Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
METHODS FOR >DENTIFYING COMPOUNDS USEFUL FOR INHIBITING
FARNESYL DIPHOSPHATE SYNTHASE
BRIEF DESCRIPTION OF THE INVENTION
The present invention relates to methods for identifying compounds
useful as inhibitors of farnesyl diphosphate synthase. More particularly, the
compounds so identified are useful for inhibiting bone resorption. The present
invention also relates to methods for inhibiting bone resorption in a mammal
comprising administering to a mammal in need thereof a therapeutically
effective
amount of a farnesyl diphosphate synthase inhibitor.
BACKGROUND OF THE INVENTION
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.
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
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imbalance in these processes is manifested in various disease states and
conditions of
the skeleton.
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.
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.
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.
The mevalonate biosynthetic pathway is an important pathway of
osteoclast function. This pathway is involved in the bisosynthesis of
cholesterol and
of isoprenoids, some of which are used in protein prenylation. The enzyme
farnesyl
disphosphate synthase (FPP synthase) mediates the synthesis of farnesyl
diphosphate
by catalyzing the sequential condensation of two molecules of isopentenyl
diphosphate (IPP) with one molecule of dimethylallyl diphosphate (DMAPP) to
produce geranyl diphosphate (GPP) and then farnesyl diphosphate (FPP).
Farnesyl diphosphate is essential for the farnesylation of several
proteins required for cytoskeletal organization and vesicular traffic.
Interference with
the function of these proteins can also lead to apoptosis, i.e. programmed
cell death.
Therefore, farnesyl diphosphate synthase, the enzyme involved in the synthesis
of
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farnesyl diphosphate, is essential for the proper biological functioning of
the
osteoclasts.
It would be highly desirable to identify and develop compounds useful
as selective inhibitors of farnesyl diphosphate synthase in the osteoclasts.
Such
inhibitors would be useful for inhibiting ostetoclast function, thereby
inhibiting
undesired bone resorption and its manifestations.
In the present invention it is surprising found that nitrogen-
containining bisphosphonates such as alendronate and risedronate are specific
nanomolar inhibitors of farnesyl diphosphate synthase. It is also surprisingly
found
that it is possible to identify other compounds useful as farnesyl
disphosphate
synthase inhibitors.
In the present invention it is also found that inhibitors of farnesyl
diphosphate synthase are useful for inhibiting bone resorption. Without being
limited
by theory, it is believed that these inhibitors are responsible for inhibiting
the bone
resorption activity of the osteoclasts.
It is an object of the present invention to provide methods for
identifying compounds useful as farnesyl diphosphate synthase inhibitors.
It is an object of the present invention to provide methods for
inhibiting farnesyl diphosphate synthase in a mammal comprising administering
to a
mammal in need thereof a therapeutically effective amount of a farnesyl
disphosphate
synthase inhibitor having an IC50 value from about 0.01 nanoM to about 1000
nanoM.
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 a farnesyl disphosphate
synthase
inhibitor having an IC50 value from about 0.01 nanoM to about 1000 nanoM.
It is another object of the present invention to provide methods for
treating or reducing the risk of contracting a disease state or condition
mediated by
farnesyl disphosphate synthase in a mammal comprising administering to a
mammal
in need thereof a therapeutically effective amount of a farnesyl disphosphate
synthase
inhibitor having an IC50 value from about 0.01 nanoM to about 1000 nanoM.
It is another object of the present invention to provide methods for
treating or reducing the risk of contracting a disease state or condition
involving or
affecting bone tissue in a mammal comprising administering to a mammal in need
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thereof a therapeutically effective amount of a farnesyl disphosphate synthase
inhibitor having an IC50 value from about 0.01 nanoM to about 1000 nanoM.
It is an object of the present invention to provide methods for
inhibiting farnesyl diphosphate synthase activity in a mammal comprising
administering to a mammal in need thereof comprising administering to a mammal
in
need thereof a therapeutically effective amount of the combination of: (a) a
farnesyl
disphosphate synthase inhibitor having an IC50 value from about 0.01 nanoM to
about 1000 nanoM, and (b) a bisphosphonate active.
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 the combination of: (a) a
farnesyl
disphosphate synthase inhibitor having an IC50 value from about 0.01 nanoM to
about 1000 nanoM, and (b) a bisphosphonate active.
It is an object of the present invention to provide methods for treating
or reducing the risk of contracting a disease state or condition mediated by
farnesyl
diphosphate synthase comprising administering to a mammal in need thereof a
therapeutically effective amount of the combination of: (a) a farnesyl
disphosphate
synthase inhibitor having an IC50 value from about 0.01 nanoM to about 1000
nanoM, and (b) a bisphosphonate active.
It is an object of the present invention to provide methods for treating
or reducing the risk of contracting a disease state or condition involving or
affecting
bone tissue in a mammal comprising administering to a mammal in need thereof a
therapeutically effective amount of the combination of: (a) a farnesyl
disphosphate
synthase inhibitor having an IC50 value from about 0.01 nanoM to about 1000
nanoM, and (b) a bisphosphonate active.
