ug of proteins per well) were loaded were quantified by optical density analysis using an image on a 10% SDSPAGE gel and resolved by standard electro digitizing software Un-Scan-Tt version 5.1 (Silk Scientific, phoresis at 90V. Proteins were then electrophoretically trans Inc., Orem, Utah). ferred to Immobilon-PPVDF membranes overnight at 32V at 0117 Statistics 4°C. Membranes were blocked for 1 hr at room temperature 0118 Statistically significant differences between groups in 10% non-fat dried milk in PBS containing 0.05% Tween 20 were determined by a one way analysis of variance (ANOVA) (PBS-T), incubated with appropriate primary antibodies followed by a Newman-Keuls post hoc analysis. against phospho-CREB (pSER', mouse monoclonal, 0119) In vivo Assays 1:2000; Cell Signaling Technology, Beverly, Mass.), CREB (rabbit polyclonal. 1:1000; Cell Signaling Technology, Bev Immature Rat Uterotrophic Bioassay for Estrogenicity Anti erly, Mass.), spinophilin (rabbit polyclonal. 1:1000; Upstate Estrogenicity Biotecholagy, Lake Placid, N.Y.), actin (mouse monoclonal, 1:1000; Santa Cruz, Biotechnology, Inc., Santa Cruz, Calif.) I0120 Antiestrogenic activity was determined by the abil or histone H1 (mouse monoclonol. 1:250; Santa Cruz Bio ity of a test compound to suppress the increase in uterine wet techology, Inc., Santa Cruz, Calif.) attemperatures and times weight resulting from the administration of 0.2 Lug 17-B- specified by the antibody providers. All primary antibodies estradiol ("E") per day. Any statistically significant were dissolved in PBS-T with 1% horse serum (for mouse decreases in uterine weight in a particular dose group as monoclonal antibody) or goat serum (for rabbit polyclonal). compared with the E control group are indicative of anti After washing in PBS-T, the membranes were incubated with estrogenicity. horseradish peroxidase-conjugated anti-mouse IgG (1:5000; I0121 One hundred forty (140) female pups (19 days old) Vector Laboratories, Inc., Burlingame, Calif.) in PBS-T with in the 35-50 g body weight range are selected for the study. On 1% horse serum or anti-rabbit IgG (1:5000; Vector Labora day 19 of age, when the pups weigh approximately 35-50 g, tories, Inc., Burlingame, Calif.) in PBS-T with 1% goat serum they are body weight-order randomized into treatment pups. for 1 hr. Immunoreactive bands were visualized by TMI3 Observations for mortality, morbidity, availability of food detection kit (Vector Laboratories. Inc., Burlingame, Calif.) and water, general appearance and signs of toxicity are made and quantified using Un-Scan-It gel image Software (Silk twice daily. Pups not used in the study are euthanized along Scientific, Inc., Orem, Utah). Following transfer, gels were with, the foster dams. Initial body weights are takenjust prior stained with Coomassie blue (Bio-rad Laboratories, Her to the start of treatment at day 19 of age. The final body cules, Calif.) to ensure equal protein loading. weights are taken at necropsy on day 22 of age. 0115 Bcl-2 and Bcl-Xl Expression 0.122 Treatment commences on day 19 of age and contin 0116 Primary hippocampal neurons were pretreated with ues until day 20 and 21 of age. Each animal is given three compounds for 48 hr before the cells were lysed by incubation Subcutaneous (“sc') injections daily for 3 consecutive days. in ice-coldlysis buffer containing: 0.005% SDS, 0.1% Igepal, Three rats in each of the control and mid- to high-level dose 0.2 mM sodium orthovanadate, 0.2 mM phenylmethylsulfo test groups are anesthetized with a ketamine/xylazine mix nylfluoride and protease inhibitor mixture in PBS at 4°C. for ture. Their blood is collected by exsanguination using a 22 45 min. Cell lysates were centrifuged at 10,000 rpm at 4°C. gauge needle and 5 ml syringe flushed with 10 USP with for 10 min, and the concentration of protein in the Supernatant Sodium heparin/ml through the descending Vena cava; and was determined using the BCA Protein Assay (Pierce Bio then transferred into a 5 ml green top plasma tube (sodium technology, Inc., Rockford, Ill.). 25 ug of total protein were heparin (freeze-dried), 72 USP units). Plasma samples are diluted in 15ul 2xSDS containing sample buffer and the final collected by centrifugation, frozen at -70° C., and analyzed volume was made 30 ul with water. After denaturalization on using mass spectrographic to determine the presence and a hot plate at 95-100° C. for 5 min, 25ul of the mixture were amount of test compound in the serum. Blood chemistry is loaded per lane on 10% SDS-polyacrylamide mini-gels fol also analyzed to determine other blood parameters. The uteri lowed by electrophoresis at 90V. The proteins were then from the rats are excised and weighed. The remaining rats are US 2012/0164122 A1 Jun. 28, 2012

