Note: Descriptions are shown in the official language in which they were submitted.
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THERAPEUTIC COMBINATIONS OI~ (SELECTIVE) ESTROGEN RECEPTOR MODULATORS (SERM)
AND GROWTH
HORMONE SECRETAGOGUES (GHS) FOR TREATING MUSCULOSKELETAL FRAILTY
13ACKGROUND OF THE INVENTION
This invention r~afates to a pharmaceutical combination of a selective
estrogen receptor modulator (SERfVI} and a growth hormone secretagogue (GHS)
that stimulates bone forrnation, increases bone mass, decreases serum lipid
levels
and increases muscle nnass. The invention also relates to kits containing such
combinations and the use of such combinations to treat musculoskeletal
frailty,
including osteoporosis, osteoporotic fracture, low bane mass, frailty, low
muscle
mass and the like in mammals, including humans. In particular, this invention
relates to a combination of (-)-cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy)-
phenyl}-
5,6,7,8-tetrahydronaphthalene-2-of or a pharmaceutically acceptable salt
thereof
and 2-amino-N-(1 (R)-(2,4-difluoro-~benzyloxymethyl)-2-oxo-2-(3-oxo-3a(R)-
pyridin-
2-ylmethyl)-2-(2,2,2-trifluoro-ethyl)-2,3,3a,4,6,7-hexahydro-pyrazolo[4,3-
c~pyridin-
5-yl)-ethyl)-2-methyl-propionamide or a pharmaceutically acceptable salt
thereof,
kits containing such a combination and the use of such a combination to treat
musculoskeletal frailty, including osteoporosis, osteoporotic fracture, low
bone
mass, frailty, low muscle mass and the like in mammals, including humans.
Osteoporosis is a systemic: skeletal disease, characterized by low bone
mass and deterioration of bone tissue, with a consequent increase in bone
fragility
and susceptibility to fracture. In the U.S., the condition affects more than
25
million people and causes more than 1.3 million fractures each year, including
500,000 spine, 250,000 hip and 240,000 wrist fractures annually. Hip fractures
are the most serious, with 5-20% of patients dying within one year, and over
50%
of survivors being incapacitated.
The elderly are at greatest risk of osteoporosis, and the problem is
therefore predicted to increase significantly with the aging of the
population.
Worldwide fracture incidence is forecast to increase three-fold over the next
60
years, and one study estimates that there will be 4.5 million hip fractures
worldwide in 2050.
Although both men and women are susceptible to musculoskeletal frailty,
including osteoporosis, women are at greater risk of osteoporosis than men.
Women experience a sharp acceleration of bone loss immediately following
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menopause. Other factors that increase bone loss leading to osteoporosis
include
smoking, alcohol abuse, a sedentaryr lifestyle and low calcium intake.
Estrogen is the agent of choice in preventing osteoporosis or post
menopausal bone loss iin women. In addition, Black, et al. in EP 0605193A1
report that estrogen, particularly when taken orally, lowers plasma levels of
LDL
and raises those of the beneficial high density lipoproteins (HDL's). Long-
term
estrogen therapy, however, has been implicated in a variety of disorders,
including
an increase in the risk of uterine cancer, endometrial cancer and possibly
breast
cancer, causing many women to either avoid this treatment or take the
medication
for only a short period of time. Although the risk of endometrial cancer is
thought
to be reduced by a concurrent use of a progesterone, there is still concern
about
possible increased risk of breast cancer with the use of estrogen. Recently
suggested therapeutic regimens, which seek to lessen the cancer risk, such as
administering combinations of progesterone and estrogen, cause the patient to
experience unacceptablE: bleeding. Furthermore, combining progesterone with
estrogen seems to blunt the serum cholesterol lowering effects of estrogen.
The
significant undesirable side effects associated with estrogen therapy support
the
need to develop alternative therapies for osteoporosis that have the desirable
beneficial effect on serunn LDL but do not cause undesirable side effects.
Recently, a numk~er of selective estrogen receptor modulators have been
proposed for treatment of osteoporosis. It has been reported (Osteoporosis
Conference Scrip No. 1812/13 April 16120, 1993, p. 29) that rafoxifene, 6-
hydroxy-
2-(4-hydroxyphenyl)-3-[4-(2-piperidinoethoxy) benzoyl] benzo[b] thiophene,
mimics
the favorable action of estrogen on bone and lipids but, unlike estrogen, has
minimal uterine stimulatory effect. [Black, L.J. et al., Raloxifene (LY139481
Hcl)
Prevents Bone Loss and Reduces Serum Cholesterol Without Causing Uterine
Hypertrophy in Ovariectamized Rats, J. Clin. Invest., 1994, 93:63-69 and
Delmas,
P.D. et al., Effects of F;aloxifene on Bone Mineral Density, Serum Cholesterol
Concentration, and Uterine Endometrium in Postmenopausal Women, New
England Journal of Medicine, 1997, 337:1641-1647].
Agents such as di°oioxifene, U.S. pat. no. 5,254,595, prevent bone
loss and
thereby reduce the risk of fracture without estrogen's side effects. However,
estrogen and estrogen acaonists alone are only expected to reduce the fracture
ri k
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by about 50% leaving approximateiy 50% of osteopenic women still at risk for
an
osteoporotic fracture.
Commonly assigned U.S. pat, no. 5,552,412, which is incorporated herein
by reference, discloses SERM compounds of the formula
,G
~Y
H
~e
r~
wherein the variables arE: defined as set forth therein.
Growth hormonE: (GH), which is secreted from the pituitary gland,
stimulates growth of all tissues of the body that are capable of growing. In
addition, GH is known to have the following basic effects on the metabolic
process
of the body:
1. Increased rate of protein synthesis in substantially all cells of the
body;
2. Decreased rate of carbohydrate utilization in cells of the body;
3. Increased mobilization of free fatty acids and use of fatty acids for
energy.
Deficiency in GH' results in a variety of medical disorders. In children, it
causes dwarfism. in adults, the consequences of acquired GH deficiency include
profound reduction in lean body mass and concomitant increase in total body
fat,
particularly in the truncal region. Decreased skeletal and cardiac muscle mass
and muscle strength lead to a significant reduction in exercise capacity. Bone
density is also reduced, Administration of exogenous GH has been shown to
reverse many of the metabolic changes. Additional benefits of therapy have
included reduction in LDL cholesterol and improved psychological well-being.