It is another object of the present invention to provide pharmaceutical
compositions comprising a therapeutically effective amount of a farnesyl
disphosphate
synthase inhibitor having an IC50 value from about 0.01 nanoM to about 1000
nanoM.
It is another object of the present invention to provide pharmaceutical
compositions comprising a therapeutically effective amount of the combination
of:
(a) a farnesyl disphosphate synthase inhibitor having an IC50 value from about
0.01
nanoM to about 1000 nanoM and (b) a bisphosphonate active.
These and other objects will become readily apparent from the detailed
description which follows.
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SUMMARY OF THE INVENTION
The present invention relates to methods for identifying compounds
useful as farnesyl diphosphate synthase inhibitors, comprising:
a). contacting a putative farnesyl diphosphate synthase inhibitor
with a farnesyl diphosphate synthase solution, and
b). determining the farnesyl diphosphate synthase activity of said
solution with a farnesyl diphosphate synthase solution not contacted with said
putative
inhibitor.
The present invention also relates to methods for inhibiting farnesyl
diphosphate synthase in a mammal comprising administering to a mammal in need
thereof a therapeutically effective amount of a farnesyl disphosphate synthase
inhibitor having an IC50 value from about 0.01 nanoM to about 1000 nanoM.
The present invention also relates to methods for inhibiting bone
resorption in a mammal comprising administering to a mammal in need thereof a
therapeutically effective amount of a farnesyl disphosphate synthase inhibitor
having
an IC50 value from about 0.01 nanoM to about 1000 nanoM.
The present invention also relates to methods for treating or reducing
the risk of contracting a disease state or condition mediated by farnesyl
disphosphate
synthase in a mammal comprising administering to a mammal in need thereof a
therapeutically effective amount of a farnesyl disphosphate synthase inhibitor
having
an IC50 value from about 0.01 nanoM to about 1000 nanoM.
The present invention also relates to methods for treating or reducing
the risk of contracting a disease state or condition involving or affecting
bone tissue in
a mammal comprising administering to a mammal in need thereof a
therapeutically
effective amount of a farnesyl disphosphate synthase inhibitor having an ICSp
value
from about 0.01 nanoM to about 1000 nanoM.
The present invention also relates to methods for inhibiting farnesyl
diphosphate synthase activity in a mammal comprising administering to a mammal
in
need thereof a therapeutically effective amount of the combination of: (a) a
farnesyl
disphosphate synthase inhibitor having an IC50 value from about 0.01 nanoM to
about 1000 nanoM, and (b) a bisphosphonate active.
The present invention also relates to methods for inhibiting bone
resorption in a mammal comprising administering to a mammal in need thereof a
therapeutically effective amount of the combination of: (a) a farnesyl
disphosphate
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synthase inhibitor having an ICSp value from about 0.01 nanoM to about 1000
nanoM, and (b) a bisphosphonate active.
The present invention also relates to methods for treating or reducing
the risk of contracting a disease state or condition mediated by farnesyl
diphosphate
synthase comprising administering to a mammal in need thereof a
therapeutically
effective amount of the combination of: (a) a farnesyl disphosphate synthase
inhibitor
having an IC50 value from about 0.01 nanoM to about 1000 nanoM, and (b) a
bisphosphonate active.
The present invention also relates to methods for treating or reducing
the risk of contracting a disease state or condition involving or affecting
bone tissue in
a mammal comprising administering to a mammal in need thereof a
therapeutically
effective amount of the combination of: (a) a farnesyl disphosphate synthase
inhibitor
having an IC50 value from about 0.01 nanoM to about 1000 nanoM, and (b) a
bisphosphonate active.
The present invention also relates to pharmaceutical compositions
comprising a therapeutically effective amount of a farnesyl disphosphate
synthase
inhibitor having an ICSp value from about 0.01 nanoM to about 1000 nanoM.
The present invention also relates to pharmaceutical compositions
comprising a therapeutically effective amount of the combination of: (a) a
farnesyl
disphosphate synthase inhibitor having an IC50 value from about 0.01 nanoM to
about 1000 nanoM and (b) a bisphosphonate active.
The present invention also relates to the use of such compositions in
the manufacture of a medicament for the methods disclosed herein.
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
The present invention relates to methods for identifying compounds
useful as farnesyl diphosphate synthase inhibitors and for inhibiting this
enzyme with
the compounds so identifited.
The mevalonate biosynthetic pathway is an important pathway of
osteoclast function. This pathway is involved in the bisosynthesis of
cholesterol and
of isoprenoids, some of which are used in protein prenylation. It would be
highly
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desirable to identify and develop compounds useful as selective inhibitors of
farnesyl
diphosphate synthase in the osteoclasts. Such inhibitors would be useful for
inhibiting ostetoclast function, thereby inhibiting undesired bone resorption
and its
manifestations in various disease states and conditions.
