US008552057B2

(12) United States Patent (10) Patent No.: US 8,552,057 B2 Brinton et al. (45) Date of Patent: *Oct. 8, 2013

(54) PHYTOESTROGENIC FORMULATIONS FOR OTHER PUBLICATIONS ALLEVATION OR PREVENTION OF Morito et al. Interaction of Phytoestrogens with Estrogen Receptors NEURODEGENERATIVE DISEASES a and b (II). Biol. Pharm. Bull. 25(1), pp. 48-52 (2002).* Kinjo et al. Interactions of Phytoestrogens with Estrogen Receptors a (75) Inventors: Roberta Diaz, Brinton, Rancho Palos and b (III). Biol. Pharm. Bull. 27(2) pp. 185-188 (2004).* Verdes, CA (US); Liqin Zhao, Los An, et al., "Estrogen receptor -selective transcriptional activity and Angeles, CA (US) recruitment of coregulators by phytoestrogens', J Biol. Chem. 276(21): 17808-14 (2001). (73) Assignee: University of Southern California, Los Avis, et al. Is there a menopausal syndrome'? Menopausal status and symptoms across racial/ethnic groups', Soc. Sci. Med., 52(3):345-56 Angeles, CA (US) (2001). Brinton, et al. Impact of estrogen therapy on Alzheimer's disease: a (*) Notice: Subject to any disclaimer, the term of this fork in the road?’ CNS Drugs, 18(7):405-422 (2004). patent is extended or adjusted under 35 Bromberger, et al., Psychologic distress and natural menopause: a U.S.C. 154(b) by 0 days. multiethnic community study’. Am. J. Public Health, 91 (9) 1435-42 (2001). This patent is Subject to a terminal dis Brookmeyer, et al., “Projections of Alzheimer's disease in the United claimer. States and the public health impact of delaying disease onset'. Am. J. Public Health, 88(9): 1337-42 (1998). (21) Appl. No.: 13/362,825 Cohen, et al. Risk for new onset of depression during the meno pausal transition: the Harvard study of moods and cycles Arch. Gen. Psychiatry. 63(4):385-90 (2006). (22) Filed: Jan. 31, 2012 Dasilva and Van Lier, “Synthesis and structure-affinity of a series of 7 alpha-undecylestradiol derivatives: a potential vector for therapy (65) Prior Publication Data and imaging of estrogen-receptor-positive cancers, J. Med, Chem. 33(1):430-4 (1990). US 2012/0164122 A1 Jun. 28, 2012 Espeland, et al., “Conjugated equine estrogens and global cognitive function in postmenopausal women: Women's Health Initiative Memory Study, JAMA, 291 (24):2959-68 (2004). Related U.S. Application Data Freeman, et al. Associations of hormones and menopausal status with depressed mood in women with no history of depression, Arch. (62) Division of application No. 1 1/777,951, filed on Jul. Gen. Psychiatry, 63(4):375-82 (2006). 13, 2007. Freeman, et al. Hormones and menopausal status as predictors of depression in women in transition to menopause Arch. Gen. Psy (60) Provisional application No. 60/819,849, filed on Aug. chiatry, 61 (462-70 (2004). 2, 2006, provisional application No. 60/889,920, filed Gao, et al., “The relationships between age, sex, and the incidence of on Feb. 14, 2007, provisional application No. dementia and Alzheimer disease: a meta analysis, Arch. Gen. Psy chiatry, 55(9):809-15 (1998). 60/943,190, filed on Jun. 11, 2007. Gustafsson, et al., “What pharmacologists can learn from recent advances in estrogen signalling, Trends Pharmacol Sci., 24(9):479 (51) Int. Cl. 85 (2003). A6 IK3I/35 (2006.01) Hadley, et al. The future of aging therapies. Cell, 120(4):557-67 (52) U.S. Cl. (2005). USPC ...... 514/454 Henderson, et al. Postmenopausal hormone therapy and Alzheimer's disease risk: interaction with age, J. Neural. NeuroSurg. (58) Field of Classification Search Psychiatry, 76(1): 103-5 (2005). USPC ...... 514/454 See application file for complete search history. (Continued) Primary Examiner — Jeffrey S. Lundgren (56) References Cited Assistant Examiner — Sara E Townsley U.S. PATENT DOCUMENTS (74) Attorney, Agent, or Firm — Pabst Patent Group LLP 5,952.374. A 9, 1999 Clarkson (57) ABSTRACT 6,335,038 B1 1/2002 Cavazza Select phytoestrogen pharmaceutical compositions and 2002/0001565 A1 1/2002 Shapiro methods of use for promoting neurological heath and preven 2004/OO72765 A1 4/2004 Kelly tion of age-related neurodegeneration, Such as AD, have been 2004/0106561 A1 6/2004 Kelly developed. These select phytoestrogen formulations are com 2005, 0004360 A1 1/2005 Gayo-Fung et al. posed of a number of plant-derived estrogenic molecules 2005/OO58709 A1 3, 2005 Fisher and/or their structural analogues and exhibit binding prefer 2005/0245492 A1 1 1/2005 Lephart ence to ERB over ERC. and agonist activity in the brain. These 2008. O108696 A1 5, 2008 Brinton et al. ERB-selective phytoestrogen formulations cross the blood brain-barrier and promote estrogen-associated neurotro FOREIGN PATENT DOCUMENTS phism and neuroprotections mechanisms in the brain, without EP O 149551 7, 1985 activating proliferative mechanisms in the reproductive tis JP 2OO1523258 11, 2001 Sues and are therefore devoid of other estrogen-associated JP 20O254.2286 11, 2002 problematic aspects. These are administered enterally, trans JP 2006504409 2, 2006 dermally, transmucosally, intranasally or parenterally, in a WO 9423716 10, 1994 WO 985OO26 11, 1998 dosage effective to prevent or alleviate neuronal damage, WO O064438 11, 2000 effect neuronal regeneration or Sustain viability, increase WO 02051821 T 2002 expression of anti-apoptotic proteins, and/or decrease indica WO 2004009035 1, 2004 tors of Alzheimer's Disease. WO 2005089567 9, 2005 6 Claims, 17 Drawing Sheets US 8,552,057 B2 Page 2

(56) References Cited Soares, et al. “Efficacy of estradiol for the treatment of depressive disorders in perimenopausal women: a double-blind, randomized, OTHER PUBLICATIONS placebo-controlled trial' Arch. Gen. Psychiatry, 58(6):529-34 Hogervorst, et al. Hormone replacement therapy for cognitive func (2001). tion in postmenopausal women, Cochrane Database Syst. Rev., Stauffer, et al. 'Pyrazole ligands: structure-affinity/activity relation 3):CD003122. (2002). ships and estrogen receptor-alpha-selective agonists' J. Med. Chem. http://www.ninds.nih.gov/disorders/disorder index.htm. National Institute of Neurological Disorders and Stroke website. 43(26):4934-4947 (2000). Kreijkamp-Kaspers, et al. Effect of soy protein containing Sun, et al. Molecular basis for the subtype discrimination of the isoflavones on cognitive function, bone mineral density, and plasma estrogen receptor-beta-selective ligand, diarylpropionitrile MoMot. lipids in postmenopausal women: a randomized controlled trial, EndocrinoL, 17(2):247-58 (2003). JAMA, 292(1):65-74 (2004). Usui, et al., Pharmaceutic& prospects of phytoestrogens. Endo Kuiper, et al., “Interaction of estrogenic chemicals and crine Journal, 53110-20 (2006). phytoestrogens with estrogen receptor”. Endocrinology, Vijg and Campisi, Puzzles, promises and a cure for ageing. Nature, 139(10):4252-63 (1998). 454 (7208):1065-71 (2008). Manson, et al. Postmenopausal hormonetherapy: new questions and Wakeling, et al., “A potent specific pure antiestrogen with clinical the case for new clinical trials'. Menopause, 13(1): 139-47 (2006). potential”, Cancer Res, 51:3867 (1991). Meyers, et al. Estrogen receptor-beta potency-selective ligands: Wassertheil-Smoller, et al. Depression and cardiovascular sequelae structure-activity relationship studies of diarylpropionitriles and in postmenopausal women. The Women's Health Initiative (WHI) their acetylene and polar analogues'.J.Med. Chem., 44(24):4230-51 Arch. Intern. Med., 164(3):289-98 (2004). (2001). Weihua, et al. Update onestrogen signaling, FEESLett., 546(1): 17 Morito, et al., “Interaction of phytoestrogens with estrogen receptors 24 (2003). a and ”, Biol. Pharm. Bull., 24(4):351-56 (2001). Yaffe, et al. “Estrogen therapy in postmenopausal women: effects on Morrison, et al. Lack of efficacy of estradiol for depression in cognitive function and dementia” JAMA, 279(9):688-95 (1998). postmenopausal women: a randomized, controlled trial Biol. Psy Zandi, et al. Hormone replacement therapy and incidence of chiatry, 15:55(4):406-12 (2004). Alzheimer disease in older women: the Cache County Study, JAMA, Mueller, et al. Phytoestrogens and Their Human Metabolites Show 288(17):2123-2129(2002). Distinct Agonistic and Antagonistic Properties on Estrogen Receptor Zhao, et al., “Neuroprotective and neurotrophic efficacy of a (ERa) and ERb in Human Cells. Tox. Sci. 80, pp. 14-25, 2004. phytoestrogens in cultured hippocampal neurons'. Exp. Biol. Med. North American Menopause Society. (2004) The menopause prac (Maywood), 27(7):509-19 (2002). tice: a clinician's guide htto://www.menopause.oro/aboutmenof Zhao, et al., “Design, synthesis, and estrogenic activity of a novel overview.htm. estrogen receptor modulator-a hybrid structure of 17beta-estradiol Olshansky, et al., Position statement on humanaging, J. Gerontol. A and vitamin E in hippocampal neurons, J. Med. Chem. Biol. Sci. Med. Sci., 57(8): B292-7 (2002). 50(18):4471-4481 (2007). Olshansky, et al., “No truth to the fountain of youth'. Scientific Zhao, et al., Estrogenic agonist activity of IC 1182,780 (Feslodex) in American, 92-95 (2002). hippocampal neurons: implications for basic science understanding Resnick, et al. Hormone therapy and risk of Alzheimer disease: a of estrogen signaling and development of estrogen modulators with a critical time, JAMA, 288(17):2170-2 (2002). dual therapeutic profile'. J. Pharmacol. Exp. Ther., 319(3):1124-32 Schmidt, et al. Alongitudinal evaluation of the relationship between (2006). reproductive status and mood in perimenopausal women, Am. J. Zhao, et al. 2004 Abstract Book: The Keystone Symposia: Nuclear Psychiatry, 161 (12):2238-44 (2004). Receptors: Steroid Sisters'. Keystone, CO; Feb. 2004. Schmidt, et al. Estrogen replacement in perimenopause-related Zweifel, et al. A meta-analysis of the effect of hormone replacement depression: apreliminary report, Am J ObstetGynecol., 183(2):414 therapy upon depressed mood Psychoneuroendocrinology, 20 (2000). 22(3): 189-212 (1997). Schmidt, et al. Mood, depression, and reproductive hormones in the Kinjo, "Phytoestrogens'.Nippon, Rinsho, 58(12):60-64 (2000). menopausal transition, Am. J. Med., 118 Suppl 12B:54-8 (2005). Hedlund, et al., “Prostatic fluid concentrations ofisoflavonoids in soy Shumaker, et al., “Conjugated equine estrogens and incidence of consumers are sufficient to inhibit growth of benign and malignant probable dementia and mild cognitive impairment in prostatic epithelial cells in vitro', Prostate, 66(5):557-66 (2006). postmenopausal women: Women's Health Initiative Memory Study.” JAMA, 291(24):2947-58 (2004). * cited by examiner U.S. Patent Oct. 8, 2013 Sheet 1 of 17 US 8,552,057 B2

