WO2000020007A1 - Method and compositions for increasing bone mass - Google Patents

Method and compositions for increasing bone mass Download PDF

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Publication number
WO2000020007A1
WO2000020007A1 PCT/US1999/023355 US9923355W WO0020007A1 WO 2000020007 A1 WO2000020007 A1 WO 2000020007A1 US 9923355 W US9923355 W US 9923355W WO 0020007 A1 WO0020007 A1 WO 0020007A1
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compound
estrogen
cells
bone
receptor
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PCT/US1999/023355
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English (en)
French (fr)
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Stavros C. Manolagas
Robert L. Jilka
Robert S. Weinstein
Teresita Bellido
Donald Bodenner
Stavroula Kousteni
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The Board Of Trustees Of The University Of Arkansas
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Priority to AU11036/00A priority Critical patent/AU1103600A/en
Priority to EP99954769A priority patent/EP1121132A4/en
Priority to JP2000573366A priority patent/JP2003535018A/ja
Priority to CA002346456A priority patent/CA2346456A1/en
Publication of WO2000020007A1 publication Critical patent/WO2000020007A1/en

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    • 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
    • A61K31/566—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 having an oxo group in position 17, e.g. estrone
    • 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
    • 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
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • 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
    • A61K31/11—Aldehydes
    • 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
    • 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
    • A61K31/567—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 substituted in position 17 alpha, e.g. mestranol, norethandrolone
    • 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
    • A61K31/568—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 substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/29—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/02—Nutrients, e.g. vitamins, minerals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/743—Steroid hormones
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/723—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • This invention is in the field of bone physiology, and in particular provides methods and compositions that include compounds to increase bone mass, i.e., to achieve bone anabolism.
  • the compounds bind to the estrogen or androgen receptor without causing significant hormonal transcriptional activation.
  • Bones consist of living cells embedded within a matrix of proteins and minerals. Bones provide support and protection to the vital organs of the animal, and give strength and form to its structure.
  • Osteoporosis is a decrease in bone mass in combination with microarchitectural deterioration which leads to bone fragility and fractures. Treatments for osteoporosis have historically focused on the prevention of further bone loss. I n contrast, a bone anabolic agent is one that substantially increases bone mass. To date, while there have been several drugs approved by the U.S. Food and Drug Administration for th e treatment of osteoporosis, it is believed that no drug has yet b een approved in the United States to be used as a bone anabolic agent, for either humans or other animals.
  • Bone is a dynamic tissue which undergoes continual resorption and formation through a remodeling process, which is accomplished by two types of cells: osteoclasts, which erode cavities, and osteoblasts that synthesize new bone matrix. Remodeling takes place mainly on the internal surfaces of bone and it is carried out not by individual cells, but rather by temporary anatomical structures, termed basic multi- cellular units (BMUs), comprising teams of osteoclasts in the front and osteoblasts in the rear. In an established BMU, bone resorption and formation happens at the same time.
  • BMUs basic multi- cellular units
  • osteoclasts After osteoclasts stop resorbing bone, they die b y apoptosis and are quickly removed by phagocytes. During th e longer lifespan of the osteoblasts (about three months, a s compared to three weeks for osteoclasts), some osteoblasts convert to lining cells that cover quiescent bone surfaces and some are entombed within the mineralized matrix as osteocytes (Parfitt, In: Bone, Telford and CRC Press, PP351-429, 1990). However, the majority (65%) of osteoblasts that originally assembled at the remodeling site die by apoptosis (Jilka et al, JBMR 13 :793-802, 1998) .
  • osteoporosis due to old age Jilka et al., JCI 97 : 1732- 1740, 1996
  • osteoporosis due to glucocorticoid-excess Weinstein et al., JCI 102:274-282, 1998; Weinstein et al, Bone, 23:S461, 1998; Bellido et al, Bone, 23:S324, 1998).
  • antiresorptive agents that reduce bone turnover by inhibiting th e activation of bone remodeling (commonly but inaccurately referred to as "antiresorptive") increase bone mass by a maximum of 6-10%, and more typically, 2-3%, as measured by Dual Energy X-Ray Absorptiometry (DEXA). Most of this increase is in the first 1-2 years and is due to contraction of the remodeling space. Modest further increases may result from more complete secondary mineralization. Improvement of focal balance due to reduction of resorption depth has been demonstrated in animal experiments, but not yet in human subjects. Regardless of the mechanism, an increase of less than 10% will in almost all cases fail to restore bone mass to its peak value and fail to reestablish trabecular connectivity so that fracture risk will remain increased.
  • DEXA Dual Energy X-Ray Absorptiometry
  • bone anabolic agents for humans as well as animals.
  • uses for bone anabolic agents in humans include the strengthening of bone in healthy subjects who engage in strenuous physical activities such as sports or manual labor, and the strengthening of bone in persons who do not have osteoporosis but might be subject to osteoporosis in the future because the person is in a risk group for that disease.
  • a bone anabolic agent in humans include the treatment of persons who fail to obtain an adequate bone mass at the completion of growth or persons who are born with unusually fragile bones, persons who have a genetic predisposition to a bone catabolic disease, or an orthopedic bone disease such as joint degeneration, non-union fractures, orthopedic problems caused b y diabetes, periimplantitis, poor responses to bone grafts, implants, fracture.
  • bone anabolic agents there are many uses for bone anabolic agents in animals. For example, it would be useful to increase th e bone mass in horses and dogs used for labor as well as those u sed in sports such as racing. It would also be useful to increase the bone mass in chickens and turkeys used in meat production to maximize the amount of meat yield per animal.
  • drugs that are u s ed in the treatment of osteoporosis: anabolic steroids, bisphosphonates, calcitonins, estrogens/progestogens, Selective Estrogen Receptor Modulators (SERMs) such as raloxifene, phytoestrogen, parathyroid hormone (“PTH”), fluoride, Vitamin D metabolites, and calcium preparations.
  • SERMs Selective Estrogen Receptor Modulators
  • Anabolic Steroids (Andro ens Anabolic steroids (androgens) have been known to build muscle mass in the host. However, there has been no reported evidence that they function as bone anabolic agents a s defined herein (Snyder et al, JCEM 84: 1966- 1972, 1999) . Androgens are typically used as a replacement therapy for male hypogonadal disorders and they are used in adolescent males with a history of delayed puberty or growth.
  • Androgens can produce significant side effects when taken over a period of time, including water retention, jaundice, decreased high density lipoprotein an d increased low density lipoprotein, hepatic toxicity (most usually associated with the 17 ⁇ -alkylated androgens), hepatic carcinoma, increased risk of cardiovascular disease, and when taken in large dosages, irrationality, psychotic episodes, violent behavior, and death.
  • U.S. Patent No. 5,565,444 discloses the use of an androgen for the treatment of bone loss or for increasing bone mass. Calcitonin
  • Endogenous calcitonin is a polypeptide hormone involved in the regulation of calcium and bone metabolism.
  • Forms used therapeutically include calcitonin (pork), extracted from pig thyroid, a synthetic human calcitonin; elcatonin, a synthetic analogue of eel calcitonin; and salcatonin, a synthetic salmon calcitonin. They all have the property of lowering plasma-calcium concentration by diminishing the rate of bone resorption.
  • Calcitonins are typically administered subcutaneously or b y intramuscular injection.
  • Bisphosphonates have been widely used to treat osteoporosis.
  • the bisphosphonate disodium etidronate has similar effects on bone mass and fractures in established osteoporosis to those of calcitonin, but cannot be given for a prolonged period because of the risk of osteomalacia.
  • Bisphosphonate alendronate treatment at a dose of 10 mg/day results in a 5% increase in spinal bone mineral density (BMD) over the first year (Dempster, Exploiting and Bypassing the Bone Remodeling Cycle to Optimize the Treatment of Osteoporosis, Journal of Bone and Mineral Research, Volume 12, Number 8, 1997, pages 1152-1154).
  • BMD spinal bone mineral density
  • Dempster (Dempster D.W., New concepts in bone remodeling, In: Dynamics of Bone and Cartilage Metabolism, Chapter 18, pp.261-273, Acad. Press, 1999) confirms that the potential for an agent that can increase bone mass and hence reverse the skeletal defect in patients with osteoporosis is great, particularly if in doing so it also repairs microarchitectural damage. He notes that estrogens and calcitonin primarily stabilize bone mass and prevent further loss of bone, although a transient small increment in mass is often reported, particularly in patients with elevated levels of bone remodeling. Dempster et al conclude that this is not a true anabolic effect but is related to the temporal effects on turnover in which resorption declines initially followed by a reduction in formation that may take several months.
  • U.S. Patent No. 4,870,063 discloses a bisphosphonic acid derivative to increase bone mass.
  • U.S. Patent Nos. 5,532,226 and 5,300,687 describe the use of trifluoromethylbenzylphosphonates to increase bone mass.
  • U.S. Patent No. 5,885,973 to Papapoulos, et al discloses a bone mass anabolic composition that includes olpandronate, which is a bisphosphonate.
  • Estrogens/progestogens as a class have not to date been shown to increase bone mass by more than 10%, but instead have been used to retard the effect of osteoporosis. Estrogens are currently the most effective method of preventing osteoporosis in postmenopausal women.
  • U.S. Patent No. 5, 183,815 discloses the use of a steroidal hormone covalently linked to a hydroxy alkyl- 1 , 1 - bisphosphonate.
  • U.S. Patent No. 5,843,934 claims that an estrogen having insubstantial sex-related activity can be administered to a patient to retard the adverse effects of osteoporosis in a male or female. The '934 patent does not address how to select a compound to increase bone mass, but instead teaches how to retard the effect of bone loss.
