U.S. patent application number 09/416733 was filed with the patent office on 2002-06-27 for methods for inhibiting bone resorption.
Invention is credited to HARADA, SHUN-ICHI, LABELLE, MARC, MACHWATE, MOHAMED, METTERS, KATHLEEN, RODAN, GIDEON A., YOUNG, ROBERT N..
Application Number | 20020082287 09/416733 |
Document ID | / |
Family ID | 26801424 |
Filed Date | 2002-06-27 |
United States Patent
Application |
20020082287 |
Kind Code |
A1 |
HARADA, SHUN-ICHI ; et
al. |
June 27, 2002 |
METHODS FOR INHIBITING BONE RESORPTION
Abstract
The present invention relates to methods for inhibiting bone
resorption in a mammal comprising administering to a mammal in need
thereof a therapeutically effective amount of an EP.sub.4 receptor
subtype antagonist.
Inventors: |
HARADA, SHUN-ICHI; (NORTH
WALES, PA) ; RODAN, GIDEON A.; (BRYN MAWR, PA)
; MACHWATE, MOHAMED; (LANSDALE, PA) ; LABELLE,
MARC; (ST. LAZARE, CA) ; METTERS, KATHLEEN;
(MONTREAL, CA) ; YOUNG, ROBERT N.; (SENNEVILLE,
CA) |
Correspondence
Address: |
MERCK AND CO INC
P O BOX 2000
RAHWAY
NJ
070650907
|
Family ID: |
26801424 |
Appl. No.: |
09/416733 |
Filed: |
October 13, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60104339 |
Oct 15, 1998 |
|
|
|
Current U.S.
Class: |
514/384 |
Current CPC
Class: |
G01N 33/88 20130101;
A61K 31/66 20130101; A61K 31/663 20130101; A61K 31/00 20130101;
A61K 2300/00 20130101; G01N 2500/10 20130101; A61K 31/66 20130101;
A61K 45/06 20130101; A61K 31/4196 20130101; G01N 2500/04
20130101 |
Class at
Publication: |
514/384 |
International
Class: |
A61K 031/47; A61K
031/41 |
Claims
What is claimed is:
1. A method for inhibiting bone resorption in a mammal in need
thereof comprising administering to said mammal a therapeutically
effective amount of an EP.sub.4 receptor subtype antagonist having
an EC.sub.50 value of from about 0.1 nanoM to about 100 microM.
2. A method according to claim 1 wherein said mammal is a
human.
3. A method for treating or reducing the risk of contracting a
disease state or condition in a mammal in need of such treatment or
prevention, comprising administering to said mammal a
theraperutically effective amount of an EP.sub.4 receptor subtype
antagonist.
4. A method according to claim 3 wherein said mammal is a
human.
5. A method according to claim 4 wherein said disease state or
condition is selected from the group consisting of osteoporosis,
glucocorticoid induced osteoporosis, Paget's disease, abnormally
increased bone turnover, periodontal disease, tooth loss, bone
fractures, rheumatoid arthritis, periprosthetic osteolysis,
osteogenesis imperfecta, metastatic bone disease, hypercalcemia of
malignancy, and multiple myeloma.
6. A method according to claim 5 wherein said disease state or
condition is selected from the group consisting of osteoporosis,
glucocorticoid induced osteroporosis, and Paget's disease.
7. A method according to claim 1 wherein said antagonist is a
non-cyclopentanone or non-hydroxycyclopentane compound.
8. A method according to claim 1 wherein said antagonist is
selected fom the group consisting of
5-butyl-2,4-dihydro-4-[[2'-[N-(3-chloro-2-thiophe-
necarbonyl)sulfamoyl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1-
,2,4-triazol-3-one potassium salt,
5-butyl-2,4-dihydro-4-[[2'-[N-(2-methyl-
-3-furoyl)sulfamoyl]biphenyl4-yl]methyl]-2-[2-(trifluoromethyl)phenyl]-1,2-
,4-triazol-3-one,
5-butyl-2,4-dihydro-4-[[2'-[N-(3-methyl-2-thiophenecarbo-
nyl)sulfamoyl]biphenyl-4-yl]methyl]-2-[(2-trifluoromethyl)phenyl]-1,2,4-tr-
iazol-3-one,
5-butyl-2,4-dihydro-4-[[2'-[N-(2-thiophenecarbonyl)sulfamoyl]-
biphenyl-4-yl]methyl]-2-[(2-trifluoromethyl)phenyl]-1,2,4-triazol-3-one,
5-butyl-2,4-dihydro-4-[[2'-[N-[2-(methylpyrrole)carbonyl]sulfamoyl]biphen-
yl-4-yl]methyl]-2-[(2-trifluoromethyl)phenyl-1,2,4-triazol-3-one,
and the pharmaceutically acceptable salts thereof, and mixtures
thereof.
9. A method for inhibiting bone resorption in a mammal in need
thereof comprising administering to said mammal a therapeutically
effective amount of an EP.sub.4 receptor subtype antagonist and a
bisphosphonate active.
10. A method according to claim 9 wherein said bisphosphonate
active corresponds to the chemical structure 3wherein n is an
integer from 0 to 7 and wherein A and X are independently selected
from the group consisting of H, OH, halogen, NH.sub.2, SH, phenyl,
C1-C30 alkyl, C3-C30 branched or cycloalkyl, C1-C30 substituted
alkyl, C1-C10 alkyl substituted NH.sub.2, C3-C10 branched or
cycloalkyl substituted NH.sub.2, C1-C10 dialkyl substituted
NH.sub.2, C1-C10 alkoxy, C1-C10 alkyl substituted thio, thiophenyl,
halophenylthio, C1-C10 alkyl substituted phenyl, pyridyl, furanyl,
pyrrolidinyl, imidazolyl, imidazopyridinyl, and benzyl; or A and X
are taken together with the carbon atom or atoms to which they are
attached to form a C3-C10 ring; and provided that when n is 0, A
and X are not selected from the group consisting of H and OH; and
the pharmaceutically acceptable salts thereof.
11. A method according to claim 9 wherein said bisphosphonate is
selected from the group consisting of alendronate, cimadronate,
clodronate, tiludronate, etidronate, ibandronate, neridronate,
olpandronate, risedronate, piridronate, pamidronate, zolendronate,
pharmaceutically acceptable salts thereof, and mixtures
thereof.
12. A method according to claim 11 wherein said bisphosphonate is
alendronate, pharmaceutically acceptable salts thereof, and
mixtures thereof.
13. A method according to claim 11 wherein said bisphosphonate is
alendronate monosodium trihydrate.
14. A pharmaceutical composition comprising a therapeutically
effective amount of an EP.sub.4 receptor subtype antagonist.
15. A pharmaceutical composition according to claim 13 which
further comprises a pharmaceutically acceptable carrier.
16. A pharmaceutical composition according to claim 14 which
further comprises a therapeutically effective amount of a
bisphosphonate active.