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is -1 US 8,552,057 B2 1. 2 PHYTOESTROGENC FORMULATIONS FOR decline and neurodegenerative disorders, such as Alzhe ALLEVATION OR PREVENTION OF imer's disease (AD), while it is not an effective treatment NEURODEGENERATIVE DISEASES Strategy. The current widely prescribed ET, conjugated equine CROSS-REFERENCE TO RELATED estrogens (“CEE), is a highly complex ET with over 200 APPLICATIONS different components. Whether CEE provides the optimal therapeutic efficacy has been questioned. Another key issue This application is a divisional of prior application U.S. challenging HT is the optimal composition. The progestin Ser. No. 1 1/777,951 filed Jul. 13, 2007, entitled Phytoestrog and its timing of administration in conjunction with ET, 10 remains to be determined. Moreover, while ET/HT has long enie Formulations for Alleviation or Prevention of Neurode been used in postmenopausal women to delay or reverse some generative Diseases', by Roberta Diaz, Brinton and Liqin of the problems associated with menopause, epidemiological Zhao, which claims priority to U.S. Ser. No. 60/819,849 and clinical studies have uncovered potential long-term risks accorded a filing date of Aug. 2, 2006, U.S. Ser. No. 60/889, related to this therapy. The recently revealed risks associated 920 filed Feb. 14, 2007, and U.S. Ser. No. 60/943,190 filed 15 with ET/HT have greatly increased interest in the develop Jun. 11, 2007, all of which are herein incorporated in their ment of estrogen alternatives that promote beneficial effects entirety by reference. of estrogen in brain, bone and the cardiovascular system, while not eliciting deleterious effects in other organs, particu BACKGROUND OF THE INVENTION larly in breast and uterine tissues. Two nuclear receptors for estrogen (ERs), ERC. and ERB, The demographics Suggest that we face a devastating have been identified. In the central nervous system, both ERC. increase in the prevalence of AD, reinforcing the immediate and ERB are expressed in the hippocampus and cortex of need for basic and translational neuroscience to develop safe rodent and human brains. Previous studies have demonstrated and efficacious ET and HT regimens for the brain. Of those that both ERC. and ERB can equivalently promote neuronal affected with AD, 68% are female and 32% are male (Brook 25 Survival by activating estrogen mechanisms of action in rat meyer et al., 1998 Am J Public Health 88:13372). Because hippocampal neurons. Increasing evidence indicates that women have a longer life expectancy than men, the absolute ERB is a key requirement for activation of mechanisms that number of women with AD exceeds that of men. However, a underlie estrogen-inducible neuronal morphological plastic double danger exists for women. Results of a meta-analysis of ity, brain development, and cognition. ERO, on the other seven sex-specific studies concluded that women are 1.5 30 hand, is more predominant in mediating the sexual character times more likely to develop AD than age-matched men (Gao istics of estrogen effects in the reproductive organs such as et al., 1998 Arch Gen Psychiatry 55:809), which was sup breast and uterus. Taken together, these data establish a poten ported by the Cache County analysis that showed a clear tial therapeutic application for ERB as a pharmacological female gender increase in the incidence of AD (Zandi et al., target to promote memory function and neuronal defense 2002 JAMA 288:21239). 35 mechanisms against age-related neurodegeneration Such as At the turn of the new millennium in the United States, Alzheimer's disease (AD), while avoiding activating unto there were nearly 42 million women over the age of 50 years wardestrogenic proliferative effects in the breast and uterus, and, of these, more than 31 million women were over the age although this might be at the cost of lower efficacy due to the of 55 years (North American Menopause Society, 2004). lack of activation of ERB in the brain. Other potential thera Worldwide, there are currently more than 470 million women 40 peutic advantages associated with ERB include regulation of aged 50 years or older, and 30% of those are projected to live estrogen vasculoprotective action and development of inter into their 80s (North American Menopause Society, 2004). ventions targeting diseases Such as depression, colon cancer, These women can anticipate spending one-third to one-half prostate cancer, obesity, leukemia, and infertility. However, a of their lifetime in the menopausal state. Reports on preva potential disadvantage of an ERB-selective ligand is the lack lence of AD vary, but of the 18 million American women in 45 of activation of ERC. in bone, as ERC. has been demonstrated their mid to late 70s, as many as 5 million may suffer from to mediate estrogen regulation of bone density. AD, and this figure increases dramatically at older ages Although there is still controversy regarding the differen (Brookmeyer et al., 1998). The projected exponential tial roles of two estrogen receptor (“ER) subtypes, ERC. increase in the prevalence of AD, along with the anticipated and/or ERB, in mediating estrogen actions in the brain and/or impact on families and Society, highlights the imperative for 50 neurons, it has been widely demonstrated that ERB plays a developing strategies to prevent or delay the onset of AD key role in regulating brain development, neurogenesis and Sooner rather than later. estrogen-induced improved neuronal plasticity and Survival. The profound disparities between the largely positive basic In addition, as compared with ERO, ERB is less effective in Science findings of gonadal steroidal action in brain and the mediating the sexual characteristics of estrogen action in adverse outcomes of recent estrogen or hormone therapy 55 reproductive tissues, avoiding activating untowardestrogenic (“ET/HT”) clinical trials in women who are either aged post proliferative effects in the breast and uterus. Therefore, ERB menopausal or postmenopausal with Alzheimer's disease represents a potentially safer therapeutic target for promoting (AD), has led to an intense reassessment of gonadal hormone memory function and neuroprotection. However, this safety action and the model systems used in basic and clinical sci may be at the cost of lower efficacy, due to the lack of acti ence. One key factor that could contribute to the negative 60 vation of Ma in the brain. Other potential advantages for results of the Women's Health Initiative Memory Study ERB-target therapeutics arise from its regulation of estro (“WHIMS) trial was the advanced age, more than ten years gen's cardioprotective effects. ERB-selective ligands may following menopause, at which ET/HT was initiated in also provide effective therapeutics for preventing or treating women. Data from both basic science analyses and clinical inflammation, depression, anxiety, colon cancer, prostate studies indicate a “healthy cell bias” of estrogen action in the 65 cancer, obesity, leukaemia, and infertility. neurons/brains, suggesting that ET/HT acts as an effective In searching for an effective ERB-selective estrogen alter preventative therapeutic strategy for age-related cognitive native replacement therapy for promoting neurological func US 8,552,057 B2 3 4 tion and preventing age-related neurodegeneration, such as agonist may also account for the reduced efficacy exerted by AD, in postmenopausal women, it is of particular interest to the combination of both agonists. identify and develop naturally occurring molecules or ana Development of an ERB-selective phytoestrogen formula logues that potentially have a less toxic profile for long-term tion could maximize the therapeutic benefits associated with administration. It is known that several plant-derived estro activation of ERB in the brain while minimizing the adverse genic molecules (referred to as “phytoestrogens') bind to effects associated with the activation of ERC. in reproductive ERC. and to ERB subtypes, and some of these molecules tissues. Moreover, selective targeting of ERB potentially possess moderate binding selectivity for ERB and exert estro reduces antagonistic actions that may occur in a complex genic effects in multiple tissues. Soy-derived preparation. This naturally occurring ideal for The therapeutic efficacy of phytoestrogens in the brain 10 mulation would have tremendous therapeutic value in pro remains controversial. On the one hand, when administered moting neurological function and preventing AD in a popu singly, phytoestrogens appeared to be moderately neuropro lation at risk for losing neurological capacity and losing tective. On the other hand, a recent clinical trial revealed that memory function, i.e., postmenopausal women. To date, no a soy protein Supplement that contains a mixture of phy Such phytoestrogen formulation exists. Thus, there is a need 15 to discover and develop a novel select phytoestrogen formu toestrogens did not show improved cognitive function in post lation, generally, and particularly, a formulation that func menopausal women, when treatment was initiated at the age tions in the brain. of 60 years or older. The clinical trial of phytoestrogens It is therefore an object of the present invention to provide reported that a soy protein Supplement containing a complex an ERB-selective phytoestrogen formulation maximizing the formulation of isoflavones did not improve cognitive function therapeutic benefits associated with activation of ERB in the in postmenopausal women when treated at the age of 60 years brain while minimizing the adverse effects associated with or older, Kreijkamp-Kaspers, et al. JAMA 2004, 292, 65-74, the activation of ERC. in reproductive tissues. also indicating that when started 10 or more years following It is a further object of the invention to provide such a menopause in postmenopausal women when age-related neu composition wherein the active ingredients are isolated from ronal reorganization has taken place, ET/HT has no benefit on 25 natural Substances. neural function. Age and hormonal “history' may be impor tant factors that were responsible for these negative results, as SUMMARY OF THE INVENTION was the case for the WHIMS trials. Another issue that can Substantially impact the efficacy of Select phytoestrogen pharmaceutical compositions and a mixture of phytoestrogens action in the brain is the formu 30 methods of use for promoting neurological health and pre lation of phytoestrogens, since when administered alone, a vention of age-related neurodegeneration, such as AD, have number of phytoestrogens were protective to neurons from been developed. These select phytoestrogen formulations are neurodegenerative insults. Zhao, et al. Exp. Biol. Med. 2002, composed of a number of plant-derived estrogenic molecules 227, 509-519. Soy extracts or soy protein supplements gen and/or their structural analogues and exhibit binding prefer erally contain multiple phytoestrogenic molecules, some of 35 ence to ERB over ERC. and agonist activity in the brain. These which may be ERC.-selective agonists, while others may be ERB-selective phytoestrogen formulations cross the blood ERB-selective agonists, and others may be ineffective in acti brain-barrier and promote estrogen-associated neurotro vating either ERC. or ERB but may function as inhibitors of phism and neuroprotection mechanisms in the brain, without ER binding of those ERC. and/or ERB phytoestrogenic ago activating proliferative mechanisms in the reproductive tis nists. The ineffectiveness of a complex formulation of phy 40 Sues and are therefore devoid of other estrogen-associated toestrogens in promoting beneficial effects of estrogen in problematic aspects. The select phytoestrogen formulations brain, such as a Soy-derived preparation, may also arise from are therapeutically useful to both women and men for sus antagonizing actions among the different phytoestrogens, in taining neurological health and preventing age-related cog addition to the possible ER antagonism, likely from the acti nitive decline and neurodegenerative disorders, such as AD. vation of both ERC. and ERR in the same context. Co-admin 45 These are administered enterally, transdermally, transmu istration of an ERC-selective agonist and an ERB-selective cosally, intranasally or parenterally, in a dosage effective to agonist is less effective than treatment with either agonist prevent or alleviate neuronal damage, effect neuronal regen alone in various neuroprotective measurements. eration or Sustain viability, increase expression of anti-apop ERC. and ERB have a yin/yang relationship in many con totic proteins, and/or decrease indicators of Alzheimer's Dis texts where one receptor may antagonize the actions of the 50 ease. The formulations preferably contain combinations of other. Weihua, et al. FEESLett. 2003, 546, 17-24: Gustafsson, compounds, and can be formulated for daily, Sustained, J. A. Trends Pharmacol. Sci. 2003, 24, 479-485. Studies delayed or weekly/monthly administration. In a preferred confirmed this observation, showing that coadministration of embodiment, these are administered to women who are in ERC.