  • WO 98/22113 filed by the University of Florida Research Foundation, Inc. discloses methods to utilize an isomer of an estrogen compound to confer cytoprotection on a population of cells associated with an ischemic event. Phytoestro gens
  • parathyroid hormone a n agent known for its role in calcium homeostasis, increases bone mass in animals and humans, as does the related PTH-related hormone PHTrP, the only other known ligand of the PTH receptor.
  • PTH parathyroid hormone
  • osteoblast apoptosis e., postponement of osteoblast apoptosis, is the principal, if not the sole, mechanism for the anabolic effects of intermittent parathyroid hormone administration on bone (Jilka et al., J. Clin. Invest. 104:439-446, 1999).
  • the anabolic effect of the drug is due to decreased prevalence of osteoblast apoptosis from 1.7-2.2% to as little as 0.1-0.4%, while the osteocytes in the newly made lamellar cancellous bone are closer together and more numerous than those found in the animals receiving vehicle alone.
  • the closely spaced, more numerous osteocytes are the predictable consequence of protecting osteoblasts from apoptosis.
  • the anti- apoptotic effect of PTH on osteoblasts as well as osteocytes has been confirmed in vitro using primary bone cell cultures and established cell lines.
  • teriparatide the 1-34 amino acid fragment of human parathyroid growth hormone
  • teriparatide administered a s daily injections has been reported to selectively increase th e trabecular bone density of the spine in osteoporotic patients.
  • U.S. Patent No. 5,510,370 discloses the use of a combination of PTH and raloxifene to increase bone mass.
  • U.S. Patent No. 4,833,125 discloses the use of PTH in combination with either a hydroxylated vitamin D derivative, or a dietary calcium supplement. Calcium Preparations
  • sodium fluoride The most thoroughly studied anabolic agent, sodium fluoride, can increase vertebral bone mass by 10% a year for at least four years but there is controversy about the quality of th e bone formed.
  • Sodium fluoride has not been approved as a bone anabolic agent. It has been difficult to establish anti-fracture efficacy because of serious qualitative abnormalities. First, much of the new bone is initially woven rather than lamellar. Second and more important, there is severe impairment of bone mineralization, in spite of sodium fluoride's effectiveness in increasing bone mass.
  • U.S. Patent No. 5,071 ,655 discloses a composition to increase bone mass that includes a fluoride source and a mitogenic hydantoin. SERMs
  • SERMs such as tamoxifen and raloxifene have also been used to treat osteoporosis.
  • U.S. Patent No. 4,970,237 discloses the use of clomiphene to increase bone mass in premenopausal women.
  • U.S. Patent No. 5,859,001 discloses the use of non- estrogen compounds having a terminal phenol group in a four-ring cyclopentanophenanthrene compound structure to confer neuroprotection to cells.
  • U.S. Patent No. 5,824,672 discloses a method for preserving tissues during transplantation procedures that includes administering an effective dose of a cyclopentanophenanthrene compound having a terminal phenol A ring.
  • WO 98/31381 filed by the University of Florida Research Foundation, Inc. discloses a method for enhancing the cytoprotective effect of polycyclic phenolic compounds on a population of cells that involves the steps of administering a combination of polycyclic phenolic compounds and anti-oxidants to achieve an enhanced effect.
  • One disclosed combination is glutathione and estrogen. It is an object of the present invention to provide a method to increase bone mass in a host by at least 10% per year without a loss in bone strength (defined by fracture incidence in vivo and mechanical strength in vitro) and/or deterioration of bone quality (as defined by abnormal collagen orientation and excessive accumulation of unmineralized bone matrix, determined, for example, with histomorphometry).
  • a method for increasing bone mass in a host at least 10% without a loss in bone strength or quality includes administering an effective amount of a compound that (i) binds to the estrogen ⁇ or ⁇ receptor (or the equivalent receptor in the host animal) with a n association constant of at least 10 8 M "1 , and preferably, at least 10 10 M "1 : (ii) (a) induces estrogenic gene transcriptional activity at a level that is no greater than 10% that of 17 ⁇ -estradiol, an d preferably no greater than 5, 1 or even 0.1% that of 17 ⁇ -estradiol when administered in vivo at a dosage of at least 0.1 ng/kg body weight or in vitro in osteoblastic or osteocytic cells with natural estrogen receptors or cells transfected with estrogen receptors or (b) induces an increase in uterine weight of no more than 10% that of 17 ⁇ -estradiol (or the equivalent compound in a host animal) ; (iii) induces the phosphoronuenta compound in
  • the compound is not a n estrogen compound, as that term is defined below.
  • the compound is an estrogen compound which is converted to a nonestrogen by attaching a substituent which prevents the compound from entering the cell but does not significantly affect the binding of the compound to the estrogen cell-surface receptor.
  • a method for increasing bone mass in a host at least 10% without a loss in bone strength or quality includes administering an effective amount of a compound that (i) binds to the androgen receptor (or the equivalent receptor in the host animal) with an association constant of at least 10 8 M "1 , and preferably, at least 10 10 M "1 : (ii) (a) induces androgenic gene transcriptional activity at a level that is no greater than 10% that of testosterone, and preferably no greater than 5, 1 or even 0.1% that of testosterone wh en administered in vivo at a dosage of at least 0.1 ng/kg body weight or in vitro in osteoblastic cells with the natural androgen receptor or cells transfected with the androgen receptor or (b) induces a n increase in muscle weight of no more than 10% that which is induced by testosterone (or the equivalent compound in a host animal); (iii) induces the phosphorylation of extracellular signal regulated kinase (ERK) when administered in
  • the compound is not a n androgen.
  • the compound is an androgen compound which is converted to a nonandrogen by attaching a substituent which prevents the compound from entering the cell but which does not significantly affect the ability of the compound to bind to the androgen cell- surface receptor.
  • the compound has a pro-apoptotic effect on osteoclasts at an in vivo dosage of at least 0.1 ng/kg body weight, or in osteoclastic cells with natural estrogen receptors or cells transfected with estrogen receptors.
  • the disclosed invention is based on the fundamental discovery that bone loss occurs because of an increase in osteoblast and perhaps osteocyte apoptosis, which can be inhibited by a compound that binds to an estrogen or androgen receptor, which induces the phosphorylation of ERKs without significant hormonal transcriptional activation.
  • the discovery of this fundamental pathway allows the selection of compounds which provide a maximum effect on bone mass and strength.
  • a method for selecting a compound that increases bone mass in a host at least 10% without a loss in bone strength or quality includes evaluating whether the compound (i) binds to th e estrogen or androgen receptor (or the equivalent receptor in the host animal) with an association constant of at least 10 8 M "1 , and preferably, at least 10 10 M "1 : (ii) (a) induces estrogenic or androgenic gene transcriptional activity at a level that is no greater than 10% that of 17 ⁇ -estradiol or testosterone, and preferably no greater than 5, 1 or even 0.1% that of 17 ⁇ -estradiol or testosterone, as appropriate, when administered in vivo at a dosage of at least 0.1 ng/kg body weight or in vitro in osteoblastic cells with the natural androgen or estrogen receptor or cells transfected with the androgen or estrogen receptor or (b) induces an increase in uterine weight of no more than 10% that which is induced by 17 ⁇ -estradiol
  • Estrogenic compounds like 17 ⁇ -estradiol and synthetic polycyclic phenols, such as estratriene-3-ol inhibit osteoblast and osteocyte apoptosis in vitro. Yet unlike the classical mechanism of estrogen receptor action that involves direct or indirect interaction with the transcriptional apparatus, the receptor-dependent anti- apoptotic effects of these compounds are nongenomic, as they are due to rapid (within 5 minutes) phosphorylation of ERKs.
  • Estratriene-3-ol increases bone mass in both estrogen-replete and estrogen-deficient mice.
  • Esstratriene-3-ol when given in low doses, has little effect on estrogenic-type activity but also h as little effect on bone mass. As the dosage increases, both effects increase.
  • the compound can also be used for the augmentation of bone mass and/ or fracture prevention in diseases characterized by low bone mas s and increased fragility.
  • the compounds can also be used to treat bone disease states in which osteoblastogenesis is decreased, such as senile osteoporosis, and glucocorticoid-induced osteoporosis-- especially in growing children and adolescents, during which time in whom interfering with bone remodeling is detrimental.
  • Figures provided herein illustrate embodiments of the invention and are not intended to limit the scope of th e invention.
  • Figure 1 provides nonlimiting examples of one class of compounds that can be used to increase bone mass without adversely affecting bone strength.
  • Figure 2 is a bar chart graph of the degree of apoptosis of osteoblasts and osteocytes in murine vertebral bone as a function of estrogen deficiency.
  • Swiss Webster mice (four months old) were ovariectomized. Twenty eight days later, th e animals were sacrificed, vertebrae were isolated, fixed an d embedded, and then undecalcified in methacrylate. The prevalence of osteoblast and osteocyte apoptosis was determined by the TUNEL method with CuS0 4 enhancement, and was found to be dramatically increased following loss of estrogen. ***p ⁇ 0.00001 ; *P ⁇ 0.0382.
  • Figure 3 is a series of bar chart graphs which illustrate the percentage of Etoposide-induced osteoblast apoptosis versus the log of the concentration of added estrogens 17 ⁇ - estradiol, 17 ⁇ -estradiol-BSA, 17 -estradiol, and estratriene-3-ol.
  • Osteoblastic cells derived from murine calvaria were pretreated with the sterols for 1 hour before the addition of the pro-apoptotic agent, etoposide. Apoptosis was determined after 6 hours b y trypan blue uptake (Jilka et al, J.Bone and Min. Res. 13 :793 : 802, 1998).