17. A pharmaceutical composition according to claim 16 wherein said
bisphosphonate active corresponds to the chemical structure
4wherein n is an integer from 0 to 7 and wherein A and X are
independently selected from the group consisting of H, OH, halogen,
NH.sub.2, SH, phenyl, C1-C30 alkyl, C3-C30 branched or cycloalkyl,
C1-C30 substituted alkyl, C1-C10 alkyl substituted NH.sub.2, C3-C10
branched or cycloalkyl substituted NH.sub.2, C1-C10 dialkyl
substituted NH.sub.2, C1-C10 alkoxy, C1-C10 alkyl substituted thio,
thiophenyl, halophenylthio, C1-C10 alkyl substituted phenyl,
pyridyl, furanyl, pyrrolidinyl, imidazolyl, imidazopyridinyl, and
benzyl; or A and X are taken together with the carbon atom or atoms
to which they are attached to form a C3-C10 ring; and provided that
when n is 0, A and X are not selected from the group consisting of
H and OH; and the pharmaceutically acceptable salts thereof.
18. A pharmaceutical composition according to claim 16 wherein said
bisphosphonate is selected from the group consisting of
alendronate, cimadronate, clodronate, tiludronate, etidronate,
ibandronate, neridronate, olpandronate, risedronate, piridronate,
pamidronate, zolendronate, pharmaceutically acceptable salts
thereof, and mixtures thereof.
19. A pharmaceutical composition according to claim 18 wherein said
bisphosphonate is alendronate, pharmaceutically acceptable salts
thereof, and mixtures thereof.
20. A pharmaceutical composition according to claim 19 wherein said
bisphosphonate is alendronate monosodium trihydrate.
21. A method for identifying a compound which antagonizes an
EP.sub.4 receptor subtype comprising: a). contacting a putative
antagonist of an EP.sub.4 receptor subtype with a cell culture; and
b). determining the antagonist activity of said putative antagonist
with a cell culture not contacted with said putative
antagonist.
22. A method for identifying a compound which antagonizes an
EP.sub.4 receptor subtype comprising: a). contacting a putative
antagonist of an EP.sub.4 receptor subtype with an EP.sub.4
receptor; and b). determining the antagonist activity of said
putative antagonist with an EP.sub.4 receptor not contacted with
said putative antagonist.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority of U.S. provisional
application Serial No. 60/104,339, filed Oct. 15, 1998.
BRIEF DESCRIPTION OF THE INVENTION
[0002] The present invention relates to methods for inhibiting bone
resorption in a mammal comprising administering to a mammal in need
thereof a therapeutically effective amount of an EP.sub.4 receptor
subtype antagonist.
BACKGROUND OF THE INVENTION
[0003] A variety of disorders in humans and other mammals involve
or are associated with abnormal bone resorption. Such disorders
include, but are not limited to, osteoporosis, glucocorticoid
induced osteoporosis, Paget's disease, abnormally increased bone
turnover, periodontal disease, tooth loss, bone fractures,
rheumatoid arthritis, periprosthetic osteolysis, osteogenesis
imperfecta, metastatic bone disease, hypercalcemia of malignancy,
and multiple myeloma. One of the most common of these disorders is
osteoporosis, which in its most frequent manifestation occurs in
postmenopausal women. Osteoporosis is a systemic skeletal disease
characterized by a low bone mass and microarchitectural
deterioration of bone tissue, with a consequent increase in bone
fragility and susceptibility to fracture. Osteoporotic fractures
are a major cause of morbidity and mortality in the elderly
population. As many as 50% of women and a third of men will
experience an osteoporotic fracture. A large segment of the older
population already has low bone density and a high risk of
fractures. There is a significant need to both prevent and treat
osteoporosis and other conditions associated with bone resorption.
Because osteoporosis, as well as other disorders associated with
bone loss, are generally chronic conditions, it is believed that
appropriate therapy will typically require chronic treatment.
[0004] Normal bone physiology involves a process wherein bone
tissue is continuously being turned over by the processes of
modeling and remodeling. In other words, there is normally an
appropriate balance between resorption of existing bone tissue and
the formation of new bone tissue. The exact mechanism underlying
the coupling between bone resorption and formation is still
unknown. However, an imbalance in these processes is manifested in
various disease states and conditions of the skeleton.
[0005] Two different types of cells called osteoblasts and
osteoclasts are involved in the bone formation and resorption
processes, respectively. See H. Fleisch, Bisphosphonates In Bone
Disease, From The Laboratory To The Patient, 3rd Edition, Parthenon
Publishing (1997), which is incorporated by reference herein in its
entirety.
[0006] Osteoblasts are cells that are located on the bone surface.
These cells secrete an osseous organic matrix, which then
calcifies. Substances such as fluoride, parathyroid hormone, and
certain cytokines such as protaglandins are known to provide a
stimulatory effect on osetoblast cells. However, an aim of current
research is to develop therapeutic agents that will selectively
increase or stimulate the bone formation activity of the
osteoblasts.
[0007] Osteoclasts are usually large multinucleated cells that are
situated either on the surface of the cortical or trabecular bone
or within the cortical bone. The osteoclasts resorb bone in a
closed, sealed-off microenvironment located between the cell and
the bone. The recruitment and activity of osteoclasts is known to
be influenced by a series of cytokines and hormones. It is well
known that bisphosphonates are selective inhibitors of osteoclastic
bone resorption, making these compounds important therapeutic
agents in the treatment or prevention of a variety of systemic or
localized bone disorders caused by or associated with abnormal bone
resorption. However, despite the utility of bisphosphonates there
remains the desire amongst researchers to develop additional
therapeutic agents for inhibiting the bone resorption activity of
osteoclasts.
[0008] Prostaglandins are alicyclic compounds related to the basic
compound prostanoic acid. A natural prostaglandin, PGE.sub.2, has
the following structure. 1
[0009] Prostaglandins such as PGE.sub.2 are known to stimulate bone
formation and increase bone mass in mammals, including man. It is
believed that four different receptor subtypes, designated
EP.sub.1, EP.sub.2, EP.sub.3, and EP.sub.4 are involved in
mediating the bone modeling and remodeling processes of the
osteoblasts and osteoclasts. The major prostaglandin receptor in
bone is EP.sub.4, which is believed to provide its effect by
signaling via cyclic AMP. However, the scientific information that
is currently known about the prostaglandin mediated bone effect is
rather limited, because the exact mechanism of action is not known.
Prostaglandins and their accosted receptors are more fully
described in for example, K. Ono et al., Important role of
EP.sub.4, a subtype of prostaglandin (PG) E receptor, in
osteoclast-like cell formation from mouse bone marrow cells induced
by PGE.sub.2, J. of Endocrinology, 158, R1-R5 (1998), C. D. Funk et
al., Cloning and Expression of a cDNA for the Human Prostaglandin E
Receptor EP! Subtype, Journal of Biological Chemistry, vol. 268,
no. 35, pp. 26767-26772 (1993), J. W. Reagan et al., Cloning of a
Novel Human Prostaglandin Receptor with Characteristics of the
Pharmacologically Defined EP.sub.2 Subtype, Molecular Pharmacology,
vol. 46, pp. 213-220 (1994), J. Yang et al., Cloning and Expression
of the EP.sub.3-Subtype of Human Receptors for Prostaglandin
E.sub.2, Biochemical Biophysical Research Communication, vol., 198,
pp. 999-1006 (1994), L. Bastien et al., Cloning, Functional
Expression and Characterization of the Human Prostaglandin E.sub.2
Receptor EP.sub.2 Subtype, Journal Biological Chemistry, vol. 269,
pp. 11873-11877 (1994), which are all incorporated by reference
herein in their entirety.