sacrificed by asphyxiation under CO. The uteri from these stored for biochemical analyses. The remaining brain tissues rats are excised, nicked, blotted to remove fluid, and weighed minus cerebellum, pineal gland, and brainstem were utilized to the nearest 0.1 mg. for mitochondrial isolations, followed immediately by mito 0123. In order to determine whether the test compound chondria respiratory activity measurements. The rest of mito significantly affected final body weight, a parametric one chondrial samples were stored for cytochrome c oxidase way analysis of variance (ANOVA) is performed (SIGMAS activity measurements. Uteri were excised, trimmed offat TAT version 2.0, available commercially from Jandel Scien and connective tissue, and both a wet weight and a dry weight tific, San Rafael, Calif.). Estrogen agonist and antagonist were recorded. activity is assessed comparing uterine wet weights across 0128 Results treatment groups using a parametric ANOVA on loglo trans I0129. The PhytoSERMs tested are shown in FIG. 1. formed data. The data are transformed to meet assumptions of normality and homogeneity of variance of the parametric I0130 Selective Binding for both ERB and ERC. AWOVA. The F value is determined and a Student-Newman I0131 FIG. 2 presents the competition binding curves of Kuels multiple range test is performed to determine the pres four known ER ligands for both ERB and ERC. The ICso ence of significant differences among the treatment groups. determined for these ligands from the binding curves are The test compound is determined to act as a mixed estrogen consistent with the previously reported values using alterna agonist/antagonist if the test compound does not completely tive methods such as radioligand assay, demonstrating the inhibit the 17-B-estradiol stimulated uterotrophic response. reliability of this assay in determining the binding profiles of 0.124. The use of animals was approved by the Institutional small molecules to both ERs. Animal Care and Use Committee at the University of South I0132 FIGS. 2A and 2B show the competition binding ern California (Protocol Number: 10780). Embryonic day 18 curves for ERC. and ERR. Data were generated with a fluo Sprague-Dawley rat (Harlan, Indianapolis, Ind.) fetuses were rescence polarization-based competitive binding assay using used to obtain primary hippocampal neuronal cultures for in full-length human ERC. and ERB, and plotted against the vitro experiments. Young adult (14 to 16-week-old, weighing logarithm of serially diluted concentrations of the test com from 270-290 g) female ovariectomized Sprague-Dawley rats pounds (or combinations). Progesterone served as a negative (Harlan) were used for in vivo experiments. control. 17 B-Estradiol served as a positive control. Combined 0.125. In vitro neuroprotection and associated mechanistic formulations were composed of equivalent molar of indi studies were conducted in primary hippocampal neurons vidual phytoSERMs included. G: genistein; D: daidzein; E: obtained from embryonic day 18 rat fetuses. Adult female equol. I: IBSO03569. 17 B-estradiol has no binding prefer ovariectomized rats were used to relate the in vitro findings to ence to ERC. or to ERB. The concentration of a test molecule in vivo environment, along with the assessment of the impact resulting in the half-maximum shift in polarization value of PhytoSERMs on brain mitochondrial functions and uterine equals its ICso. Non-convergence within the dose range, pre weight. dicts that either the molecule does not bind to the receptor or 0126. During a 2-week surgery recovery following ova that the binding affinity is very low. riectomy, but before treatment, rats were placed on a phy (0.133 Table 2 shows the binding data for ERC. and ERB.