  • Figure 4 is a series of bar chart graphs of the inhibition of etoposide-induced apoptosis of osteocytes (MLO-Y4) by 17 ⁇ -estradiol, 17 ⁇ -estradiol-BSA, 17 ⁇ -estradiol, and estratriene-3-ol.
  • Cells were pretreated with the indicated concentrations of the compounds for 1 hour before the addition of the pro-apoptotic agent etoposide.
  • Apoptosis was determined after 6 hour by trypan blue uptake as described in Figure 3.
  • * indicates p ⁇ 0.05 versus etoposide alone, by ANOVA (Student- Newman-Keuls method).
  • Figure 5 is a series of bar chart graphs that indicates that the anti-apoptotic effect of 17 ⁇ -estradiol, 17 ⁇ -estradiol-BSA, 17 ⁇ -estradiol, and estratriene-3-ol (E-3-ol) on etoposide-induced apoptosis of osteoblasts is abrogated by the estrogen receptor antagonist, ICI182,780.
  • Osteoblastic cells derived from murine calvaria were pretreated for 1 hour with the pure receptor antagonist ICI182,780 ( 10 ⁇ 7 M) before the addition of the test agents ( 10 8 M).
  • Apoptosis was induced and quantified a s described in Figure 3. * indicates p ⁇ 0.05 versus etoposide alone, by ANOVA (Student-Newman-Keuls method).
  • Figure 6 is a series of bar chart graphs that indicates that the anti-apoptotic effect of 17 ⁇ -estradiol, 17 ⁇ -estradiol-BSA, 17 ⁇ -estradiol, and estratriene-3-ol (E-3-ol) on MLO-Y4 osteocytic cells is abrogated by the estrogen receptor antagonist, ICI182,780.
  • MLO-Y4 cells were pretreated for 1 hour with the pure receptor antagonist ICI182,780 ( 10 ⁇ 7 M) before the addition of the test agents ( 10 ⁇ 8 M).
  • Apoptosis was induced and quantified as described in Figure 3. * indicates p ⁇ 0.05 versus etoposide alone, by ANOVA (Student-Newman-Keuls method).
  • Figure 7 is a series of bar chart graphs which demonstrate that estrogen receptor ⁇ or ⁇ is required for the anti- apoptotic effects of 17 ⁇ -estradiol, 17 ⁇ -estradiol, and estratriene- 3-ol on the etoposide-induced apoptosis of osteoblasts.
  • CMV promoter alone and CMV promoter-driven cDNA for mER ⁇ or mEr ⁇ were stably transfected into HeLa cells.
  • Subconfluent cultures were treated for 1 hr with 10 "8 M 17 ⁇ -estradiol, 17 ⁇ -estradiol, or estratriene-3-ol followed by a 6 hr incubation with etoposide (5x l 0 5 M). Cells were trypsinized, pelleted and trypan blue positive cells enumerated.
  • Each bar represents mean of duplicate experiments ⁇ SEM. *P ⁇ 0.02 versus etoposide alone.
  • Figure 8 is Western blot which demonstrates that 17 ⁇ -estradiol, 17 -estradiol, 17 ⁇ -estradiol-BSA or estratriene-3-ol activate the extracellular signal regulated kinases (ERKs).
  • MLO-Y4 osteocytic cells were incubated for 25 minutes in serum-free medium. Subsequently, 17 ⁇ -estradiol, 17 ⁇ -estradiol, 17 ⁇ - estradiol-BSA or estratriene-3-ol ( 10 ⁇ 8 M) were added and cells incubated for an additional 5, 15, or 30 min. Cell lysates were prepared and proteins were separated by electrophoresis in polyacrylamide gels and transferred to PVDF membranes .
  • Figure 9 is a Western blot which demonstrates th at the effect of estrogenic compounds on the activation of ERK1/2 is blocked by the specific inhibitor of ERK kinase, PD98059.
  • MLO-Y4 cells were incubated for 25 minutes in serum-free medium in the presence or absence of 50 ⁇ M PD98059. Subsequently, 17 ⁇ - estradiol, 17 ⁇ -estradiol, 17 ⁇ -estradiol-BSA or estratriene-3-ol ( 10 " 8 M) were added and cells incubated for an additional 5 min. Cell lysates were prepared and proteins were separated b y electrophoresis in polyacrylamide gels and transferred to PVDF membranes. Western blotting was performed using a specific antibody recognizing phosphorylated ERKs 1 and 2, followed b y reblotting with an antibody recognizing total ERKs. Blots were developed by enhanced chemiluminescence.
  • Figure 10 is a series of bar chart graphs which demonstrate that the specific inhibitor of ERK activation, PD98059, abolishes the anti-apoptotic effect of 17 ⁇ -estradiol and related compounds.
  • MLO-Y4 osteocytic cells were pretreated for 1 hour with 50 ⁇ M PD98059 before the addition of 10 "8 M 17 ⁇ -estradiol, 17 ⁇ -estradiol, or 17 ⁇ -estradiol-BSA.
  • Apoptosis was induced b y incubation with the pro-apoptotic agent dexamethasone for 6 hour and quantified as described in Figure 3.
  • * indicates p ⁇ 0.05 versus the corresponding control group without dexamethasone, b y ANOVA (Student-Newman-Keuls method).
  • Figure 11 illustrates that unlike 17 ⁇ estradiol, estratriene-3-ol does not transactivate an estrogen response element through ER ⁇ .
  • the human ER was overexpressed in 293 cells lacking ER ⁇ along with a reporter construct containing 3 copies of an estrogen response element driving the luciferase gene.
  • Light units were counted and normalized to coexpressed b - galactosidase activity to control for differences in transfection efficiency. Results represent percent stimulation compared to ER ⁇ transfected cells, but not treated with the two agents.
  • Each b ar represents mean of duplicate experiments +/- SEM. *p ⁇ 0.001 vs. cells not exposed to the sterols.
  • Figure 12 is an illustration of the chemical structures of certain 3-ring compounds: [2S-(2a,4a ⁇ , 10a ⁇ )]- l ,2,3,4,4a,9, 10, 10a-octahydro-7-hydroxy-2-methyl-2- phenanthrenemethanol (PAM) and [2S-(2a,4a ⁇ , 10a ⁇ )]- l ,2,3 ,4,4a,9, 10, 10a-octahydro-7-hydroxy-2-methyl-2- phenanthrenecarboxaldehyde (P AC A) .
  • Figure 13 illustrates the generalized core ring structures with numbered carbons (Figure 13a) 4-ring structure, ( Figure 13 b ) 3-ring structure, (Figure 13c) 2-ring structure (fused), and ( Figure 13d) 2-ring structure (non-fused).
  • Figure 14 is an illustration of three mechanisms of estrogen activity: Figure 14 A (anti-apoptotic effect of estrogen), Figure 14B (anti-remodeling effect of estrogen) and Figure 14 C
  • Figure 15 compares the activity of the antiresorptive (e.g., 17 ⁇ -estradiol) versus non-anti-resorptive agents [e.g., estratriene-3-ol or intermittent PTH] on osteoblast and osteocyte apoptosis.
  • Bone formation occurs only on sites of previous osteoclastic bone resorption, i.e., on sites undergoing remodeling.
  • Each remodeling cycle is a transaction that, once consummated, is irrevocable.
  • agents with anti-apoptotic properties that do not have anti- resorptive/anti-remodeling properties rebuild more bone and therefore, increase the overall bone mass because they will not decrease the number of the remodeling units (i.e., the number of transactions).
  • both classical antiresorptive agents like 17 ⁇ -estradiol and agents that are not anti-resorptives are expected to have anti-fracture efficacy over and above that resulting from their effects on bone mass .
  • Figure 16 A is a table of examples of RI and R2 substitutions on the compound illustrated in Figure 1.
  • Figure 16B provides the molecular structures of ⁇ and ⁇ estradiol.
  • Figure 17 provides the chemical structures of estratrienes with anti-apoptotic properties.
  • Figure 18 provides the chemical structures of estradiol, phenol and diphenols with anti-apoptotic properties.
  • Figure 19 depicts the effect of 17 ⁇ estradiol on the transcriptional activity of a minimal ERE containing gene promoter and the blockade of this effect by a peptide (all) recognizing the ligand-induced specific conformational change of the estrogen receptor protein.
  • 293, human kidney cells were transiently transfected with a plasmid carrying the ER-specific all peptide with the GAL4-DNA binding domain inserted upstream of the peptide sequence, an ERE/IL-6 promoter-driven luciferase reporter plasmid and a ⁇ -galactosidase ( ⁇ -gal)-containing plasmid.
  • the ERE-luciferase construct carried three copies of the Xenopus vitellogenin ERE driving the luciferase gene in the pGL3-Basic vector (Promega). * indicates p ⁇ 0.05 versus cells transfected with the peptide all, by ANOVA (Student-Newman-Keuls method).
  • Figure 20 depicts the effect of 17 ⁇ estradiol on the transcriptional activity of the IL-6 promoter and the blockade of this effect by a peptide (all) recognizing the ligand-induced specific conformational change of the estrogen receptor protein.
  • the IL-6-luciferase plasmid carried 225bp of the proximal IL- 6 promoter cloned upstream of the luciferase gene in pGL3-Basic.
  • the all peptide inhibited the transcriptional effects of estrogen on the ERE-dependent transcription model, all was also shown to block transcription when mediated via protein/protein interaction between the ER and another transcription factor on the IL-6 gene model.
  • Figure 21 demonstrates the anti-apoptotic effect of 17 ⁇ estradiol conjugated with BSA and the lack of inhibition of this particular effect by the conformation sensitive peptide all.
