WO2008016768A1 - Formulations phyto-œstrogéniques pour soulager ou prévenir des maladies neurodégénératives - Google Patents

Formulations phyto-œstrogéniques pour soulager ou prévenir des maladies neurodégénératives Download PDF

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WO2008016768A1
WO2008016768A1 PCT/US2007/073505 US2007073505W WO2008016768A1 WO 2008016768 A1 WO2008016768 A1 WO 2008016768A1 US 2007073505 W US2007073505 W US 2007073505W WO 2008016768 A1 WO2008016768 A1 WO 2008016768A1
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estrogen
erβ
formulation
women
brain
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PCT/US2007/073505
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WO2008016768A8 (fr
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Roberta Diaz Brinton
Liqin Zhao
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Roberta Diaz Brinton
Liqin Zhao
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Priority to CA002659905A priority Critical patent/CA2659905A1/fr
Priority to EP07812924A priority patent/EP2046325A1/fr
Priority to JP2009522917A priority patent/JP2009545605A/ja
Priority to AU2007281381A priority patent/AU2007281381A1/en
Publication of WO2008016768A1 publication Critical patent/WO2008016768A1/fr
Publication of WO2008016768A8 publication Critical patent/WO2008016768A8/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/12—Drugs for genital or sexual disorders; Contraceptives for climacteric disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/24—Drugs for disorders of the endocrine system of the sex hormones
    • A61P5/30—Oestrogens

Definitions

  • ET/HT acts as an effective preventative therapeutic strategy for age-related cognitive decline and neurodegenerative disorders, such as Alzheimer's disease (“AD”), while it is not an effective treatment strategy.
  • AD Alzheimer's disease
  • the current widely prescribed ET, conjugated equine estrogens (“CEE”) is a highly complex ET with over 200 different components. Whether CEE provides the optimal therapeutic efficacy has been questioned.
  • Another key issue challenging HT is the optimal composition. The progestin and its timing of administration in conjunction with ET, remains to be determined.
  • ET/HT has long been used in postmenopausal women to delay or reverse some of the problems associated with menopause
  • epidemiological and clinical studies have uncovered potential long-term risks related to this therapy.
  • the recently revealed risks associated with ET/HT have greatly increased interest in the development of estrogen alternatives that promote beneficial effects of estrogen in brain, bone and the cardiovascular system, while not eliciting deleterious effects in other organs, particularly in breast and uterine tissues.
  • Two nuclear receptors for estrogen (ERs), ER ⁇ and ER ⁇ have been identified. In the central nervous system, both ER ⁇ and ER ⁇ are expressed in the hippocampus and cortex of rodent and human brains.
  • ER ⁇ is a key requirement for activation of mechanisms that underlie estrogen-inducible neuronal morphological plasticity, brain development, and cognition.
  • ER ⁇ is more predominant in mediating the sexual characteristics of estrogen effects in the reproductive organs such as breast and uterus.
  • ER ⁇ As a pharmacological target to promote memory function and neuronal defense mechanisms against age-related neurodegeneration such as Alzheimer's disease (AD), while avoiding activating untoward estrogenic proliferative effects in the breast and uterus, although this might be at the cost of lower efficacy due to the lack of activation of ER ⁇ in the brain.
  • Other potential therapeutic advantages associated with ER ⁇ include regulation of estrogen vasculoprotective action and development of interventions targeting diseases such as depression, colon cancer, prostate cancer, obesity, leukemia, and infertility.
  • a potential disadvantage of an ER ⁇ -selective ligand is the lack of activation of ER ⁇ in bone, as ER ⁇ has been demonstrated to mediate estrogen regulation of bone density.
  • ER ⁇ estrogen receptor subtypes
  • ER ⁇ and/or ER ⁇ are two estrogen receptor subtypes
  • ER ⁇ plays a key role in regulating brain development, neurogenesis and estrogen-induced improved neuronal plasticity and survival.
  • ER ⁇ is less effective in mediating the sexual characteristics of estrogen action in reproductive tissues, avoiding activating untoward estrogenic proliferative effects in the breast and uterus. Therefore, ER ⁇ represents a potentially safer therapeutic target for promoting memory function and neuroprotection.
  • this safety may be at the cost of lower efficacy, due to the lack of activation of ER ⁇ in the brain.
  • ER ⁇ -target therapeutics arise from its regulation of estrogen's cardioprotective effects.
  • ER ⁇ -selective ligands may also provide effective therapeutics for preventing or treating inflammation, depression, anxiety, colon cancer, prostate cancer, obesity, leukaemia, and infertility.
  • phytoestrogens bind to ER ⁇ and to ER ⁇ subtypes, and some of these molecules possess moderate binding selectivity for ER ⁇ and exert estrogenic effects in multiple tissues.
  • the therapeutic efficacy of phytoestrogens in the brain remains controversial. On the one hand, when administered singly, phytoestrogens appeared to be moderately neuroprotective.
  • Soy extracts or soy protein supplements generally contain multiple phytoestrogenic molecules, some of which may be ER ⁇ -selective agonists, while others may be ER ⁇ -selective agonists, and others may be ineffective in activating either ER ⁇ or ER ⁇ but may function as inhibitors of ER binding of those ER ⁇ and/or ER ⁇ phytoestrogenic agonists.
  • the ineffectiveness of a complex formulation of phytoestrogens in promoting beneficial effects of estrogen in brain, such as a soy-derived preparation may also arise from antagonizing actions among the different phytoestrogens, in addition to the possible ER antagonism, likely from the activation of both ER ⁇ and ER ⁇ in the same context.
  • Co-administration of an ER ⁇ -selective agonist and an ER ⁇ -selective agonist is less effective than treatment with either agonist alone in various neuroprotective measurements.
  • ER ⁇ and ER ⁇ have a yin/yang relationship in many contexts where one receptor may antagonize the actions of the other.
  • the different ratio and distinct function of homodimer and heterodimer induced by co-administration of an ER ⁇ - selective agonist and an ER ⁇ -selective agonist may also account for the reduced efficacy exerted by the combination of both agonists.
  • ER ⁇ -selective phytoestrogen formulation could maximize the therapeutic benefits associated with activation of ER ⁇ in the brain while minimizing the adverse effects associated with the activation of ER ⁇ in reproductive tissues. Moreover, selective targeting of ER ⁇ potentially reduces antagonistic actions that may occur in a complex soy- derived preparation. This naturally occurring ideal formulation would have tremendous therapeutic value in promoting neurological function and preventing AD in a population at risk for losing neurological capacity and losing memory function, i.e., postmenopausal women. To date, no such phytoestrogen formulation exists. Thus, there is a need to discover and develop a novel select phytoestrogen formulation, generally, and particularly, a formulation that functions in the brain.
  • Select phytoestrogen pharmaceutical compositions and methods of use for promoting neurological health and prevention of age-related neurodegeneration, such as AD have been developed.
  • These select phytoestrogen formulations are composed of a number of plant-derived estrogenic molecules and/or their structural analogues and exhibit binding preference to ER ⁇ over ER ⁇ and agonist activity in the brain.
  • These ER ⁇ - selective phytoestrogen formulations cross the blood-brain-barrier and promote estrogen-associated neurotrophism and neuroprotection mechanisms in the brain, without activating proliferative mechanisms in the reproductive tissues and are therefore devoid of other estrogen-associated problematic aspects.
  • the select phytoestrogen formulations are therapeutically useful to both women and men for sustaining neurological health and preventing age- related cognitive decline and neurodegenerative disorders, such as AD.
  • the formulations preferably contain combinations of compounds, and can be formulated for daily, sustained, delayed or weekly/monthly administration. In a preferred embodiment, these are administered to women who are in menopause or post menopausal, most preferably early in menopausal.
