U.S. patent application number 14/282177 was filed with the patent office on 2015-04-23 for methods of diagnosing, preventing, and treating bone mass diseases.
The applicant listed for this patent is Patricia F. Ducy, Gerard Karsenty, Donald Landry, Yuli Xie, Vijay Yadav. Invention is credited to Patricia F. Ducy, Gerard Karsenty, Donald Landry, Yuli Xie, Vijay Yadav.
Application Number | 20150111908 14/282177 |
Document ID | / |
Family ID | 41135912 |
Filed Date | 2015-04-23 |
United States Patent
Application |
20150111908 |
Kind Code |
A1 |
Karsenty; Gerard ; et
al. |
April 23, 2015 |
METHODS OF DIAGNOSING, PREVENTING, AND TREATING BONE MASS
DISEASES
Abstract
The present invention provides methods and therapeutic agents
for lowering or increasing serum serotonin levels in a patient in
order to increase or decrease bone mass, respectively. In preferred
embodiments, the patient is known to have, or to be at risk for, a
low bone mass disease such as osteoporosis and the agents are TPH1
inhibitors or serotonin receptor antagonists.
Inventors: |
Karsenty; Gerard; (New York,
NY) ; Ducy; Patricia F.; (New York, NY) ; Xie;
Yuli; (Shanghai, CN) ; Landry; Donald; (New
York, NY) ; Yadav; Vijay; (Cambridge, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Karsenty; Gerard
Ducy; Patricia F.
Xie; Yuli
Landry; Donald
Yadav; Vijay |
New York
New York
Shanghai
New York
Cambridge |
NY
NY
NY |
US
US
CN
US
GB |
|
|
Family ID: |
41135912 |
Appl. No.: |
14/282177 |
Filed: |
May 20, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12935651 |
Dec 21, 2010 |
8759364 |
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PCT/US09/38817 |
Mar 30, 2009 |
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14282177 |
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61072596 |
Mar 31, 2008 |
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61090940 |
Aug 22, 2008 |
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Current U.S.
Class: |
514/272 |
Current CPC
Class: |
A61K 31/513 20130101;
A61P 35/04 20180101; A61K 31/505 20130101; A61P 43/00 20180101;
G01N 33/942 20130101; A61K 31/357 20130101; A61P 19/10 20180101;
A61P 19/00 20180101; G01N 2800/50 20130101; A61K 45/06 20130101;
A61K 31/357 20130101; G01N 2800/108 20130101; A61K 31/513 20130101;
A61P 25/24 20180101; A61P 19/08 20180101; A61K 2300/00 20130101;
A61K 2300/00 20130101 |
Class at
Publication: |
514/272 |
International
Class: |
A61K 31/505 20060101
A61K031/505 |
Goverment Interests
STATEMENT OF GOVERNMENTAL INTEREST
[0002] This invention was made with Government support under
NIH-5R01 DK 067936 and March of Dimes grant 6FY05-1260. The
Government has certain rights in the invention.
Claims
1-16. (canceled)
17. A method of treating osteoporosis in a patient in need thereof
comprising administering to the patient a therapeutically effective
amount of a TPH1 inhibitor that lowers the level of peripheral
serotonin in the patient; where the TPH1 inhibitor is selected from
the group consisting of: ##STR00044## and pharmaceutically
acceptable salts thereof, wherein: A is optionally substituted
cycloalkyl, aryl, or heterocycle; X is a bond (i.e., A is directly
bound to D), --O--, --S--, --C(O)--, --C(R4)=, .dbd.C(R.sub.4)--,
--C(R.sub.3R.sub.4)--, --C(R.sub.4).dbd.C(R.sub.4)--,
--C.ident.C--, --N(R.sub.5)--, --N(R.sub.5)C(O)N(R.sub.5)--,
--C(R.sub.3R.sub.4)N(R.sub.5)--, --N(R.sub.5)C(R.sub.3R.sub.4)--,
--ONC(R.sub.3)--, --C(R.sub.3)NO--, --C(R.sub.3R.sub.4)O--,
--OC(R.sub.3R.sub.4)--, --S(O.sub.2)--, --S(O.sub.2)N(R.sub.5)--,
--N(R.sub.5)S(O.sub.2)--, --C(R.sub.3R.sub.4)S(O.sub.2)--, or
--S(O.sub.2)C(R.sub.3R.sub.4)--; D is optionally substituted aryl
or heterocycle; R.sub.1 is hydrogen or optionally substituted
alkyl, alkyl-aryl, alkyl-heterocycle, aryl, or heterocycle; R.sub.2
is hydrogen or optionally substituted alkyl, alkyl-aryl,
alkyl-heterocycle, aryl, or heterocycle; R.sub.3 is hydrogen,
alkoxy, amino, cyano, halogen, hydroxyl, or optionally substituted
alkyl; R.sub.4 is hydrogen, alkoxy, amino, cyano, halogen,
hydroxyl, or optionally substituted alkyl or aryl; each R.sub.5 is
independently hydrogen or optionally substituted alkyl or aryl; and
n is 0-3; and ##STR00045## and pharmaceutically acceptable salts
thereof, wherein: A is optionally substituted cycloalkyl, aryl, or
heterocycle; X is a bond (i.e., A is directly bound to D), --O--,
--S--, --C(O)--, --C(R4)=, .dbd.C(R.sub.4)--,
--C(R.sub.3R.sub.4)--, --C(R.sub.4).dbd.C(R.sub.4)--,
--C.ident.C--, --N(R.sub.5)--, --N(R.sub.5)C(O)N(R.sub.5)--,
--C(R.sub.3R.sub.4)N(R.sub.5)--, --N(R.sub.5)C(R.sub.3R.sub.4)--,
--ONC(R.sub.3)--, --C(R.sub.3)NO--, --C(R.sub.3R.sub.4)O--,
--OC(R.sub.3R.sub.4)--, --S(O.sub.2)--, --S(O.sub.2)N(R.sub.5)--,
--N(R.sub.5)S(O.sub.2)--, --C(R.sub.3R.sub.4)S(O.sub.2)--, or
--S(O.sub.2)C(R.sub.3R.sub.4)--; D is optionally substituted aryl
or heterocycle; E is optionally substituted aryl or heterocycle;
R.sub.1 is hydrogen or optionally substituted alkyl, alkyl-aryl,
alkyl-heterocycle, aryl, or heterocycle; R.sub.2 is hydrogen or
optionally substituted alkyl, alkyl-aryl, alkyl-heterocycle, aryl,
or heterocycle; R.sub.3 is hydrogen, alkoxy, amino, cyano, halogen,
hydroxyl, or optionally substituted alkyl; R.sub.4 is hydrogen,
alkoxy, amino, cyano, halogen, hydroxyl, or optionally substituted
alkyl or aryl; each R.sub.5 is independently hydrogen or optionally
substituted alkyl or aryl; and n is 0-3.
18-45. (canceled)
46. The method of claim 17 where the TPH1 inhibitor is selected
from the group consisting of: ##STR00046## and pharmaceutically
acceptable salts thereof; and ##STR00047## and pharmaceutically
acceptable salts thereof.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/935,651, which is a 371 of International
Patent Application No. PCT/US2009/038817, filed Mar. 30, 2009,
which claims priority to U.S. Provisional Patent Application Ser.
No. 61/072,596, filed Mar. 31, 2008 and U.S. Provisional Patent
Application Ser. No. 61/090,940, filed Aug. 22, 2008. The contents
of these applications are hereby incorporated by reference in their
entireties.
FIELD OF THE INVENTION
[0003] The invention is in the field of diagnosis and therapy of
bone diseases associated with higher than normal or lower than
normal bone mass.
BACKGROUND OF THE INVENTION
[0004] Bone remodeling, the mechanism whereby vertebrates renew
bone tissues throughout adulthood, comprises two phases: resorption
of preexisting mineralized bone matrix by a specialized cell type,
the osteoclast, followed by de novo bone formation by another
specialized cell type, the osteoblast. Genetic and molecular
studies have shown that local effectors (cytokines and growth
factors) and systemic effectors (hormones and neuromediators)
modulate both phases of bone remodeling.
[0005] One of the most intensively studied genes regulating bone
remodeling is LDL-receptor related protein 5 (LRP5).
Loss-of-function mutations in LRP5 result in osteoporosis
pseudoglioma (OPPG), a disease characterized by severe bone loss
due to a decrease in bone formation and by the persistence of
embryonic vascularization of the eyes, causing blindness. By
contrast, gain-of-function mutations in LRP5 cause another bone
disease, high bone mass syndrome. The involvement of Lrp5 in two
human diseases of opposite nature underscores the importance of the
pathways controlled by this gene in the regulation of bone
formation. However, the mechanism by which LRP5 affects bone
development is not known.
SUMMARY OF THE INVENTION
[0006] It has been discovered that elevated levels of serum
serotonin, due to overexpression of tryptophan hydroxylase 1, the
enzyme responsible for the first step of serotonin synthesis in
enterochromaffin cells of the duodenum, and possibly in
osteoblasts, causes decreased bone mass in LRP5 loss of function
mutants. Thus, certain embodiments of the invention are directed to
methods for treating or preventing low bone mass diseases such as
osteoporosis and OPPG by administering a therapeutic agent that
inhibits serotonin synthesis or inhibits TPH1, the enzyme necessary
for serotonin synthesis in duodenum, or by administering
antagonists of the serotonin receptor HT1B that is the receptor
mediating the effect of serotonin on osteoblasts.
[0007] Certain other embodiments of the invention are directed to
pharmaceutical compositions for increasing bone mass that include
therapeutic agents that decrease serum serotonin levels including
one or more TPH 1 inhibitors, or one or more serotonin receptor
antagonists, or both, for use in treating or preventing low bone
mass diseases. In some embodiments, the present invention includes
a pharmaceutical composition for treating or preventing anxiety or
depression where the pharmaceutical composition includes both an
SSRI and a drug that reduces the level of serum serotonin, in order
to prevent patients treated with serotonin reuptake inhibitors from
developing osteoporosis or to treat osteoporosis in patients taking
SSRIs.
[0008] In other embodiments, the present invention provides methods
of treating patients for anxiety or depression where an SSRI and a
drug that reduces the level of serum serotonin are administered to
a patient via separate pharmaceutical compositions.
[0009] Diseases associated with abnormally high bone mass can be
treated by increasing the levels of peripheral serotonin, either by
administering serotonin itself, or serotonin reuptake inhibitors
that act in the periphery, HT1B agonists, activators of TPH1, or
combinations thereof.
[0010] U.S. Provisional Patent Application Ser. No. 60/976,403,
filed Sep. 28, 2007, and incorporated by reference herein in its
entirety, discloses that brain-derived serotonin (hereafter
abbreviated BDS) has the opposite effect of peripheral serotonin.
Elevated BDS increases bone mass by acting through HT2C receptors
on target neurons in the hypothalamus. Thus, some embodiments of
the present invention include administering a combination of
therapeutic agents that includes agents that decrease peripheral
serotonin and agents that increase BDS. BDS can be increased by
increasing the activity of TPH2, the enzyme responsible for the
first step of serotonin synthesis in neurons of the brain stem, and
by administering agonists of the HT2C serotonin receptor in the
brain.
[0011] Other methods disclosed herein are directed to diagnosing a
person at risk of developing a low bone mass disease such as
osteoporosis by determining if the serum level of serotonin in the
periphery is abnormally high (about 25% or more) compared to normal
individuals, taking into account the age, gender, or other factors
that affect serum serotonin levels. Such a person at risk may be
treated with therapeutic agents that decrease serum serotonin to
prevent the low bone mass disease from developing. Those of skill
in the art will understand that serum serotonin levels may vary
among individuals depending on certain factors and will be able to
take those factors into account to determine whether a person has
abnormally high serum serotonin levels. One possible range which
those skilled in the art may consider to be normal serum serotonin
levels is 101-283 ng/ml (nanograms per milliliter).
[0012] Since elevated serum serotonin may not be the only cause of
diseases associated with low bone mass, methods other than those
measuring serum serotonin levels may also be used to determine if a
person having a low bone mass disease such as osteoporosis should
be treated with drugs that decrease serum serotonin.
[0013] The present invention provides a method of lowering serum
serotonin levels in a patient known or suspected to be in need of
lowering of serum serotonin levels comprising administering to the
patient known or suspected to be in need of lowering of serum
serotonin levels a TPH1 inhibitor or a serotonin receptor
antagonist.
[0014] The present invention also provides a method of treating or
preventing a low bone mass disease in a patient known or suspected
to be in need of such treatment or prevention comprising
administering to the patient known or suspected to be in need of
such treatment or prevention a therapeutically effective amount of
an agent that lowers the level of serum serotonin.
[0015] In certain embodiments, the agent is a TPH1 inhibitor or a
serotonin receptor antagonist. In preferred embodiments, the agent
is a TPH1 inhibitor that does not cross the blood brain barrier. In
other embodiments, the agent is a TPH1 inhibitor that does not
significantly inhibit TPH2.
[0016] In certain embodiments, the agent is a TPH1 inhibitor
selected from the group consisting of:
##STR00001##
and pharmaceutically acceptable salts and solvates thereof,
wherein: A is optionally substituted cycloalkyl, aryl, or
heterocycle; X is a bond (i.e., A is directly bound to D), --O--,
--S--, --C(O)--, --C(R4)=, .dbd.C(R.sub.4)--,
--C(R.sub.3R.sub.4)--, --C(R.sub.4).dbd.C(R.sub.4)--,
--C.ident.C--, --N(R.sub.5)--, --N(R.sub.5)C(O)N(R.sub.5)--,
--C(R.sub.3R.sub.4)N(R.sub.5)--, --N(R.sub.5)C(R.sub.3R.sub.4)--,
--ONC(R.sub.3)--, --C(R.sub.3)NO--, --C(R.sub.3R.sub.4)O--,
--OC(R.sub.3R.sub.4)--, --S(O.sub.2)--, --S(O.sub.2)N(R.sub.5)--,
--N(R.sub.5)S(O.sub.2)--, --C(R.sub.3R.sub.4)S(O.sub.2)--, or
--S(O.sub.2)C(R.sub.3R.sub.4)--; D is optionally substituted aryl
or heterocycle; R.sub.1 is hydrogen or optionally substituted
alkyl, alkyl-aryl, alkyl-heterocycle, aryl, or heterocycle; R.sub.2
is hydrogen or optionally substituted alkyl, alkyl-aryl,
alkyl-heterocycle, aryl, or heterocycle; R.sub.3 is hydrogen,
alkoxy, amino, cyano, halogen, hydroxyl, or optionally substituted
alkyl; R.sub.4 is hydrogen, alkoxy, amino, cyano, halogen,
hydroxyl, or optionally substituted alkyl or aryl; each R.sub.5 is
independently hydrogen or optionally substituted alkyl or aryl; and
n is 0-3;
##STR00002##
and pharmaceutically acceptable salts and solvates thereof,
wherein: A is optionally substituted cycloalkyl, aryl, or
heterocycle; X is a bond (i.e., A is directly bound to D), --O--,
--S--, --C(O)--, --C(R4)=, .dbd.C(R.sub.4)--,
--C(R.sub.3R.sub.4)--, --C(R.sub.4).dbd.C(R.sub.4)--,
--C.ident.C--, --N(R.sub.5)--, --N(R.sub.5)C(O)N(R.sub.5)--,
--C(R.sub.3R.sub.4)N(R.sub.5)--, --N(R.sub.5)C(R.sub.3R.sub.4)--,
--ONC(R.sub.3)--, --C(R.sub.3)NO--, --C(R.sub.3R.sub.4)O--,
--OC(R.sub.3R.sub.4)--, --S(O.sub.2)--, --S(O.sub.2)N(R.sub.5)--,
--N(R.sub.5)S(O.sub.2)--, --C(R.sub.3R.sub.4)S(O.sub.2)--, or
--S(O.sub.2)C(R.sub.3R.sub.4)--; D is optionally substituted aryl
or heterocycle; E is optionally substituted aryl or heterocycle;
R.sub.1 is hydrogen or optionally substituted alkyl, alkyl-aryl,
alkyl-heterocycle, aryl, or heterocycle; R.sub.2 is hydrogen or
optionally substituted alkyl, alkyl-aryl, alkyl-heterocycle, aryl,
or heterocycle; R.sub.3 is hydrogen, alkoxy, amino, cyano, halogen,
hydroxyl, or optionally substituted alkyl; R.sub.4 is hydrogen,
alkoxy, amino, cyano, halogen, hydroxyl, or optionally substituted
alkyl or aryl; each R.sub.5 is independently hydrogen or optionally
substituted alkyl or aryl; and n is 0-3;
##STR00003##
where R is hydrogen or lower alkyl; and n is 1, 2, or 3;
##STR00004##
where R is hydrogen or lower alkyl; and n is 1, 2, or 3;
##STR00005##
where R is hydrogen or lower alkyl; R.sub.1, R.sub.2, and R.sub.3,
are independently: [0017] hydrogen; [0018] halogen; [0019] lower
alkyl; [0020] alkoxy; or [0021] amino; and n is 1, 2, or 3;
##STR00006##
[0021] where R is hydrogen or lower alkyl; R.sub.1, R.sub.2, and
R.sub.3, are independently: [0022] hydrogen; [0023] halogen; [0024]
lower alkyl; [0025] alkoxy; or [0026] amino; and n is 1, 2, or
3;
##STR00007##
[0026] where R is hydrogen, lower alkyl, or cycloalkyl;
##STR00008##
where R is hydrogen, lower alkyl, or cycloalkyl;
##STR00009##
where R.sub.1 and R.sub.2 are independently hydrogen, lower alkyl,
or cycloalkyl;
##STR00010##
where R is hydrogen, lower alkyl, or cycloalkyl;
##STR00011##
where R.sub.1 and R.sub.2 are independently hydrogen, lower alkyl,
cycloalkyl, F, Cl, or OH;
##STR00012##
where R.sub.1 and R.sub.2 are independently hydrogen, lower alkyl,
or cycloalkyl; including any racemic mixtures and individual
enantiomers of the agents, esters, and salts of the agents with a
physiologically acceptable acid.