[0010] In the present invention it is found that antagonists of the
EP.sub.4 subtype receptor are useful for inhibiting bone
resorption. Without being limited by theory, it is believed that
these antagonists are responsible for inhibiting the bone
resorption activity of the osteoclasts.
[0011] It is an object of the present invention to provide methods
for inhibiting bone resorption in a mammal comprising administering
to a mammal in need thereof a therapeutically effective amount of
an EP.sub.4 receptor subtype antagonist.
[0012] It is another object of the present invention to provide
methods for treating or reducing the risk of contracting a disease
state or condition in a mammal in need of such treatment or
prevention, comprising administering to said mammal a
therapeutically effective amount of an EP.sub.4 receptor subtype
antagonist.
[0013] It is another object of the present invention to provide
methods for inhibiting bone resorption in a mammal in need thereof
comprising administering to said mammal a therapeutically effective
amount of an EP.sub.4 receptor subtype antagonist and a
bisphosphonate active.
[0014] It is another object of the present invention to provide
pharmaceutical compositions comprising a therapeutically effective
amount of an EP.sub.4 receptor subtype antagonist.
[0015] It is another object of the present invention to provide
pharmaceutical compositions comprising a therapeutically effective
amount of an EP.sub.4 receptor subtype antagonist and a
bisphosphonate active.
[0016] It is another object of the present invention to identify
EP.sub.4 receptor subtype antagonists useful for inhibiting bone
resorption.
[0017] These and other objects will become readily apparent from
the detailed description which follows.
SUMMARY OF THE INVENTION
[0018] The present invention relates to methods for inhibiting bone
resorption in a mammal comprising administering to a mammal in need
thereof a therapeutically effective amount of an EP.sub.4 receptor
subtype antagonist having an EC.sub.50 value of from about 0.1
nanoM to about 100 microM.
[0019] In further embodiments, the present invention relates to
methods for treating or reducing the risk of contracting a disease
state or condition involving bone tissue in a mammal in need of
such treatment or risk reduction, comprising administering to said
mammal a therapeutically effective amount of an EP.sub.4 receptor
subtype antagonist.
[0020] In further embodiments, the present invention relates to
methods for inhibiting bone resorption in a mammal in need thereof
comprising administering to said mammal a therapeutically effective
amount of an EP.sub.4 receptor subtype antagonist and a
bisphosphonate active.
[0021] In further embodiments, the present invention relates to
pharmaceutical compositions comprising a therapeutically effective
amount of an EP.sub.4 receptor subtype antagonist.
[0022] In further embodiments, the present invention relates to
pharmaceutical compositions comprising a therapeutically effective
amount of an EP.sub.4 receptor subtype antagonist and a
bisphosphonate active.
[0023] In further embodiments, the present invention relates to a
method for identifying antagonists of an EP.sub.4 receptor
subtype.
[0024] In further embodiments, the present invention relates to the
use of a composition in the manufacture of a medicament for
inhibiting bone resorption in a mammal comprising administering to
a mammal in need thereof a therapeutically effective amount of an
EP.sub.4 receptor subtype antagonist.
[0025] All percentages and ratios used herein, unless otherwise
indicated, are by weight. The invention hereof can comprise,
consist of, or consist essentially of the essential as well as
optional ingredients, components, and methods described herein.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The present invention relates to methods for inhibiting bone
resorption in a mammal comprising administering to a mammal in need
thereof a therapeutically effective amount of an EP.sub.4 receptor
subtype antagonist having an EC.sub.50 value of from about 0.1
nanoM to about 100 microM.
[0027] Prostaglandins E (especially PGE.sub.2) stimulate bone
formation and increase bone mass in several species, including man.
The mechanism of this effect, the target cells and the receptors
involved are not completely known. Specific cell-surface receptors
for PGE.sub.2, such as EP.sub.1-4, which employ different secondary
messenger systems have been cloned and characterized. It is
believed that cyclic AMP may have a role in osteogenesis induced by
PGE.sub.2. The expression of the EP.sub.2 and EP.sub.4 receptors is
found to be involved in cAMP production in the bone tissue of young
adult rats (where PGE.sub.2 is markedly anabolic), and in various
osteoblastic cell lines. Osteoblastic cell lines, RCT-1, RCT-3,
TRAB-11 and RP-1, as well as osteoblastic cells harvested from
fetal rat bones express EP.sub.4 mRNA but not EP.sub.2 mRNA. In
addition, EP.sub.4 mRNA is expressed in tibiae and calvariae of
5-week-old rats while EP.sub.2 is not. Treatment of periosteal
cells (RP-1) in vitro with 10.sup.-6 M PGE.sub.2 increases the
level of EP.sub.4 mRNA which peaks at 2 hours. Similarly, systemic
administration of an anabolic dose of PGE.sub.2 (3-6 mg/kg) to
young adult rats upregulates the expression of EP.sub.4 in tibiae
and calvariae, an effect which peaks at 1-2 hours. Using in-situ
hybridization it is found that the increased expression of EP.sub.4
mRNA in the tibial metaphysis following systemic PGE.sub.2
treatment is localized to bone marrow cells.
[0028] EP.sub.4 is expressed in osteoblastic cells in vitro and in
bone marrow putative osteoprogenitor cells in vivo and is
upregulated by its ligand, PGE.sub.2. Given the presence of
EP.sub.4 expression in the cells examined and in bone tissue, it is
believed that EP.sub.4 is the receptor subtype which mediates the
anabolic effects of PGE.sub.2.
[0029] Prostaglandins (especially PGE.sub.2) have multiple effects
on bone, stimulating both resorption and formation. Systemic
administration of PGE.sub.2 or E.sub.1 to infants and to animals is
clearly anabolic, stimulating bone formation and increases bone
mass. Also local administration of PGE.sub.2 into long bones
stimulates new bone formation, suggesting that that PGE.sub.2 acts
directly on bone tissue to induce osteogenesis. Histological
analysis of bones treated with PGE.sub.2 indicates that PGE.sub.2
increases the number of osteoblasts present on the bone surface,
suggesting that prostaglandins act by recruiting osteoblasts from
their precursors.
