WO2002022645A2 - Procede de preparation de d'utilisation de 7alpha-methyl-11beta-methoxy estradiol - Google Patents

Procede de preparation de d'utilisation de 7alpha-methyl-11beta-methoxy estradiol Download PDF

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Publication number
WO2002022645A2
WO2002022645A2 PCT/US2001/042154 US0142154W WO0222645A2 WO 2002022645 A2 WO2002022645 A2 WO 2002022645A2 US 0142154 W US0142154 W US 0142154W WO 0222645 A2 WO0222645 A2 WO 0222645A2
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Prior art keywords
estrogenic
bioactivity
molecules
solvent
modulatory
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PCT/US2001/042154
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English (en)
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WO2002022645A3 (fr
Inventor
Lawrence B. Hendry
Lewis W. Roach, Jr.
Marc W. Andersen
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Accelerated Pharmaceuticals, Inc.
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Priority to AU2001296872A priority Critical patent/AU2001296872A1/en
Publication of WO2002022645A2 publication Critical patent/WO2002022645A2/fr
Publication of WO2002022645A3 publication Critical patent/WO2002022645A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J51/00—Normal steroids with unmodified cyclopenta(a)hydrophenanthrene skeleton not provided for in groups C07J1/00 - C07J43/00
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J1/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, not substituted in position 17 beta by a carbon atom, e.g. estrane, androstane
    • C07J1/0051—Estrane derivatives
    • C07J1/0066—Estrane derivatives substituted in position 17 beta not substituted in position 17 alfa
    • C07J1/007—Estrane derivatives substituted in position 17 beta not substituted in position 17 alfa the substituent being an OH group free esterified or etherified

Definitions

  • the present invention relates generally to molecules that possess estrogenic bioactivity or modulate estrogenic bioactivity, novel methods of synthesizing these molecules, and new uses for these molecules to provide or modulate estrogenic bioactivity.
  • Estrogen and deficiencies in estrogen bioactivity or estrogen levels have been implicated in numerous conditions including osteoporosis, myocardial infarction, stroke, infertility, angiogenesis and cancer. These conditions affect millions of people each year at significant cost to the individual and to society. As life span increases and the population ages, the number of individuals affected by conditions implicating estrogen is increasing dramatically. Accurate and rapid identification of safe, economical and effective estrogenic molecules is a critical step in saving drug development costs and accelerating access to new and effective molecules useful as estrogenic molecules or modulators of estrogenic bioactivity.
  • Molecules that modulate the biological activity of estrogens or possess estrogenic biological activity are used in the treatment of a variety of conditions involving estrogen replacement therapy and the modulation of the effects of estrogen on biological target sites. These conditions include, but are not limited to the following: bone mineral density, osteopenia, osteoporosis and other conditions related to bone synthesis and metabolism; conditions affecting the cardiovascular system including heart disease, stroke, angiogenesis, serum cholesterol concentrations, serum levels of high density lipoprotein (HDL), serum levels of low density lipoprotein (LDL), the relative ratio of serum levels of HDL to LDL, and proliferation of endothelial cells; conditions affecting the nervous system, including Alzheimer's disease; conditions affecting endocrine systems and neuroendocrine systems, including the hypothalamo-pituitary- ovarian reproductive system, further including conditions affecting the onset and duration of puberty, fertility, infertility, contraception, conception, lactation, successful implantation, menstruation, menopause, endometriosis, alopecia,
  • the present invention provides new uses for molecules to provide or modulate estrogenic bioactivity. These uses include, but are not limited to, administration to an animal or human to provide or modulate estrogenic bioactivity.
  • the estrogenic and estrogenic modulatory molecules of the present invention may also be administered in vitro.
  • the present invention provides new uses for the molecule ll ⁇ -methoxy-7 ⁇ -methylestra-l,3,5(10)-triene-3,17 ⁇ -diol, which displays greater estrogenic bioactivity than 17 ⁇ estradiol in some test systems described herein.
  • the present invention provides a variety of uses for l l ⁇ - methoxy-7 ⁇ -methylestra-l,3,5(10)-triene-3,17 ⁇ -diol to provide or modulate estrogenic bioactivity.
  • the present invention also provides a novel method for synthesizing 11 ⁇ -methoxy-7 ⁇ -methylestra- 1,3,5(10)-triene-3 , 17 ⁇ -diol and related molecules.
  • estrogenic bioactivity Two types of bioactivity with relevance to the present invention are estrogenic bioactivity and modulation of estrogenic bioactivity. It is to be understood that a molecule which possesses estrogenic bioactivity will show "estrogen-like" effects when administered with a pharmaceutically acceptable carrier to an animal or a human. It is to be understood that a molecule which possesses the ability to modulate estrogen bioactivity, will modulate the effects of "estrogenic molecules" when administered with a pharmaceutically acceptable carrier to an animal or a human. Such estrogenic modulatory activity may be evident in an enhanced or reduced response to an estrogenic molecule following administration of the estrogenic modulator. Estrogenic modulatory activity may also be evident in an enhanced or reduced response to an anti-estrogenic molecule following administration of the estrogenic modulator.
  • Estrogenic bioactivity and estrogenic modulatory activity may also be evident in vitro in a variety of test systems and conditions commonly known to one of skill in the art. Such systems and conditions include, but are not limited to the following: tissue culture; cell culture; cell free systems; estrogen responsive transcription of nucleic acids; receptor binding systems; cell growth; cell death; and cell differentiation.
  • the molecules of the present invention may be combined with pharmaceutically acceptable carriers and administered as compositions in vitro and in vivo.
  • the molecules of the present invention may be combined with cosmetically acceptable carriers and administered as compositions in vitro and in vivo.
  • the molecules of the present invention may be administered in combination with one or more other compounds to treat conditions in which estrogenic bioactivity or modulation of estrogenic bioactivity is desired.
  • the administration ofthe molecules ofthe present invention may occur before, during and/or after administration of the additional compound(s).
  • the additional compounds, and the appropriate dosage and administration schedules, are known to one of ordinary skill in the art to treat specific conditions.
  • compositions of the present invention may be useful to provide estrogenic bioactivity or modulation of estrogen bioactivity in conditions in which estrogenic bioactivity or modulation of estrogen bioactivity is desirable, such as hormone replacement therapy.
  • conditions include, but are not limited to, the following: conditions related to bone synthesis and metabolism including, but not limited to, bone mineral density, osteoporosis, and osteopenia; conditions affecting the cardiovascular system including, but not limited to, heart disease, myocardial infarction, stroke, angiogenesis, serum cholesterol concentrations, serum levels of high density lipoprotein (HDL), serum levels of low density lipoprotein (LDL), the relative ratio of HDL to LDL, atherosclerosis, and the proliferation of endothelial cells; conditions affecting the nervous system, including, but not limited to, sexual differentiation of the brain, mood, behavior, sexual motivation, libido, Alzheimer's disease, cognition, neuroprotection, neuronal cell death; retention of memory; conditions affecting endocrine systems and neuroendocrine systems, including but not limited to the hypothal
  • Yet another object of the present invention is to provide new uses for a molecule to provide or modulate estrogenic bioactivity, wherein the molecule is encompassed within the generic structure shown in Figure 1, further wherein R 2 is CH 3 and R 2 is CH,.