Farnesyl diphosphate synthase is also known by the following names:
prenyltransferase, dimethylallyltransferase, and dimethylallyl
diphosphate:isopentenyl
diphosphate dimethylallyltransferase.
Alendronate (4-amino-1-hydroxybutylidene-l,l-bisphosphonate) is a
potent inhibitor of bone resorption, used in the treatment and prevention of
osteoporosis and other bone diseases. Without being limited by theory, it is
believed
that alendronate and other bisphosphonates are readily adsorbed onto the bone
surface
and are selectively taken up by osteoclasts during bone resorption. It is
generally
accepted that at the cellular level bisphosphonates act by inhibiting
osteoclast
activity. The effects of alendronate monosodium trihydrate and of the HMG-CoA
reductase inhibitor, lovastatin, on osteoclasts in culture is known.
Osteoclast
formation and bone resorption are inhibited by alendronate monosodium
trihydrate
and by lovastatin. Mevalonic acid lactone or geranylgeraniol reverse the
effects of
lovastatin but only geranylgeraniol reverses the effects of alendronate,
thereby
supporting the hypothesis that alendronate monosodium trihydrate induces
apoptosis
by inhibiting protein prenylation via inhibition of the mevalonate pathway
prior to the
formation of geranylgeranyl diphosphate.
It is known that several nitrogen-containing bisphosphonates, including
YM 175, EB 1053 and PHPBP, are potent, nanomolar inhibitors of rat liver
squalene
synthase. See, Amin D, Cornell SA, Gustafson SK, Needle SJ, Ullrich JW, Bilder
GE,
and Perrone MH (1992) J. Lipid Res. 33: 1657-1663, which is incorporated by
reference herein in its entirety. On the other hand, alendronate and
pamidronate, two
other nitrogen containing bisphosphonates, have comparatively little effect on
squalene synthase. Alendronate and pamidronate, however, block sterol
synthesis, as
14
measured by the incorporation of C-MVA into sterol in a rat liver-cell free
system,
with respective IC50's of 168 nM and 420 nM, suggesting that these compounds
inhibit another enzyme in the pathway. Without being limited by theory, it is
therefore
believed that ntirogen-containing bisphosphonates are potent inhibitors of any
of
several enzymes involved in isoprenoid synthesis.
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The synthesis of geranylgeranyl diphosphate from mevalonate involves
six enzymes, mevalonate (MVA) kinase (EC 2.7.1.36), phosphomevalonate (MVAP)
kinase (EC 2.7.4.2), mevalonate diphosphate (MVAPP) decarboxylase, isopentenyl
diphosphate (IPP) isomerase (EC 5.3.3.2), farnesyl diphosphate (FPP) synthase
(EC
2.5.1.1), and geranylgeranyl diphosphate (GGPP) synthase. Farnesyl protein
transferase (FTase), geranylgeranyl protein transferase I (GGTase I) and
geranylgeranyl protein transferase II (GGTase II) are the enzymes responsible
for
prenylating proteins. These transferases are not inhibited by alendronate. It
is found
in the present invention that nitrogen-containing bisphosphonates such as
alendronate
monosodium trihydrate are specific and potent inhibitors of farnesyl
diphosphate
synthase. This specificity is seen in that high micromolar concentrations of
alendronate monosodium trihydrate do not inhibit any other enzyme in the
mevalonate
pathway.
Alendronate inhibition of osteoclast activity in vitro is prevented by
geranylgeraniol, consistent with alendronate inhibition of the mevalonate
pathway,
resulting in a decrease in cellular GGPP. The surprising findings in the
present
invention show that alendronate, which is a nitrogen-containing
bisphosphonate, is a
specific inhibitor of FPP synthase and that it does not inhibit any other
enzymes
involved in the conversion of MVA to GGPP. The present invention also
surprisingly
demonstrates that alendronate and other nitrogen-containing bisphosphonates
inhibit
farnesyl diphosphate synthase and lower the concentration of the
isoprenylation
substrate farnesyl diphosphate and the downstream product geranylgeranyl
diphosphate. These lipids are essential for the prenylation of several
proteins
including GTP binding proteins of around 20 KDa, including those belonging to
the
rho, rac, CdC42 and rab families. These proteins are essential for
cytoskeletal
organization and vesicular traffic. Inactivation of rhoA, for example, causes
osteoclast inactivation, and rab is implicated in vesicular fusion to
membranes, which
is impaired following alendronate administration. Interference with the
function of
these proteins also leads to apoptosis. The ICSp for alendronate inhibition of
farnesyl
disphosphate synthase is 340 nanoM and for pamidronate inhibition of farnesyl
disphosphate synthase is 500 nanoM.
Other nitrogen-containing bisphosphonates are found to inhibit prenyl
transferases involved in isoprenoid synthesis. It is therefore surprising, in
view of the
similarity of these enzymatic reactions, that the alendronate inhibition is
specific for
farnesyl diphosphate synthase to the exclusion of geranylgeranyl diphosphate
synthase
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and squalene synthase. The data show that although these enzymes are closely
related, their interaction with bisphosphonates is distinctly different.