[0030] PGEs act on various cells via specific cell-surface
receptors divided into 4 subtypes, EP.sub.1-4, according to their
relative sensitivity to selective agonists and antagonists. The
receptor subtypes all belong to the G-protein-coupled receptor
family and activate different secondary messenger systems such as
adenylate cyclase or phospholipase C. Of these 4 receptors,
EP.sub.4 and EP.sub.2 activate adenylate cyclase, EP.sub.1
activates phospholipase C, and EP.sub.3 either lowers intracellular
cAMP levels or activates phospholipase C, depending on the specific
spliced variant.
[0031] In osteoblastic cells in vitro, PGE.sub.2 stimulates both
phosphatidylinositol and cyclic AMP transduction pathways. Both
EP.sub.1 and EP.sub.4, found in osteoblastic MC3T3-E.sub.1 cells
are believed to play a role in the biological action of PGE.sub.2
in bone tissue. Also PGE.sub.1, a potent inducer of bone formation
in humans and other species, increases intracellular cyclic AMP but
has no effect on phosphatidylinositol turnover in osteoblastic
cells. It is therefor believed that PGE receptors coupled to
adenylate cyclases, EP.sub.2 and/or EP.sub.4, are involved in
osteogenesis. It is also believed that the cyclic AMP pathway is
involved in the recruitment of osteoblasts from bone marrow cells.
Initial characterization of in vivo expression of EP receptors by
in situ hybridization shows that in embryonic and neonatal mice
EP.sub.4 is the major form found in bone tissue, especially in
preosteoblasts. See Ikeda T, Miyaura C, Ichikawa A, Narumiya S,
Yoshiki S and Suda T, 1995, In situ localization of three subtypes
(EP.sub.1, EP.sub.3 and EP.sub.4) of prostaglandin E receptors in
embryonic and newborn mice. J Bone Miner Res 10 (sup 1):S 172,
which is incorporated by reference herein in its entirety.
[0032] Also, it is found that EP.sub.4 but not EP.sub.2 mRNA is
expressed in adult rat bone tissue and bone-derived cell lines and
that expression is stimulated by PGE.sub.2.
[0033] Analysis of the in vivo expression of PGE receptors shows
that EP4 but not EP.sub.2 is expressed in total RNA from adult rat
tibiae and calvariae. EP.sub.4 is believed to be the major
adenylate cyclase-coupled PGE.sub.2 receptor expressed in
osteoblastic cells and in bone tissue. Also, the EP.sub.4 receptor
subtype is expressed in the bone tissue of young adult rats, in
which PGE.sub.2 is strongly anabolic.
[0034] EP.sub.4 mRNA is expressed in osteoblast precursor cells. It
is also found in less differentiated bone cell lines such as RCT-1,
TRAB-11 and the RP-1 periosteal cells, but not in fibroblasts. It
is highly expressed in bone marrow cells that include osteoblast
precursor cells, but not in fully mature osteoblasts on the bone
surface. It is believed that PGE.sub.2 induces osteogenesis via an
increase in the number of active osteoblasts present on the bone
surface, resulting from the recruitment of osteoblast precursor
cells rather than the enhancement of the activity of existing
osteoblasts.
[0035] It is found that osteoblast precursors are the major target
cells for the anabolic effect of PGE.sub.2 and that its action in
these cells is mediated by EP.sub.4. The EP.sub.4 receptor subtype
is believed to be the major receptor which mediates the effects of
PGE.sub.2 in bone tissue rats. Induction of EP.sub.4 by PGE.sub.2
further supports its biological role in the bone tissue and points
to a mechanism of autoamplification of PGE action.
Methods of Inhibiting Bone Resorption
[0036] The present invention relates to methods for inhibiting bone
resorption in a mammal comprising administering to a mammal in need
thereof a therapeutically effective amount of an EP.sub.4 receptor
subtype antagonist.
[0037] The methods and compositions of the present invention are
useful for both treating and reducing the risk of disease states or
conditions associated with abnormal bone resorption. Such disease
states or conditions include, but are not limited to, osteoporosis,
glucocorticoid induced osteoporosis, Paget's disease, abnormally
increased bone turnover, periodontal disease, tooth loss, bone
fractures, rheumatoid arthritis, periprosthetic osteolysis,
osteogenesis imperfecta, metastatic bone disease, hypercalcemia of
malignancy, and multiple myeloma.
[0038] In further embodiments, the methods comprise administering a
therapeutically effective amount of an EP.sub.4 receptor subtype
antagonist and a bisphosphonate active. Both concurrent and
sequential administration of the EP.sub.4 receptor subtype
antagonist and the bisphosphonate active are deemed within the
scope of the present invention. With sequential administration, the
antagonist and the bisphosphonate can be administered in either
order. In a subclass of sequential administration the antagonist
and bisphosphonate are typically administered within the same 24
hour period. In yet a further subclass, the antagonist and
bisphosphonate typically administered within about 4 hours of each
other.
[0039] The term "therapeutically effective amount", as used herein,
means that amount of the EP.sub.4 receptor subtype antagonist, or
other actives of the present invention, that will elicit the
desired therapeutic effect or response or provide the desired
benefit when administered in accordance with the desired treatment
regimen. A prefered therapeutically effective amount is a bone
resorption inhibiting amount.
[0040] "Pharmaceutically acceptable" as used herein, means
generally suitable for administration to a mammal, including
humans, from a toxicity or safety standpoint.
[0041] In the present invention, the agonist is typically
administered for a sufficient period of time until the desired
therapeutic effect is achieved. The term "until the desired
therapeutic effect is achieved", as used herein, means that the
therapeutic agent or agents are continuously administered,
according to the dosing schedule chosen, up to the time that the
clinical or medical effect sought for the disease or condition
being mediated is observed by the clinician or researcher. For
methods of treatment of the present invention, the compounds are
continuously administered until the desired change in bone mass or
structure is observed. In such instances, achieving an increase in
bone mass or a replacement of abnormal bone structure with normal
bone structure are the desired objectives. For methods of reducing
the risk of a disease state or condition, the compounds are
continuously administered for as long as necessary to prevent the
undesired condition. In such instances, maintenance of bone mass
density is often the objective.
[0042] Nonlimiting examples of administration periods can range
from about 2 weeks to the remaining lifespan of the mammal. For
humans, administration periods can range from about 2 weeks to the
remaining lifespan of the human, preferably from about 2 weeks to
about 20 years, more preferably from about 1 month to about 20
years, more preferably from about 6 months to about 10 years, and
most preferably from about 1 year to about 10 years.
[0043] Methods of Identifying Antagonists of The EP.sub.4 Receptor
Subtype
[0044] The present invention also relates to methods for
identifying compounds useful as antagonists of the EP.sub.4
receptor subtype. Compounds so identified are useful for inhibiting
bone resorption.
[0045] The present invention relates to a method for identifying
compounds which antagonize an EP.sub.4 receptor subtype
comprising:
[0046] a). contacting a putative antagonist of an EP.sub.4 receptor
subtype with a cell culture; and
[0047] b). determining the antagonist activity of said putative
agonist with a cell culture not contacted with said putative
antagonist.
[0048] Compositions of The Present Invention
[0049] The pharmaceutical compositions of the present invention
comprise a therapeutically effective amount of an EP.sub.4 receptor
antagonist.