  • Figure 1 shows the chemical Structures of 17 ⁇ -estradiol and the phytoSERMs genistein, daidzein, equol, and IBSO03569.
  • Figures 2A and 2B show the competition binding curves for ER ⁇ and ER ⁇ (molar concentration versus fluorescence polarization (mP)) of G, D, E, I or combinations: G+D, G+D+E, or G+D+E+I.
  • Figure 3 is a graph showing the neuroprotective efficacy of four ER ⁇ -selective phytoestrogenic molecules when administered alone at concentrations that elicited the maximal neuroprotective effects as revealed from the dose-response analyses (100 nM for all four molecules Genistein (G), Daidzein (D), Equol (E) and IBSO03569 (I)), or co-administered: G+D f G+D+E, or G+D+E+I, against supraphysiological glutamate (100 ⁇ M)- induced neurotoxicity in primary hippocampal neurons by measurement of calcein AM staining.
  • Figures 4A and 4B are graphs showing the effect of four ER ⁇ - selective phytoestrogenic molecules when co-administered (100 nM for all four molecules) as G+D, G+D+E, or G+D+E+I, on the expression of the anti-apoptotic proteins, Bcl-2 and Bcl-xL, in primary hippocampal neurons.
  • Figure 5 is a graph illustrating the effect of four ER ⁇ -selective phytoestrogenic molecules when co-administered (100 nM for all four molecules), G+D, G+D+E, or G+D+E+I, on the expression of the anti- ⁇ - amyloid protein, insulin-degrading enzyme ("IDE”), in primary hippocampal neurons.
  • IDE insulin-degrading enzyme
  • Figure 6 is a graph illustrating the effect of four ER ⁇ -selective phytoestrogenic molecules when co-administered (100 nM for all four molecules): G+D, G+D+E, or G+D+E+I, on the expression of the spine marker, spinophilin, in primary hippocampal neurons.
  • Figures 7A-7D are graphs shows the neuroprotective efficacy of G, D, E, and I, alone and in combination: G+D, G+D+E, or G+D+E+I, against (7A) glutamate- and (7B) ⁇ -amyloid 1-42-induced neurotoxicity in rat primary hippocampal neurons, controls live/dead cells (7C); dead cells (7D).
  • Figures 8A-8C are graphs showing the effects of G, D, E, and I, alone and in combination: G+D, G+D+E, and G+D+E+I, on insulin- degrading enzyme (IDE) expression on neprilysin (NEP) expression in hippocampal tissues derived from adult ovariectomized rats.
  • IDE insulin- degrading enzyme
  • NEP neprilysin
  • Figures 9A-9E are graphs showing the effects of G, G+D+E, and G+D+E+I on forebrain mitochondrial cytochrome c oxidase (COX) activity in adult ovariectomized rats.
  • Figures 10A-10E are graphs showing the effects of G, G+D+E, and G+D+E+I on percent increase forebrain mitochondrial respiratory activity in adult ovariectomized rats.
  • FIGS 11 A-IlC are schematics showing estrogen mechanisms of action that lead to neurotrophic and neuroprotective outcomes.
  • 1 IA, 17- ⁇ - Estradiol (E2) acting via a membrane-associated site (mER) activates a cascade required for multiple responses that lead to enhanced neural plasticity, morphogenesis, neurogenesis, and neural survival.
  • E2 17- ⁇ - Estradiol
  • mER membrane-associated site
  • the signaling sequence induced by E2 at the membrane site is as follows: (1) E2 binding to mER, (2) E2-mER complexes with p85 to activate PBK, (3) activating calcium-independent PKC, (4) phosphorylating the L-type calcium channel, (5) inducing calcium influx, (6) activating calcium-dependent PKCs 5 (7) activating Src kinase, (8) activating the MEK/ERKl/2 pathway, (9) ERK translocates to the nucleus, (10) activating and phosphorylating CREB, (11) enhancing transcription of antiapoptotic genes Bcl-2 and Bcl-xl, which enhance mitochondrial vitality, and spinophilin, which encourages synaptic growth, (12) simultaneously, estrogen activation of PBK leads to activation of Akt, which phosphorylates and inhibits the proapoptotic protein BAD.
  • Estrogen-induced neuroprotective mechanisms converge on mitochondria.
  • Estrogen-activated cellular signaling cascade promotes enhanced mitochondrial function, leading to increased calcium load tolerance, enhanced electron transport chain efficiency, and promotion of antioxidant defense mechanisms. These actions are mediated by the regulation of both nuclear and mitochondrial encoded genes initiated by the activation of second-messenger signaling cascades.
  • 11 C Conceptual schematic of NeuroSERM design and therapeutic use. Consistent with the healthy cell bias of estrogen benefit hypothesis, selective molecules would be administered before neurodegenerative insult while neurons are still healthy. NeuroSERM exposure would lead to enhanced neural survival mechanisms, represented as mitochondria with Bcl-2 additions, that promote neural defense against neurodegenerative insults associated with age-associated diseases such as Alzheimer's and Parkinson's.
  • AMPAR AMPA receptor
  • C cytochrome oxidase
  • F 0 , F 1 ATPase subunits
  • LTD long-term depression
  • LTP long-term potentiation
  • NAD nicotinamide adenine dinucleotide
  • NADH nicotinamide adenine dinucleotide
  • VDCC voltage-dependent calcium channel.
  • Estrogen Receptor refers to any protein in the nuclear receptor gene family that binds estrogen, including, but not limited to, any isoforms and variants thereof.
  • Human estrogen receptors include the alpha- and beta-isoforms (referred to herein as "ER ⁇ ” and "ER ⁇ ").
  • Estrogen Receptor Modulator refers to a compound that can act as an estrogen receptor agonist or antagonist of an estrogen receptor or estrogen receptor isoform having an IC 50 or EC 50 with respect to ER ⁇ , ER ⁇ and/or other estrogen receptor isoforms of no more than about 50 ⁇ M as determined using the ER ⁇ , and/or ER ⁇ transactivation assay described herein. More typically, estrogen receptor modulators have IC 50 or ECso values (as agonists or antagonists) of not more than about 10 ⁇ M. Representative compounds are predicted to exhibit agonist or antagonist activity via an estrogen receptor.
  • Compounds preferably exhibit an antagonist or agonist IC 50 or EC 50 with respect to ER ⁇ and/or ER ⁇ of about 10 ⁇ M, more preferably, about 500 nM, even more preferably about 1 nM, and most preferably, about 500 pM, as measured in the ER ⁇ and/or ER ⁇ transactivation assays.
  • IC 50 is that concentration of compound which reduces or inhibits the activity of a target (e.g., ER ⁇ or ERp) to half-maximal level.
  • EC 50 is that concentration of compound which provides half- maximum effect
  • Selective Estrogen Receptor Modulator refers to a compound that exhibits activity as an agonist or antagonist of an estrogen receptor (e.g., ER ⁇ , ER ⁇ or other estrogen receptor isoform) in a tissue-dependent or receptor dependent manner.
  • an estrogen receptor e.g., ER ⁇ , ER ⁇ or other estrogen receptor isoform
  • compounds that function as SERMs can act as estrogen receptor agonists in some tissues, e.g., bone, brain, and/or cardiovascular, and as antagonists in other tissue types, e.g., the breast and/or uterine tissue.
  • “Phytoestrogen” refers to a naturally occurring compound of plants, such as soybeans, or plant products, such as whole grain cereals, that acts like estrogen or binds to an estrogen receptor.
  • NeuroSERM refers to compounds that target the membrane site of estrogen action.
  • the term “PhytoSERM” refers to natural source compounds that preferentially target estrogen receptor beta.