  • the effect of the peptide on apoptosis was assayed using etoposide as the apoptotic stimulus. Upon etoposide treatment, cells that had been transfected with the ER and treated with 17 ⁇ - BSA were protected from apoptosis.
  • the invention as disclosed provides a method to increase bone mass without compromising bone quality, through the administration to a host of an effective amount of a compound that binds to the estrogen or androgen receptor so as to trigger the anti-apoptotic signalling pathway, but with minimal or no resultant transcriptional activity.
  • a n anabolic effect will be established by demonstrating increased bone formation, assessed by double tetracycline labeling (Weinstein R.S. In Disorders of Bone and Mineral Metabolism (eds. Coe and Favus) Raven Press, 1992, pp. 455-474) and a continuous increase in BMD, assessed by DEXA (Jilka et al. J. Clin. Invest. 97: 1732- 1740, 1996) for at least five years, along with increased, or at least no decreased quality or strength.
  • This invention is based on the fundamental discovery that bone loss occurs because of an increase in osteoblast apoptosis, which can be inhibited by a compound that binds to a n estrogen or androgen receptor (which induces the phosphorylation of ERKs) with minimal or no resultant transcriptional activity.
  • the discovery of this fundamental pathway allows the selection of compounds which provide a maximum effect on bone mass and strength.
  • a method for increasing bone mass in a host at least 10% without a loss in bone quality or strength includes administering a n effective amount of a compound that (i) binds to the estrogen ⁇ or ⁇ receptor (or the equivalent receptor in the host animal) with a n association constant of at least 10 8 M "1 , and preferably, at least 10 10 M "1 ; (ii) (a) induces estrogenic gene transcriptional activity a t a level that is no greater than 10% that of 17 ⁇ -estradiol, and preferably no greater than 5, 1 or even 0.1% that of 17 ⁇ -estradiol when administered in vivo at a dosage of at least 0.1 ng/kg body weight or in vitro in cells with natural estrogen receptors or transfected with estrogen receptors or (b) induces an increase in uterine weight of no more than 10% that of estrogen (or the equivalent compound in a host animal); (iii) induces the phosphorylation of extracellular signal regulated kina
  • the compound is not an estrogen compound, as that term is defined herein.
  • the compound is an estrogen compound which is converted to a nonestrogen b y attaching a substituent which prevents the compound from entering the cell, but which does not significantly affect the binding of the compound to the estrogen cell-surface estrogen receptor.
  • a method for increasing bone mass in a host at least 10% per year without a loss in bone strength or quality includes administering a n effective amount of a compound that (i) binds to the androgen receptor (or the equivalent receptor in the host animal) with a n association constant of at least 10 8 M "1 , and preferably, at least l O ⁇ M ' ⁇ ii) (a) induces androgenic gene transcriptional activity a t a level that is no greater than 10% that of testosterone, and preferably no greater than 5, 1 or even 0.1% that of testosterone when administered in vivo at a dosage of at least 0.1 ng/kg body weight or in vitro in cells with the natural androgen receptor o r transfected with the androgen receptor or (b) induces an increase in muscle weight of no more than 10% that which is induced b y testosterone (or the equivalent compound in a host animal); (iii) induces the phosphorylation of extracellular signal regulated kinas
  • the compound is not an androgen.
  • the compound is an androgen compound which is converted to a nonandrogen by attaching a substituent which prevents the compound from entering the cell containing the cell-surface androgen receptor.
  • the compound also has a pro-apoptotic effect on osteoclasts at an in vivo dosage of at least 0.1 ng/kg body weight or in vitro in cells with the natural androgen receptor or transfected with the androgen receptor.
  • a method for selecting a compound that increases bone mass in a host at least 10% without a loss in bone strength or quality includes evaluating whether the compound (i) binds to the estrogen or androgen receptor (or the equivalent receptor in the host animal) with an association constant of at least 10 8 M "1 , an d preferably, at least 10 10 M "!
  • (ii) induces estrogenic or androgenic gene transcriptional activity at a level that is no greater than 10% that of testosterone or 17 ⁇ -estradiol, an d preferably no greater than 5, 1 or even 0.1% that of 17 ⁇ -estradiol or testosterone, as appropriate, when administered in vivo at a dosage of at least 0.1 ng/kg body weight or at a concentration of 10 " 11 to 10 "7 M or in vitro in cells with the natural androgen or estrogen receptor or transfected with the androgen or estrogen receptor or (b) induces an increase in uterine or muscle weight, a s appropriate, of no more than 10% that which is induced by 17 ⁇ - estradiol or testosterone (or the equivalent compound in a host animal); (iii) induces the phosphorylation of extracellular signal regulated kinase (ERK) when administered in vivo at a dosage of at least 0.1 ng/kg body weight or at a concentration of 10 "11 to 10 "7 M vitro in cells with
  • Compounds selected according to the criteria provided herein can also be used as for the augmentation of bone mass and/or fracture prevention in diseases characterized by low bone mass and increased fragility.
  • the compounds can be used to treat bone disease states in which osteoblastogenesis is decreased, such as senile osteoporosis, and glucocorticoid-induced osteoporosis-- especially in growing children and adolescents, in whom interfering with bone remodeling is detrimental.
  • An estrogen compound refers to a four ring steroidal compound which possesses the biological activity of an estrus-producing hormone, or its conjugated and esterified derivative, or a derivative thereof of same chemical composition and structure but which does not possess the biological activity of the active form because it exhibits a different stereochemistry from the active form.
  • Nonlimiting examples of estrogens include broparestrol, chlorotrianisene, dienoestrol, epimestrol, equilin, estrapronicate, estropipate, ethinylestradiol, fosfestrol, hydroxyesetrone, mestranol, estradiol, estriol, conjugated and esterified estrogens, estrone, polyestradiol, promestriene, quinestradol, quinestrol, stilbestrol, and zeranol.
  • An androgen compound refers to a four ring steroidal compound which can be produced in the testis or adrenal cortex, or is a synthetic hormone, which acts to regulate masculine secondary sexual characteristics, or a derivative thereof of same chemical composition and structure but which does not possess the biological activity of the active form because it exhibits a different stereochemistry from the active form.
  • Nonlimiting examples include boldenone, clostebol, danazol, drosstanolone, epitiostanol, ethylestrenol, fluoxymesterone, formebolone, furazabol, mepitiostane, mesterolone, methandienone, methenolone, methyltestosterone, nandrolone, norethandrolone, oxabolone, oxymetholone, prasterone, quinbolone, staolone, stanozolol, testosterone, and trenbolone.
  • estrogens and androgens have chiral carbons, and thus can exist in a number of stereochemical configurations.
  • the 17 ⁇ hydroxy estrogens have biological activity while the 17 ⁇ hydroxy estrogens have very little effect on sexual characteristics (and induce little hormone-like gene transcriptional activation).
  • any stereochemical configuration including either the biologically active or the biologically inactive or less active structure, can be used, as long as the compound satisfies the specifically itemized criteria of the invention.
  • the catalog can be obtained by contacting the company and is also currently available on the internet at http://www.steraloids.com.
  • bone mass refers to the mass of bone mineral and is typically determined by Dual-Energy X-Ray Absorb tiometry (DEXA).
  • bone strength refers to resistance to mechanical forces and can be measured by any known method, including vertebrae compression strength or three point -bending of long bones.
  • bone quality refers to normal collagen orientation without excessive accumulation of unmineralized bone matrix, and can be measured by any known method, including undecalcified bone histomorphometry.
  • bone anti-resorption agent refers to a compound that blocks bone resorption by suppressing remodeling or the activity and/or lifespan of osteoclasts.
  • osteoopenia refers to decreased bone mas s below a threshold which compromises structural integrity.
  • metabolic bone disease refers to decreased bone mas s below a threshold which compromises structural integrity.
  • apoptosis refers to programmed cell death characterized by nuclear fragmentation and cell shrinkage as detected by morphological criteria and Terminal Uridine Deoxynucleotidal Transferase Nick End Labeling (TUNEL) staining.
  • host refers to any bone- containing animal, including, but not limited to humans, other mammals, canines, equines, felines, bovines (including chickens, turkeys, and other meat producing birds), cows, and bulls. II .
  • Estrogen compounds that bind to the estrogen ⁇ or ⁇ receptor with an association constant of at l e as t 10 8 M 1 , and preferably, at least 1 0 10 M x , but whi ch exhibit little transcriptional activation
  • a compound should be selected that binds to the estrogen ⁇ or ⁇ receptor (or the equivalent receptor in the host animal) with an association constant of at least 10 8 M "1 , and preferably, at least 10 10 M "1 .
  • This constant can be measured b y any known technique, including receptor binding assays whereby ligand binding affinities are determined by competitive radiometric binding assays using 10 nM [ 3 H] estradiol as tracer, purified estrogen receptor preparations, or cell cytosol preparations, or intact cells, during one hour incubation at room temperature or overnight at 4°. Bound receptor-ligand complex is absorbed using hydroxylapatite.
  • the estrogen ⁇ and ⁇ receptor subtypes have significantly different primary sequences in their ligand binding and transactivation domains.
  • ER ⁇ and ER ⁇ show a 56% amino acid homology in the hormone binding domain/activation function- 1 region, and only 20% homology in their A/B domain/activation function-1 region.
  • the difference between ER ⁇ and ER ⁇ structure suggests that some compounds might bind ER ⁇ or ER ⁇ , but not both. All such selectively binding compounds are considered to fall within the scope of this invention.
