U.S. patent application number 13/249248 was filed with the patent office on 2012-01-26 for method of treating osteoporosis.
This patent application is currently assigned to NIIKI PHARMA INC.. Invention is credited to Hooshmand SHESHBARADARAN.
Application Number | 20120022036 13/249248 |
Document ID | / |
Family ID | 42828672 |
Filed Date | 2012-01-26 |
United States Patent
Application |
20120022036 |
Kind Code |
A1 |
SHESHBARADARAN; Hooshmand |
January 26, 2012 |
Method of treating osteoporosis
Abstract
A method for treating osteoporosis and related methods are
disclosed. The methods generally comprise administering to a
patient in need of treatment an effective amount of
tris(8-quinolinolato)gallium(III) or an analog thereof.
Inventors: |
SHESHBARADARAN; Hooshmand;
(Hoboken, NJ) |
Assignee: |
NIIKI PHARMA INC.
Hoboken
NJ
|
Family ID: |
42828672 |
Appl. No.: |
13/249248 |
Filed: |
September 30, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US2010/029285 |
Mar 30, 2010 |
|
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13249248 |
|
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61164856 |
Mar 30, 2009 |
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Current U.S.
Class: |
514/187 |
Current CPC
Class: |
C07F 5/00 20130101; A61K
31/555 20130101; A61K 31/00 20130101; A61P 19/08 20180101; A61K
31/47 20130101; A61P 19/10 20180101; C07F 5/003 20130101; A61K
33/00 20130101; A61K 33/24 20130101; A61P 35/04 20180101 |
Class at
Publication: |
514/187 |
International
Class: |
A61K 31/555 20060101
A61K031/555; A61P 19/10 20060101 A61P019/10; A61P 35/00 20060101
A61P035/00; A61P 19/00 20060101 A61P019/00 |
Claims
1. A method of reducing osteoclastic bone resorption in a patient,
comprising administering to the patient an effective amount of a
compound according to Formula (I) ##STR00005## wherein R.sup.1
represents hydrogen, a halogen or a sulfono group SO.sub.3M, in
which M is a metal ion, and R.sup.2 represents hydrogen, or R.sup.1
is Cl and R.sup.2 is I, or a pharmaceutically acceptable salt
thereof.
2. A method of treating or preventing osteoporosis, comprising
administering to a patient in need of treatment an effective amount
of a compound according to Formula (I) ##STR00006## wherein R.sup.1
represents hydrogen, a halogen or a sulfono group SO.sub.3M, in
which M is a metal ion, and R.sup.2 represents hydrogen, or R.sup.1
is Cl and R.sup.2 is I, or a pharmaceutically acceptable salt
thereof.
3. A method of treating or preventing or reducing the risk of low
bone mineral density, bone loss, skeletal-related events including
bone fractures, or increasing bone mass, comprising administering
to a patient in need of treatment an effective amount of a compound
according to Formula (I) ##STR00007## wherein R.sup.1 represents
hydrogen, a halogen or a sulfono group SO.sub.3M, in which M is a
metal ion, and R.sup.2 represents hydrogen, or R.sup.1 is Cl and
R.sup.2 is I, or a pharmaceutically acceptable salt thereof.
4. A method of treating or delaying the onset of localized bone
loss associated with periodontal diseases or with periprosthetic
osteolysis, comprising administering to a patient an effective
amount of a compound according to Formula (I) ##STR00008## wherein
R.sup.1 represents hydrogen, a halogen or a sulfono group
SO.sub.3M, in which M is a metal ion, and R.sup.2 represents
hydrogen, or R.sup.1 is Cl and R.sup.2 is I, or a pharmaceutically
acceptable salt thereof.
5. A method of treating or preventing Paget's disease, comprising
administering to a patient in need of treatment an effective amount
of a compound of Formula (I) ##STR00009## wherein R.sup.1
represents hydrogen, a halogen or a sulfono group SO.sub.3M, in
which M is a metal ion, and R.sup.2 represents hydrogen, or R.sup.1
is Cl and R.sup.2 is I, or a pharmaceutically acceptable salt
thereof.
6. A method for preventing, or delaying the onset of, bone
metastasis of cancer in a patient, comprising administering to the
patient an effective amount of a compound of Formula (I)
##STR00010## wherein R.sup.1 represents hydrogen, a halogen or a
sulfono group SO.sub.3M, in which M is a metal ion, and R.sup.2
represents hydrogen, or R.sup.1 is Cl and R.sup.2 is I, or a
pharmaceutically acceptable salt thereof.