[0050] These compositions can further comprise a pharmaceutically-
acceptable carrier. In further embodiments these compositions also
comprise a bisphosphonate active.
[0051] EP.sub.4 Receptor Subtype Antagonist
[0052] The methods and compositions of the present invention
comprise an EP.sub.4 receptor subtype antagonist.
[0053] The term "antagonist" as used herein, is used in its
standard meaning to mean a chemical substance that opposed the
physiological effects of another substance. In other words, an
antagonist is a chemical substance that opposes the
receptor-associated responses normally induced by another bioactive
agent.
[0054] The antagonists useful herein generally have an EC.sub.50
value from about 0.1 nM to about 100 microM, although antagonists
with activities outside this range can be useful depending upon the
dosage and route of administration. In a subclass of the present
invention, the antagonists have an EC.sub.50 value of from about
0.01 microM to about 10 microM. In a further subclass of the
present invention, the antagonists have an EC.sub.50 value of from
about 0.1 microM to about 10 microM. EC.sub.50 is a common measure
of antagonist activity well known to those of ordinary skill in the
art and is defined as the concentration or dose of an antagonist
that is needed to produce half, i.e. 50%, of the maximal effect.
See also, Goodman and Gilman's, The Pharmacologic Basis of
Therapeutics, 9th edition, 1996, chapter 2, E. M. Ross,
Pharmacodynamics, Mechanisms of Drug Action and the Relationship
Between Drug Concentration and Effect, which is incoroporated by
reference herein in its entirety.
[0055] Nonlimiting examples of antagonists useful herein are
selected fom the group consisting of
[0056]
5-butyl-2,4-dihydro-4-[[2'-[N-(3-chloro-2-thiophenecarbonyl)sulfamo-
yl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-one
potassium salt,
[0057]
5-butyl-2,4-dihydro-4-[[2'-[N-(2-methyl-3-furoyl)sulfamoyl]biphenyl-
4-yl]methyl]-2-[2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-one,
[0058]
5-butyl-2,4-dihydro-4-[[2'-[N-(3-methyl-2-thiophenecarbonyl)sulfamo-
yl]biphenyl-4-yl]methyl]-2-[(2-trifluoromethyl)phenyl]-1,2,4-triazol-3-one-
,
[0059]
5-butyl-2,4-dihydro-4-[[2'-[N-(2-thiophenecarbonyl)sulfamoyl]biphen-
yl-4-yl]methyl]-2-[(2-trifluoromethyl)phenyl]-1,2,4-triazol-3-one,
[0060]
5-butyl-2,4-dihydro-4-[[2'-[N-[2-(methylpyrrole)carbonyl]sulfamoyl]-
biphenyl-4-yl]methyl]-2-[(2-trifluoromethyl)phenyl-1
,2,4-triazol-3-one,
[0061] and the pharmaceutically acceptable salts thereof, and
mixtures thereof.
[0062] In the present invention, the antagonists useful herein are
compounds that do not contain a cyclopentanone or
hydroxycyclopentane ring. In other words, these are
non-cyclopentanone and non-hydroxycyclopentane structures.
[0063] Bisphosphonates
[0064] The methods and compositions of the present invention, can
further comprise a bisphosphonate active. The bisphosphonates of
the present invention correspond to the chemical formula 2
[0065] wherein n is an integer from 0 to 7 and wherein A and X are
independently selected from the group consisting of H, OH, halogen,
NH.sub.2, SH, phenyl, C1-C30 alkyl, C3-C30 branched or cycloalkyl,
C1-C30 substituted alkyl, C1-C10 alkyl substituted NH.sub.2, C3-C10
branched or cycloalkyl substituted NH.sub.2, C1-C10 dialkyl
substituted NH.sub.2, C3-C10 branched or cycloalkyl disubstituted
NH.sub.2, C1-C10 alkoxy, C1-C10 alkyl substituted thio, thiophenyl,
halophenylthio, C1-C10 alkyl substituted phenyl, pyridyl, furanyl,
pyrrolidinyl, imidazolyl, imidazopyridinyl, and benzyl, such that
both A and X are not selected from H or OH when n is 0; or A and X
are taken together with the carbon atom or atoms to which they are
attached to form a C3-C10 ring.
[0066] In the foregoing chemical formula, the alkyl groups can be
straight, branched, or cyclic, provided that sufficient atoms are
selected for the chemical formula. The C1-C30 substituted alkyl can
include a wide variety of substituents, nonlimiting examples which
include those selected from the group consisting of phenyl,
pyridyl, furanyl, pyrrolidinyl, imidazonyl, NH.sub.2, C1-C10 alkyl
or dialkyl substituted NH.sub.2, OH, SH, and C1-C10 alkoxy.
[0067] The foregoing chemical formula is also intended to encompass
complex carbocyclic, aromatic and hetero atom structures for the A
and/or X substituents, nonlimiting examples of which include
naphthyl, quinolyl, isoquinolyl, adamantyl, and
chlorophenylthio.
[0068] A non-limiting class of structures useful in the instant
invention are those in which A is selected from the group
consisting of H, OH, and halogen, X is selected from the group
consisting of C1-C30 alkyl, C1-C30 substituted alkyl, halogen, and
C1-C10 alkyl or phenyl substituted thio, and n is 0.
[0069] A non-limiting subclass of structures useful in the instant
invention are those in which A is selected from the group
consisting of H, OH, and C1, X is selected from the group
consisting of C1-C30 alkyl, C1-C30 substituted alkyl, C1, and
chlorophenylthio, and n is 0.
[0070] A non-limiting example of the subclass of structures useful
in the instant invention is when A is OH and X is a 3-aminopropyl
moiety, and n is 0, so that the resulting compound is a
4-amino-1,-hydroxybutylidene-1,- 1-bisphosphonate, i.e.
alendronate.
[0071] Pharmaceutically acceptable salts and derivatives of the
bisphosphonates are also useful herein. Nonlimiting examples of
salts include those selected from the group consisting alkali
metal, alkaline metal, ammonium, and mono-, di, tri-, or
tetra-C1-C30-alkyl-substituted ammonium. Preferred salts are those
selected from the group consisting of sodium, potassium, calcium,
magnesium, and ammonium salts. Nonlimiting examples of derivatives
include those selected from the group consisting of esters,
hydrates, and amides.
[0072] It should be noted that the terms "bisphosphonate" and
"bisphosphonates", as used herein in referring to the therapeutic
agents of the present invention are meant to also encompass
diphosphonates, biphosphonic acids, and diphosphonic acids, as well
as salts and derivatives of these materials. The use of a specific
nomenclature in referring to the bisphosphonate or bisphosphonates
is not meant to limit the scope of the present invention, unless
specifically indicated. Because of the mixed nomenclature currently
in use by those or ordinary skill in the art, reference to a
specific weight or percentage of a bisphosphonate compound in the
present invention is on an acid active weight basis, unless
indicated otherwise herein. For example, the phrase "about 5 mg of
a bisphosphonate selected from the group consisting of alendronate,
pharmaceutically acceptable salts thereof, and mixtures thereof, on
an alendronic acid active weight basis" means that the amount of
the bisphosphonate compound selected is calculated based on 5 mg of
alendronic acid. For other bisphosphonates, the amount of
bisphosphonate is calculated based on the corresponding
bisphosphonic acid.