  • Estrogen compounds include those described in the 11th Edition of "Steroids" from Steraloids Inc., Wilton, N. H., which bind to the estrogen receptor with an association constant of at least 10 8 M "1 , and preferably, at least 10 10 M "1 . 2 . Minimal effect on estro gen -in prised transcriptional activation
  • an estrogen compound is selected that has a minimal effect on estrogen-induced transcriptional activation (or suppression).
  • the basis for this requirement is th at it has been discovered that apoptosis of osteoblasts is decreased by receptor binding, in the absence of transcriptional activation by estrogen-type compounds. Therefore, to provide a maximum therapeutic efficacy on bone without causing unrelated an d undesired side estrogen-related effects, estrogen receptor ligands with minimal transcriptional effects should be used.
  • a compound should be selected that induces estrogenic gene transcriptional activity at a level that is no greater than 10% that of 17 ⁇ -estradiol, and preferably no greater than 5, 1 or even 0.1% that of 17 ⁇ -estradiol when administered in vivo at a dosage of at least 0.1 ng/kg body weight or in vitro in cells with natural estrogen receptors or transfected with estrogen receptors or which induces an increase in uterine weight of no more than 10% that of estrogen (or the equivalent compound in a host animal).
  • surrogate markers of estrogenic transcriptional activation include, but are not limited to, the expression of the complement C-3 gene and lactoferin in the uterus.
  • the level of estrogen- induced transcriptional activity can be assessed in vitro.
  • genes induced or repressed by estrogen include, but are not limited to, complement C-3, lactoferin, or interleukin-6.
  • a preferred marker gene for estrogenic transcriptional activity is a minimal gene containing one or more copies of the ERE driving a reporter gene such as luciferase.
  • cell lines examples include human uterine HeLa cells, human embryonic kidney cells 293, murine osteocytic MLO-Y4 cells and murine osteoblastic calvaria derived cells.
  • Preferred compounds induce an increase in uterine weight of no more than approximately 10% that of estrogen (or the equivalent compound in a host animal). This can be easily tested according to known protocols. For example, in experimental mice, uteri are removed and cleaned of adjacent ligaments and fat. Wet weight is determined on a Mettler PB303 microgram balance (Toledo) and compared to total body weight (mg/lOOg BW) as an index of the estrogenic status of the animals. In women, similar assessment can be performed by uterine ultrasound.
  • the selected compound should induce the phosphorylation of ERKs at a concentration of 10 " ⁇ to 10 "7 M in vitro in cells with natural estrogen receptors or transfected with estrogen receptors using any known method, including but not limited to, the method set out in Figures 8 and 9 and Examples 7 - 9.
  • the phosphorylation of ERKs is easily assessed in vitro using osteoblastic or osteocytic cells with natural estrogen receptors or cells transfected with estrogen receptors. Examples of the evaluation of the phosphorylation of ERK in MLO-Y4 cells are provided in Figures 8 and 9 and Examples 7-9.
  • Other appropriate cell models include osteoblastic cells isolated from neonatal murine calvaria.
  • the anti-apoptotic effect on osteoblasts in vivo can b e assessed by any known method, including by the method described in Figure 2 and Example 3.
  • the anti-apoptotic effect in vitro can be assessed by any known method including the methods described in Figures 3-7 and 10, and Examples 2-6 and 9.
  • a nonestrogen compound refers to a compound other than an estrogen, as that term is defined above, which binds to the estrogen ⁇ or ⁇ receptor with an association constant of at least 10 8 M "1 and preferably, at least 10 10 M ⁇ ⁇ There are a number of reported compounds which are not estrogens but which bind to the estrogen receptor.
  • Examples include the aryl-substituted pyrazole described by Sun et al., Novel Ligands that Function as Selective Estrogens or Antiestrogens for Estrogen Receptor- ⁇ or Estrogen Receptor- ⁇ , Endocrinology, Volume 140, No. 2 (1999), one example of which is illustrated below.
  • an estrogen or nonestrogen compound is covalently linked to a second moiety that does not significantly interfere with the binding to the estrogen receptor but which does substantially prevent th e estrogen from entering the cell.
  • the second moiety is a protein such as bovine serum albumin, polyethelene glycol or dextran or liposomes.
  • th e second moiety is not a protein or peptide, but for polar, steric, or other reasons, prevents cell penetration. Examples of these type s of moieties include carboxylate, ammonium, and sulfide.
  • linking moiety is any divalent group that links two chemical residues, including but not limited to alkyl, alkenyl, alkynyl, aryl, polyalkyleneoxy (for example, -[(CH 2 ) n O-] n -), - alkyl-, -NR 3 -, and
  • the linking moiety can be a bifunctional linker moiety of the formula X-(CH 2 ) n -Y, wherein X and Y are functional groups capable of linking, including those independently selected from the group consisting of hydroxyl, sulfhydryl, carboxyl and amine groups, and n can be any integer between one and twenty four.
  • Androgen compounds that bind to the androgen receptor with an association constant of a t least 1 0 8 M ⁇ and preferably, at least 1 0 10 M ⁇ but which exhibit little transcriptional activation
  • a compound should be selected that binds to th e androgen receptor (or the equivalent receptor in the host animal) with an association constant of at least 10 8 M _1 , and preferably, a t least 10 10 M "1 .
  • the androgen receptor binding association constant is defined as the concentration of the ligand capable of saturating 50% of the unoccupied receptors. This constant can be measured by any known technique, including receptor binding assays whereby ligand binding affinities are determined by competitive radiometric binding assays using 10 nM [ 3 H] of the synthetic androgen RU1881 as tracer, purified androgen receptor preparations, or cell cytosol preparations, or intact cells, during one hour incubation at room temperature or overnight at 4°C.
  • Bound receptor-ligand complex is absorbed using hydroxylapatite.
  • Androgen compounds include those described in the 11th Edition of "Steroids" from Steraloids Inc., Wilton, N.H., which bind to th e androgen receptor with an association constant of at least 10 8 M "1 , and preferably, at least 10 10 M 1 .
  • an androgen compound is selected that has a minimal effect on androgen-induced transcriptional activation.
  • the basis for this requirement is that it has been discovered that apoptosis of osteoblasts is decreased by receptor binding in the absence of transcriptional activation by androgen- type compounds. Therefore, to provide a maximum therapeutic efficacy on bone without causing unrelated and undesired androgen-related effects, androgen receptor ligands with minimal transcriptional activity should be used.
  • a compound should be selected that induces androgenic transcriptional activity at a level that is no greater than 10% that of testosterone, and preferably no greater than 5, 1 or even 0.1% that of testosterone when administered in vivo at a dosage of at least 0.1 ng/kg body weight or in vitro in cells with natural androgen receptors or transfected with androgen receptors or induces an increase in prostate specific antigen (PSA) prostatic serum androgen of no more than 10% that of testosterone (or the equivalent compound in a host animal).
  • PSA prostate specific antigen
  • surrogate markers of androgenic transcriptional activation include, but are not limited to prostate specific antigen (PSA).
  • the level of androgen- induced transcriptional activity can be assessed in vitro in osteoblastic or osteocytic cells with natural androgen receptors or traf calvaria cells, ML0-Y4 osteocytic cells and HeLa cells.
  • PSA serum levels after administration of the selected compound.
  • Appropriate compounds induce an increase in PSA cells transfected with androgen receptors. Examples of such cell types include, primary cultures of PSA of no more than approximately 10% that of testosterone (or the equivalent compound in a host animal). This can be easily tested according to known protocols.
  • Examples of androgenic compounds that do not induce significant androgenic-like transcriptional activity include, but are not limited to, testosterone 17 ⁇ -hemisuccinate conjugated with BSA.
  • the selected compound should induce the phosphorylation of ERKs when administered in vivo at a dosage of at least 0.1 ng/kg body weight or at a concentration of 10 "11 to 10 "7
  • the phosphorylation of ERK in a host can be assessed in biopsies, for example from bone, using immunohistostaining with specific antibodies against phosphorylated ERKs.
  • the phosphorylation of ERK is also easily assessed in vitro using osteoblastic or osteocytic cells with natural androgen receptors or cells transfected with androgen receptors. Examples of the evaluation of the phosphorylation of ERK in MLO-Y4 cells are provided Figures 8 and 9 and Examples 7-9.
  • the anti-apoptotic effect on osteoblasts and osteocytes can be assessed in vivo by any known method, including the method described in Figure 2 and Example 1; and in vitro by any known method, including the method described in Figures 3-7 an d
  • Nonandrogen compounds that bind to t h e androgen receptor with an association constant of a t least 1 0 8 M ⁇ and preferably, at least 1 0 10 M *, b u t which exhibit little transcriptional activation
  • a nonandrogenic compound refers to a compound other than an androgen, as that term is defined above, which binds to the androgenic receptor with an association constant of at least 10 8 M "1 and preferably, at least 10 10 M "1 .
  • an association constant of at least 10 8 M "1 and preferably, at least 10 10 M "1 .
  • an androgen compound is covalently linked to a second moiety that does not significantly interfere with the binding to the androgen receptor but which does substantially prevent the androgen from entering the cell.
  • the second moiety is a protein such as bovine serum albumin.
  • the second moiety is not a protein or peptide, but for polar, steric, or other reasons, prevents cell penetration. Examples of these types of moieties include dextran or plyethelene glycol.
  • Nonlimiting examples of compounds that can b e used in the present invention to increase bone mass include those having a terminal phenyl ring and at least a second carbon ring.
  • the compound may have a number of R groups attached to any available site on the phenyl ring or elsewhere. These R groups may be selected from inorganic or organic atoms or moieties. Representative R groups are provided, although the invention is not to be limited by these examples: (a) The R i or R 2 groups may include a hydroxyl group or an inorganic R group including any of a halogen, a n amide, a sulfate, a nitrate, fluoro, chloro, or bromo groups. Additionally, R, or R 2 groups such as sodium, potassium and/or ammonium salts may be attached to the alpha or beta positions to replace hydrogen on any available carbon in the structure.