7. A method of treating a cancer metastasized to bone, comprising
administering to a patient having a cancer with bone metastasis an
effective amount of a compound of Formula (I) ##STR00011## wherein
R.sup.1 represents hydrogen, a halogen or a sulfono group
SO.sub.3M, in which M is a metal ion, and R.sup.2 represents
hydrogen, or R.sup.1 is Cl and R.sup.2 is I, or a pharmaceutically
acceptable salt thereof.
Description
RELATED PATENT APPLICATIONS
[0001] This application is a continuation of PCT/US2010/029285
filed on Mar. 30, 2010 which claims the benefit and priority of
U.S. Provisional Application No. 61/164,856 filed on Mar. 30, 2009,
the content of both being incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0002] The present invention generally relates to methods of
treating diseases, and particularly to a method of treating
osteoporosis and related disorders.
BACKGROUND OF THE INVENTION
[0003] Osteoporosis is a major health problem afflicting millions
of people worldwide. It is most prevalent in postmenopausal women,
but also occurs in a significant portion of men over the age of 50.
In patients on glucocorticoids, and those undergoing hormone
ablation therapy for either prostate or breast cancer, bone loss
and osteoporosis are especially significant. In osteoporosis
patients, the decrease of bone mineral density (BMD) and bone mass
content (BMC) result in increased bone fragility and risk of bone
fracture. Osteoporosis may significantly affect life expectancy and
quality of life.
[0004] Estrogen replacement therapy was the main approach for a
long time in postmenopausal women for preventing osteoporosis until
it was discovered to be associated with an increased incidence of
cancer. See e.g., Prentice et al., Am. J. Epidemiol., 170(1):12-23
(2009). Bisphosphonates were first developed in mid 1990s and have
become the main pharmaceutical measures for osteoporosis. However,
oral bisphosphonates are poorly absorbed and are often associated
with esophagial inflammation. Recently, RANKL-targeting antibodies
such as denosumab have shown some promise as effective agents in
reducing bone resorption. See e.g., McClung et al., N. Engl. J.
Med., 354(8):821-31 (2006).
SUMMARY OF THE INVENTION
[0005] It has been surprisingly discovered that
tris(8-quinolinolato)gallium(III) is effective in inhibiting
osteoclastic bone resorption, reducing bone loss, and treating
osteoporosis. Accordingly, in a first aspect, the present invention
provides a method of reducing osteoclastic bone resorption in a
patient comprising administering to a patient in need of treatment
an osteoclastic bone resorption-inhibiting amount of a compound
according to Formula (I) below or a pharmaceutically acceptable
salt thereof (e.g., tris(8-quinolinolato)gallium(III)).
[0006] In a second aspect, a method of treating osteoporosis is
provided comprising administering to a patient in need of treatment
a therapeutically effective amount of a compound according to
Formula (I) below or a pharmaceutically acceptable salt thereof
(e.g., tris(8-quinolinolato)gallium(III)). In one embodiment, the
method is used to treat or prevent postmenopausal osteoporosis
(PMO) in women. In another embodiment, the method is used to treat,
prevent, or reduce the risk or incidence of, bone loss or
skeletal-related events (such as bone fractures) in patients
undergoing hormone ablation therapy for either prostate or breast
cancer. In yet another embodiment, the method is used for the
prevention and treatment of glucocorticoid-induced osteoporosis
(GIO) in men and women who are either initiating or continuing
systemic glucocorticoid treatment.
[0007] In yet another aspect, the present invention provides a
method of treating Paget's disease by administering to a patient in
need of treatment a therapeutically effective amount of a compound
according to Formula (I) below or a pharmaceutically acceptable
salt thereof (e.g., tris(8-quinolinolato)gallium(III)).
[0008] Additionally, the present invention also provides a method
of preventing, or delaying the onset of, bone metastasis of cancer
comprising administering an effective amount of a compound of
Formula (I) or a pharmaceutically acceptable salt thereof (e.g.,
tris(8-quinolinolato)gallium(III)) to a patient identified as, or
diagnosed of, having cancer (e.g., lung cancer, breast cancer,
prostate cancer, lymphoma or multiple myeloma) but without bone
metastasis, thereby preventing, or delaying the onset of, cancer
bone metastasis.