[0073] Nonlimiting examples of bisphosphonates useful herein
include the following:
[0074] Alendronic acid,
4-amino-l-hydroxybutylidene-1,1-bisphosphonic acid.
[0075] Alendronate (also known as alendronate sodium or alendronate
monosodium trihydrate),
4-amino-1-hydroxybutylidene-1,1-bisphosphonic acid monosodium
trihydrate.
[0076] Alendronic acid and alendronate are described in U.S. Pat.
Nos. 4,922,007, to Kieczykowski et al., issued May 1, 1990;
5,019,651, to Kieczykowski et al., issued May 28, 1991; 5,510,517,
to Dauer et al., issued Apr. 23, 1996; 5,648,491, to Dauer et al.,
issued Jul. 15, 1997, all of which are incorporated by reference
herein in their entirety.
[0077] Cycloheptylaminomethylene-1,1-bisphosphonic acid, YM 175,
Yamanouchi (cimadronate), as described in U.S. Pat. No. 4,970,335,
to Isomura et al., issued Nov. 13, 1990, which is incorporated by
reference herein in its entirety.
[0078] 1,1-dichloromethylene-1,1-diphosphonic acid (clodronic
acid), and the disodium salt (clodronate, Procter and Gamble), are
described in Belgium Patent 672,205 (1966) and J. Org. Chem 32,
4111 (1967), both of which are incorporated by reference herein in
their entirety.
[0079] 1-hydroxy-3-(1-pyrrolidinyl)-propylidene-1,1-bisphosphonic
acid (EB-1053).
[0080] 1-hydroxyethane-1,1-diphosphonic acid (etidronic acid).
[0081]
1-hydroxy-3-(N-methyl-N-pentylamino)propylidene-1,1-bisphosphonic
acid, also known as BM-210955, Boehringer-Mannheim (ibandronate),
is described in U.S. Pat. No. 4,927,814, issued May 22, 1990, which
is incorporated by reference herein in its entirety.
[0082] 6-amino-1-hydroxyhexylidene-1,1-bisphosphonic acid
(neridronate).
[0083] 3-(dimethylamino)-1-hydroxypropylidene-1,1-bisphosphonic
acid (olpadronate).
[0084] 3-amino-1-hydroxypropylidene-1,1-bisphosphonic acid
(pamidronate).
[0085] [2-(2-pyridinyl)ethylidene]-1,1-bisphosphonic acid
(piridronate) is described in U.S. Pat. No. 4,761,406, which is
incorporated by reference in its entirety.
[0086] 1-hydroxy-2-(3-pyridinyl)-ethylidene-1,1-bisphosphonic acid
(risedronate).
[0087] (4-chlorophenyl)thiomethane-1,1-disphosphonic acid
(tiludronate) as described in U.S. Pat. No. 4,876,248, to Breliere
et al., Oct. 24, 1989, which is incorporated by reference herein in
its entirety.
[0088]
1-hydroxy-2-(1.sup.H-imidazol-1-yl)ethylidene-1,1-bisphosphonic
acid (zolendronate).
[0089] A non-limiting class of bisphosphonates useful in the
instant invention are selected from the group consisting of
alendronate, cimadronate, clodronate, tiludronate, etidronate,
ibandronate, neridronate, olpandronate, risedronate, piridronate,
pamidronate, zolendronate, pharmaceutically acceptable salts
thereof, and mixtures thereof.
[0090] A non-limiting subclass of the above-mentioned class in the
instant case is selected from the group consisting of alendronate,
pharmaceutically acceptable salts thereof, and mixtures
thereof.
[0091] A non-limiting example of the subclass is alendronate
monosodium trihydrate.
[0092] Other Components of the Pharmaceutical Compositions
[0093] The EP.sub.4 receptor subtype antagonists, and in further
embodiments the bisphosphonate actives and any other additional
actives are typically administered in admixture with suitable
pharmaceutically acceptable diluents, excipients, or carriers,
collectively referred to herein as "carrier materials", suitably
selected with respect to the mode of administration. Nonlimiting
examples of product forms include tablets, capsules, elixirs,
syrups, powders, suppositories, nasal sprays, liquids for ocular
administration, formulations for transdermal administration, and
the like, consistent with conventional pharmaceutical practices.
For example, for oral administration in the form of a tablet,
capsule, or powder, the active ingredient can be combined with an
oral, non-toxic, pharmaceutically acceptable inert carrier such as
lactose, starch, sucrose, glucose, methyl cellulose, magnesium
stearate, mannitol, sorbitol, croscarmellose sodium and the like.
For oral administration in liquid form, e.g., elixirs and syrups,
the oral drug components can be combined with any oral, non-toxic,
pharmaceutically acceptable inert carrier such as ethanol,
glycerol, water and the like. Moreover, when desired or necessary,
suitable binders, lubricants, disintegrating agents and coloring
agents can also be incorporated. Suitable binders can include
starch, gelatin, natural sugars such a glucose, anhydrous lactose,
free-flow lactose, beta-lactose, and corn sweeteners, natural and
synthetic gums, such as acacia, guar, tragacanth or sodium
alginate, carboxymethyl cellulose, polyethylene glycol, waxes, and
the like. Lubricants used in these dosage forms include sodium
oleate, sodium stearate, magnesium stearate, sodium benzoate,
sodium acetate, sodium chloride and the like. An example of a
tablet formulation is that described in U.S. Pat. No. 5,358,941, to
Bechard et al, issued Oct. 25, 1994, which is incorporated by
reference herein in its entirety. The compounds used in the present
method can also be coupled with soluble polymers as targetable drug
carriers. Such polymers can include polyvinylpyrrolidone, pyran
copolymer, polyhydroxylpropyl- methacrylamide, and the like.
[0094] The following Examples are presented to better illustrate
the invention.
EXAMPLES
[0095] 1. Animal Procedures
[0096] For mRNA localization experiments, 5-week old Sprague-Dawley
rats (Charles River) are euthanized by CO.sub.2, their tibiae and
calvariae are excised, cleaned of soft tissues and frozen
immediately in liquid nitrogen. For EP.sub.4 regulation
experiments, 6-week old rats are given a single injection of either
vehicle (7% ethanol in sterile water) or an anabolic dose of
PGE.sub.2 (Cayman Chemical, Ann Arbor, Mich.), 3-6 mg/kg in the
same vehicle) intraperitoneally. Animals are euthenized at several
time points post-injection and their tibiae and calvariae, as well
as samples from lung and kidney tissues are frozen in liquid
nitrogen.