-selective agonist PPT and ERB-selective agonist DPN menopause or post menopausal, most preferably early in was less efficacious than either PPT or DPN alone in protect 55 menopausal. ing hippocampal neurons against excitotoxic insults. Based on this analysis, a presumption can be made that the ineffec BRIEF DESCRIPTION OF THE DRAWINGS tiveness of administering a mixture of phytoestrogens (i.e. a soy protein Supplement) may partly come from the antago FIG. 1 shows the chemical Structures of 17 B-estradiol and nizing actions among different phytoestrogens, which may be 60 the phytoSERMs genistein, daidzein, equol, and IBSO03569. ERC. selective or ERB selective. These findings indicate that FIGS. 2A and 2B show the competition binding curves for although both ERC. and ERB contribute to estrogen promo ERC. and ERB (molar concentration versus fluorescence tion of neuronal survival, simultaneous activation of both ER polarization (mP)) of G, D, E, I or combinations: G+D, G+D+ subtypes, ERC. and ERB, in the same context may diminish E, or G+D+E+I. the efficacy. In addition, the different ratio and distinct func 65 FIG. 3 is a graph showing the neuroprotective efficacy of tion of homodimer and heterodimer induced by co-adminis four ERB-selective phytoestrogenic molecules when admin tration of an ERO-selective agonist and an ERB-selective istered alone at concentrations that elicited the maximal neu US 8,552,057 B2 5 6 roprotective effects as revealed from the dose-response analy insult while neurons are still healthy. NeuroSERM exposure ses (100 nM for all four molecules Genistein (G), Daidzein would lead to enhanced neural Survival mechanisms, repre (D), Equol (E) and IBSO03569 (I)), or co-administered: sented as mitochondria with Bcl-2 additions, that promote G+D, G+D+E, or G+D+E+I, against Supraphysiological neural defense against neurodegenerative insults associated glutamate (100 LM)-induced neurotoxicity primary hippoc with age-associated diseases such as Alzheimer's and Parkin ampal neurons by measurement of calcein AM staining. sons. Designer NeuroSERM molecules target the membrane FIGS. 4A and 4B are graphs showing the effect of four site of estrogen action, whereas PhytoSERM molecules pref ERB-selective phytoestrogenic molecules when co-adminis erentially target estrogen receptor?. Abbreviations: AMPAR, tered (100 nM) for all four molecules) as G+D, G+D+E, or AMPA receptor; C, cytochrome oxidase: F, F, ATPase sub G+D+E+I, on the expression of the anti-apoptotic proteins, 10 units; LTD. long-term depression; LTP. long-term potentia Bcl-2 and Bcl-XL, in primary hippocampal neurons. tion; NAD, nicotinamide adenine dinucleotide:NADH, nico FIG. 5 is a graph illustrating the effect of four ERB-selec tinamide adenine dinucleotide; VDCC, Voltage-dependent tive phytoestrogenic molecules when co-administered (100 calcium channel. nM for all four molecules), G+D, G+D+E, or G+D+E+I, on the expression of the anti-B-amyloid protein, insulin-degrad 15 DETAILED DESCRIPTION OF THE INVENTION ing enzyme ("IDE'), in primary hippocampal neurons. FIG. 6 is a graph illustrating the effect of four ERB-selec I. Definitions tive phytoestrogenic molecules when co-administered (100 “Estrogen Receptor, as used herein, refers to any protein nM for all four molecules): G+D, G+D+E, or G+D+E+I, on in the nuclear receptor gene family that binds estrogen, the expression of the spine marker, spinophilin, in primary including, but not limited to, any isoforms and variants hippocampal neurons. thereof. Human estrogen receptors include the alpha- and FIGS. 7A-7D are graphs shows the neuroprotective effi beta-isoforms (referred to herein as “ERC and “ERB'). cacy of G, D, E, and I, alone and in combination: G+D. “Estrogen Receptor Modulator, as used herein, refers to a G+D+E, or G+D+E+I, against (7A) glutamate- and (7B) compound that can act as an estrogen receptor agonist or B-amyloid1-42-induced neurotoxicity in rat primary hippoc 25 antagonist of an estrogen receptor or estrogen receptor iso ampal neurons, controls live/dead cells (7C); dead cells (7D). form having an ICs or ECso with respect to ERO, ERB and/or FIGS. 8A-8C are graphs showing the effects of G, D, E, and otherestrogen receptor isoforms of no more than about 50LM I, alone and incombination: G+D, G+D+E, and G+D+E+I, on as determined using the ERC., and/or ERB transactivation insulin-degrading enzyme (IDE) expression on neprilysin assay described herein. More typically, estrogen receptor (NEP) expression in hippocampal tissues derived from adult 30 modulators have ICs or ECso values (as agonists or antago ovariectomized rats. nists) of not more than about 10 uM. Representative com FIGS.9A-9E are graphs showing the effects of G, G+D+E, pounds are predicted to exhibit agonist or antagonist activity and G+D+E+I on forebrain mitochondrial cytochrome c oxi via an estrogen receptor. Compounds preferably exhibit an dase (COX) activity in adult ovariectomized rats. antagonistoragonist ICso or ECso with respect to ERC. and/or FIGS. 10A-10E are graphs showing the effects of G, G+D+ 35 ERB of about 10 uM, more preferably, about 500 nM, even E. and G+D+E+I on percent increase forebrain mitochondrial more preferably about 1 nM, and most preferably, about 500 respiratory activity in adult ovariectomized rats. pM, as measured in the ERC. and/or ERB transactivation FIGS. 11A-11C are schematics showing estrogen mecha assays. “ICso is that concentration of compound which nisms of action that lead to neurotrophic and neuroprotective reduces or inhibits the activity of a target (e.g., ERC. or ERB) outcomes. 11A, 17-f-Estradiol (E2) acting via a membrane 40 to half-maximal level. “ECs” is that concentration of com associated site (mER) activates a cascade required for mul pound which provides half-maximum effect. tiple responses that lead to enhanced neural plasticity, mor “Selective Estrogen Receptor Modulator” (or “SERM), as phogenesis, neurogenesis, and neural Survival. The signaling used herein, refers to a compound that exhibits activity as an sequence induced by E2 at the membrane site is as follows: agonist orantagonist of an estrogen receptor (e.g., ERO, ERB (1) E2 binding to mER, (2) E2-mER complexes with p85 to 45 or other estrogen receptor isoform) in a tissue-dependent or activate PI3K, (3) activating calcium-independent PKC, (4) receptor dependent manner. Thus, as will be apparent to those phosphorylating the L-type calcium channel, (5) inducing of skill in the biochemistry, molecular biology and endocri calcium influx, (6) activating calcium-dependent PKCS, (7) nology arts, compounds that function as SERMs can act as activating Src kinase, (8) activating the MEK/ERK 1/2 path estrogen receptor agonists in Some tissues, e.g., bone, brain, way, (9) ERK translocates to the nucleus, (10) activating and 50 and/or cardiovascular, and as antagonists in other tissue types, phosphorylating CREB, (11) enhancing transcription of anti e.g., the breast and/or uterine tissue. apoptotic genes Bcl-2 and Bcl-X1, which enhance mitochon "Phytoestrogen' refers to a naturally occurring compound drial vitality, and spinophilin, which encourages synaptic of plants, such as Soybeans, or plant products, such as whole growth, (12) simultaneously, estrogen activation of PI3K grain cereals, that acts like estrogen or binds to an estrogen leads to activation of Akt, which phosphorylates and inhibits 55 receptor. the proapoptotic protein BAD. 11B, Estrogen-induced neu As used herein, the term “NeuroSERM' refers to com roprotective mechanisms converge on mitochondria. Estro pounds that target the membrane site of estrogen action. gen-activated cellular signaling cascade promotes enhanced As used herein, the term “PhytoSERM' refers to natural mitochondrial function, leading to increased calcium load Source compounds that preferentially target estrogen receptor tolerance, enhanced electron transport chain efficiency, and 60 beta. promotion of antioxidant defense mechanisms. These actions As used herein, the term “analogue' refers to a chemical are mediated by the regulation of both nuclear and mitochon compound with a structure similar to that of another (refer drial encoded genes initiated by the activation of second ence compound) but differing from it in respect to a particular messenger signaling cascades. 11C, Conceptual schematic of component, functional group, atom, etc. NeuroSERM design and therapeutic use. Consistent with the 65 As used herein, the term "derivative' refers to compounds healthy cell bias of estrogen benefit hypothesis, selective which are formed from a parent compound by chemical reac molecules would be administered before neurodegenerative tion(s). US 8,552,057 B2 7 8 II. Compositions include diethylamine, ethylenediamine, ethanolamine, Compositions containing one or more phytoestrogens are diethanolamine, and piperazine. described herein. A number of phytoestrogens have been Appropriate carriers can be added that assist the com isolated and identified and additional analogs created, all of pounds to cross the blood-brain-barrier. which have estrogen receptor binding selectivity. In one 5 B. Additional Active Agents embodiment, of the composition contains two or more plant While the compounds can be administered as the sole derived estrogenic molecules and/or structural analogues, active pharmaceutical agent, they can also be used in combi which possess ERB-binding selectivity and exhibit neuropro nation with one or more other compound as described herein, tective activity when administered individually. These com and/or in combination with other agents used in the treatment positions are useful for preventing estrogen-deficiency asso 10 and/or prevention of estrogen receptor-mediated disorders. ciated symptoms and disorders, particularly age-related Alternatively, the compounds can be administered sequen cognitive decline and neurodegenerative diseases, such as tially with one or more Such agents to provide Sustained Alzheimer's disease (AD). therapeutic and prophylactic effects. Suitable agents include, A. PhytoSERMs 15 but are not limited to, other SERMs as well as traditional The compositions described herein contain one or more estrogen agonists and antagonists. phytoestrogens or natural Source selective estrogen receptor Representative agents useful in combination with the com modulators (SERMs) exhibiting a binding preference for pounds for the treatment of estrogen receptor-mediated dis ERB. PhytoSERMs can be identified as described in Example orders include, for example, tamoxifen, 4-hydroxytarnox 1. Suitable phytoSERMs include, but are not limited to, ifen, raloxifene, toremifene, droloxifene, TAT-59, idoxifene, genistein, daidzein, equol. IBSO03569 and combinations RU 58,688, EM 139, ICI 164,384, ICI 182,780, clomiphene, thereof. The structures of genistein, daidzein, equol, and MER-25, DES, nafoxidene, CP-336,156, GW5638, LY IBSO03569 are shown in FIG.1. Others are listed in Table 1. 139481, LY353581, Zuclomiphene, enclomiphene, ethamox Preferred compounds cross the blood brain barrier. ytriphetol, delmadinone acetate, bisphosphonate. Other As demonstrated by Example 2, combinations of two or 25 agents that can be combined with one or more of the com more PhytoSERMS are more effective than administration of pounds include aromatase inhibitors such as, but not limited one PhytoSERM. to, 4-hydroxymdrostenedione, plomestane, exemestane, ami The compounds can be used in the form of salts derived nogluethimide, rogletimide, fadrozole, Vorozole, letrozole, from inorganic or organic acids. These salts include, but are and anastrozole. not limited to, the following: acetate, adipate, alginate, cit 30 Still other agents useful in combination with the com rate, aspart ate, benzoate, benzenesulfonate, bisulfate, pounds described herein include, but are not limited to anti butyrate, camphorate, camphorsulfonate, digluconate, cyclo neoplastic agents, such as alkylating agents, antibiotics, hor pentanepro-pionate, dodecylsulfate, ethanesulfonate, gluco monal antineoplastics and antimetablites. An example heptanoate, glycerophosphate, hemi-sulfate, heptanoate, includes the compounds used to treat or prevent osteoporosis. hexamate, fumarate, hydrochloride, hydrobromide, hydroio 35 Other ingredients include vitamins, nutritional Supplements, dide, 2-hydroxyethanesulfonate, lactate, maleate, methane anti-oxidant agents, coenzymes, etc. Sulfonate, nicotinate, 2-napthalenesulfanate, oxalate, pamo The additional active agents may generally be employed in ate, pectinate, Sulfate, 3-phenylpropionate, picrate, pivalate, therapeutic amounts as indicated in the PHYSICIANS propionate. Succinate, tartrate, thiocyanate, p-toluene DESK REFERENCE (PDR) 53rd Edition (2003), or such Sulfonate and undecanoate. Also, any basic nitrogen-contain 40 therapeutically useful amounts as would be known to one of ing groups can be quaternized with agents such as lower alkyl ordinary skill in the art. The compounds and the other thera halides, such as methyl, ethyl, propyl, and butyl chloride, peutically active agents can be administered at the recom bromides, and iodides; dialkylsulfates like dimethyl, diethyl, mended maximum clinical dosage or at lower doses. Dosage dibutyl, and diamylsulfates, long chainhalides Such as decyl levels of the active compounds in the compositions may be lauryl, myristyl and Stearyl chlorides, bromides and iodides, 45 varied to obtain a desired therapeutic response depending on aralkyl halides like benzyl and phenethyl bromides, and oth the route of administration, severity of the disease and the ers. Wafer or oil-soluble or dispersible products are thereby response of the patient. The combination can be administered obtained. as separate compositions or as a single dosage form contain Examples of acids which may be employed to form phar ing both agents. When administered as a combination, the maceutically acceptable acid addition salts include such inor 50 therapeutic agents can be formulated as separate composi ganic acids as hydrochloric acid, Sulfuric acid, and phospho tions that are given at the same time or different times, or the ric acid, and organic acids Such as Oxalic acid, maleic acid, therapeutic agents can be given as a single composition. Succinic acid and citric acid. Basic addition salts can be C. Pharmaceutical Compositions prepared in situ during the final isolation and purification of The compounds can be administered enterally, transder the compounds, or separately