[0009] The present invention further provides a method of treating,
preventing, or reducing the risk or incidence of, bone loss or
skeletal-related events (such as bone fractures) in patients having
cancer with bone metastasis comprising identifying a patient having
a cancer that has metastasized to bone, and administering a
therapeutically effective amount of a compound of Formula (I) or a
pharmaceutically acceptable salt thereof (e.g.,
tris(8-quinolinolato)gallium(III)) to the patient.
[0010] In yet another aspect, the present invention provides a
method of treating, preventing, or reducing the risk or incidence
of, bone loss or skeletal-related events (such as bone fractures)
in patients having neuroendocrine tumors by administering to a
patient in need of treatment a therapeutically effective amount of
a compound according to Formula (I) below or a pharmaceutically
acceptable salt thereof (e.g.,
tris(8-quinolinolato)gallium(III)).
[0011] The present invention further provides a method of treating
cancer with bone metastasis comprising identifying a patient having
a cancer that has metastasized to bone, and administering a
therapeutically effective amount of a compound of Formula (I) or a
pharmaceutically acceptable salt thereof (e.g.,
tris(8-quinolinolato)gallium(III)) to the patient.
[0012] In another aspect, the present invention provides a use of a
compound according to Formula (I) below or a pharmaceutically
acceptable salt thereof (e.g., tris(8-quinolinolato)gallium(III))
for the manufacture of a medicament useful for reducing
osteoclastic bone resorption, treating, preventing or delaying the
onset of osteoporosis, treating or preventing or delaying the onset
of Paget's disease, or treating, preventing, or delaying the onset
of, bone metastasis of cancer, or treating a cancer with bone
metastasis.
[0013] The foregoing and other advantages and features of the
invention, and the manner in which the same are accomplished, will
become more readily apparent upon consideration of the following
detailed description of the invention taken in conjunction with the
accompanying examples, which illustrate preferred and exemplary
embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 is a graph demonstrating that treatment with
tris(8-quinolinolato)gallium(III) inhibited osteoclast
differentiation;
[0015] FIG. 2 shows tris(8-quinolinolato)gallium(III) ("drug")
significantly inhibited cathepsin K, NFAT2 and TRAP gene
expression;
[0016] FIG. 3 is a bar graph showing daily administration of
tris(8-quinolinolato)gallium(III) ("daily drug") in ovariectomized
rats was associated with increased bone mineral density when
compared to ovariectomized untreated rats; and
[0017] FIG. 4 is a bar graph showing that
tris(8-quinolinolato)gallium(III) treatment ("drug daily") in
ovariectomized rats was significantly associated with reduced serum
osteocalcin when compared to ovariectomized untreated rats.
DETAILED DESCRIPTION OF THE INVENTION
[0018] The present invention is in part based on the discovery that
the compound tris(8-quinolinolato)gallium(III) is particularly
effective in inhibiting osteoclast maturation or formation,
reducing bone resorption, preventing bone loss, thereby maintaining
or increasing bone mineral density in mammals.
[0019] Accordingly, in a first aspect, the present invention
provides a method of reducing osteoclastic bone resorption in a
patient comprising treating a patient in need of treatment, with an
osteoclastic bone resorption-inhibiting amount of a gallium complex
of Formula (I)
##STR00001##
wherein R.sup.1 represents hydrogen, a halogen or a sulfono group
SO.sub.3M, in which M is a metal ion, and R.sup.2 represents
hydrogen, or R.sup.1 is Cl and R.sup.2 is I, or a pharmaceutically
acceptable salt thereof. The method can result in the reduction of
osteoclastic bone resorption and alleviation of the associated
symptoms, such as skeletal-related events (e.g., bone loss and/or
decrease of bone mineral density). That is, the present invention
is directed to the use of an effective amount of a compound
according to Formula (I) or a pharmaceutically acceptable salt
thereof for the manufacture of medicaments for reducing
osteoclastic bone resorption, preventing or slowing bone loss,
preventing or slowing the decrease of bone mineral density, or
preventing or reducing the incidence of skeletal-related events
(e.g., bone fractures, bone loss, etc.) in mammals including
humans.
[0020] In preferred embodiments, the gallium complex is
tris(8-quinolinolato)gallium(III)
##STR00002##
[0021] or a pharmaceutically acceptable salt thereof.