[0027] The present invention provides pharmaceutical compositions
comprising a therapeutically effective amount of a TPH1 inhibitor
disclosed above and at least one pharmaceutically acceptable
excipient.
[0028] In certain embodiments, the agent is a serotonin receptor
antagonist, preferably an HT1B, HT2A or HT2B serotonin receptor
antagonist, and most preferably an HT serotonin receptor
antagonist. In certain embodiments, the serotonin receptor
antagonist is an HT1B serotonin receptor antagonist listed in Table
3.
[0029] The present invention also provides methods where the
patient is administered both a TPH1 inhibitor and a serotonin
receptor antagonist. The TPH1 inhibitor and the serotonin receptor
antagonist may be administered together in a single pharmaceutical
composition.
[0030] In certain embodiments of the present invention, the low
bone mass disease is osteoporosis, osteoporosis pseudoglioma
syndrome (OPPG), osteopenia, osteomalacia, renal osteodystrophy,
faulty bone formation or resorption, Paget's disease, fractures and
broken bones, or bone metastasis. Preferably, the low bone mass
disease is osteoporosis.
[0031] In other embodiments of the invention, the patient is being
treated with an SSRI, a bisphosphonate, or a beta blocker in
addition to an agent that lowers the level of serum serotonin. In
some embodiments, the methods of the present invention also
comprise administering an SSRI, a bisphosphonate, or a beta blocker
in addition to an agent that lowers the level of serum
serotonin.
[0032] In certain embodiments, the patient is being treated with an
agent that increases the level of serum serotonin (e.g., an SSRI)
or the patient has a condition associated with an increased level
of serum serotonin. In certain embodiments, the method also
comprises treating the patient with an agent that increases the
level of serum serotonin (e.g., an SSRI).
[0033] In certain embodiments, the patient's level of serum
serotonin is measured prior to administering the agent that lowers
the level of serum serotonin. In other embodiments, the patient's
level of serum serotonin is measured after administering the agent
that lowers the level of serum serotonin. In some embodiments, the
patient's level of serum serotonin is measured before and after
administering the agent that lowers the level of serum
serotonin.
[0034] In certain embodiments, the agent that lowers the level of
serum serotonin is repeatedly administered to the patient and the
patient's level of serum serotonin is measured until the patient's
level of serum serotonin is reduced by at least about 10% compared
to the level measured prior to the first administration of the
agent that lowers the level of serum serotonin.
[0035] In certain embodiments, the patient has been identified as
having a serum serotonin level that is more than 25% higher than
the normal level of serum serotonin.
[0036] In certain embodiments, the patient is administered an agent
that increases brain derived serotonin in addition to the agent
that lowers the level of serum serotonin. In preferred embodiments,
the agent that increases brain derived serotonin is an agent that
increases TPH2 activity.
[0037] In certain embodiments, the patient's level of serum
serotonin is lowered by at least about 10% compared to the level
before administering the agent that lowers the level of serum
serotonin.
[0038] In certain embodiments, the agent that lowers the level of
serum serotonin is administered in an amount of from about 1 mg/day
to about 2 g/day.
[0039] The present invention provides a pharmaceutical composition
comprising an amount of an agent that lowers the level of serum
serotonin in a patient to whom the composition is administered by
at least about 10%. In preferred embodiments, the agent is a TPH1
inhibitor or a serotonin receptor antagonist.
[0040] The present invention provides a pharmaceutical composition
comprising a therapeutically effective amount of an agent that
lowers the level of serum serotonin in a patient to whom the
composition is administered. In preferred embodiments, the agent is
a TPH1 inhibitor or a serotonin receptor antagonist.
[0041] In some embodiments, the pharmaceutical composition
comprises an agent that lowers the level of serum serotonin and an
agent that raises the level of brain-derived serotonin.
[0042] In some embodiments, the pharmaceutical composition
comprises an agent that lowers the level of serum serotonin and an
SSRI, a bisphosphonate, or a beta blocker. In preferred
embodiments, the agent is a TPH1 inhibitor or a serotonin receptor
antagonist. In certain embodiments, the serotonin receptor
antagonist is an HT1B, HT2A or HT2B serotonin receptor antagonist,
preferably an HT1B serotonin receptor antagonist.
[0043] In certain embodiments, the pharmaceutical composition
comprises both a TPH1 inhibitor and a serotonin receptor
antagonist.
[0044] The present invention also provides a method for identifying
a subject at risk of developing a disease associated with low bone
mass, comprising,
a) determining the level of serum serotonin in biological samples
taken from the patient and from a normal subject, b) concluding
that the patient is at risk of developing the disease if the level
of serum serotonin in the sample from the patient is elevated by at
least about 25% above the serum serotonin level in the sample from
the normal subject.
[0045] In certain embodiments, the method comprises, after step
(b), administering to the patient at risk of developing the disease
an agent that lowers the level of serum serotonin.
BRIEF DESCRIPTION OF THE FIGURES
[0046] FIG. 1. Lrp5-/- mice have low bone mass (A) with no change
in osteoclast surface (B) but decreased osteoblast numbers (C).
Real-time PCR analysis of Lrp5-/- molecular signature. Lrp5-/-
osteoblasts do not show changes in osteoblast-specific gene
expression (D) but have decreased Cyclin gene expression (E).
[0047] FIG. 2. Real-time PCR analysis of cell cycle marker genes in
.beta.-cat.sub.ob -/- bones (ob-/-).
[0048] FIG. 3. Microarray analysis of Lrp5-/- bones reveals an
increased expression of Tryptophan hydroxylase 1 (Tph1) gene
expression compared to wt bones. Green and red bars indicate a
decrease and an increase in gene expression, respectively. Genes
including and to the left of Hk2 showed decreased expression while
genes including and to the right of Spna1 showed increased
expression.
[0049] FIG. 4A. Tph1 expression is increased in the duodenum of
Lrp5-/- mice. FIG. 4B Tph1 expression is 1000 fold higher in
duodenum than in bone in Lrp5-/- mice. FIG. 4C Tph1 expression in
duodenum and FIG. 4D serum serotonin levels increase progressively
with age in Lrp5-/- mice. Neither Tph2 expression (FIG. 4E) nor
serotonin levels (FIG. 4F) are altered in the brain of Lrp5-/-
mice.
[0050] FIG. 5. 5Htt-/- mice have low bone mass and decreased
osteoblast numbers (A). Real-time PCR analysis of gene expression
in bone revealed a decreased expression of cyclins in 5Htt-/- mice
while no changes in the expression of osteoblast differentiation
markers or type I collagen genes can be detected (B).
[0051] FIG. 6. Histologic and histomorphometric comparison of
Lrp5/5Htt (Htt) compound mice. Lrp5+/-; Htt+/- double heterozygous
mice have a more severe decrease in bone mass than the Lrp5+/- or
5Htt+/- single heterozygous mice. This is also true for the
decrease in osteoblast numbers.
[0052] FIG. 7A. Tryptophan hydroxylase inhibitor (pCPA) treatment
normalizes serum serotonin levels and corrects bone abnormalities
observed in Lrp5 -/- mice. Treatment regimen for the pCPA
treatment, FIG. 7B histomorphometric analysis of bone phenotype,
FIG. 7C serum serotonin levels, FIG. 7D gut Tph1 expression levels,
FIG. 7E brain Tph2 expression upon vehicle and pCPA treatment,
brain Tph2 expression upon vehicle and pCPA treatment.
[0053] FIG. 8. Real-time PCR analysis of the expression of known
serotonin receptors in WT osteoblasts (A) and the expression of
cyclins and osteoblast-specific genes in primary osteoblasts
treated with serotonin or vehicle (B).
[0054] FIG. 9A. Western blot analysis of CREB phosphorylation and
FIG. 9B ChIP analysis of CREB binding to the Cyclin D1 promoter in
primary osteoblasts treated with serotonin for the indicated
times.
[0055] FIG. 10. Real-time PCR analysis of the expression of Tph1
(left panel) and Lrp5 (right panel) in duodenum of WT mice at the
indicated ages.
[0056] FIG. 11. Oral feeding of CBMIDA reduces peripheral
serotonin.
[0057] FIG. 12. Mice engineered to express in both alleles of their
Lrp5 genes of their duodenal cells a mutation that in humans leads
to high bone mass show a higher bone mass than wild-type (WT) mice.
Vertebrae were embedded in plastic medium, sectioned at 5
micrometers and stained with the von Kossa/Van gieson reagent. The
bone matrix was stained in black.
[0058] FIG. 13. Regimen for the oral feeding of CBMIDA and other
compounds in Example 11.
[0059] FIG. 14. Changes in serotonin levels upon oral deeding of
CBMIDA and other compounds in Example 11.
[0060] FIG. 15. Effect of CBMIDA and pCPA on peripheral serotonin
production in ovariectomized mice.
DETAILED DESCRIPTION OF THE INVENTION
[0061] Diseases associated with low bone density ("low bone mass
diseases"), as used herein, refers to any bone disease or state
that results in or is characterized by loss of health or integrity
to bone due to abnormally low bone mass, and includes, but is not
limited to, osteoporosis, osteoporosis pseudoglioma syndrome
(OPPG), osteopenia, osteomalacia, renal osteodystrophy, faulty bone
formation or resorption, Paget's disease, fractures and broken
bones, and bone metastasis. More particularly, bone diseases that
can be treated and/or prevented in accordance with the present
invention include bone diseases characterized by a decreased bone
mass relative to that of corresponding non-diseased bone.
[0062] Diseases associated with high bone density, as used herein,
refers to any bone disease or state which results in or is
characterized by an abnormally high bone density, such as high bone
mass syndrome.
[0063] Prevention of bone disease means actively intervening as
described herein prior to overt disease onset to prevent the
disease or minimize the extent of the disease or slow its course of
development.
[0064] Treatment of bone disease means actively intervening after
onset to slow down, ameliorate symptoms of, or reverse the disease
or situation in a patient who is known or suspected of having a
bone disease, particularly a low bone mass disease. More
specifically, treating refers to a method that modulates bone mass
to more closely resemble that of corresponding non-diseased bone
(that is a corresponding bone of the same type, e.g., long,
vertebral, etc.) in a non-diseased state.
[0065] Unless otherwise indicated, a "therapeutically effective
amount" of a compound is an amount that provides a therapeutic
benefit in the treatment or management of a disease or condition,
delays or minimizes one or more symptoms associated with the
disease or condition, or enhances the therapeutic efficacy of
another therapeutic agent. An agent is said to be administered in a
"therapeutically effective amount" if the amount administered
results in a desired change in the physiology of a recipient
mammal, (e.g., increases bone mass in a mammal having or at risk of
developing a low bone mass disease) compared to pre-treatment
levels, or decreases bone mass in an animal having or at risk of
developing a high bone mass disease (compared to pre-treatment
levels). That is, drug therapy results in treatment, i.e.,
modulates bone mass to more closely resemble that of corresponding
non-diseased bone (such as a corresponding bone of the same type,
e.g., long, vertebral, etc.) in a non-diseased state. For example,
a therapeutically effective amount of a TPH1 inhibitor or agent
that reduces serotonin synthesis includes an amount that reduce
serum serotonin levels to a level that is at least about 10% less
than the level before drug treatment.
[0066] A therapeutic agent such as a TPH1 inhibitor significantly
reduces serum serotonin if the post-treatment level of serotonin is
reduced at least about 10% or more compared to pre-treatment
levels. A patient is at risk of developing a low bone mass disease
if his or her serum serotonin levels are elevated by about 25% or
more compared to serum serotonin levels in normal subjects.
Alternatively, A patient is at risk of developing a high bone mass
disease if his or her serum serotonin levels are reduced by about
25% or more compared to serum serotonin levels in normal
subjects.
[0067] A "patient" is a mammal, preferably a human, but can also be
companion animals such as dogs or cats, or farm animals such as
horses, cattle, pigs, or sheep.
[0068] A patient in need of treatment or prevention for a bone
disease includes a patient known or suspected of having or being at
risk of developing a bone disease. Such a patient in need of
treatment could be, e.g., a person known to have osteoporosis. A
patient at risk of developing a bone disease could include the
elderly, post-menopausal women, patients being treated with
glucocorticoids, patients being treated with SSRIs, and patients
having bone density outside the normal range. Other persons in need
of treatment or prevention by the methods of the present invention
include persons who are known to be in need of therapy to decrease
serum serotonin levels in order to treat or prevent a bone disease,
e.g., osteoporosis. Such persons might include persons who have
been identified as having a serum serotonin level that is about 25%
or more above that of serum serotonin levels in normal
subjects.
[0069] A patient in need of treatment or prevention for a bone
disease by the methods of the present invention does not include a
patient being treated with a TPH1 inhibitor, a serotonin HT1B
antagonist, or other agent that decreases serum serotonin levels
where the patient is being treated with the TPH1 inhibitor,
serotonin HT1B antagonist, or other agent that decreases serum
serotonin levels for a purpose other than to treat a bone disease.
Thus, patient in need of treatment or prevention for a bone disease
by the methods of the present invention does not include a patient
being treated with a TPH1 inhibitor for the purpose of treating
chemotherapy-induced emesis or gastrointestinal disorders such as
irritable bowel syndrome.
[0070] A "small organic molecule" is meant organic compounds of
molecular weight of more than 100 and less than about 2,500
daltons, and preferably less than 500 daltons.
[0071] A "TPH1 inhibitor" is a substance that reduces the amount of
5-hydroxytryptophan produced from tryptophan by TPH1 by at least
about 10% in a suitable assay, as compared to the amount of
5-hydroxytryptophan produced from tryptophan by TPH1 in the assay
in the absence of the substance. Assays for determining the level
of TPH1 inhibition of an agent are described in International
Patent Publication WO 2007/089335.
[0072] A "TPH2 inhibitor" is a substance that reduces the amount of
5-hydroxytryptophan produced from tryptophan by TPH2 by at least
about 10% in a suitable assay, as compared to the amount of
5-hydroxytryptophan produced from tryptophan by TPH2 in the assay
in the absence of the substance.
[0073] Techniques for measuring bone mass include those techniques
well known to those of skill in the art including, but not limited
to, skeletal X-rays, which show the lucent level of bone (the lower
the lucent level, the higher the bone mass); classical bone
histology (e.g., bone volume, number and aspects of
trabiculi/trabiculations, numbers of osteoblasts relative to
controls and/or relative to osteoclasts); and dual energy X-ray
absorptiometry (DEXA) (Levis & Altman, 1998, Arthritis and
Rheumatism, 41:577-587) which measures bone mass and is commonly
used in osteoporosis. BFR means bone formation rate. Any method
known in the art can be used to diagnose a person at risk of
developing high or low bone mass diseases, or to determine the
efficacy of drug therapy.
[0074] Selective serotonin reuptake inhibitors (SSRIs) mean a class
of antidepressants used in the treatment of depression, anxiety
disorders, and some personality disorders. They are also typically
effective and used in treating premature ejaculation problems.