  • This molecule is also referred to herein as 1 l ⁇ - methoxy-7 ⁇ -methylestra-l,3,5(10)-triene-3,17 ⁇ -diol or PDC-7 and is shown in Figure 2.
  • Another object of the present invention is to use molecules encompassed within the generic structure shown in Figure 1, further wherein R ⁇ is CH 3 , C 2 H 5 , HCO, or CH 3 CO and R, is CH 3 , to treat conditions wherein estrogenic bioactivity or modulation of estrogenic bioactivity is desired. It is another object of the present invention to use ll ⁇ -methoxy-
  • Another object of the present invention is to provide new uses for ll ⁇ -methoxy-7 ⁇ -methylestra-l,3,5(10)-triene-3,17 ⁇ -diol and structurally related molecules ofthe present invention for providing desired estrogenic or estrogenic modulatory activity in conditions including, but not limited to, the following: conditions related to bone synthesis and metabolism including but not limited to bone mineral density, osteoporosis, and osteopenia; conditions affecting the cardiovascular system including but not limited to heart disease, myocardial infarction, stroke, angiogenesis, serum cholesterol concentrations, serum levels of high density lipoprotein (HDL), serum levels of low density lipoprotein (LDL), the relative ratio of HDL to LDL, atherosclerosis, and the proliferation of endothelial cells; conditions affecting the nervous system, including but not limited to sexual differentiation ofthe brain, mood, behavior, sexual motivation, libido, Alzheimer's disease, neuroprotection, neuronal cell death, cognition, retention of memory; conditions affecting endocrine systems and neuroendoc
  • Another object ofthe present invention is to provide compositions comprising pharmaceutically or cosmetically acceptable carriers combined with the novel molecules ofthe present invention.
  • a related object of the present invention is to administer these compositions in vivo to humans or animals or in vitro when estrogenic bioactivity or modulation of estrogenic bioactivity is desired, and/or to treat the conditions described above.
  • a specific object of the present invention is to provide molecules possessing estrogenic bioactivity which is greater than the bioactivity of estrogen at one or more biological sites of estrogenic bioactivity.
  • Yet another object ofthe present invention is to provide a method for making molecules encompassed within the generic structure shown in Figure 1, wherein R ( is CF ⁇ , C 2 H 5 , HCO or CH 3 CO and R 2 is CH 3 or H.
  • a specific object of the present invention is to provide a method for making molecules encompassed within the generic structure shown in Figure 1, wherein R is CH 3 and R 2 is CH 3 , also called ll ⁇ -methoxy-7 ⁇ -methylestra- l,3,5(10)-triene-3,17 ⁇ -diol as shown in Figure 2.
  • Figure 1 shows a generic structure for a class of estrogenic molecules, wherein
  • R is CH 3 , C 2 H 5 , HCO or CH 3 CO, and R j is CH 3 or H. Dashed lines represent groups located below the plane ofthe molecule. A solid triangular line without a designated group represents CH .
  • Figure 2 is a schematic representation of ll ⁇ -methoxy-7 ⁇ -methylestra-l,3,5(10)- triene-3,17 ⁇ -diol, a species ofthe genus displayed in Figure 1, wherein R x is CH 3 and R 2 is CH 3 . Dashed lines represent groups located below the plane of the molecule. A solid triangular line without a designated group represents CH .
  • Figure 3 is a schematic diagram of a general method of making the compounds represented by the generic structure shown in Figure 1, wherein R ⁇ s CH 3 , C 2 H 3 , HCO or CH 3 CO and R, is CH or H.
  • Figure 4 is a schematic diagram of a method of making l l ⁇ -methoxy-7 ⁇ - methylestra-l,3,5(10)-triene-3,17 ⁇ -diol.
  • Figure 5 is a schematic representation of the dose response of uterine weight/body weight as a function of estradiol dose (picomoles, closed circles) or l l ⁇ -methoxy-7 ⁇ -methylestra-l,3,5(10)-triene-3,17 ⁇ -diol (picomoles, closed squares).
  • the lower dashed line represents a value for the uterine weight divided by the body weight of ovariectomized rats.
  • the upper dashed line represents two times the level represented by the lower line.
  • Statistically significant differences between corresponding dosages of estradiol and ll ⁇ -methoxy-7 ⁇ -methylestra- l,3,5(10)-triene-3,17 ⁇ -diol at p ⁇ 0.05 are represented by asterisks.
  • the present invention provides new uses for molecules to provide or modulate estrogenic bioactivity.
  • the present invention also provides new uses for known molecules, wherein these molecules have been predicted and found herein to possess estrogenic bioactivity or estrogenic modulatory bioactivity.
  • the present invention also provides compositions comprising cosmetically or pharmaceutically acceptable carriers combined with the molecules ofthe present invention. These compositions may be administered in vivo or in vitro. Several methods of assessing the estrogenic bioactivity or estrogenic modulatory bioactivity of these novel molecules are provided herein.
  • the present invention also provides methods of using compositions including the molecules of the present invention in pharmaceutically or cosmetically acceptable carriers to treat conditions wherein estrogen bioactivity or modulation of estrogenic bioactivity is desired.
  • the molecules of the present invention may display estrogenic bioactivity or estrogenic modulatory bioactivity at one or more biological sites.
  • estrogenic modulatory bioactivity includes modulatory bioactivity that either enhances or reduces the biological activity of estrogenic molecules at a specific biological target site. Examples of molecules of the present invention that provide estrogenic bioactivity or estrogenic modulatory bioactivity are shown in Figure 1, wherein R ; is CH 3 , C 2 H 5 , HCO, or CH 3 CO and R 2 is CH 3 .
  • One preferred molecule of the present invention that provides estrogenic bioactivity or estrogenic modulatory bioactivity is referred to interchangeably herein as l l ⁇ -methoxy-7 ⁇ -methylestra- 1,3,5 (10)-triene-3,17 ⁇ - diol, 7 ⁇ -methyl-l l ⁇ -methoxy estradiol, or PDC-7 and is shown in Figure 2. It is a species ofthe genus displayed in Figure 1, wherein R j is CH 3 and R 2 is CH 3
  • the present invention also provides methods of making molecules encompassed within the structure shown in Figure 1, wherein R 2 is CH 3 , CH 5 , HCO or CH 3 CO, and R 2 is CH 3 or H.
  • One preferred molecule made with the method ofthe present invention is ll ⁇ -methoxy-7 ⁇ -methylestra- 1,3,5(10)-triene- 3,17 ⁇ -diol and is shown in Figure 2. It is a species of the genus displayed in Figure 1, wherein Rj is CH 3 and R 2 is CH 3 .
  • the design of the molecules of the present invention to possess estrogenic bioactivity or to modulate estrogenic bioactivity was accomplished using the estrogen pharmacophore and methods described in U.S. Patent Nos: 5,705,335, 5,888,738 and 5,888,741 to Hendry. These methods provide an efficient and cost-effective means to evaluate a molecule for suspected bioactivity or to design a molecule to possess a desired bioactivity.