TABLE 2 Binding data for ERC and ERB ERC ERB ICso RBA ICso RBA Selectivity Compounds (IM) (%)- R2B (IM) (%)- R2B (B.C.) Progesterome Non-Binding Non-Binding 17B-Estradiol O.O2S3 100.0 O.9791 O.O325 100.0 O.9611 O.78 Genistein 4.735 O.S343 O.9811 O.O789 41.12 O.99.08 6O.O Daidzein 26.65 O.O949 O.7876 1.738 1867 O.9883 1427 Equol 5.876 O.4306 0.9948 O.S825 S.S71 O.9986 10.09 IBSQ03569 1695 O.OO15 O.9917 7.8.19 0.415 0.9959 -100 G - D 9.896 O.2557 0.986S O1574 20.62 0.997O 52.57 G +D+E 15.71 O.1610 O.992S O.1902 17.06 0.9969 8260 G - D - E + I 13.85 O.1596 O.9932 O.2615 12.41 0.9891 60.61 RBA (%) refers to the relative binding affinity of the test compound (combination) that is expressed as the percent if the binding affinity of 17 B-estradiol (RBA = 100%). R’ refers to goodness offit 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, toSERM-reduced diet, TD.96155 (Harlan Teklad). Rats were I0134) Neuroprotective Effect given, once daily, 2 subcutaneous injections of vehicle (con- I0135 Table 3 and FIG.3 show the dose-dependent neuro trol), 17 B-estradiol (70 ug/kg BW), genistein (6 mg/kg BW), protective effects of four ERB-selective phytoestrogenic mol or phytoSERM combinations (6 mg/kg BW). Dosages used ecules against Supraphysiological glutamate (100 uM)-in here are commensurate with those used in humans. duced neurotoxicity in primary hippocampal neurons by 0127. Following the second injection, animals fasted for measurement of LDH release. "P-0.01 compared to vehicle 24 hours prior to sacrifice and brain dissection. Hippocampal alone-treated cultures; * P<0.05 and ** P-0.01 compared to and cortical tissues were collected from one hemisphere and glutamate alone-treated cultures. US 2012/0164122 A1 Jun. 28, 2012 15

ecules, when administered individually, are concentration TABLE 3 dependent and are protective against excitotoxic glutamate induced neurotoxicity in primary neurons, these effects are Dose-dependent effects of individual phytoSERMs against glutamate-induced neurotoxicity in primary moderate and arise from the weaker binding to the estrogen hippocampal neurons by LDH measurements receptor compared to the endogenous estrogen 173-estradiol (E2). FIG.3 demonstrates that co-administration of 3 or 4 of LDH Release (% of Control) these phytoestrogens afforded much greater neuroprotective efficacy compared to administration of single phytoestrogens Treatment Genistein or a combination of 2 phytoestrogens. Control 100.00 3.09 Glutamate alone 410.99 it 8.27f Expression of Anti-Apoptotic Proteins Bcl-2 and 1 nM 361.037.71*.* 10 nM 350.028.21** Bcl-XL 100 nM 347.24 16.96** 1 M 356.79 11.15** 0.138. These outcomes are paralleled by the results derived 10 M 377.848.45** from the western analyses of the expression of anti-apoptotic Treatment Daidzein proteins, Bcl-2 and Bcl-XL, in primary neurons. FIGS. 4A-4B Control 1OOOO 428 shows the effects on Bcl-2 and Bcl-XL expression in rat Glutamate alone 378.26 - 11.95* primary hippocampal neurons and hippocampal tissues 1 nM 338.39 - 16.49 derived from adult ovariectomized rats. Primary hippocam 10 nM 333.98 9.10 pal neurons grown for 7 divisions were treated with the test 100 nM 301.427.70** 1 M 3.18.49 15.92** compounds (or combinations) for 48 hr followed by Western 10 M 325.41 26.12* blot analyses. Adult ovariectomized rats were given, once Treatment Equol daily, 2 Subcutaneous injections of the test compounds (or combinations). Rats were sacrificed 24 h later following the Control 1OOOO 14.95 Glutamate alone 460.27+ 12.20" 2nd injection. Hippocampal tissues were homogenized fol 1 nM 453.S.O. 23.37 lowed by Western blot analyses. Combined formulations 10 nM 403.78 17.02* were composed of equivalent molar in (A) and equivalent 100 nM 331.599.67** weight in (B) of individual phytoSERMs including G: 1 M 381.80 - 12.01** 10 M 390.219.40** genistein; daidzein; E: equol; and I: IBSO003569. Treatment IBSO03569 0.139 Incubation of neurons with a combination of four phytoestrogens for 48 hours induces a significantly increased Control 1OOOO 2.05 expression of both proteins comparable to those induced by Glutamate alone 281.17 6.77f 1 nM 26241 - 10.60 E2. This is illustrated in FIG.4, which shows the effect of four 10 nM 270.86 - 12.94 ERB-selective phytoestrogenic molecules when co-adminis 100 nM 220.56 6.80** tered (100 nM for all four molecules) on the expression of the 1 M 