SSRIs increase the extracellular level of the neurotransmitter
serotonin by inhibiting its reuptake into the presynaptic cell,
increasing the level of serotonin available to bind to the
postsynaptic receptor. They have varying degrees of selectivity for
the other monoamine transporters, having little binding affinity
for the noradrenaline and dopamine transporters. The first class of
psychotropic drugs to be rationally designed, SSRIs are the most
widely prescribed antidepressants in many countries. SSRIs include:
citalopram (CELEXA.RTM., CIPRAMIL.RTM., EMOCAL.RTM., SEPRAM.RTM.,
SEROPRAM.RTM.); escitalopram oxalate (LEXAPRO.RTM., CIPRALEX.RTM.,
ESERTIA.RTM.); fluoxetine (PROZAC.RTM., FONTEX.RTM., SEROMEX.RTM.,
SERONIL.RTM., SARAFEM.RTM., FLUCTIN.RTM. (EUR), FLUOX.RTM. (NZ));
fluvoxamine maleate (LUVOX.RTM., FAVERIN.RTM.); paroxetine
(PAXIL.RTM., SEROXAT.RTM., AROPAX.RTM., DEROXAT.RTM., REXETIN.RTM.,
XETANOR.RTM., PAROXAT.RTM.); sertraline (ZOLOFT.RTM., LUSTRAL.RTM.,
SERLAIN.RTM.), and dapoxetine (no known trade name).
Lrp5 Regulates Bone Development Through More than One Mechanism
[0075] The extreme conservation of gene function between mouse and
human when it comes to skeletal biology explains why skeletal
biology, and especially the study of bone remodeling and
homeostasis, has been profoundly influenced by mouse and human
genetic studies. Although gene inactivation experiments in mice or
molecular cloning of disease genes in humans were designed
initially to identify genes important during embryonic development,
results of these studies went further than this initial goal by
also shedding new light on the molecular bases of skeletal biology
after birth. Among the genes identified either through gene
deletion experiments or through human genetic studies that turned
out to be important for the maintenance of bone mass in adults, one
can cite the vitamin D receptor, Interleukin 6, Estrogen receptor
.alpha. and LDL receptor related protein 5 (Lrp5) (Gong et al.,
2001, Cell 107: 513-523; Boyden et al., 2002, N. Engl. J. Med.
346:1513-1521; Yoshizawa et al., 1997, Nat. Genet. 16: 391-396;
Ohshima et al., 1998, Proc. Natl. Acad. Sci. USA 95:8222822-6;
Windahl et al., 2002, Trends Endocrinol. Metab. 13:195-200).
[0076] The identification of Lrp5 as a regulator of post-natal bone
formation is one of the most vivid examples of how developmental
studies can profoundly affect the understanding of physiology
because this receptor is expressed during development but its
function only becomes apparent post-natally. Indeed,
loss-of-function mutations in Lrp5 cause osteoporosis pseudoglioma
syndrome (OPPG) in humans, a pediatric disease, and
gain-of-function mutations in Lrp5 cause high bone mass, a
phenotype most often appearing only in adolescents and persisting
into adulthood (Gong et al., 2001, Cell 107: 513-523; Boyden et
al., 2002, N. Engl. J. Med. 346:1513-1521; Johnson et al., 1997,
Am. J. Hum. Genet. 60:1326-1332). Likewise, skeletogenesis is
normal in Lrp5-/- mice and their low bone mass phenotype only
develops post-natally (Kato et al., 2002, J. Cell. Biol. 157:
303-314).
[0077] The LDL receptor related protein 5 (LRP5) is required for
normal bone mass, and a low bone mass phenotype is caused by Lrp5
inactivation in humans and mice (Gong et al., 2001, Cell
107:513-523; Kato et al., 2002, J. Cell. Biol. 157: 303-314).
Lrp5-/- mice have low bone mass with no change in osteoclast
surface but decreased osteoblast numbers. Real-time PCR analysis of
Lrp5-/- molecular signature shows that Lrp5-/- osteoblasts do not
show changes in osteoblast-specific gene expression but have
decreased expression of cyclin genes (FIG. 1). Lrp5 and its closest
relative Lrp6 are the vertebrate homologues of the Drosophila gene
arrow that encodes a surface receptor functioning as a co-receptor
for Wingless, the drosophila homologue of the Wnt proteins (Wehrli
et al., 2000, Nature 407:527-530; Tamai et al., 2000, Nature
407:530-535). In vertebrate cells, Wnt signaling is mainly mediated
by .beta.-catenin. Upon binding of a Wnt ligand to its receptor,
.beta.-catenin is translocated to the nucleus where it cooperates
with Lef/Tcf transcription factors to activate gene expression
(Logan et al., 2004, Annu Rev. Cell Dev. Biol. 20:781-810; Mao et
al., 2001, Mol. Cell, 7:801-809). According to this canonical
model, co-transfection of Lrp5 increases the ability of Wnt
proteins to enhance the activity of a Tcf-dependent promoter such
as the TopFlash promoter (Gong et al., 2001, Cell 107: 513-523;
Boyden et al., 2002, N. Engl. J. Med. 346:1513-1521; Mao et al.,
2001, Mol. Cell, 7:801-809). Together, the homology of sequence
between arrow and Lrp5 and the ability of Lrp5 to favor Wnt
signaling through its canonical pathway have led to a model whereby
Wnt signaling would regulate bone mass post natally and during
adulthood by regulating osteoblast proliferation and function.
There is no reason to question the notion that Lrp5 may be a
co-receptor for Wnts and that Wnt signaling is involved in the
regulation of bone formation (Glass et al., 2005, Dev. Cell
8:751-764; Holmen et al., 2005, J. Biol. Chem. 280:21162-21168; Day
et al., 2005, Dev. Cell, 8:739-750; Hu et al., 2005, Development
132:49-60). Nevertheless, there may be additional mechanisms that
explain the bone abnormalities observed in either Lrp5 loss- or
gain-of-function models.
Lrp5 Regulates Bone Mass in the Periphery Through Serotonin
[0078] TPH1 encodes the first enzyme in the biochemical pathway
resulting in serotonin synthesis outside the central nervous
system. It is viewed as a cell-specific gene mostly expressed in
the enterochromaffin cells of the duodenum (Gershon and Tack,
Gastroenterology, 2007, 132:397-414). By contrast, serotonin
synthesis in the brain relies on TPH2, which is encoded by a
different gene expressed in the central nervous system (CNS).
[0079] In an effort to elucidate the molecular mechanisms whereby
Lrp5 inactivation affects bone formation, a microarray analysis in
WT and Lrp5-/- bones was performed. Tryptophan hydroxylase 1 (TPH1)
was identified as the gene most highly over expressed in Lrp5-/-
bones having low bone mass disease. This result was surprising
since it is the opposite of what would be expected given the role
of serotonin in the brain, where it increases bone mass.
Remarkably, TPH1 expression was normal in mice lacking
.beta.-catenin in osteoblasts only (Glass et al., 2005, Dev. Cell
8:751-764).
[0080] It was shown that TPH1 is overexpressed not only in bone,
but also in the duodenum in Lrp5-/- mice, where TPH1 expression is
more than 1300-fold higher than in osteoblasts. It was further
discovered that serum serotonin levels are normal in newborn
Lrp5-/- mice but increase steadily with age as their bone phenotype
develops. This is consistent with the fact that the low bone mass
phenotype in Lrp5-/- mice is not present at birth but appears later
during development. Further discoveries showed that treating
Lrp5-/- mice with an inhibitor of serotonin synthesis called pCPA
corrects their low bone phenotype (FIG. 7). Finally, it was
discovered that TPH1 expression is increased in aging animals,
i.e., when bone mass is well-known to decrease. Based on these and
additional data described below, it can be conclude that LRP5,
through yet unknown mechanisms, is a negative regulator of
serotonin synthesis in the duodenum, and that increasing serum
serotonin signaling negatively impacts osteoblast proliferation and
function.
Serotonin, a Multifaceted Molecule
[0081] Serotonin (5-hydroxytryptamine, 5-HT) is a biogenic amine
that functions both as a neurotransmitter in the mammalian central
nervous system and as a hormone in the periphery, where most of it
is produced (Gershon et al., 1990, Neuropsychopharmacology,
3:385-395). Serotonin is generated through an enzymatic cascade in
which L-tryptophan is converted into L-5-hydroxytryptophan by an
enzyme called tryptophan hydroxylase (TPH). This intermediate
product is then converted to serotonin by an aromatic L-amino acid
decarboxylase. There are two TPH encoding genes, TPH1 and TPH2,
which are 71% identical in amino acid sequence and about 90%
similar in the catalytic domain. While TPH1 controls serotonin
synthesis in the periphery, TPH2 is responsible for serotonin
synthesis in the brain (Walther et al., 2003, Science 299:76).
Given that serotonin cannot cross the blood-brain barrier, these
two genes are therefore solely responsible for regulating the level
of this molecule in the periphery and in the brain, respectively.
As a consequence, designing TPH1 inhibiting compounds that cannot
cross the blood brain barrier is one of the ways to achieve
selective inhibition of TPH1 in the periphery and decrease
serotonin levels in this physiologic compartment.
[0082] TPH1 is expressed almost exclusively in cells of the
duodenum, and it is responsible for the synthesis of peripheral
serotonin, which represents 95% of total serotonin (Gershon &
Tack, 2007, 132:397-414). TPH1 expression in any tissues other than
duodenum is at least 100-1000 fold lower. Thus, TPH1 can be viewed
as a duodenum-specific gene and peripheral serotonin production as
a duodenum-specific process.
[0083] Besides its role as a neuromediator, and because of its
abundance in the general circulation, serotonin has been implicated
in a variety of developmental and physiological processes in
peripheral tissues, including heart development, gastrointestinal
movement, liver regeneration and mammary gland development
(Lesurtel et al., 2006, Science, 312:104-107; Matsuda et al., 2004,
Dev. Cell, 6:193-203; Nebigil et al., 2000, Proc. Natl. Acad. Sci.
USA 97:9508-9513). To carry out its functions, serotonin can bind
to at least 14 receptors, most of them being G-protein coupled
receptors (GPCRs). One or several serotonin receptors are present
in most cell types, including osteoblasts (Westbroek et al., 2001,
J. Biol. Chem. 276:28961-28968).
Type 1 Collagen, Osteocalcin, Regulatory Genes Affecting Osteoblast
Differentiation and/or Extracellular Matrix Protein Synthesis
(Runx2 and Osterix and Atf4) and Osteoclast Differentiation (RankL
and Osteoprotegrin) are Normal in Lrp5-Deficient Mice
[0084] Lrp5-/- mice are indistinguishable by all accounts from WT
mice at birth, but afterward progressively develop a significant
low bone mass phenotype (Kato et al., 2002, J. Cell. Biol. 157:
303-314). Histological and histomorphometric analyses established
that this low bone mass phenotype is due to a decrease in bone
formation while bone resorption is unaffected. Importantly,
osteoblast differentiation is not affected in the mutant mice while
osteoblast proliferation is decreased two fold in the absence of
Lrp5. See FIG. 1, which shows that Lrp5-/- mice have low bone mass
(A) with no change in osteoclast surface (B) but have decreased
osteoblast numbers (C).
Real-Time PCR Analysis of Lrp5-/- Molecular Signature.
[0085] To delineate the molecular signature of the disruption of
Lrp5 signaling, the expression of multiple genes characterizing
either the osteoblast lineage or determining cell proliferation was
studied using Lrp5-/- mice (Kato et al., 2002, J. Cell. Biol. 157:
303-314). The expression of genes particularly relevant to bone
formation was first analyzed. Expression of type I collagen and
Osteocalcin, two genes highly expressed in osteoblasts, is normal
in Lrp5-/- bones (data not shown). This finding is important as it
establishes that the bone phenotype of the Lrp5-/- mice is not
caused by a defect in type I collagen synthesis, the main
constituent of the bone extracellular matrix (ECM). Expression of
regulatory genes affecting osteoblast differentiation and/or
extracellular matrix protein synthesis was also studied. Expression
of Runx2 and Osterix and Atf4, the three known osteoblast-specific
transcription factors, was unaltered in Lrp5 -/- bones (FIG. 1D).
Likewise, expression of RankL and Osteoprotegerin (OPG), two
regulators of osteoclast differentiation expressed by osteoblasts
is unaffected by Lrp5 deletion (FIG. 1D). This latter feature
distinguishes Lrp5 -/- from .beta.-catenin osteoblast-specific
deficient (.beta.catob-/-) bones (Glass et al., 2005, Dev. Cell
8:751-764; Holmen et al., 2005, J. Biol. Chem.
280:21162-21168).
[0086] Given the decrease in osteoblast proliferation
characterizing Lrp5-/- bones, the expression of marker genes of
cell cycle progression was also studied. Expression of Cyclin D1,
D2 and E1, three genes necessary for the transition from the G1 to
S phase of the cell cycle, was decreased in the Lrp5-/- bones (FIG.
1E). Based on these results, it appears that at the molecular level
the low bone mass phenotype caused by the absence of Lrp5 is purely
a cell proliferation defect while expression of type I collagen,
the main protein constituent of the bone extracellular matrix
(ECM), and of all 3 known osteoblast-specific transcription factors
is normal.
Low Bone Phenotype in Lrp5-/- Mice is not Due to Abnormal Wnt
Signaling
[0087] Given the sequence homology and convincing experimental
arguments suggesting that Lrp5 could be a co-receptor for Wnt and
may be part of the Wnt canonical signaling pathway, whether the
bone phenotype of Lrp5-/- mice was due to abnormal Wnt signaling
was investigated. To that end, mice lacking .beta.-catenin in
osteoblasts only were analyzed (Glass et al., 2005, Dev. Cell
8:751-764). It had been shown earlier that mice lacking
.beta.-catenin only in osteoblasts developed a low bone mass
phenotype and that this phenotype was caused by a totally different
mechanism than the one operating in the Lrp5-/- mice. Indeed,
.beta.-cat.sub.ob -/- mice have a normal number of osteoblasts, an
increase of the number of osteoclasts and an increase in
elimination of deoxypyridinoline, abnormalities that are secondary
to a decrease in OPG expression (Glass et al., 2005, Dev. Cell
8:751-764). In addition, unlike in Lrp5-/- bones, expression of the
cell cycle markers Cyclin D1, D2 and E1 was normal in the in
.beta.-cat.sub.ob -/- bones (FIG. 2). Thus, the cellular and
molecular bases of the .beta.-cat.sub.ob -/- and Lrp5-/- mice bone
phenotype appear to be quite different. Although these unexpected
results do not rule out that Lrp5 could act as a Wnt co-receptor,
there was still a possibility that other mechanisms could explain
how the loss of Lrp5 could affect bone formation so specifically.
To that end, a microarray analysis looking for genes abnormally
expressed in Lrp5-/- compared to WT bones was performed.
TPH1 is Overexpressed in Bone and Duodenum in Lrp5-/- Mice
[0088] A microarray analysis of Lrp5-/- bones surprisingly showed
that one of the genes most highly over expressed was TPH1 (FIG. 3).
It is important to emphasize that TPH1 expression is normal in
.beta.-cat.sub.ob -/- bones and osteoblasts, further underscoring
the molecular differences that exist between these two mutant mouse
strains. Given the rather confined pattern of expression of TPH1 in
WT mice, where it is restricted to the duodenum, its overexpression
in Lrp5-/- bones was surprising and raised the question whether it
was an osteoblast-specific feature.
[0089] To answer this question, TPH1 expression in all tissues of
WT and Lrp5-/- mice was analyzed by qPCR. It was found that TPH1
expression was also increased 3 fold in duodenum of Lrp5-/-
compared to WT mice (FIG. 4A). However, TPH1 expression remained
more than 1300 fold higher in duodenum than in osteoblasts in
Lrp5-/- mice (FIG. 4B). These latter data suggested for the first
time that the bone phenotype observed in Lrp5-/- mice may primarily
have a gut origin. The increase in expression of TPH1 was also
observed, albeit as expected to a lower level, in Lrp5+/- mice
(FIG. 4C). This is an important observation since heterozygous
Lrp5+/- mice also have a low bone mass phenotype. Importantly, in
agreement with the absence of a bone phenotype in newborn Lrp5-/-
mice, TPH1 expression was not elevated in newborn mice (FIG. 4C).
The changes in TPH1 expression were reflected in increased serum
serotonin levels in both Lrp5+/- and Lrp5-/- mice (FIG. 4D); which
were absent at birth but present at 2, 4 and 8 weeks of age.
Moreover these changes preceded the appearance of the bone
phenotype in Lrp5-/- mice.
[0090] By contrast, the expression of TPH2 in the brain was not
affected in Lrp5-/- mice and serotonin content in the brain was
similar in WT and Lrp5-/- mice (FIGS. 4E and 4F). This observation
is consistent with the fact that serotonin does not cross the blood
brain barrier (Mann et al., 1992, Arch. Gen. Psychiatry,
49:442-446) and indicates that the link between Lrp5 function and
serotonin biology has to be with peripheral serotonin.