In cases where increased levels of GH were desired, the problem was
generally solved by providing exogenous GH or by administering an agent which
stimulated GH production and/or release. In either case the peptidyf nature of
the
compound necessitated that it be ~~dministered by injection. Initially the
source of
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GH was the extraction of the pituitary glands of cadavers. This resulted in an
expensive product, and carried with it the risk that a disease associated with
the
source of the pituitary gland could be transmitted to the recipient of the GH
(e.g.,
Jacob-Creutzfeld disease). Recently, recombinant GH has become available
which, while no longer carrying any risk of disease transmission, is still a
very
expensive product which must be given by injection or by a nasal spray.
Mast GH deficiencies are <;aused by defects in GH release, not primary
defects in pituitary synthesis of GH. Therefore, an alternative strategy for
normalizing serum GH levels is by stimulating its release from somatotrophs.
Increasing GH secretion can be achieved by stimulating or inhibiting various
neurotransmitter systems in the brain and hypothalamus. As a result, the
development of synthetic GH-releasing agents to stimulate pituitary GH
secretion
are being pursued, and may have several advantages over expensive and
inconvenient GH replac;ement therapy. By acting along physiologic regulatory
pathways, the most desirable agents would stimulate puisatile GH secretion,
and
excessive levels of GH that have been associated with the undesirable side
effects of exogenous GH administration would be avoided by virtue of intact
negative feedback loops..
Physiologic and pharmacologic stimulators of GH secretion include
arginine, L-3,4-dihydro~:yphenylalanine {L-DOPA), glucagon, vasopressin, and
insulin induced hypoglycemia, as well as activities such as sleep and
exercise,
indirectly cause GH to be released from the pituitary by acting in some
fashion on
the hypothalamus perhaps either to decrease somatostatin secretion or to
increase the secretion o~f the known secretagogue GH releasing factor (GHRF)
or
an unknown endogenous GH-releasing hormone or all of these.
Commonly assigned International Patent Application Publication Number
W097/24369, designating, infer alia, the U.S., discloses GH secretagogues of
the
formula
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O X4
R~ ~~ R3 I
Y (CH~e (CH?~ N~C~C,/ NBC /R\N/R7
I 1 II ~R8
2/N\N~ (CH~w R4 O
R
wherein the variables are defined as set forth therein. International Patent
Application Publication Number W097124369 is incorporated herein by reference.
Tang et al., Rest.orina and Maintaining Sone in Osteoaenic Female Rat
Skeleton: I. Chan es in Etone Mass and Structure, J. Bone Mineral Research 7
(9),
p1093-1104, 1992 discloses data for the lose, restore and maintain (LRM)
concept, a practical approach for reversing existing osteoporosis. The LRM
concept uses anabolic agents to restore bone mass and architecture {+ phase)
and then switches to an agent with the established ability to maintain bone
mass,
to keep the new bone (+,/- phase). The rat study utilized PGE2 and
risedronate, a
bisphosphonate, to show that most of the new cancellous and cortical bone
induced by PGE2 can be maintained for at least 60 days after discontinuing
PGE2
by administering risedron~ate.
Shen et al., Effe~~ts of Reciprocal Treatment with Estrogen and Estrogen
plus Parathyroid Hormone on Bone Structure and Strength in Ovariectomized
Rats, J. Clinical Investigation, 1995, 96:2331-2338 discloses data for the
combination and/or sequential use of anti-resorptive agents and anabolic
agents
for the treatment of osteoporosis.
Commonly assigned International Patent Application Publication Number
W097I31640, designating, inter ayia, the U.S., discloses the use of certain GH
secretagogues in combination with certain SERMS to treat osteoporosis.
International Patent Application Publication Number W097/31640 is incorporated
herein by reference.
SUMMARY OF THE INVENTION
This invention is directed to a pharmaceutical composition comprising:
a. a first compound, said first compound being (-)-cis-6-phenyl-5-(4-(2-
pyrrolidin-1-yl-ethoxy)-phenyl)-5,6,7,8-tetrahydronaphthalene-2-of or a
pharmaceutically acceptable salt thereof; and
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b. a second compound, said second compound being 2-amino-N-(1 (R)-
(2,4-difluoro-benzyloxym~ethyl)-2-oxo-2-(3-oxo-3a{R}-pyridin-2-ylmethyl)-2-
(2,2,2-
trifluoro-ethyl)-2,3,3a,4,6,7-hexahydro-pyrazolo[4,3-c]pyridin-5-yi)-ethyl)-2-
methyl-
propionamide or a pharmaceutically acceptable salt therecaf.
This invention is further directed to a pharmaceutical composition as recited
in the immediately preceding paragraph additionally comprising a
pharmaceutical
carrier.
This invention is ;still further directed to a composition as set forth in
either
of the first two paragraphs of this summary wherein said first compound is {-}-
cis-6-
phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy)-phenyl)-5,6,7,8-tetrahydronaphthalene-2-
of
D-tartrate and said second compound is 2-amino-N-(1 {R)-(2,4-difluoro-
benzyloxymethyl)-2-axo-2-(3-oxo-3a(R)-pyridin-2-ylmethyl)-2-(2,2,2-trifluoro-
ethyl)-
2,3,3a,4,6,7-hexahydro-pyrazolo[4,3-c]pyridin-5-yl)-ethyl}-2-methyl-
propionamide
L-tartrate.
This invention is still further directed to a method, designated Method A, for
treating a mammal suffering from rnusculoskeletal frailty comprising
administering
to said mammal a pharrnaceutical composition as recited in any of the first
three
paragraphs of this summary.
A preferred method within Method A, designated Method B, is wherein said
mammal is suffering from osteoporosis.
Another preferred method within Method A, designated Method C, is
wherein said mammal is suffering from osteotomy, childhood idiopathic bone
loss
or bone loss associated 'with periodontitis.
This invention is still further directed to a method, designated Method A',
for treating a mammal suffering from musculoskeletal frailty comprising
administering to said mammal
a. a first compound, said first compound being {-)-cis-6-phenyl-5-(4-(2-
pyrrolidin-1-yl-ethoxy)-phenyl)-5,6,7,8-tetrahydronaphthafene-2-of or a
pharmaceutically acceptable salt thereof; and
b. a second compound, said second compound being 2-amino-N-(1(R)-
(2,4-difluoro-benzyloxymethyl)-2-oxo-2-(3-oxo-3a(R)-pyridin-2-ylmethyl)-2-
(2,2,2-
trifluoro-ethyl)-2,3,3a,4,6~,7-hexahydro-pyrazolo(4,3-c]pyridin-5-yl)-ethyl)-2-
methyl-
propionamide or a pharmaceutically acceptable salt thereof.