  • molecules such as ll ⁇ -methoxy-7 ⁇ -methylestra-l,3,5(10)-triene-3,17 ⁇ -diol, were evaluated for potential estrogenic bioactivity or estrogenic modulatory bioactivity using methods described in these patents and using additional methods described elsewhere herein.
  • l l ⁇ -methoxy-7 ⁇ -methylestra-l,3,5(10)- triene-3,17 ⁇ -diol is also called 7 -methyl-ll ⁇ -methoxy estradiol and PDC-7 in the present application.
  • carbon atoms in the steroid backbone are numbered according to the conventional numbering system known to one of ordinary skill in the art.
  • the estrogen pharmacophore was created, as previously described, by docking estradiol and highly active steroidal and non-steroidal estrogens into DNA using optimum van der Waals and hydrogen bonding interactions.
  • the resulting composite or pharmacophore is represented by an enclosed three dimensional surface bordered by point charges which reflect the positions of hydrogen bond acceptors. Quantitation of fit of any given molecule into the pharmacophore was approximated by the sum of the volume of the molecule measured in A 3 which fits within the pharmacophore, and the electrostatic attraction between the hydrogen bonding functional groups and the point charges on the pharmacophore. In order to compare the approximate fit of candidate molecules, the data were normalized to estradiol which was given a value of 100% fit (Table 1). The estrogen pharmacophore was employed to screen existing compounds for estrogenic activity as well as to design new molecular structures as potential estrogens or modulators of estrogenic bioactivity.
  • Estrogenic Biological Activity or Estrogenic Modulatory Biological Activity ofthe Molecules ofthe Present Invention Once a molecule has been designed to possess estrogenic bioactivity or to modulate estrogenic bioactivity using the estrogen pharmacophore, these biological activities may be verified. Biological assessment of predicted estrogenic bioactivity of designed molecules may be performed using currently available assays known to one of ordinary skill in the art. It is to be understood that the molecules ofthe present invention may display estrogenic bioactivity at one site and little or no estrogenic bioactivity at another site. It is also to be understood that the molecules of the present invention may display estrogenic bioactivity at one site and anti-estrogenic bioactivity at another site. A variety of assay methods exist for evaluating various forms of estrogenic bioactivity or estrogenic modulatory bioactivity.
  • the assays in these references provide means to assess estrogenic bioactivity and/or the modulation of estrogenic bioactivity in a variety of test systems, including but not limited to the following test systems: effects on uterine thickness, weight and histology, effects on bone mineral density, bone growth and thickness; effects on serum cholesterol and adipose tissue distribution and quantity; effects on serum luteinizing hormone levels; effects on pituitary weight and luteinizing hormone content and release from the pituitary gland; effects on hypothalamic content of luteinizing hormone-releasing hormone; effects on choline acetyltransferase, high affinity choline uptake and neurotrophin levels; effects on glutamate-induced neurotoxicity and effects of transforming growth factor (TGF) secretion from astrocytes.
  • TGF transforming growth factor
  • Assessment of estrogenic bioactivity and estrogenic modulatory bioactivity may include examination ofthe effects of these molecules on the following non-inclusive list of biological target sites: uterine cellular proliferation, thicl ⁇ iess and histology; uterine eosinophil peroxidase activity; serum and pituitary gonadotropin levels; gonadotropin release from the pituitary gland; serum and pituitary luteinizing hormone levels; pituitary weight; hypothalamic content of luteinizing hormone-releasing hormone; the preovulatory luteinizing hormone surge; ovulation; estrus and menstrual cycles; puberty onset; vaginal opening in non-human animals; libido; fertility; ovulation; menarche; menopause; treatment of menopausal and post- menopausal symptoms; cytology of the vaginal epithelium; endometriosis; alopecia; induction of progesterone receptors; growth of estrogen-responsive tumors; angiogenesis
  • Breast cancer is another biological parameter useful for evaluation of the estrogenic bioactivity of the molecules of the present invention.
  • Other useful biological parameters include, but are not limited to, the following: cell cycle time; growth of estrogen-responsive cells, including but not limited to MCF-7 cells and ZR-75-1 cells; growth of mammary tumors; and effects on endometrial tumors.
  • estrogenic bioactivity of these molecules are the following: bone mineral density; osteoporosis; osteopenia; serum osteocalcin levels; serum lipids; serum high density lipoprotein (HDL); serum low density lipoprotein (LDL); the ratio of HDL to LDL; cholesterol synthesis; serum cholesterol levels; adipose tissue distribution and quantity; growth factor expression, including but not limited to expression of genes for growth factors such as TGF-beta.
  • the molecules of the present invention may also be examined for their ability to modulate the binding of estrogen or related estrogen receptor ligands to the estrogen receptor.
  • Estrogen responsive genes are also used to assess estrogenic bioactivity of the molecules of the present invention. Additional testing may be performed in cell culture systems or in tissue or membrane preparations to assess potential competition of these novel molecules with radiolabeled estradiol, for example [ 3 H]-17 ⁇ -estradiol, and other estrogen analogs for binding to the estrogen receptor.
  • radiolabeled estradiol for example [ 3 H]-17 ⁇ -estradiol, and other estrogen analogs for binding to the estrogen receptor.
  • the modulatory capability of these estrogenic modulatory molecules for the binding of estrogen or estrogenic agonists to the estrogen receptor may be evaluated.
  • bioactivity of these estrogenic molecules may be assessed in terms of their abilities to augment, inhibit or otherwise modulate the bioactivity of estrogenic and anti-estrogenic molecules in a variety of test systems that are commonly known to one of ordinary skill in the art.
  • the activity of these estrogenic compositions may be evaluated in terms of alleviating the symptoms of menopause, in causing or preventing ovulation and in increasing bone density.
  • Estrogen responsive genes may also be employed to assess estrogenic activity of these molecules using techniques known to one of skill in the art of molecular endocrinology. Additional testing may be performed in cell culture systems or in tissue and membrane preparations to assess potential competition of these predicted or designed estrogenic compositions with radiolabeled estrogen analogs for binding to the estrogen receptor.
  • the references cited herein contain information useful to one of skill in the art concerning methods and routes of delivery of estrogenic compositions including, but not limited to, topical, oral, anal, parenteral, aerosol, vaginal, subcutaneous, implants, rings, intrauterine devices, and skin patches.
  • the estrogenic molecules of the present invention may also be administered in combination with other substances, for example hormones such as estrogen, estradiol, progesterone, glucocorticoids, or analogs, agonists and antagonists thereof, and combinations thereof.
  • hormones such as estrogen, estradiol, progesterone, glucocorticoids, or analogs, agonists and antagonists thereof, and combinations thereof.
  • Such hormonal combinations may be useful in various hormone replacement therapies, for example therapies related to contraception, conception, cancer, menopause, inflammation, mood, and libido.
  • estrogenic modulatory bioactivities may be evaluated. These include, but are not limited to evaluations of estrogenic modulatory bioactivities in vitro and in vivo.