246.307.70** anti-apoptotic proteins, Bcl-2 and Bcl-XL, in primary hippoc 10 M 307.532.62 ampal neurons. **P<0.01 compared to vehicle alone-treated “Primary hippocampal neurons grown for 7DIV were pretreated with the test phytoSERMs cultures. By comparison, a combination of two phytoestro at serially diluted concentrations for 48 h, followed by a 5-min exposure to 100 mM lutamate. The amount of LDH released into the culture media was measured 24 h later, gens is not sufficient to induce a significant increase in the Data are derived from a single experiment and are representative of at lease three indepen expression of both proteins, as also illustrated in FIGS. 4A dent experiments. Results are presented as the percent of LDH release from vehicletreated control cultures and expressed as means + S.E.M., n 6. and 4B. FP < 0.01 compared to vehicle-treated control cultures, 0140. Up-regulation of the Bcl-2 family anti-apoptotic *P < 0.05 and **P<0.01 compared to glutamate alone-treated cultures; P × 0.05 and "P-0.01 compared proteins have been associated with the neuroprotective to cultures treated with 10 nM phytoSERMs; "Pix 0.05 and "P-0.01 compared to cultures treated with 1 MphytoSERMs; P<0.05 and P - 0.01 compared to cultures treated with mechanism elicited by E2. These data indicate that a com 10 mM phytoSERMs. bined used of multiple ERB-selective phytoestrogens is effec tive to activate the neuroprotective mechanism leading to 0.136 FIG. 3 shows the neuroprotective efficacy of four improved neuronal Survival against neurodegenerative ERB-selective phytoestrogenic molecules when administered insults. Estrogen receptor interaction with p85/PI3K also alone at concentrations that elicited the maximal neuropro enhances pAkt, which phosphorylates the proapoptotic pro tective effects as revealed from the dose-response analyses tein Bcl-2-associated death protein (BAD) to prevent het (100 nM for all four molecules), or co-administered, against erodimerization with, and inactivation of, Bcl-2. In cortical Supraphysiological glutamate (100LLM)-induced neurotoxic neurons, estradiol induced pAkt translocation to the nucleus. ity in primary hippocampal neurons by measurement of cal Recent analyses indicate that estradiol, via the PI3K signaling cein AM staining. Results are presented in terms of neuro pathway, activates both the Akt and the ERK1/2 cascades in protective efficacy the same population of cortical and hippocampal neurons. NE(reatment glutamate) (control-glutamate)"100%, (1) Simultaneous activation of two pathways that prevent mito chondria from activating cell-death cascades is likely to pro where V, is the individual value from phytoestrogen mote neuron Survival. treated cultures, V is a mean value from glutamate alone-treated cultures, and V is a mean value from Increased Expression of the Anti-B-Amyloid Protein, vehicle-treated control cultures. "P-0.01 compared to IDE vehicle alone-treated cultures; * PK0.05 and **P<0.01 com pared to glutamate alone-treated cultures. 0141 FIG. 5 illustrates the effect of four ERB-selective 0137 Data presented in FIG. 3 and Table 3 demonstrate phytoestrogenic molecules when co-administered (100 nM that although the four ERB-selective phytoestrogenic mol for all four molecules) on the expression of the anti-B-amy US 2012/0164122 A1 Jun. 28, 2012

loid protein, insulin-degrading enzyme ("IDE') in primary where V, is the individual value from the test com hippocampal neurons. **P<0.01 compared to vehicle alone pounds (or combinations)-treated cultures, V., is a treated cultures. FIG. 5 demonstrates the effects of these mean value from glutamate or B-amyloid alone-treated various combinations of phytoestrogens along with E2 on the cultures, and V is a mean value from vehicle-treated expression of the anti-B-amyloid (anti-A?) protein, insulin control cultures. "PK0.01 compared to vehicle-treated con degrading enzyme (IDE) in primary neurons. Data showed trol cultures: *P<0.05 and **P<0.01 compared to glutamate that all three combinations composed of two, three or four or f3-amyloid, alone treated cultures;P<0.05 compared to phytoestrogens significantly increases IDE expression in E2-treated cultures; P-0.05 compared to genistein-treated neurons. Among them, a combination of three phytoestrogens cultures; "P-0.05 and "P<0.01 compared to combination induced the greatest neuronal response, with an efficacy (G+D)-treated cultures; "P-0.05 compared to combination (G+D+E+I)-treated cultures. Combined formulations were greater than E2 as well as a combination of two phytoestro composed of equivalent molar of individual phytoSERMs genS. included. G: genistein; D: daidzein; E: equol. I: IBSO03569. 