  • the term “analogue” refers to a chemical compound with a structure similar to that of another (reference compound) but differing from it in respect to a particular component, functional group, atom, etc.
  • compositions refers to compounds which are formed from a parent compound by chemical reaction(s). I ⁇ .
  • compositions containing one or more phytoestrogens are described herein.
  • a number of phytoestrogens have been isolated and identified and additional analogs created, all of which have estrogen receptor binding selectivity
  • of the composition contains two or more plant-derived estrogenic molecules and/or structural analogues, which possess ER ⁇ -binding selectivity and exhibit neuroprotective activity when administered individually.
  • These compositions are useful for preventing estrogen-deficiency associated symptoms and disorders, particularly age- related cognitive decline and neurodegenerative diseases, such as Alzheimer's disease ("AD").
  • AD Alzheimer's disease
  • A. PhytoSERMs The compositions described herein contain one or more phytoestrogens or natural source selective estrogen receptor modulators (SERMs) exhibiting a binding preference for ER ⁇ .
  • PhytoSERMs can be identified as described in Example 1. Suitable phytoSERMs include, but are not limited to, genistein, daidzein, equol, IBSO03569 and combinations thereof. The structures of genistein, daidzein, equol, and IB S 003569 are shown in Figure 1. Others are listed in Table 1. Preferred compounds cross the blood brain barrier.
  • the compounds can be used in the form of salts derived from inorganic or organic acids. These salts include, but are not limited to, the following: acetate, adipate, alginate, citrate, aspart ate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, cyclopentanepro-pionate, dodecylsulfate, ethanesulfonate, glucoheptanoate, glycerophosphate, hemi-sulfate, heptanoate, hexamate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2- hydroxyethanesulfonate, lactate, maleate, methanesulfonate, nicotinate, 2- napthalenesulfan
  • any basic nitrogen- containing groups can be quaternized with agents such as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides, and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl, and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides, and others. Wafer or oil-soluble or dispersible products are thereby obtained.
  • lower alkyl halides such as methyl, ethyl, propyl, and butyl chloride, bromides, and iodides
  • dialkyl sulfates like dimethyl, diethyl, dibutyl, and diamyl sulfates
  • acids which may be employed to form pharmaceutically acceptable acid addition salts include such inorganic acids as hydrochloric acid, sulfuric acid, and phosphoric acid, and organic acids such as oxalic acid, maleic acid, succinic acid and citric acid.
  • Basic addition salts can be prepared in situ during the final isolation and purification of the compounds, or separately by reacting carboxylic acid moieties with a suitable base such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation or with ammonia, or an organic primary, secondary or tertiary amine.
  • Pharmaceutically acceptable salts include, but are not limited to, cations based on the alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, and aluminum salts, as well as non-toxic ammonium, quaternary ammonium, and mine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.
  • Other representative organic amines useful for the formation of base addition salts include diethylamine, ethylenediamine, ethanolamine, diethanolamine, and piperazine.
  • Appropriate carriers can be added that assist the compounds to cross the blood-brain-barrier.
  • the compounds can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more other compound as described herein, and/or in combination with other agents used in the treatment and/or prevention of estrogen receptor-mediated disorders. Alternatively, the compounds can be administered sequentially with one or more such agents to provide sustained therapeutic and prophylactic effects. Suitable agents include, but are not limited to, other SERMs as well as traditional estrogen agonists and antagonists.
  • agents useful in combination with the compounds for the treatment of estrogen receptor-mediated disorders include, for example, tamoxifen, 4-hydroxytamoxifen, raloxifene, toremifene, droloxifene, TAT- 59, idoxifene, RU 58,688, EM 139, ICI 164,384, ICI 182,780, clomiphene, MER-25, DES, nafoxidene, CP-336,156, GW5638, LY 139481, LY353581, zuclomiphene, enclomiphene, ethamoxytriphetol, delmadinone acetate, bisphosphonate.
  • aromatase inhibitors such as, but not 1 imited to, 4- hydroxymdrostenedione, plomestane, exemestane s aminogluethimide, rogletimide, fadrozole, vorozole, letrozole, and anastrozole .
  • agents useful in combination with the compounds described herein include, but are not limited to antineoplastic agents, such as alkylating agents, antibiotics, hormonal antineoplastics and antimetablites.
  • An example includes the compounds used to treat or prevent osteoporosis.
  • Other ingredients include vitamins, nutritional supplements, anti-oxidant agents, coenzymes, etc.
  • the additional active agents may generally be employed in therapeutic amounts as indicated in the PHYSICIANS' DESK REFERENCE (PDR) 53rd Edition (2003), or such therapeutically useful amounts as would be known to one of ordinary skill in the art.
  • the compounds and the other therapeutically active agents can be administered at the recommended maximum clinical dosage or at lower doses. Dosage levels of the active compounds in the compositions may be varied to obtain a desired therapeutic response depending on the route of administration, severity of the disease and the response of the patient.
  • the combination can be administered as separate compositions or as a single dosage form containing both agents. When administered as a combination, the therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition.
  • the compounds can be administered enterally, transdermally, transmucisally, intranasally or parenterally.
  • Excipients for oral formulation are known to those skilled in the art, as discussed briefly below, and can be used to provide immediate, sustained, delayed, or pulsed release.
  • the compounds can also be administered via a transdermal patch, a depo, vaginally or rectally using a topical carrier such as a gel, lotion, ointment, liposomal formulation, suspension, foam, spray or supposity, via the pulmonary or nasal route, buccally or sublingual via the mucosal membranes of the mouth.
  • the appropriate excipients for all of these formulations are known.
  • the compounds may be dissolved or suspended in saline, sterile water or phosphate buffered saline, or a suitable oil for injection iv, im, subcu, or ip.
  • Suitable pharmaceutically acceptable excipients include processing agents and drug delivery modifiers and enhancers, such as, for example, calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose, dextrose, hydroxypropyl-.beta.-cyclodextrin, polyvinylpyrrollidone, low melting waxes, and ion exchange resins, as well as combinations of any two or more thereof.
  • processing agents and drug delivery modifiers and enhancers such as, for example, calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose, dextrose, hydroxypropyl-.beta.-cyclodextrin, polyvinylpyrrollidone, low melting waxes, and ion exchange resins, as well as combinations of any
  • compositions containing estrogen receptor modulating compounds may be in any form suitable for the intended method of administration, including, for example, a solution, a suspension, or an emulsion.
  • Liquid carriers are typically used in preparing solutions, suspensions, and emulsions.
  • Liquid carriers contemplated for use include, for example, water, saline, pharmaceutically acceptable organic solvent(s), pharmaceutically acceptable oils or fats, as well as mixtures of two or more thereof.
  • the liquid carrier may contain other suitable pharmaceutically acceptable additives such as solubilizers, emulsifiers, nutrients, buffers, preservatives, suspending agents, thickening agents, viscosity regulators, or stabilizers.
  • Suitable organic solvents include, for example, monohydric alcohols, such as ethanol, and polyhydric alcohols, such as glycols.
  • Suitable oils include, for example, soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil.
  • the carrier can also be an oily ester such as ethyl oleate, isopropyl myristate.
  • Compositions may also be in the form of microparticles, microcapsules, liposomal encapsulates, as well as combinations of any two or more thereof.
  • the compounds may be administered orally, parenterally, sublingually, by inhalation spray, rectally, vaginally, or topically in dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired.
  • Topical administration may also involve the use of transdermal administration such as transdermal patches or ionophoresis devices.
  • parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal inj ection, or infusion techniques.
  • sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3 -propanediol.
  • acceptable vehicles and solvents that may be employed are water; Ringer's solution, and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid can be useful in the preparation of injectables.