[0091] Expression of the TPH1 gene was decreased compared to wild
type (WT) in mice engineered with a mutation causing high bone mass
in humans in one allele (Lrp5+/act duo) or both alleles (Lrp5 act
duo) of the mouse Lrp5 gene specifically in cells of the duodenum.
RNA was extracted from duodenum of one-month-old mice and
expression of the TPH1 gene quantified by real-time PCR (FIG.
12).
TABLE-US-00001 TABLE 1 WT Lrp5+/act duo Lrp5 act duo Relative Tph1
1 0.77 .+-. 0.000 0.54 .+-. 0.005 expression
[0092] Mice engineered with a mutation causing high bone mass in
human in the Lrp5 gene specifically in cells of the duodenum (Lrp5
act duo) show a higher bone mass than wild type mice (FIG. 12).
[0093] Taken together, the results of these analyses indicated that
the increase in TPH expression caused by Lrp5 deficiency was
restricted to TPH1 (and therefore to peripheral serotonin) and that
it occurs both in osteoblasts and duodenal cells although its
expression is at least 1300-fold higher in duodenum. This result
raises two questions: is the increase in serum serotonin the cause
of the Lrp5-/- mice bone phenotype and is this an endocrine effect
mediated by the production of serotonin by duodenal cells and/or an
autocrine effect related to the local production of serotonin by
osteoblasts?
Lrp5-/- and 5Htt-/- Mice have Identical Bone Phenotypes
[0094] If the bone phenotype of the Lrp5-/- mice is secondary to an
increase in the level of serum serotonin, then a mouse model
characterized by an increase in serum serotonin should have not
only the same histological bone phenotype as the Lrp5-/- mice but
also the same molecular signature defined previously, i.e.,
decreased cyclin gene expression and normal type I collagen
expression (FIG. 1). This is what was observed.
The Serotonin Synthesis Inhibitor (pCPA) Rescues the Bone Phenotype
of Lrp5-/- Mice
[0095] Consistent with the conclusion that the increase in serum
serotonin level is responsible fully or partly for the bone
phenotype of the Lrp5-/- mice is the discovery that pCPA, a
serotonin synthesis inhibitor (Eldridge et al., 1981, Ann. Rev.
Physiol. 43:121-135), prevented the appearance of the Lrp5-/- bone
phenotype by decreasing serotonin production. WT and Lrp5-/- mice
were treated with 300 mg/kg pCPA intraperitoneally three times per
week, from 3 weeks to 12 weeks of age (FIG. 7A) and the changes in
serum serotonin levels, TPH1 expression in gut and TPH2 expression
in brain stem were analyzed. Bone histomorphometry was also
performed. As shown in FIG. 7B, pCPA treatment corrected the bone
abnormalities observed in Lrp5-/- mice without overtly affecting
bone mass in WT mice. This rescue of the Lrp5-/- phenotype was
achieved by normalization of the gut TPH1 mRNA and of serum
serotonin levels (FIGS. 7C and 7D). Brain TPH2 mRNA levels were not
affected in the treated mice, further demonstrating that the
phenotype observed in Lrp5-/- bones is directly caused by changes
in serum, not brain, serotonin levels.
Serotonin Binds to Specific Serotonin Receptors in Osteoblasts
[0096] From the working hypothesis that Lrp5 acts on bone formation
through serum serotonin, a third inference was tested: osteoblasts
should express some serotonin receptors, and serotonin treatment of
osteoblasts should blunt the expression of Cyclin D1, D2 and E1
without affecting the expression of .alpha.(I) collagen, Runx2 or
Osteocalcin. To address the first part of this point, the
expression of each of the known serotonin receptors was analyzed by
qPCR in WT osteoblasts. The expression of three different serotonin
receptors in osteoblasts, all belonging to the G-protein coupled
receptor superfamily was detected (Noda et al., 2004, Mol.
Neurobiol. 29:31-39). HT1B was the most highly expressed receptor.
It is coupled to G.sub.i-type G proteins and inhibits adenylyl
cyclase activity. HT2B is the second most abundant receptor and is
coupled to the G proteins that activate a
phosphatidyl-inositol-calcium second messenger system. Lastly, HT2A
is the third receptor significantly expressed in osteoblasts. Like
HT2B, it is coupled to the G proteins that activate a
phosphatidylinositol-calcium second messenger system. Remarkably,
HT1B, the most highly expressed serotonin receptor in osteoblasts,
is also more highly expressed in these cells than in any other
cells. Thus, there is at least a partially cell-specific signaling
pathway occurring in osteoblasts that could be able to specifically
transduce serotonin signaling in these cells. See FIG. 8 which
shows real-time PCR analysis of the expression of known serotonin
receptors expression in WT osteoblasts (A) and of the expression of
cyclins and osteoblast-specific genes in primary osteoblasts
treated with serotonin or vehicle (B).
[0097] To test whether serotonin regulates the expression of
cyclins in osteoblasts, a real-time PCR analysis of cyclin
expression in primary osteoblasts treated with serotonin or vehicle
was performed. As shown in FIG. 8B, expression of Cyclin D1 and D2
was decreased in the presence of serotonin. In contrast, expression
of Runx2, Osteocalcin and Type I collagen was not modified (FIG.
8B). That the molecular signature of serotonin treatment of
osteoblasts is similar to the one displayed in absence of Lrp5
further strengthens the hypothesis of a functional link between
Lrp5 and serotonin signaling in osteoblasts.
[0098] Decreased expression of Cyclin D1 is a major feature of both
Lrp5 deficiency and serotonin treatment of osteoblasts (FIGS. 1 and
8). One transcription factor that is known to modulate the
expression of cyclin genes and is expressed in osteoblasts is CREB
(Fu et al., 2005, Cell 122:803-815). Therefore, whether serotonin
could decrease CREB activity in these cells was tested. As shown in
FIG. 9A, serotonin treatment significantly decreased CREB
phosphorylation in primary osteoblasts. Furthermore, a CREB binding
site in the Cyclin D1 mouse promoter was identified and it was
shown using ChIP assays that serotonin decreased binding of CREB to
this promoter (FIG. 9B). These two observations raise the
hypothesis that CREB could be mediating serotonin action on
osteoblasts.
TPH1 Expression is Increased in Aging Animals
[0099] It has been shown in C. elegans that TPH1 expression
increases with age (Murakami et al., 2007 Feb. 28 [Epub ahead of
print], Neurobiol Aging). To test if this was also the case in
mammals, TPH1 expression in aging mice was analyzed. Using real
time PCR, it was shown that, while expression of Lrp5 remained
stable with age, expression of TPH1 doubled in 1 year-old compared
to 2 month-old mice (FIG. 10). Since serum serotonin acts as a
negative regulator of bone formation, such an increase in TPH1
expression with age exacerbates the bone loss associated with aging
and therefore is a target for therapeutic intervention for
age-related bone loss.
Methods of Diagnosis
[0100] The results disclosed herein show that elevated serum
serotonin decreases bone mass and low serum serotonin increases it.
Thus, certain embodiments of the invention are directed to methods
for diagnosing persons at risk of developing high or low bone mass
diseases and to methods for treating or preventing diseases
associated with abnormally low bone mass (such as osteoporosis and
OPPG) and abnormally high bone mass (such as high bone mass
syndrome) by administering drugs that either decrease or increase,
respectively, the level of peripheral serum serotonin. Other
embodiments are directed to new pharmaceutical compositions for
treating or preventing bone diseases of high or low bone mass.
[0101] One embodiment of the invention is directed to a method for
determining if a patient is at risk of developing a bone disease by
determining the patient's level of serum serotonin. If the
patient's level is significantly lower (at least about 25% lower)
than the level in a normal subject, then the patient is at risk of
developing abnormally high bone mass and serotonin can be
administered (preferably intravenously) to normalize serum
serotonin, thereby preventing high bone mass from developing.
Alternatively, if the patient level of serum serotonin is
significantly higher (more than about 25% higher) than the level in
a normal subject, then the patient is at risk of developing
abnormally low bone mass and TPH1 inhibitors or other therapeutic
agents that reduce serotonin synthesis, or serotonin receptor
antagonists (that target HT1B, HT2A and/or HT2B) can be
administered to reduce (and preferably normalize) serum serotonin
levels, thereby preventing low bone mass from developing. Patient
monitoring will determine if an abnormal serum serotonin profile is
chronic. If it is chronic, then the patient may need to continue
treatment to normalize serum serotonin.
[0102] In this context, when a patient's level of serum serotonin
is compared to the level of serum serotonin in a normal subject, it
should be understood that "normal subject" refers to a person who
is matched to the patient in those characteristics that would be
expected to affect serum serotonin levels, e.g., gender, age,
general health, medications being taken, etc.
Methods of Treatment and Prevention of Low and High Bone Mass
Diseases
[0103] The present invention provides a method of preventing or
treating a low bone mass disease in a patient known or suspected to
be in need of such prevention or treatment comprising administering
to the patient a therapeutically effective amount of an agent that
decreases serum serotonin levels.
[0104] In certain embodiments, the agent that decreases serum
serotonin levels is a TPH1 inhibitor or a serotonin receptor
antagonist.
[0105] In certain embodiments, the agent that decreases serum
serotonin levels is a TPH1 inhibitor that reduces serum serotonin
to a level that is at least about 10% less than the level before
treatment with the TPH1 inhibitor. In certain embodiments, the TPH1
inhibitor reduces serum serotonin to a level that is about 10%
less, about 20% less, about 30% less, about 40% less, about 50%
less, about 60% less, about 70% less, about 80% less, or about 90%
less, than the level before treatment with the TPH1 inhibitor.
[0106] In certain embodiments, the agent is a TPH1 inhibitor
selected from the group consisting of: [0107] pCPA; [0108] CBMIDA;
[0109] LP-533401; [0110] LP-615819; [0111]
(S)-2-Amino-3-(4-(4-amino-6-((R)-1-(naphthalen-2-yI)ethylamino)-1,3,5-tri-
azin-2-yl)phenyl)propanoic acid; [0112]
(S)-2-Amino-3-(4-(4-amino-6-((4'-methylbiphenyl-4-yl)methylamino-1,3,5-tr-
iazin-2-yl)phenyl)propanoic acid; [0113]
(S)-2-Amino-3-(4-(4-morpholino-6-(naphthalen-2-ylmethyIamino)-1,3,5-triaz-
in-2-yl)phenyl)propanoic acid; [0114]
(2S)-2-Amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(2-(trifluoromethyl)pheny-
l)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid; [0115]
(2S)-2-Amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-p-tolylethoxy)pyrimidin-4-
-yl)phenyl)propanoic acid; [0116]
(2S)-2-Amino-3-(4-(2-amino-6-(1-cyclohexyl-2,2,2-trifluoroethoxv)pyrimidi-
n-4-yl(phenyl)propanoic acid; [0117]
(S)-2-Amino-3-(4-(6-(2-fluorophenoxyl)pyrimidin-4-yl)phenyl)propanoic
acid; [0118]
(2S)-2-Amino-3-(4-(4-(3-(4-chlorophenyl)piperidin-1-yl)-1,3,5-triazin-2-y-
l)phenyl)propanoic acid; [0119]
(2S)-2-Amino-3-(4-(4-amino-6-(2,2,2-trifluoro-1-phenylethoxy)-1,3,5-triaz-
in-2-yI)phenyl)propanoic acid; [0120]
(S)-2-Amino-3-(5-(4-amino-6((R)-(naphthalen-2-yl)ethylamino)-1,3,5-triazi-
n-2-yl)pyridin-2-yl)propanoic acid; [0121]
(S)-2-Amino-3-(3-(4-ainino-6-(R)-1-(naphthalen-2-yl)ethylamino)-1,3,5-tri-
azin-2-yl)-1H-pyrazol-1-yl)propanoic acid; [0122]
(S)-2-Amino-3-(4'-(3-(cyclopentyloxy)-4-methoxybenzylamino)biphenyl-4-yl)-
propanoic acid; [0123]
(S)-2-Amino-3-(4-(6-(3-(cyclopentyloxy)-4-methoxybenzylamino)pyrimidin-4--
yl)phenyl)propanoic acid; [0124]
(S)-2-Amino-3-(4-(6-(3-(cyclopentyIoxy)-4-methoxybenzylamino)pyrazin-2-yl-
)phenyl)propanoic acid; [0125]
(S)-2-Amino-3-(4-(5-((4'-methylbiphenyl-2-yl)methylamino)pyrazin-2-yl)phe-
nyl)poropanoic acid; [0126]
(2S)-2-Amino-3-(4-(6-(2,2,2-trifluoro-1-phenylethoxy)-pyrimidin-4-yl)phen-
yl)propanoic acid; [0127]
(2S)-2-Amino-3-(4-(6-(1-(3,4-difluorophenyl)-2,2,2-trifluoroethoxy)pyriin-
idin-4-yl)phenyl)propanoic acid; [0128]
(S)-2-Amino-3-(4-(5-(3-(cyclopentyloxy)-4-methoxvbenzvlamino)-pyrazin-2-y-
l)phenyl)propanoic acid; [0129]
(S)-2-Amino-3-(4-(5-((3-(cyclopentyloxy)-4-methoxybenzyl)-(methyl)amino)p-
vrazin-2-yl)phenyl)propanoic acid; [0130]
(S)-2-Amino-3-(4-(5-((1,3-dimethyl-1H-pyrazol-4-yl)methylamino)pyrazin-2--
yl)phenyl)propanoic acid; [0131]
(S)-2-Amino-3-(4-(4-amino-6-((S)-1-(naphthalen-2-yl)ethylamino)-1,3,5-tri-
azin-2-yloxy)phenyl)propanoic acid; [0132]
(S)-2-Amino-3-(4-(4-amino-6-((R)-1-(biphenyl-2-yl)-2,2,2-trifluoroethoxy)-
-1,3,5-triazin-2-yl)phenyl)propanoic acid; [0133]
(2S)-2-Amino-3-(4-(4-amino-6-(1-(6,8-difluoronaphthalen-2-yl)ethylamino)--
1,3,5-triazin-2-yl)phenyl)propanoic acid; [0134]
(2S)-2-Amino-3-(4-(4-amino-6-(2,2,2-trifluoro-1-(3'-methylbiphenyl-2-yl)e-
thoxy)-1,3,5-triazin-2-yl)phenv)propanoic acid; [0135]
(S)-2-Amino-3-(4-(5-(3,4-dimethoxyphenylcarbamoyl)-pyrazin-2-yl)phenyl)pr-
opanoic acid; [0136]
(S)-2-Amino-3-(4-(2-amino-6-(4-(2-(trifluoromethyl)phenyl)-piperidin-1-yl-
)pyrimidin-4-yl)phenyl)propanoic acid; [0137]
(S)-2-Amino-3-(4-(2-amino-6-((R)-1-(naphthalen-2-yl)ethylamino)pyrimidin--
4-yl)phenyl)propanoic acid; [0138]
(S)-2-Amino-3-(4-(2-amino-6-(methyl(R)-1-(naphthalen-2-yl)ethyl)amino)pyr-
imidin-4-yl)phenyl)propanoic acid; [0139]
(S)-2-Amino-3-(4-(2-amino-6-((S)-2,2,2-trifluoro-1-(6-methoxynaphthalen-2-
-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid; [0140]
(S)-2-Amino-3-(4-(5-(biphenyl-4-ylmethylamino)pyrazin-2-yl)phenyl)propano-
ic acid; [0141]
(S)-2-Amino-3-(4-(5-(naphthalen-2-ylmethylamino)pyrazin-2-yl)phenyl)propa-
noic acid; [0142]
(S)-2-(Tert-butoxycarbonylamino)-3-(4-(5-(naphthalen-2-ylmethylamino)pyra-
zin-2-yl)phenyl)propanoic acid; [0143] (S)-2-Morpholinoethyl
2-amino-3-(4-(5-(naphthalen-2-ylmethylamino)pyrazin-2-yl)phenyl)propanoat-
e; [0144]
(S)-2-Amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(3'-fluorobipheny-
l-4-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid; [0145]
(S)-2-Amino-3-(4-(2-amino-6-(benzylthio)pyrimidin-4-yl)phenyl)propanoic
acid; [0146]
(S)-2-Amino-3-(4-(2-amino-6-(naphthalen-2-ylmethylthio)pyrimidin-4-yl)phe-
nyl)propanoic acid; [0147]
(2S)-2-Amino-3-(4-(2-amino-6-(1-(3,4-difluorophenyl)-2,2,2-trifluoroethox-
y)pyrimidin-4-yl)phenyl)propanoic acid; [0148]
(2S)-2-Amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(3'-methylbiphenyl-2-yl)e-
thoxy)pyrimidin-4-yl)phenyl)propanoic acid; [0149]
(S)-2-Amino-3-(4-(5-(3-(cyclopentyloxy)-4-methoxybenzylamino)pyridin-3-yl-
)phenyl)propanoic acid; [0150]
2-Amino-3-(3-(4-amino-6-((R)-1-(naphthalen-2-yl)ethylamino)-1,3,5-triazin-
-2-yl)phenyl)propanoic acid; [0151]
2-Amino-3-(4-(4-amino-6-((R)-1-(naphthalen-2-yl)ethylamino)-1,3,5-triazin-
-2-yl)-2-fluorophenyl)propanoic acid; [0152]
(2S)-2-Amino-3-(4-(4-amino-6-(1-(adamantyl)ethylamino)-1,3,5-triazin-2-yl-
)phenyl)propanoic acid; [0153]
(S)-2-Amino-3-(4-(5-fluoro-4-((R)-1-(naphthalen-2-yl)ethylamino)pyrimidin-
-2-yl)phenyl)propanoic acid; [0154]
(S)-2-Amino-3-(4-(2-amino-6-(4-(trifluoromethyl)-benzylamino)pyrimidin-4--
yl)phenyl) propanoic acid; [0155]
2-Amino-3-(5-(5-phenylthiophen-2-yl)-1H-indol-3-yl)propanoic acid;
[0156]
(S)-2-Amino-3-(4-(4-(4-phenoxyphenyl)-1H-1,2,3-triazol-1-yl)phenyl)propan-
oic acid; [0157]
(S)-2-Amino-3-(4-(4-(4-(thiophene-2-carboxamido)phenyl)-1H-1,2,3-triazoI--
1-yl)phenyl)propanoic acid; and
[0158]
(S)-2-Amino-3-(4-(2-amino-6-(phenylethynyl)pyrimidin-4-yl)phenyl)pr-
opanoic acid;
[0159] as well as racemic mixtures and individual enantiomers of
said compounds, and salts of said compounds with a physiologically
acceptable acid.