0142. It is clear that one neuropathological hallmark of (0.148. Effect on IDE/NEP Expression AD is a significant deposition of extracellular AB peptide, as 014.9 FIGS. 8A-8C show the effects on insulin-degrading referred to AB plaque. Impaired AB clearance and/or degra enzyme (IDE)/neprilysin (NEP) expression in (A) rat primary dation has been demonstrated to contribute in part to AB hippocampal neurons and (B) hippocampal tissues derived plaque formation in AD brain. Besides degrading insulin and from adult ovariectornized rats. (A) Primary hippocampal several regulatory peptides, IDE, a metalloprotease enzyme, neurons grown for 7 DIV were treated with the test com has been demonstrated to play a key role in degrading AB pounds (or combinations) for 48 hr followed by Western blot peptide monomer in the brain. Choronic upregulation of IDE analyses. (B) Adult ovariectomized rats were given, once represents a novel efficacious therapeutic approach to lower daily, 2 Subcutaneous injections of the test compounds (or ing the steady-state AB level in the brain and eventually combinations). Rats were sacrificed 24 h later following the preventing the occurrence of Alzheimer-type pathology. second injection. Hippocampal tissues were homogenized Therefore, these data indicate that coadministration of mul followed by western blot analyses. Results are presented as tiple ERB-selective phytoestrogens have the potential to acti the fold increase in protein expression and expressed as the vate the anti-AB mechanism, and as a result, maintain the percent of control, n24. *P<0.05 and **P<0.01 compared to brain in a long-term healthy status. vehicle-treated control cultures or animals. P-0.05 and 0143 Upregulation of Spinophilin P<0.01 compared to E2 treated cultures; "P<0.01 com 014.4 FIG. 6 illustrates the effect of four ERB-selective pared to combination (G+D) or genisteintreated cultures; phytoestrogenic molecules when co-administered (100 nM 'PK0.05 compared to combination (G+D+E+I)-treated cul for all four molecules) on the expression of the spine marker, tures. Combined formulations were composed of equivalent spinophilin, in primary hippocampal neurons. **P<0.01 molar in (A) and equivalent weight in (B) of individual phy compared to vehicle alone-treated cultures. Spinophilin, a toSERMs including G: genistein; D: daidzein; E: equal; and I: protein that is enriched in the heads of neuronal dendritic IBSOO3569. spines, has been demonstrated to play a significant role in 0150. Effect on Forebrain Mitochondria modulating both dendritic morphology and glutamatergic 0151 FIGS. 9A-9E show the effects on forebrain mito synaptic activity. Upregulation of spinophilin has been cor chondrial cytochrome c oxidase (COX) activity in adult ova related with estrogen regulation of neuronal synaptic plastic riectomized rats. Rats were given, once daily, 2 subcutaneous ity. Therefore, theses results indicate that these phytoestrogen injections of the test compounds (or combinations). Rats were combinations are effective to promote neurotrophism, sacrificed 24 h later following the 2nd injection. Forebrain thereby Sustaining the brain staying in a synaptically active mitochondria were isolated followed by a spectrophotometric status, and prevent cognitive decline and memory loss. measurement of COX activity using an immunocapture 0145 Neuroprotection Against Glutamate method. Colorimetric absorbance at 550 nm was recorded 0146 FIGS. 7A-7D shows the neuroprotective efficacy of every 5 min for 115 min. COX activity is presented as the the compounds against glutamate (FIG. 7A) and amyloid initial rate of oxidation of reduced cytochrome c, and deter 42-induced neurotoxicity in rat primary hippocampal neu mined by calculating the initial slope between two time points rons. Primary hippocampal neurons grown for 7 divisions (<20 min) within the linear region. (Upper Panel) Time-lapse were pretreated with the test compounds (or combinations) change in absorbance; (Lower Panel) 96 increase in mito for 48 h, followed by a 5-min exposure to 100 mM glutamate. chondria COX activity, n24; *P<0.05 and **P<0.01 com Neurons were incubated for an additional 24 h prior to neu pared to vehicle-treated control animals: "P-0.05 compared ronal viability analyses by calcein AM staining. Following to genistein-treated animals. Combined formulations were pretreatment with the compounds (or combinations) for 48 hr, composed of equivalent weight of individual phytoSERMs neurons were exposed to 3 mM 3-amyloid for 2d. Neu including E2: 17bestradiol; G: genistein; D: daidzein; E: ronal viability was analyzed by fluorometric measurements equal; I: IBSO03569. 