Note: Descriptions are shown in the official language in which they were submitted.
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
METHOD AND COMPOSITIONS FOR INCREASING BONE MASS
BACKGROUND OF THE INVENTION
Federal Funding
This invention was funded in part through a grant
from the National Institutes of Health. Therefore, the federal
government has certain rights in this invention.
Field of the Invention
This invention is in the field of bone physiology, and i n
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.
Descr,~' ption of the Related A r t
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
1
CA 02346456 2001-04-06
WO OOI20007 PCT/US99/23355
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 dru g s
approved by the U.S. Food and Drug Administration for the
treatment of osteoporosis, it is believed that no drug has yet been
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, b a t
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.
After osteoclasts stop resorbing bone, they die b y
apoptosis and are quickly removed by phagocytes. During the
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).
Most metabolic disorders of the adult skeleton result
from an imbalance between the resorption of old bone b y
2
CA 02346456 2001-04-06
WO 00/20007 PCTNS99/23355
osteoclasts and its subsequent replacement by osteoblasts.
Changes in cell numbers, as opposed to individual cell activity
(Manolagas and Jilka, NEJM 332:305-311, 1995), appear to be the
cause of most metabolic bone diseases, including the three most
common forms of osteoporosis: osteoporosis due to sex steroid
deficiency in females and males (Jilka et al., Science 257:88-91,
1992; Jilka et al., JCI 101:1942-1950, 1998; Bellido et al., JCI .
95:2886-2895, 1995; Weinstein et. al., Endocrinology 138:4013-
4021, 1997); osteoporosis due to old age (Jilka et al., JCI 97:1732-
1740, 1996); and osteoporosis due to glucocorticoid-excess
(Weinstein et al., JCI 102:274-282, 1998; Weinstein et al, Bone,
23:S461, 1998; Bellido et al, Bone, 23:5324, 1998).
Agents that reduce bone turnover by inhibiting the
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.
There are a wide variety of needs for bone anabolic
agents for humans as well as animals. Examples of uses for bone
anabolic agents in humans, besides patients with osteoporosis,
3
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
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 h av a
osteoporosis but might be subject to osteoporosis in the future
because the person is in a risk group for that disease. Other a s a s
for 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.
Likewise, there are many uses for bone anabolic
agents in animals. For example, it would be useful to increase the
bone mass in horses and dogs used for labor as well as those asad
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.
There are currently ten classes of drugs that are a s a d
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. No compound within th a s a
classes has been approved as a bone anabolic agent.
Anabolic Steroids (Androgens)
4
CA 02346456 2001-04-06
WO 00/20007 PCT/US99123355
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 and
increased low density lipoprotein, hepatic toxicity (most usually
associated with the 17 a-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 (po.rk), 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.
B~ ,s~hosphonates
Bisphosphonates have been widely used to treat
osteoporosis. The bisphosphonate disodium etidronate has similar
5
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
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 i n
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
continues to increase, albeit at a slower rate, at this site during the
second and third years of treatment. The magnitude and duration
of the increase in BMD has led to speculation that alendronate i s
doing more than simply reducing remodeling space and that i t
may possess anabolic activity. The bisphosphonate etidronate
reduced resorption depth in human iliac trabecular bone b y
almost 30% after one year of treatment, but no such data are y a t
available for alendronate. Etidronate did not change the thickness
of trabecular packets, but recent studies in osteoporotic women
suggest that this is increased after two years of alendronate
treatment at 10 and 20 mg/day. This result was not confirmed
after three years of treatment.
In another article, 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 a n d
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
6
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
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.
Albeit, bisphosphonates have anti-apoptotic effects o n
osteoblasts and osteocytes (Plotkin et al. Bone, 23:S157, 1998).
Significantly, the anti-apoptotic effect of bisphosphonates in vitro
is achieved with doses 100-1000 lower than the doses at which
IO these same agents inhibit osteoclast activity; and additionally can
be demonstrated with bisphosphonates that do not block
osteoclast activity at all (compound IG9204). 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
Estrogens/progestogens (anti-remodeling and anti-
resorptive compounds) as a class have not to date been shown to
increase bone mass by more than 10%, but instead have b a a n
used to retard the effect of osteoporosis. Estrogens are currently
the most effective method of preventing osteoporosis i n
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
7
CA 02346456 2001-04-06
WO 00/20007 . PCT/US99/23355
having insubstantial sex-related activity can be administered to a
patient to retard the adverse effects of osteoporosis in a male o r
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~e~ns_
Little is known about the actions of phytoestrogens on
bone (Fitzpatrick, L.A., Mayo Clinic Proceedings, 74:601-607,
1999). Soy protein did not prevent increased bone turnover in
cynomolgus monkeys; they actually increased it. However, BMD
declined after two years in postmenopausal women taking only
calcium but did not change in those receiving ipriflavone.
Isoflavone significantly increased spinal BMD in postmenopausal
women after 6 months of 40 mg/day of soy protein
supplementation (containing 90 mg isoflavones) but not with
lower doses (56 mg/day) (Feinkel, E. Lancet, 352:762, 1998).
Parathyroid Hormone fPTHI _
Daily injections of parathyroid hormone (PTH), 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.
Whereas increased prevalence of apoptosis of osteoblasts a n d
osteocytes are key pathogenic mechanisms for osteoporosis
(Weinstein et al., J Clin Invest, 102:274-282, 1998; Weinstein et al,
8
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
Bone, 23:S461, 1998; Bellido et al, Bone, 23:5324, 1998), the
reverse, i. 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 increased bone mineral density,
osteoblast perimeter and bone formation rate that occur with
intermittent PTH administration in mice happen without a change
in osteoblast production. Instead, 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 h a s
been confirmed in vitro using primary bone cell cultures and
established cell lines.
The use of teriparatide (the 1-34 amino acid fragment
of human parathyroid growth hormone) to stimulate bone
formation has also been investigated; teriparatide administered as
daily injections has been reported to selectively increase the
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.
9
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/Z3355
calcium Preparations
Calcium preparations, while useful as a dietary
supplement for persons who are calcium deficient, have not b a a n
shown effective to increase bone mass. However, they may reduce
the rate of bone loss. U.S. Patent No. 5,618,549 (a calcium salt)
describes the use of calcium.
Fluor' .
The most thoroughly studied anabolic agent, sodium
fluoride, can increase vertebral bone mass by 10% a year for a t
least four years but there is controversy about the quality of the
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 i n
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. A recent study carried out with
raloxifene indicated that after three years of treatment, women o n
raloxifene had 30-50% fewer spinal fractures, and had 2-3%
increase in bone density in their hips and spine, but showed n o
fewer nonspinal fractures, a category that includes hip fractures
(Ettinger, B., JAMA, 282:637-645, 1999).
CA 02346456 2001-04-06
WO 00/20007 PG"T/US99/23355
U.S. Patent 4,970,237 discloses the use of
No.
clomiphene to increase mass in premenopausalwomen.
bone
Vitamin D deriva tives
There have been conflicting reports the value of
about
Vitamin D or its derivativeson bone loss and bone
anabolism.
Some studies on the hormonal D, calcitrioi,
metabolite
of vitamin
have reported an increase spinal bone density, others h a v
in but a .
found no effect.
The following patents describe the use of Vitamin D
derivatives to treat bone disease: U.S. Patent Nos. 4,973,584;
5,750,746; 5,593,833; 5,532,391; 5,414,098; 5,403,831; 5,260,290;
5,104,864; 5,001,118; 4,973,584; 4,619,920; and 4,588,716.
Other Compounds
The following patents disclose the use of other
compounds for the treatment of bone disease: U.S. Patent Nos.
5,753,649 and 5,593,988 (azepine derivative); 5,674,844
(morphogen); 5,663,195 (cyclooxygenase-2 inhibitor); 5,604,259
(ibuprofen or flurbiprofen); 5,354,773 (bafilomycine); 5,208,219
(activin); 5,164,368 (growth hormone releasing factor); an d
5,118,667, 4,870,054 and 4,710,382 (administration of a bone
growth factor and an inhibitor of bone resorption).
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
11
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
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 i n
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).
It is another object of the present invention to provide
a method to rebuild strong bones instead of preventing further
loss of bone.
It is a further object of the present invention to
provide a method to select compounds that increase bone mass in
a host at least 10% per year without a loss in bone strength or
quality.
It is a still further object of the present invention to
provide a method to increase bone strength by at least 20%.