  • Estrogenic molecules include estrogens such as estradiol, 17 ⁇ -estradiol, and any other molecule known to possess estrogenic bioactivity. Modulation includes any effect of the estrogenic modulator to alter estrogenic bioactivity at any biological target site such effects including enhancing, reducing or eliminating estrogenic bioactivity.
  • an estrogenic modulatory molecule may partially overcome anti-estrogenic effects of an estrogenic antagonist.
  • Another estrogenic modulatory molecule may partially inhibit the estrogenic effects of an estrogenic agonist.
  • test systems are known to those of ordinary skill in the art and may be employed to evaluate the estrogenic bioactivity or estrogenic modulatory bioactivity of a designed molecule or a molecule predicted to possess such bioactivity.
  • These methods include the use of experimental animals, such as rats, which have been bilaterally ovariectomized and then administered a test compound, such as l l ⁇ -methoxy-7 -methylestra-l,3,5(10)-triene-3,17 ⁇ -diol or any of the structures shown in Figures 1 and 2, or a derivative thereof, in combination with, before, or after the administration of an estrogenic or anti- estrogenic molecule.
  • the activity of the test compound to modulate the biological activity of the estrogenic or anti-estrogenic molecule is assessed.
  • the biological activities of the molecules of the present invention are compared to effects of known estrogenic molecules, such as 17 ⁇ -estradiol, in order to assess the relative estrogenic activity of the molecules.
  • Means of making such comparisons are known to one of ordinary skill in the art and include comparative evaluations of activity at similar dosages, calculation of EC 50 values, and other determinations of efficacy.
  • the effects of the test compound are examined on a variety of biological targets including, but not limited to, the uterus (for example, mitotic index, uterine weight), bone mineral density, serum lipids, lipoprotein ratios, body fat type and distribution, pituitary hormonal response (for example, luteinizing hormone-releasing hormone induced luteinizing hormone secretion), plasma and pituitary concentrations of various hormones (for example, luteinizing hormone, follicle stimulating hormone), plasma levels, tissue levels and biosynthesis of peptides (for example, luteinizing hormone-releasing hormone, neuropeptide Y, neurotrophic factors), hypophysiotropic hormones and releasing factors (for example, luteinizing hormone-releasing hormone), plasma levels, tissue levels and biosynthesis of growth factors, plasma levels, tissue levels and biosynthesis of neurotransmitters (for example, dopamine, norepinephrine, epinephrine, acetylcholine, serotonin) and their synthetic enzymes in tissues and body fluids, transmitter uptake
  • the estrogenic molecules of the present invention may be combined with pharmaceutically or cosmetically acceptable vehicles or carriers for administration as compositions to humans or animals.
  • the molecules of the present invention may be administered, alone or in combination with other compounds, as hormone replacement therapy (HRT).
  • HRT hormone replacement therapy
  • the molecules of the present invention may also be administered in vitro.
  • the molecules of the present invention may be combined with pharmaceutically or cosmetically acceptable carriers and administered in the form of injections, solutions, creams, gels, implants, pumps, ointments, pastes, patches, tablets, sprays or other means familiar to one of ordinary skill in the art.
  • Such pharmaceutically or cosmetically acceptable carriers are commonly known to one of ordinary skill in the art.
  • Pharmaceutical formulations of the present invention can be prepared by procedures known in the art using well known and readily available ingredients.
  • the compounds can be formulated with common excipients, diluents, or carriers, and formed into tablets, capsules, suspensions, powders, and the like.
  • excipients, diluents, and carriers that are suitable for such formulations include the following: fillers and extenders (e.g., starch, sugars, mannitol, and silicic derivatives); binding agents (e.g., carboxymethyl cellulose and other cellulose derivatives, alginates, gelatin, and polyvinyl-pyrrolidone); moisturizing agents (e.g., glycerol); disintegrating agents (e.g., calcium carbonate and sodium bicarbonate); agents for retarding dissolution (e.g., paraffin); resorption accelerators (e.g., quaternary ammonium compounds); surface active agents (e.g., cetyl alcohol, glycerol monostearate); adsorptive carriers (e.g., kaolin and bentonite); e
  • the compounds can also be formulated as elixirs or solutions for convenient oral administration or as solutions appropriate for parenteral administration, for example, by intramuscular, subcutaneous or intravenous routes. Additionally, the compounds are well suited to formulation as sustained release dosage forms and the like.
  • the formulations can be so constituted that they release the active ingredient only or preferably in a particular physiological location, possibly over a period of time.
  • the coatings, envelopes, and protective matrices may be made, for example, from polymeric substances or waxes.
  • the pharmaceutical composition (or formulation) for application may be packaged in a variety of ways depending upon the method used for administering the composition.
  • an article for distribution includes a container which contains the pharmaceutical formulation in an appropriate form.
  • Suitable containers are well-known to those skilled in the art and include materials such as bottles (plastic and glass), sachets, ampules, plastic bags, metal cylinders, and the like.
  • the container may also include a tamper-proof assemblage to prevent indiscreet access to the contents of the package.
  • the container has deposited thereon a label which describes the contents ofthe container. The label may also include appropriate warnings.
  • pharmaceutically or cosmetically acceptable carrier or “pharmaceutically or cosmetically acceptable vehicle” are used herein to mean any liquid including but not limited to water or saline, a gel, cream, salve, solvent, diluent, fluid ointment base, ointment, paste, implant, liposome, micelle, giant micelle, and the like, which is suitable for use in contact with living animal or human tissue without causing adverse physiological or cosmetic responses, and which does not interact with the other components of the composition in a deleterious manner.
  • Other pharmaceutically or cosmetically acceptable carriers or vehicles known to one of skill in the art may be employed to make compositions for delivering the molecules ofthe present invention.
  • compositions may conveniently be presented in unit dosage form and may be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers. Suitable carriers are known to one of ordinary skill in the art and include, but are not limited to, oils such as sesame oil or corn oil, ethanol, saline, and propylene glycol.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti- oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition ofthe sterile liquid carrier, for example, water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets commonly used by one of ordinary skill in the art.
  • Preferred unit dosage formulations are those containing a dose or unit, or an appropriate fraction thereof, ofthe administered ingredient. It should be understood that in addition to the ingredients, particularly mentioned above, the formulations of the present invention may include other agents commonly used by one of ordinary skill in the art.
  • the molecules ofthe present invention may be administered in the form of creams, gels, tablets, ointments, pastes, patches, tablets, implants, injections, sprays, transdermal delivery devices, osmotic pumps, or other means familiar to one of ordinary skill in the art.
  • estrogenic modulatory compositions ofthe present invention include, but are not limited to the following: oral including buccal and sublingual (pill, liquid, syrup, suspension), transdermal (gel, cream, paste, patch, spray, gel), parenteral, intramuscular, rectal, anal, aerosol, intranasal aerosol, injection, suppository, topical, intradermal, subcutaneous administration (implants, depot, needles, SILASTIC (Dow Corning, Midland MI) capsules, osmotic pump), microparticles, liposomes, emulsions, intrauterine, vaginal (creams, rings). Many of these methods are described by Sturdee et al, Br. J. Obstet. Gynaecol., 104:1109-1115, 1997.