Without being limited by theory, it is believed that nitrogen-containing
bisphosphonates have a different mechanism of action from non-nitrogen-
containing
bisphosphonates. Three nitrogen-containing bisphosphonates, alendronate,
risedronate, and pamidronate effectively inhibit farnesyl diphosphate
synthase,
wherease the two non-nitrogen-containing bisphosphonate, etidronate and
clodronate,
have little or no effect on farnesyl diphosphate synthase. Without being
limited by
theory, these findings are consistent with the notion that the pharmacological
action of
the nitrogen-containing bisphosphonates is based on a similar mechanism:
osteoclast
inactivation and/or apoptosis resulting from interference with protein
prenylation, due
to reduced cellular geranylgeranyl diphosphate levels, caused by farnesyl
diphosphate
synthase inhibition.
Methods of Identifying Inhibitors of Farnes 1 Dipho~hate S nthase
The present invention relates to a method for identifying an inhibitor of
farnesyl diphosphate synthase comprising:
a). contacting a putative farnesyl diphosphate synthase inhibitor
with a farnesyl diphosphate synthase assay solution, and
b). determining, i.e. comparing, the farnesyl diphosphate synthase
activity of said assay solution with a farnesyl diphosphate synthase assay
solution not
contacted with said putative inhibitor, in order to determine the amount of
inhibition.
In these methods the farnesyl diphosphatesynthase assay solution is
typically an aqueous solution. The inhibition effect is measured with respect
to the
catalysis of an appropriate reaction that one of ordinary skill in the art can
select.
Typical substrates include dimethylallyl diphosphate, isopentenyl diphosphate,
and
geranyl diphosphate. Reaction times, conditions, quatitation methods, and
other
variables are chosen for convenience to obtain a readily quantitated system
for
measuring the inhibition of the farnesyl diphosphate synthase.
Additionally, in these methods of identifying inhibitors of farnesyl
diphosphate synthase, the enzyme can be used in a crude, unpurified state,
from
various tissues sources, e.g., liver. Alternatively, the enzyme can be used in
a partially
purified state, a purified state, or as an expressed form of the enzyme, e.g.,
the
expressed human enzyme.
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Methods Of Inhibiting Bone Resorption
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 a farnesyl diphosphate synthase inhibitor.
The methods and compositions of the present invention are useful for
both treating and reducing the risk of contracting disease states or
conditions
involving or 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. The methods and compositions are also useful for both
teating
and reducing the risk of contracting other disease states or conditions
mediated by
farnesyl disphosphate synthase.
In further embodiments, the methods comprise administering a
therapeutically effective amount of the combination of (a) a farnesyl
diphosphate
synthase inhibitor, which can itself be a bisphosphonate active, and (b) an
additional
bisphosphonate active. Both concurrent and sequential administration of the
farnesyl
disphosphate synthase inhibitor and the additional bisphosphonate active are
deemed
within the scope of the present invention. With sequential administration, the
farnesyl
diphosphate synthase inhibitor and the additional bisphosphonate can be
administered
in either order. In a subclass of sequential administration the farnesyl
diphosphate
synthase inhibitor and the additional bisphosphonate are typically
administered within
the same 24 hour period. In yet a further subclass, the farnesyl diphosphate
synthase
inhibitor and the additional bisphosphonate are typically administered within
about 4
hours of each other.
The term "therapeutically effective amount", as used herein, means that
amount of the farnesyl diphosphate synthase inhibitor, 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.
"Pharmaceutically acceptable" as used herein, means generally suitable
for administration to a mammal, including humans, from a toxicity or safety
standpoint.
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In the present invention, the farnesyl diphosphate synthase inhibitor 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.
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.
Compositions Of The Present Invention
The pharmaceutical compositions of the present invention comprise a
therapeutically effective amount of a farnesyl diphosphate synthase inhibitor.
These compositions can further comprise a pharmaceutically-
acceptable Garner.
In further embodiments these compositions can also comprise an
additional active.
Farnesyl Diphosphate Synthase Inhibitor
The methods and compositions of the present invention comprise a
farnesyl diphosphate synthase inhibitor. These inhibitors can in themselves be
bisphosphonates.
The farensyl diphosphate synthase inhibitors useful herein generally
have an IC50 value from about 0.01 nM to about 1000 nanoM, although inhibitors
with activities outside this range can be useful depending upon the dosage and
route
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of administration. In a subclass of the present invention, the inhibitors have
an IC50
value of from about 0.01 nM to about 100 nM. In a further subclass of the
present
invention, the inhibitors have an IC50 value of from about 0.01 nM to about 1
nM.
IC50 is a common measure of inhibition activity well known to those of
ordinary skill
in the art and is defined as the concentration of the inhibitor needed to
obtain a 50%
reduction in the activity of the farnesyl disphosphate synthase.