12
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
SUMMARY OF THE INVENTION
In a first embodiment, a method for increasing bone
mass in a host at least 10% without a loss in bone strength o r
quality is provided that includes administering an effective
amount of a compound that (i) binds to the estrogen a or B
receptor (or the equivalent receptor in the host animal) with a n
association constant of at least 108 M-', and preferably, at least
10'° M-': (ii) (a) induces estrogenic gene transcriptional activity at
a level that is no greater than 10% that of 17B-estradiol, and
preferably no greater than 5, 1 or even O.l.% that of 1713-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
1 S (b) induces an increase in uterine weight of no more than 10% that
of 1713-estradiol (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 in vitro in osteoblastic cells with natural
estrogen receptors or cells transfected with estrogen receptors;
and (iv) has an anti-apoptotic effect on osteoblasts and osteocytes
at an in vivo 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 the estrogen receptor. In another aspect
of this first embodiment of this invention, the compound is not a n
estrogen compound, as that term is defined below. In yet another
aspect of this first embodiment, the compound is an estrogen
compound which is converted to a nonestrogen by attaching a
13
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
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.
In a second embodiment, a method for increasing b o n a
mass in a host at least 10% without a loss in bone strength o r
quality is provided that 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 108 M'', and preferably, at least 10'° M'': (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 w h a n
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 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; and (iv) has an anti-apoptotic effect o n
osteoblasts and osteocytes at an in vivo dosage of at least 0.1
ng/kg body weight or in vitro in osteoblastic cells with the natural
androgen receptor or transfected with the androgen receptor. I n
another aspect of the second embodiment, the compound is not a n
androgen. In yet another aspect of this second embodiment, the
compound is an androgen compound which is converted to a
14
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
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.
In other aspects of the first or second embodiment of
this invention, the compound has a pro-apoptotic effect o n
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 i n
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.
Therefore, in a third embodiment, a method for
selecting a compound that increases bone mass in a host at least
10% without a loss in bone strength or quality is provided that
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 10g M-1, and
preferably, at least 10' ° M-' : (ii) (a) induces estrogenic o r
androgenic gene transcriptional activity at a level that is no
greater than 10% that of 173-estradiol or testosterone, and
preferably no greater than 5, 1 or even 0.1% that of 17(3-estradiol
or testosterone, as appropriate, when administered in vivo at a
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
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 173-estradiol or 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 i n
vivo at a dosage of at least 0.1 ng/kg body weight or in vitro in
osteoblastic or osteocytic cells with the natural androgen o r
estrogen receptor or cells transfected with the androgen o r
estrogen receptor; and (iv) has an anti-apoptotic effect o n
osteoblasts and osteocytes at an in vivo dosage of at least 0.1
ng/kg body weight or in vitro in osteoblastic and osteocytic cells
with the natural androgen or estrogen receptor or cells transfected
with the androgen or estrogen receptor.
Estrogenic compounds like 17a-estradiol and synthetic
polycyclic phenols, such as estratriene-3-of 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 S minutes) phosphorylation of ERKs.
Estratriene-3-of 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 has
little effect on bone mass. As the dosage increases, both effects
increase. To optimize the use of this compound or others
16
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
exhibiting this type of activity, one can derivatize the compound
to preserve the estrogen-binding activity and decrease the
transcriptional activity as described in detail herein, including b y
attaching a substituent or moiety that inhibits cell penetration.
Compounds selected according to the criteria provided
herein can also be used for the augmentation of bone mass a n d / o r
fracture prevention in diseases characterized by low bone mass
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.
BRIEF DESCRIPTION OF THE DRAWINGS
The Figures provided herein illustrate embodiments of
the invention and are not intended to limit the scope of the
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 a
animals were sacrificed, vertebrae were isolated, fixed a n d
embedded, and then undecalcified in methacrylate. The
17
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
prevalence of osteoblast and osteocyte apoptosis was determined
by the TUNEL method with CuS04 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 Vii-
estradiol, 173-estradiol-BSA, 17a-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). * indicates p<0.05 versus etoposide alone, by analysis of
variance (ANOVA) (Student-Newman-Keuls method).
Figure 4 is a series of bar chart graphs of the
inhibition of etoposide-induced apoptosis of osteocytes (MLO-Y4)
by 173-estradiol, 173-estradiol-BSA, 17a-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~i-estradiol, 173-estradiol-BSA,
17a-estradiol, and estratriene-3-of (E-3-ol) on etoposide-induced
apoptosis of osteoblasts is abrogated by the estrogen receptor
antagonist, ICI182,780. Osteoblastic cells derived from murine
18
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
calvaria were pretreated for 1 hour with the pure receptor
antagonist ICI182,780 (10-' M) before the addition of the test
agents ( 10-g 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(3-estradiol, 17~i-estradiol-BSA,
17a-estradiol, and estratriene-3-of (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-' 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 7 is a series of bar chart graphs which
demonstrate that estrogen receptor a or ~i is required for the anti-
apoptotic effects of 17~i-estradiol, 17a-estradiol, and estratriene-
3-0l on the etoposide-induced apoptosis of osteoblasts. CMV
promoter alone and CMV promoter-driven cDNA for mERa or mEr~i
were stably transfected into HeLa cells. Subconfluent cultures
were treated for 1 hr with 10-g M 17 a-estradiol, 17 (3-estradiol, o r
estratriene-3-of followed by a 6 hr incubation with etoposide
(5x10-5 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 th at
17~i-estradiol, 17a-estradiol, 17~-estradiol-BSA or estratriene-3-of
activate the extracellular signal regulated kinases (ERKs). MLO-Y4
19
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
osteocytic cells were incubated for 25 minutes in serum-free
medium. Subsequently, 17~i-estradiol, 17a-estradiol, 17~-
estradiol-BSA or estratriene-3-of ( 10-g M) were added and cells
incubated for an additional 5, I5, or 30 min. Cell lysates were
prepared and proteins were separated by electrophoresis i n
polyacrylamide gels and transferred to PVDF membranes.
Western blotting was performed using a specific antibody .
recognizing phosphorylated ERKs I and 2, followed by reblotting
with an antibody recognizing total ERKs. Blots were developed b y
enhanced chemiluminescence.
Figure 9 is a Western blot which demonstrates that
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 (3-
estradiol, 17a-estradiol, 17~i-estradiol-BSA or estratriene-3-of ( 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~i-estradiol,
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
17a-estradiol, or 17~i-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 a estradiol,
estratriene-3-of does not transactivate an estrogen response
element through ERa. The human ERa was overexpressed in 2 9 3
cells lacking ERa 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 ERa
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,4aa, l0a(3)]-
1,2,3,4,4a,9,10, l0a-octahydro-7-hydroxy-2-methyl-2-
phenanthrenemethanol (PAM) and [2S-(2a,4aa, l0a~i)]-
1,2,3,4,4a,9,10, l0a-octahydro-7-hydroxy-2-methyl-2-
phenanthrenecarboxaldehyde {PACA).
Figure 13 illustrates the generalized core ring
structures with numbered carbons (Figure 13a) 4-ring structure,
(Figure 13b) 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 14A (anti-apoptotic effect of estrogen),
21
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
Figure 14B (anti-remodeling effect of estrogen) and Figure 14 C
(feminizing effect of estrogen).
Figure 15 compares the activity of the anti
resorptive (e.g., 17~i-estradiol) versus non-anti-resorptive agents
[e.g., estratriene-3-of 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. As shown in the right panel, 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). In addition, by decreasing the prevalence of
osteoblast apoptosis, the active compounds expand the pool of
mature osteoblasts at sites of new bone formation and allow th a s a
cells more time to make bone. Moreover, by upholding th a
osteocyte-canalicular network by preventing osteocyte apoptosis,
both classical antiresorptive agents like 173-estradiol and agents
that are not anti-resorptives are expected to have anti-fracture
efficacy over and above that resulting from their effects on b o n a
mass.
Figure 16A is a table of examples of R1 and R2
substitutions on the compound illustrated in Figure 1.
Figure 16B provides the molecular structures of a
and ~i estradiol.
Figure 17 provides the chemical structures of
estratrienes with anti-apoptotic properties.
22
CA 02346456 2001-04-06
WO 00/20007 PC'T/US99/23355
Figure 18 provides the chemical structures of
estradiol, phenol and diphenols with anti-apoptotic properties.