  • Suppositories for rectal or vaginal administration of the drug can be prepared by mixing the drug with a suitable nonirritating excipient such as cocoa butter and polyethylene glycols that are solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum and release the drug.
  • Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules.
  • the active compound may be admixed with at least one inert diluent such as sucrose lactose or starch.
  • Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate.
  • the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.
  • Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water. Such compositions may also comprise adjuvants, such as wetting agents, emulsifing and suspending agents, cyclodextrins, and sweetening, flavoring, and perfuming agents.
  • adjuvants such as wetting agents, emulsifing and suspending agents, cyclodextrins, and sweetening, flavoring, and perfuming agents.
  • the compounds can also be administered in the form of lipsomes.
  • liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multilamellar hydrated liquid crystals that are dispersed in an aqueous medium.
  • any nontoxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used.
  • the present compositions in liposome form can contain, in addition to a compound, stabilizers, preservatives, excipients.
  • the preferred lipids are the phospholipids and phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art (Prescott 1976).
  • Transdermal patches are well known for delivery of nicotine, nitroglycerin and birth control. These can be utilized with these formulations as well. Depos that are implanted under the skin or ip can also be used, similarly to the manner of delivering birth control. III.
  • Compounds can be administered in a variety of ways including enteral, parenteral, pulmonary, nasal, mucosal and other topical or local routes of administration.
  • suitable modes of administration include oral, subcutaneous, transdermal, transmucosal, iontophotetic, intravenous, intramuscular, intraperitoneal, intranasal, subdural, rectal, vaginal and inhalation.
  • an effective amount of the compound or composition is administered to treat and/or prevent an estrogen receptor-mediated disorder in a human or animal subject.
  • Modulation of estrogen receptor activity results in a detectable suppression or up-regulation of estrogen receptor activity either as compared to a control or as compared to expected estrogen receptor activity.
  • Effective amounts of the compounds generally include any amount sufficient to detectably modulate estrogen receptor activity by any of the assays described herein, by other activity assays known to those having ordinary skill in the art, or by detecting prevention and/or alleviation of symptoms in a subject afflicted with an estrogen receptor-mediated disorder.
  • Estrogen/hormone therapy has been associated with the reduced risk of developing AD when treated at the menopausal transition in women Brinton, R. D. Impact of estrogen therapy on Alzheimer's disease: a fork in the road? CNS Drugs 2004, 18, 405-422.
  • results of the Cache County Study indicate that women who receive ET/HT at the time of menopause and continue for 10 years have a 3- fold lower risk of developing AD, Zandi, et al. JAMA 2002, 288, 2123-2129, whereas the recent data from the Women's Health Initiative Memory Study indicate that women who begin the therapy late in menopause have a greater risk of developing AD , Espeland, et al.
  • Estrogen receptor-mediated disorders that may be treated include any biological or medical disorder in which estrogen receptor activity is implicated or in which the inhibition of estrogen receptor potentiates or retards signaling through a pathway that is characteristically defective in the disease to be treated.
  • the condition or disorder may either be caused or characterized by abnormal estrogen receptor activity.
  • Representative estrogen receptor-mediated disorders include, for example, osteoporosis, atherosclerosis, estrogen-mediated cancers (e.g., breast and endometrial cancer), Turner's syndrome, benign prostate hyperplasia (i.e., prostate enlargement),, prostate cancer, elevated cholesterol, restenosis, endometriosis, uterine fribroid disease, hot flashes, and skin and/or vagina atrophy.
  • Other estrogen receptor-mediated conditions that may be treated include neurological diseases and disorders including memory loss and dementia, and neurodegenerative disease, including Alzheimer's disease.
  • MCI mild cognitive impairment
  • Successful treatment of a subject may result in the prevention, inducement of a reduction in, or alleviation of symptoms in a subject afflicted with an estrogen receptor-mediated medical or biological disorder.
  • treatment can result in a reduction in breast or endometrial tumors and/or various clinical markers associated with such cancers.
  • Treatment of Alzheimer's disease can result in a reduction in rate of disease progression, detected, for example, by measuring a reduction in the rate of increase of dementia.
  • endocrine events are suggested by the stage of the menopause transition (i.e., late) during which depressions appeared.
  • the late transition is characterized by more prolonged hypogonadism than the early perimenopause, during which estradiol secretion may be increased.
  • estradiol withdrawal and/or recent-onset of prolonged hypogonadism suggest an endocrine mechanism related to the perimenopause (estradiol withdrawal and/or recent-onset of prolonged hypogonadism) in the pathophysiology of perimenopausal depression.
  • estradiol significantly decreased depression scores compared with both baseline and placebo conditions.
  • short-term administration (3 weeks) of estradiol significantly decreased depression scores compared with both baseline and placebo conditions.
  • a full or partial therapeutic response was seen in 80% of perimenopausal women on estradiol compared with 22% of those on placebo (Schmidt et al., 2000).
  • the efficacy of ET in perimenopausal depression is consistent with the observed effect size (0.69) in a recent meta-analysis of studies examining the effects of estrogen on mood (Zweifel and O'Brien, 1997 Psychoneuroendocrinology 22: 189).
  • the therapeutic response to estradiol was observed in both major and minor depression as well as in women with and without hot flushes.
  • the efficacy of ET in perimenopausal depression is not solely a product of its ability to reduce the distress of hot flushes.
  • the administration of estradiol under similar conditions failed to improve mood in depressed women who were 5 years postmenopause (Morrison et al., 2004).
  • the effects of estradiol on depression may be limited to perimenopausal women.
  • the stage of reproductive aging at which women present and/or commence ET might modify the observed outcomes.
  • the amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the estrogen-mediated disease, the host treated and the particular mode of administration. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity of the particular disease undergoing therapy. The prophylactically or therapeutically effective amount for a given situation can be readily determined by routine experimentation and is within the skill and judgment of the ordinary clinician.
  • a prophylactically or therapeutically effective dose will generally be from about 0.01 mg/kg/day to about 100 mg/kg/day, preferably from about 0.1 mg/kg/day to about 2 0 mg/kg/day, and most preferably from about 1 mg/kg/day to about 10 mg/kg/day of a estrogen receptor modulating compound , which may be administered in one or multiple doses.
  • IV. Kits Kits may be provided which contain the formulation to be administered. The formulation may be administered once a day or more than once a day. The formulation can be administered enterally, parenterally, or topically.
  • the kits typically contain the active agent(s) to be administered, excipients and carriers, and instructions for administration of the formulation.
  • the kits may also contain equipment/devices used to administer the formulation, such as syringes.
  • ER ⁇ has been associated with estrogen-induced promotion of memory function and neuronal survival.
  • computer-aided structure-based virtual screening against a natural source chemical database was conducted to determine the occurrence of plant-based ER ⁇ -selective ligands. Twelve representative hits derived from database screening were assessed for their binding profiles to both ERs, three of which displayed over 100-fold binding selectivity to ER ⁇ over ER ⁇ .
  • the receptor-docking site was defined based on the binding position of genistein in the receptor and specified as all atoms within 10 A of the center carbon of genistein.
  • GOLD 2.0 Genetic Optimization for Ligand Docking
  • CCDC Cambridge Crystallographic Data Center
  • validation test Prior to the database screening, initial validation using genistein as the test ligand was conducted.
  • the aim of the validation test was to evaluate the effectiveness of the algorithm of the docking program in identifying the experimentally observed binding mode of the ligand in the receptor, to determine whether the program is applicable to the specific target system in this study.
  • the validation test was used to determine the optimal parameter settings for the later database screening. Twenty docking runs were carried out on the test complex, using the fastest default generic algorithm parameters optimized for virtual library screening, and the GoldScore fitness function was applied.