The combination of two or more farnesyl diphosphate synthase
inhibitors are also deemed as within the scope of the present invention.
The precise dosage of the farnesyl diphosphate synthase inhibitor will
vary with the dosing schedule, the particular compound chosen, the age, size,
sex and
condition of the mammal or human, the nature and severity of the disorder to
be
treated, and other relevant medical and physical factors. Thus, a precise
pharmaceutically effective amount cannot be specified in advance and can be
readily
determined by the caregiver or clinician. Appropriate amounts can be
determined by
routine experimentation from animal models and human clinical studies.
Generally,
an appropriate amount is chosen to obtain an inhibition of the farnesyl
diphosphate
synthase activity so as to obtain a bone resorption inhibiting effect.
For humans, an effective oral dose of the farnesyl diphosphate synthase
inhibitor is about 1 ~g/kg to about 1000 p,g/kg, preferably about 10 pg/kg,
for a
human subject.
For the farnesyl diphosphate synthase inhibitor, human doses which
can be administered are generally in the range of about 0.1 mg/day to about 10
mg/day, preferably from about 0.25 mg/day to about 5 mg/day, and more
preferably
from about 0.5 mg/day to about 1.5 mg/day, based on a geranylgeraniol active
weight
basis. A typical nonlimiting dosage amount would be about 0.75 mg/day. The
pharmaceutical compositions herein comprise from about 0.1 mg to about 10 mg,
preferably from about 0.25 mg to about 5 mg, and more preferably from about
0.5 mg
to about 1.5 mg of the farnesyl diphosphate synthase inhibitor. A typical
nonlimiting
amount is about 0.75 mg.
Bisphosphonates
The methods and compositions of the present invention, can further
comprise a bisphosphonate active or a pharmaceutically acceptable salt
thereof.
These bisphosphonate actives are defined herein to be distinct from and not to
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included the farnesyl diphosphate synthase inhibitors of the present
invention, because
certain nitrogen-containing bisphosphonates, e.g., alendronate are found to
have
activity as farnesyl diphosphate synthase inhibitors. In other words, the
present
invention can include the combination of a farnesyl diphosphate synthase
inhibitor
which happens to have a bisphosphonate structure and an additional
bisphosphonate
active which does not necessarily have activity as a farnesyl diphosphate
synthase
inhibitor.
The term "nitrogen-containing" as used herein means that the
bisphosphonate compound or pharmaceutically acceptable salt thereof comprises
at
least one nitrogen atom in the bisphosphonate portion of the molecule. In
other
words, for a pharmaceutically-acceptable salt of the bisphosphonate, any
nitrogen
atom contained in the positive counter ion of such a salt, e.g., the nitrogen
atom of an
ammonium counter ion, would not be considered in meeting the "nitrogen-
containing"
definition. For example, alendronic acid, i.e. 4-amino-1-hydroxybutylidene-1,1-
bisphosphonic acid is an example of a nitrogen-containing bisphosphonate.
However,
the ammonium salt of the unsubstituted 1-hydroxybutylidene-1,1-bisphosphonic
acid
would not be a nitrogen-containing bisphosphonate as defined herein.
The bisphosphonates useful in certain embodiments of the present
invention correspond to the chemical formula
P03H2
A-(CH2)n-C-X
P03H2
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, NH2, SH, phenyl, C1-C30 alkyl, C3-
C30 branched or cycloalkyl, C1-C30 substituted alkyl, Cl-C10 alkyl substituted
NH2
C3-C10 branched or cycloalkyl substituted NH2~ C1-C10 dialkyl substituted NH2
C3-C10 branched or cycloalkyl disubstituted NH2~ C1-C10 alkoxy, C1-C10 alkyl
substituted thio, thiophenyl, halophenylthio, Cl-C10 alkyl substituted phenyl,
pyridyl,
furanyl, pyrrolidinyl, imidazolyl, imidazopyridinyl, and benzyl, such that
both A and
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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.
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, NH2, C1-C10 alkyl or
dialkyl
substituted NH2, OH, SH, and C1-C10 alkoxy.
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.
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.
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 Cl, X is
selected
from the group consisting of C1-C30 alkyl, C1-C30 substituted alkyl, Cl, and
chlorophenylthio, and n is 0.
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.
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.
It should be noted that the terms "bisphosphonate" and
"bisphosphonates", as used herein in refernng 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
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specific nomenclature in refernng 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.
Nonlimiting examples of bisphosphonates useful herein include the
following:
Alendronic acid, 4-amino-1-hydroxybutylidene-1,1-bisphosphonic
acid.
Alendronate (also known as alendronate sodium or alendronate
monosodium trihydrate), 4-amino-1-hydroxybutylidene-1,1-bisphosphonic acid
monosodium trihydrate.
Alendronic acid and alendronate are described in U.S. Patents
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
April
23, 1996; 5,648,491, to Dauer et al., issued July 15, 1997, all of which are
incorporated by reference herein in their entirety.