  • Intramuscular injections may range in volume from about 0.1 ml to 1.0 ml.
  • the molecules of the present invention may be dissolved in an acceptable carrier and administered in vitro for any use wherein estrogenic bioactivity or modulation of estrogenic bioactivity is desired.
  • Such applications may include, but are not limited to the following: cell culture, organ culture, growth of artificial organs such as skin and bone, cell-free systems, growth of blast cells and stem cells including but not limited to neuroblasts, osteoblasts, fibroblasts, endothelial cells, and bone marrow cells.
  • Other applications may include use in cell-free systems and various assay systems in which estrogenic bioactivity or modulation of estrogenic bioactivity is desired.
  • concentrations of the estrogenic modulatory molecules ofthe present invention may be employed depending on the method of administration and target tissue. For example, one of skill in the art might wish to achieve plasma levels of these estrogenic modulatory molecules in the micromolar ( ⁇ M) to femtomolar (fM) range. By adjusting the concentration of the estrogenic modulatory molecule in a subcutaneously implanted SILASTIC tube, for example, one of skill in the art could produce plasma levels in this range. Desirable in vitro concentrations may range from picomolar (pM) to millimolar (mM).
  • estrogenic compositions of the present invention include, but are not limited to the following: oral (pill, liquid, syrup, suspension); transdermal (gel, cream, paste, patch, spray, gel); anal; aerosol; intranasal aerosol; injection; suppository; subcutaneous administration (implants, depot, needles, SILASTIC capsules, osmotic pump); microparticles; liposomes; emulsions; intrauterine; and vaginal (creams, rings). Many of these methods are described by Sturdeeet al, Br. J. Obstet. Gynaecol, 104:1109-1115, 1997, and in U.S. Patent No. 6,083,528.
  • the estrogenic and estrogenic modulatory molecules of the present invention may be administered in combination with other substances, including but not limited to hormones, immunomodulators, cytokines, neurotransmitters, anti-depressants, nutrients, nutraceuticals, minerals, vitamins, ions, drugs, anti-cancer drugs, substances which affect metabolism, especially weight loss or gain and fat deposition, metabolic inhibitors, anti-mitotic drugs, and agonists thereof, antagonists thereof, and combinations thereof.
  • the estrogenic and estrogenic modulatory molecules of the present invention may also be administered in combination with other modulators of the effects of estrogen on biological targets known to one of ordinary skill in the art.
  • the estrogenic molecules of the present invention may be administered in combination with estrogen, estradiol, progesterone, glucocorticoids or analogs, agonists and antagonists thereof.
  • a preferred combination is progesterone administered with the estrogenic molecules or the estrogenic modulatory molecules of the present invention encompassed within formula I, wherein R 2 is CH 3 .
  • Another preferred combination is progesterone administered with l l ⁇ -methoxy-7 ⁇ -methylestra- l,3,5(10)-triene-3,17 ⁇ -diol.
  • Such hormonal combinations may be useful in various hormone replacement therapies, for example therapies related to puberty, contraception, conception, fertility, lactation, menopause, inflammation, mood, libido, alopecia, memory, senility, Alzheimer's disease, cell division, cancer, bone mineral density, osteoporosis, osteopenia, lipid metabolism, angiogenesis, and heart disease.
  • the estrogenic and estrogenic modulatory molecules of the present invention may be administered in a dose range of from about 0.1 ⁇ g to 10 mg, preferably 1 ⁇ g to 5 mg depending on the route of administration and the condition of the recipient.
  • a clinician skilled in the art of hormone replacement therapy may chose specific dosages and dose ranges, and frequency of administration, as required by the circumstances.
  • a clinician skilled in the art of hormone replacement therapy may chose specific dosages and dose ranges, and frequency of administration, for compounds, such as progesterone, to be administered in combination with the estrogenic and estrogenic modulatory molecules of the present invention as required by the circumstances.
  • progesterone, and other progestins known to one of skill in the art may be administered in amounts ranging from about 50 ⁇ g to 300 mg, preferably 100 ⁇ g to 200 mg, more preferably 1 mg to 100 mg.
  • Specific dosages and combinations of dosages of estrogenic and estrogenic modulatory molecules and progestins will depend on the route and frequency of administration, and also on the condition to be treated.
  • each dosage unit may preferably contain l ⁇ g to 5 mg of estrogenic and estrogenic modulatory molecules and 50 ⁇ g to 300 mg of progesterone.
  • U.S. Patent No. 4,900,734 provides additional examples of acceptable dose combinations of estrogenic molecules and progestins.
  • the estrogenic and estrogenic modulatory molecules of the present invention may also be administered in combination with other compounds as described in the following non-limiting list: hormones such as other steroids (estrogens, androgens, progesterones, glucocorticoids, thyroid hormones), pituitary hormones such as gonadotropins, hypothalamic hormones and various hypophysiotropic factors, metabolic hormones such as parathyroid hormone, calcitonin, insulin, glucagon, somatostatin, and pancreatic polypeptide, and analogs and antagonists thereof; cytokines, monokines, and analogs and antagonists thereof; metabolic inhibitors, anti-cancer drugs, including but not limited to, tamoxifen, taxol, metalloproteinase inhibitors, cisplatin, and DNA related drugs and analogs and antagonists thereof; drugs which affect lipid levels; drugs which affect angiogenesis; drugs which affect mood such as anti- depressants, neurotransmitter reuptake inhibitors, catecholaminergic and serot
  • compositions may be stored at temperatures of from about 4°C to -100°C.
  • the compositions may also be stored in a lyophilized state at different temperatures including room temperature.
  • the compositions may be sterilized through conventional means known to one of ordinary skill in the art. Such means include, but are not limited to filtration, radiation and heat.
  • compositions of the present invention may also be combined with bacteriostatic agents, such as thimerosal or other bacteriostatic agents known to one of skill in the art.
  • the estrogenic bioactivity of a molecule is tested in vivo by bilaterally ovariectomizing rats and two weeks later providing a daily subcutaneous injection of the molecules of the present invention in a pharmaceutically acceptable vehicle for three days.
  • the dosages for subcutaneous injection are from about 1 pg to 1 mg.
  • Control animals receive only injection of vehicle. The animals are sacrificed by cervical dislocation, decapitated and the trunk blood collected. Following clot formation at 4°C, aliquots of the serum are analyzed in an LH assay, preferably a radioimmunoassay. The uterus and uterine tubes of each animal are removed and weighed.
  • Decreased serum LH levels and increased uterine weights relative to ovariectomized animals receiving vehicle indicate estrogenic bioactivity of a molecule.
  • the ability of the estrogenic molecules of the present invention to stimulate the uterus in an ovariectomized rat is an index of the estrogenic bioactivity of the molecule. Such effects may be compared on a dose level to a stimulatory dose of 17 ⁇ -estradiol to compare efficacy.
  • the rat uterotrophic assay also provides a means for assessingthe biological activity ofthe molecules ofthe present invention.
  • a stimulating dose of 17 ⁇ -estradiol may be employed which is intermediate between a maximally stimulatory dose and a non-stimulating dose.