by reacting carboxylic acid 55 mally, transmucisally, intranasally or parenterally. Excipients moieties with a suitable base such as the hydroxide, carbonate for oral formulation are known to those skilled in the art, as orbicarbonate of a pharmaceutically acceptable metal cation discussed briefly below, and can be used to provide immedi or with ammonia, oran organic primary, secondary or tertiary ate, Sustained, delayed, or pulsed release. The compounds can amine. Pharmaceutically acceptable salts include, but are not also be administered via a transdermal patch, a depo, vagi limited to, cations based on the alkali and alkaline earth 60 nally or rectally using a topical carrier Such as a gel, lotion, metals, such as Sodium, lithium, potassium, calcium, magne ointment, liposomal formulation, Suspension, foam, spray or sium, and aluminum salts, as well as non-toxic ammonium, Suppository, via the pulmonary or nasal route, buccally or quaternary ammonium, and mine cations, including, but not sublingual via the mucosal membranes of the mouth. The limited to ammonium, tetramethylammonium, tetraethylam appropriate excipients for all of these formulations are monium, methylamine, dimethylamine, trimethylamine, tri 65 known. The compounds may be dissolved or Suspended in ethylamine, ethylamine, and the like. Other representative saline, sterile water orphosphate buffered saline, or a suitable organic amines useful for the formation of base addition salts oil for injection iv, im, Subcu, or ip. US 8,552,057 B2 9 10 Suitable pharmaceutically acceptable excipients include agents such as magnesium Stearate. In the case of capsules, processing agents and drug delivery modifiers and enhancers, tablets, and pills, the dosage forms may also comprise buff Such as, for example, calcium phosphate, magnesium Stear ering agents. Tablets and pills can additionally be prepared ate, talc, monosaccharides, disaccharides, starch, gelatin, cel with enteric coatings. lulose, methyl cellulose, Sodium carboxymethyl cellulose, Liquid dosage forms for oral administration may include dextrose, hydroxypropyl-..beta.-cyclodextrin, polyvinylpyr pharmaceutically acceptable emulsions, Solutions, Suspen rollidone, low melting waxes, and ion exchange resins, as sions, syrups, and elixirs containing inert diluents commonly well as combinations of any two or more thereof. Other suit used in the art, such as water. Such compositions may also able pharmaceutically acceptable excipients are described in comprise adjuvants, such as wetting agents, emulsifing and Remington’s Pharmaceutical Sciences, Mack Pub.Co., New 10 Suspending agents, cyclodextrins, and Sweetening, flavoring, Jersey (1991). Pharmaceutical compositions containing estrogen receptor and perfuming agents. modulating compounds may be in any form Suitable for the The compounds can also be administered in the form of intended method of administration, including, for example, a lipsomes. As is known in the art, liposomes are generally Solution, a suspension, or an emulsion. Liquid carriers are 15 derived from phospholipids or other lipid substances. Lipo typically used in preparing solutions, Suspensions, and emul Somes are formed by mono- or multilamellar hydrated liquid sions. Liquid carriers contemplated for use include, for crystals that are dispersed in an aqueous medium. Any non example, water, Saline, pharmaceutically acceptable organic toxic, physiologically acceptable and metabolizable lipid Solvent(s), pharmaceutically acceptable oils or fats, as well as capable of forming liposomes can be used. The present com mixtures of two or more thereof. The liquid carrier may positions in liposome form can contain, in addition to a com contain other Suitable pharmaceutically acceptable additives pound, stabilizers, preservatives, excipients. The preferred Such as solubilizers, emulsifiers, nutrients, buffers, preserva lipids are the phospholipids and phosphatidylcholines (leci tives, Suspending agents, thickening agents, Viscosity regula thins), both natural and synthetic. Methods to form liposomes tors, or stabilizers. Suitable organic solvents include, for are known in the art (Prescott 1976). example, monohydric alcohols, such as ethanol, and polyhy 25 Transdermal patches are well known for delivery of nico dric alcohols, such as glycols. Suitable oils include, for tine, nitroglycerin and birth control. These can be utilized example, soybean oil, coconut oil, olive oil, safflower oil, with these formulations as well. Depos that are implanted cottonseed oil. For parenteral administration, the carrier can under the skin orip can also be used, similarly to the manner also be an oily ester Such as ethyl oleate, isopropyl myristate. of delivering birth control. Compositions may also be in the form of microparticles, 30 microcapsules, liposomal encapsulates, as well as combina III. Methods of Administration tions of any two or more thereof. Compounds can be administered in a variety of ways The compounds may be administered orally, parenterally, including enteral, parenteral, pulmonary, nasal, mucosal and Sublingually, by inhalation spray, rectally, vaginally, or topi other topical or local routes of administration. For example, cally in dosage unit formulations containing conventional 35 Suitable modes of administration include oral, Subcutaneous, nontoxic pharmaceutically acceptable carriers, adjuvants, transdermal, transmucosal, iontophoretic, intravenous, intra and vehicles as desired. Topical administration may also muscular, intraperitoneal, intranasal, Subdural, rectal, vaginal involve the use of transdermal administration Such as trans and inhalation. dermal patches or ionophoresis devices. The term parenteral An effective amount of the compound or composition is as used herein includes Subcutaneous injections, intravenous, 40 administered to treat and/or prevent an estrogen receptor intramuscular, intrasternal injection, or infusion techniques. mediated disorder in a human or animal Subject. Modulation Injectable preparations, for example, Sterile injectable of estrogen receptor activity results in a detectable Suppres aqueous or oleaginous Suspensions may be formulated sion or up-regulation of estrogen receptor activity either as according to the known art using Suitable dispersing or wet compared to a control or as compared to expected estrogen ting agents and Suspending agents. The sterile injectable 45 receptor activity. Effective amounts of the compounds gen preparation may also be a sterile injectable solution or Sus erally include any amount sufficient to detectably modulate pension in a nontoxic parenterally acceptable diluent or Sol estrogen receptor activity by any of the assays described vent, for example, as a solution in 1,3-propanediol. Among herein, by other activity assays known to those having ordi the acceptable vehicles and solvents that may be employed nary skill in the art, or by detecting prevention and/or allevia are water, Ringer's Solution, and isotonic sodium chloride 50 tion of symptoms in a Subject afflicted with an estrogen recep Solution. In addition, Sterile, fixed oils are conventionally tor-mediated disorder. employed as a solvent or Suspending medium. For this pur The effective amount will also be determined based on pose, any bland fixed oil may be employed including Syn when the compounds are administered. Estrogen/hormone thetic mono- or diglycerides. In addition, fatty acids such as therapy (ET/HT) has been associated with the reduced risk of oleic acid can be useful in the preparation of injectables. 55 developing AD when treated at the menopausal transition in Suppositories for rectal or vaginal administration of the women Brinton, R. D. Impact of estrogen therapy on Alzhe drug can be prepared by mixing the drug with a suitable imer's disease: a fork in the road'? CNS Drugs 2004, 18, nonirritating excipient such as cocoa butter and polyethylene 405-422. For example, results of the Cache County Study glycols that are solidat ordinary temperatures but liquid at the indicate that women who receive ET/HT at the time of meno rectal temperature and will therefore melt in the rectum and 60 pause and continue for 10 years have a 3-fold lower risk of release the drug. developing AD, Zandi, et al. JAMA 2002, 288, 2123-2129, Solid dosage forms for oral administration may include whereas the recent data from the Women's Health Initiative capsules, tablets, pills, powders, and granules. In Such solid Memory Study indicate that women who begin the therapy dosage forms, the active compound may be admixed with at late in menopause have a greater risk of developing AD, least one inert diluent such as Sucrose lactose or starch. Such 65 Espeland, et al. Women's Health Initiative Memory Study. dosage forms may also comprise, as is normal practice, addi JAMA 2004, 291, 2959-2968; Shumaker, et al., JAMA 2004, tional Substances other than inert diluents, e.g., lubricating 291, 2947-2958. These clinical observations are consistent US 8,552,057 B2 11 12 with basic science analyses of estrogen-inducible molecular ing the menopause transition and hot flushes: both occur at mechanisms in the brain, indicating a healthy cell bias of this stage of life, but depression is not simply caused by hot estrogen action. flushes (Avis et al., 2001 Soc SciMedS2:345). Second, recent Estrogen receptor-mediated disorders that may be treated longitudinal studies that followed women with no past history include any biological or medical disorder in which estrogen of depression demonstrated an increased risk of first-onset receptor activity is implicated or in which the inhibition of depressions during the late menopause transition (Schmidt et estrogen receptor potentiates or retards signaling through a al., 2004 Am J Psychiatry 161:22384; Cohen et al., 2006 Arch pathway that is characteristically defective in the disease to be Gen Psychiatry 63:385; Freeman et at, 2006 Arch Gen Psy treated. The condition or disorder may either be caused or chiatry 61:62). Finally, both major and minor depressions are characterized by abnormal estrogen receptor activity. Repre 10 clinically significant to women at midlife, because both are sentative estrogen receptor-mediated disorders include, for associated with an increased risk for several other medical example, osteoporosis, atherosclerosis, estrogen-mediated conditions (Wassertheil-Smoller et al., 2004 Arch Intern Med cancers (e.g., breast and endometrial cancer), Turner's Syn 164:289) relevant to the health of women at midlife (e.g., drome, benign prostate hyperplasia (i.e., prostate enlarge cardiovascular disease, dementia, and the metabolic Syn ment), prostate cancer, elevated cholesterol, restenosis, 15 drome). endometriosis, uterine fribroid disease, hot flashes, and skin The majority of women do not develop depression during and/or vagina atrophy. Other estrogen receptor-mediated the menopause transition, and, therefore, reproductive aging conditions that may be treated include neurological diseases is not uniformly associated with either depressive symptoms and disorders including memory loss and dementia, and neu or the syndrome of depression. Nonetheless, despite numer rodegenerative disease, including Alzheimer's disease. ous studies concluding that the menopause is not associated In addition to the potential beneficial effects of estrogen on with an increased risk for developing depression in women, episodic memory, some evidence Suggests that HT reduced several other longitudinal, community-based studies reported the risks of both dementia (including AD) and mild cognitive an association between the menopause transition and an impairment (MCI). MCI is a condition thought to representa increased risk for depression (Schmidt, 2005 Am J Med 118: transitional state between normal cognition and dementia in 25 54). Indeed, five recent longitudinal studies all have docu some individuals, with a 12% conversion rate from MCI to mented an increased risk for depression during the meno dementia each year. Observational studies repeatedly docu pause transition, with odds ratios ranging from 1.8 to 2.9 ment that women taking HT enjoy an 30% reduced risk for compared with the premenopause (Bromberger et al., 2001 dementia compared with women not taking HT odds ratio Am J Public Health91: 14352: Freeman et al., 2004 Arch Gen range, 0.306 (Yaffe et al., 1998JAMA279:688; Hogervorstet 30 Psychiatry 61:62, 2006 Arch Gen Psychiatry 63:375; al., 2003 Cochrane Database Syst Rev CD003122). Thus, Schmidt et al., 2004 Am J Psychiatry 161:22384; Cohen et al., observational studies suggest that declining reproductive 2006 Arch Gen Psychiatry 63:385). These data suggest that function could be a modifiable risk factor for dementia or that events Surrounding the final menstrual period may predispose HT/ET could serve a protective role against some of the risks Some women to develop clinically significant depressive ill for developing dementia. 