0152 FIGS. 10A-10E show the effects on forebrain mito ofactivities of the LDH and dead-cell protease released in the chondrial respiratory activity in adult ovariectomized rats. culture media, and the live-cell protease exclusively entering Rats were treated as above. Forebrain mitochondria were intact viable neurons. isolated followed immediately by a polygraphical measure 0147 Results are presented as neuroprotective efficacy ment of respiratory activity using an oxygen electrode. Fol (NE), which is defined as the percentage of neurotoxin-in lowing a basal recording, mitochondrial state 4 respiration duced toxicity prevented by the test compounds (or combi was measured following the addition of Substrates, malate/ nations) and quantitated by the equation: glutamate. State 3 respiration was measured following the NE(reatment-eurotoxin)/(control, eurotoxin)"100% addition of ADP Respiratory control ratio (RCR) was calcu US 2012/0164122 A1 Jun. 28, 2012 17 lated as the ratio between the rate of oxygen uptake at state 3 effective than single administrations and alternative com and the rate of oxygen uptake at state 4. (FIGS. 12A-12D) bined formulations. In particular, the present study suggests Time-lapse oxygen uptake: (FIG. 12E) '% increase in mito the potential of the combination of genistein, daidzein and chondrial respiratory activity, n24; *P<0.05 and *P<0.01 equol, at an equivalent weight, for prevention of neurodegen compared to vehicle-treated control animals; 'P<0.05 com eration and AD, along with management of climacteric Symp pared to genistein-treated animals. Combined formulations toms in postmenopausal women. were composed of equivalent weight of individual phy toSERMs including E2: 17b-estradiol; G: genistein; D: daid 0157 FIGS. 11A-11C are schematics showing estrogen Zein; E: equol; and I: IBSO03569; Mito: mitochondria; Mal/ mechanisms of action that lead to neurotrophic and neuro Glut: malate? glutamate. protective outcomes. 17-B-Estradiol (E2) acting via a mem 0153. Effect on Uterine Weight brane-associated site (mER) activates a cascade required for 0154 Table 4 shows the effects on uterine weight in adult multiple responses that lead to enhanced neural plasticity, ovariectomized rats. Changes in uterine weight in response to morphogenesis, neurogenesis, and neural Survival. The sig estrogenic stimulation can be used to evaluate the estrogenic naling sequence induced by E2 at the membrane site is as characteristics of test compounds on uterine tissues. In one follows: (1) E2 binding to mER, (2) E2-mER complexes with example, described below, immature female rats having low p85 to activate PI3K, (3) activating calcium-independent endogenous levels of estrogen are dosed with test compound PKC, (4) phosphorylating the L-type calcium channel, (5) (Subcutaneously) daily for 3 days. Compounds are formu inducing calcium influx, (6) activating calcium-dependent lated as appropriate for Subcutaneous injection. As a control, PKCS, (7) activating Src kinase, (8) activating the MEK/ 17-B estradiol is administered alone to one dose group. ERK 1/2 pathway, (9) ERK translocates to the nucleus, (10) Vehicle control dose groups are also included in the study. activating and phosphorylating CREB, (11) enhancing tran Twenty-four hours after the last treatment, the animals are Scription of antiapoptotic genes Bcl-2 and Bcl-X1, which necropsied, and their uteri excised, nicked, blotted and enhance mitochondrial vitality, and spinophilin, which weighed. Any statistically significant increases in uterine encourages synaptic growth, (12) simultaneously, estrogen weight in a particular dose group as compared to the vehicle activation of PI3K leads to activation of Akt, which phospho control group demonstrate evidence of estrogenicity. rylates and inhibits the proapoptotic protein BAD.