Cycloheptylaminomethylene-l,l-bisphosphonic acid, YM 175,
Yamanouchi (cimadronate), as described in U.S. Patent 4,970,335, to Isomura et
al., issued November 13, 1990, which is incorporated by reference herein in
its
entirety.
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.
1-hydroxy-3-(1-pyrrolidinyl)-propylidene-1,1-bisphosphonic acid
(EB-1053).
1-hydroxyethane-l,l-diphosphonic acid (etidronic acid).
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1-hydroxy-3-(N-methyl-N-pentylamino)propylidene-l, l-
bisphosphonic acid, also known as BM-210955, Boehringer-Mannheim
(ibandronate), is described in U.S. Patent No. 4,927,814, issued May 22, 1990,
which is incorporated by reference herein in its entirety.
6-amino-1-hydroxyhexylidene-l,l-bisphosphonic acid
(neridronate).
3-(dimethylamino)-l-hydroxypropylidene-1,1-bisphosphonic acid
(olpadronate).
3-amino-1-hydroxypropylidene-1,1-bisphosphonic acid
(pamidronate).
[2-(2-pyridinyl)ethylidene]-1,1-bisphosphonic acid (piridronate) is
described in U.S. Patent No. 4,761,406, which is incorporated by reference in
its
entirety.
1-hydroxy-2-(3-pyridinyl)-ethylidene-l,l-bisphosphonic acid
(risedronate).
(4-chlorophenyl)thiomethane-l,l-disphosphonic acid (tiludronate)
as described in U.S. Patent 4,876,248, to Breliere et al., October 24, 1989,
which
is incorporated by reference herein in its entirety.
H
1-hydroxy-2-(1 -imidazol-1-yl)ethylidene-1,1-bisphosphonic acid
(zolendronate).
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.
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.
A non-limiting example of the subclass is alendronate monosodium
trihydrate.
It is recognized that mixtures of two or more of the bisphosphonate
actives can be utilized.
The precise dosage of the bisphosphonate will vary with the dosing
schedule, the particular bisphosphonate chosen, the age, size, sex and
condition of the
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mammal or human, the nature and severity of the disorder to be treated, and
other
relevant medical and physical factors. Thus, a precise therapeutically
effective
amount cannot be specified in advance and can be readily determined by the
caregiver
or clinician. Appropriate amounts can be determined by routine experimentation
from
animal models and human clinical studies. Generally, an appropriate amount of
bisphosphonate is chosen to obtain a bone resorption inhibiting effect, i.e. a
bone
resorption inhibiting amount of the nitrogen-containing bisphosphonate is
administered. For humans, an effective oral dose of nitrogen-containing
bisphosphonate is typically from about 1.5 to about 6000 ~.g/kg body weight
and
preferably about 10 to about 2000 p.g/kg of body weight.
For the bisphosphonate, alendronate monosodium trihydrate, common
human doses which are administered are generally in the range of about 2
mg/day to
about 40 mg/day, preferably about 5 mg/day to about 40 mg/day. In the U.S.
presently
approved dosages for alendronate monosodium trihydrate are 5 mg/day for
preventing
osteoporosis, 10 mg/day for treating osteoporosis, and 40 mg/day for treating
Paget's
disease.
In alternative dosing regimens, the bisphosphonate can be administered
at intervals other than daily, for example once-weekly dosing, twice-weekly
dosing,
biweekly dosing, and twice-monthly dosing. In such dosing regimens,
appropriate
multiples of the bisphosphonate dosage would be administered. For example, in
a
once weekly dosing regimen, alendronate monosodium trihydrate would be
administered at dosages of 35 mg/week or 70 mg/week in lieu of seven
consecutive
daily dosages of 5 mg or 10 mg.
The pharmaceutical compositions herein comprise from about 1 mg to
about 100 mg of bisphosphonata, preferably from about 2 mg to 70 mg, and more
preferably from about 5 mg to about 70, on a bisphosphonic acid basis. For the
bisphosphonate alendronate monosodium trihydrate, the pharmaceutical
compositions
useful herein comprise about 2.5 mg, 5 mg, 10 mg, 35, mg, 40 mg, or 70 mg of
the
active on an alendronic acid active weight basis.
See also, U.S. Patent 4,610,077, to Rosini et al., issued November 4,
1986; U.S. Patent 5,358,941, to Bechard et al., issued October 25, 1994; and
PCT
application number WO 99/04773, to Daifotis et al., published February 4,
1999; all
of which are incorporated by reference herein in their entirety.
Other Bone A_~ents
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Further embodiments of the methods and compositions of the present
invention can comprise additional bone agents useful for inhibiting bone
resorption
and providing the desired therapeutic benefits of the invention. Examples of
such
agents include those selected from the group consisting of calcitonin,
estrogens,progesterone, androgens, calcium supplements, fluoride, growth
hormone
secretagogues, vitamin D analogues, and selective estrogen receptor
modulators. The
calcitonins useful herein can be from human or nonhuman sources, e.g. salmon
calcitonin. Nonlimiting examples of estrogens include estradiol. Nonlimiting
examples of selective estrogen receptor modulators include raloxifene,
iodoxifene,
and tamoxifene. Growth horomone secretagogues are described in U.S. Patent No.