  • the R j or R 2 groups may be organic or may include a mixture of organic molecules and ions.
  • Organic Rj or R 2 groups may include alkanes, alkenes or alkynes containing up to six carbons in a linear or branched array.
  • additional R t or R 2 group substituents may include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, dimethyl, isobutyl, isopentyl, tert-butyl, sec-butyl, isobutyl, methylpentyl, neopentyl, isohexyl, hexenyl, hexadiene, l ,3-hexadiene-5-yne, vinyl, allyl, isopropenyl, ethynyl, ethylidine, vinylidine, isopropylidene, methylene, sulfate, mercap
  • R j or R 2 groups may be attached to any of the constituent rings to form a pyridine, pyrazine, pyrimidine, or v-triazine. Additional R j or R 2 group substituents may include any of the six- member or five-member rings itemized in section (b) below.
  • any compound having, in addition to the terminal phenyl group, at least one heterocyclic carbon ring (shown as R 3 in Figure 1), which may be an aromatic or non- aromatic phenolic ring with any of the substitutions described in section (a) above, and further may be, for example, one or more of the following structures: phenanthrene, naphthalene, naphthols, diphenyl, benzene, cyclohexane, 1 ,2-pyran, 1 ,4-pyran, 1 ,2-pyrone, 1 ,4-pyrone, 1 ,2-dioxin, 1 ,3-dioxin (dihydro form), pyridine, pyridazine, pyrimidine, pyrazine, piperazine, s-triazine, as - triazine, v-triazine, 1 ,2,4-oxazine, 1 ,3,2-oxazine, 1 ,3,6-ox
  • any of the above carbon ring structure may be linked directly, or via a linkage group, to any further heterocyclic aromatic or non aromatic carbon ring including: furan, thiophene (thiofuran), pyrrole (azole), isopyrrole (isoazole), 3 - isopyrrole (isoazole), pyrazole (1,2 diazole), 2-isoimidazole ( 1 ,3- isodiazole), 1,2,3-triazole, 1,2,4-triazole, 1,2-dithiazole, 1 ,2,3- oxathiazole, isoxazole (furo(a) monozole), oxazole (furo(b) monazole), thiazole, isothiazole, 1 ,2,3-oxathiazole, 1 ,2,4-oxadiazole, 1,2,5-oxadiazole, 1,3,5-oxadiazole, 1,2,3,4-oxatriazole, 1 ,2,3,
  • th at may form a cyclopentanophen(a)anthrene ring compound and which, for example, may be selected from the group consisting of l ,3,5( 10),6,8-estrapentaene, 1 ,3 , 5( 10), 6, 8, 1 1 -estrapentaene, l ,3,5(10),6,8, 15-estrapentaene, 1 ,3 , 5( 10), 6-estratetraene,
  • Any compound in the form of a prodrug th at may be metabolized to form an active polycyclic-phenolic compound having bone protective activity. II I .
  • Methods for Using the Active Compounds The active compounds which satisfy the criteria set out in detain herein can be used to treat a wide variety of medical conditions, including any condition in which it is helpful or necessary to build bone mass. Because of the discovery of the fundamental basis for bone loss (inappropriate osteoblastic apoptosis), one can for the first time envision the building of healthy bone as opposed to merely treating bone loss.
  • the active compounds can be used as bone anabolic agents in a host, including a human, to strengthen bone for strenuous physical activities such as sports or manual labor, and to strengthen bone in persons or other hosts who do not h ave osteoporosis but might be subject to osteoporosis in the future because the host is in a risk group for that disease.
  • Other uses for a bone anabolic agent in humans include the treatment of hosts, including persons who are born with naturally thin, small, or unusually fragile bones, including weak teeth, persons who have a genetic predisposition to a bone catabolic disease, or an orthopedic bone disease such as joint degeneration, non-union fractures, orthopedic problems caused by diabetes, periimplantitis, poor responses to bone grafts, implants, or fracture.
  • These compounds can be used to increase the bone mass in horses and dogs used for labor as well as those used in sports such as racing.
  • the compounds can also be used to increase the bone mass in chickens and turkeys used in meat production to increase the ease of processing.
  • Representative metabolic bone diseases are postmenopausal osteoporosis, senile osteoporosis in males and females, glucocorticoid-induced osteoporosis, immobilization- induced osteoporosis, weightlessness-induced osteoporosis (as in space flights), post-transplantation osteoporosis, migratory osteoporosis, idiopathic osteoporosis, juvenile osteoporosis, Paget' s Disease, osteogenesis imperfecta, chronic hyperparathyroidism, hyperthyroidism, rheumatoid arthritis, Gorham-Stout disease, McCune-Albright syndrome and osteolytic metastases of various cancers or multiple myeloma.
  • the present invention provides a method of screening for compounds that possess bone anabolic effects, comprising the steps of: a) contacting a sample of osteoblast cells with a compound; and b) comparing the number of osteoblast cells undergoing apoptosis in the compound-treated cells with th e number of osteoblast cells undergoing apoptosis in an untreated sample of osteoblast cells.
  • a lower number of apoptotic cells following contact with the compound indicates that the compound possesses bone anabolic effects.
  • Preferred compounds also inhibit apoptosis of osteocytes.
  • the compound may b e contacted with the sample either in vitro, e.g., in cell culture or in vivo, e.g., in an animal model.
  • Typical methods of determining apoptosis are nuclear morphologic criteria, DNA end-labeling, DNA fragmentation analysis and immunohistochemical analysis.
  • a method for selecting a compound that increases bone mass at least 10% in a host without a loss in bone strength or quality includes evaluating whether the compound (i) binds to the estrogen or androgen receptor (or the equivalent receptor in the host animal) with an association constant of at least 10 8 M "1 , and preferably, a t least 10 10 M "1 : (ii) (a) induces estrogenic or androgenic gene transcriptional activity at a level that is no greater than 10% that of testosterone or 17 ⁇ -estradiol, and preferably no greater than 5 , 1 or even 0.1% that of 17 ⁇ -estradiol or testosterone, a s appropriate, when administered in vivo at a dosage of at least 0.1 ng/kg body weight or in vitro at concentrations of 10 "u to 10 "7 M in cells with the natural androgen or estrogen receptor o r transfected with the androgen or estrogen receptor or (b) induces an increase in uterine or muscle weight or increase virilization in
  • a method for screening for compounds that bind to the estrogen or androgen receptor and activate the anti-apoptotic signalling pathway, without resultant transcriptional activation is provided.
  • This method is based on the fundamental discovery that the ligand-induced conformational changes of the estrogen receptor protein required for prevention of apoptosis, are distinct from the conformational changes required for transcriptional activity ( Figures 19 - 21 ).
  • This discovery allows for selecting compounds, from a large library of small molecules, which have anti-apoptotic, but not transcriptional, activity. Selection is accomplished using small peptides that can specifically block the transcriptional activity of ligand activated receptor, but do not interfere with the ability of the receptor to initiate the anti-apoptotic signalling cascade.
  • cells are transfected with the estrogen or androgen receptor with or without a peptide th at recognizes the conformation of the protein required for transcriptional activation, but not anti-apoptosis.
  • compounds that induce conformational changes resulting in both transcriptional and anti-apoptosis compatible conformations can be distinguished from compounds that only induce the latter conformational changes.
  • Nonlimiting examples of this method of screening include peptide binding assays for ER ⁇ or ER ⁇ whereby th e purified receptor protein is immobilized on streptavidin-coated plates using biotinylated vitellogenin ERE according to previously described methods of affinity selection (Sparks AB, Adey NB, Cwirla S, Kay BK. Screening phage-displayed peptide libraries. I n Phage Display of Peptides and Proteins, A Laboratory Manual, eds. Kay BK, Winter J and McCafferty J. (Academic, San Diego), pp .227- 253, 1996).
  • one of the active compounds described herein can be administered to a host to increase bone mass in combination with a second pharmaceutical agent.
  • the second pharmaceutical agent can be a bone anti- resorption agent, a second bone mass anabolizing agent, a n antioxidant, a dietary supplement, or any other agent that increases the beneficial effect of the active compound on bone structure, strength, density, or mass.
  • any member of the ten classes of drugs described in the Background of the Invention that are used in the treatment of osteoporosis can be administered in combination with the primary active agent, including: an anabolic steroid, a bisphosphonate, a calcitonin, an estrogen or progesterone, an anti-estrogens such a s raloxifene or tamoxifene, parathyroid hormone ("PTH”), fluoride, Vitamin D or a derivative thereof, or a calcium preparations.
  • the primary active agent including: an anabolic steroid, a bisphosphonate, a calcitonin, an estrogen or progesterone, an anti-estrogens such a s raloxifene or tamoxifene, parathyroid hormone ("PTH”), fluoride, Vitamin D or a derivative thereof, or a calcium preparations.
  • PTH parathyroid hormone
  • Nonlimiting examples of suitable agents for combination include, but are not limited to, alendronic acid, disodium clondronate, disodium etidronate, disodium medronate, disodium oxidronate, disodium pamidronate, neridronic acid, risedronic acid, teriparatide acetate, tiludronic acid, ipriflavone, potassium bicarbonate, progestogen, a thiazide, gallium nitrate, NSAIDS, plicamycin, aluminum hydroxide, calcium acetate, calcium carbonate, calcium, magnesium carbonate, and sucralfate.