  • the validation test demonstrated that, based on the specified parameter settings, GOLD was effective in capturing the contributive hydrogen bond donor (NDl in His 475) crucial to the binding and reproducing the nearly coincident solution in terms of both the binding orientation and conformation of genistein as observed in the experimental measurement (see Figure 1).
  • the root-mean-square (RMS) deviations were computed between the observed experimental position and the GOLD solutions, with RMSD 0.3299 and 0.4483 compared to top-ranked and worst solutions, respectively.
  • the average RMSD of all solutions was 0.3566, which is regarded as a good prediction based on the subjective classifications defined by the program developer (refer to the program manual), suggesting that this program is reliable and applicable to the database screening toward ER ⁇ .
  • the 3D natural source chemical database was input and docked into the prepared ER ⁇ binding site in a flexible docking manner (full ligand and partial protein) and scored based on the GoldScore fitness function. Five hundred resultant top-scoring molecules were filtered via visual screening in the context of the receptor in Insightll. Based on visual analysis, 100 molecules underwent further analysis by Affinity, a more complex and predictive ligand docking program to refine the binding modes predicted by GOLD.
  • the criteria used for the selection of candidate molecules for investigation included the following (a) formation of hydrogen bond with donor atom NDl in His 475; (b) hydrophobic and hydrophilic balance appearing in the structure (e.g., the molecule should potentially have two relatively hydrophilic sides and a hydrophobic center to enhance both the steric and electrostatic complementarity with the receptor); (c) bound pose of the molecule in the receptor; and d) structural diversity. Finally, molecules that met the above criteria were computationally predicted for their drug-likeness (Lipinski's Rule of Five) and blood-brain barrier (BBB) penetration properties.
  • BBB blood-brain barrier
  • the ligand binding domains of the human ER ⁇ and ER ⁇ are approximately 60% homologous. Structural modeling and mutational analyses indicate that two variant amino acid residues along the ligand binding pocket, Leu 384 and Met 421 in ER ⁇ , which are replaced with Met 336 and He 373, respectively, in ER ⁇ , are the key molecular constituents underlying discriminative binding of selective ligands to either receptor subtypes. Sun, et al. MoI. Endocrinol, 2003, 17, 247-258. This slight structural variance serves as the foundation for both design and discovery of ER specific ligands. The similarities in the chemical features of both pairs of residues presents a substantial challenge to discover a selective ligand based on this difference. Of the known natural source ER ⁇ -selective ligands, genistein remains the most selective. However, an increasing number of synthetic compounds are emerging showing greater selectivity than genistein for ER ⁇ , as evidenced by the compound DPN developed in
  • the binding affinity and selectivity of candidate molecules yielded from database screening were determined by a fluorescent polarization competitive binding assay using purified baculovirus-expressed human ER ⁇ or ER ⁇ and a fluorescent estrogen ligand EL Red (PanVera Corp.). Test molecules were serially diluted to a 2 ⁇ concentration in assay buffer (200 ⁇ M to 200 pM). Fifty microliters of preincubated 2 ⁇ complex of ER ⁇ (30 nM) or ER ⁇ (60 nM) and EL Red (2 nM) was added to each well in a 96- well Non-binding Surface black microplate (Corning Life Sciences) for a final volume of 100 ⁇ L.
  • Negative controls containing ER and EL Red (equivalent to 0% inhibition) and positive controls containing only free EL Red (equivalent to 100% inhibition) were included.
  • the polarization values were measured using a Tecan GENios Pro reader at 535 nm/590 run excitation/emission and plotted against the logarithm of the test molecule concentration.
  • IC50 values concentration of test molecule that displaces half of the EL Red from ER
  • Table 1 summarizes the IC 5O binding results of test molecules to both ER ⁇ and ER ⁇ as well as the binding selectivity of representative molecules selected from four categories.
  • the negative control steroid does not bind to either ER.
  • genistein was found to bind to ER ⁇ with a 47.2-fold greater binding selectivity over ER ⁇ , but at an affinity one-fourth of 17 ⁇ -estradiol.
  • five molecules, 1, 2, 5, 7, and 8 showed binding selectivity to ER ⁇ over ER ⁇ , 3 of which, 2, 5, and 8, displayed the selectivity over 100-fold.
  • Preliminary structure and binding activity relationship analyses revealed that both the central hydrophobic skeletal structure and the connected two polar 'arms' contribute to the binding affinity of ligands to both ERs.
  • the enhanced VDW contact derives mainly from the central hydrophobic feature of the molecule.
  • the number of rings increases the binding affinity of molecules to the receptor, as indicated by the VDW value of 17 ⁇ - estradiol (-67.98) versus that of genistein (-60.75) and molecule 9 (-58.04), which are well correlated with their order-different binding affinities.
  • the hydrogen bonds derived from the two polar "arms" of the molecule are essential for the binding as well.
  • ERb-selective PhytoSERMs when administered singly or in combination on neuronal survival and molecular/functional markers associated with prevention of neurodegeneration and Alzheimer's disease (AD) was investigated.
  • 17 ⁇ -Estradiol was purchased from Steraloids (Newport, RI). Genistein, daidzein and equol were purchased from Indofine Chemical (H ⁇ lsborough, NJ). IBSO03569 was purchased from InterBioScreen (Moscow, Russia). The structures of these compounds are shown in Figure 1.
  • Test compounds were first dissolved in analytically pure DMSO (10 mM) and diluted in Neurobasal medium to the working concentrations right before treatments.
  • Test compounds were first dissolved in analytically pure DMSO and diluted in corn oil (50 ml of DMSO in 950 ml of corn oil) to the working concentrations at 100 mg/ml for 17 ⁇ -estradiol and 10 mg/ml for phytoSERMs.
  • DMSO DMSO in 950 ml of corn oil
  • ER ⁇ receptor (about.0.2 mg/ml, Affinity Bioreagents) is diluted to about 2 x 10 3 mg/ml in phosphate-buffered saline ( 11 PBS") at a pH of 7.4. Fifty microliters of the EPa -PBS solution is then added to each of the wells of a fiashplate. The plates are sealed and stored in the dark at 4 0 C for 16-18 hours. The buffered receptor solution is removed just prior to use, and the plates are washed 3 times with 200 microliters per well of PBS. The washing is typically performed using a slow dispense of reagent into the wells to avoid stripping the receptor from the well surface.
  • a total of 150 microliters from each of the wells is added to the corresponding wells of the pre-coated ER ⁇ plates.
  • the plates are sealed and the components in the wells are incubated either at room temperature for 4 hours or at 4°C overnight.
  • the receptor bound Hgand is read directly after incubation using a scintillation counter.
  • the amount of receptor bound ligand is determined directly, i.e., without separation of bound from free ligand. If estimates of both bound and free ligand are required, the supernatant is removed from the wells, liquid scintillant is added, and the wells are counted separately in a liquid scintillation counter.
  • ERg receptor (.about.0.2 mg/ml, Affinity Bioreagents) is diluted to about 2 xlO 3 mg/ml in phosphate-buffered saline ("PBS") at a pH of 7.4. Fifty microliters of the ER ⁇ -PBS solution is then added to each the wells of a flashplate. The plates are sealed and are stored in the dark at 4°C for 16-18 hours. The buffered receptor solution is removed just prior to use, and the plates are washed 3 times with 200 microliters per well of PBS. The washing is typically performed using a slow dispense of reagent into the wells to avoid stripping the receptor from the well surface.
  • PBS phosphate-buffered saline
  • a total of 150 microliters from each of the wells is added to the corresponding wells of the pre-coated ER ⁇ plates.
  • the plates are sealed and the components in the wells are incubated at room temperature either for 4 hours or at 4°C overnight.
  • the receptor bound Hgand is read directly after incubation using a scintillation counter.