Figure 19 depicts the effect of 17 (3 estradiol on th a
transcriptional activity of a minimal ERE containing gene promoter
and the blockade of this effect by a peptide (aII) 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 aII peptide
with the GAL4-DNA binding domain inserted upstream of the
peptide sequence, an ERE/IL-6 promoter-driven luciferase
reporter plasmid and a ~3-galactosidase ((3-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 aII, by ANOVA (Student-Newman-Keuls method).
Figure 2 0 depicts the effect of 17 ~i estradiol on t h a
transcriptional activity of the IL-6 promoter and the blockade of
this effect by a peptide (aII) 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 aII peptide inhibited the transcriptional effects of estrogen on
the ERE-dependent transcription model. aII 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. * indicates p<0.05 versus cells transfected with the peptide
aII, by ANOVA (Student-Newman-Keuls method).
23
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
Figure 21 demonstrates the anti-apoptotic effect of
17[3 estradiol conjugated with BSA and the lack of inhibition of
this particular effect by the conformation sensitive peptide aII.
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 (3-
BSA were protected from apoptosis. Following co-transfection of
the GAL4-driven peptide, cells remained resistant to etoposide-
induced apoptosis indicating that the peptide did not inhibit the
protective, anti-apoptotic action of the ER (Figure 21). * indicates
p<0.05 versus cells transfected with the peptide aII, by ANOVA
(Student-Newman-Keuls method).
DETAILED DESCRIPTION OF THE INVENTION
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 n o
resultant transcriptional activity.
In an optimal embodiment using this invention, 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.
24
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
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.
Therefore, in a first embodiment, a method for
increasing bone mass in a host at least 10% without a loss in bone
quality or strength is provided that includes administering a n
effective amount of a compound that (i) binds to the estrogen a o r
13 receptor (or the equivalent receptor in the host animal) with a n
association constant of at least 108 M-', and preferably, at least
10'° M-'; (ii) (a) induces estrogenic gene transcriptional activity a t
a level that is no greater than 10% that of 1713-estradiol, and
preferably no greater than 5, 1 or even 0.1% that of 1713-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 i n
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 kinase (ERK)
when administered in vivo at a dosage of at least 0.1 ng/kg body
weight or at a concentration of 10-" to 10'' M in vitro in cells with
natural estrogen receptors or transfected with estrogen receptors.;
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
and (iv) has an anti-apoptotic effect on osteoblasts and osteocytes
at an in vivo dosage of at least 0.1 ng/kg body weight or at a
concentration of 10-" to 10'' M in vitro in cells with natural
estrogen receptors or transfected with estrogen receptors. I n
another aspect of this first embodiment of this invention, the
compound is not an estrogen compound, as that term is defined
herein. In another aspect of this first embodiment, 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.
In a second embodiment, a method for increasing b o n a
mass in a host at least 10% per year without a loss in bone
strength or quality is provided that 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 108 M-', and preferably, at least
10 '° M-1: (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 or
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
kinase (ERK) when administered in vivo at a dosage of at least 0.1
26
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
ng/kg body weight, or at a concentration of 10-" to 10'' M in vitro
in cells with the natural androgen receptor or transfected with the
androgen receptor; and (iv) has an anti-apoptotic effect o n
osteoblasts and osteocytes at an in vivo dosage of at least 0.1
ng/kg body weight or at a concentration of 10-" to 10-' M in vitro
in cells with the natural androgen receptor or transfected with the
androgen receptor. In another aspect of the second embodiment,
the compound is not an androgen. In another aspect of this
second embodiment, 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.
In other aspects of the first or second embodiment of
this invention, the compound also has a pro-apoptotic effect o n
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.
Therefore, in a third embodiment, a method for
selecting a compound that increases bone mass in a host at least
10% without a loss in bone strength or quality is provided that
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 108 M-', and
preferably, at least 10 '° M-': (ii) (a) induces estrogenic o r
androgenic gene transcriptional activity at a level that is no
greater than 10% that of testosterone or 17(3-estradiol, and
preferably no greater than 5, 1 or even 0.1% that of 17~i-estradiol
or testosterone, as appropriate, when administered in vivo at a
27
CA 02346456 2001-04-06
WO 00/20007 PCT/CTS99/23355
dosage of at least 0.1 ng/kg body weight or at a concentration of
10-11 to 10-' 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 (3-
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-" to 10-'
M in vitro in cells with the natural androgen or estrogen receptor
or transfected with the androgen or estrogen receptor; and (iv)
has an anti-apoptotic effect on osteoblasts at an in vivo dosage of
at least 0.1 ng/kg body weight or in vitro in cells with the natural
androgen or estrogen receptor or transfected with the androgen or
estrogen receptor.
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.
I. Definitions
An estrogen compound, as used herein, 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
28
CA 02346456 2001-04-06
WO 00/20007 PCT/IJS99/23355
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, as used herein, 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 i t
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.
As known, estrogens and androgens have chiral
carbons, and thus can exist in a number of stereochemical
configurations. Typically, for example, the 17~i hydroxy estrogens
have biological activity while the 17a hydroxy estrogens have
very little effect on sexual characteristics (and induce little
hormone-like gene transcriptional activation). For the purpose of
this specification, any stereochemical configuration, including
29
CA 02346456 2001-04-06
WO 00/20007 PCTNS99/23355
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 catalogue entitled "Steroids" from Steraloids Inc.,
Wilton, N.H., provides a list of over 3000 steroids, with numerous
estrogen and androgen derivatives. The catalog can be obtained
by contacting the company and is also currently available on the
Internet at http://www.steraloids.com. One can select and
purchase compounds from this library, which are all commercially
available and thus easy to obtain and evaluate, for use in this
invention. One can also use known estrogen and androgen
receptor binding compounds.
The term "bone mass" refers to the mass of bone
mineral and is typically determined by Dual-Energy X-Ray
Absorbtiometry (DEXA).
The term "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.
The term "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.
The term "bone anti-resorption agent" refers to a
compound that blocks bone resorption by suppressing remodeling
or the activity and/or lifespan of osteoclasts.
The term "osteopenia" refers to decreased bone m a s s
below a threshold which compromises structural integrity.
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
As used herein, the terms "metabolic bone disease",
"orthopedic bone disease" or "dental disease" are defined a s
conditions characterized by decreased bone mass and/or
structural deterioration of the skeleton and/or teeth.
As used herein, the term "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.
The term "host", as used herein, 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. Compounds Useful in the Invention
A. Estrogen compounds that bind to the estrogen a
or (3 receptor with an association constant of at 1 a a s t
10 8 M~1, and preferably, at least 101 ° M '', but w h i c h
exhibit little transcriptional activation
According to the present invention, one can easily
select estrogen compounds that significantly increase bone mass
by evaluating them according to the disclosed criteria.
1 . Binding to the estrogen a or ~i receptor
A compound should be selected that binds to th a
estrogen a or 13 receptor (or the equivalent receptor in the host
animal) with an association constant of at least 10g M-', and
preferably, at least 10'° M-' . This constant can be measured b y
any known technique, including receptor binding assays whereby
31
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
ligand binding affinities are determined by competitive
radiometric binding assays using 10 nM [3H] 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 a and 13 receptor subtypes have
significantly different primary sequences in their ligand binding
and transactivation domains. ERa and E1t13 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 ERa and ER13 structure
suggests that some compounds might bind ERa or E1t13, 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 a t
least 108 M-', and preferably, at least 10'° M-'.
2. Minimal effect on estrogen-induced
transcriptional activation
In this embodiment, an estrogen compound is selected
that has a minimal effect on estrogen-induced transcriptional
activation (or suppression). 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 estrogen-type compounds. Therefore, to provide a maximum
therapeutic efficacy on bone without causing unrelated and
32
CA 02346456 2001-04-06
WO 00/20007 PCT/IJS99/23355
undesired side estrogen-related effects, estrogen receptor ligands
with minimal transcriptional effects should be used.
To accomplish this separation of receptor binding a n d
transcriptional activity, a compound should be selected that
induces estrogenic gene transcriptional activity at a level that is
no greater than 10% that of 1713-estradiol, and preferably n o
greater than 5, 1 or even 0.1 % that of 1713-estradiol w h a n .
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).
One can determine whether a selected compound
induces estrogenic transcriptional activity at a level that is n o
greater than 10% that of 17(3-estradiol, and preferably no greater
than 5, 1 or even 0.1 % that of 17 (3-estradiol when administered i n
vivo at a dosage of at least 0.1 ng/kg body weight, b y
administering the selected compound to a host, and then
monitoring the level of induction or suppression of a surrogate
marker of estrogenic transcriptional activity. Nonlimiting
examples of 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.