[0097] 2. Cell Cultures
[0098] RP-1 periosteal cells are spontaneously immortalized from
primary cultures of periosteal cells from tibae of 4-week old
Sprague-Dawley rats and are cultured in DMEM (BRL, Gaithersburg,
Md.) with 10% fetal bovine serum (JRH Biosciences, Lenexa, Kans.).
These cells do not express osteoblastic phenotypic markers in early
culture, but upon confluence, express type I collagen, alkaline
phosphatase and osteocalcin and produce mineralized extracellular
matrix.
[0099] RCT-1 and RCT-3 are clonal cell lines immortalized by SV-40
large T antigen from cells released from fetal rat calvair by a
cmbination collagenase/hyaluronidase digestion. RCT-1 cells,
derived from cells released during the first 10 minutes of
digestion (fraction I), are cultured in RPMI 1640 medium (BRL) with
10% fetal bovine serum and 0.4 mg/ml G418 (BRL). These cells
differentiate and express osteoblastic features upon retinoic acid
treatment. RCT-3 cells, immortalized from osteoblast-enriched
fraction III cells, are cultured in F-12 medium (BRL) with 5% Fetal
bovine serum and 0.4 mg/ml G418. TRAB-11 cells are also
immortalized by SV40 large T antigen from adult rat tibia and are
cultured in RPMI 1640 medium with 10% FBS and 0.4 mg/ml G418. ROS
17/2.8 rat osteosarcoma cells are cultured in F-12 containing 5%
FBS. Osteoblast-enriched (fraction III) primary fetal rat calvaria
cells are obtained by collagenase/hyaluronidase digestion of
calvariae of 19 day-old rat fetuses. See Rodan et al., Growth
stimulation of rat calvaria osteoblastic cells by acidic FGF,
Endocrinology, 121, 1919-1923 (1987), which is incorporated by
reference herein in its entirety. Cells are released during 30-50
minutes digestion (fraction III) and are cultured in F-12 medium
containing 5% FBS.
[0100] P815 (mouse mastocytoma) cell, cultured in Eagles MEM with
10% FBS, and NRK (normal rat kidney fibroblasts) cells, cultured in
DMEM with 10% FBS, are used as positive and negative controls for
the expression of EP.sub.4, respectively. See Abramovitz et al.,
Human prostanoid receptors: cloning and characterization. In:
Samulesson B. et al. ed) Advances in prostaglandin, Thrombosznes
and leukotriene research, vol. 23, pp. 499-504 (1995) and de Larco
et al., Epithelioid and fibroblastic rat kidney cell clones: EGF
receptors and the effect of mouse sarcoma virus transformation,
Cell Physiol., 94, 335-342 (1978), which are both incorporated by
reference herein in their entirety.
[0101] 3. Northern Blot Analysis
[0102] Total RNA is extracted from the tibial metaphysis or
diaphysis and calvaria using a guanidinium
isothiocyanate-phenol-chloroform method after pulverizing frozen
bone samples by a tissue homogenizer. See P. Chomczynski et al.,
Single-step method of RNA isolation by acid guanidium
thiocyanate-phenol-chloroform extraction., Analyt Biochem, 162,
156-159 (1987), which is incorporated by reference herein in its
entirety. RNA samples (20 mg) are separated on 0.9%
agarose/formaldehyde gels and transferred onto nylon membranes
(Boehringer Mannheim, Germany). Membranes are prehybridized in
Hybrisol I (Oncor, Gaithersburg, Md.) and 0.5 mg/ml sonicated
salmon sperm DNA (Boehringer) at 42.degree. C. for 3 hours and are
hybridized at 42.degree. C. with rat EP.sub.2 and mouse EP.sub.4
cDNA probes labeled with [.sup.32P]-dCTP (Amersham,
Buckinghamshire, UK) by random priming using the rediprime kit
(Amersham). After hybridization, membranes are washed 4 times in
2.times.SSC+0.1% SDS at room temperature for a total of 1 hour and
once with 0.2.times.SSC+0.1% SDS at 55.degree. C. for 1 hour and
then exposed to Kodak XAR 2 film at -70.degree. C. using
intensifying screens. After developing the films, bound probes are
removed twice with 0.1% SDS at 80.degree. C. and membranes are
hybridized with a human GAPDH (Glyceraldehyde 3-Phosphate
Dehydrogenase) cDNA probe (purchased from Clontech, Palo Alto,
Calif.) for loading control.
[0103] 4. In-Situ Hybridization
[0104] Frozen tibiae are sectioned coronally at 7 mm thickness and
sections are mounted on charged slides (Probe On Plus, Fisher
Scientific, Springfield, N.J.) and are kept at -70.degree. C. until
hybridization. cRNA probes are labeled with .sup.35S-UTPgS (ICN,
Costa Mesa, Calif.) using a Riboprobe II kit (Promega Madison,
Wis.). Hybridization is performed overnight at 50.degree. C. See M.
Weinreb et al., Different pattern of alkaline phosphatase,
osteopontin and osteocalcin expression in developing rat bone
visualized by in-situ hybridization, J. Bone Miner Res., 5, 831-842
(1990) and D. Shinar et al., Expression of alphav and beta3
integrin subunits in rat osteoclasts in situ, J. Bone Miner. Res.,
8, 403-414 (1993), which are both incorporated by reference herein
in their entirety. Following hybridization and washing, sections
are dipped in Ilford K5 emulsion diluted 2:1 with 6% glycerol in
water at 42.degree. C. and exposed in darkness at 4.degree. C. for
12-14 days. Slides are developed in Kodak D-19 diluted 1:1 with
water at 15.degree., fixed-, washed in distilled water and mounted
with glycerol-gelatin (Sigma) after hematoxylin staining. Stained
sections are viewed under the microscope (Olympus, Hamburg,
Germany), using either bright-field or dark-field optics.
[0105] 5. Expression of EP.sub.4 in Osteoblastic Cell Lines and in
Bone Tissue
[0106] The expression of EP.sub.4 and EP.sub.2 mRNA is examined in
various bone derived cells including osteoblast-enriched primary
rat calvaria cells, immortalized osteoblastic cell lines from fetal
rat calvaria or from adult rat tibia and an osteoblastic
osteosarcoma cell line. Most of the osteoblastic cells and cell
lines show significant amounts of 3.8 kb EP.sub.4 mRNA, except for
the rat osteosarcoma cell line ROS 17/2.8. Consistent with this
finding, in ROS 17/2.8 cells PGE.sub.2 has no effect on
intracellular cAMP, which is markedly induced in RCT-3 and TRAB-1 1
cells. Treatment of RCT-1 cells with retinoic acid, which promotes
their differentiation, reduces the levels of EP.sub.4 mRNA. NRK
fibroblasts do not express EP.sub.4 mRNA, while P815 mastocytoma
cells, used as positive controls, express large amounts of EP.sub.4
mRNA. In contrast to EP.sub.4 mRNA, none of the osteoblastic cells
and cell lines express detectable amounts of EP.sub.2 mRA in total
RNA samples. Expression of EP.sub.4 mRNA in osteoblastic cells,
EP.sub.4 is also expressed in total RNA isolated from tibiae and
calvariae of 5-week-old rats. In contrast, no EP.sub.2 mRNA is
found in RNA from tibial shafts.