  • the estrogenic modulatory activity of a molecule is assessed by administering the molecule to the same rat before, during, or after, or some combination thereof, the administration of the stimulatory dose of 17 ⁇ -estradiol.
  • TGF transforming growth factor
  • estradiol transforming growth factor- ⁇ ! and this secretion is enhanced by estradiol (Buchanan et al., Biology of Reproduction 62:1710-1721, 2000).
  • the stimulation of TGF- ⁇ , by estradiol is mediated through the estrogen receptor.
  • Brain astrocytes possess both estrogen receptor- ⁇ and estrogen receptor- ⁇ .
  • the enhancement of TGF-fi, secretion by brain astrocytes by estradiol is a specific action of estradiol as the estrogen receptor antagonist 1C1 182,870 block this effect.
  • Estrogens also enhance the secretion of TGF- ⁇ 2 specifically from brain astrocytes.
  • Estradiol also increases TGF- ⁇ type II receptors in the hypothalamus.
  • the conditioned medium from brain astrocytes prevents the cell death of GT1-7 cells in culture that are serum starved as well as death of rat brain neurons treated with a toxic dose of glutamate in vitro (Buchanan et al., Biology of Reproduction 62:1710-1721, 2000).
  • This effect of conditioned medium is abolished if antibodies to TGF- ⁇ are added to inactivate the secreted TGF- ⁇ .
  • TGF- ⁇ added to GT1-7 cells that are serum starved, in quantities found in the astrocyte conditioned medium are also able to prevent cell death.
  • TGF- ⁇ also stimulates neurite outgrowth in GT1-7 cells.
  • the estrogen-astrocyte-TGF- ⁇ link is an important signaling link in neuroprotection.
  • the molecules ofthe present invention are tested for estrogenic bioactivity to prevent osteoporosis. These molecules are evaluated for this specific type of bioactivity using techniques l ⁇ iown to one of ordinary skill in the art. Some of these techniques are revealed in the publications by Jardine et al, Ann. Reports in Medicinal Chem., J.A. Bristol ed., 31:211-220, 1996, and Delmas et al, New England J. Med., 337:1641-1647, 1997. Such techniques include, but are not limited to the following, evaluation of bone density, evaluation of bone mineral density and measurement of various biomarkers related to bone physiology.
  • Bone density may be determined by evaluating the density of selected bones such as the vertebrae, tibia, femur, pelvis, radius, ulna, humerus or any other bone useful for measuring bone density. Imaging techniques such as radiographs, computerized assisted tomography, magnetic resonance imaging or positron assisted tomography, commonly known to one of ordinary skill in the art, are employed to measure bone density.
  • bone mineral density is evaluated by dual- energy x-ray absorptiometry as taught by Delmas et al, New England J. Med., 337:1641-1647, 1997.
  • biochemical markers of bone turnover such as serum osteocalcin, bone-specific alkaline phosphatase, and the ratio of urinary type I collagen C-telopeptide to creatinine are measured as taught by Delmas et al, New England J. Med., 337:1641-1647, 1997, and also in selected references cited therein.
  • Increased bone density following administration of a molecule of the present invention indicates bone-protective effects of a molecule.
  • Decreased bone density following administration of a molecule of the present invention indicates potential osteoporotic or bone- wasting effects of a molecule. It is to be understood that the biological activity of the molecules of the present invention may be evaluated using other biological markers related to bone physiology.
  • angiogenic activity of a compound is performed using currently available assays known to one of ordinary skill in the art. These assays and methods include, but are not limited to the following: the chick chorioallantoic (CAM) assay (Crum et al, Science 230: 1375-1378, 1985, Gagliardi et al, Cancer Research 52: 5073-5075, 1992, and Gagliardi et al, Cancer Research 53: 533-535, 1993); inhibition or proliferation of capillary endothelial cells or fibroblasts (Fotsis et al, Nature 368: 237-239, 1994); the human umbilical vein endothelial cell assay (Morales et al, Circulation 91: 755-763, 1995); in vivo vascularization of Matrigel plugs (Morales et al, Circulation 91
  • 11 ⁇ -Nitro estradiol derivatives (I) prepared by methods known in the art, such as those of Peters et al, (J. Med. Chem. 32: 2306-2310, 1989) are useful intermediates in the synthesis of 11 -substituted estradiol compounds of formula 1, wherein R ⁇ is CH 3 , C 2 H 5 , HCO or CH,CO, and R 2 is CH 3 or H.
  • Example 2 the values listed in Example 2 for temperature conditions, amounts of reagents, concentrations, and reaction times, for the synthesis of 11 ⁇ -methoxy- 7 ⁇ -methylestra-l,3,5(10)-triene-3,17 ⁇ -diol, may be used in a range approximately 10%, and preferably 5% above and below the stated value, not only for the synthesis of ll ⁇ -methoxy-7 ⁇ -methylestra-l,3,5(10)-triene-3,17 ⁇ - diol, but also for synthesis of the other 11 -substituted estradiol compounds of formula 1, ' wherein R, 1 is CH 3,' C 2H 5,' HCO or CH 3 ->CO,' and R 2, is CH 3 or H.
  • the general method of synthesis is schematically represented in
  • a suitable hydride base such as sodium hydride, or alkoxide bases such as potassium tert-butoxide.
  • the displacement reaction may be carried out in a suitable solvent such as dimethylformamide, dioxane or tetrahydrofuran, at a temperature of from about 0°C to 70°C, preferably 0°C to 24°C, more preferably at about 20°C to 24°C.
  • a suitable solvent such as dimethylformamide, dioxane or tetrahydrofuran
  • Removal of RPG, where RPG is benzyl may be performed by hydrogenation in the presence of a catalyst such as palladium on carbon, or by the use of alkali bases such as sodium hydroxide, in a suitable solvent such as methanol, ethanol, or tetrahydrofuran.
  • RPG wherein RPG is trialkylsilyl
  • fluoride ion or mild mineral acids such as hydrochloric acid
  • suitable solvent such as tetrahydrofuran, dioxane, or alcohol solvents at temperatures of about 20°C to 100°C, preferably about 20°C to 24°C.
  • Preferred compounds made with the method of the present invention include those encompassed within the generic structure of Figure 1, wherein R ⁇ is CH 3 , C 2 H 5 , HCO or CIICO, and R 2 is CH 3 or H.
  • Especially preferred compounds made with the method of the present invention include structures encompassed within the generic structure shown in Figure 1, wherein R, is CB ⁇ or C 2 H 5J and R- 2 i s CH 3 or H.
  • a more preferred compound made with the method of the present invention is shown in Figure 2 wherein R, is CH, and R 2 is CH 3 .
  • PDC-7 ll ⁇ - methoxy-7 ⁇ -methylestra-l,3,5(10)-triene-3,17 ⁇ -diol
  • PDC-7 demonstrated desired improvements in fit over estradiol.
  • PDC-7 had a slightly lower electrostatic fit (99.6%) but an improved volume fit by 40 A 3 (115.4 %) (Table 1).
  • the total normalized fit of PDC-7 was 107.5%.
  • PDC-7 was predicted to be more active than estradiol.