35 ness. Although several factors could precipitate depression in Several recent observational studies have identified that the these women, endocrine events are Suggested by the stage of stage of reproductive aging at which HT/ET is started modi the menopause transition (i.e., late) during which depressions fies the risk of dementia. In these studies, women who take appeared. The late transition is characterized by more pro HT/ET during the late menopause transition or early post longed hypogonadism than the early perimenopause, during menopause have a lower risk of dementia than those starting 40 which estradiol Secretion may be increased. Thus, the timing HT/ET later (Zandiet al., 2002 JAMA288:21239; Henderson of appearance of the depressions observed suggest an endo et al., JNeurol Neurosurg Psychiatry 76:103 2005). Thus, the crine mechanism related to the perimenopause (estradiol timing of starting HT/ET relative to the menopause has been withdrawal and/or recent-onset of prolonged hypogonadism) proposed to be one factor explaining the otherwise discordant in the pathophysiology of perimenopausal depression. observations between the observational studies and the RCTs 45 Efforts to investigate the potential role of declining ovarian (Resnick and Henderson, 2002 JAMA 288:21702; Mansonet hormone secretion in the onset of depression have examined al., 2006 Menopause 13:139). Recent preclinical studies the effects on mood of administering HT/ET in women with reviewed below highlight the importance of timing of ET in perimenopausal and postmenopausal depression. The antide this report. pressant efficacy of estradiol has been examined in three Successful treatment of a subject may result in the preven 50 relatively recent RCTs of women meeting standardized diag tion, inducement of a reduction in, or alleviation of symptoms nostic criteria for major and minor depression, who were in a Subject afflicted with an estrogen receptor-mediated randomly assigned to enter double-blind, placebo-controlled medical or biological disorder. Thus, for example, treatment trials (Schmidt et al., 2000; Soares et al., 2001 Arch Gen can result in a reduction in breast or endometrial tumors Psychiatry 58:529; Morrison et al., 2004 Biol. Psychiatry and/or various clinical markers associated with Such cancers. 55 55:406). In perimenopausal women, short-term administra Treatment of Alzheimer's disease can result in a reduction in tion (3 weeks) of estradiol significantly decreased depression rate of disease progression, detected, for example, by mea scores compared with both baseline and placebo conditions. Suring a reduction in the rate of increase of dementia. In one study, a full or partial therapeutic response was seen in Historically, there has been a presumption that declining 80% of perimenopausal women on estradiol compared with reproductive function plays no role in the onset of mood 60 22% of those on placebo (Schmidt et al., 2000). The efficacy disorders that occur during midlife in women. The symptoms of ET in perimenopausal depression is consistent with the of depression during the menopause transition also were observed effect size (0.69) in a recent meta-analysis of studies assumed to be transient and of Such minor severity that they examining the effects of estrogen on mood (Zweifel and were dismissed to be of little clinical consequence. Recent O'Brien, 1997 Psychoneuroendocrinology 22:189). The studies, however, Suggest that these presumptions are incor 65 therapeutic response to estradiol was observed in both major rect. First, several community-based longitudinal studies and minor depression as well as in women with and without have reported the relative independence of depressions dur hot flushes. Thus, the efficacy of ET in perimenopausal US 8,552,057 B2 13 14 depression is not solely a product of its ability to reduce the screening were assessed for their binding profiles to both distress of hot flushes. In contrast to these studies in peri ERs, three of which displayed over 100-fold binding selec menopausal depression, the administration of estradiol under tivity to ERB over ERC. similar conditions failed to improve mood in depressed Materials and Methods women who were 5 years postmenopause (Morrison et al., Identification of Molecules 2004). Thus, the effects of estradiol on depression may be Identification of Compounds in Database limited to perimenopausal women. Additionally, as with the All computational work was performed on a SOT Octane potential effects of estrogen on the course of dementia, the workstation equipped with the IRIX 6.5 operating system stage of reproductive aging at which women present and/or (Silicon Graphic Inc.). First, the 3D crystallographic struc 10 ture of human ERB LBD complexed with genistein was commence ET might modify the observed outcomes. downloaded from the Protein Data Bank (PDB ID: 1GKM). In Summary, the majority of women do not develop depres The complex structure was fixed and energy minimized with sion during or after the menopause transition. Nevertheless, the Accelrys molecular modeling software package Insight I recent prospective studies monitoring both reproductive sta 2000 (Accelrys Inc.). An in-house 2D natural source chemi tus and mood have documented that, for Some women, peri 15 cal collection containing approximately 25 000 plant-based menopause-related events increase the risk for the onset of natural molecules or derivatives was converted to a 3D mul depression. The role of ovarian function in these episodes of ticonformational database with the Accelry's modeling soft depression is suggested by both the timing of their onset ware package Catalyst 9.8 (Accelrys Inc.). relative to the last menstrual period and the antidepressant The receptor-docking site was defined based on the binding efficacy of short-term ET. position of genistein in the receptor and specified as all atoms The amount of active ingredient that may be combined within 10 A of the center carbon of genistein. GOLD 2.0 with the carrier materials to produce a single dosage form will (Genetic Optimization for Ligand Docking), an automated vary depending upon the estrogen-mediated disease, the host ligand docking program distributed by CCDC (Cambridge treated and the particular mode of administration. It will be Crystallographic Data Center), was applied to calculate and understood, however, that the specific dose level for any par 25 rank the molecules based on their complementarities with the ticular patient will depend upon a variety of factors including receptor binding site, on both geometrical and chemical fea the activity of the specific compound employed, the age, body tures. weight, general health, sex, diet, time of administration, route Prior to the database screening, initial validation using of administration, rate of excretion, drug combination, and genistein as the test ligand was conducted. The aim of the the severity of the particular disease undergoing therapy. The 30 validation test was to evaluate the effectiveness of the algo prophylactically or therapeutically effective amount for a rithm of the docking program in identifying the experimen given situation can be readily determined by routine experi tally observed binding mode of the ligand in the receptor, to mentation and is within the skill and judgment of the ordinary determine whether the program is applicable to the specific clinician. target system in this study. In addition, the validation test was 35 used to determine the optimal parameter settings for the later For exemplary purposes, a prophylactically or therapeuti database screening. Twenty docking runs were carried out on cally effective dose will generally be from about 0.01 mg/kg/ the test complex, using the fastest default generic algorithm day to about 100 mg/kg/day, preferably from about 0.1 parameters optimized for virtual library Screening, and the mg/kg/day to about 20 mg/kg/day, and most preferably from GoldScore fitness function was applied. The validation test about 1 mg/kg/day to about 10 mg/kg/day of aestrogen recep 40 demonstrated that, based on the specified parameter settings, tor modulating compound, which may be administered in one GOLD was effective in capturing the contributive hydrogen or multiple doses. bond donor (ND1 in His 475) crucial to the binding and IV. Kits reproducing the nearly coincident Solution in terms of both Kits may be provided which contain the formulation to be the binding orientation and conformation of genistein as administered. The formulation may be administered once a 45 observed in the experimental measurement (see FIG. 1). The day or more than once a day. The formulation can be admin root-mean-square (RMS) deviations were computed between istered enterally, parenterally, or topically. The kits typically the observed experimental position and the GOLD solutions, contain the active agent(s) to be administered, excipients and with RMSD 0.3299 and 0.4483 compared to top-ranked and carriers, and instructions for administration of the formula worst solutions, respectively. The average RMSD of all solu tion. The kits may also contain equipment/devices used to 50 tions was 03566, which is regarded as a good prediction based administer the formulation, Such as Syringes. on the Subjective classifications defined by the program The present invention will be further understood by refer developer (refer to the program manual), Suggesting that this ence to the following non-limiting examples. program is reliable and applicable to the database screening toward ERB. EXAMPLES 55 Using the parameter settings determined in the validation test, the 3D natural source chemical database was input and Example 1 docked into the prepared ERB binding site in a flexible dock ing manner (full ligand and partial protein) and scored based Identification of PhytoSERMS on the GoldScore fitness function. Five hundred resultant 60 top-scoring molecules were filtered via visual screening in ERB has been associated with estrogen-induced promotion the context of the receptor in Insight|I. Based on visual analy of memory function and neuronal Survival. Based on the sis, 100 molecules underwent further analysis by Affinity, a optimized complex structure of human ERB LBD bound with more complex and predictive ligand docking program to genistein, computer-aided structure-based virtual screening refine the binding modes predicted by GOLD. The criteria against a natural Source chemical database was conducted to 65 used for the selection of candidate molecules for investigation determine the occurrence of plant-based ERB-selective included the following (a) formation of hydrogen bond with ligands. Twelve representative hits derived from database donoratom ND1 in His 475; (b) hydrophobic and hydrophilic US 8,552,057 B2 15 16 balance appearing in the structure (e.g., the molecule should baculovirus-expressed human ERB or ERC. and a fluorescent potentially have two relatively hydrophilic sides and a hydro estrogen ligand EL Red (PanVera Corp.). Test molecules phobic center to enhance both the steric and electrostatic were serially diluted to a 2x concentration in assay buffer complementarity with the receptor); (c) bound pose of the (200 uM to 200 uM). Fifty microliters of preincubated 2x molecule in the receptor; and d) structural diversity. Finally, complex of ERB (30 nM) or ERB (60 nM) and EL Red (2nM) molecules that met the above criteria were computationally was added to each well in a 96-well Non-binding Surface predicted for their drug-likeness (Lipinski’s Rule of Five) and black microplate (Corning Life Sciences) for a final volume blood-brain barrier (BBB) penetration properties. of 100 uL. Negative controls containing ER and EL Red The ligand binding domains of the human ERC. and ERB (equivalent to 0% inhibition) and positive controls containing are approximately 60% homologous. Structural modeling 10 and mutational analyses indicate that two variant amino acid only free EL Red (equivalent to 100% inhibition) were residues along the ligand binding pocket, Leu 384 and Met included. After a 2-h incubation period at room temperature, 421 in ERC, which are replaced with Met 336 and Ile 373, the polarization values were measured using a Tecan GENios respectively, in ERB, are the key molecular constituents Pro reader at 535 nm/590 nm excitation/emission and plotted underlying discriminative binding of selective ligands to 15 against the logarithm of the test molecule concentration. ICso either receptor subtypes. Sun, et al. Mol. Endocrinol. 2003, values (concentration of test molecule that displaces half of 17, 247-258. This slight structural variance serves as the the EL Red from ER) were determined from the plot using a foundation for both design and discovery of ER specific nonlinear least-squares analysis. ligands. The similarities in the chemical features of both pairs Results of residues presents a Substantial challenge to discover a 31 molecules that can form a hydrogen bond with ND1 in selective ligand based on this difference. Of the known natu His 475 were selected and grouped into three categories based ral source ERB-selective ligands, genistein remains the most upon the chemical features that favored both the van der selective. However, an increasing number of synthetic com Waals (VDW) contact (the number of the rings in the struc pounds are emerging showing greater selectivity than ture) and electrostatic interactions (the number of the hydro genistein for ERB, as evidenced by the compound DPN devel 25 gen bonds) with the receptor. 10 molecules that have strong oped in Katzellenebogen's laboratory. Computer-aided struc VDW interactions with the receptor, but without contributive ture-based virtual database screening provides an efficient hydrogen bonding, were grouped in Category IV. These mol approach to rationally highlight a small group of lead candi ecules contain three or four five- or six-membered rings in dates from a large number of compounds for investigation at their structures that could promote the hydrophobic interac the bench. 30 tions with the center of the receptor binding site as observed in endogenous estrogen 173-estradiol that consists of four Determination of Binding Affinity and Selectivity rings in its structure and binds to the estrogen receptor with a high affinity. The binding affinity and selectivity of candidate molecules Table 1 Summarizes the ICso binding results of test mol yielded from database screening were determined by a fluo ecules to both ERC. and ERB as well as the binding selectivity rescent polarization competitive binding assay using purified of representative molecules selected from four categories. TABLE 1.