5,536,716, to Chen et al., issued July 16, 1996, which is incorporated by
reference
herein in its entirety.
Other Components Of The Pharmaceutical Compositions
The farensyl diphosphate synthase inhibitors, 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
Garner
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 are combined
with any
oral, non-toxic, pharmaceutically acceptable inert Garner 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
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chloride and the like. , An example of a tablet formulation is that described
in U.S.
Patent No. 5,358,941, to Bechard et al, issued October 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.
The following Examples are presented to better illustrate the invention.
EXAMPLE 1
Isopentenyl diphosphate (IPP), dimethylallyl-diphosphate (DMAPP),
farensyl diphosphate (FPP), and geranylgeranyl diphosphate (GPP) are obtained
from
14 3
Echelon, Salt Lake City, Utah. [1- C]IPP (55 mCi/mmol), and [5- H]MVL (50
Ci/mmol) are obtained from ARC (St. Louis, MO).
Human recombinant farnesyl diphosphate synthase is expressed and
purified as described by Ding et al., Biochem. J., 275, pp. 61-65 (1991),
which.
Alternatively, the crude expressed enzyme in an E. coli 5100 fraction can be
used.
EXAMPLE 2
The farnesyl diphosphate synthase assay is based on the method of
Rilling HS, (1985) Methods in Enzymology 110: 145-152, which is incorporated
by
reference herein in its entirety. Geranyl diphosphate is used as the allylic
substrate
14
with [1- C] isoprenyl isopentenyl diphosphate as the second substrate. Hepes
is used
at 100 mM, geranylgeranyl diphosphate is used at 40 ~.M, isopentenyl
diphosphate is
used at 20 ~,M, and heptane is used for extractions. Alternatively, DMAPP is
used as
14
the allylic substrate. Control assays are run with [1- C]IPP without allylic
substrate,
to correct for IPP isomerase activity. For assays using the human recombinant
FPP
synthase, 1% BSA is added to stabilize the enzymatic activity.
EXAMPLE 3
3
Labeling of osteoclasts with H-MVL: Osteoclast formation, murine
co-cultures of osteoblasts and marrow cells are prepared using the methods of
Wesolowski et al., Exp. Cell Res., 219:679-686 (1995), which is incorporated
by
reference herein in its entirety. Cells are harvested from the bone marrow of
6-week-
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old male Balb/C mice 'and suspended in: a-MEM supplemented with fetal calf
serum
(10% v/v) and 1,25-(Ovitamim D3 (10 nM). Bone marrow cells are then added to
sub-
confluent monolayers of osteoblastic MB 1.8 cells and cultured for 7 days at
37C in
the presence of 5% C02. Co-cultures are treated (1 hr at 37C) with type 1
collagenase
(Wako Pure Chemical Industries, Osaka; Japan) at a concentration of 1 mg/ml in
phosphate buffered saline. Suspended osteoblasts are gently aspirated, leaving
an
enriched mixture of prefusion osteoclasts and remaining MB 1.8 osteoblasts.
These are
released with EDTA (0.2 g/I in PBS) for 20 min at 37C. Cells are then re-
plated in 6-
well dishes using a-MEM supplemented with fetal calf serum (10% v/v) and 1,25-
(OH)2 vitamin D3 (10 nM) and cultured for an additional three days. Osteoclast
co-
cultures generated in 6-well plates are treated with Type XI Collagenase
(Sigma) in
PBS to remove all osteoblasts, followed by EDTA to remove prefusion
osteoclasts.
Osteoclasts L95% purity) are then maintained in a-MEM supplemented with fetal
calf serum (10% v/v) and 1;25-(OH)2 vitamin D3 (10 nM )and M-CSF (5 ng/ml).
For the labeling of the non-saponifiable lipids, osteoclasts are treated
with alendronate (0-60 pM) for 2 hours before the addition of 200 pCi of R,S-
[5-
3
H]MVL per dish. After 3 hours of labeling, media are removed, cells are washed
twice with PBS, and then the cells are scraped into 2 ml of 1 M NaOH. Wells
are
rinsed with an additional equal volume of 1 M NaOH. Two volumes of 40% NaOH
and one volume of methanol are added to the pooled NaOH extracts and are
heated at
65°C for 3 hours to saponify the lipids. Non-saponifiable lipids are
extracted with
heptane and backwashed with 1 M NaOH. The radioactive content of the non-
saponifiable lipid extract is determined by scintillation counting. TLC of
these lipids
is performed on LK6D silica gel 60 A° plates (Whatman, Fairfield, NJ)
using
hexane:EtZO:acetic acid (70:30:3). After developing the chromatograph, the
dried
3
plate is sprayed with En Hance (NEN, Boston, MA) and exposed to XAR2 film
(Sigma, St. Louis, MO).