[0022] In another aspect, the present invention provides a method
of treating, preventing or delaying the onset of, osteoporosis. The
method comprises treating a patient in need of the treating,
preventing or delaying the onset, with a therapeutically effective
amount of a gallium complex of Formula (I)
##STR00003##
wherein R.sup.1 represents hydrogen, a halogen or a sulfono group
SO.sub.3M, in which M is a metal ion, and R.sup.2 represents
hydrogen, or R.sup.1 is Cl and R.sup.2 is I, or a pharmaceutically
acceptable salt thereof. That is, the present invention is directed
to the use of an effective amount of a compound according to
Formula (I) or a pharmaceutically acceptable salt thereof for the
manufacture of medicaments for treating, preventing or delaying the
onset of osteoporosis.
[0023] In preferred embodiments, the gallium complex is
tris(8-quinolinolato)gallium(III)
##STR00004##
[0024] or a pharmaceutically acceptable salt thereof.
[0025] Thus, the present invention provides a method of treating
osteoporosis or osteopenia comprising treating a patient in need of
treatment with a therapeutically effective amount of
tris(8-quinolinolato)gallium(III). The present invention also
provides a method of slowing down the decrease of bone mineral
density, delaying the onset of osteoporosis, or increasing bone
mineral density in a patient in need of such treatment (e.g., those
having a tendency or increased risk of developing osteoporosis)
comprising treating the patient with
tris(8-quinolinolato)gallium(III) at an amount sufficient to
slowing down the decrease of bone mineral density, increasing bone
mineral density, increasing bone mass content, or delaying the
onset of osteoporosis.
[0026] Osteoporosis is generally characterized by generalized bone
loss or low bone mineral density. The methods of the present
invention is useful in treating or delaying the onset of any
generalized bone loss or low mineral density, including, but not
limited to, osteoporosis, such as postmenopausal osteoporosis,
steroid- or glucocorticoid-induced osteoporosis, age-related
osteoporosis, osteoporosis induced by rheumatoid arthritis or by
cancer, osteomalacia, idiopathic osteoporosis, or Paget's
disease.
[0027] In various embodiments of the various methods of the present
invention, optionally a step of identifying a patient in need of
treatment or prevention can be included. For example, patients
having osteoporosis or osteopenia or low bone mineral density can
be diagnosed by any diagnosis methods or criteria in the art, e.g.,
by measuring the bone mineral density (BMD), using, e.g., dual
energy X-ray absorptiometry (DXA or DEXA), serum markers, X-rays,
etc.
[0028] Also, the identification of patients at risk of developing
osteoporosis or generalized or local bone loss is generally known
in the art. For example, patients having risk factors that are
typically associated with an increased likelihood of bone loss and
of developing osteoporosis can be identified. Known risk factors
for osteoporosis include, but are not limited to, post-menopause,
steroid or glucocorticoid use, age particularly in females,
diseases such as rheumatoid arthritis, osteomalacia, and Paget's
disease, periodontal disease, bone fracture, and periprosthetic
osteolysis. In addition, patients having certain types of cancer
(e.g., lung cancer, breast cancer, prostate cancer, multiple
myeloma or neuroendocrine tumors) with or without bone metastasis,
and patients undergoing hormone ablation therapy for either
prostate or breast cancer, are all at risk of bone loss, bone
fractures, increased frequency of skeletal-related events, and
osteoporosis.
[0029] Thus, in some embodiments, the methods of the present
invention are used for treating, or preventing or delaying the
onset of, postmenopausal osteoporosis (PMO) in women by
administering a compound of Formula (I) (e.g.,
tris(8-quinolinolato)gallium(III)) to a women in need of such
treatment, prevention or delay. In other embodiments, the methods
are used to reduce the risk or incidence of bone fractures,
skeletal-related events, or to reduce bone loss or increase bone
mass in patients with osteoporosis.
[0030] In other embodiments, the methods are used to treat, prevent
or delay the onset of, glucocorticoid-induced osteoporosis (GIO) or
skeletal-related events in men and women, particularly in those who
are either initiating or continuing systemic glucocorticoid
treatment (e.g., daily dosage equivalent to 7.5 mg or greater of
prednisone) for chronic diseases. The method comprises a step of
administering a compound of Formula (I) (e.g.,
tris(8-quinolinolato)gallium(III)) to a patient in need of such
treatment, prevention or delay.