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

ICso Selectivity

Compd Structure ERC ERB (ERC/ERB)

Progesteron O CH NC: NC

genistein OH 4.68 LM 98.7 nM 47.2 OH O

HO O US 8,552,057 B2 17 18 TABLE 1-continued Binding Affinity (ICs) and Selectivity of Representative Molecules for Estrogen Receptor C. and ICso Selectivity Compd Structure ERC ERB (ERC/ERB)

75.7 M 18.6 nM 4.07

0.68 M >100 2 cC OH NC HO O

3 HO O O 120 nM 250 nM O.48

NC NC

5 OH NC 2.80 IM >100

HO COO 1. NC NC

43.0 M 1.99

OH K) 85.7 M

HO US 8,552,057 B2 19 20 TABLE 1-continued Binding Affinity (ICs) and Selectivity of Representative Molecules for Estrogen Receptor C. and ICso Selectivity Compd Structure ERC ERB (ERC/ERB)

8 O NC 4.48 M >100

HO

O O O N

OH

9 O NC NC OroCICCl 10 uCl NC NC O o1 O O

O

11 2.32 IM NC <0.01

O N

\ N O O O O

12 NC NC

N

O *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. As expected, the negative control steroid, progesterone, contribute to the binding affinity of ligands to both ERs. The does not bind to either ER. As a positive natural source estro enhanced VDW contact derives mainly from the central gen control, genistein was found to bind to ERB with a 47.2- 60 hydrophobic feature of the molecule. For example, the num fold greater binding selectivity over ERC, but at an affinity ber of rings increases the binding affinity of molecules to the one-fourth of 17f-estradiol. Among 12 molecules tested, five receptor, as indicated by the VDW value of 17 B-estradiol molecules, 1,2,5,7, and 8, showed binding selectivity to ERB (-67.98) versus that of genistein (-60.75) and molecule 9 over ERC., 3 of which, 2, 5, and 8, displayed the selectivity (-58.04), which are well correlated with their order-different over 100-fold. Preliminary structure and binding activity 65 binding affinities. Meanwhile, the hydrogen bonds derived relationship analyses revealed that both the central hydropho from the two polar"arms of the molecule are essential for the bic skeletal structure and the connected two polar arms binding as well. The lack of one “arm” of the hydrogen bond, US 8,552,057 B2 21 22 as represented by molecule 4 and 6, or two arms, as repre dilutions of estradiol, centered on the ICs of 1-2 nM, are also sented by 10 and 12, even though the latter two molecules can added to individual wells to generate a standard curve. The elicit strong VDW interactions with the receptor, with the plates are gently shaken to mix the reagents. A total of 150 VDW value of -72.58 and -69.19, respectively, leads to microliters from each of the wells is added to the correspond either very weak or no binding. With respect to the binding 5 ing wells of the pre-coated ERC. plates. The plates are sealed selectivity, as demonstrated in the modeling complex struc and the components in the wells are incubated either at room tures of a synthetic ERB-selective agonist, PPT, Stauffer, et al. temperature for 4 hours or at 4°C. overnight. The receptor J. Med. Chem. 2000, 43, 4934-4947 and a synthetic ERFB bound ligand is read directly after incubation using a scintil selective agonist, DPN, Meyers, et al., J. Med. Chem. 2001, lation counter. The amount of receptor bound ligand is deter 44, 4230-4251, with both ERs, Zhao, et al. 2004 Abstract 10 mined directly, i.e., without separation of bound from free Book: The Keystone Symposia: Nuclear Receptors: Steroid ligand. If estimates of both bound and free ligand are Sisters, Keystone, Colo.; February 2004, relatively larger required, the Supernatant is removed from the wells, liquid molecular size favors the binding selectivity for ERB over Scintillant is added, and the wells are counted separately in a ERC, as represented by molecule 3 and 11. liquid Scintillation counter. These analyses shed light on the future searchand design of 15 ERB Binding Assays more active and selective ER subtype-selective ligands. Fur ERB receptor (about.0.2 mg/ml, Affinity Bioreagents) is ther, 3 out of 12 representative molecules yielded from data diluted to about 2x10 mg/ml in phosphate-buffered saline base searching displayed over 100-fold selectivity toward (“PBS) at a pH of 7.4. Fifty microliters of the ERB-PBS ERB over ERC, demonstrating the effectiveness of this com solution is then added to each the wells of a flashplate. The puter-aided virtual screening approach applied in the present 20 plates are sealed and are stored in the dark at 4°C. for 16-18 study in the discovery of potential molecules that preferen hours. The buffered receptor solution is removed just prior to tially interact with ERB. use, and the plates are washed 3 times with 200 microliters per well of PBS. The washing is typically performed using a slow Example 2 dispense of reagent into the wells to avoid stripping the recep 25 tor from the well surface. Preclinical Identification of ERB-Selective For library screening, 150 microliters of 1 nMH-estradiol PhytoSERM Combinations for Prevention of (New England Nuclear, Boston, Mass.) in 20 mM Tris-HCl, 1 Neurodegeneration mM EDTA, 10% glycerol, 6 mM monothioglycerol, 5 mM KC1, pH 7.8 was mixed with 50 microliters of the test com The impact of ERb-selective PhytoSERMs when adminis- 30 pound (in same buffer) in a 96 well microtiter plate, resulting tered singly or in combination on neuronal Survival and in a final estradiol concentration of 0.6 nM. In addition, molecular/functional markers associated with prevention of several dilutions of estradiol, centered on the ICs of 1-2 nM neurodegeneration and Alzheimer's disease (AD) was inves is also added to individual wells to generate a standard curve. tigated. The plates are then gently shaken to mix the reagents. A total Materials and Methods 35 of 150 microliters from each of the wells is added to the 17 B-Estradiol was purchased from Steraloids (Newport, corresponding wells of the pre-coated ERB plates. The plates R.I.). Genistein, daidzein and equol were purchased from are sealed and the components in the wells are incubated at Indofine Chemical (Hillsborough, N.J.). IBSO03569 was room temperature either for 4 hours or at 4°C. overnight. The purchased from InterBioScreen (Moscow, Russia). The struc receptor bound ligand is read directly after incubation using a tures of these compounds are shown in FIG. 1. 40 Scintilation counter. The amount of receptor bound ligand is In Vitro Treatments: Test compounds (or combinations) determined directly, i.e., without separation of bound from were first dissolved in analytically pure DMSO (10 mM) and free ligand. If estimates of both bound and free ligand are diluted in Neurobasal medium to the working concentrations required, the Supernatant is removed from the wells, liquid right before treatments. Scintillant is added, and the wells are counted separately in a In Vivo Treatments: Test compounds (or combinations) 45 liquid Scintillation counter. were first dissolved in analytically pure DMSO and diluted in ERC/ERB Transactivation Assays corn oil (50 ml of DMSO in 950 ml of corn oil) to the working Construction of Transfected CHO Cells concentrations at 100 mg/ml for 17 B-estradiol and 10 mg/ml Transfected CHO cells were derived from CHO KI cells for phytoSERMs. obtained from the American Type Culture Collection In vitro Assays 50 (“ATCC, Rockville, Md.): The transfected cells were modi ERC. Binding Assays fied to contain the following four plasmid vectors: (1) pKCRE ERC. receptor (about 0.2 mg/ml, Affinity Bioreagents) is with DNA for the human estrogen receptor, (2) p.AG-60-neo diluted to about 2x10 mg/ml in phosphate-buffered saline with DNA for the protein leading to neomycin resistance, (3) (“PBS) at a pH of 7.4. Fifty microliters of the EPO-PBS pRO-LUC with DNA for the rat oxytocin promoter and for solution is then added to each of the wells of a flashplate. The 55 firefly luciferase protein, and (4) pl)R with DNA for the plates are sealed and stored in the dark at 4° C. for 16-18 protein leading to hygromycine resistance. All transforma hours. The buffered receptor solution is removed just prior to tions with these genetically modified CHO cells are per use, and the plates are washed 3 times with 200 microliters per formed under rec-VMT containment according to the guide well of PBS. The washing is typically performed using a slow lines of the COGEM (Commissie Genetische Modificatie). dispense of reagent into the wells to avoid stripping the recep- 60 Screening is performed either in the absence of estradiol tor from the well surface. (estrogenicity) or in the presence of estradiol (anti-estroge For library screening, 150 microliters of 1 nMH-estradiol nicity). (New England Nuclear, Boston, Mass.) in 20 mM Tris-HCl, 1 Assays to Assess Neuronal Function mM EDTA, 10% glycerol, 6 mM monothioglycerol, 5 mM Neuronal Culture Preparation KC1, pH 7.8 is mixed with 50 microliters of the test compound 65 Primary cultures of hippocampal neurons were obtained (in same buffer) in a 96 well microtiter plate, resulting in a from Embryonic Day 18 (E18d) rat fetuses. Briefly, after final estradiol concentration of 0.6 nM. In addition, several dissected from the brains of the rat fetuses, the hippocampi US 8,552,057 B2 23 24 were treated with 0.02% trypsin in Hank's balanced salt Biotecholagy, Lake Placid, N.Y.), actin (mouse monoclonal, solution (137 mMNaCl, 5.4 mM KC1, 0.4 mMKHPO, 0.34 1:1000; Santa Cruz, Biotechnology, Inc., Santa Cruz, Calif.) mM NaHPO.7H2O, 10 mM glucose, and 10 mM HEPES) or histone H1 (mouse monoclonol. 1:250; Santa Cruz Bio for 5 min at 37° C. and dissociated by repeated passage techology, Inc., Santa Cruz, Calif.) attemperatures and times through a series of fire-polished constricted Pasteur pipettes. 5 specified by the antibody providers. All primary antibodies Between 2x10 and 4x10" cells were plated onto poly-D- were dissolved in PBS-T with 1% horse serum (for mouse lysine (10 ug/ml)-coated 22 mm coverslips in covered 35 mm petri dishes for morphological analysis, and 1x10 cells/ml monoclonal antibody) or goat serum (for rabbit polyclonal). were plated onto poly-D-lysine-coated 24-well, 96-well cul After washing in PBS-T, the membranes were incubated with ture plates or 3-5x10 cells/ml onto 0.1% polyethylenimine- 10 horseradish peroxidase-conjugated anti-mouse IgG (1:5000; coated 60 mm petri dishes for biochemical analyses. Nerve Vector Laboratories, Inc., Burlingame, Calif.) in PBS-T with cells were grown in phenol-red free Neurobasal medium 1% horse serum or anti-rabbit IgG (1:5000; Vector Labora (NBM, Invitrogen Corporation, Carlsbad, Calif.) supple tories, Inc., Burlingame, Calif.) in PBS-T with 1% goat serum mented with B27. 5 U/ml penicillin, 5 g/ml streptomycin, for 1 hr. Immunoreactive bands were visualized by TMI3 0.5 mM glutamine and 25uM glutamate at 37°C. in a humidi- 15 detection kit (Vector Laboratories. Inc., Burlingame, Calif.) fied 10% CO, atmosphere at 37° C. for the first 3 days and and quantified using Un-Scan-It gel image software (Silk NBM without glutamate afterwards. Cultures grown in Scientific, Inc., Orem, Utah). Following transfer, gels were serum-free Neurobasal medium yield approximately 99.5% stained with Coomassie blue (Bio-rad Laboratories, Her neurons and 0.5% glial cells. cules, Calif.) to ensure equal protein loading. Neuroprotection Measurements 2O Bcl-2 and Bcl-Xl Expression Glutamate Exposure Primary hippocampal neurons were pretreated with com Primary hippocampal neurons were pretreated with com pounds for 48 hr before the cells were lysed by incubation in pounds for 48 hr followed by exposure to 100 glutamate for 5 ice-cold lysis buffer containing: 0.005% SDS, 0.1% Igepal, min at room temperature in HEPES buffer containing 100 0.2 mM sodium orthovanadate, 0.2 mM phenylmethylsulfo mMNaCl, 2.0 mMKC1, 2.5 mM CaCl, 1.0 mM MgSO 1.0 25 nylfluoride and protease inhibitor mixture in PBS at 4°C. for mMNaH2PO4.2 mMNaHCOs, 10.0 mM glucose and 12.5 45 min. Cell lysates were centrifuged at 10,000 rpm at 4°C. mMT-LEPES. Immediately following glutamate exposure, for 10 min, and the concentration of protein in the Supernatant cultures were washed once with HEPES buffer and replaced was determined using the BCA Protein Assay (Pierce Bio with fresh Neurobasal medium containing the test com technology, Inc., Rockford, Ill.). 25ug of total protein were pounds. Cultures were returned to the culture incubator and 30 diluted in 15ul 2xSDS containing sample buffer and the final allowed to incubate for 24 hr prior to cell viability measure volume was made 30 ul with water. After denaturalization on ments on the following day. a hot plate at 95-100° C. for 5 min, 25ul of the mixture were Western Immunoblotting loaded per lane on 10% SDS-polyacrylamide mini-gels fol CREB Phosphorylation lowed by electrophoresis at 90V. The proteins were then Nuclear lysates were prepared as following: Briefly, hip- 35 electro-transferred to polyvinylidene difluoride membranes pocampal neurons grown on poly-D-lysine coated culture (Millipore Corp., Bedford, Mass.) from the gels, Nonspecific dishes were treated with compounds for appropriate periods, binding sites were blocked with 5% nonfat dry milk in PBS washed with cold PBS once and scraped into 1 ml PES. Cells containing 0.05% TweenTM-20 (PBS-TweenTM). Membranes were then centrifuged at 5,000 rpm for 5 min, and the pellet were incubated with the primary monoclonal antibody was dissolved in Cytoplasm Extraction buffer (10 mM 40 against Bcl-2 (Zymed Laboratories, Inc., S. San Francisco, HEPES, 1 mM EDTA, 60 mM KC1, 0.075% gepal and pro Calif.) diluted 1:250 in PBS-Tween TMwith 1% horse serum tease and phosphatase inhibitor cocktail) and Suspended by (Vector Laboratories, Inc., Burlingame, Calif.) overnight at passage through a 200 ul pipette tip. After 30-45 RPM of 4°C., then incubated with the secondary horseradish peroxi incubation at 4°C., the samples were centrifuged at 5,000 dase (HRP)-conjugated horse anti-mouse IgG (Vector Labo rpm for 5 min to generate the cytoplasmic extract in the 45 ratories, Inc, Burlingame, Calif.) diluted 1:5,000 in PBS Supernatant. The Supernatant cytoplasmic extract was TweenTM with 1% horse serum for 2 hr at room temperature, removed, and Nuclear Extraction buffer (20 mM Tris HCl, 1.5 and Bcl-2 proteins were visualized by developing the mem mM MgCl, 420 mM. NaCl, 0.2 mM EDTA, 25% glycerol, branes with TAB substrate for peroxidase (Vector Laborato 0.5% Igepal and protease and phosphatase inhibitor cocktail) ries, Inc., Burlingame, Calif.). B-Actin (Santa Cruz Biotech was added to the pellet followed by 5 M NaCl to break the 50 nology, Inc., Santa Cruz, Calif.) level was determined to nuclear membrane. Following 30-45 RPM of incubation at 4 ensure equal protein loading, and high-range Precision Pro C., the samples were centrifuged at 12,000 rpm for 10 minto tein Standards (Bio-Rad Laboratories, Hercules, Calif.) was generate a Supernatant containing the nuclear extract. used to determine protein sizes. Relative intensities of bands Protein concentration was determined by the BCA method. were quantified by optical density analysis using an image An appropriate volume of 2x sample buffer was added to the 55 digitizing software Un-Scan-Tt version 5.1 (Silk Scientific, protein samples, and samples were boiled at 95°C. for 5 min. Inc., Orem, Utah). Samples (25 ug of proteins per well) were loaded on a 10% Statistics SDSPAGE gel and resolved by standard electrophoresis at Statistically significant differences between groups were 90V. Proteins were then electrophoretically transferred to determined by a one way analysis of variance (ANOVA) Immobilon-PPVDF membranes overnight at 32 V at 4° C. 60 followed by a Newman-Keuls post hoc analysis. Membranes were blocked for 1 hr at room temperature in In vivo Assays 10% non-fat dried milk in PBS containing 0.05% Tween 20 Immature Rat Uterotrophic Bioassay for Estrogenicity Anti (PBS-T), incubated with appropriate primary antibodies Estrogenicity against phospho-CREB (pSER', mouse monoclonal, Antiestrogenic activity was determined by the ability of a 1:2000; Cell Signaling Technology, Beverly, Mass.), CREB 65 test compound to Suppress the increase in uterine wet weight (rabbit polyclonal. 1:1000; Cell Signaling Technology, Bev resulting from the administration of 0.2 ug 17-3-estradiol erly, Mass.), spinophilin (rabbit polyclonal. 1:1000; Upstate (“E”) per day. Any statistically significant decreases in uter US 8,552,057 B2 25 26 ine weight in a particular dose group as compared with the E In vitro neuroprotection and associated mechanistic stud control group are indicative of anti-estrogenicity. ies were conducted in primary hippocampal neurons obtained One hundred forty (140) female pups (19 days old) in the from embryonic day 18 rat fetuses. Adult female ovariecto 35-50 g body weight range are selected for the study. On day mized rats were used to relate the in vitro findings to in vivo 19 of age, when the pups weigh approximately 35-50 g, they environment, along with the assessment of the impact of are body weight-order randomized into treatment pups. PhytoSERMs on brain mitochondrial functions and uterine Observations for mortality, morbidity, availability of food weight. and water, general appearance and signs of toxicity are made During a 2-week Surgery recovery following ovariectomy, twice daily. Pups not used in the study are euthanized along but before treatment, rats were placed on a phytoSERM with, the foster dams. Initial body weights are takenjust prior 10 reduced diet, TD.96155 (Harlan Teklad). Rats were given, to the start of treatment at day 19 of age. The final body once daily, 2 Subcutaneous injections of vehicle (control), 17 weights are taken at necropsy on day 22 of age. B-estradiol (70 ug/kg BW), genistein (6 mg/kg BW), or phy Treatment commences on day 19 of age and continues until toSERM combinations (6 mg/kg BW). Dosages used here are day 20 and 21 of age. Each animal is given three Subcutaneous commensurate with those used in humans. (“sc') injections daily for 3 consecutive days. Three rats in 15 each of the control and mid- to high-level dose test groups are Following the second injection, animals fasted for 24 hours anesthetized with a ketamine/xylazine mixture. Their blood is prior to sacrifice and brain dissection. Hippocampal and cor collected by exsanguination using a 22 gauge needle and 5 ml tical tissues were collected from one hemisphere and stored syringe flushed with 10 USP with sodium heparin/ml through for biochemical analyses. The remaining brain tissues minus the descending vena cava; and then transferred into a 5 ml cerebellum, pineal gland, and brainstem were utilized for green top plasma tube (sodium heparin (freeze-dried), 72 mitochondrial isolations, followed immediately by mito USP units). Plasma samples are collected by centrifugation, chondria respiratory activity measurements. The rest of mito frozen at -70° C., and analyzed using mass spectrographic to chondrial samples were stored for cytochrome c oxidase determine the presence and amount of test compound in the activity measurements. Uteri were excised, trimmed offat serum. Blood chemistry is also analyzed to determine other 25 and connective tissue, and both a wet weight and a dry weight blood parameters. The uteri from the rats are excised and were recorded. weighed. The remaining rats are sacrificed by asphyxiation Results under CO. The uteri from these rats are excised, nicked, The PhytoSERMs tested are shown in FIG. 1. blotted to remove fluid, and weighed to the nearest 0.1 mg. Selective Binding for both ERB and ERC. In order to determine whether the test compound signifi 30 FIG. 2 presents the competition binding curves of four cantly affected final body weight, a parametric one-way known ER ligands for both ERB and ERC. The ICs deter analysis of variance (ANOVA) is performed (SIGMASTAT mined for these ligands from the binding curves are consistent version 2.0, available commercially from Jandel Scientific, with the previously reported values using alternative methods San Rafael, Calif.). Estrogen agonist and antagonist activity Such as radioligandassay, demonstrating the reliability of this is assessed comparing uterine wet weights across treatment 35 assay in determining the binding profiles of Small molecules groups using a parametric ANOVA on loglo transformed data. to both ERS. The data are transformed to meet assumptions of normality FIGS. 2A and 2B show the competition binding curves for and homogeneity of variance of the parametric AWQVA. The ERC. and ERR. Data were generated with a fluorescence F value is determined and a Student-Newman-Kuels multiple polarization-based competitive binding assay using full range test is performed to determine the presence of signifi 40 length human ERC. and ERB, and plotted against the loga cant differences among the treatment groups. The test com rithm of serially diluted concentrations of the test compounds pound is determined to act as a mixed estrogen agonist/an (or combinations). Progesterone served as a negative control. tagonist if the test compound does not completely inhibit the 17B-Estradiol served as a positive control. Combined formu 17-B-estradiol stimulated uterotrophic response. lations were composed of equivalent molar of individual phy The use of animals was approved by the Institutional Ani 45 toSERMs included. G: genistein; D: daidzein, E: equol; I: mal Care and Use Committee at the University of Southern IBSO03569. 17 B-estradiol has no binding preference to ERC. California (Protocol Number: 10780). Embryonic day 18 or to ERB. The concentration of a test molecule resulting in Sprague-Dawley rat (Harlan, Indianapolis, Ind.) fetuses were the half-maximum shift in polarization value equals its ICso. used to obtain primary hippocampal neuronal cultures for in Non-convergence within the dose range, predicts that either vitro experiments. Young adult (14 to 16-week-old, weighing 50 the molecule does not bind to the receptor or that the binding from 270-290 g) female ovariectomized Sprague-Dawley rats affinity is very low. (Harlan) were used for in vivo experiments. 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) (%)- R2 (IM) (%) R2 (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 US 8,552,057 B2 27 28 TABLE 2-continued Binding data for ERC and ERB ERC ERB ICso RBA ICso RBA Selectivity Compounds (IM) (%)- R2B (IM) (%)- R2B (B.C.) G +D+E 15.71 O.1610 O.992S O.1902 17.06 0.9969 82.60 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,