TABLE 4 Effects on uterine weight in adult ovariectomized rats Uterine Weight Treatment Wet Weight (mg) Increase (%) Dry Weight (mg) Increase (%) Control 127.62 10.75 O.OO 842 26.42 2.45 O.OO 927 (Vehicle) 17B-Estradiol 281.0632.00**P 120.23 - 25.07**P 46.70 - 4.13**P 76.74 15.63**EP (70 g/kg BW) Genistein 14.4.11 - 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 - 0.93 23.71 - 0.04 -1026. O.13 (6 mg/kg BW) G - D - E + I 146.99 - 1845 15.17 14.46 28.733.67 8.73 - 13.90 (6 mg/kg BW) Adult ovariectomized rats were given, daily once, 2 subcutaneous injections of the test compounds (or combinations) (n 24 for each group). Rats were sacrificed 24h later following the 2nd injection. Uteriwere immediately excised and a wet weight was corded Uteri were then air dried for 1 hour followed by at 70° C. overnight, and the dry weight was recorded. Increase in uterine weight compared with vehicle-treated control animals and expressed as the percent of control (set as 0), Combined formulations were composed of equivalent weight of individual phytoSERMs included for a total amount of6 mg/kg BW given to animals, G: genistein; D: daidzein; E: equol; I: DBSO03569. P:B-001 compared to any other treatment groups,

Summary 0158 Estrogen-induced neuroprotective mechanisms 0155 Both in vitro and in vivo analyses demonstrated that converge on mitochondria. Estrogen-activated cellular sig combined use of select test phytoSERMs provided signifi naling cascade promotes enhanced mitochondrial function, cantly increased efficacy in Sustaining neuronal Survival leading to increased calcium load tolerance, enhanced elec when challenged with neurotoxins, promoting expression of tron transport chain efficiency, and promotion of antioxidant proteins as key players in neuroprotection and metabolism/ defense mechanisms. These actions are mediated by the regu clearance off-amyloid in neurons/brain, and enhancing brain lation of both nuclear and mitochondrial encoded genes ini mitochondrial functions. In particular, combined use of tiated by the activation of second-messenger signaling cas genistein, daidzein and equolatan equivalent weight afforded cades. the maximal efficacy comparable or greater than 17b-estra 0159. These mechanisms and the data herein demonstrate diol in neuronal/brain assays. In contrast, Such a combination that, consistent with the healthy cell bias of estrogen benefit showed no impact on uterine weight, which however was hypothesis, selective molecules can be administered before markedly increased by 17b-estradiol. neurodegenerative insult while neurons are still healthy and 0156 The present study indicates that combined use of that phytoSERM exposure will lead to enhanced neural Sur select En-selective PhytoSERMs can be more therapeutically vival mechanisms, represented as mitochondria with Bcl-2 US 2012/0164122 A1 Jun. 28, 2012 additions, that promote neural defense against neurodegen receptor beta are selected from the group consisting of erative insults associated with age-associated diseases such as genistein, daidzein, equol. IBSO03569, and combinations Alzheimer's and Parkinson's. thereof. 0160 These studies exemplify the therapeutic promise of 22. The method of claim 20, wherein the formulation con select ERB-selective phytoestrogens when used in combina sists essentially of equal, genistein and daidzein. tion for Sustaining memory function and preventing age-re 23. The method of claim 20, wherein the formulation con lated neurodegenerative insults and AD. These ERB-selective sists essentially of equol, genistein, daidzein and phytoestrogen formulations, which optimize activation of IBSOO3569. 24. The method of claim 6, wherein the formulation com ERB while minimizing or avoiding activating ERC, should prising one or more excipients or carriers. serve as an effective estrogen alternative replacement therapy 25. The method of claim 6, wherein the formulation com for Sustaining neurological health, function and prevention of prising one or more additional active agents selected from the AD without induction of proliferative responses in the repro group consisting of antineoplastic agents, alkylating agents, ductive tissues as seen with the current ET/HT. Moreover, in antibiotics, antimetablites, anti-osteoporosis drugs, vitamins, light of the most recent data indicating that activation of ERB nutritional Supplements, antioxidants and coenzymes. significantly reduces both ApoE mRNA and protein expres 26. The method of claim 6, wherein the formulation is sion in neurons, ERB-selective phytoestrogen