[0031] In yet some other embodiments, the methods are applied to
treat, prevent or delay the onset of, or reduce the risk of,
osteoporosis, particularly bone loss or bone fractures or
skeletal-related events in patients receiving hormone-deprivation
therapy for either prostate or breast cancer, by administering a
compound of Formula (I) (e.g., tris(8-quinolinolato)gallium(III))
to such patients.
[0032] In yet some other embodiments, the methods are applied to
treat, prevent or delay the onset of, or reduce the risk of,
osteoporosis or low bone mineral density or bone loss or
skeletal-related events such as bone fractures, associated with
cancer (e.g., lung cancer, breast cancer, prostate cancer,
lymphoma, multiple myeloma or neuroendocrine tumors). In
particular, the compounds used in the present invention can be
administered to patients identified as having cancer (e.g., lung
cancer, breast cancer, prostate cancer, lymphoma, multiple myeloma
or neuroendocrine tumors), either with or without bone metastasis,
thereby treating, preventing or delaying the onset of, or reducing
the risk of, osteoporosis, particularly bone loss, low bone mineral
density, bone fractures, or skeletal-related events (e.g., severe
bone pain, pathological fracture, spinal cord compression). In
particular, neuroendocrine tumors often result in abnormal
parathyroid hormone levels, and are thus frequently associated with
low bone mineral density, bone loss, osteoporosis or
skeletal-related events. Thus, in specific embodiments, the present
invention provides a method of treating, preventing, or reducing
the risk or incidence of, low bone mineral density, bone loss,
osteoporosis or skeletal-related events (such as bone fractures) in
patients having neuroendocrine tumors.
[0033] In addition, the present invention is also useful in
treating or delaying the onset of any localized bone loss, e.g.,
associated with periodontal disease, with bone fractures, with
periprosthetic osteolysis.
[0034] In yet another embodiment, the present invention is used for
treating or preventing Paget's disease by administering to a
patient in need of treatment a therapeutically effective amount of
a compound according to Formula (I) below or a pharmaceutically
acceptable salt thereof (e.g.,
tris(8-quinolinolato)gallium(III)).
[0035] Moreover, inhibition of osteoclasts and bone resorption can
result in prevention or inhibition of tumor cell migration to bone
and tumor growth in bone. Therefore, the present invention also
provides a method of preventing, or delaying the onset of, bone
metastasis of cancer (e.g., lung cancer, breast cancer, prostate
cancer, lymphoma or multiple myeloma), which comprises
administering an effective amount of a compound of Formula (I) or a
pharmaceutically acceptable salt thereof (e.g.,
tris(8-quinolinolato)gallium(III)) to a patient identified as
having, or diagnosed of, cancer (e.g., lung cancer, breast cancer,
prostate cancer, lymphoma or multiple myeloma) but without bone
metastasis, thereby preventing or delaying the onset of cancer bone
metastasis, and/or prolonging bone metastasis-free survival. In one
embodiment, the method further includes a step of identifying a
patient having cancer (e.g., lung cancer, breast cancer, prostate
cancer, lymphoma, multiple myeloma) but without bone
metastasis.
[0036] In addition, a method of treating a cancer with bone
metastasis is also provided, comprising identifying a patient
having a cancer (e.g., lung cancer, breast cancer, prostate cancer,
lymphoma or multiple myeloma) that has metastasized to bone, and
administering a therapeutically effective amount of a compound of
Formula (I) or a pharmaceutically acceptable salt thereof (e.g.,
tris(8-quinolinolato)gallium(III)) to the patient.
[0037] In accordance with the methods of the present invention, a
therapeutically effective amount of a compound of Formula (I) or a
pharmaceutically acceptable salt thereof (e.g.,
tris(8-quinolinolato)gallium(III)) can be used alone as a single
agent, or alternatively in combination with one or more other
agents (e.g., anti-cancer agents and anti-osteoporosis agents). For
example, the compound of the present invention may be administered
to patients who also receive supplemental calcium and/or vitamin
D.
[0038] As used herein, the phrase "treating . . . with . . . "
means either administering a compound to a patient or causing the
formation of a compound inside a patient.
[0039] In accordance with the present invention, it is provided a
use of a compound having a compound of Formula (I) or a
pharmaceutically acceptable salt thereof (e.g.,
tris(8-quinolinolato)gallium(III)) for the manufacture of a
medicament useful for reducing osteoclastic bone resorption, and
for treating or preventing osteoporosis, low mineral density or
skeletal-related events such as bone fractures.