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This invention is particularly directed to a method of Method A' wherein the
first compound and the second compounds are administered substantially
simultaneously.
This invention is also particularly directed to a method of Method A';
hereinafter termed Method D, wherein the second compound is administered for a
period of from about three months to about three years.
This invention is more particularly directed to a method of Method D
followed by administration of the first compound for a period of from about
three
months to about three years without the administration of the second compound
during the period of from about three months to about three years.
This invention is also more particularly directed to a method of Method D
followed by administration of the first compound for a period greater than
about
three years without the administration of the second compound during the
greater
than about three year period.
This invention is also directed to a method, hereinafter termed Method E,
for treating a mammal suffering from musculoskeletal frailty comprising
administering to said mammal a therapeutically effective amount of a
composition
as recited in any of the first threee paragraphs of this summary.
A preferred method within Method E is wherein bone healing following
facial reconstruction, maxillary reconstruction or mandibular reconstruction
is
enhanced, vertebral synostosis is induced, long bone extension is enhanced,
the
healing rate of a bone graft or a long bone fracture is enhanced or prosthetic
ingrowth is enhanced.
This invention is .also directed to a method for increasing muscle mass in a
mammal comprising administering to said mammal a muscle mass increasing
effective amount of a composition as recited in any of the first three
paragraphs of
this summary.
In all of the methods of this invention, it is particularly preferred that the
mammal is a human.
This invention is ;also directed to a kit comprising a treatment for a mammal
suffering from muscuioskeletal fraili;y comprising:
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a. (-)-cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy}-phenyl)-5,6,7,8-
tetrahydronaphthalene-2-01 or a pharmaceutically acceptable salt thereof and a
pharmaceutically acceptable carrier in a first unit dosage form;
b. 2-amino-N-(1 (R)-(2,4-difluoro-benzyloxymethyl}-2-oxo-2-{3-oxo-3a(R)-
pyridin-2-ylmethyl)-2-{2,2,2-trifluoro-ethyl)-2,3,3a,4,6,7-hexahydro-
pyrazolo[4,3-
c]pyridin-5-yl)-ethyl}-2-methyl-propionamide or a pharmaceutically acceptable
salt
thereof and a pharmaceutically acceptable carrier in a second unit dosage
form;
and
c. a container.
This invention is particularly directed to a kit as described in the
immediately preceding paragraph wherein said first unit dosage form comprises
(-
-cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy)-phenyl}-5,6,7,8-
tetrahydronaphthalene-2-of D-tartrate and said second unit dosage form
comprises
2-amino-N-(1 (R)-(2,4-difluioro-benzyloxymethyl)-2-oxo-2-(3-oxo-3a{R)-pyridin-
2-
ylmethyl}-2-(2,2,2-trifluoro-ethyl)-2,3,3a,4,6,7-hexahydro-pyrazolo[4,3-
c]pyridin-5-
yl)-ethyl)-2-methyl-propionamide L-tartrate.
In all of the compositions, methods and kits of this invention, it is
particularly preferred that the D-tartrate salt of (-)-cis-6-phenyl-5-(4-{2-
pyrrolidin-1-
yl-ethoxy)-phenyl)-5,6,7,8-tetrahydronaphthalene-2-of is used and that the L-
tartrate salt of 2-amino-N-(1 (R)-(2,4-difluoro-benzyloxymethyl}-2-oxo-2-(3-
oxo-
3a(R)-pyridin-2-ylmethyl}-2-(2,2,2-triouoro-ethyl)-2,3,3x,4,6,7-hexahydro-
pyrazofo[4,3-c]pyridin-5-y~l)-ethyl)-2-methyl-propionamide is used.
The phrase "condition which presents with law bone mass" refers to a
condition where the IevE:l of bone mass is below the age specific normal as
defined in standards by v~the World Health Organization "Assessment of
Fracture
Risk and its Application to Screening for Postmenopausal Osteoporosis (1994},
Report of a World Health Organization Study Group. World Health Organization
Technical Series 843". Childhood idiopathic and primary osteoporosis are also
included. included in the treatrnent of osteoporosis is the prevention or
attenuation of long term complicatuons such as curvature of the spine, loss of
height, prosthetic surgery, and prevention of prostate malfunctioning. Also
included is increasing the bone fracture healing rate and enhancing the rate
of
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successful bone grafts. Also included is periodontal disease and alveolar bone
loss.
The phrase "condition which presents with low bone mass" also refers to a
mammal known to have .a significantly higher than average chance of developing
such diseases as are described above including osteoporosis (e.g., post-
menopausal women, me;n over the age of 60, and persons being treated with
drugs known to cause osteoporosis as a side effect (such as glucocorticoid)).
Those skilled in l:he art will recognize that the term bone mass actually
refers to bone mass pf:r unit area which is sometimes (although not strictly
correctly) referred to as bone mineral density.
The phrase "musc:uloskeletal frailty" refers to a condition wherein a subject
has low bone mass and/or low muscle mass, and includes such diseases,
disorders and conditions such as, but not limited to, conditions which present
with
low bone mass, osteoporosis, conditions which present with low muscle mass,
osteotomy, childhood idiopathic bone loss, bone loss associated with
periodontitis,
bone healing following facial reconstruction, maxillary reconstruction,
mandibular
reconstruction and bone fracture. Further, musculoskeletal frailty encompasses
such conditions as interfaces between newly attached prostheses and bone which
require bone ingrowth.
The term "treating", "treat" or "treatment" as used herein includes curative,
preventative (e.g., prophylactic) and palliative treatment.
The parenthetical negative or positive sign used herein in the nomenclature
denotes the direction plane polarized light is rotated by the particular
stereoisomer.
The compositions of this invention may include hydrates of the compounds
used therein.