In an alternative embodiment, the level of estrogen
induced transcriptional activity can be assessed in vitro. One can
determine whether a selected compound induces transcriptional
activity at a level that is no greater than 10% that of 1713-estradiol,
and preferably no greater than 5, 1 or even 0.1% that of 1713
33
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
estradiol in vitro using cells with natural estrogen receptors or
transfected with estrogen receptors, by monitoring the level of
induction or suppression of a surrogate marker. Nonlimiting
examples of genes induced or repressed by estrogen include, b a t
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.
Examples of cell lines that can be used include h a m a n
uterine HeLa cells, human embryonic kidney cells 293, murine
osteocytic MLO-Y4 cells and murine osteoblastic calvaria derived
cells.
One can assess the increase in uterine weight after
administration of the selected compound in vivo. 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
pro~ocols. 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/100g BW) as an index of the
estrogenic status of the animals. In women, similar assessment can
be performed by uterine ultrasound.
Examples of estrogen compounds that do not induce
significant estrogen-like transcriptional activity include, but are
not limited to estratriene-3-ol, 17a-estradiol, 17[3-estradiol
conjugated with BSA.
34
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
3 . Induction of the phosphorylation o f
extracellular signal regulated kinase (ERK)
The selected compound should induce t h a
phosphorylation of ERKs at a concentration of 10-" to 10-' 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.
4 . Anti-apoptotic effect on osteoblasts at an i n
v i v o dosage of at least 0.1 ng/kg body weight or a t
an in vitro concentration of 10'11 to 10-' M or less.
25
The anti-apoptotic effect on osteoblasts in vivo can b a
assessed by any known method, including by the method
described in Figure 2 and Example 3. The anti-apoptotic effect i n
vitro can be assessed by any known method including the
methods described in Figures 3-7 and 10, and Examples 2-6 and 9.
B. Nonestrogen compounds that bind to t h a
estrogen a or ~3 receptor with an association c o n s t a n t
CA 02346456 2001-04-06
WO 00/20007 PC"T/US99/23355
of at least 10 8 M m and preferably, at least 10' ° M -1,
but which exhibit little transcriptional activation
1. Nonestrogen compound which binds to t h a
estrogen a or (3 receptor
A nonestrogen compound, as used herein, refers to a
compound other than an estrogen, as that term is defined above,
which binds to the estrogen a or (3 receptor with an association
constant of at least 108 M'' and preferably, at least 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-a or Estrogen
Receptor-(3, Endocrinology, Volume 140, No. 2 (1999), one example
of which is illustrated below.
In an alternative embodiment, an estrogen o r
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 the
estrogen from entering the cell. In one example, the second
moiety is a protein such as bovine serum albumin, polyethelene
glycol or dextran or liposomes. In another embodiment, the
second moiety is not a protein or peptide, but for polar, steric, o r
other reasons, prevents cell penetration. Examples of these types
of moieties include carboxylate, ammonium, and sulfide. A
"linking moiety" as used herein, is any divalent group that links
two chemical residues, including but not limited to alkyl, alkenyl,
36
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
alkynyl, aryl, polyalkyleneoxy (for example, -[(CH2)"O-]"-), -
C,_6alkoxy-C,_,oalkyl-, -C,_6alkylthio-C1_,o alkyl-, -NR3-, and -
(CHOH)"CH20H, wherein n is independently 0, 1, 2, 3, 4, 5, or 6,
which can be attached at either end of the linking moiety to t h a
structures of interest by any suitable functional groups. In a n
alternative embodiment, the linking moiety can be a bifunctional
linker moiety of the formula X-(CH2)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.
C. Androgen compounds that bind to the a n d r o g a n
receptor with an association constant of a t
least 10 8 M '', and preferably, at least 10'° M -~,
but which exhibit little transcriptional
activation
According to the present invention, one can also easily
select androgenic compounds that significantly increase bone m a s s
by evaluating them according to the disclosed criteria.
1. Binding to the androgen receptor
A compound should be selected that binds to the
androgen receptor (or the equivalent receptor in the host animal)
with an association constant of at least 1 Og M-1, and preferably, a t
least 10'° M''. The androgen receptor binding association constant
is defined as the concentration of the ligand capable of saturating
SO% of the unoccupied receptors. This constant can be measured
by any known technique, including receptor binding assays
37
CA 02346456 2001-04-06
WO 00/20007 PCT/LTS99/23355
whereby ligand binding affinities are determined by competitive
radiometric binding assays using 10 nM [3H] 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 4C.
Bound receptor-ligand complex is absorbed using hydroxylapatite.
Androgen compounds include those describ ed in the 11th Edition
of "Steroids" from Steraloids Inc., Wilton,
N.H., which bind to the
androgen receptor with an association constant
of at least 108 M-',
and preferably, at least 10' M-' .
2. Minimal effect on androgen-induced
transcriptional activation
In this embodiment, an androgen compound is selected
that has a minimal effect on androgen-induced transcriptional
activation. The basis for this requirement, is that it has b a a n
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.
To accomplish this separation of receptor binding a n d
transcriptional activity, a compound should be selected that
induces androgenic transcriptional activity at a level that is n o
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
38
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
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).
One can determine whether a selected compound
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 w h a n
administered in vivo at a dosage of at least 0.1 ng/kg body weight,
by administering the selected compound to a host, and then
monitoring the level of induction or suppression of a surrogate
marker of androgenic transcriptional activity. Nonlimiting
examples of surrogate markers of androgenic transcriptional
activation, include, but are not limited to prostate specific antigen
(PSA).
In an alternative embodiment, the level of androgen-
induced transcriptional activity can be assessed in vitro in
osteoblastic or osteocytic cells with natural androgen receptors o r
traf calvaria cells, MLO-Y4 osteocytic cells and HeLa cells.
Alternatively, one can assess the increase in 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
39
CA 02346456 2001-04-06
WO 00/20007 PGT/US99/23355
not limited to, testosterone 17~i-hemisuccinate conjugated with
BSA.
3 . Induction of the phosphorylation o f
extracellular signal regulated kinase (ERK)
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-" to 10''
M in vitro in cells with natural androgenic receptors or transfected
with androgenic receptors.
The phosphorylation of ERK in a host can be assessed
in biopsies, for example from bone, using immunohistostaining
with specific antibodies against phosphorylated ERKs.
Alternatively, the phosphorylation of ERK is also easily assessed i n
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.
4 . Anti-apoptotic effect on osteoblasts a n d
osteocytes at an in vivo dosage of at least 0.1 n g / k g
body weight or at an in v i tro concentration of 10 -1 ~
to 10'' M or less.
The anti-apoptotic effect on osteoblasts and osteocytes
can be assessed in vivo any known method, including the
by
method described in Figure and Example 1; and in vitro any
2 by
known method, including method described in Figures and
the 3-7
10 and Examples 2-6 and 9.
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
D. Nonandrogen compounds that bind to t h a
androgen receptor with an association constant of a t
least 10 8 M -1, and preferably, at least 101 ° M -', b a t
which exhibit little transcriptional activation
A nonandrogenic compound, as used herein, 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 10g M-' and preferably, at least 10'° M-'..
There are a number of reported compounds which are not
androgens but which bind to the androgen receptor. Examples
include testosterone 17~i-hemisuccinate conjugated with BSA.
In an alternative embodiment, 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. In
one example, the second moiety is a protein such as bovine s a r a m
albumin. In another embodiment, . 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.
E. Other compounds that can be used to increase
bone mass.
Other nonlimiting examples of compounds that can b a
used in the present invention to increase bone mass include those
having a terminal phenyl ring and at least a second carbon ring.