[0040] The pharmaceutical compounds of Formula (I) can be
administered through intradermal, intramuscular or intravenous
injection, or oral administration or any other suitable means at an
amount of from 0.01 mg to 1000 mg per kg of body weight of the
patient based on total body weight. The active ingredients may be
administered at predetermined intervals of time, e.g., four times a
day. It should be understood that the dosage ranges set forth above
are exemplary only and are not intended to limit the scope of this
invention. The therapeutically effective amount of the active
compound can vary with factors including, but not limited to, the
activity of the compound used, stability of the active compound in
the patient's body, the severity of the conditions to be
alleviated, the total weight of the patient treated, the route of
administration, the ease of absorption, distribution, and excretion
of the active compound by the body, the age and sensitivity of the
patient to be treated, and the like, as will be apparent to a
skilled artisan. The amount of administration can be adjusted as
the various factors change over time.
[0041] For example, the compound tris(8-quinolinolato)gallium(III)
can be orally delivered in the form of enclosed gelatin capsules or
compressed tablets. Capsules and tablets can be prepared in any
conventional techniques with suitable pharmaceutically acceptable
carriers such as binders, excipients, lubricants, and sweetening or
flavoring agents.
[0042] Suitable oral formulations can also be in the form of
suspension, syrup, chewing gum, wafer, elixir, and the like. If
desired, conventional agents for modifying flavors, tastes, colors,
and shapes of the special forms can also be included. In addition,
for convenient administration by enteral feeding tube in patients
unable to swallow, the active compounds can be dissolved in an
acceptable lipophilic vegetable oil vehicle such as olive oil, corn
oil and safflower oil. Injectable forms are generally known in the
art, e.g., in buffered solution or suspension.
[0043] In accordance with another aspect of the present invention,
a pharmaceutical kit is provided comprising in a container a unit
dosage form of a compound of Formula (I) or a pharmaceutically
acceptable salt thereof (e.g., tris(8-quinolinolato)gallium(III));
and optionally instructions for using the kit in the method in
accordance with the present invention. As will be apparent to a
skilled artisan, the amount of a therapeutic compound in the unit
dosage form is determined by the dosage to be used on a patient in
the method of the present invention. In the kit, for example,
tris(8-quinolinolato)gallium(III) can be in tablet or capsule or
any other suitable form at an amount of, e.g., 0.01 mg to about
3000 mg per unit dosage form.
EXAMPLES
1. In Vitro Effects of Tris(8-quinolinolato)gallium(III) on
Osteoclasts
[0044] Mouse bone marrow cells were flushed out from femora and
tibiae of two 1-2-month old mice (C57BL/6), plated in culture
medium (alpha MEM supplemented with 10% heat-inactivated fetal
bovine serum, 100 international units/ml penicillin, 100 .mu.g/ml
streptomycin, and 2 mM L-glutamine) in a 100-mm dish, and incubated
at 37.degree. C. in 5% CO.sub.2 overnight. The next day, the
non-adherent cells were pelleted and seeded at a density of 200,000
cells/cm.sup.2. Then the osteoclast precursors were cultured in the
presence of sRANKL (30 ng/ml) and M-CSF (30 ng/ml), and were
treated with either tris(8-quinolinolato)gallium(III) or GaNO.sub.3
or DMSO (as a control). The medium was changed at day 3 to obtain
mature osteoclasts at day 6. For treatment with
tris(8-quinolinolato)gallium(III), a drug stock solution prepared
by dissolving tris(8-quinolinolato)gallium(III) in DMSO was added
to the cell cultures to arrive at final concentrations of 0.2 nM, 2
nM, 0.02 .mu.M, 0.2 .mu.M, 2 .mu.M, 20 .mu.M, 50 .mu.M, or 100
.mu.M. At the end of the treatment, the osteoclast preparations
were stained for TRAP activity using a leukocyte acid phosphatase
kit from Sigma and counted for TRAP-staining multinucleated cells.
Specifically, TRAP staining was performed and five areas of each
well were photographed. The number of cells having more than 3
nuclei was counted. FIG. 1 shows that treatment with
tris(8-quinolinolato)gallium(III) significantly inhibits the fusion
of osteoclast precursors into multinucleated cells, and thus
inhibits osteoclast differentiation/formation (Mean.+-.SD of three
samples; p<0.05 compared with DMSO and GaNO.sub.3 (3.9 .mu.M, 39
.mu.M)).