[0160] Additional TPH1 inhibitors that may be used in the present
invention are listed in the table below.
TABLE-US-00002 TABLE 2 (S)-2-amino-3-(4-(5-(2-fluoro-4,5-
dimethoxybenzylamino)pyrazin-2-yl)phenyl)propanoic acid
(S)-2-amino-3-(4-(2-amino-6-(4-(2-methoxyphenyl)piperidin-1-
yl)pyrimidin-4-yl)phenyl)propanoic acid
(S)-2-amino-3-(4-(6-(3-(cyclopentyloxy)-4-
methoxybenzylamino)-2-(dimethylamino)pyrimidin-4-
yl)phenyl)propanoic acid
(S)-2-amino-3-(4-(5-(3,4-dimethylbenzylamino)pyrazin-2-
yl)phenyl)propanoic acid
(S)-2-amino-3-(4-(5-(biphenyl-2-ylmethylamino)pyrazin-2-
yl)phenyl)propanoic acid (S)-ethyl 2-amino-3-(4-(2-amino-6-(4-
(trifluoromethyl)benzylamino)pyrimidin-4-yl)phenyl)propanoate
(S)-2-amino-3-(4-(5-(cyclopentylmethylamino)pyrazin-2-
yl)phenyl)propanoic acid (2S)-2-amino-3-(4-(2-amino-6-(3-(2-
(trifluoromethyl)phenyl)pyrrolidin-1-yl)pyrimidin-4-
yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(1,2,3,4-tetrahydronaphthalen-1-
ylamino)pyrimidin-4-yl)phenyl)propanoic acid
(S)-2-amino-3-(4-(2-amino-6-((R)-1-(naphthalen-2-
yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(1,2-
diphenylethylamino)pyrimidin-4-yl)phenyl)propanoic acid
(S)-2-amino-3-(4-(2-amino-6-((R)-1-(4-(benzo[b]thiophen-3-
yl)phenyl)ethylamino)pyrimidin-4-yl)phenyl)propanoic acid
(S)-2-amino-3-(4-(4-amino-6-((R)-1-(4'-methoxybiphenyl-4-
yl)ethylamino)-1,3,5-triazin-2-yl)phenyl)propanoic acid
2-amino-3-(1-(4-amino-6-((R)-1-(naphthalen-2-yl)ethylamino)-
1,3,5-triazin-2-yl)piperidin-4-yl)propanoic acid
(2S)-2-amino-3-(4-(4-amino-6-(1-(4-fluoronaphthalen-1-
yl)ethylamino)-1,3,5-triazin-2-yl)phenyl)propanoic acid
(S)-2-amino-3-(4-(4-amino-6-((3'-fluorobiphenyl-4-
yl)methylamino)-1,3,5-triazin-2-yl)phenyl)propanoic acid
2-amino-3-(4-(4-amino-6-((R)-1-(naphthalen-2-yl)ethylamino)-
1,3,5-triazin-2-yl)-2-fluorophenyl)propanoic acid
(S)-2-amino-3-(4-(2-amino-6-((R)-2,2,2-trifluoro-1-(3'-
methoxybiphenyl-4-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(4-amino-6-(2,2,2-trifluoro-1-(3'-
fluorobiphenyl-2-yl)ethoxy)-1,3,5-triazin-2-yl)phenyl)propanoic
acid (2S)-2-amino-3-(4-(4-amino-6-(1-(4-tert-
butylphenyl)ethylamino)-1,3,5-triazin-2-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(3'-
fluorobiphenyl-4-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(4-amino-6-(6,7-dihydroxy-1-methyl-3,4-
dihydroisoquinolin-2(1H)-yl)-1,3,5-triazin-2- yl)phenyl)propanoic
acid (2S)-2-amino-3-(4-(4-amino-6-(2,2,2-trifluoro-1-(3'-
methylbiphenyl-4-yl)ethoxy)-1,3,5-triazin-2- yl)phenyl)propanoic
acid (S)-2-amino-3-(4-(4-amino-6-((R)-1-(naphthalen-2-
yl)ethylamino)pyrimidin-2-yl)phenyl)propanoic acid
(S)-2-amino-3-(4-(2-amino-6-(benzylthio)pyrimidin-4-
yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(4'-
fluorobiphenyl-4-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(6-(3-(4-chlorophenoxy)piperidin-1-
yl)pyrimidin-4-yl)phenyl)propanoic acid
(S)-3-(4-(4-amino-6-((R)-1-(naphthalen-2-yl)ethylamino)-1,3,5-
triazin-2-yl)phenyl)-2-(2-aminoacetamido)propanoic acid
(S)-2-amino-3-(4-(6-((R)-1-(naphthalen-2-yl)ethylamino)-2-
(trifluoromethyl)pyrimidin-4-yl)phenyl)propanoic acid
(S)-2-amino-3-(4-(2-amino-6-(4-(3-chlorophenyl)piperazin-1-
yl)pyrimidin-4-yl)phenyl)propanoic acid
(S)-2-amino-3-(4-(2-amino-6-((R)-2,2,2-trifluoro-1-
phenylethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(1,4-
diphenylbutylamino)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(6-(1-(3'-chlorobiphenyl-2-yl)-2,2,2-
trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(4-amino-6-(1-(biphenyl-4-yl)-2,2,2-
trifluoroethoxy)-1,3,5-triazin-2-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(2,2,3,3,3-pentafluoro-1-(3-
fluoro-4-methylphenyl)propoxy)pyrimidin-4- yl)phenyl)propanoic acid
(S)-ethyl 2-amino-3-(4-(2-amino-6-((R)-2,2,2-trifluoro-1-(3'-
methoxybiphenyl-4-yl)ethoxy)pyrimidin-4- yl)phenyl)propanoate
(S)-2-amino-3-(4-(2-amino-6-((S)-2,2,2-trifluoro-1-(3'-
methoxybiphenyl-4-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(3-fluoro-3'-
methoxybiphenyl-4-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(1-(3'-(dimethylamino)biphenyl-
2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(3'-methoxy-5-
methylbiphenyl-2-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(4'-methoxy-5-
methylbiphenyl-2-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(3'-methoxy-3-
(methylsulfonyl)biphenyl-4-yl)ethoxy)pyrimidin-4-
yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(1-(2-(cyclopropylmethoxy)-4-
fluorophenyl)-2,2,2-trifluoroethoxy)pyrimidin-4-
yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(6-(1-(2-(cyclopropylmethoxy)-4-
fluorophenyl)-2,2,2-trifluoroethoxy)pyrimidin-4-
yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(2-
(isopentyloxy)phenyl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(5-(2,2,2-trifluoro-1-(3'-fluorobiphenyl-4-
yl)ethoxy)pyrazin-2-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(4'-
methoxybiphenyl-2-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(1-(3'-carbamoylbiphenyl-2-yl)-
2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(1-(4'-carbamoylbiphenyl-2-yl)-
2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(4-(2-
methoxyphenoxy)phenyl)ethoxy)pyrimidin-4- yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(6-(2,2,2-trifluoro-1-(4-(2-
methoxyphenoxy)phenyl)ethoxy)pyrimidin-4- yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(6-(2,2,2-trifluoro-1-(2-
(isopentyloxy)phenyl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-3-(4-(6-(1-(3'-acetamidobiphenyl-2-yl)-2,2,2-
trifluoroethoxy)-2-aminopyrimidin-4-yl)phenyl)-2- aminopropanoic
acid (2S)-3-(4-(6-(1-(4'-acetamidobiphenyl-2-yl)-2,2,2-
trifluoroethoxy)-2-aminopyrimidin-4-yl)phenyl)-2- aminopropanoic
acid (2S)-2-amino-3-(4-(2-amino-6-(1-(4-cyanophenyl)-2,2,2-
trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid (S)-ethyl
2-amino-3-(4-(2-amino-6-((R)-2,2,2-trifluoro-1-p-
tolylethoxy)pyrimidin-4-yl)phenyl)propanoate
(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(1-
methoxybicyclo[2.2.2]oct-5-en-2-yl)ethoxy)pyrimidin-4-
yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(1-(4-(cyclopentyloxy)phenyl)-
2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(6-(1-(4-(cyclopentyloxy)phenyl)-2,2,2-
trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(4-(3-
methoxyphenoxy)phenyl)ethoxy)pyrimidin-4- yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(1-(4,5-dimethoxybiphenyl-2-yl)-
2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(1-(4,5-dimethoxy-3'-
methylbiphenyl-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-
yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(5-(2,2,2-trifluoro-1-(2'-methylbiphenyl-2-
yl)ethoxy)pyrazin-2-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(6-(2,2,2-trifluoro-1-(4-(3-
methoxyphenoxy)phenyl)ethoxy)pyrimidin-4- yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(1-(2-(3,5-
difluorophenoxy)phenyl)-2,2,2-trifluoroethoxy)pyrimidin-4-
yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(4-(4-
methoxyphenoxy)phenyl)ethoxy)pyrimidin-4- yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(1-(4'-((S)-2-amino-2-
carboxyethyl)biphenyl-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-
yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(1-(2-bromophenyl)-2,2,2-
trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(5-(2,2,2-trifluoro-1-(3'-methylbiphenyl-2-
yl)ethoxy)pyrazin-2-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(4-
methoxybiphenyl-2-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(5-(2,2,2-trifluoro-1-(2-(4-methylthiophen-3-
yl)phenyl)ethoxy)pyrazin-2-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(4-methoxy-3'-
methylbiphenyl-2-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(3'-
(hydroxymethyl)biphenyl-2-yl)ethoxy)pyrimidin-4-
yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(1-(3'-cyanobiphenyl-2-yl)-2,2,2-
trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(6-(1-(2-(3,5-difluorophenoxy)phenyl)-2,2,2-
trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(6-(2,2,2-trifluoro-1-(4-(4-
methoxyphenoxy)phenyl)ethoxy)pyrimidin-4- yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(2-(4-
methylthiazol-2-yl)thiophen-3-yl)ethoxy)pyrimidin-4-
yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(5-(4-
methoxyphenyl)isoxazol-3-yl)ethoxy)pyrimidin-4- yl)phenyl)propanoic
acid (2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(1-phenyl-5-
(trifluoromethyl)-1H-pyrazol-4-yl)ethoxy)pyrimidin-4-
yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(1-(2-(cyclohexyloxy)-4-
methylphenyl)-2,2,2-trifluoroethoxy)pyrimidin-4-
yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(1-(2-(cyclopentyloxy)-4-
methylphenyl)-2,2,2-trifluoroethoxy)pyrimidin-4-
yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(1-(benzo[d]thiazol-6-yl)-2,2,2-
trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(1-methyl-1H-
imidazol-5-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(6-(1-(2-(cyclopentyloxy)-4-methylphenyl)-
2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(6-(1-(2-(cyclohexyloxy)-4-methylphenyl)-
2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(pyridin-3-
yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(1-(1,3-dimethyl-1H-pyrazol-5-
yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(S)-2-amino-3-(4-(2-amino-6-(3-hydroxyphenyl)pyrimidin-4-
yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(3'-
hydroxybiphenyl-2-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(S)-2-amino-3-(4-(2-amino-6-(3,5-difluorophenyl)pyrimidin-4-
yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(1-(3',5'-difluorobiphenyl-2-yl)-
2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(6-(2,2,2-trifluoro-1-(3'-fluorobiphenyl-3-
yl)ethoxy)pyrazin-2-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(1-(5-ethoxy-2-methyl-2,3-
dihydrobenzofuran-6-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-
yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(1-(benzofuran-5-yl)-2,2,2-
trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(2-m-
tolylfuran-3-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(S)-ethyl 3-(4-(2-amino-6-((R)-2,2,2-trifluoro-1-(3'-
methoxybiphenyl-4-yl)ethoxy)pyrimidin-4-yl)phenyl)-2-(2-
aminoacetamido)propanoate
(2S)-2-amino-3-(4-(6-(2,2,2-trifluoro-1-(2-(4-methylthiophen-3-
yl)phenyl)ethoxy)pyrazin-2-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(5-methyl-3-
phenylisoxazol-4-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(S)-2-amino-3-(4-(2-amino-6-(3-(methylthio)phenyl)pyrimidin-
4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(3'-
(methylthio)biphenyl-2-yl)ethoxy)pyrimidin-4- yl)phenyl)propanoic
acid (2S)-2-amino-3-(4-(2-amino-6-(1-(3'-
((dimethylamino)methyl)biphenyl-2-yl)-2,2,2-
trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(S)-2-amino-3-(4-(2-amino-6-(3-
(trifluoromethoxy)phenyl)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(3'-
(trifluoromethoxy)biphenyl-2-yl)ethoxy)pyrimidin-4-
yl)phenyl)propanoic acid
(S)-3-(4-(2-amino-6-((R)-2,2,2-trifluoro-1-(3'-methoxybiphenyl-
4-yl)ethoxy)pyrimidin-4-yl)phenyl)-2-(2- aminoacetamido)propanoic
acid (2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(1-methyl-5-
phenyl-1H-pyrazol-4-yl)ethoxy)pyrimidin-4- yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(4-
(methylsulfonyl)phenyl)ethoxy)pyrimidin-4- yl)phenyl)propanoic acid
(S)-2-amino-3-(4-(2-amino-6-((R)-1-(3'-
(dimethylamino)biphenyl-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-
4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(1-(2-chloro-4-
(methylsulfonyl)phenyl)-2,2,2-trifluoroethoxy)pyrimidin-4-
yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(3-(furan-2-
yl)thiophen-2-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(1-(2-(cyclopentyloxy)-4-
fluorophenyl)-2,2,2-trifluoroethoxy)pyrimidin-4-
yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(2-(3-
methoxyphenyl)cyclohex-1-enyl)ethoxy)pyrimidin-4-
yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(pyrimidin-5-
yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(5-(2,2,2-trifluoro-1-(3'-methoxybiphenyl-3-
yl)ethoxy)pyrazin-2-yl)phenyl)propanoic acid
(S)-2-amino-3-(4-(2-amino-6-((S)-1-(3'-
(dimethylamino)biphenyl-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-
4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(2-(furan-2-
carboxamido)phenyl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(1-(4-chloro-2-
(methylsulfonyl)phenyl)-2,2,2-trifluoroethoxy)pyrimidin-4-
yl)phenyl)propanoic acid (S)-isopropyl
2-amino-3-(4-(2-amino-6-((R)-2,2,2-trifluoro-1-
(3'-methoxybiphenyl-4-yl)ethoxy)pyrimidin-4- yl)phenyl)propanoate
(2S)-2-amino-3-(4-(6-(1-(2-(cyclopentyloxy)-4-fluorophenyl)-
2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(6-(1-(2-(cyclohexyloxy)-4-fluorophenyl)-
2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(1-(thiophen-2-
yl)cyclohexyl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-(2,2,2-trifluoro-1-(3'-methoxybiphenyl-4-
yl)ethoxy)thiazol-5-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(1-(2-(cyclohexyloxy)-4-
fluorophenyl)-2,2,2-trifluoroethoxy)pyrimidin-4-
yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(1-(4-
methoxyphenyl)cyclohexyl)ethoxy)pyrimidin-4- yl)phenyl)propanoic
acid (2S)-2-amino-3-(4-(6-(2,2,2-trifluoro-1-(4-fluoro-2-
methylphenyl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(4-fluoro-2-
methylphenyl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(oxazol-2-
yl(phenyl)methoxy)pyrimidin-4-yl)phenyl)propanoic acid
(S)-2-amino-3-(4-(2-amino-6-(1-cyclohexyl-2,2,2-
trifluoroethylideneaminooxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(1-(2-(3-
(dimethylamino)phenyl)furan-3-yl)-2,2,2-
trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(5-
phenylthiophen-2-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(S)-phenyl 2-amino-3-(4-(2-amino-6-((R)-2,2,2-trifluoro-1-(3'-