In addition to these required structures, the compound may h av a
a number of R groups attached to any available site on the phenyl
41
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
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 th a s a
examples:
(a) The R, or RZ 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 R2 groups such as sodium, potassium a n d / o r
ammonium salts may be attached to the alpha or beta positions to
replace hydrogen on any available carbon in the structure. The Rl
or R2 groups may be organic or may include a mixture of organic
molecules and ions. Organic R, or R2 groups may include alkanes,
alkenes or alkynes containing up to six carbons in a linear o r
branched array. For example, additional R, or RZ group
substituents may include methyl, ethyl, propyl, butyl, pentyl,
hexyl, heptyl, dimethyl, isobutyl, isopentyl, tert-butyl, sec-butyl,
isobutyl, methylpentyl, neopentyl, isohexyl, hexenyl, hexadiene,
1,3-hexadiene-5-yne, vinyl, allyl, isopropenyl, ethynyl, ethylidine,
vinylidine, isopropylidene, methylene, sulfate, mercapto,
methylthio, ethylthio, propylthio, methylsulfinyl, methylsulfonyl,
thiohexanyl, thiobenzyl, thiophenol, thicyanato, sulfoethylamide,
thionitrosyl, thiophosphoryl, p-toluenesulfonate, amino, imino,
cyano, carbamoyl, acetamido, hydroxyamino, nitroso, nitro,
cyanato, selecyanato, arccosine, pyridinium, hydrazide,
semicarbazone, carboxymethylamide, oxime, hydrazone,
sulfurtrimethylammonium, semicarbazone, o -
carboxymethyloxime, aldehyde hemiacetate, methylether,
ethylether, propylether, butylether, benzylether, methylcarbonate,
42
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
carboxylate, acetate, chloroacetate, trimethylacetate,
cyclopentylpropionate, propionate, phenylpropionate, carboxylic
acid methylether, formate, benzoate, butyrate, caprylate,
cinnamate, decylate, heptylate, enanthate, glucosiduronate,
succinate, hemisuccinate, palmitate, nonanoate, stearate, tosylate,
valerate, valproate, decanoate, hexahydrobenzoate, laurate,
myristate, phthalate, hydroxyl, ethyleneketal, diethyleneketal,
formate, chloroformate, formyl, dichloroacetate, keto,
difluoroacetate, ethoxycarbonyl, trichloroformate,
hydroxymethylene, epoxy, peroxy, dimethyl ketal, acetonide,
cyclohexyl, benzyl, phenyl, diphenyl, benzylidene, and cyclopropyl
groups. R, or R2 groups may be attached to any of the constituent
rings to form a pyridine, pyrazine, pyrimidine, or v-triazine.
Additional R, or RZ group substituents may include any of the six-
member or five-member rings itemized in section (b) below.
( b ) Any compound having, in addition to t h a
terminal phenyl group, at least one heterocyclic carbon ring
(shown as R~ 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, a s -
triazine, v-triazine, 1,2,4-oxazine, 1,3,2-oxazine, 1,3,6-oxazine
(pentoxazole), 1,2,6-oxazine, 1,4-oxazine, o-isoxazine, p-isoxazine,
1,2,5-oxathiazine, 1,2,6-oxathiazine, 1,4,2-oxadiazine, 1,3,5,2-
oxadiazine, morpholine (tetrahydro-p-isoxazine), any of the six-
43
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
ringed structures listed above being a terminal group in the
compound. Additionally, 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, 1,2,3,5-
oxatriazole, 1,2,3-dioxazole, 1,2,4-dioxazole, 1,3,2-dioxazole, 1,3,4-
dioxazole, 1,2,5-oxathiazole, 1,3-oxathiazole, cyclopentane. These
compounds, in turn, may have associated R1 or R2 groups selected
from section (a) or section (b) above that are substituted on t h a
carbon ring at any of the available sites.
(c) Any compound, including those listed above, that
may form a cyclopentanophen(a)anthrene ring compound and
which, for example, may be selected from the group consisting of
1,3,5(10),6,8-estrapentaene, 1,3,5(10),6,8,1I-estrapentaene,
1,3,5(10),6,8,15-estrapentaene, 1,3,5(10),6-estratetraene,
I,3,5(10),7-estratetraene, 1,3,5(10),8-estratetraene, 1,3,5(10),16-
estratetraene, 1,3,5(10),15-estratetraene, I,3,5(10)-estratriene,
1,3,5( 10),15-estratriene.
( d ) Any compound including precursors o r
derivatives selected from raloxifen, tamoxifen, androgenic
compounds, and their salts, where an intact phenol ring is present
with a hydroxyl group present on carbons 1, 2, 3 and 4 of the
terminal phenol ring.
44
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
(e) Any compound in the form of a prodrug that
may be metabolized to form an active polycyclic-phenolic
compound having bone protective activity.
III. 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 o r
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 av a
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, o r
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 i n
sports such as racing. The compounds can also be used to increase
CA 02346456 2001-04-06
WO 00/20007 PCTIUS99/23355
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. Characteristics of the orthopedic
bone diseases are loss of bone mass, general bone fragility, joint
degeneration, non-union fractures, orthopedic and dental
problems caused by diabetes, periimplantitis, poor responses to
bone grafts/implants/bone substitute materials, periodontal
diseases, and skeletal aging and its consequences.
I V . Method for Screening for Compounds that I n c r a a s a
Bone Mass
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 the
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
46
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
apoptosis of osteocytes. Generally, the compound may b a
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.
In another embodiment, a method for selecting a
compound that increases bone mass at least 10% in a host without
a loss in bone strength or quality is provided that includes
evaluating whether the compound (i) binds to the estrogen o r
androgen receptor (or the equivalent receptor in the host animal)
with an association constant of at least 1 Og M'', and preferably, a t
least 10'° M'': (ii) (a) induces estrogenic or androgenic gene
transcriptional activity at a level that is no greater than 10% that
of testosterone or 173-estradiol, and preferably no greater than 5,
1 or even 0.1 % that of 17 ~i-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'" to 10'' 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 i n
females, as appropriate, of no more than 10% that which is
induced by 17(3-estradiol or testosterone (or the equivalent
compound in a host animal); (iii) induces the phosphorylation of
extracellular signal regulated kinase (ERK) when administered i n
vivo at a dosage of at least 0.1 ng/kg body weight or in vitro in
cells with the natural androgen or estrogen receptor or transfected
with the androgen or estrogen receptor; and (iv) has an anti-
apoptotic effect on osteoblasts at an in vivo dosage of at least 0.1
47
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
ng/kg body weight or in vitro in cells with the natural androgen
or estrogen receptor or transfected with the androgen or estrogen
receptor.
In another embodiment, 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.
To accomplish this, cells are transfected with the
estrogen or androgen receptor with or without a peptide that
recognizes the conformation of the protein required for
transcriptional activation, but not anti-apoptosis. Using this
method, 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 ERa or ER(3 whereby th a
purified receptor protein is immobilized on streptavidin-coated
48
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
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). Following incubation with various ligands, the peptide
is added and after 30 min bound peptide is detected using a n
anti-M 13 antibody coupled to horseradish peroxidase. Compounds
that bind to the receptor and induce conformational changes
recognized by the peptide (i.e. the peptide binds to the receptor)
will be discarded. The remaining compounds are then screened
for anti-apoptotic potency.
V . Combination Therapy
In one aspect of the invention, 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 i n
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.
49
CA 02346456 2001-04-06
WO 00/20007 PGT/US99/23355
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 the
compounds of the present invention. As used herein, the term
antioxidant refers to a substance that prevents the oxidation of a n
oxidizable compound under physiological conditions. In one
embodiment, 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. As nonlimiting examples, 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. The term antioxidant includes, but is not limited
to, the following classes of compounds:
A) Dithiocarbamates: 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,
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
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. ~ It appears that the -SC(S)-
moiety is essential for therapeutic activity, and that A and B c an
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.
B) N-Acetyl Cysteine and its Derivatives
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) is the 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 a
L and D enantiomers. The term "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.
51
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
In one embodiment, 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). 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, Vo1.66, pp. 56-61)
N-acetylcysteine exhibits low toxicity in vivo, and is
significantly less toxic than deprenyl (for example, the LDso 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.
52
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
C) Scavengers ding but not limited to
of Peroxides,
inclu
catalase and pyruvate.
D) T hiols includingdithiothreitoland 2-mercaptoethanol.
E) Antioxidants which are inhibitors of lipid
peroxidation,including not limited to TroloxTM, BI3A, BI3'f,
but
aminosteroid antioxidants,tocopherol and its analogs, and
lazaroids.
F) Dietary 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 a t
not limited to ubiquinols and thiol antioxidants, such as, and
including glutathione, Se, and lipoic acid.
I ) Synthetic Phenolic Antioxidants: inducers of Phase I
and II drug-metabolizing enzymes.
V I . Pharmaceutical Compositions
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,
53
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
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 a
enclosed in gelatin capsules or compressed into tablets. For the
purpose of oral therapeutic administration, the compound can b a
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, o r
orange flavoring. When the dosage unit form is a capsule, it can
contain, in addition to material of the above type, a liquid carrier
such as a fatty oil. In addition, dosage unit forms can contain
various other materials which modify the physical form of the
dosage unit, for example, coatings of sugar, shellac, or other
enteric agents.