The pharmaceutical compositions and methods of this invention result in a
more rapid and higher magnitude bone mass gain than is achievable with the
same Bases of (-)-c:is-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-
5,6,7,8-
tetrahydro-naphthalene-2'-of as described above alone or 2-amino-N-(1 (R)-(2,4-
difluoro-benzyloxymethyl)-2-oxo-2-(3-oxo-3a(R)-pyridin-2-ylmethyl)-2-(2,2,2-
trifluoro-ethyl)-2,3,3a,4,6,7-hexahydro-pyrazolo[4,3-c]pyridin-5-yl)-ethyl)-2-
methyl-
propionamide as described above alone. Thus, these combinations increase bone
mass and decrease fracture rates to a greater extent than is achievable
through
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use of either agent alone. Further, these combinations increase bone density
and
muscle mass while at the same time reducing fat mass and total serum
cholesterol. This invention makes a significant contribution to the art by
providing
compositions and methods that increase and maintain bone mass resulting in
prevention, retardation, andlor regression of osteoporosis and related bone
disorders.
Other features and advantages will be apparent from the specification and
claims which describe the: invention.
DETAILED DESCRIPTION OF THE IN1/ENTION
The first compound of this invention is (-}-cis-6-phenyl-5-[4-(2-pyrrolidin-1-
yl-ethoxy}-phenyl]-5,8,7,8-tetrahydro-naphthalene-2-of, or a ~
pharmaceutically
acceptable salt thereof, which has the structure of Formula I:
n ~N-
Et0
(-}-Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydro-
naphthalene-2-of and the: pharmaceutically acceptable salts thereof are
prepared
as described in commonly assigned US Patent Number 5,552,412, which is
referenced above.
(-}-Cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydro-
naphthalene-2-oI D-tartrate is prepared as set forth in the immediately
preceding
paragraph or, alternatively, as set forth in Intemationai Patent Application
Publication Number WO97/18434, designating the United States and which is
incorporated herein by rs:ference.
The second compound of this invention is 2-amino-N-(1 (R}-(2,4-difluoro-
benzyloxymethyl}-2-oxo-:2-(3-oxo-3a(R)-pyridin-2-ylmethyl)-2-(2,2,2-trifluoro-
ethyl)-
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2,3,3a,4,6,7-hexahydro-pyrazolo[4,3-c]pyridin-5-yl)-ethyl)-2-methyl-
propionamide
or a pharmaceutically acceptable salt thereof, which has the structure of
Formula
F
N v
F C~~'N ' 1 O
~N NHz
:w
..-~-N O H Me Me
ll
2-Amino-N-(1 (R)-(2,4-difluoro-benzyloxymethyl}-2-oxo-2-(3-oxo-3a(R)-pyridin-2-
yimethyl)-2-(2;2,2-trifiuoro-ethyl)-2,3,3a,4,6,7-hexahydro-pyrazolo[4,3-
c]pyridin-5-
yl)-ethyl)-2-methyl-propionamide and the pharmaceutically acceptable salts
thereof
are prepared as set forth in commonly assigned International Patent
Application
Publication Number W097124369, designating, interalia, the United States,
which
is referenced above.
2-Amino-N-(1 (R)-(2,4-difluoro-benzyloxymethyl)-2-oxo-2-(3-oxo-3a(R}-
pyridin-2-ylmethyl)-2-(2,2;,2-trifluoro-ethyl)-2, 3, 3a,4,6,7-hexahydro-
pyrazolo[4,3-
c]pyridin-5-yl)-ethyl)-2-methyl-propionamide L-tartrate is prepared as set
forth in
International Patent Application Publication Number W097124369, referenced
above. Alternatively, 2-amino-N-(1 (R}-(2,4-difluoro-benzyloxymethyl)-2-oxo-2-
(3-
oxo-3a(R)-pyridin-2-ylmethyl)-2-(2,2,2-trifluoro-ethyl)-2,3,3a,4,6,7-hexahydro-
pyrazolo[4,3-c]pyridin-5-yl)-ethyl)-2-methyl-propionamide L-tartrate is
prepared as
described in Example Orte herein.
In addition, when the compounds and pharmaceutically acceptable salts
thereof used in the compositions and methods of this invention form hydrates
or
solvates such hydrates or solvates are also within the scope of the invention.
The pharmaceutiical combinations and methods of this invention are all
adapted to therapeutic use as agents that either activate bone turnover or
prevent
bone resorption or increase bone formation in mammals, particularly humans.
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Since these functions are: closely related to the development of osteoporosis
and
bone related disorders, these combinations, by virtue of their action on bone,
prevent, arrest, regress or reverse osteoporosis.
The utility of the compositions and methods of the present invention as
medical agents in the trE:atment of musculoskeletal frailty (e.g., conditions
which
present with low bone mass or low muscle mass including osteoporosis) in
mammals {e.g. humans) is demonstrated by the activity of the compounds of this
invention in conventional assays as set faith in U.S. Patent Number 5,552,412
and
International Patent Application Publication Number W097124369. Further
evidence of the utility of the instant combination is set forth in Example Two
below.
Such assays also provide a means whereby the activities of the compounds of
this invention can be compared between themselves and with the activities of
other known compounds. The results of these comparisons are useful for
determining dosage Ieve;Is in mammals, including humans, for the treatment of
such diseases.
Administration of the compounds of this invention can be via any method
which delivers a compound of the combination of this invention systemically
and/or
locally. These methods include oral routes, parenteral, intraduodenal routes,
etc.
Generally, the compounds of this invention are administered orally, but
parenteral
administration (e:g., intravenous, intramuscular, transcutaneous, subcutaneous
or
intramedullary) may be: utilized, far example, where oral administration is
inappropriate for the instant target or where the patient is unable to ingest
the
drug. The two different compounds of this invention can be co-administered
simultaneously or sequentially in any order, or a single pharmaceutical
composition comprising a first compound as described above and a second
compound as described above in a pharmaceutically acceptable carrier can be
administered.
In any event the amount and timing of compounds administered will, of
course, be dependent on the subject being treated, on the severity of the
affliction,
on the manner of administration and on the judgment of the prescribing
physician.
Thus, because of patie~~nt to patient variability, the dosages given below are
a
guideline and the physician may titrate doses of the drug to achieve the
activity
(e.g., bone mass augmE;ntation) that the physician considers appropriate for
the
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individual patient. In considering the degree of activity desired, the
physician must
balance a variety of factors such as bone mass starting level, age of the
patient,
presence of preexisting .disease, as well as presence of other diseases (e.g.,
cardiovascular). For e~;ample, the administration of (-}-cis-6-phenyl-5-(4-(2-
pyrrolidin-1-yl-ethoxy}-phenyl)-5,6,7,8-tetrahydronaphthaiene-2-o1 can provide
cardiovascular benefits, particularly for post-menopausal women. The following
paragraphs provide preferred dosage ranges for the various components of this
invention.