methoxybiphenyl-4-yl)ethoxy)pyrimidin-4- yl)phenyl)propanoate
(S)-2-amino-3-(4-(2-amino-6-((R)-1-(3'-
((dimethylamino)methyl)biphenyl-4-yl)-2,2,2-
trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(S)-2-amino-3-(4-(1-(3-methoxybenzoyl)-1H-pyrazol-4-
yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(6-(2,2,2-trifluoro-1-(5-phenylfuran-2-
yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(1-(4-chloro-2-fluorophenyl)-
2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(S,E)-2-amino-3-(4-(2-amino-6-(4-
(trifluoromethyl)styryl)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(1-(3,4-dichlorophenyl)-2,2,2-
trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(1-(4-chloro-3-fluorophenyl)-
2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(S)-2-amino-3-(4-(2-amino-6-((R)-1-(3'-
(dimethylamino)biphenyl-4-yl)-2,2,2-trifluoroethoxy)pyrimidin-
4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(1-chloro-2,2,2-trifluoro-1-(4-
methoxybiphenyl-2-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(6-(2,2,2-trifluoro-1-(5-phenylthiophen-2-
yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid
(S)-2-amino-3-(4-(5-(4-phenoxyphenyl)-1H-1,2,3-triazol-1-
yl)phenyl)propanoic acid
(S,E)-2-amino-3-(4-(2-amino-6-(2-(biphenyl-4-
yl)vinyl)pyrimidin-4-yl)phenyl)propanoic acid
(S)-2-amino-3-(4-(4-amino-6-((R)-2,2,2-trifluoro-1-(3'-
methoxybiphenyl-4-yl)ethoxy)pyrimidin-2-yl)phenyl)propanoic acid
(S)-2-amino-3-(4-(4'-methoxybiphenyl-4-
ylsulfonamido)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(6-(3-
methoxyphenyl)pyridin-3-yl)ethoxy)pyrimidin-4- yl)phenyl)propanoic
acid
(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(6-(2-fluoro-3-
methoxyphenyl)pyridin-3-yl)ethoxy)pyrimidin-4- yl)phenyl)propanoic
acid 2-amino-3-(5-(4'-methylbiphenyl-4-yl)-1H-indol-3-yl)propanoic
acid 2-amino-3-(5-m-tolyl-1H-indol-3-yl)propanoic acid
(2S)-2-amino-3-(4-(2-(2-methoxyphenyl)furan-3-
carboxamido)phenyl)propanoic acid
2-amino-3-(5-(1-benzyl-1H-pyrazol-4-yl)-1H-indol-3- yl)propanoic
acid
(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(6-(thiophen-2-
yl)pyridin-3-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid
2-amino-3-(6-(1-benzyl-1H-pyrazol-4-yl)-1H-indol-3- yl)propanoic
acid (S)-2-amino-3-(4-((2-(4-(trifluoromethyl)phenyl)thiazol-4-
yl)methylamino)phenyl)propanoic acid
(S)-2-amino-3-(4-((4'-methoxybiphenyl-4-
ylsulfonamido)methyl)phenyl)propanoic acid
(S)-2-amino-3-(4-(3-(2-methoxydibenzo[b,d]furan-3-
yl)ureido)phenyl)propanoic acid (S)-2-amino-3-(4-(3-(2,2-
diphenylethyl)ureido)phenyl)propanoic acid
(S)-2-amino-3-(4-(phenylethynyl)phenyl)propanoic acid
(S)-2-amino-3-(4-(2-amino-6-((5-(1-methyl-5-(trifluoromethyl)-
1H-pyrazol-3-yl)thiophen-2-yl)methoxy)pyrimidin-4-
yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(1,1,1-trifluoro-3-((R)-2,2,3-
trimethylcyclopent-3-enyl)propan-2-yloxy)pyrimidin-4-
yl)phenyl)propanoic acid (2S)-2-amino-3-(4-(2-amino-6-(3-(2-
hydroxyethylcarbamoyl)piperidin-1-yl)pyrimidin-4-
yl)phenyl)propanoic acid
(2S)-2-amino-3-(4-(2-amino-6-(3-(pyridin-2-yloxy)piperidin-1
yl)pyrimidin-4-yl)phenyl)propanoic acid
(S)-2-amino-3-(4-(2-amino-6-(4-chloro-3-(piperidine-1-
carbonyl)phenyl)pyrimidin-4-yl)phenyl)propanoic acid
[0161] Additional TPH1 inhibitors that may be used in the present
invention include: [0162] N-[(1R,4R,9aS)-4-phenyl
octahydropyrido[2,1-c][1,4]oxazin-1-yl]3,4,5-trimethoxybenzamide;
[0163] 2,6-Piperidinedione,
3-[3-(dimethylamino)propyl]-3-(3-methoxyphenyl)-4,4-dimethyl-,
monohydrochloride; and
[0164] Triptosine (CAS registry number 86248-47-7; U.S. Pat. No.
4,472,387).
[0165] Methods of making many of the therapeutic agents disclosed
in the preceding paragraphs are disclosed in International Patent
Publication WO 2007/089335. Such disclosures are incorporated
herein by reference.
[0166] In certain embodiments, the therapeutic agent is a TPH1
inhibitor having the structure:
##STR00013##
[0167] In certain embodiments, the therapeutic agent is a TPH1
inhibitor having the structure:
##STR00014##
[0168] In certain embodiments, the therapeutic agent is a TPH1
inhibitor having the structure:
##STR00015##
where R is hydrogen or lower alkyl, where lower alkyl refers to a
straight-chain or branched-chain hydrocarbon group having 1 to 6
carbon atoms (e.g., methyl, ethyl, propyl, isopropyl, butyl,
isobutyl, pentyl and hexyl); and
[0169] n is 1, 2, or 3.
[0170] In certain embodiments, the therapeutic agent is a TPH1
inhibitor having the structure:
##STR00016##
where R is hydrogen or lower alkyl, where lower alkyl refers to a
straight-chain or branched-chain hydrocarbon group having 1 to 6
carbon atoms (e.g., methyl, ethyl, propyl, isopropyl, butyl,
isobutyl, pentyl and hexyl); and n is 1, 2, or 3.
[0171] In certain embodiments, the therapeutic agent is a TPH1
inhibitor having the structure:
##STR00017##
where R is hydrogen or lower alkyl, where lower alkyl refers to a
straight-chain or branched-chain hydrocarbon group having 1 to 6
carbon atoms (e.g., methyl, ethyl, propyl, isopropyl, butyl,
isobutyl, pentyl and hexyl); R.sub.1, R.sub.2, and R.sub.3, are
independently:
[0172] hydrogen;
[0173] halogen (preferably F or Cl);
[0174] lower alkyl, where lower alkyl refers to a straight-chain or
branched-chain hydrocarbon group having 1 to 6 carbon atoms (e.g.,
methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl and
hexyl);
[0175] alkoxy, where alkoxy refers to a group R'--O--, where R' is
lower alkyl as defined above;
[0176] amino; or
[0177] nitro;
and n is 1, 2, or 3.
[0178] In certain embodiments, the therapeutic agent is a TPH1
inhibitor having the structure:
##STR00018##
where R is hydrogen or lower alkyl, where lower alkyl refers to a
straight-chain or branched-chain hydrocarbon group having 1 to 6
carbon atoms (e.g., methyl, ethyl, propyl, isopropyl, butyl,
isobutyl, pentyl and hexyl); R.sub.1, R.sub.2, and R.sub.3, are
independently:
[0179] hydrogen;
[0180] halogen (preferably F or Cl);
[0181] lower alkyl, where lower alkyl refers to a straight-chain or
branched-chain hydrocarbon group having 1 to 6 carbon atoms (e.g.,
methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl and
hexyl);
[0182] alkoxy, where alkoxy refers to a group R'--O--, where R' is
lower alkyl as defined above;
[0183] amino; or
[0184] nitro;
and
[0185] n is 1, 2, or 3.
[0186] In certain embodiments, the therapeutic agent is a TPH1
inhibitor having the structure:
##STR00019##
where R is hydrogen; lower alkyl, where lower alkyl refers to a
straight-chain or branched-chain hydrocarbon group having 1 to 6
carbon atoms (e.g., methyl, ethyl, propyl, isopropyl, butyl,
isobutyl, pentyl and hexyl); or cycloalkyl, where cycloalkyl refers
a cyclic hydrocarbon group having 3 to 8 carbon atoms.
[0187] In certain embodiments, the therapeutic agent is a TPH1
inhibitor having the structure:
##STR00020##
where R is hydrogen; lower alkyl, where lower alkyl refers to a
straight-chain or branched-chain hydrocarbon group having 1 to 6
carbon atoms (e.g., methyl, ethyl, propyl, isopropyl, butyl,
isobutyl, pentyl and hexyl); or cycloalkyl, where cycloalkyl refers
a cyclic saturated hydrocarbon group having 3 to 8 carbon
atoms.
[0188] In certain embodiments, the therapeutic agent is a TPH1
inhibitor having the structure:
##STR00021##
where R.sub.1 and R.sub.2 are, independently, hydrogen; lower
alkyl, where lower alkyl refers to a straight-chain or
branched-chain hydrocarbon group having 1 to 6 carbon atoms (e.g.,
methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl and
hexyl); or cycloalkyl, where cycloalkyl refers a cyclic saturated
hydrocarbon group having 3 to 8 carbon atoms.
[0189] In certain embodiments, the therapeutic agent is a TPH1
inhibitor having the structure:
##STR00022##
where R is hydrogen; lower alkyl, where lower alkyl refers to a
straight-chain or branched-chain hydrocarbon group having 1 to 6
carbon atoms (e.g., methyl, ethyl, propyl, isopropyl, butyl,
isobutyl, pentyl and hexyl); or cycloalkyl, where cycloalkyl refers
a cyclic saturated hydrocarbon group having 3 to 8 carbon
atoms.
[0190] In certain embodiments, the therapeutic agent is a TPH1
inhibitor having the structure:
##STR00023##
where R.sub.1 and R.sub.2 are, independently, hydrogen; lower
alkyl, where lower alkyl refers to a straight-chain or
branched-chain hydrocarbon group having 1 to 6 carbon atoms (e.g.,
methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl and
hexyl); cycloalkyl, where cycloalkyl refers a cyclic saturated
hydrocarbon group having 3 to 8 carbon atoms; F, Cl, or OH.
[0191] In certain embodiments, the therapeutic agent is a TPH1
inhibitor having the structure:
##STR00024##
where R.sub.1 and R.sub.2 are, independently, hydrogen; lower
alkyl, where lower alkyl refers to a straight-chain or
branched-chain hydrocarbon group having 1 to 6 carbon atoms (e.g.,
methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl and
hexyl); or cycloalkyl, where cycloalkyl refers a cyclic saturated
hydrocarbon group having 3 to 8 carbon atoms.
[0192] In certain embodiments where the agent is a TPH1 inhibitor
that is administered without also administering another
pharmaceutically active substance (e.g., an SSRI, a beta blocker,
or a serotonin receptor antagonist), the TPH1 inhibitor is not
pCPA, CBMIDA, or a compound as follows:
##STR00025##
where n is 2, 3, or 5; and R is, independently, OCH.sub.3,
CH.sub.2O.sub.2, CH.sub.3, NO.sub.2, or Cl.
[0193] The structure of pCPA is:
##STR00026##
[0194] The structure of CBMIDA is:
##STR00027##
[0195] The ability of selective TPH1 inhibitor CBMIDA to reduce
peripheral serotonin measured in serum was tested. Either 250 or
500 mg/kg doses of CBMIDA were administered orally twice in 20
hours to 4 week old mice, 4-5 mice per group. As a control, some
mice were untreated and some received 250 mg/kg pCPA orally. The
results showed that there was a dose response to CBMIDA
administration such that 250 mg/kg caused about a 45% reduction of
peripheral serotonin, and 500 mg/kg reduced peripheral serotonin by
about 80%. pCPA (250 mg/kg) caused about a 50% reduction of serum
serotonin. These results showed that pCPA was more effective than
CBMIDA and at the amounts used (250 mg/kg), pCPA did not cross the
blood brain barrier. Therefore, pCPA did not decrease serotonin in
the brain, where serotonin has the opposite effect of peripheral
serotonin. CBMIDA has been reported to be an EDTA analog that
increases osteoid volume in beagle dogs and induces proliferation
of rat calvarial-derived osteoblasts in vitro (Xie, et al.,
Bioorganic & Medicinal Chemistry Letters 15(2005) 3267-3270,
incorporated herein by reference in its entirety).
[0196] It should be understood that the present invention may
encompass the use of pCPA and CBMIDA when those agents are used to
decrease serum serotonin levels together with another
pharmaceutically active substance, where the other pharmaceutically
active substance may be used for another purpose (e.g., an SSRI
used to treat depression) or where the other pharmaceutically
active substance is used to decrease serum serotonin by a method
that does not involve inhibition of TPH1.
[0197] The present invention also may further encompass the use of
a compound as follows:
##STR00028##
where n is 2, 3, or 5; and R is, independently, OCH.sub.3,
CH.sub.2O.sub.2, CH.sub.3, NO.sub.2, or Cl, where the compound is
used with another pharmaceutically active substance that decreases
serum serotonin levels.
[0198] Methods of making the compounds described in the preceding
paragraphs can be found in Xie, et al., Bioorganic & Medicinal
Chemistry Letters 15(2005) 3267-3270, incorporated herein by
reference in its entirety.
[0199] The present invention also encompasses the use of certain
derivatives of the TPH1 inhibitors disclosed herein. For example,
prodrugs of the TPH1 inhibitors could be produced by esterifying
the carboxylic acid functions of the TPH1 inhibitors with a lower
alcohol, e.g., methanol, ethanol, propanol, isopropanol, butanol,
etc. The use of prodrugs of the TPH1 inhibitors that are not esters
is also contemplated. For example, pharmaceutically acceptable
carbonates, thiocarbonates, N-acyl derivatives, N-acyloxyalkyl
derivatives, quaternary derivatives of tertiary amines, N-Mannich
bases, Schiff bases, amino acid conjugates, phosphate esters, metal
salts and sulfonate esters of the TPH1 inhibitors are also
contemplated. In some embodiments, the prodrugs will contain a
biohydrolyzable moiety (e.g., a biohydrolyzable amide,
biohydrolyzable carbamate, biohydrolyzable carbonate,
biohydrolyzable ester, biohydrolyzable phosphate, or
biohydrolyzable ureide analog). Guidance for the preparation of
prodrugs of the TPH1 inhibitors disclosed herein can be found in
publications such as Design of Prodrugs, Bundgaard, A. Ed.,
Elsevier, 1985; Design and Application of Prodrugs, A Textbook of
Drug Design and Development, Krosgaard-Larsen and H. Bundgaard,
Ed., 1991, Chapter 5, pages 113-191; and Bundgaard, H., Advanced
Drug Delivery Review, 1992, 8, pages 1-38.
[0200] In certain embodiments, the TPH1 inhibitor inhibits TPH1
without significantly affecting the level of brain-derived
serotonin. Methods of obtaining such inhibitors include: (1)
screening for compounds that inhibit TPH1 to a much greater extent
than TPH2; and (2) screening for compounds that, while they inhibit
both TPH1 and TPH2, cannot cross the blood brain barrier and thus
are effectively specific for TPH1 when administered to the patient
outside the central nervous system. Of course, compounds that both
inhibit TPH1 to a much greater extent than TPH2 and cannot cross
the blood brain barrier are also suitable. Preferably, compounds
that inhibit TPH1 to a much greater extent than TPH2 have an
IC.sub.50 for TPH2 that is at least about 10-fold greater than
their IC.sub.50 for TPH1.