54
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
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 (3-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. 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 (EI)TA); buffers such a s
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. If administered
intravenously, preferred carriers are physiological saline o r
phosphate buffered saline (PBS).
CA 02346456 2001-04-06
WO 00/20007 . PCT/US99/23355
In a preferred embodiment, 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. Biodegradable, biocompatible polymers can be used,
such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Methods for ..
preparation of such formulations will be apparent to those skilled
in the art.
Liposomal suspensions (including liposomes targeted
with monoclonal antibodies to surface antigens of specific cells)
are also pharmaceutically acceptable carriers. These may b a
prepared according to methods known to those skilled in the art,
for example, as described in U.S. Patent No. 4,522,811 (which is
incorporated herein by reference in its entirety}. For example,
liposome formulations may be prepared by dissolving appropriate
lipids) (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. An aqueous solution of the active compound or its
monophosphate, diphosphate, and/or triphosphate derivatives) is
then introduced into the container. The container is then swirled
by hand to free lipid material from the sides of the container and
to disperse lipid aggregates, thereby forming the liposomal
suspension.
The dose and dosage regimen will depend upon the
nature of the metabolic bone disease, the characteristics of the
56
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
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 m g / k g
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.
For parenteral administration, 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. Such vehicles 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 10
mg/ml.
The 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 i n
the art. It is to be noted that dosage values will also vary with the
57
CA 02346456 2001-04-06
WO 00/20007 PCT1US99/23355
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. It is to be further understood that for any particular
patient, specific dosage regimens should be adjusted over time
according to the individual need and the professional judgment of
the person administering or supervising the administration of the
compositions, and that the concentration ranges set forth herein
are exemplary only and are not intended to limit the scope o r
practice of the claimed composition.
V I I . Illustrative Examples
The following examples are illustrations of the
embodiments of the invention as described above, but are not
intended to limit its scope.
As one example, 17~-estradiol, the synthetic steroid
estratriene-3-ol, which is a potent neuroprotective compound, and
17a-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-relaxed activity, and i n
particular, a-estrogens such as 17a-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
58
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
for the treatment of osteoporosis, but has not approved any drugs
to date as bone anabolic agents.
17~i-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.
It is noteworthy that (a) the anti-apoptotic effect of
17~i-estradiol on both osteoblasts and osteocytes are reproduced
with a membrane impermeable 17~i-estradiol - BSA conjugate; (b)
the anti-apoptotic effects of these compounds are diminished b y
ICI 182780, a pure estrogen receptor antagonist; and (c) that the
anti-apoptotic effects of all these compounds cannot be shown i n
HeLa cells unless these cells are stably transfected with either the
estrogen receptor a or the estrogen receptor ~3.
The following examples are given for the purpose of
illustrating various embodiments of the invention and are n o t
meant to limit the present invention in any fashion.
59
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
EX MPLE 1
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.
However, increased remodeling alone cannot explain th a
progressive bone loss that lasts long after the rate of bone
remodeling has slowed. Indeed, in addition to changes in the
number of osteoblast and osteoclast cells during/following
estrogen deficiency, a qualitative abnormality also occurs;
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. The deeper
erosion is explained by loss of estrogen's effect to promote
apoptosis of osteoclasts (Hughes et al, Nature Med. 1996; 2:1132-
1136; Kameda et al, J Exp Med. 1997; 186:489-495; Raisz, Nature
Med. 1996; 2:1077-1078). 17 (3-estradiol increased the apoptosis
of osteoclasts from approximately 0.5% to as much as 2.7%. This
change could prolong the lifespan of osteoclasts and increase their
numbers two- to three-fold, thus accounting for the perforation of
trabeculae and grinding away of endocortical margins.
To determine whether the role of estrogen deficiency
affects osteoblast and osteocyte apoptosis, the prevalence of these
cells in murine vertebrae removed 28 days after ovariectomy was
determined. In these experiments, four month old Swiss Webster
mice were ovariectomized and 28 days later, the animals were
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
sacrificed an d the vertebrae were isolated, and embedded
fixed
undecalcified in methacrylate. As shown in Figure 2, the
prevalence of determined b y
osteoblast
and osteocyte
apoptosis,
TUNEL with CuS04 enhancement, increased ten-and four-fold,
respectively.These results indicate that the
accelerated loss of
bone that occurs not only to a n
after estrogen
deficiency
is due
increase in osteoclast number and lifespan, but also to a .
premature reduction in the lifespan (work hours) of the
osteoblasts. The increase in osteocyte apoptosis
could further
weaken the skeleton by impairment of the
osteocyte-canalicular
mechanosensory network.
EXAMPLE 2
Consistent with the in vivo data described under
Example 1, 17 ~i-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 (3-estradiol conjugated with bovine serum albumin, a
membrane impermeable compound. The same effect could also b a
shown with 17 a-estradiol, a compound heretofore thought to b a
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 the
addition of the pro-apoptotic agent, etoposide.
61
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
EXAMPLE 3
In agreement with the in vivo results indicating that
estrogen loss increases both osteoblast and osteocyte apoptosis,
17 (3-estradiol, 17 ~i-estradiol conjugated with BSA, 17a-estradiol,
and estratriene-3-of dose-dependently inhibited also the
apoptosis of an established osteocytic cell line (Figure 4). In this
experiment, MLO-Y4 cells were pretreated with the indicated
concentrations of the various compounds for 1 h before th a
addition of the pro-apoptotic agent, etoposide. Apoptosis was
determined after 6 h by trypan blue uptake as described in Figure
3.
EXAMPLE 4
As shown in Figure 5, the anti-apoptotic effect of 10-8
M 17~i-estradiol, 17[3-estradiol-BSA, 17a-estradiol, or estratriene-
3-0l (E-3-ol) on osteoblastic cells was abrogated when the cells
were pretreated for 1 h with the pure receptor antagonist
ICI182,780 ( 10-' M) before the addition of the estrogenic
compounds.
EXAMPLE 5
As in the case of the antiapoptotic effect of 17~3-
estradiol, 17~i-estradiol-BSA, 17a-estradiol, or estratriene-3-of (E-
3-0l) on osteoblastic cells, their antiapoptotic effect on osteocytes
was abrogated when the cells were pretreated for 1 h with the
62
CA 02346456 2001-04-06
WO OOI20007 ~ PCT/US99/23355
pure receptor antagonist ICI182,780 ( 10-' M). Collectively, th a
results of examples 4 and 5 strongly suggest that the anti-
apoptotic effects of these compounds on osteoblasts and osteocytes
are mediated via the estrogen receptor (ER).
S
EXAMPLE 6
Definitive demonstration of the requirement of the
estrogen receptor for the anti-apoptotic effects of 17~i-estradiol
and the related compounds tested herein was provided by the
results of the experiment shown in Figure 7. In this experiment,
instead of calvaria cells, human HeLa cells which contain
undetectable, if any, estrogen receptor were used. HeLa cells were
stably transfected with either a CMV promoter-driven cDNA for
the murine estrogen receptor-alpha (mERa) or a CMV promoter-
driven cDNA for the murine estrogen receptor-beta (mER~i).
Subconfluent cultures of stable transfectants were treated for 1 h
with 17[3-estradiol, or 17a-estradiol, estratriene-3-of (10-8 M),
followed by a 6 hour incubation with etoposide (5x10-5 M). Cells
were trypsinized, pelleted and trypan blue positive cells were
enumerated. As shown in Figure 7, none of the three compounds
had any effect on the apoptosis of the wild type HeLa cells, b a t
they potently inhibited etoposide-induced apoptosis in HeLa cells
transfected with the estrogen receptor a or estrogen receptor Vii.
EXAMPLE 7
63
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
The mechanism of the anti-apoptotic effect of the
estrogenic compounds described herein was established b y
demonstrating that 17a-estradiol, 17~i-estradiol, 17~i-estradiol-
BSA or estratriene-3-ol, at 10-8 M concentrations, activated
extracellular signal regulated kinases (ERKs). In this experiment,
MLO-Y4 osteocytic cells were incubated for 25 minutes in serum-
free medium. Subsequently, 17 a-estradial, 17 (3-estradiol, 17 ~3-
estradiol-BSA or estratriene-3-of ( 10-8 M) were added and cells
incubated for an additional 5, 15, or 30 minutes. CeII 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 by reblotting with an antibody recognizing total
extracellular signal regulated kinases. Blots were developed b y
enhanced chemiluminescence. As shown in Figure 8, all these
compounds specifically increased the phosphorylated fraction of
ERKl/2 without affecting the total amount of ERK1/2. This effect
is too rapid to be accounted for by the classical mechanism of
estrogen action. Instead, it is consistent with a non-genomic
action mediated via membrane-associated estrogen receptors, as
suggested by the experiments presented in Examples 4, 5 and 6.