  • the amount of receptor bound ligand is determined directly, i.e., without separation of bound from free ligand. If estimates of both bound and free Hgand are required, the supernatant is removed from the wells, liquid scintillant is added, and the wells are counted separately in a liquid scintillation counter.
  • ERa/ER ⁇ Transactivation Assays Construction of Transfected CHO Cells
  • Transfected CHO cells were derived from CHO KI cells obtained from the American Type Culture Collection ("ATCC", Rockville, Md.). The transfected cells were modified to contain the following four plasmid vectors: (1) pKCRE with DNA for the human estrogen receptor, (2) pAG-60- neo with DNA for the protein leading to neomycin resistance, (3) pRO-LUC with DNA for the rat oxytocin promoter and for firefly luciferase protein, and (4) pDR 2 with DNA for the protein leading to hygromycine resistance.
  • hippocampal neurons Primary cultures of hippocampal neurons were obtained from Embryonic Day 18 (El 8d) rat fetuses. Briefly, after dissected from the brains of the rat fetuses, the hippocampi were treated with 0.02% trypsin in Hank's balanced salt solution (137 mM NaCl, 5.4 raM KCl, 0.4 mM KH2PO4, 0.34 mM Na2HP0 4 .7H2 ⁇ , 10 mM glucose, and 10 mM HEPES) for 5 min at 37°C and dissociated by repeated passage through a series of fire-polished constricted Pasteur pipettes.
  • Hank's balanced salt solution 137 mM NaCl, 5.4 raM KCl, 0.4 mM KH2PO4, 0.34 mM Na2HP0 4 .7H2 ⁇ , 10 mM glucose, and 10 mM HEPES
  • Nerve cells were grown in phenol-red free Neurobasal medium (NBM, Invitrogen Corporation, Carlsbad, CA) supplemented with B27, 5 U/ml penicillin, 5 ⁇ g/ml streptomycin, 0.5 niM glutamine and 25 ⁇ M glutamate at 3 7°C in a humidified 10% CO2 atmosphere at 37°C for the first
  • Nuclear lysates were prepared as following: Briefly, hippocampal neurons grown on poly-D-lysine coated culture dishes were treated with compounds for appropriate periods, washed with cold PBS once and scraped into 1 ml PBS. Cells were then centrifuged at 5,000 rpm for 5 min, and the pellet was dissolved in Cytoplasm Extraction buffer (10 mM HEPES, 1 mM EDTA, 60 mM KCl, 0.075% Igepal and protease and phosphatase inhibitor cocktail) and suspended by passage through a 200 ⁇ l pipette tip.
  • Cytoplasm Extraction buffer (10 mM HEPES, 1 mM EDTA, 60 mM KCl, 0.075% Igepal and protease and phosphatase inhibitor cocktail
  • Igepal and protease and phosphatase inhibitor cocktail was added to the pellet followed by 5M NaCl to break the nuclear membrane. Following 30- 45 RPM of incubation at 4 0 C, the samples were centrifuged at 12,000 rpm for 10 min to generate a supernatant containing the nuclear extract.
  • Protein concentration was determined by the BCA method. An appropriate volume of 2X sample buffer was added to the protein samples, and samples were boiled at 95°C for 5 rain. Samples (25 ⁇ g of proteins per well) were loaded on a 10% SDSPAGE gel and resolved by standard electrophoresis at 90V. Proteins were then electrophoretically transferred to Immobilon-P PVDF membranes overnight at 32 V at 4 0 C.
  • Membranes were blocked for 1 hr at room temperature in 10% non-fat dried milk in PBS containing 0.0 5% Tween 20 (PBS-T), incubated with appropriate primary antibodies against phospho-CREB (pSER ⁇ ; mouse monoclonal, 1:2000; Cell Signaling Technology, Beverly, MA), CREB (rabbit polyclonal, 1 : 1000; Cell Signaling Technology, Beverly, MA), spinophilin (rabbit polyclonal, 1:1000; Upstate Biotecholagy, Lake Placid, NY), actin.
  • pSER ⁇ mouse monoclonal, 1:2000; Cell Signaling Technology, Beverly, MA
  • CREB goat polyclonal, 1 : 1000
  • spinophilin rabbit polyclonal, 1:1000; Upstate Biotecholagy, Lake Placid, NY
  • Membranes were incubated with the primary monoclonal antibody against Bcl-2 (Zymed Laboratories, Inc., S. San Francisco, CA) diluted 1:250 in PBS-TweenTM with 1% horse serum (Vector Laboratories, Inc., Burlingame, CA) overnight at 4 "C, then incubated with the secondary horseradish peroxidase (HRP)-conjugated horse anti-mouse IgG (Vector Laboratories, Inc, Burlingame, CA) diluted 1:5,000 in PBS-TweenTM with 1% horse serum for 2 hr at room temperature, and Bcl-2 proteins were visualized by developing the membranes with TMB substrate for peroxidase (Vector Laboratories, Inc., Burlingame, CA).
  • HRP horseradish peroxidase
  • ⁇ - Actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) level was determined to ensure equal protein loading, and high-range Precision Protein Standards (Bio-Rad Laboratories, Hercules, CA) was used to determine protein sizes. Relative intensities of bands were quantified by optical density analysis using an image digitizing software Un-Scan-Tt version 5.1 (Silk Scientific, Inc., Orem, UT). Statistics
  • Antiestrogenic activity was determined by the ability of a test compound to suppress the increase in uterine wet weight resulting from the administration of 0.2 ⁇ g 17- ⁇ -estradiol ("E2") per day. Any statistically significant decreases in uterine weight in a particular dose group as compared with the E2 control group are indicative of anti-estrogenicity .
  • One hundred forty (140) female pups (19 day s old) in the 35-50 g body weight range are selected for the study. On day 19 of age, when the pups weigh approximately 35-50 g, they are body weight-order randomized into treatment pups. Observations for mortality, morbidity, availability of food and water, general appearance and signs of toxicity are made twice daily. Pups not used in the study are euthanized along with the foster dams. Initial body weights are taken just prior to the start of treatment at day 19 of age. The final body weights are taken at necropsy on day 22 of age. Treatment commences on day 19 of age and continues until day 20 and 21 of age. Each animal is given three subcutaneous (“sc”) injections daily for 3 consecutive days.
  • sc subcutaneous
  • Three rats in each of the control and mid- to high-level dose test groups are anesthetized with a ketamine/xylazine mixture.
  • Their blood is collected by exsanguination using a 22 gauge needle and 5 ml syringe flushed with 10 USP with sodium heparin/ml through the descending vena cava; and then transferred into a 5 ml green top plasma tube (sodium heparin (freeze-dried), 72 USP units).
  • Plasma samples are collected by centrifugation, frozen at -70 0 C, and analyzed using mass spectrographic to determine the presence and amount of test compound in the serum .
  • Blood chemistry is also analyzed to determine other blood parameters.
  • the uteri from the rats are excised and weighed.
  • the remaining rats are sacrificed by asphyxiation under CO2-
  • the uteri from these rats are excised,, nicked, blotted to remove fluid, and weighed to the nearest 0.1 mg.
  • a parametric one-way analysis of variance is performed (SIGMASTAT version 2.0, available commercially from Jandel Scientific, San Rafael, Calif). Estrogen agonist and antagonist activity is assessed comparing uterine wet weights across treatment groups using a parametric ANOVA on loglo transformed data. The data are transformed to meet assumptions of normality and homogeneity of variance of the parametric AWQVA. The F value is determined and a Student-
  • Newman-Kuel s multiple range test is performed to determine the presence of significant differences among the treatment groups.
  • the test compound is determined to act as a mixed estrogen agonist/antagonist if the test compound does not completely inhibit the 17.- ⁇ -estradiol stimulated uterotrophic response.