An effective dosage for (-)-cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-
phenyl]-5,6,7,8-tetrahydro~-naphthalene-2-of is in the range of 0.0001 to 100
mglkg/day, preferably 0Ø01 to 10 mglkg/day.
An effective dosage for 2-amino-N-(1 (R)-(2,4-difluoro-benzyloxymethyl)-2-
oxo-2-(3-oxo-3a ( R)-pyri d i n-2-yl methyl)-2-(2, 2, 2-trifi uo ro-ethyl}-2,
3, 3a, 4, 6, 7-
hexahydro-pyrazolo[4,3-c;~pyridin-5-yi)-ethyl)-2-methyl-propionamide is in the
range
of 0.0001 to 100 mg/kg/day, preferably 0.01 to 5 mglkglday.
Where the tartrate: salt or other pharmaceutically acceptable salt of either
of the above compounds is used in this invention, the skilled person will be
able to
calculate effective dosage: amounts by calculating the molecular weight of the
salt
form and performing simple stoichiometric ratios.
The compounds of the present invention are generally administered in the
form of a pharmaceutical composition comprising at least one of the compounds
or
pharmaceutically acceptable salts thereof of this invention together with a
pharmaceutically acceptable carrier or diluent. Thus, the compounds and
pharmaceutically acceptable salts thereof of this invention can be
administered
separately or together in any conventional oral, parenteral or transdermal
dosage
form. When administered separately, the administration of the other compound
or
pharmaceutically acceptable salt thereof of the invention follows.
For oral administration a pharmaceutical composition can take the form of
solutions, suspensions, tablets, pills, capsules, powders, and the like.
Tablets
containing various excipients such as sodium citrate, calcium carbonate and
calcium phosphate are employed along with various disintegrants such as starch
and preferably potato or tapioca starch and certain complex silicates,
together with
binding agents such as polyvinylpyrrolidone, sucrose, gelatin and acacia.
CA 02335112 2000-12-14
WO 99/65488 '1~" PCT/IB9910~796
Additionally, lubricating agents such as magnesium stearate, sodium lauryl
sulfate
and talc are often useful' for tabletting purposes. Solid compositions of a
similar
type are also employed as fillers in soft and hard-filled gelatin capsules;
preferred
materials in this connection also include lactose or milk sugar as well as
high
molecular weight polyethylene glycols. When aqueous suspensions and/or elixirs
are desired for oral administration, the compounds or pharmaceutically
acceptable
salts thereof of this invention can be combined with various sweetening
agents,
flavoring agents, coloring agents, emulsifying agents andlor suspending
agents,
as well as such diluents as water, ethanol, propylene glycol, glycerin and
various
like combinations thereof.
For purposes of parenterai administration, solutions in sesame or peanut oil
or in aqueous propylene glycol can be employed, as well as sterile aqueous
solutions of the corresponding water soluble salts. Such aqueous solutions may
be suitably buffered, if nE:cessary, and the liquid diluent first rendered
isotonic with
sufficient saline or glucose. These aqueous solutions are especially suitable
for
intravenous, intramuscular, subcutaneous and intraperitoneal injection
purposes.
In this connection, the sterile aqueous media employed are all readily
obtainable
by standard techniques uvell-known to those skilled in the art.
For purposes of transdermal (e.g.,topical) administration, dilute sterile,
aqueous or partially aqueous solutions (usually in about 0.1 % to 5%
concentration), otherwise: similar to the above parenteral solutions, are
prepared.
Methods of preparing various pharmaceutical compositions with a certain
amount of each active ingredient are known, or will be apparent in fight of
this
disclosure, to those skilled in this art. For examples, see Reminaton's
Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 19th Edition
(1990).
Pharmaceutical compositions according to the invention may contain 0.1 %-
95% of a combination of the compounds or pharmaceutically acceptable salts
thereof of this invention, preferablly 1 %-70%. In any event, the composition
or
formulation to be administered Hrill contain a quantity of the compounds or
pharmaceutically accepfiable salts thereof of the invention in an amount
effective
to treat the diseaselcondlition of the subject being treated.
CA 02335112 2000-12-14
WO 99/65488 -15- PCT/IB99/00796
Since the present invention relates to treatment with a combination of the
two active ingredients which may be administered separately, the invention
also
relates to combining separate pharmaceutical compositions in kit form. The kit
includes two separate pharmaceutical compositions: (-)-cis-6-phenyl-5-(4-(2-
pyrrolidin-1-yl-ethoxy)-phE:nyl}-5,8,7,8-tetrahydronaphthalene-2-of or a
pharmaceutically acceptable salt thereof and 2-amino-N-(1 (R)-(2,4-difiuoro-
benzyloxymethyl)-2-oxo-2-(3-oxo-3a(R)-pyridin-2-ylmethyl)-2-(2,2,2-trifluoro-
ethyf)-
2,3,3a,4,6,7-hexahydro-pyrazolo[4,3-c]pyridin-5-yl)-ethyl)-2-methyl-
propionamide
or a pharmaceutically ae:ceptable salt thereof. The kit includes a container
for
containing the separate compositions such as a divided bottle or a divided
foil
packet, however, the separate compositions may also be contained within a
single,
undivided container. Typically the kit includes directions for the
administration of
the separate components. The kit form is particularly advantageous when the
separate components arE: preferably administered in different dosage forms
(e.g.,
ora! and parenteral), are administered at different dosage intervals, or when
titration of the individual components of the combination is desired by the
prescribing physician.
An example of such a kit is a so-called blister pack. Blister packs are well
known in the packaging industry and are being widely used for the packaging of
pharmaceutical unit dosaige forms (tablets, capsules, and the like). Blister
packs
generally consist of a sheet of relatively stiff material covered with a foil
of a
preferably transparent pliastic material. During the packaging process
recesses
are formed in the plastic foil. The recesses have the size and shape of the
tablets
or capsules to be packed. Next, the tablets or capsules are placed in the
recesses
and the sheet of relativelvy stiff material is sealed against the plastic foil
at the face
of the foil which is opposite from the direction in which the recesses were
formed.
As a result, the tablets or capsules are sealed in the recesses between the
plastic
foil and the sheet. Preferably the strength of the sheet is such that the
tablets or
capsules can be removed from the blister pack by manually applying pressure on
the recesses whereby an opening is formed in the sheet at the place of the
recess.