EXAMPLE 8
The ability of 17 a-estradiol, 17 ~3-estradiol, 17 [3-
estradiol-BSA or estratriene-3-of to activate ERKs was abrogated
in the presence of the specific inhibitor of ERK kinase, PD98059.
64
CA 02346456 2001-04-06
WO 00/2000? PCT/US99/23355
In this experiment, MLO-Y4 osteocytic cells were incubated for 2 S
minutes in serum-free medium in the presence or absence of 5 0
p.M PD98059. Subsequently, 17 a-estradiol, 17 ~i-estradiol, 17 ~3
estradiol-BSA or estratriene-3-of ( 10-8 M) were added and cells
incubated for another 5 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.
EXAMPLE 9
That indeed the anti-apoptotic effect of all the
compounds tested herein was mediated via activation of ERKs w a s
established by the results of the experiments shown in Figure 10.
In this experiment, MLO-Y4 osteocytic cells were pretreated for 1
hour with the specific inhibitor of ERKs activation, PD98059,
before the addition of 108 M 17 a-estradiol, 17 ~i-estradiol, or 17 (3-
estradiol-BSA. Apoptosis was induced by incubation with the pro-
apoptotic agent dexamethasone for 6 hours and quantified a s
described in Figure 3. PD98059 prevented the anti-apoptotic
effect of all three compounds tested in this experiment.
In conclusion, the results of the examples provided
above demonstrate that loss of estrogen irc vivo leads to several-
fold increase in the prevalence of apoptosis of osteoblasts and
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
osteocytes. Consistent with the in vivo findings, 17a-estradiol, as
well as 17(3-estradiol, 17(3-estradiol-BSA and estratriene-3-of
inhibit the apoptosis of osteoblastic cells derived from murine
calvaria or osteocytes, represented herein by the cell line MLO-Y4.
The anti-apoptotic effect of all these compounds requires the
presence of either estrogen receptor a or estrogen receptor ~i and
is mediated via the ability of these compounds to activate specific
MAP kinases, namely the extracellular signal regulated kinases
(ERKs).
EXAMPLE 10
Similar to the results with estrogenic compounds,
androgenic compounds also inhibited apoptosis of osteoblastic cells
derived from murine calvaria induced by etoposide (Table 3). I n
these experiments, cells were pretreated with the indicated
concentrations of the various compounds for 1 hour, in the
absence or presence of the androgen receptor antagonist
flutamide, before the addition of the proapoptotic agent etoposide.
Apoptosis was determined after 6 hours by trypan blue uptake a s
described in Figure 3. Notably, as in the case of estrogenic
compounds, all these effects were apparently mediated by the
androgen receptor, as evidenced by the inhibition of the anti-
apoptotic effects of the androgenic compounds by a specific
androgen receptor antagonist. Moreover, and as in the case of
estrogens, the androgen receptor-mediated protection of
etoposide-induced apoptosis was seen with a membrane
impermeable androgen (testosterone-17(3-hemisuccinate
66
CA 02346456 2001-04-06
WO 00/20007 PCTNS99/23355
conjugated with BSA), strongly suggesting the existence of a
membrane-associated androgen receptor, analogous to the
membrane-associated estrogen receptor.
able 3
Inhibition of etoposide-induced
osteoblast apoptosis
by androgens and
progestins
ompound Lowest Suppression by 10'g M
Effective Flutamide
Concentration
Testosterone 10'9 M yes
Testosterone 17(3- 10-g M y a s
Hemisuccinate: BSA
5-a- 10-9 M y a s
dihydrotestosterone
5-(3- 10-' M yes
dihydrotestosterone
Dehydroisoandroste 10-g M no*
rone-3-sulfate
(DHES)
4-androstene-3,17- 10-8 M yes
dione
5-androstene-3(3- 10-a M yes
17 -diol
RU1881 10-8 M yes
* Flutamide did block the anti-apoptotic effect of l~ti~ at higher
( 10-' M) concentration.
EXAMPLE 11
67
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
That the anti-apoptotic effects of estrogenic
compounds is dissociated from their transcriptional activity w a s
established by demonstrating that even though estratriene-3-of
was as potent as 17 ~i estradiol in inhibiting apoptosis, unlike 17 [3
estradiol, it did not transactivate an estrogen response element
through the estrogen receptor a. In this experiment, hERa was
overexpressed in 293 cells (which lack constitutive ERa) 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 ~i-galactosidase activity to
control for differences in transfection efficiency.
EXAMPLE 12
Herein, a general experimental protocol for studies
aiming to evaluate compounds with anti-apoptotic efficacy, b a t
decreased transcriptional activity (e.g., estratriene-3-ol) on
osteoblasts and osteocytes in animal models is provided.
According to this design, 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)), or animal models of glucocorticoid
excess (e.g., Weinstein et al. J Clin Invest, 102:274-282, 1998) are
administered estratriene-3-of or other test compound to
determine whether they can suppress osteoblast and osteocyte
apoptosis and whether changes in apoptosis would be associated
68
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
with changes in BMD, bone formation rate, or cancellous bone
volume.
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 the
experiment to establish that peak adult bone mass has been
attained. A subset of mice are then ovariectomized. Intact a n d
ovariectomized mice are treated with vehicle, or 20, 200 or 2 0 0 0
ng/g body weight estratriene-3-of or another test compound.
Ovariectomized mice are also treated with 20 ng/g body weight
17 ~i-estradiol for comparison purposes.
Stock solutions of the test agents (10,000 p,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. For each animal injection, 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.
TABLE 1
Treatment Injection
69
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
(steroid + sesame oil)
vehicle 100 pl 95% ethanol + 1900 p.l
20 ng/g estratriene-3-of 100 p.l 100 ~g/ml stock + 1900 p,l
200 ng/g estratriene-3-of 100 p.l 1000 ~g/ml stock + 1900 p.l
2000 ng/g estratriene-3-of 100 p,l 10,000 p.g/ml stock + 1900 p.l
20 ng/g 173-estradiol 50 p.l 100 p,g/ml stock + 950 p.l
During the 28 day experiment, BMD is determined i n
live animals at day 0, 14 and 28. Following animal sacrifice at the
end of the experiment, the vertebral bones Ll-L4 are collected for
fixation and embedded undecalcified in methylmethacrylate
plastic for the determination of the prevalence of osteoblast and
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 th a s a
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.
As an example, the results of an experiment w h a r a b y
2000 ng/g body weight of estratriene-3-of was administered for
28 days to estrogen-replete (intact) or estrogen-deficient
(ovariectomized) mice are shown in Table 2.