formulations formulated for enteral, parenteral, or topical administration. may serve as a particular viable strategy for reducing a major 27. A method for alleviating or preventing or treating neu risk factor of AD in ApoE4 carriers. rological diseases or disorders in a patient comprising admin 1-5. (canceled) istering to the patient an effective amount of a composition 6. A method for alleviating or preventing one or more comprising an active agent, wherein the active ingredient symptoms associated with estrogen deficiency in a patient consists of one or more phytoestrogens, analogues, or deriva comprising administering to the patient an effective amount tives thereof that selectively preferentially bind to estrogen ofa formulation consisting of two or more naturally occurring receptor B and not to estrogen receptor C. phytoestrogen compounds that selectively bind to estrogen 28. The method of claim 27, wherein the patient is a post menopausal woman. receptor beta and cross the blood brain barrier, in a pharma 29. The method of claim 27, wherein the one or more ceutically acceptable excipient, symptoms associated with estrogen deficiency comprises a wherein the two or more naturally occurring compounds neurological disease or disorder. are present in an effective amount to alleviate one or 30. The method of claim 29, wherein the neurological more symptoms of a menopausal woman. disease is a neurodegenerative disease. 7. The method of claim 6, wherein the patient is a post 31. The method of claim 30, wherein the neurodegenera menopausal woman. tive disease is cognitive deficit or memory loss. 8. The method of claim 6, wherein the one or more symp 32. The method of claim 30, wherein the neurodegenera toms associated with estrogen deficiency comprises a neuro tive disease is Alzheimer's disease. logical disease or disorder. 33. The method of claim 27, wherein the composition is 9. The method of claim 8, wherein the neurological disease administered in an effective amount to treat or prevent hot is a neurodegenerative disease. flashes. 10. The method of claim 9, wherein the neurodegenerative 34. The method of claim 27, wherein the one or more disease is cognitive deficit or memory loss. symptoms associated with estrogen deficiency comprises 11. The method of claim 9, wherein the neurodegenerative cognitive decline. disease is Alzheimer's disease. 35. The method of claim 27, wherein the one or more 12. The method of claim 6, wherein the formulation is symptoms associated with estrogen deficiency comprises administered in an effective amount to treat or prevent hot osteoporosis. flashes. 36. The method of claim 27, wherein one or more phy 13. The method of claim 6 wherein the one or more symp toestrogens, analogues, or derivatives thereof are selected toms associated with estrogen deficiency comprises cognitive from the group consisting of genistein, daidzein, equal, decline. IBSO03569, and combinations thereof. 14. The method of claim 6 wherein the one or more symp 37. The method of claim 27, wherein the composition toms associated with estrogen deficiency comprises comprises genistein and daidzein. osteoporosis. 38. The method of claim 37, wherein the composition 15. The method of claim 6, wherein one or more phy further comprises equol. toestrogens, analogues, or derivatives thereof are selected 39. The method of claim 38, wherein the composition from the group consisting of genistein, daidzein, equol. further comprises IBSO03569. IBSO03569, and combinations thereof. 40. The method of claim 27, wherein the composition 16. The method of claim 6, wherein the formulation com comprising one or more excipients or carriers. prises genistein and daidZein. 41. The method of claim 27, wherein the composition 17. The method of claim 16, wherein the formulation fur comprising one or more additional active agents selected ther comprises equol. from the group consisting of antineoplastic agents, alkylating 18. The method of claim 17, wherein the formulation fur agents, antibiotics, antimetablites, anti-osteoporosis drugs, ther comprises IBSO03569. Vitamins, nutritional Supplements, antioxidants and coen 20. The method of claim 6, wherein the formulation con Zymes. sists essentially of equol and one or more phytoestrogen 42. The method of claim 27, wherein the composition is compounds that selectively bind to estrogen receptor beta. formulated for enteral, parenteral, or topical administration. 21. The method of claim 20, wherein the one or more phytoestrogen compounds that selectively bind to estrogen c c c c c