The tablet or capsule care then be removed via said opening.
It is desirable to provide a memory aid on a card insert, e.g., in the form of
numbers next to the tablsas or capsules whereby the numbers correspond with
the
___. ., _ _ __ . ~ ~_ . ._m m_ .. _ . _,~~_..~M; v__ ._.. . _ . _~. ~. -_
CA 02335112 2000-12-14
WO 99/65488 -16- PCTIIB99/00796
days of the regimen which the tablets or capsules so specified should be
ingested.
Another example of such a memory aid is a calendar printed on the card e.g.,
as
follows "First Week, IVlonday, Tuesday, ...etc.... Second Week, Monday,
Tuesday,..:" etc. Other variations of memory aids will be readily apparent. A
"daily
dose" can be a single tablet or capsule or several pills or capsules to be
taken on
a given day. Afso a daily dose of SERM can consist of one tablet or capsule
while
a daily dose of a GH sec~retagogue can consist of several tablets or capsules.
The
memory aid should reflect this.
In another specifJc embodiment of the invention a dispenser designed to
dispense the daily dosEa ane at a time in the order of their intended use is
provided. Preferably, the dispenser is equipped with a memory-aid, so as to
further facilitate compliance with the regimen. An example of such a memory-
aid
is a mechanical counter which indicates the number of daily doses that has
been
dispensed. Another example of such a memory-aid is a battery-powered micro
chip memory coupled with a liquid crystal readout, or audible reminder signal
which, for example, reads out the date that the last daily dose has been taken
and/or reminds one when the next dose is to be taken.
Examlale 1
2-Amino-N ~1-(2.4-difluoro-benzvloxvmethvl)-_ 2-oxo-2-(3-oxo-3a-pvridin-2-
vlmethvl-
~2.2.2-trifluoro-ethrlZ2,3~3a,4.6,7-hexahvdro-oyrazolo(4 3-clpyridin-5-yl}-
ethy~i -2-
methvl-aropionamide L-(v tartrate
A. 4-Oxo-3-pvridin-2,rlmethyl-c~iperidine-1 3-dicarboxylic acid 1-Pert but rLl
ester
3-ethyl ester
To a solution of 4-oxo-piperidine-1,3-dicarboxylic acid 1-tent butyl ester 3
ethyl ester (10.34 g, 38.:2 mmol) in DMF (40 mL) at about 0 °C was
added picolyl
chloride hydrochloride (:5.7 g, 34.7 mmol), potassium carbonate (14.4 g, 104.1
mmol) and potassium iodide (5.76 g, 34.7 mmol). After stirring at about 0
°C for
about 2 hours, the ice bath was removed and DABCO (973 mg, 8.68 mmol) was
added. The reaction mixture was stirred for about 30 min. and poured into a
mixture of water and IF~E. The organic layer was separated and washed with
saturated aqueous NaHC03 and saturated aqueous NaCI, dried over Na2S04 and
concentrated in vacuo. 'The crude residue was crystallized from hexanes to
give a
white solid (8.19 g, yield 65%). 'H-NMR (CDCI3) 8 1.17 (t, 3H), 1.48 { s, 9H),
1.55
CA 02335112 2000-12-14
WO 99/65488 -17- PCTfIB99100796
(s, 2H), 2.67 (m, 1 H), 2.71 (m, 1 H), 3.31-3.50 (m, 3H), 4.11 (d, 2H), 4.49
(d, 1 H),
7.06 (br s, 1 H}, 7.17(d, 1 IH), 7.54 (m, 1 H}, 8.40 (s, 1 H).
B. 3-Oxo-Sap ridin-;2-Ylmethyl-2-(2,2,2-trifluoro-ethyl)-2 3 3a 4 6 7-
hexahydro-
p)rrazolol4,3-clpyridine-5-carboxylic acid tert butyl ester
A 70% aqueous solution of CF3CHZNHNH2 (325 mL, 1.986 mot) (obtained
from Aldrich) was extracted with toluene (3 x 1200 ml_). To a solution of the
product made according to step A (600 g, 1.655 mol) in toluene (900 mt_) was
first
added the combined toluene extracts containing the anhydrous 2,2,2-
trifluoroethyl
hydrazine, followed by acetic acid {121.4 g, 1.986 mol). The reaction mixture
was
heated at about 70 °C for about 2 hours , then another toluene
extraction of 70%
aqueous 2,2,2-trifluoroei:hyl hydrazine (50 g) was added. The reaction mixture
was heated at about 80 °C for about 3.5 hours, cooled to room
temperature and
diluted with saturated aqueous NaHC03 (2 L). The toluene layer was separated
and washed with saturated aqueous NaCI, dried over Na2S04 and concentrated in
vacuo to give an oil (754.8 g). Crystallization from methanollwater afforded
the
desired product as a white solid (609.5 g). 'H-NMR (CDCI~) b 1.50 (s, 9H),
2.53 (d,
1 H}, 2.70 {br s, 2H), 2:88 (br s, 1 H), 3.31 (m, 2H), 3.97 (m, 1 H), 4.19 (m,
1 H), 4.46
(br s, 1 H), 4.63 {br s, 1 H}, 7.06 (m, 2H), ?.51 (m, 1 H), 8.34 (m, 1 H).
C. 3a-Pvridin-2-ylme~thyl-2-(2.2:2-trifluoroeth~l-2,3a.4 5 6 7-hexahydro-
pyrazolof4,3-cltayridin-3-one
Methanesulfonic .acid (11.6 g, 121 mmol) was added dropwise to a solution
of the product from step B (10 g, 24.2 mmol) in CHZCIZ (100 mL) over about 30
minutes. The reaction mixture was stirred for' about 1 hour, then cooled to
about 0
°C, and then triethylamine (18.6 mi_, 133.1 mmol) was added through an
addition
funnel. The mixture was allowed to warm to room temperature over about 1 hour,
diluted with additional C;HzCiz and washed with saturated aqueous NaCI, dried
over Na2S04, filtered and concentrated in vacuo to afford the product as a
white
solid (7.2 g). 'H-NMR (CDCI3) b: 2.51-2.72 (m, 4H), 3.35 {m, 2H), 3.49 (m,
2H),
4.03 (m, 1 H), 4.25 {m, 111), 7.08 {d, 2H), 7.51 {t, 1 H), 8.37 (d, 1 H).