  • Rats were placed on a phytoSERM-reduced diet, TD.96155 (Harlan Teklad). Rats were given, once daily, 2 subcutaneous injections of vehicle (control), 17 ⁇ -estradiol (70 ⁇ g/kg BW), genistein (6 mg/kg BW), or phytoSERM combinations (6 mg/kg BW). Dosages used here are commensurate with those used in humans.
  • mice fasted for 24 hours prior to sacrifice and brain dissection.
  • Hippocampal and cortical tissues were collected from one hemisphere and stored for biochemical analyses. The remaining brain tissues minus cerebellum, pineal gland, and brainstem were utilized for mitochondrial isolations, followed immediately by mitochondrial respiratory activity measurements. The rest of mitochondrial samples were stored for cytochrome c oxidase activity measurements. Uteri were excised, trimmed of fat and connective tissue, and both a wet weight and a dry weight were recorded. Results
  • Figures 2 A and 2B show the competition binding curves for ER ⁇ and ER ⁇ .
  • Data were generated with a fluorescence polarization-based competitive binding assay using full-length human ER ⁇ and ER ⁇ , and plotted against the logarithm of serially diluted concentrations of the test compounds (or combinations).
  • Progesterone served as a negative control.
  • 17 ⁇ -Estradiol served as a positive control.
  • Combined formulations were composed of equivalent molar of individual phytoSERMs included.
  • 17 ⁇ -estradiol has no binding preference to ER ⁇ or to ER ⁇ .
  • the concentration of a test molecule resulting in the half-maximum shift in polarization value equals its IC 50 Non-convergence within the dose range, predicts that either the molecule does not bind to the receptor or that the binding affinity is very low.
  • Neuroprotective Effect Table 3 and Figure 3 show the dose-dependent neuroprotective effects of four ER ⁇ -selective phytoestrogenic molecules against supraphysiological glutamate (100 ⁇ M)-induced neurotoxicity in primary hippocampal neurons by measurement of LDH release. ## P ⁇ 0.01 compared to vehicle alone-treated cultures; * P ⁇ 0.05 and ** P ⁇ 0.01 compared to glutamate alone-treated cultures.
  • ⁇ Data are derived from a single experiment and are representative of at lease three independent experiments. Results are presented as the percent of LDH release from vehicletreated control cultures and expressed as means ⁇ S.E.M., n 6.
  • Figure 3 shows the neuroprotective efficacy of four ER ⁇ -selective phytoestro genie molecules when administered alone at concentrations that elicited the maximal neuroprotective effects as revealed from the dose- response analyses (100 nM for all.four molecules), or co-administered, against supraphysiological glutamate (100 ⁇ M)-induced neurotoxicity in primary hippocampal neurons by measurement of calcein AM staining.
  • V t reatm ent is the individual value from phytoestrogen-treated cultures
  • Vgiutamate is a mean value from glutamate alone-treated cultures
  • Ventral is a mean value from vehicle-treated control cultures.
  • FIG. 4A-4B shows the effects on Bcl-2 and BcI-XL expression in rat primary hippocampal neurons and hippocampal tissues derived from adult ovariectomized rats.
  • Primary hippocampal neurons grown for 7 divisions were treated with the test compounds (or combinations) for 48 hr followed by Western blot analyses.
  • Adult ovariectomized rats were given, once daily, 2 subcutaneous injections of the test compounds (or combinations). Rats were sacrificed 24 h later following the 2nd injection.
  • Hippocampal tissues were homogenized followed by Western blot analyses. Combined formulations were composed of equivalent molar in (A) and equivalent weight in (B) of individual phytoSERMs including G: genistein; D: daidzein; E: equol; and I: IBSO03569. Incubation of neurons with a combination of four phytoestrogens for
  • estradiol via the PI3K signaling pathway, activates both the Akt and the ERK 1/2 cascades in the same population of cortical and hippocampal neurons. Simultaneous activation of two pathways that prevent mitochondria from activating cell-death cascades is likely to promote neuron survival.
  • FIG. 5 illustrates the effect of four ER ⁇ -selective phytoestrogenic molecules when co-administered (100 nM for all four molecules) on the expression of the anti- ⁇ -amyloid protein, insulin-degrading enzyme ("IDE") in primary hippocampal neurons. **P ⁇ 0.01 compared to vehicle alone- treated cultures.
  • Figure 5 demonstrates the effects of these various combinations of phytoestrogens along with E2 on the expression of the anti- ⁇ -amyloid (anti-A ⁇ ) protein, insulin-degrading enzyme (IDE) in primary neurons. Data showed that all three combinations composed of two, three or four phytoestrogens significantly increases IDE expression in neurons. Among them, a combination of three phytoestrogens induced the greatest neuronal response, with an efficacy greater than E2 as well as a combination of two phytoestrogens.
  • AD one neuropathological hallmark of AD is a significant deposition of extracellular A ⁇ peptide, as referred to A ⁇ plaque.
  • Impaired A ⁇ clearance and/or degradation has been demonstrated to contribute in part to A ⁇ plaque formation in AD brain.
  • IDE a metalloprotease enzyme
  • Choronic upregulation of IDE represents a novel efficacious therapeutic approach to lowering the steady-state A ⁇ level in the brain and eventually preventing the occurrence of Alzheimer-type pathology. Therefore, these data indicate that coadministration of multiple ER ⁇ -selective phytoestrogens have the potential to activate the anti-A ⁇ mechanism, and as a result, maintain the brain in a long-term healthy status.
  • Upregulation of Spinophilin Upregulation of Spinophilin
  • Figure 6 illustrates the effect of four ER ⁇ -selective phytoestrogens molecules when co-administered (100 nM for all four molecules) on the expression of the spine marker, spinophilin, in primary hippocampal neurons. **P ⁇ 0.01 compared to vehicle alone-treated cultures.
  • FIGS. 7A-7D shows the neuroprotective efficacy of the compounds against glutamate ( Figure 7A) and amyloids-induced neurotoxicity in rat primary hippocampal neurons.
  • Primary hippocampal neurons grown for 7 divisions were pretreated with the test compounds (or combinations) for 48 h f followed by a 5-min exposure to 100 mM glutamate. Neurons were incubated for an additional 24 h prior to neuronal viability analyses by calcein AM staining. Following pretreatment with the compounds (or combinations) for 48 hr, neurons were exposed to 3 mM ⁇ -amyloidi.42 for 2 d. Neuronal viability was analyzed by fluorometric measurements of activities of the LDH and dead-cell protease released in the culture media, and the live-cell protease exclusively entering intact viable neurons.
  • NE neuroprotective efficacy
  • Vtreaiment -V neuro toxin Vtreaiment -V neuro toxin
  • Vcontrol - V neuro to ⁇ in 100%
  • V tr eatment is the individual value from the test compounds (or combinations)-treated cultures
  • V neuro t 0 ⁇ i ⁇ is a mean value from glutamate or ⁇ -amyloidi-42 alone-treated cultures
  • V CO ntroi is a mean value from vehicle-treated control cultures.
  • IDE vascular endothelial fibroblasts
  • NEP neuropeptide expression in (A) rat primary hippocampal neurons and (B) hippocampal tissues derived from adult ovariectomized rats.
  • Figures 9A-9E show the effects on forebrain mitochondrial cytochrome c oxidase (COX) activity in adult ovariectomized rats. Rats were given, once daily, 2 subcutaneous injections of the test compounds (or combinations). Rats were sacrificed 24 h later following the 2nd injection. Forebrain mitochondria were isolated followed by a spectrophotometric measurement of COX activity using an immunocapture method. Colorimetric absorbance at 550 nm was recorded every 5 min for 115 min. COX activity is presented as the initial rate of oxidation of reduced cytochrome c, and determined by calculating the initial slope between two time points ( ⁇ 20 min) within the linear region.