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
T B~ LE 2
Increased BMD by estratriene-3-of administration
intact-vehicle:
global global hindquart
1 hindquartpine 2 spine
1 2
Ø0552 0.0585 0.0599 0.0533 0.0581 0.0595
0.0535 0.0586 0.0575 0.0546 0.0571 0.0599
0.0516 0.0557 0.0559 0.0503 0.0560 0.0544
0.0516 0.0513 0.0569 0.0492 0.0499 0.0527 ,
0.0552 0.0553 0.0589 0.0492 0.0521 0.0531
0.0475 0.0494 0.0525 0.0480 0.0450 0.0539
0.0535 0.0524 0.0574 0.0524 0.0592 0.0553
0.0526 0.0544 0.0570 0.0510 0.0539 0.0555 (mean)
0.0027 0.0036 0.0024 0.0025 0.0052 0.0030 (std)
0.0552 0.0586 0.0599 0.0546 0.0592 0.0599 (max)
0.0475 0.0494 0.0525 0.0480 0.0450 0.0527 (min)
intact-2000 3-0l:
ng/g
_ lhindquar>apine global hindquart
global 1 2 spine
2
_ 0.0509 0.0511 0.0550 0.0585 0.0602
0.0486
0.0511 0.0565 0.0560 0.0543 0.0603 0.0604
0.0543 0.0605 0.0593 0.0541 0.0619 0.0601
0.0537 0.0577 0.0584 0.0568 0.0629 0.0613
0.0533 0.0546 0.0571 0.0568 0.0640 0.0620
0.0521 0.0544 0.0555 0.0537 0.0554 0.0588
0.0560 0.0587 0.0623 0.0574 0.0605 0.0632
0.0527 0.0562 0.0571 0.0554 0.0605 0.0609 (mean)
0.0024 0.0032 0.0035 0.0015 0.0029 0.0014 (std)
0.0560 0.0605 0.0623 0.0574 0.0640 0.0632 (max)
0.0486 0.0509 0.0511 0.0537 0.0554 0.0588 (min)
__vehicle vs 2000 ng/g:
t-test 0.0036 0.0077 0.0037
Ovx-vehicle:
global lhindquarkpine global hindquart spine 2
1 2
_ 0.0539 0.0550 0.0493 0.0507 0.0548
0.0525
0.0479 0.0484 0.0538 0.0497 0.0569 0.0545
0.0510 0.0543 0.0565 0.0483 0.0509 0.0545
0.0538 0.0548 0.0583 0.0483 0.0515 0.0536
71
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
0.0567 0.0632 0.0620 0.0538 0.0584 0.0588
0.0533 0.0533 0.0572 0.0504 0.0507 0.0559
0.0543 0.0592 0.0583 0.0491 0.0526 0.0545
0.0490 0.0518 0.0551 0.0475 0.0487 0.0534
0.0523 0.0550 0.0570 0.0496 0.0528 0.0550 (mean)
0.0029 0.0049 0.0026 0.0019 0.0035 0.0017 (std)
0.0567 0.0632 0.0620 0.0538 0.0584 0.0588 (max)
Ovx-2000 ng/g 3-0l:
global global hindquart spine
lhindquar>,pine 2 Z
1
0.0505 0.0527 0.0547 0.0565 0.0602 0.0608
0.0542 0.0588 0.0581 0.0557 0.0632 0.0585
0.0496 0.0504 0.0542 0.0548 0.0599 0.0584
0.0540 0.0596 0.0586 0.0545 0.0624 0.0598
0.0526 0.0547 0.0580 0.0564 0.0598 0.0610
0.0569 0.0604 0.0628 0.0568 0.0647 0.0625
0.0565 0.0591 0.0603 0.0550 0.0630 0.0578
0.0528 0.0582 0.0568 0.0539 0.0599 0.0605
0.0534 0.0573 0.0579 0.0555 0.0618 0.0599 (mean)
0.0026 0.0036 0.0028 0.0011 0.0020 0.0016 (std)
0.0569 0.0604 0.0628 0.0568 0.0647 0.0625 (max)
0.0496 0.0504 0.0542 0.0539 0.0598 0.0578 (min)
ovx vs 2000 n /
t-test 0.0000 0.0001 0.0001
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. Global =
BMD of the entire skeleton minus the head and tail; hindquarters =
the mean BMD of both hindlimbs; spine = the BMD of cervical,
thoracic and lumbar spine.
72
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
EXAl~!~ LP E 13
Herein, a general experimental protocol evaluating the
anti-fracture efficacy of compounds like estratriene-3-of is
provided. According to this design, 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)), or animal models
of glucocorticoid excess (e.g., Weinstein et al. J Clin Invest,
102:274-282, 1998) are administered estratriene-3-of to
determine whether they can increase bone strength.
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 been
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-of or another ANGEL compound.
Ovariectomized mice are also treated with 20 ng/g body weight
17 ~3-estradiol for comparison purposes. Ultimate load bearing
properties of the fifth lumbar murine vertebrae (L5) i s
determined. This is done using a servohydraulic axial-torsional
material testing machine (Model MTS 810 Bionx; MTS Systems
Corp., Eden Prairie, MN) and a Lebow load cell (Eaton Products,
Troy, MI). Data are recorded and analyzed using the LabVIEW
software package and an acquisition/signal conditioning board
(Model NB-MIO-16, National Instruments Corporation, Austin, TX).
73
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
The L5 specimens that is used for ultimate load bearing i s
cleaned of surrounding soft tissue and the length and diameter
recorded with a digital caliper at a resolution of 0.01 m m
(Mitutoyo Model #500-196, Ace Tools, Ft. Smith, AR). The
vertebrae are wrapped in saline-soaked gauge throughout
preparation and testing and stored overnight at 4°C before testing.
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.
As an example, the results of an experiment whereby
2000 ng/g body weight of estratriene-3-of was administered for
28 days to estrogen-replete (intact) or estrogen-deficient
(ovariectomized) mice (from the same animals shown in Example
12) is shown in Table 4.
Ta le
4
Changes in Compression Strength (VCS*),
Vertebral
Induced by In v i v on of E-3-of
o Administrati
Demonstration CVS than BMD
of Greater
Increase
in
{n - 7 per group}
Vertebral Global BMD (g/cm2)
Compression
(Newtons)
Intact-
v a h i c 66.78 17.47 0.0508 0.0026
l a
50.3 7.58
Ovx- 96.26 15.92 (p<0.006)0.0486 0.0011
vehicle 85.57 10.17
0.0554 0.0015 (p<0.002)
Intact-E- (P<0.00001)
3-0l
0.0555 0.0011
74
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
Ovx-E-3- ~ ~ (p<0.00001)
of
*Each value represents the mean from seven animals.
The BMD values shown for comparison here are from the
experiment described in Example 12.
iEXAMPLE 14
To determine whether the anti-apoptotic effects of
estrogenic compounds are mechanistically dissociable from their
transcriptional effects, specific conformational changes of the
receptor protein leading to prevention of apoptosis versus
transcriptional activity were sought. The rationale behind these
studies was based on recent evidence that the transcriptional
activity of the ER is greatly dependent on ligand-induced
conformational changes of the receptor protein. Indeed, using
phage display libraries, McDonnell and co-workers have recently
screened for and isolated four classes of small (11 amino acids)
peptides that recognize distinct conformational changes of the
estrogen receptor, and can either selectively block transcription
from specific ligands (e.g., estradiol but not tamoxifen and vice
versa) or selectively block ERa but not ER~i -mediated
transcription, and vice versa, when tested on a consensus )~~ERE
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
(Norris et al. Science 285:744-746, 1999). The first class contains
the LX~~.L motif and can interact with both estradiol-activated
ERa and ER~3. The second class displays specific interaction w i th
estradiol- and tamoxifen-activated ERa, whereas the third class
can interact specifically with tamoxifen-activated ER~3. Yet a fourth
class with a SREWFXXXL conserved motif was found to complex t o
tamoxifen-activated ERa and ER~i. Indeed, when fusion proteins
made with these peptides and the Gal4-DNA binding domain and
were co-expressed with ER in HeLa cells they functioned a s
ligand-receptor complex-specific antagonists, demonstrating that
ligand activation triggers transcriptional activity by conferring
specific conformational changes on the receptor protein (Paige LA,
Christensen DJ, Gron H, Norris JD, Gottlin EB, Padilla KM, Change C-
Y, Ballas LM, Hamilton PT, McDonnell DP, Fowlkes DM. Estrogen
receptor (ER) modulators each induce distinct conformational
changes in ERa and ERj3. Proc. Natl. Acad. Sci 96:3999-4004, 1999).
Based on the findings that estrogenic compounds like
the conjugated 17-~i estradiol with BSA have, at least as potent
anti-apoptotic effects as estrogen while have significantly
decreased transcriptional activity, the hypothesis that the non
genomic anti-apoptotic effects of estrogen can be initiated b y
distinct ligand-dependent conformational changes of the ER, as
compared to the conformational changes required for the
transcriptional effects of the ER was tested. It was found that
indeed there is dissociation of conformational changes. Based on
this, one can explain the mechanistic basis of the apparent
dissociation of the two sets of actions. This knowledge forms th a
basis for the design of the screening strategies described herein for
76
CA 02346456 2001-04-06
WO 00/20007 PCT/US99/23355
ligands which display non-transcriptional effects, but lack the
ability to initiate transcriptional activation.
One skilled in the art will readily appreciate that the
present invention is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those
objects, ends and advantages inherent herein. The present
examples, along with the methods, procedures, treatments,
molecules, and specific compounds described herein are presently
representative of preferred embodiments, are exemplary, and are
not intended as limitations on the scope of the invention. Changes
therein and other uses will occur to those skilled in the art which
are encompassed within the spirit of the invention as defined b y
the scope of the claims.
77