D. 3a-Pvridin-2-~mEahvl-2-(2.2,2-trifluoroethyl)-2.3a 4 5 6 7-hexahydro-
~yrazolof4,3-cl~yridin-3-one (D)-tartrate
In a dry and nitrogen purged 5 L round bottom flask equipped with a
mechanical stirrer, D-(-) tartaric acid (129 g, 0.86 mol) was added to the
CA 02335112 2000-12-14
WO 99165488 -1$- PCT/IB99/00796
compound made according to step C (243 g, 0.78 mol) in acetoneiwater (9:1,
2430 mL) at about 17 °C. The mixture was stirred at room temperature
overnight,
filtered, the solid was collected and washed with cold acetone and dried under
vacuum. The product was obtained as a yellow solid (284 g, yield 78.8%).
E. 2-tent-Butoxvcarbonylamino-3-(2,4-difluoro-benzyloxy)-aropionic acid
To a solution of N-Boc-{D)-serine (452 g, 2.2026 mol) in a mixture of THF
(7 L) and DMF (3 L) at about 0 °C was added potassium tent-butoxide
solution
(515.8 g, 4.5963 mol}. 'f'he reaction mixture was stirred at about 0 °C
for about 30
min., then 2,4-difluorobenzyl bromide {456.5 g, 2.2051 mol) was added. After
warming to room tempeirature, the reaction mixture was concentrated in vacuo
to
remove the THF. Partitioned the reaction mixture between 4.5 L Hz0 and 4.5 L
IPE. Separated the layers and adjusted the pH of the aqueous layer with 1 N
HCI
to about 3. The aqueous layer was extracted twice with 4 L each of 1PE. The
organic solution was dried over Na2S04, and concentrated in vacuo to yield a
yellow waxy solid (518.0 g, yield: 70.9 %). 'H-NMR (CDCIa) 8 1.44 (s, 9H),
3.73 {m,
1 H), 3.94 (d, 1 H), 4.44 (br s, 1 H), 4.,54 (s, 2H), 5.34 (m, 1 H), 6.78 (m,
1 H), 6.84 {m,
1 H), 7.30 (m, 1 H).
F. 2-Amino-3-{2,4-difluoro-benzvloxy)-~ro~aionic acid methanesulfonic acid
salt
To a solution of the product from step E (1.19 g, 3.59 mmol) in CHZCIZ/ IPE
{1:1, 12 mL) was added methanesulfonic acid (1.72 g, 77.95 mmol) through a
syringe over about 10 minutes. A solid immediately precipitated out of
solution.
After about 1 hour, the solid was filtered and washed with a CH2CI2/IPE
mixture
(1:1) to afford 939 mg of product (yield 80 %).
G. 2-(2-tert-Butaxyc,arbonvlamino-2-methyl-proaionylamino)-3~2 4-difluoro-
benzyloxvLpropionic acid
To a solution of the product from step F (520 mg, 1.46 mmol) in THF/water
(4:1, 10 mL) was added 2-tart butoxycarbonylamino-2-methyl-propionic acid-2,5-
dioxo-pyrrolidin-1-yl ester (438 mg, 1.46 mmol) and triethylamine (369 mg,
3.65
mmol). The reaction mixture was stirred at room temperature for about 1 hour
and
quenched with a 10% aqueous citric acid solution (10 mL). After about 15 min.,
ethyl acetate (50 mL) was added and the organic layer was separated and washed
with saturated aqueous NaCI, dried over Na2S04 and concentrated in vacuo to
CA 02335112 2000-12-14
WO 99/65488 -19- PCT/IB99/0079b
give a foam (534.1 mg, yield 88 %). 'H-NMR (CD3OD): b 1.38 (br s, 15H), 3.77
(d,
1 H), 3.92 (d, 1 H), 4.52 {nn, 3H), 6.92 (m, 1 H), 7.41 {m, 1 H), 7.58 (d, 1
H).
H. j1-~[1-(2,4-Difluorcs-benzylox)rmethyl)-2-oxo-2-[3-oxo-3a-pyridin-2-
ylmethyl-
2-(2,2~2-trifluoro-ethy~3.3a.4.6,7-hexahydro-pyrazolo[4,3-clpyridin-5-vll-
ethvlcarbamoyl)~1-methyl-ethyl)-carbamic acid tent butyl ester
(a) To the compound made according to step D (517 g, 1.12 mol) was
added at about -F °C to ethyl acetate (5170 mL) in a dry and nitrogen
purged 12 L
round bottom flask equipped with a~mechanical stirrer. The solution was cooled
to
about -40 °C, then triethylamine (398 mL, 2.86 mot) was added over
about 45
minutes. The reaction mixture was stirred for about 90 min. at a temperature
between about -50 °C and about -~40 °C, filtered into a 22 L
round bottom flask
purged with nitrogen and washed ~nrith ethyl acetate (2068 mL, pre-cooled to
about
-50 °C) to give the free base as a white solid.
(b) The compound made according to step G (425 g, 1.02 mol ) was added
at about -30 °C to an ethyl acetate solution containing the product
from step H(a),
triethylamine (654 mL, 4.69 mol) and PPAA (1-propanephosphonic acid cyclic
anhydride) (50% in ethyl acetate, 916 mL, 1.53 mo1). The reaction mixture was
stirred for about 1 hour, washed with water and saturated aqueous NaCI, dried
over Na2SO4 and concentrated in vacuo to give the product as an oil (636 g,
yield:
87.8%).
1. 2-Amino-N-~~1-~2s4-difluoro-benzyloxymethyl)-2-oxo-2-f 3-oxo-3a=pyridin-2-
ylmethyl-~2,2,2-trifluoro-ethyl)-2.3.3a,4.6.7-hexahydro:pyrazolo[4 3-clpyridin-
5-
yll-ethyl-2-rnethyl ~~ropionamide
Methanesulfonic acid (258.3 mL, 3.98 mol) was added dropwise at about
15 °C over about 55 minutes to the product from step H (566 g, 0.796
mol) in
CH2CI2 (11,320 mL) in a dry and nitrogen purged 22 L round bottom flask
equipped with a mechanical stirrer. The mixture was stirred for about 40
minutes
at about 20 °C, then saturated aqueous NaHC03 (8,490 mL) was added
until the
pH was about 7.8. Ttre organic layer was separated, washed with water and
saturated aqueous NaC;I, dried over Na2S04, and concentrated in vacuo to
afford
an oily product (388.8 g" yield 80%).