[0045] RNA was also isolated for gene expression analysis of
osteoclast marker genes (TRAP, cathepsin K and NFAT2). Total RNA
was extracted from osteoclasts treated with DMSO or
tris(8-quinolinolato)gallium(III), and mRNAs were measured using
real time RT-PCR. The relative levels of mRNAs were normalized to
.beta.-actin and then expressed as fold stimulation over
DMSO-treated cells. FIG. 2 shows tris(8-quinolinolato)gallium(III)
("drug") significantly inhibited cathepsin K, NFAT2 and TRAP gene
expression.
[0046] In conclusion, tris(8-quinolinolato)gallium(III) is very
effective in inhibiting osteoclast differentiation/maturation.
2. In Vivo Effects of Tris(8-quinolinolato)gallium(III)
[0047] The Sprague-Dawley female rats (100-125 gram, about 12 weeks
old) were purchased from Taconic (Hudson, N.Y.). They were divided
into 5 groups of 8 rats each. Four groups were ovariectomized, and
one group was sham-operated. The rats were treated in the following
manner: Group 1 ovariectomized rats were orally administered day
one through day five of each week with 15 mg/kg
tris(8-quinolinolato)gallium(III) ("daily"); Group 2 ovariectomized
rats were treated with 75 mg/kg tris(8-quinolinolato)gallium(III)
on day one of each week ("weekly"); Group 3 ovariectomized rats
were treated with 38 mg/kg Ga(NO.sub.3).sub.3 on day one of each
week ("weekly"); Group 4 ovariectomized rats were treated with
vehicle only (0.1% carboxymethylcellulose); Group 5 rats were
sham-operated and were administered with vehicle only (0.1%
carboxymethylcellulose). At the end of the experiment (32 days
after the first administration of the compounds), the animals were
anesthetized. Areal bone mineral density (BMD) and bone mineral
content (BMC) were measured for the tibiae by dual-energy X-ray
absorptiometry using an X-ray pixiMUS mouse densitometer. As shown
in FIG. 3, daily administration of
tris(8-quinolinolato)gallium(III) ("daily drug") to ovariectomized
rats was associated with increased bone mineral density when
compared to ovariectomized untreated rats ("vehicle").
[0048] Blood was drawn from the rats and left at room temperature
for at least 30 minutes before centrifuging at 200.times.g for 10
minutes to separate sera. Serum was used for the measurement of
serum markers including osteocalcin. Osteocalcin was measured by
ELISA. Osteocalcin is produced only by osteoblast in the course of
bone remodeling. It is the most abundant non-collagenous protein of
the bone extracellular matrix. See Weinreb, M., Shinar, D. &
Rodan, G. A. J. Bone Miner. Res. 5, 831-842 (1990). It has been
shown that osteocalcin-deficient mice exhibit higher bone mass and
bones of improved functional quality. Ducy et al., Nature. 382,
448-452 (1996). Osteocalcin is thus a negative regulator of bone
formation, and reduction of serum osteocalcin can lead to increased
bone mass. As shown in FIG. 4, treatment of ovariectomized rats
with tris(8-quinolinolato)gallium(III) ("drug daily") was
significantly associated with reduced serum osteocalcin when
compared to ovariectomized rats treated with vehicle only
("vehicle"). Thus, this further confirms that
tris(8-quinolinolato)gallium(III) is useful for osteoporosis
treatment.
3. Quantitative Effects of Tris(8-quinolinolato)gallium(III) in
Bone Models
[0049] Mouse bone marrow cells derived as described in section 2 is
plated onto 16-well BD BioCoat.TM. Osteologic.TM. Discs seeded at a
density of 200,000 cells/cm.sup.2 in culture medium plus sRANKL (30
ng/ml) and M-CSF (30 ng/ml). Cells are treated with varying
concentrations of tris(8-quinolinolato)gallium(III) (0.0002, 0.002,
0.02, 0.2, 2, 20 .mu.M). Similar medium changes are performed every
2 days until day 8 for quantitation of resorption areas using SPOT
Advanced software by microscopy.
[0050] All publications and patent applications mentioned in the
specification are indicative of the level of those skilled in the
art to which this invention pertains. All publications and patent
applications are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference. The mere mentioning of the publications and patent
applications does not necessarily constitute an admission that they
are prior art to the instant application.
[0051] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be apparent that certain changes and
modifications may be practiced within the scope of the appended
claims.
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