  • Reducing agents such as glutathione or other antioxidants may also be useful in combination with any of th e compounds of the present invention.
  • the term antioxidant refers to a substance that prevents the oxidation of a n oxidizable compound under physiological conditions.
  • a compound is considered an antioxidant for purposes of this disclosure if it reduces endogenous oxygen radicals in vitro.
  • the antioxidant can be added to a cell extract under oxygenated conditions and the effect on an oxidizable compound evaluated.
  • antioxidants scavenge oxygen, superoxide anions, hydrogen peroxide, superoxide radicals, lipooxide radicals, hydroxyl radicals, or bind to reactive metals to prevent oxidation damage to lipids, proteins, nucleic acids, etc.
  • antioxidant includes, but is not limited to, the following classes of compounds:
  • Dithiocarbamates have been extensively described in patents and in scientific literature. Dithiocarbamates and related compounds have been reviewed extensively for example, by G. D. Thorn et al., entitled “The Dithiocarbamates and Related Compounds," Elsevier, New York, 1962. Dithiocarboxylates are compounds of the structure, A - SC(S)-B, which are members of the general class of compounds known as thiol antioxidants, and are alternatively referred to a s carbodithiols or carbodithiolates.
  • a and B can be any group that does not adversely affect the efficacy or toxicity of the compound.
  • a and B can be selected by one of ordinary skill in the art to impart desired characteristics to the compound, including size, charge, toxicity, and degree of stability, (including stability in an acidic environment such as the stomach, or basic environment such as the intestinal tract). The selection of A and B will also have an important effect on the tissue-distribution and pharmacokinetics of the compound. The compounds are preferably eliminated by renal excretion.
  • Cysteine is an amino acid with one chiral carbon atom. It exists as an L-enantiomer, a D-enantiomer, or a racemic mixture of the L- and D-enantiomers. The L-enantiomer is the naturally occurring configuration. N-acetylcysteine (acetamido-mercaptopropionic acid,
  • NAC N-acetylated derivative of cysteine. It also exists as a n L-enantiomer, a D-enantiomer, an enantiomerically enriched composition of one of the enantiomers, or a racemic mixture of th e L and D enantiomers.
  • enantiomerically enriched composition or compound refers to a composition or compound that includes at least 95%, and preferably, at least 97% by weight of a single enantiomer of the compound. Any of these forms of NAC can be delivered as an antioxidant in the present invention.
  • a single isomer of a thioester or thioether of NAC or its salt, and most preferably, the naturally occurring L- enantiomer, is used in the treatment process.
  • N-acetylcysteine exhibits antioxidant activity (Smilkstein, Knapp, Kulig and Rumack, N. Engl. J. Med. 1988, Vol. 319, pp. 1557-62; Knight, K.R., MacPhadyen, K., Lepore, D.A., Kuwata, N., Eadie, P. A., O'Brien, B. Clinical Sci., 1991, Vol. 81, pp. 31-36; Ellis, E.F., Dodson, L.Y., police, R.J., J. Neurosurg. , 1991, Vol. 75, pp. 774-779).
  • the sulfhydryl functional group is a well characterized, highly reactive free radical scavenger.
  • N- acetylcysteine is known to promote the formation of glutathione (a tri-peptide, also known as g-glutamylcysteinylglycine), which is important in maintaining cellular constituents in the reduced state (Berggren, M., Dawson, J., Moldeus, P. FEBS Lett. , 1984, Vol. 176, pp. 189-192).
  • glutathione a tri-peptide, also known as g-glutamylcysteinylglycine
  • the formation of glutathione may enhance the activity of glutathione peroxidase, an enzyme which inactivates hydrogen peroxide, a known precursor to hydroxyl radicals (Lalitha, T., Kerem, D., Yanni, S., Pharmacology and Toxicology, 1990, Vol.66, pp.
  • N-acetylcysteine exhibits low toxicity in vivo, and is significantly less toxic than deprenyl (for example, the LD 50 in rats has been measured at 1140 and 81 mg/kg intravenously, for N- acetylcysteine and deprenyl, respectively).
  • N-acetyl cysteine and derivatives thereof are described, for example, in WO/95/26719. Any of the derivatives described in this publication can be used in accordance with this invention.
  • Scavengers of Peroxides including but not limited to catalase and pyruvate.
  • Antioxidants which are inhibitors of lipid peroxidation, including but not limited to TroloxTM, BHA, BHT, aminosteroid antioxidants, tocopherol and its analogs, and lazaroids.
  • antioxidants including antioxidant vitamins (vitamin C or E or synthetic or natural prodrugs or analogs thereof), either alone or in combination with each other, flavanoids, phenolic compounds, caratenoids, and alpha lipoic acid.
  • G Inhibitors of lipoxygenases and cyclooxygenases, including but not limited to nonsteriodal antiinflammatory drugs, COX-2 inhibitors, aspirin-based compounds, and quercetin.
  • H Antioxidants manufactured by the body, including b u t not limited to ubiquinols and thiol antioxidants, such as, and including glutathione, Se, and lipoic acid.
  • An active compound or its pharmaceutically acceptable salt, selected according to the criteria described in detail herein, can be administered in an effective amount to treat any of the conditions described herein, optionally in a pharmaceutically acceptable carrier or diluent.
  • the active materials can be administered by any appropriate route for systemic, local or topical delivery, for example, orally, parenterally, intravenously, intradermally, subcutaneously, buccal, intranasal, inhalation, vaginal, rectal or topically, in liquid or solid form.
  • Methods of administering the compound of the invention may be by specific dose or b y controlled release vehicles.
  • a preferred mode of administration of the active compound is oral.
  • Oral compositions will generally include a n inert diluent or an edible carrier.
  • the active compound can b e enclosed in gelatin capsules or compressed into tablets.
  • the compound can b e incorporated with excipients and used in the form of tablets, troches, or capsules.
  • Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; and/or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • dosage unit forms can contain various other materials which modify the physical form of th e dosage unit, for example, coatings of sugar, shellac, or other enteric agents.
  • the compound can be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like.
  • a syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.
  • the compound or a pharmaceutically acceptable derivative or salts thereof can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, such as classical estrogen like 17 ⁇ -estradiol or ethinyl estradiol; bisphosphonates like alendronate, etidronate, pamidronate, risedronate, tiludronate, zoledronate, cimadronate, clodronate, ibandronate, olpadronate, neridronate, EB-1053; calcitonin of salmon, eel or human origin; and anti-oxidants like glutathione, ascorbic acid or sodium bisulfite.
  • other active materials that do not impair the desired action, or with materials that supplement the desired action, such as classical estrogen like 17 ⁇ -estradiol or ethinyl estradiol; bisphosphonates like alendronate, etidronate, pamidronate, risedronate,
  • Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the parental preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • preferred carriers are physiological saline or phosphate buffered saline (PBS).
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, poly anhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • Liposomal suspensions are also pharmaceutically acceptable carriers.
  • liposome formulations may be prepared by dissolving appropriate lipid(s) (such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and/or cholesterol) in an inorganic solvent that is then evaporated, leaving behind a thin film of dried lipid on the surface of the container.
  • appropriate lipid(s) such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidyl choline, and/or cholesterol
  • an inorganic solvent that is then evaporated, leaving behind a thin film of dried lipid on the surface of the container.
  • An aqueous solution of the active compound or its monophosphate, diphosphate, and/or triphosphate derivative(s) is then introduced into the container.
  • the container is then swirled by hand to free lipid material from the sides of the container
  • the dose and dosage regimen will depend upon the nature of the metabolic bone disease, the characteristics of the particular active compound, e.g., its therapeutic index, the patient, the patient's history and other factors.
  • the amount of an activator of non-genomic estrogen-like signaling compound administered will typically be in the range of about 1 pg/kg to about 10 mg/kg of patient weight.
  • the schedule will be continued to optimize effectiveness while balanced against negative effects of treatment. See Remington's Pharmaceutical Science, 17th Ed. (1990) Mark Publishing Co., Easton, Penn.; and Goodman and Gilman's: The Pharmacological Basis of Therapeutics 8th Ed (1990) Pergamon Press.
  • the active compound will most typically be formulated in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle.
  • a pharmaceutically acceptable parenteral vehicle are preferably non-toxic and non-therapeutic. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as fixed oils and ethyl oleate may also be used. Liposomes may be used a s carriers.
  • the vehicle may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives.
  • an activator of non-genomic estrogen-like signaling compound will typically be formulated in such vehicles at concentrations of about 10 pg/ml to about 1 0 mg/ml.
  • concentration of the compound in the drug composition will depend on absorption, inactivation, and excretion rates of the drug as well as other factors known to those of skill in the art. It is to be noted that dosage values will also vary with the severity of the condition to be alleviated. Additionally, the active ingredient may be administered at once, or may be divided into a number of smaller doses to be administered at varying intervals of time.
  • 17 ⁇ -estradiol the synthetic steroid estratriene-3-ol, which is a potent neuroprotective compound, and 17 ⁇ -estradiol, have potent anti-apoptotic effects on osteoblastic cells in vitro.
  • U.S. Patent No. 5,843,934 to Simpkins discloses that a n estrogen having insubstantial sex-related activity, and in particular, ⁇ -estrogens such as 17 ⁇ -estradiol, can be administered to a patient to retard the adverse effects of osteoporosis in a male or female.
  • the '934 patent does not address how to select a compound to increase bone mass opposed to treat osteoporosis.
  • Increasing bone mass is a different indication from the treatment of bone loss, as dramatically illustrated by the fact that the U.S. Food and Drug Administration has approved a number of drugs for the treatment of osteoporosis, but has not approved any drugs to date as bone anabolic agents.