[0201] Several facts suggest that CBMIDA does not cross the blood
brain barrier and is therefore TPH2 selective. First, the structure
of CBMIDA is EDTA based and when given orally it is poorly
transport into the circulation (only 3-5%). Second, EDTA-based
compounds in general have poor transport across the blood brain
barrier.
[0202] In certain embodiments, the agent is a TPH1 inhibitor that
does not significantly affect the level of expression of Type 1
collagen, osteocalcin, Runx2, Osterix, or Atf4 in osteoblasts. In
certain embodiments, the agent is a TPH1 inhibitor that decreases
the expression of Cyclin D1, D2 and E1 in osteoblasts.
[0203] In certain embodiments, the agent is a TPH1 inhibitor having
the structure:
##STR00029##
and pharmaceutically acceptable salts and solvates thereof,
wherein: A is optionally substituted cycloalkyl, aryl, or
heterocycle; X is a bond (i.e., A is directly bound to D), --O--,
--S--, --C(O)--, --C(R4)=, .dbd.C(R.sub.4)--,
--C(R.sub.3R.sub.4)--, --C(R.sub.4).dbd.C(R.sub.4)--,
--C.ident.C--, --N(R.sub.5)--, --N(R.sub.5)C(O)N(R.sub.5)--,
--C(R.sub.3R.sub.4)N(R.sub.5)--, --N(R.sub.5)C(R.sub.3R.sub.4)--,
--ONC(R.sub.3)--, --C(R.sub.3)NO--, --C(R.sub.3R.sub.4)O--,
--OC(R.sub.3R.sub.4)--, --S(O.sub.2)--, --S(O.sub.2)N(R.sub.5)--,
--N(R.sub.5)S(O.sub.2)--, --C(R.sub.3R.sub.4)S(O.sub.2)--, or
--S(O.sub.2)C(R.sub.3R.sub.4)--; D is optionally substituted aryl
or heterocycle; R.sub.1 is hydrogen or optionally substituted
alkyl, alkyl-aryl, alkyl-heterocycle, aryl, or heterocycle; R.sub.2
is hydrogen or optionally substituted alkyl, alkyl-aryl,
alkyl-heterocycle, aryl, or heterocycle; R.sub.3 is hydrogen,
alkoxy, amino, cyano, halogen, hydroxyl, or optionally substituted
alkyl; R.sub.4 is hydrogen, alkoxy, amino, cyano, halogen,
hydroxyl, or optionally substituted alkyl or aryl; each R.sub.5 is
independently hydrogen or optionally substituted alkyl or aryl; and
n is 0-3. Such TPH1 inhibitors are described in International
Patent Publication WO 2007/089335, where they are disclosed as
being useful for treatment of carcinoid syndrome as well as
gastrointestinal diseases and disorders.
[0204] In certain embodiments, the agent is a TPH1 inhibitor having
the structure:
##STR00030##
and pharmaceutically acceptable salts and solvates thereof,
wherein: A is optionally substituted cycloalkyl, aryl, or
heterocycle; X is a bond (i.e., A is directly bound to D), --O--,
--S--, --C(O)--, --C(R4)=, .dbd.C(R.sub.4)--,
--C(R.sub.3R.sub.4)--, --C(R.sub.4).dbd.C(R.sub.4)--,
--C.ident.C--, --N(R.sub.5)--, --N(R.sub.5)C(O)N(R.sub.5)--,
--C(R.sub.3R.sub.4)N(R.sub.5)--, --N(R.sub.5)C(R.sub.3R.sub.4)--,
--ONC(R.sub.3)--, --C(R.sub.3)NO--, --C(R.sub.3R.sub.4)O--,
--OC(R.sub.3R.sub.4)--, --S(O.sub.2)--, --S(O.sub.2)N(R.sub.5)--,
--N(R.sub.5)S(O.sub.2)--, --C(R.sub.3R.sub.4)S(O.sub.2)--, or
--S(O.sub.2)C(R.sub.3R.sub.4)--; D is optionally substituted aryl
or heterocycle; E is optionally substituted aryl or heterocycle;
R.sub.1 is hydrogen or optionally substituted alkyl, alkyl-aryl,
alkyl-heterocycle, aryl, or heterocycle; R.sub.2 is hydrogen or
optionally substituted alkyl, alkyl-aryl, alkyl-heterocycle, aryl,
or heterocycle; R.sub.3 is hydrogen, alkoxy, amino, cyano, halogen,
hydroxyl, or optionally substituted alkyl; R.sub.4 is hydrogen,
alkoxy, amino, cyano, halogen, hydroxyl, or optionally substituted
alkyl or aryl; each R.sub.5 is independently hydrogen or optionally
substituted alkyl or aryl; and n is 0-3. Such TPH1 inhibitors are
described in International Patent Publication WO 2007/089335, where
they are disclosed as being useful for treatment of carcinoid
syndrome as well as gastrointestinal diseases and disorders.
[0205] In the compounds disclosed in the two paragraphs immediately
above:
"cycloalkyl" means a cyclic hydrocarbon having from 1 to 20 (e.g.,
1 to 10 or 1 to 4) carbon atoms. Cycloalkyl moieties may be
monocyclic or multicyclic, and examples include cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, and adamantyl; "aryl" means an
aromatic ring or an aromatic or partially aromatic ring system
composed of carbon and hydrogen atoms. An aryl moiety may comprise
multiple rings bound or fused together. Examples of aryl moieties
include anthracenyl, azulenyl, biphenyl, fluorenyl, indan, indenyl,
naphthyl, phenanthrenyl, phenyl, 1,2,3,4-tetrahydro-naphthalene,
and tolyl; "heterocycle" refers to an aromatic, partially aromatic
or non-aromatic monocyclic or polycyclic ring or ring system
comprised of carbon, hydrogen and at least one heteroatom (e.g., N,
O or S). A heterocycle may comprise multiple (i.e., two or more)
rings fused or bound together. Heterocycles include heteroaryls.
Examples include benzo[1,3]dioxolyl,
2,3-dihydro-benzo[1,4]dioxinyl, cinnolinyl, furanyl, hydantoinyl,
morpholinyl, oxetanyl, oxiranyl, piperazinyl, piperidinyl,
pyrrolidinonyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl,
tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl,
tetrahydrothiopyranyl and valerolactamyl; "alkyl" means a straight
chain, branched and/or cyclic ("cycloalkyl") hydrocarbon having
from 1 to 20 (e.g., 1 to 10 or 1 to 4) carbon atoms. Examples of
alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl,
t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl,
4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl,
undecyl and dodecyl. Cycloalkyl moieties may be monocyclic or
multicyclic, and examples include cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, and adamantyl. Additional examples of
alkyl moieties have linear, branched and/or cyclic portions (e.g.,
1-ethyl-4-methyl-cyclohexyl). The term "alkyl" includes saturated
hydrocarbons as well as alkenyl and alkynyl moieties; "alkyl-aryl"
means an alkyl moiety bound to an aryl moiety; "alkyl-heteroaryl"
means an alkyl moiety bound to a heteroaryl moiety; alkoxy" means
an --O-alkyl group. Examples of alkoxy groups include --OCH.sub.3,
--OCH.sub.2CH.sub.3, --O(CH.sub.2).sub.2CH.sub.3,
--O(CH.sub.2).sub.3CH.sub.3, --O(CH.sub.2).sub.4CH.sub.3, and
--O(CH.sub.2).sub.5CH.sub.3;
[0206] In certain embodiments, the agent is a TPH1 inhibitor having
the structure:
##STR00031##
[0207] LP-533401 and LP-615819 are described in Liu et al., J.
Pharmacol. Exp. Ther., 2008, January 11 [Epub ahead of print]
#132670, incorporated herein by reference in its entirety.
LP-615819 is a prodrug (i.e., the ethyl ester) of LP-533401. As
described in Liu et al., both LP-533401 and LP-615819 were
administered to mice twice per day over a 3-4 day period in amounts
of from about 30-90 mg/kg. Serotonin levels in the small intestine
were significantly lowered after just six consecutive doses, while
brain-derived serotonin was unchanged. Even though LP-533401
inhibits TPH2 as effectively as TPH1, it did not cross the
blood-brain barrier in vivo, and therefore did not cause a decrease
in brain-derived serotonin. Liu et al. disclosed that LP-533401 and
LP-615819 may be useful for treating chemotherapy-induced emesis
and irritable bowel syndrome.
[0208] LP-533401 and LP-615819 selectively reduce peripheral as
opposed to brain-derived serotonin. In animal studies, LP-533401
reduced serotonin in the GI tract to less than about 2/3 normal
levels using 135 mg/kg, po, qd. The effect followed a dose response
curve. In the amounts administered to mice, brain serotonin levels
were not affected. Lexicon Pharmaceuticals Incorporated developed
these TPH1 inhibitors specifically to treat diarrhea or irritable
bowel syndrome. LP-533401 is presently being evaluated in Phase I
clinical trials by Lexicon Pharmaceuticals. In a single dose
regimen, 250 mg to 2,000 mg/day was administered orally. LP-533401
was also administered in multiple doses orally in amounts from
250-1,000 mg/day. In one format, the drug was given as a 500 mg
dose twice per day or as a 500 mg dose four times per day for over
14 days Infrequent adverse events were reported at all dose levels.
These TPH1 inhibitors can be used in the present invention for
treating or preventing low bone mass diseases including
osteoporosis and osteoporosis pseudoglioma.
[0209] Various other TPH1 inhibitors related to LP-533401 and
LP-615819, including multicyclic amino acid derivatives, are
described in U.S. Patent Application Publication 2007/191370, and
in related applications including International Patent Publication
WO 2007/089335, which references are incorporated herein by
reference in their entirety. These inhibitors can also be used in
the methods of the present invention to treat or prevent low bone
mass diseases.
[0210] In certain embodiments of the invention, a therapeutically
effective amount of one or more of the compounds described in the
preceding paragraphs is administered alone or in combination with
other compounds that are known to increase bone mass to a subject
who has or is at risk of developing a low bone mass disease in
order to treat or prevent such disease.
[0211] The efficacy of low bone density therapy by administering
TPH1 inhibitors can be monitored by measuring bone density changes
before and over time after treatment to determine drug
efficacy.
[0212] The present invention provides a method of preventing or
treating a low bone mass disease in a patient known or suspected to
be in need of such prevention or treatment comprising administering
to the patient a therapeutically effective amount of serotonin
receptor antagonist.
[0213] In certain embodiments, the serotonin receptor antagonist is
an HT1B, HT2A or HT2B receptor antagonist. In preferred
embodiments, the serotonin receptor antagonist is an HT1B
antagonist.
[0214] The serotonin receptor antagonist may be one of the many
known antagonists of peripheral serotonin receptors HT1B, HT2A or
HT2B that are present on osteoblasts. Antagonists that are
selective for HT1B, HT2A or HT2B receptors are preferred. The
efficacy of low bone density therapy by administering HT1B, HT2A or
HT2B antagonists can be monitored by measuring bone density changes
before and over time after treatment to determine drug efficacy.
Diseases associated with low bone mass can be treated with HT1B
antagonists such as those listed in Table 3 below.
TABLE-US-00003 TABLE 3 selective 5-HT1B antagonist GR 55562 Mlinar
and Corradetti, Neurosci., 2003, 18: 1559-1571 elzasonan U.S.
Patent Application AZD1134 Publication 2005/0203130 AR-A2 trazodone
hydrochloride (antidepressant) U.S. Pat. No., 7,198,914 highly
selective 5-HT 1B antagonist U.S. Patent Application (SB216641)
Publication 2006/0135415 the selective antagonist at terminal
5-HT.sub.1B receptors, Rojas-Corrales et al., Eur. J.
N-[3-(2-dimethylamino) ethoxy-4-methoxyphenyl]-2'- Pharmacol., 511:
21-26 methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)-(1,1'-
biphenyl)-4-carboxamide (SB216641, 0.1-0.8 mg/kg) GR 127,935 Naunyn
Schmiedebergs Arch. Mixed HT1B/1D antagonist Pharmacol., 1997, 355:
423-430; Wurch, et al., British J. Pharmacol., 1997, 120: 153-159
Cyanopindolol J. Neurochem., 2000, 75: 2113-2122 -GR 125,743
2'-Methyl-4-(5-methyl- Methiothepin [1,2,4]oxadiazol-3-yl)-
ketanserin biphenyl-4-carboxylic acid [4-
methoxy-3-(4-methyl-piperazin- 1-yl)-phenyl]-amide (GR 127,935),
ketanserin and methiothepin and each behaved as silent, competitive
antagonists at rb 5-HT1B receptors British Journal of Pharmacology
(1997) 120, 153 .+-. 159 ICS 205-930 (Sandoz) is a selective
antagonist at 5- Br J Clin Pharmacol. 1989 hydroxytryptamine3
receptors and exerts marked September; 28(3): 315-322 effects on
gastrointestinal motility in animalsGut specific pindolol Pindolol
is also a nonselective a beta-adrenoceptor
blocker/5-hydroxytryptamine.sub.1A/1B beta blocker; rapidly and
well receptor antagonist absorbed from the GI tract AR-A000002 - A
Novel Selective 5-HT.sub.1B Antagonist Journal of Pharmacology And
anxiolytic and antidepressant potential of the selective
Experimental Therapeutics Fast 5-HT.sub.1B antagonist, AR-A000002
((R)--N-[5-Methyl-8- Forward
(4-methylpiperazin-1-yl)-1,2,3,4-tetrahydro-2- First published on
Nov. naphthyl]-4-morpholinobenzamide). AR-A000002 25, 2002;
functions as a 5-HT.sub.1B antagonist in vivo cyanopindolol,
5-HT-moduline and methiothepin Daws, et al., Neuroscience Letters,
1999, 266: 165-168; Daws, et al., J. Neurochem., 2000, 75:
2113-2122 GR 55562, a selective 5-HT1B antagonist British Journal
of selective 5-HT.sub.1B receptor antagonist 3-[3- Pharmacology
(2003) 138, 71-80 (dimethylamino)propyl]-4-hydroxy-N-[4-(4-
pyridinyl)phenyl]benzamide dihydrochloride (GR 55562; K.sub.B
.apprxeq. 100 nM) SB224289 Brain Res. 2004 May 8; 1007(1-2): 86-97
SB 216641 Roca-Vinardell et al., Anesthesiology, 2003, 98: 741-747
Nonselective 5-HT(1B/D) receptor antagonists such as ketanserin,
ritanserin and methiothepin
[0215] In certain embodiments, the agent that increases peripheral
serum serotonin levels is a small organic molecule, an antibody,
antibody fragment, a protein, or polypeptide.
[0216] The present invention also provides a method of preventing
or treating a low bone mass disease in a patient known or suspected
to be in need of such prevention or treatment comprising
administering to the patient both a TPH1 inhibitor and a serotonin
receptor antagonist.
[0217] In certain embodiments, the TPH1 inhibitor and the serotonin
receptor antagonist are administered together in a single
pharmaceutical composition. In other embodiments, the TPH1
inhibitor and the serotonin receptor antagonist are administered in
separate pharmaceutical compositions.
[0218] In certain embodiments of the methods described herein, the
low bone mass disease is osteoporosis, osteoporosis pseudoglioma
syndrome (OPPG), osteopenia, osteomalacia, renal osteodystrophy,
faulty bone formation, faulty bone resorption, Paget's disease,
bone fracture, broken bones, or bone metastasis. In preferred
embodiments, the low bone mass disease is osteoporosis.
[0219] The amount of therapeutic agent to be used depends on many
factors, as discussed herein. However, in humans, for example, the
amount ranges from about 1 mg/day to about 2 g/day; preferably from
about 15 mg/day to about 500 mg/day; or from about 20 mg/day to
about 250 mg/day; or from about 40 mg/day to about 100 mg/day.
Other preferred dosages include about 2 mg/day, about 5 mg/day,
about 10 mg/day, about 15 mg/day, about 20 mg/day, about 25 mg/day,
about 30 mg/day, about 40 mg/day, about 50 mg/day, about 60 mg/day,
about 70 mg/day, about 80 mg/day, about 90 mg/day, about 100
mg/day, about 125 mg/day, about 150 mg/day, about 175 mg/day, about
200 mg/day, about 250 mg/day, about 300 mg/day, about 350 mg/day,
about 400 mg/day, about 500 mg/day, about 600 mg/day, about 700
mg/day, about 800 mg/day, and about 900 mg/day. Routine
experimentation will determine the appropriate value for each
patient by monitoring the compound's effect on serum serotonin
levels, which can be frequently and easily monitored. The agent can
be administered once or multiple times per day. Serum serotonin
levels can be monitored before and during therapy to determine the
appropriate amount of TPH1 inhibitor to administer to lower serum
serotonin levels or bring serum serotonin levels to normal and to
maintain normal levels over extended periods of time. In a
preferred embodiment, a patient is tested to determine if his/her
serum serotonin levels are significantly elevated above normal
levels (about 25% above) before administering treatment with TPH1
inhibitors and/or HT1B, HT2A or HT2B receptor antagonists. The
frequency of administration may vary from a single dose per day to
multiple doses per day. Preferred routes of administration include
oral, intravenous and intraperitoneal, but other forms of
administration may be chosen as well.