15 Neuroprotective Effect TABLE 3-continued Table 3 and FIG.3 show the dose-dependent neuroprotec Dose-dependent effects of individual phytoSERMs tive effects of four ERB-selective phytoestrogenic molecules against glutamate-induced neurotoxicity in primary against Supraphysiological glutamate (100LM)-induced neu hippocampal neurons by LDH measurements' rotoxicity in primary hippocampal neurons by measurement of LDH release. "P-0.01 compared to vehicle alone-treated LDH Release (% of Control) cultures; * P<0.05 and ** P-0.01 compared to glutamate 1 M 246.307.70** alone-treated cultures. 10 M 307.532.62 Primary hippocampal neurons grown for 7DIV were pretreated with the test phytoSERMs 25 at serially diluted concentrations for 48 h, followed by a 5-min exposure to 100 mM TABLE 3 glutamate. The amount of LDH released into the culture media was measured 24 h later, Data are derived from a single experiment and are representative of at lease three indepen dent experiments. Results are presented as the percent of LDH release from vehicletreated Dose-dependent effects of individual phytoSERMs control cultures and expressed as means + S.E.M., n 6. against glutamate-induced neurotoxicity in primary FP < 0.01 compared to vehicle-treated control cultures, *P < 0.05 and hippocampal neurons by LDH measurements' **P<0.01 compared to glutamate alone-treated cultures; P × 0.05 and "P-0.01 compared 30 to cultures treated with 10 nM phytoSERMs; "P3 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 LDH Release (% of Control) 10 mM phytoSERMs.

Treatment Genistein FIG. 3 shows the neuroprotective efficacy of four ERB selective phytoestrogenic molecules when administered Control 100.00 3.09 35 alone at concentrations that elicited the maximal neuropro Glutamate alone 410.99 + 8.27" tective effects as revealed from the dose-response analyses 1 nM 361.037.71*.* (100 nM for all four molecules), or co-administered, against 10 nM 350.028.21** Supraphysiological glutamate (100LM)-induced neurotoxic 100 nM 347.24 16.96** ity in primary hippocampal neurons by measurement of cal 1 M 356.79 11.15** 40 cein AM staining. Results are presented in terms of neuro 10 M 377.848.45** protective efficacy Treatment Daidzein NE(reatment glutamate)/('control-glutamate)"100%, (1) Control 1OOOO 428 where V, is the individual value from phytoestrogen Glutamate alone 378.26 - 11.95* 45 treated cultures, V is a mean value from glutamate 1 nM 338.39 - 16.49 alone-treated cultures, and V is a mean value from 10 nM 333.98 9.10 vehicle-treated control cultures. "P-0.01 compared to 100 nM 301.427.70** vehicle alone-treated cultures; * PK0.05 and **P<0.01 com 1 M 3.18.49 15.92** pared to glutamate alone-treated cultures. 10 M 325.41 26.12* 50 Data presented in FIG. 3 and Table 3 demonstrate that Treatment Equol although the four ERB-selective phytoestrogenic molecules, when administered individually, are concentration-depen Control 1OOOO 14.95 dent and are protective against excitotoxic glutamate-induced Glutamate alone 460.27+ 12.20" neurotoxicity in primary neurons, these effects are moderate 1 nM 453.S.O. 23.37 55 and arise from the weaker binding to the estrogen receptor 10 nM 403.78 17.02* compared to the endogenous estrogen 17 B-estradiol (E2). 100 nM 331.599.67** FIG.3 demonstrates that co-administration of 3 or 4 of these 1 M 381.80 - 12.01** phytoestrogens afforded much greater neuroprotective effi 10 M 390.219.40** cacy compared to administration of single phytoestrogens or Treatment IBSO03569 60 a combination of 2 phytoestrogens.