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WO 99/65488 -2~- PCT/IB99/00796
J. 2-Amino-N-f1-(2.~1-difluoro-benzvloxymethyl)-2-oxo-2-[3-oxo-3a-pain-2-
ly methv~2.2.2-trifluoro-ethvlL2,3.3a.4,6,7-hexahydro-p~rrazolo(4 3-clt~yridin-
5-
yll-ethyl}-2-methyl-propionamide L-(+) tartrate
To a solution of the product from step 1 (370 g, 0.6 mol) in methanol (4,070
mL) in a 12 L round bottom flask epuipped with a mechanical stirrer was added
L
(+) tartaric acid (90 g, 0.~6 mol). The reaction mixture was stirred for about
90 min.
at about 22_°C, filtered and concentrated. The crude residue was
diluted with ethyl
acetate (4,560 mL), heated at about 70 °C and slowly allowed to cool to
room
temperature over about 17 hours. The solid was filtered and dried to give
white
crystals, mp 188-189 °C (348.46 g, yield 76%). 'H NMR (MeOH, d4) 8:
8.28 (d,
1 H}, 7.59 (t, 1 H), 7.41-T.39 (m, 1 H}, 7.18-7.13 (m, 1 H), 6.92 (t, 1 H),
5.2 (t, 1 H),
4.56 {bs, 3H), 4.36 (s, :?H), 4.31-4.25 (m, 1 H), 4.13-4.06 (m, 1 H), 3.78 (d,
2H),
3.21 (t, 1 H), 3.18-2.96 (gym, 2H}, 2.65-2.55 (m, 2H), 1.57 (d, 6H). MS: MH+
611.
[a]~°9 +22.03 (c=11.9, Me;OH).
The following assay can be used to show that the combination and
methods of this invention increases lean body mass and decreases fat body mass
whereas the GH secretagogue alone would be expected to decrease fat body
mass with no change in lean body mass and the SERM alone would be expected
to increase both lean and fat body mass. Further, the combination increases
bone
density and decreases total serum cholesterol.
Examale Two
Female S-D rats (Harlan) are sham-operated or ovariectomized (OVX) at
3.5 months of age. Drud administration starts when the rats are 9 months of
age
and 5.5 months post-surgery. The sham-operated rats receive daily gavage of
vehicle (10% ethanol in wafer), while the OVX rats receive daily gavage of
vehicle,
or 2-amino-N-(1 (R)-(2,4.-difiuoro-benzyloxymethyl)-2-oxo-2-(3-oxo-3a(R)-
pyridin-2-
ylmethyl)-2-(2,2,2-trifluoro-ethyl)-2,3,3a,4,6,7-hexahydro-pyrazolo[4,3-
cjpyridin-5-
y!)-ethyl)-2-methyl-propionamide at 5 mglkg/d alone, or (-)cis-6-phenyl-5-(4-
(2-
pyrrolidin-1-yl-ethoxy)-phenyl)-5,6,7,8-tetrahydronaphthalene-2-of at 0.1
mglkgld
alone, or co-treatment of 2-amino-N-(1 {R)-(2,4-difluoro-benzyloxymethyl)-2-
oxo-2-
(3-oxo-3a(R)-pyridin-2-ylmethyl)-2-(2,2,2-trifluoro-ethyl)-2,3,3a,4,6,7-
hexahydro-
pyrazolo[4,3-cjpyridin-5-yl)-ethyl)-2-methyl-propionamide and (-)cis-6-phenyl-
5-(4-
(2-pyrrolidin-1-yl-ethoxy)-phenyl)-5,6,7,8-tetrahydronaphthalene-2-of for 4
weeks.
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WO 99/b5488 -21- . PCT/IB99/00796
In the combination group, 2-amino-N-(1 (R)-(2,4-difluoro-benzyloxymethyl}-2-
oxo-2-
(3-oxo-3a(R)-pyridin-2-ylmethyl)-2-(2,2,2-trifluoro-ethyl)-2,3,3a,4,6;7-
hexahydro-
pyrazolo[4,3-c]pyridin-5-yll)-ethyl)-2-methyl-propionamide is given 2 hours
prior to
(-)cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy)-phenyl)-5,6,7,8-
tetrahydronaphthalene-2-oI. There are 8 to 10 rats per each subgroup. All rats
are
given subcutaneous injections with 10 mg/kg of calcein (Sigma Chemical Co.,
St.
Louis, MO) on 13 and 3 plays before autopsy. It will be recognized by those
skilled
in the art that the compounds used in this assay may be administered in the
form
of a pharmaceutically acceptable salt and that the dosage amount can be
readily
determined by calculation of the molecular weight of the salt form and
performing
simple ratios.
Before autopsy on the terminal day of the assay, all rats under
ketamine/xylazine anesthesia undergo dual-energy X-ray absorptiometry (DXA,
QDR-1000IW, Hologic In~c., Waltham, MA) equipped with Rat Whole Body Scan
software (Hologic Inc., Ullaltham, MA) for lean and fat body mass
determination.
The rats are then autopsied and blood is obtained by cardiac puncture. Total
serum cholesterol is determined using a high performance cholesterol
colorometic
assay {Boehringer Mannheim Biochemicals, Indianapolis, IN). The body weight
gain is calculated as body weight at autopsy minus body weight at day 0. The
uterine wet weight is determined immediately at autopsy.
The right femur from each rat is removed at autopsy and scanned using
dual energy x-ray absorptiometry (DXA, QDR 10001VV, Hologic Inc., Waltham, MA)
equipped with "Regional High Resolution Scan" software (Hologic Inc., Waltham,
MA). The scan field size its 5.08 x 1.902 cm, resolution is 0.0254 x 0.0127 cm
and
scan speed is 7.25 mm/s,econd. The femoral scan images are analyzed and total
femoral bone area, bone miners! content, and bone mineral density are
determined according to the method described in H. Z. I~e et al., Droloxifene,
a
New Estrogen AntagonistlAgonist, Prevents Bone Loss in Ovariectomized Rats.
ENDOCRINOLOGY 136;2435-2441, 1995.
It should be undE:rstood that the invention is not limited to the particular
embodiments described herein, but that various changes and modifications may
be made without departing from the spirit and scope of this novel concept as
defined by the following claims.