  • PDC-7 was synthesized and tested for estrogenic activity in the rat uterotropic assay using estradiol as the reference estrogen. The assay measured the dose resulting in a doubling of uterine weight.
  • Electrostatic Fit* 100 (-43.969 kcal) 99.6 (-43.802 kcal)
  • ⁇ Values in parentheses represent the electrostatic interaction of the positive charge on the proton donor of hydroxyl groups and point charges on the pharmacophore.
  • R2 is CH3.
  • 3,17 ⁇ -diol was chemically synthesized according to the following procedure.
  • the synthetic procedure used the starting material 3,l l ⁇ ,17 ⁇ -trihydroxy-7 ⁇ - methylestra-l,3,5(10)-triene-ll -nitrate ester prepared by the synthetic procedure described by Peters et al. (J. Med. Chem. 32: 2306-2310, 1989).
  • the remaining synthetic steps are novel subject matter ofthe present invention.
  • the resulting estradiol analog is optionally blocked using appropriate protecting groups, preferably trialkylsilyl groups.
  • Reduction of the 11-nitro using zinc and acetic acid then provides the C3, C17-blocked l l ⁇ - hydroxy steroid, 3 , 11 ⁇ , 17 ⁇ -trihydroxy-7 ⁇ -methylestra- 1,3,5(10)-triene- 11- nitrate ester.
  • the hydroxyl group is methylated using methods known to one of skill in the art, for example, by deprotonation with a suitable base, preferably a hydride base, followed by alkylation with methyl iodide or an equivalent reactive methylating agent.
  • the silyl groups are deprotected under acidic conditions resulting in ll ⁇ -methoxy-7 ⁇ -methylestra- l,3,5(10)-triene-3,17 ⁇ -diol.
  • Immature female Sprague-Dawley rats were obtained from Harlan Laboratories (Harlan, Indianapolis, IN). On day 24 of life, the animals were anesthetized with ether and the ovaries were excised. After recovery from the anesthesia, the animals were randomly divided into groups consisting of five to six animals each. Within twenty-four hours following surgery the treatment regimen began. Each animal was treated for four days with subcutaneous injections containing varying doses ranging from 1 ng to 100 ng per day of either ll ⁇ -methoxy-7 ⁇ -methylestra-l,3,5(10)-triene-3,17 ⁇ -diol (PDC-7) or 17B- estradiol in 200 ⁇ l corn oil.
  • PDC-7-7 ll ⁇ -methoxy-7 ⁇ -methylestra-l,3,5(10)-triene-3,17 ⁇ -diol
  • Intact and ovariectomized control animals received vehicle only. Following treatment, the animals were sacrificed by cervical dislocation. Uterine weight measurements were used as a determinate of estrogenic activity. At the time of sacrifice, the uteri were removed and the weights were recorded. To obtain dry weights, the uteri were placed in a 90° C oven for two hours before weighing.
  • the uterotropic activity is presented as the mean ⁇ standard error of the mean (SEM) of the dry uterine weight expressed per gram body weight ( Figure 5 and Table 1). The results were analyzed by two-way analysis of variance followed by post hoc analysis using Student-Neuman-Keuls test. At the dosages of 30.7, 76.8 and 153.7 picomoles, PDC-7 showed a significantly greater uterotropic response (PO.05) than estradiol administered at 36.7, 91.8 and 183.6 picomoles. The results demonstrated that l l ⁇ -methoxy-7 ⁇ -methylestra- l,3,5(10)-triene-3,17 ⁇ -diol was more potent than estradiol in stimulating uterine weight gain.
  • Serum cholesterol is determined using a Boehringer Mannheim Diagnostics high performance cholesterol assay. Briefly, the cholesterol is oxidized to cholest-4-en-3-one and hydrogen peroxide. The hydrogen peroxide is then reacted with phenol and 4-aminophenazone in the presence of peroxidase to produce a p-quinone imine dye, which is read spectrophotemetrically at 500 nm. Cholesterol concentration is then calculated against a standard curve. The entire assay is automated using a Biomek Automated Workstation.
  • the rats are treated daily for 35 days (6 rats per treatment group) and sacrificed by carbon dioxide asphyxiation on the 36th day.
  • the 35 day time period is sufficient to allow maximal reduction in bone density, measured as described herein.
  • the uteri are removed, dissected free of extraneous tissue, and the fluid contents are expelled before determination of wet weight in order to confirm estrogen deficiency associated with complete ovariectomy.
  • Uterine weight is routinely reduced about 75% in response to ovariectomy.
  • the uteri are then placed in 10% neutral buffered formalin to allow for subsequent histological analysis.
  • the right femurs are excised and digitilized x-rays generated and analyzed by an image analysis program (National Institutes of Health image program) at the distal metaphysis.
  • image analysis program National Institutes of Health image program
  • the proximal aspect of the tibiae from these animals are also scanned by quantitative computed tomography.
  • Methods of the current invention for the treatment or prevention of Alzheimer's disease, especially in postenenopausal women, may be demonstrated by means ofthe following assays.
  • Assay 1 An animal model of neuronal damage may be used to demonstrate the methods ofthe current invention. For example, between twenty and thirty rats are utilized in a manner similar to that described above in Example 4 except that a neuronal lesion is produced in the brain of the test animals. Neuronal damage, similar to the eventual damage seen in afflicted patients, can be induced with a well known technique of occluding the four vessels feeding the brain for short period of time, usually five to fifteen minutes. This occlusion causes a global ischemia which in turn causes neuronal damage. After the occlusion, the animals are allowed to rest for several days, during which time the brain lesions develop. The animals are sacrificed and damage is assessed by standard histologic techniques.
  • the activity of the compounds represented by generic structure I (Fig. 1) and the compound shown in Figure 2 is illustrated by a decrease in the neuronal damage, especially, to the hippocampus and striatum.
  • Assay 2 Ten to fifty women are selected for a clinical study. The selection criteria are: at least one year post-menopausal, in reasonably good health, and diagnosed with early stages Alzheimer's Disease (AD). Further, these patients are staged in their disease, such that there is a good expectation that during the course of the study, most patients will experience a marked increase in the severity of pathologic symptoms. The patients are divided into two groups, one group is given a placebo, while the test group is given the compound of the current invention, once a day, via the oral route.
  • AD Alzheimer's Disease
  • the study is continued for six to thirty-six months in duration. All patients are given a complete mental profile at the beginning, each six months, and at termination of the study.
  • This profile used to evaluated the extent of the disease, includes capacity factors such as memory, cognition, reasoning ability, self-sufficiency, and the like.
  • objective parameters such as changes in brain structure as measured by CAT scanning techniques. Such methodologies and mental evaluations may be found in many standard texts on the subject. The results are compared both between groups at various time points and the changes in each patient versus time. A positive result is demonstrated by an inhibition in the type or severity ofthe degenerative symptoms in the test group given a formulation of the present invention, in contrast to those patients given the placebo.
  • the specific dose ofthe compounds represented by the generic structure shown in Figure 1, or by the structure shown in Figure 2 will, of course, be determined by the particular circumstances surrounding the case.