  • 17 ⁇ -Estradiol is used in these illustrative examples even though it is a potent activator of estrogen-like gene transcription, because it tightly binds to the estrogen receptor and inhibits osteoblastic apoptosis.
  • the compound must be modified to fall within the selection criteria for the present invention b y altering it in such a way that it cannot enter the cell to induce gene transcription. Such modifications can occur, for example, b y covalently attaching, either directly or through a linking moiety, a second moiety that prevents or limits cell penetration. Any other estrogen or androgen that binds appropriately to the relevant receptor can be likewise modified for use to increase bone mass.
  • the increased rate of bone remodeling that follows loss of estrogen should cause a transient acceleration of mineral loss because bone resorption is faster than bone formation and the bone made by new BMUs are less dense than older ones.
  • increased remodeling alone cannot explain the progressive bone loss that lasts long after the rate of bone remodeling has slowed.
  • osteoclasts erode deeper than normal cavities. This frequently leads to penetration through a trabecular structure causing removal of some cancellous elements entirely; the remainder are more widely separated and less well connected.
  • Example 1 17 ⁇ -estradiol prevented apoptosis of osteoblastic cells isolated from murine calvaria, in a dose dependent manner. Strikingly, inhibition of osteoblast apoptosis could also be shown by 17 ⁇ -estradiol conjugated with bovine serum albumin, a membrane impermeable compound. The same effect could also b e shown with 17 ⁇ -estradiol, a compound heretofore thought to b e inactive. Moreover, inhibition of etoposide-induced osteoblastic cell apoptosis was demonstrated by estratriene-3-ol, an estrogenic compound thought to lack feminizing properties (Figure 3). In this experiment, osteoblastic cells were derived from murine calvaria and were pretreated with the sterols for 1 hour before th e addition of the pro-apoptotic agent, etoposide. EXAMPLE 3
  • EXAMPLE 7 The mechanism of the anti-apoptotic effect of the estrogenic compounds described herein was established b y demonstrating that 17 ⁇ -estradiol, 17 ⁇ -estradiol, 17 ⁇ -estradiol- BSA or estratriene-3-ol, at 10 "8 M concentrations, activated extracellular signal regulated kinases (ERKs).
  • MLO-Y4 osteocytic cells were incubated for 25 minutes in serum- free medium.
  • 17 ⁇ -estradiol, 17 ⁇ -estradiol, 17 ⁇ - estradiol-BSA or estratriene-3-ol 10 ⁇ 8 M were added and cells incubated for an additional 5, 15, or 30 minutes.
  • Cell lysates were prepared and proteins were separated by electrophoresis in polyacrylamide gels and transferred to PVDF membranes.
  • Western blotting was performed using a specific antibody recognizing phosphorylated extracellular signal regulated kinases 1 and 2, followed b y reblotting with an antibody recognizing total extracellular signal regulated kinases. Blots were developed by enhanced chemiluminescence.
  • MAP kinases namely the extracellular signal regulated kinases
  • EXAMPLE 11 That the anti-apoptotic effects of estrogenic compounds is dissociated from their transcriptional activity w as established by demonstrating that even though estratriene-3-ol was as potent as 17 ⁇ estradiol in inhibiting apoptosis, unlike 17 ⁇ estradiol, it did not transactivate an estrogen response element through the estrogen receptor ⁇ .
  • hER ⁇ was overexpressed in 293 cells (which lack constitutive ER ⁇ ) along with a reporter construct containing 3 copies of an estrogen response element driving the luciferase gene. Light units were counted and normalized to coexpressed ⁇ -galactosidase activity to control for differences in transfection efficiency.
  • estratriene-3-ol or other test compound are administered estratriene-3-ol or other test compound to determine whether they can suppress osteoblast and osteocyte apoptosis and whether changes in apoptosis would be associated with changes in BMD, bone formation rate, or cancellous bone volume.
  • mice In a representative experiment of this sort, six 4 - 5 month old female mice per group are screened twice for BMD in a four week period immediately prior to the initiation of th e experiment to establish that peak adult bone mass has been attained. A subset of mice are then ovariectomized. Intact an d ovariectomized mice are treated with vehicle, or 20, 200 or 2000 ng/g body weight estratriene-3-ol or another test compound. Ovariectomized mice are also treated with 20 ng/g body weight 17 ⁇ -estradiol for comparison purposes.
  • test agents 10,000 ⁇ g/ml are maintained in approximately 2.0 ml of 95% ethanol. These stocks are diluted in 95% ethanol to make 1000 ⁇ g/ml and 100 ⁇ g/ml concentrations. The concentration of the stocks is checked spectrophotometrically.
  • the test agent is diluted in sesame oil and sonicated. Test agents are administered for 28 days by subcutaneous injections on alternative days. The mice are weighed weekly and serum samples are collected a t appropriate times for analysis of bone biochemical markers, such as osteocalcin or collagen cross-links. Tetracycline labeling is performed by administration of the antibiotic (30 mg/kg) at 2 and 8 days prior to the end of each experiment. Table 1 shows a representative example of 25 g mice divided into 5 groups with each animal receiving 100 ⁇ l of the test agent per injection.
  • BMD is determined in live animals at day 0, 14 and 28.
  • the vertebral bones L1-L4 are collected for fixation and embedded undecalcified in methylmethacrylate plastic for the determination of the prevalence of osteoblast an d osteocyte apoptosis and other static and dynamic histomorphometric measurements.
  • L5 vertebrae are isolated for determining anti-fracture efficacy of the compounds by assaying compression, 3 point bending and other appropriate biomechanical tests.
  • Results confirming the expected efficacy of these compounds show decreased prevalence of osteoblast and/or osteocyte apoptosis, and/or positive BMD changes, and/or increased cancellous bone area, and/or increased rate of bone formation, and/or increased biomechanical strength.
  • Ovx-vc shicle global lhindquarfcpine 1 global 2 hindquart : spine 2
  • Each row represents values for individual animals.
  • the first three sets of numbers represent the initial BMD measurements (by dual-energy x-ray absorptiometry with Hologic QDR2000 plus, using customized software) at day 0 and the last three BMD measurements at the end of the experiment.
  • estratriene-3-ol a general experimental protocol evaluating the anti-fracture efficacy of compounds like estratriene-3-ol.
  • estrogen-replete or estrogen- deficient mice, rats, dogs, primates, etc., or animals representing models of involutional osteoporosis and/or defective osteoblastogenesis e.g., the senescence accelerated mouse, SAMP6: (Jilka et al., J Clin Invest 97: 1732- 1740, 1996)
  • animal models of glucocorticoid excess e.g., Weinstein et al. J Clin Invest, 102:274-282, 1998) are administered estratriene-3-ol to determine whether they can increase bone strength.
  • mice In a representative experiment of this sort, seven 4 - 5 month old female mice per group are screened twice for BMD in a four week period immediately prior to the initiation of the experiment to establish that peak adult bone mass has b een attained. A subset of mice are then ovariectomized. Intact and ovariectomized mice are treated with vehicle, or 20, 200 or 2000 ng/g body weight estratriene-3-ol or another ANGEL compound. Ovariectomized mice are also treated with 20 ng/g body weight 17 ⁇ -estradiol for comparison purposes. Ultimate load bearing properties of the fifth lumbar murine vertebrae (L5) is determined.
  • Vertebrae are individually compressed between parallel loading platens along the cephalocaudad axis until failure and the ultimate load (in Newtons) and displacement (in mm) are recorded.
  • mice (ovariectomized) mice (from the same animals shown in Example
  • V ertebral Global BMD (g/cm2)
  • McDonnell and co-workers have recently screened for and isolated four classes of small (11 amino acids) peptides that recognize distinct conformational changes of th e estrogen receptor, and can either selectively block transcription from specific ligands (e.g., estradiol but not tamoxifen and vice versa) or selectively block ER ⁇ but not ER ⁇ -mediated transcription, and vice versa, when tested on a consensus ERE (Norris et al. Science 285 :744-746, 1999).
  • the first class contains the LXXLL motif and can interact with both estradiol-activated ER ⁇ and ER ⁇ .
  • the second class displays specific interaction with estradiol- and tamoxifen- activated ER ⁇ , whereas the third class can interact specifically with tamoxifen-activated ER ⁇ .
  • a fourth class with a SREWFXXXL conserved motif was found to complex to tamoxifen-activated ER ⁇ and ER ⁇ .

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WO2001096605A2 (en) * 2000-06-13 2001-12-20 The Board Of Trustees For The University Of Arkansas Methods of dissociating nongenotropic from genotropic activity of steroid receptors
US6660468B1 (en) 1998-10-27 2003-12-09 Board Of Trustees Of The University Of Arkansas Vitro and in vivo models for screening compounds to prevent glucocorticoid-induced bone destruction

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US5189212A (en) * 1990-09-07 1993-02-23 University Of Georgia Research Foundation, Inc. Triarylethylene carboxylic acids with estrogenic activity
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US5362720A (en) * 1991-06-28 1994-11-08 Endorecherche, Inc. Methods of treating or preventing breast or endometrial cancer with low dose non-masculinizing androgenic compounds

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US6660468B1 (en) 1998-10-27 2003-12-09 Board Of Trustees Of The University Of Arkansas Vitro and in vivo models for screening compounds to prevent glucocorticoid-induced bone destruction
WO2001096605A2 (en) * 2000-06-13 2001-12-20 The Board Of Trustees For The University Of Arkansas Methods of dissociating nongenotropic from genotropic activity of steroid receptors
WO2001096605A3 (en) * 2000-06-13 2003-09-04 Univ Arkansas Methods of dissociating nongenotropic from genotropic activity of steroid receptors

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