For studying the labeling of prenylated proteins, the osteoclasts are
treated with 15 p,M lovastatin and alendronate (0-60 p,M) for 2 hours before
the
3
addition of 200 pCi of R,S-[5- H]MVL per dish. After 3 hours of labeling,
media are
removed, cells are washed twice with PBS, and then scraped into 200 pl of SDS
sample buffer. SDS gel electrophoresis is performed on 15% gels (Gel
electrophoresis sytem, gels, and buffers from Bio Rad, Hercules, CA). The gel
is
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fixed in 12% acetic acid/50°7o MeOH and soaked in Enlightening (NEN),
dried and
put under XAR2 film for 10 days before developing.
EXAMPLE 4
Alendronate effects on FPP synthase: FPP synthase catalyzes the
sequential condensation of two molecules of IPP with one molecule of DMAPP to
produce GPP and then FPP. The FPP synthase assay, is run with a 15 minute
preincubation and shows that alendronate inhibits FPP synthase with an IC50 of
460
nM (0.15 p,g/ml). Because the inhibition of FPP synthase by alendronate is
time-
dependent, IC50 varies with preincubation time and assay length.
EXAMPLE 5
Inhibition of FPP synthase by other bisphosphonates: four other
bisphosphonates are tested for their inhibitory effect of FPP synthase. All
three
nitrogen-containing bisphosphonates examined (alendronate, pamidronate, and
risedronate) inhibit FPP synthase. Pamidronate has an (IC50 = 500 nM),
risedronate
has (IC50 = 3.9 nanoM). For the non-nitrogen containing bisphosphonate,
etidronate
the IC50 values is 80 ~.M. For the non-nitrogen containing bisphosphonate
clodronate, no inhibition observed.
EXAMPLE 6
Effect of alendronate on protein prenylation and the synthesis of
mevalonate-derived lipids in osteoclasts: alendronate inhibition of the
mevalonate
pathway and of protein prenylation is demonstrated in the osteoclasts. A major
branch point in isoprenoid metabolism occurs at FPP, which is used for sterol
synthesis via squalene synthase, for prenylation of proteins via farnesyl
protein
transferase, for GGPP synthesis, and for the synthesis of dolichol and
ubiquinone, via
3
cis and trans prenyl transferases, respectively. Osteoclasts are labeled with
H-MVL,
and the effects of alendronate on the incorporation of the label into non-
saponifiable
lipids and into prenylated proteins are examined.
3
The effects of alendronate on the incorporation of label from H-MVL
into prenylated proteins extracted from osteoclasts is studied. In the absence
of
alendronate, a series of proteins between 18-25 kD and another one of 44 kD,
are
labeled. With increasing alendronate concentrations, labeling decreases and
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essentially disappears at 60 ~.M, with an IC50 of approximately 15 ~,M. A band
of
about 18 kDa is not affected by alendronate.
The incorporation of label from MVL into the non-saponifiable lipids
is lowered by up to 80°Io by alendronate with an IC50 of around 15 ~M.
Analysis of
these non-saponifiable lipids by TLC shows three major and at least five minor
bands
labeled. The incorporation into all bands is lowered by alendronate without
bias.
Individual bands co-migrated with squalene, lanosterol, and sterols (including
cholesterol, desmosterol and 7-dehydrocholesterol). A diffuse light area of
labeling
just under the putative lanosterol band, where farnesol, geranylgeraniol, and
dolichols
migrate, is also observed.
The incorporation of label from MVL both into prenylated proteins and
into non-saponifiable lipids is inhibited by 50°Io at 15 ~,M,
consistent with FPP
synthase being the target for the action of alendronate.
EXAMPLE 7
Pharmaceutical tablets: the tablets are prepared using standard mixing
and formation techniques.
Tablets containing about 1 to 100 mg of a farnesyl diphosphate
synthase inhibitor are prepared using the following relative weights of
ingredients.
Ingredient Per Tablet
Farnesyl Diphosphate Synthase Inhibitor0.10 to 10 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
The resulting tablets are useful for administration in accordance with
the methods of the present invention for inhibiting bone resorption.
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,
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clodronate, tiludronate, etidronate, ibandronate, neridronate, olpandronate,
risedronate, piridronate, pamidronate, zolendronate, and pharmaceutically
acceptable
salts thereof.
EXAMPLE 8
Liquid formulation: liquid formulations are prepared using standard
mixing techniques.
A liquid formulation containing about 1 to about 100 mg of a farnesyl
diphosphate synthase inhibitor is prepared using the following relative
weights of
ingredients.
In redient Weight
Farnesyl Diphosphate Synthase Inhibitor0.10 to 10 mg
Sodium Propylparaben 22.5 mg
Sodium Butylparaben 7.5 mg
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
The resulting liquid formulation is useful for administration for
inhibiting bone resorption.
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.
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