  • the route of administration is a factor determined by the specifics of each case.
  • the exact dose and route of administration a-e best determined by the attending physician.
  • Hard gelatin capsules are prepared using the following: Ingredient Quantity (mg/capsule)
  • a tablet formulation is prepared using the ingredients shown below:
  • the components are blended and compressed to form tablets.
  • the active ingredient, starch, and cellulose are passed through a No. 45 mesh U.S. sieve and mixed thoroughly.
  • the solution of polyvinylpyrrolidone is mixed with the resultant powders which are then passed through a No. 14 mesh U.S. sieve.
  • the granules so produced are dried at 50°C - 60°C and passed through a No. 18 mesh U.S. sieve.
  • the sodium carboxymethyl starch, magnesium stearate, and talc previously passed through a No. 60 U.S. sieve, are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets.
  • the medicament is passed through a No. 45 mesh U.S. sieve and mixed with the sodium carboxymethyl cellulose and syrup to form a smooth paste.
  • the benzoic acid solution, flavor, and color are diluted with some of the water and added, with stirring. Sufficient water is then added to produce the required volume.
  • the active ingredient, starch, and cellulose are passed through a
  • the active ingredient, starch, and cellulose are passed through a No. 45 mesh U.S. sieve and mixed thoroughly.
  • the solution of polyvinylpyrrolidone is mixed with the resultant powders which are then passed through a No. 14 mesh U.S. sieve
  • the granules so produced are dried at 50C to 60°C and passed through a No. 18 mesh U.S. sieve.
  • the sodium carboxymethyl starch, magnesium stearate, and talc, previously passed through a No. 60 U.S. sieve, are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets.
  • Some formulations presently used as oral contraceptives contain 0.02 to 0.05 mg of ethinyl estradiol or mestranol and various amounts of a progestin, and are taken for 21 days. The next course is started 7 days after the last dose or 5 days after the onset of the menstrual flow. It should be noted that ethinyl estradiol is approximately twice as potent as mestranol.
  • l l ⁇ -methoxy-7 ⁇ -methylestra-l,3,5(10)-triene-3-17 ⁇ -diol is included in a contraceptive formulation in an amount of from about 0.001 to 0.08 mg, together with amounts of progestins commonly found in oral contraceptive pills. Sequential preparations are formulated to be taken in two
  • biphasic or three (triphasic) continuous phases.
  • a fixed-dose combination of ll ⁇ -methoxy-7 ⁇ -methylestra-l,3,5(10)-triene-3-17 ⁇ - diol and progestin is taken for 10 days, followed by a different fixed-dose combination of l l ⁇ -methoxy-7 -methylestra-l,3,5(10)-triene-3-17 ⁇ -diol and progestin for 11 days.
  • the pills are discontinued for 7 days before the cyclic administration is resumed.
  • Triphasic preparations contain the same or different quantities of l l ⁇ -methoxy-7 ⁇ -methylestra-l,3,5(10)-triene-3-17 ⁇ -diol and variable quantities of a progestin in three sets of tablets. Each set is taken for 5 to 10 days, depending upon the specific formulation. After 21 days of administration, the medication is discontinued for 7 days before the cycle is resumed.
  • rat brain astrocytes were incubated with 1 l ⁇ - methoxy-7 ⁇ -methylestra-l,3,5(10)-triene-3-17 ⁇ -diol for 6 hours, the conditioned media from the astrocytes reduced significantly the death of rat brain neurons induced to die using 500 ⁇ M glutamate.
  • Astrocyte Cultures Cultures of astrocytes were employed for two purposes: (1) to measure TGF- ⁇ j and TGF- ⁇ 2 production; and, (2) to produce conditioned media for treatment of cortical neurons in neuroprotection studies. The isolation and culture protocol is a modification of a published method by McCarthy (J. Cell Biology (1980) 85:890-902).
  • Cortical astrocytes were isolated from two day-old rat pups and cultured in 75 cm 2 flask. The astrocytes were allowed to attach for 5-6 days and were then purified by shaking. The cells grew to approximately 80% confluency and were split onto 6 well plates at approximately 500,000 cells per well. At approximately 80% confluency, the cells were treated with the following compounds: estradiol (lOnM), Tamoxifen (1 ⁇ M), ll ⁇ -methoxy-7 ⁇ -methylestra-l,3,5(10)-triene-3-17 ⁇ -diol (lOnM), and EM 652 (5 ⁇ M). Untreated cells served as control. Astrocytes were exposed to the respective compound for 18 hours and afterwards the media samples were collected.
  • the collected media was either used for TGF- ⁇ [ and TGF- ⁇ 2 measurements or frozen until a later time and used to treat cortical neurons. Additional detail concerning some methods employed in this example are provided in Buchanan et al., Biology of Reproduction 62:1710-1721, 2000.
  • Neuroprotection Studies The neuronal isolation culture procedure was based upon the protocol that accompanies Neurobasal media (Gibco BRL; Cat # 21103). Cortical neurons were isolated from embryonic day 18 rat pups. The neurons were cultured for approximately ten days in 24 well plates. The cells were exposed to either the compound of interest directly (direct effect) or astrocyte conditioned media (see above; indirect effect). Untreated cells served as controls. For the direct effect experiments, neurons were treated with the compounds of interest for durations of 24 hours before, 24 hours after or 24 hours before and after glutamate exposure. In the indirect effect experiments, neurons were incubated with the astrocyte conditioned media for 24 hours before glutamate treatment.
  • L-glutamate 500 ⁇ M
  • LDH lactate dehydrogenase
  • Rat astrocytes were cultured either without steroids or with various agents for 6 hours and the conditioned medium was incubated with rat brain neurons for 24 hours. The neurons were then treated with 500 ⁇ M glutamate for 15 minutes. Neuronal death was measured by LDH activity. A dose of 10 nM of ll ⁇ -methoxy-7 ⁇ -methylestra-l,3,5(10)-triene-3-17 ⁇ -diol demonstrated significant neuroprotection by decreasing the percentage of neuronal cell death (PO.05 compared to controls).

Abstract

L'invention concerne des molécules conçues au moyen du pharmacophore d'oestrogène et possédant une activité biologique prédite et démontrée oestrogénique et/ou de modulation oestrogénique. Elle concerne également un procédé de synthèse chimique de 11β-méthoxy-7α-méthylestra-1,3,5(10)-triène-3,17β-diol et de molécules apparentées. Elle concerne également de nouvelles utilisations de 11β-méthoxy-7α-méthylestra-1,3,5(10)-triène-3,17β-diol afin d'exercer une bioactivité oestrogénique ou une activité biologique de modulation oestrogénique, quand on administre ces molécules avec un véhicule acceptable sur le plan pharmaceutique ou cosmétique. On peut utiliser ces molécules afin de traiter des états nécessitant une modulation de l'activité biologique oestrogénique.
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US8629130B2 (en) 2005-09-30 2014-01-14 Endece, Llc 6-substituted estradiol derivatives and methods of use
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US10723757B2 (en) 2005-09-30 2020-07-28 Endece Llc 6-substituted estradiol derivatives and methods of use

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