Control 1OOOO 2.05 Expression of Anti-Apoptotic Proteins Bcl-2 and Glutamate alone 281.17 6.77f Bcl-XL 1 nM 26241 - 10.60 10 nM 270.86 - 12.94 65 These outcomes are paralleled by the results derived from 100 nM 220.56 6.80** the western analyses of the expression of anti-apoptotic pro teins, Bcl-2 and Bcl-XL, in primary neurons. FIGS. 4A-4B US 8,552,057 B2 29 30 shows the effects on Bcl-2 and Bcl-XL expression in rat monomer in the brain. Choronic upregulation of IDE repre primary hippocampal neurons and hippocampal tissues sents a novel efficacious therapeutic approach to lowering the derived from adult ovariectomized rats. Primary hippocam steady-state AB level in the brain and eventually preventing pal neurons grown for 7 divisions were treated with the test the occurrence of Alzheimer-type pathology. Therefore, these compounds (or combinations) for 48 hr followed by Western data indicate that coadministration of multiple ERB-selective blot analyses. Adult ovariectomized rats were given, once phytoestrogens have the potential to activate the anti-AB daily, 2 subcutaneous injections of the test compounds (or mechanism, and as a result, maintain the brain in a long-term combinations). Rats were sacrificed 24 h later following the healthy status. 2nd injection. Hippocampal tissues were homogenized fol Upregulation of Spinophilin lowed by Western blot analyses. Combined formulations 10 FIG. 6 illustrates the effect of four ERB-selective phy were composed of equivalent molar in (A) and equivalent toestrogenic molecules when co-administered (100 nM for all weight in (B) of individual phytoSERMs including G: four molecules) on the expression of the spine marker, spino genistein; daidzein; E: equol; and I: IBSO003569. philin, in primary hippocampal neurons. **P<0.01 compared Incubation of neurons with a combination of four phy to vehicle alone-treated cultures. Spinophilin, a protein that is toestrogens for 48 hours induces a significantly increased 15 enriched in the heads of neuronal dendritic spines, has been expression of both proteins comparable to those induced by demonstrated to play a significant role in modulating both E2. This is illustrated in FIG.4, which shows the effect of four dendritic morphology and glutamatergic synaptic activity. ERB-selective phytoestrogenic molecules when co-adminis Upregulation of spinophilin has been correlated with estro tered (100 nM for all four molecules) on the expression of the gen regulation of neuronal synaptic plasticity. Therefore, the anti-apoptotic proteins, Bcl-2 and Bcl-XL, in primary hippoc ses results indicate that these phytoestrogen combinations are ampal neurons. **P<0.01 compared to vehicle alone-treated effective to promote neurotrophism, thereby Sustaining the cultures. By comparison, a combination of two phytoestro brain staying in a synaptically active status, and prevent cog gens is not sufficient to induce a significant increase in the nitive decline and memory loss. expression of both proteins, as also illustrated in FIGS. 4A Neuroprotection Against Glutamate and 4B. 25 FIGS. 7A-7D shows the neuroprotective efficacy of the Up-regulation of the Bcl-2 family anti-apoptotic proteins compounds against glutamate (FIG. 7A) and amyloid have been associated with the neuroprotective mechanism induced neurotoxicity in rat primary hippocampal neurons. elicited by E2. These data indicate that a combined used of Primary hippocampal neurons grown for 7 divisions were multiple ERB-selective phytoestrogens is effective to activate pretreated with the test compounds (or combinations) for 48 the neuroprotective mechanism leading to improved neuronal 30 h, followed by a 5-min exposure to 100 mM glutamate. Neu Survival against neurodegenerative insults. Estrogen receptor rons were incubated for an additional 24 h prior to neuronal interaction with p85/PI3K also enhances pAkt, which phos viability analyses by calcein AM staining. Following pre phorylates the proapoptotic protein Bcl-2-associated death treatment with the compounds (or combinations) for 48 hr, protein (BAD) to prevent heterodimerization with, and inac neurons were exposed to 3 mM 3-amyloid for 2 d. Neu tivation of Bcl-2. In cortical neurons, estradiol induced pakt 35 ronal viability was analyzed by fluorometric measurements translocation to the nucleus. Recent analyses indicate that ofactivities of the LDH and dead-cell protease released in the estradiol, via the PI3K signaling pathway, activates both the culture media, and the live-cell protease exclusively entering Akt and the ERK1/2 cascades in the same population of intact viable neurons. cortical and hippocampal neurons. Simultaneous activation Results are presented as neuroprotective efficacy (NE), of two pathways that prevent mitochondria from activating 40 which is defined as the percentage of neurotoxin-induced cell-death cascades is likely to promote neuron Survival. toxicity prevented by the test compounds (or combinations) and quantitated by the equation: Increased Expression of the Anti-B-Amyloid Protein, IDE NE-(reatment-eurotoxin)/(controleurotoxin)"100% 45 where V, is the individual value from the test com FIG. 5 illustrates the effect of four ERB-selective phy pounds (or combinations)-treated cultures, V., is a toestrogenic molecules when co-administered (100 nM for all mean value from glutamate or f3-amyloid alone-treated four molecules) on the expression of the anti-B-amyloid pro cultures, and V is a mean value from vehicle-treated tein, insulin-degrading enzyme ("IDE') in primary hippoc control cultures. "P-0.01 compared to vehicle-treated con ampal neurons. **P<0.01 compared to vehicle alone-treated 50 trol cultures: *P<0.05 and **P<0.01 compared to glutamate cultures. FIG. 5 demonstrates the effects of these various or f3-amyloid, alone treated cultures;P<0.05 compared to combinations of phytoestrogens along with E2 on the expres E2-treated cultures; PK0.05 compared to genistein-treated sion of the anti-B-amyloid (anti-A?) protein, insulin-degrad cultures; "P-0.05 and "P<0.01 compared to combination ing enzyme (IDE) in primary neurons. Data showed that all (G+D)-treated cultures: 'P-0.05 compared to combination three combinations composed of two, three or four phy 55 (G+D+E+I)-treated cultures. Combined formulations were toestrogens significantly increases IDE expression in neu composed of equivalent molar of individual phytoSERMs rons. Among them, a combination of three phytoestrogens included. G: genistein; D: daidzein; E: equol. I: IBSO03569. induced the greatest neuronal response, with an efficacy Effect on IDE/NEP Expression greater than E2 as well as a combination of two phytoestro FIGS. 8A-8C show the effects on insulin-degrading genS. 60 enzyme (IDE)/neprilysin (NEP) expression in (A) rat primary It is clear that one neuropathological hallmark of AD is a hippocampal neurons and (B) hippocampal tissues derived significant deposition of extracellular AB peptide, as referred from adult ovariectornized rats. (A) Primary hippocampal to AB plaque. Impaired AB clearance and/or degradation has neurons grown for 7 DIV were treated with the test com been demonstrated to contribute in part to AB plaque forma pounds (or combinations) for 48 hr followed by Western blot tion in AD brain. Besides degrading insulin and several regu 65 analyses. (B) Adult ovariectomized rats were given, once latory peptides, IDE, a metalloprotease enzyme, has been daily, 2 Subcutaneous injections of the test compounds (or demonstrated to play a key role in degrading AB peptide combinations). Rats were sacrificed 24 h later following the US 8,552,057 B2 31 32 second injection. Hippocampal tissues were homogenized piratory activity, n>4; *P<0.05 and *P<0.01 compared to followed by western blot analyses. Results are presented as vehicle-treated control animals: 'P<0.05 compared to the fold increase in protein expression and expressed as the genistein-treated animals. Combined formulations were percent of control, n>4. *P<0.05 and *P<0.01 compared to composed of equivalent weight of individual phytoSERMs vehicle-treated control cultures or animals. P-0.05 and 5 including E2: 17b-estradiol; G: genistein; D: daidzein; E: P<0.01 compared to E2 treated cultures: "P<0.01 com equol; and I: IBSO03569; Mito: mitochondria; Mal/Glut: pared to combination (G+D) or genisteintreated cultures; malate? glutamate. Effect on Uterine Weight "P<0.05 compared to combination (G+D+E+I)-treated cul Table 4 shows the effects on uterine weight in adult ova tures. Combined formulations were composed of equivalent riectomized rats. Changes in uterine weight in response to molar in (A) and equivalent weight in (B) of individual phy 10 estrogenic stimulation can be used to evaluate the estrogenic toSERMs including G: genistein; D: daidzein; E: equal; and I: characteristics of test compounds on uterine tissues. In one IBSOO3569. example, described below, immature female rats having low Effect on Forebrain Mitochondria endogenous levels of estrogen are dosed with test compound FIGS. 9A-9E show the effects on forebrain mitochondrial (Subcutaneously) daily for 3 days. Compounds are formu cytochrome c oxidase (COX) activity in adult ovariectomized 15 lated as appropriate for Subcutaneous injection. As a control, rats. Rats were given, once daily, 2 subcutaneous injections of 17-B estradiol is administered alone to one dose group. the test compounds (or combinations). Rats were sacrificed Vehicle control dose groups are also included in the study. 24h later following the 2nd injection. Forebrain mitochon Twenty-four hours after the last treatment, the animals are dria were isolated followed by a spectrophotometric mea necropsied, and their uteri excised, nicked, blotted and Surement of COX activity using an immunocapture method. 20 weighed. Any statistically significant increases in uterine Colorimetric absorbance at 550 nm was recorded every 5 min weight in a particular dose group as compared to the vehicle for 115 min. COX activity is presented as the initial rate of control group demonstrate evidence of estrogenicity. 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 8.42 26.42 2.45 O.OO 9.27 (Vehicle) 17B-Estradiol 281.0632.00**P 120.23 - 25.07**EP 46.704.13**EP 76.74 15.63**EP (70 g/kg BW) Genistein 144.11 - 10.18 12.92 7.97 28.14 - 2.04 6.49 7.71 (6 mg/kg BW) G +D+E 119.84 - 1.19 -6.1O. O.93 23.71 - 0.04 -10.26 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 & 4 for each group), Rats were sacrificed 24h later following the 2n injection, Uteri were immediately excised and a wet weight was recorded. 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 of 6 mg/kg BW given to animals, G; genistein; D: daidzein; E: equol; I: BSOO3569, Ps is -0.01 compared to any other treatment groups, oxidation of reduced cytochrome c, and determined by cal- 45 Summary culating the initial slope between two time points (<20 min) within the linear region. (Upper Panel) Time-lapse change in Both in vitro and in vivo analyses demonstrated that com absorbance: (Lower Panel).9% increase in mitochondria COX bined use of select test phytoSERMs provided significantly activity, n>4; *P<0.05 and **P<0.01 compared to vehicle increased efficacy in Sustaining neuronal Survival when chal treated control animals; 'P<0.05 compared to genistein 50 lenged with neurotoxins, promoting expression of proteins as treated animals. Combined formulations were composed of key players in neuroprotection and metabolism/clearance of equivalent weight of individual phytoSERMs including E2: B-amyloid in neurons/brain, and enhancing brain mitochon 17bestradiol; G: genistein; D: daidzein, E: equal; I: drial functions. In particular, combined use of genistein, daid IBSOO3569. 55 Zein and equol at an equivalent weight afforded the maximal FIGS. 10A-10E show the effects on forebrain mitochon efficacy comparable or greater than 17b-estradiol in neuronal/ drial respiratory activity in adult ovariectomized rats. Rats brain assays. In contrast, Such a combination showed no were treated as above. Forebrain mitochondria were isolated impact on uterine weight, which however was markedly followed immediately by a polygraphical measurement of increased by 17b-estradiol. respiratory activity using an oxygen electrode. Following a 60 The present study indicates that combined use of select basal recording, mitochondrial state 4 respiration was mea En-selective PhytoSERMs can be more therapeutically effec Sured following the addition of Substrates, malate?glutamate. tive than single administrations and alternative combined for State 3 respiration was measured following the addition of mulations. In particular, the present study suggests the poten ADP Respiratory control ratio (RCR) was calculated as the tial of the combination of genistein, daidzein and equol, at an ratio between the rate of oxygen uptake at state 3 and the rate 65 equivalent weight, for prevention of neurodegeneration and of oxygen uptake at state 4. (FIGS. 12A-12D) Time-lapse AD, along with management of climacteric symptoms in oxygen uptake: (FIG. 12E)% increase in mitochondrial res postmenopausal women. US 8,552,057 B2 33 34 FIGS. 11A-11C are schematics showing estrogen mecha rodegenerative insults and AD. These ERB-selective phy nisms of action that lead to neurotrophic and neuroprotective toestrogen formulations, which optimize activation of ERB outcomes. 17-f-Estradiol (E2) acting via a membrane-asso while minimizing or avoiding activating ERO, should serve as ciated site (mER) activates a cascade required for multiple an effective estrogen alternative replacement therapy for sus responses that lead to enhanced neural plasticity, morphogen taining neurological health, function and prevention of AD esis, neurogenesis, and neural survival. The signaling without induction of proliferative responses in the reproduc sequence induced by E2 at the membrane site is as follows: tive tissues as seen with the current ET/HT. Moreover, in light (1) E2 binding to mER, (2) E2-mER complexes with p85 to of the most recent data indicating that activation of ERB activate PI3K, (3) activating calcium-independent PKC, (4) significantly reduces both ApoE mRNA and protein expres phosphorylating the L-type calcium channel, (5) inducing 10 Sion in neurons, ERf8-selective phytoestrogen formulations calcium influx, (6) activating calcium-dependent PKCs, (7) may serve as a particular viable strategy for reducing a major activating Src kinase, (8) activating the MEK/ERK 1/2 path risk factor of AD in ApoE4 carriers. way, (9) ERK translocates to the nucleus, (10) activating and We claimed: phosphorylating CREB, (11) enhancing transcription of anti 1. A method for alleviating or preventing hot flashes in a apoptotic genes Bcl-2 and Bcl-X1, which enhance mitochon 15 patient comprising administering to the patient an effective drial Vitality, and spinophilin, which encourages synaptic amount of a formulation consisting of two or more phy growth, (12) simultaneously, estrogen activation of PI3K toestrogen compounds or analogues thereof that selectively leads to activation of Akt, which phosphorylates and inhibits bind to estrogen receptor beta and cross the blood brain bar the proapoptotic protein BAD. rier, in a pharmaceutically acceptable excipient, wherein Estrogen-induced neuroprotective mechanisms converge the patient is a menopausal or post-menopausal woman; on mitochondria. Estrogen-activated cellular signaling cas the two or more phytoestrogen compounds are selected cade promotes enhanced mitochondrial function, leading to from the group consisting of genistein, daidzein, equol. increased calcium load tolerance, enhanced electron trans IBSO03569, and combinations thereof, and are admin port chain efficiency, and promotion of antioxidant defense istered in an effective amount from about 1 mg/kg/day to mechanisms. These actions are mediated by the regulation of 25 about 10 mg/kg/day; and both nuclear and mitochondrial encoded genes initiated by the phytoestrogen compounds are more effective in com the activation of second-messenger signaling cascades. bination than the same amount of the individual phy These mechanisms and the data herein demonstrate that, toestrogen compounds. consistent with the healthy cell bias of estrogen benefit 2. The method of claim 1, wherein the phytoestrogen com hypothesis, selective molecules can be administered before 30 pounds are genistein and daidzein. neurodegenerative insult while neurons are still healthy and 3. The method of claim 1, wherein one of the phytoestrogen that phytoSERM exposure will lead to enhanced neural sur compounds is equol. vival mechanisms, represented as mitochondria with Bcl-2 4. The method of claim3, wherein the phytoestrogen com additions, that promote neural defense against neurodegen pounds are equol, genistein and daidzein. erative insults associated with age-associated diseases such as 35 5. The method of claim3, wherein the phytoestrogen com Alzheimer's and Parkinson's. pounds are equol, genistein, daidzein and IBSO03569. These studies exemplify the therapeutic promise of select 6. The method of claim 1, wherein the formulation is for ERB-selective phytoestrogens when used in combination for mulated for enteral, parenteral, or topical administration. Sustaining memory function and preventing age-related neu ck -k, -k : :