[0107] 6. PGE.sub.2 Induces the Expression of EP mRNA in RP-1
Periosteal Cells and in Adult Rat Tibiae
[0108] PGE.sub.2 enhances its own production via upregulation of
cyclooxygenase 2 expression in osteoblasts and in bone tissue thus
autoamplifying its own effects. The effect of PGE.sub.2 on the
levels of EP.sub.4 mRNA. RP-1 cells are immortalized from a primary
culture of adult rat tibia periosteum is examined. These cells
express osteoblast phenotypic makers upon confluence and form
mineralized bone matrix when implanted in nude mice. Similar to the
other osteoblastic cells examined, RP-1 periosteal cells express a
3.8 kb EP.sub.4 transcript. Treatment with PGE.sub.2 (10-.sup.6 M)
rapidly increases EP.sub.4 mRNA levels peaking at 2 hours after
treatment. PGE.sub.2 has no effect on EP.sub.4 mRNA levels in the
more differentiated RCT-3 cells. Cell-type specific regulation of
EP.sub.4 expression by PGE.sub.2. EP.sub.2 mRNA is not expressed in
RP-1 cells before or after treatment with PGE.sub.2.
[0109] To examine if PGE.sub.2 regulates EP.sub.4 mRNA levels in
vivo in bone tissue, week-old male rats are injected with PGE.sub.2
(3-6 mg/Kg). Systemic administration of PGE.sub.2 rapidly increased
EP.sub.4 mRNA levels in the tibialt diaphysis peaking at 2 h after
injection. A similar effect of PGE.sub.2 on EP.sub.4 mRNA is
observed in the tibial metaphysis and in calvaria. PGE.sub.2
induces EP.sub.4 mRNA levels in vitro in osteogenic periosteal
cells and in vivo in bone tissue in a cell-specific and
tissue-specific manner. PGE.sub.2 does not include EP.sub.2 mRNA in
RP-1 cells nor in bone tissue.
[0110] 7. Localization of EP.sub.4 mRNA Expression in Bone
Tissue
[0111] In situ hybridization is used in order to localize cells
expressing EP.sub.4 in bone. In control experiment
(vehicle-injected) rats, low expression of EP.sub.4 was detected in
bone marrow cells. Administration of a single anabolic dose of
PGE.sub.2 increasesd the expression of EP.sub.4 in bone marrow
cells. The distribution of silver grains over the bone marrow is
not uniform and occurs in clumps or patches in many areas of the
metaphysis. Within the tibial metaphysis, EP.sub.4 expression is
restricted to the secondary spongiosa area and is not seen in the
primary spongiosa. Hybridization of similar sections with a sense
probe (negative control) did not show any signal.
[0112] EP.sub.4 is expressed in osteoblastic cells in vitro and in
bone marrow cells in vivo and is upregulated by its ligand,
PGE.sub.2.
[0113] 8. Antagonists of the Present Invention
[0114] Using standard methods for measuring antagonist activity,
the following compounds are evaluated in cell cultures and in
EP.sub.4 receptor cell-free systems to determine the antagonist
activity of the compounds in terms of their EC.sub.50 value:
[0115]
5-butyl-2,4-dihydro-4-[[2'-[N-(3-chloro-2-thiophenecarbonyl)sulfamo-
yl]biphenyl-4-yl]methyl]-2-{2-(trifluoromethyl)phenyl]-1
,2,4-triazol-3-one potassium salt,
[0116]
5-butyl-2,4-dihydro-4-[[2'-[N-(2-methyl-3-furoyl)sulfamoyl]biphenyl-
4-yl]methyl]-2-[2-(trifluoromethyl)phenyl]-1,2,4-triazol-3-one,
[0117]
5-butyl-2,4-dihydro-4-[[2'-[N-(3-methyl-2-thiophenecarbonyl)sulfamo-
yl]biphenyl-4-yl]methyl]-2-[(2-trifluoromethyl)phenyl]-1,2,4-triazol-3-one-
,
[0118]
5-butyl-2,4-dihydro-4-[[2'-[N-(2-thiophenecarbonyl)sulfamoyl]biphen-
yl-4-yl]methyl]-2-[(2-trifluoromethyl)phenyl]-1,2,4-triazol-3-one,
and
[0119]
5-butyl-2,4-dihydro-4-[[2'-[N-[2-(methylpyrrole)carbonyl]sulfamoyl]-
biphenyl-4-yl]methyl]-2-[(2-trifluoromethyl)phenyl-1,2,4-triazol-3-one.
[0120] 9. Pharmaceutical Tablets
[0121] Pharmaceutical tablets are prepared using standard mixing
and formation techniques.
[0122] Tablets containing about 1 to 100 mg of an EP.sub.4 receptor
subtype antagonist are prepared using the following relative
weights of ingredients.
1 Ingredient Per Tablet EP.sub.4 Receptor Subtype Antagonist 1 to
100 mg Anhydrous Lactose, NF 71.32 mg Magnesium Stearate, NF 1.0 mg
Croscarmellose Sodium, NF 2.0 mg Microcrystalline Cellulose, NF QS
200 mg
[0123] The resulting tablets are useful for administration in
accordance with the methods of the present invention for inhibiting
bone resorption.
[0124] In further embodiments, tablets are prepared that also
contain 5 or 10 mg of a bisphosphonate active, on a bisphosphonic
acid active basis, of a bisphosphonate selected from the group
consisting of alendronate cimadronate, clodronate, tiludronate,
etidronate, ibandronate, neridronate, olpandronate, risedronate,
piridronate, pamidronate, zolendronate, and pharmaceutically
acceptable salts thereof.
[0125] 10. Liquid Formulation
[0126] Liquid formulations are prepared using standard mixing
techniques.
[0127] A liquid formulation containing about 1 to about 100 mg of
an EP.sub.4 receptor subtype antagonist is prepared using the
following relative weights of ingredients.
2 Ingredient Weight EP.sub.4 Receptor Subtype Antagonist 1-100 mg
Sodium Propylparaben 22.5 mg Sodium Butylparaben 7.5 mg
[0128]
3 Sodium Citrate Dihydrate 1500 mg Citric Acid Anhydrous 56.25 mg
Sodium Saccharin 7.5 mg Water qs 75 mL 1 N Sodium Hydroxide (aq) qs
pH 6.75
[0129] The resulting liquid formulation is useful for
administration for inhibiting bone resorption.
[0130] In further embodiments solutions are prepared also
containing 5 or 10 mg of a bisphosphonate active, on a
bisphosphonic acid active basis, of a bisphosphonate selected from
the group consisting of alendronate cimadronate, clodronate,
tiludronate, etidronate, ibandronate, neridronate, olpandronate,
risedronate, piridronate, pamidronate, zolendronate, and
pharmaceutically acceptable salts thereof.
* * * * *