  • COX forebrain mitochondrial cytochrome c oxidase
  • Figures 10A- 1OE show the effects on forebrain mitochondrial respiratory activity in adult ovariectomized rats. Rats were treated as above. Forebrain mitochondria were isolated followed immediately by a polygraphical measurement of respiratory activity using an oxygen electrode. Following a basal recording, mitochondrial state 4 respiration was measured following the addition of substrates, malate/glutamate. State 3 respiration was measured following the addition of ADP. Respiratory control ratio (RCR) was calculated as the ratio between the rate of oxygen uptake at state 3 and the rate of oxygen uptake at state 4.
  • RCR Respiratory control ratio
  • Table 4 shows the effects on uterine weight in adult ovariectomized rats. Changes in uterine weight in response to estrogenic stimulation can be used to evaluate the estrogenic characteristics of test compounds on uterine tissues.
  • immature female rats having low endogenous levels of estrogen are dosed with test compound (subcutaneously) daily for 3 days. Compounds are formulated as appropriate for subcutaneous injection. As a control, 17- ⁇ estradiol is administered alone to one dose group. Vehicle control dose groups are also included in the study. Twenty-four hours after the last treatment, the animals are necropsied, and their uteri excised, nicked, blotted and weighed. Any statistically significant increases in uterine weight in a particular dose group as compared to the vehicle control group demonstrate evidence of estrogenicity.
  • Rats were given, daily once, 2 subcutaneous injections of the test compounds (or combinations) (n > 4 for each group). Rats were sacrificed 24 h later following the 2nd injection. Uteri were immediately excised and a wet weight was recorded. Uteri were then air dried for 1 hour followed by at 70 0 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).
  • the present study indicates that combined use of select ER ⁇ -selective PhytoSERMs can be more therapeutically effective than single administrations and alternative combined formulations.
  • the present study suggests the potential of the combination of genistein, daidzein and equol, at an equivalent weight, for prevention of neurodegeneration and AD, along with management of climacteric symptoms in postmenopausal women.
  • FIGS 1 IA- 11 C are schematics showing estrogen mechanisms of action that lead to neurotrophic and neuroprotective outcomes.
  • 17- ⁇ - Estradiol (E2) acting via a membrane-associated site (mER) activates a cascade required for multiple responses that lead to enhanced neural plasticity, morphogenesis, neurogenesis, and neural survival.
  • the signaling sequence induced by E2 at the membrane site is as follows: (1) E2 binding to mER, (2) E2-mER complexes with ⁇ 85 to activate PI3K, (3) activating calcium-independent PKC, (4) phosphorylating the L-type calcium channel, (5) inducing calcium influx, (6) activating calcium-dependent PKCs, (7) activating Src kinase, (8) activating the MEK/ERKl/2 pathway, (9) ERK translocates to the nucleus, (10) activating and phosphorylating CREB, (11) enhancing transcription of antiapoptotic genes Bcl-2 and Bcl-xl, which enhance mitochondrial vitality, and spmophilin, which encourages synaptic growth, (12) simultaneously, estrogen activation of PI3K leads to activation of Akt, which phosphorylates and inhibits the proapoptotic protein BAD.
  • Estrogen-induced neuroprotective mechanisms converge on mitochondria.
  • Estrogen-activated cellular signaling cascade promotes enhanced mitochondrial function, leading to increased calcium load tolerance, enhanced electron transport chain efficiency, and promotion of antioxidant defense mechanisms. These actions are mediated by the regulation of both nuclear and mitochondrial encoded genes initiated by the activation of second-messenger signaling cascades.
  • ER ⁇ -selective phytoestrogen formulations which optimize activation of ER ⁇ while minimizing or avoiding activating ER ⁇ 5 should serve as an effective estrogen alternative replacement therapy for sustaining neurological health, function and prevention of AD without induction of proliferative responses in the reproductive tissues as seen with the current ET/HT.
  • ER ⁇ -selective phtoestrogen formulations may serve as a particular viable strategy for reducing a major risk factor of AD in ApoE4 carriers.

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Abstract

L'invention concerne des compositions pharmaceutiques de phyto-œstrogènes sélectionnés et des procédés d'utilisation pour favoriser la santé neurologique et la prévention de la neurodégénérescence liée à l'âge, telles que la maladie d'Alzeihmer. Ces formulations de phyto-œstrogènes sélectionnés sont composées d'un nombre de molécules œstrogéniques d'origine végétale et/ou de leurs analogues structuraux et présentent une préférence de liaison à ERβ sur ERα et une activité agoniste dans le cerveau. Ces formulations de phyto-œstrogènes sélectives de ERβ traversent la barrière entre le sang et le cerveau et favorisent les mécanismes de neurotrophisme et de neuroprotection associés aux œstrogènes dans le cerveau, sans activer de mécanismes de prolifération dans les tissus reproducteurs et sont par conséquent exemptes d'autres aspects problématiques associés aux œstrogènes. Les formulations de phyto-œstrogènes sélectionnés sont thérapeutiquement utiles à la fois pour les femmes et pour les hommes pour entretenir la santé neurologique et prévenir un déclin cognitif lié à l'âge et des troubles neurodégénératifs, tels que la maladie d'Alzeihmer. Ces formulations sont administrées par voie entérale, transdermique, transmucosale, intranasale ou parentérale, dans un dosage efficace pour prévenir ou soulager un dommage neuronal, effectuent une régénération neuronale ou entretiennent la viabilité, augmentent l'expression de protéines anti-apoptotiques et/ou diminuent les indicateurs de la maladie d'Alzheimer. Les formulations contiennent, de préférence, des combinaisons de composés, et peuvent être formulées pour une administration quotidienne, entretenue, retardée ou hebdomadaire/mensuelle. Dans un mode de réalisation préféré, elles sont administrées à des femmes ménopausées ou post-ménopausées, préférablement tôt dans la ménopause.
PCT/US2007/073505 2006-08-02 2007-07-13 Formulations phyto-œstrogéniques pour soulager ou prévenir des maladies neurodégénératives WO2008016768A1 (fr)

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US9078888B2 (en) 2007-01-22 2015-07-14 Gtx, Inc. Nuclear receptor binding agents
US9333192B2 (en) 2010-02-15 2016-05-10 Sinoveda Canada, Inc Phytoestrogen product of red clover and pharmaceutical uses thereof
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JP2019526612A (ja) * 2016-09-12 2019-09-19 スティーブン・ホフマン 認知症を治療するための組成物

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Publication number Priority date Publication date Assignee Title
US9078888B2 (en) 2007-01-22 2015-07-14 Gtx, Inc. Nuclear receptor binding agents
US9604931B2 (en) 2007-01-22 2017-03-28 Gtx, Inc. Nuclear receptor binding agents
US9623021B2 (en) 2007-01-22 2017-04-18 Gtx, Inc. Nuclear receptor binding agents
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WO2013050930A1 (fr) * 2011-10-02 2013-04-11 Sinoveda Canada, Inc, Développement d'un produit de phytoestrogène de trèfle des prés pour la prévention ou le traitement de l'ostéoporose
WO2018022604A3 (fr) * 2016-07-26 2018-03-22 Ausio Pharmaceuticals, Llc Méthodes de diagnostic et de traitement de la maladie d'alzheimer par s-équol
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WO2022071670A1 (fr) * 2020-09-29 2022-04-07 주식회사 뉴로바이오넷 Dérivé de 3-phényl-2h-chromène et composition pharmaceutique le contenant pour la prévention ou le traitement de la maladie d'alzheimer

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