[0220] Another embodiment of the present invention is directed to
pharmaceutical formulations of TPH1 inhibitors or serotonin
synthesis inhibitors combined with SSRIs for administration to a
subject being treated with long term SSRI administration, in order
to prevent bone loss or to maintain or increase normal bone
mass.
[0221] In certain embodiments, the therapeutic agents of the
invention act selectively on peripheral serotonin or are
administered in an amount that decreases serum serotonin without
increasing brain-derived serotonin.
[0222] In other embodiments, the TPH1 inhibitors and serotonin
receptor antagonists are formulated and administered together with
bisphosponates such as FOSAMAX.RTM. (alendronate sodium), FOSAMAX
PLUS D.TM. (alendronate sodium/cholecalciferol) or other bone
building drugs, vitamins or minerals to potentiate their effects on
increasing bone mass.
[0223] Monitoring the therapeutic efficacy of TPH1 inhibitors and
serotonin synthesis inhibitors is straightforward, as one can
administer the inhibitors in an amount and for a duration that
reduces peripheral serum serotonin levels, and over time increases
bone mass. Both serum serotonin and bone mass can be easily
measured. Example 1 provides the details of one immunoassay for
monitoring the level of serum serotonin. Example 3 provides further
assays for serum serotonin that may be used. Monitoring serum
serotonin is simple and can be done frequently during the course of
therapy to establish the appropriate dose for each patient. Any
method known in the art for assaying serum serotonin can be used.
Increased bone mass can be measured as described herein using
various means of measuring bone density and markers of bone growth
or can be measured by other methods known in the art.
[0224] In another embodiment, low bone mass diseases are treated by
administering anti-serotonin antibodies or antibody fragments,
preferably by intravenous or intraperitoneal injections. Such
antibodies will not cross the blood brain barrier, and will
neutralize serotonin, thereby preventing it from reducing bone
mass. Antibodies or antibody fragments that recognize and
inactivate TPH1 or a serotonin receptor (e.g., HT1B), and thus
lower serum serotonin levels, can also be used in the present
invention.
[0225] The terms "antibody" and "antibodies" include polyclonal
antibodies, monoclonal antibodies, humanized or chimeric
antibodies, single chain Fv antibody fragments, Fab fragments, and
F(ab').sub.2 fragments. Polyclonal antibodies are heterogeneous
populations of antibody molecules that are specific for a
particular antigen, while monoclonal antibodies are homogeneous
populations of antibodies to a particular epitope contained within
an antigen. Monoclonal antibodies are particularly useful in the
present invention.
[0226] Antibody fragments that have specific binding affinity for a
target of interest (e.g., TPH1 or HT1B) can be generated by known
techniques. Such antibody fragments include, but are not limited
to, F(ab').sub.2 fragments that can be produced by pepsin digestion
of an antibody molecule, and Fab fragments that can be generated by
reducing the disulfide bridges of F(ab').sub.2 fragments.
Alternatively, Fab expression libraries can be constructed. See,
for example, Huse et al., 1989, Science 246:1275-1281. Single chain
Fv antibody fragments are formed by linking the heavy and light
chain fragments of the Fv region via an amino acid bridge (e.g., 15
to 18 amino acids), resulting in a single chain polypeptide. Single
chain Fv antibody fragments recognizing a target of interest can be
produced through standard techniques, such as those disclosed in
U.S. Pat. No. 4,946,778.
[0227] Once produced, antibodies or fragments thereof can be tested
for recognition of the target of interest by standard immunoassay
methods including, for example, enzyme-linked immunosorbent assay
(ELISA) or radioimmunoassay assay (RIA). See, Short Protocols in
Molecular Biology, eds. Ausubel et al., Green Publishing Associates
and John Wiley & Sons (1992).
[0228] The amount of antibody or antibody fragment to administer
will depend, inter alia, on how high the level of peripheral
serotonin is compared to normal levels. Monoclonal and polyclonal
anti-serotonin antibodies known in the art, or newly designed, can
be used and can be administered as a single therapy or as
combination therapy with TPH1 inhibitors and/or HT1B
antagonists.
[0229] U.S. Provisional Patent Application Ser. No. 60/976,403,
filed Sep. 28, 2007, and incorporated by reference herein in its
entirety, discloses that brain-derived serotonin increases bone
mass and decreases sympathetic tone. Another embodiment of the
present invention for treating or preventing low bone mass diseases
is directed to methods for treating or preventing low bone mass by
administering agents that decrease sympathetic tone, such as beta
blockers, together with a TPH 1 inhibitor, serotonin synthesis
inhibitor, HT1B, HT2A or HT2B antagonist and/or anti-serotonin
antibodies, either in a single formulation or separately. The use
of any compound that decreases sympathetic tone comes within the
scope of the invention. Preferably the compound is a beta-2
receptor antagonist, many of which are described in the art. Among
the beta blockers that can be used are three beta-2 specific
blockers that can be used to reduce sympathetic tone and increase
bone mass, alone or in combination with other therapeutic agents
described herein: IPS339, ICI118,551, and Sandoz L1 32-468 (Br. J.
Ophthalmol. 1984 April; 68(4): 245-247). Butaxamine is also a
beta-2 blocker that may be used in the present invention.
Non-selective beta blockers include: metipranolol, nadol (a
beta-specific sympatholytic which non-selectively blocks beta-2
adrenergic receptors); oxprenolol (a lipophilic beta blocker which
passes the blood-brain barrier more easily than water soluble beta
blockers), penbutolol, pindolol (a beta blocker that acts on
serotonin 5-HT1A receptors in the brain, resulting in increased
postsynaptic serotonin concentrations), and propranolol (known to
readily cross the blood-brain barrier, timolol and sotalol. The
beta blockers can be administered together with agents that
directly or indirectly increase brain-derived serotonin, including
HT2C receptor agonists, agents that increase TPH2 activity or
expression, and agents that specifically decrease reuptake of
BDS.
[0230] Certain other embodiments of the invention are directed to a
pharmaceutical composition that includes a TPH1 inhibitor; HT1B,
HT2A or HT2B antagonist; and/or anti-serotonin antibodies,
individually or in combination. More than one type of TPH1
inhibitor, HT1B, HT2A or HT2B antagonist, or anti-serotonin
antibody can be administered together for treating diseases
associated with low bone mass, and certain embodiments include
corresponding pharmaceutical compositions comprising these
compounds. In other embodiments, the different types of agents are
administered separately at one or more times on the same day, or
over a period of days, sometimes alternating administration of the
various respective agents.
[0231] Some embodiments are directed to pharmaceutical compositions
for treating or preventing anxiety or depression that include both
SSRIs and drugs that reduce the level of serum serotonin (e.g.,
TPH1 inhibitors or HT1B antagonists) in order to prevent patients
who take serotonin reuptake inhibitors from developing
osteoporosis. These preparations would permit the SSRIs to elevate
brain-derived serotonin to treat anxiety without increasing
peripheral serotonin, which can cause low bone mass diseases like
osteoporosis.
[0232] Elevated brain-derived serotonin increases bone mass by
acting through HT2C receptors on target neurons in the
hypothalamus. Thus, some embodiments of the present invention
include administering combination drug therapy using
pharmaceuticals that decrease peripheral serotonin and increase
brain-derived serotonin. For example, an HT2C agonist may be
combined with a TPH1 inhibitor or an HT antagonist.
[0233] Diseases associated with abnormally high bone mass such as
high bone mass syndrome can be treated by administering serotonin,
serotonin reuptake inhibitors, TPH1 activators, TPH2 inhibitors,
serotonin receptor agonists, or combinations thereof, to increase
serum serotonin, which will in turn decrease bone mass. HT1B
agonists, or other serotonin receptor agonists, can be used to
activate the receptors on osteoblasts to decrease bone mass. TPH1
inhibitors, HT1B agonists, HT2A agonists or HT2B agonists can be
administered together with serotonin. Thus, certain embodiments of
the present invention are directed to methods for treating high
bone mass disease by administering serotonin, preferably orally,
intraperitoneally or intravenously, alone or together with TPH1
inhibitors, HT1B agonists, HT2A agonists or HT2B agonists to
decrease bone mass, preferably to normal levels.
[0234] In certain embodiments, the methods of the present invention
comprise the step of identifying a patient in need of therapy for a
bone disease. Thus, the present invention provides a method
comprising:
[0235] (a) identifying a patient in need of therapy for a bone
disease;
[0236] (b) administering to the patient a therapeutically effective
amount of an agent that increases or decreases serum serotonin
levels.
[0237] In certain embodiments, the present invention provides a
method comprising:
[0238] (a) identifying a patient in need of therapy for a low bone
mass disease;
[0239] (b) administering to the patient a therapeutically effective
amount of an agent that decreases serum serotonin levels.
[0240] In certain embodiments, the present invention provides a
method comprising:
[0241] (a) identifying a patient in need of therapy for a high bone
mass disease;
[0242] (b) administering to the patient a therapeutically effective
amount of an agent that increases serum serotonin levels.
[0243] The present invention encompasses the use of a TPH1
inhibitor or a serotonin receptor antagonist (e.g., an HT1B
antagonist) for the manufacture of a medicament for preventing or
treating a bone disease (e.g., a low bone mass disease such as
osteoporosis). The present invention encompasses the use of a TPH1
inhibitor or a serotonin receptor antagonist (e.g., an HT1B
antagonist) for preventing or treating a bone disease (e.g., a low
bone mass disease such as osteoporosis).
Pharmaceutical Compositions
[0244] Therapeutic agents such as the TPH1 inhibitors, serotonin
receptor antagonists, serotonin receptor agonists, SSRIs, and beta
blockers described herein may be formulated into pharmaceutical
compositions. The therapeutic agents may be present in the
pharmaceutical compositions in the form of salts of
pharmaceutically acceptable acids or in the form of bases. The
therapeutic agents may be present in amorphous form or in
crystalline forms, including hydrates and solvates. Preferably, the
pharmaceutical compositions comprise a therapeutically effective
amount of a TPH1 inhibitor or serotonin receptor antagonist.
[0245] Pharmaceutically acceptable derivatives of any of the TPH1
inhibitors, serotonin receptor antagonists, or serotonin receptor
agonists described herein come within the scope of the invention. A
"pharmaceutically acceptable derivative" of a TPH1 inhibitor,
serotonin receptor antagonist, or serotonin receptor agonist means
any non-toxic derivative of a TPH1 inhibitor, serotonin receptor
antagonist, or serotonin receptor agonist described herein that,
upon administration to a recipient, exhibits that same or similar
biological activity with respect to reducing or increasing serum
serotonin expression as the TPH1 inhibitor, serotonin receptor
antagonist, or serotonin receptor agonists described herein.
[0246] Pharmaceutically acceptable salts of the therapeutic agents
described herein for use in treating or preventing bone diseases
associated with abnormally high or abnormally low bone mass,
include those salts derived from pharmaceutically acceptable
inorganic and organic acids and bases. Examples of suitable acid
salts include acetate, adipate, alginate, aspartate, benzoate,
benzenesulfonate, bisulfate, butyrate, citrate, camphorate,
camphorsulfonate, cyclopentanepropionate, digluconate,
dodecylsulfate, ethanesulfonate, formate, fumarate,
glucoheptanoate, glycerophosphate, glycolate, hemisulfate,
heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide,
2-hydroxyethanesulfonate, lactate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oxalate, palmoate, pectinate, persulfate, 3-phenylpropionate,
phosphate, picrate, pivalate, propionate, salicylate, succinate,
sulfate, tartrate, thiocyanate, tosylate and undecanoate salts.
Other acids, such as oxalic, while not in themselves
pharmaceutically acceptable, may be employed in the preparation of
salts useful as intermediates in obtaining the compounds of the
invention and their pharmaceutically acceptable acid addition
salts.
[0247] Salts derived from appropriate bases include alkali metal
(e.g., sodium and potassium), alkaline earth metal (e.g.,
magnesium), ammonium and N.sup.+(C.sub.1-4 alkyl).sub.4 salts. This
invention also envisions the quaternization of any basic
nitrogen-containing groups of the therapeutic agents disclosed
herein. Water or oil-soluble or dispersible products may be
obtained by such quaternization.
[0248] The therapeutic agents of the present invention are also
meant to include all stereochemical forms of the therapeutic agents
(i.e., the R and S configurations for each asymmetric center).
Therefore, single enantiomers, racemic mixtures, and diasteromers
of the therapeutic agents are within the scope of the invention.
Also within the scope of the invention are steric isomers and
positional isomers of the therapeutic agents. The therapeutic
agents of the present invention are also meant to include compounds
which differ only in the presence of one or more isotopically
enriched atoms. For example, therapeutic agents in which a molecule
of hydrogen is replaced by deuterium or tritium, or the replacement
of a carbon molecule by .sup.13C- or .sup.14C-enriched carbon are
within the scope of this invention.
[0249] In a preferred embodiment, the therapeutic agents of the
present invention are administered in a pharmaceutical composition
that includes a pharmaceutically acceptable carrier, adjuvant, or
vehicle. The term "pharmaceutically acceptable carrier, adjuvant,
or vehicle" refers to a non-toxic carrier, adjuvant, or vehicle
that does not destroy or significantly diminish the pharmacological
activity of the compound with which it is formulated.
Pharmaceutically acceptable carriers, adjuvants or vehicles that
may be used in the compositions of this invention encompass any of
the standard pharmaceutically accepted liquid carriers, such as a
phosphate-buffered saline solution, water, as well as emulsions
such as an oil/water emulsion or a triglyceride emulsion. An
example of an acceptable triglyceride emulsion useful in the
intravenous and intraperitoneal administration of the compounds is
the triglyceride emulsion commercially known as INTRALIPID.RTM..
Solid carriers may include excipients such as starch, milk, sugar,
certain types of clay, stearic acid, talc, gums, glycols, or other
known excipients. Carriers may also include flavor and color
additives or other ingredients.
[0250] In the practice of the invention, the pharmaceutical
compositions of the present invention are preferably administered
orally. However, the pharmaceutical compositions may be
administered parenterally, by inhalation spray, topically,
rectally, nasally, buccally, vaginally or via an implanted
reservoir. Preferably, the pharmaceutical compositions are
administered orally, intraperitoneally or intravenously. Sterile
injectable forms of the pharmaceutical compositions may be aqueous
or oleaginous suspensions. These suspensions may be formulated
according to techniques known in the art using suitable dispersing
or wetting agents and suspending agents. The sterile injectable
preparation may also be a sterile injectable solution or suspension
in a non-toxic parenterally acceptable diluent or solvent, for
example as a solution in 1,3-butanediol. Among the acceptable
vehicles and solvents that may be employed are water, Ringer's
solution and isotonic sodium chloride solution. In addition,
sterile, fixed oils are conventionally employed as a solvent or
suspending medium.
[0251] For this purpose, any bland fixed oil may be employed
including synthetic mono- or di-glycerides. Fatty acids, such as
oleic acid and its glyceride derivatives are useful in the
preparation of injectables, as are natural
pharmaceutically-acceptable oils such as olive oil or castor oil,
especially in their polyoxyethylated versions. These oil solutions
or suspensions may also contain a long-chain alcohol diluent or
dispersant, such as carboxymethyl cellulose or similar dispersing
agents that are commonly used in the formulation of
pharmaceutically acceptable dosage forms including emulsions and
suspensions. Other commonly used surfactants, such as Tweens, Spans
and other emulsifying agents or bioavailability enhancers which are
commonly used in the manufacture of pharmaceutically acceptable
solid, liquid, or other dosage forms may also be used for the
purposes of formulation.
[0252] The pharmaceutical compositions of this invention may be
orally administered in any orally acceptable dosage form including,
but not limited to, solid forms such as capsules and tablets. In
the case of tablets for oral use, carriers commonly used include
microcrystalline cellulose, lactose and corn starch. Lubricating
agents, such as magnesium stearate, are also typically added. When
aqueous suspensions are required for oral use, the active
ingredient may be combined with emulsifying and suspending agents.
If desired, certain sweetening, flavoring or coloring agents may
also be added.
[0253] The pharmaceutical compositions of this invention may also
be administered by nasal aerosol or inhalation. Such pharmaceutical
compositions are prepared according to techniques well-known in the
art of pharmaceutical formulation and may be prepared as solutions
in saline, employing benzyl alcohol or other suitable
preservatives, absorption promoters to enhance bioavailability,
fluorocarbons, and/or other conventional solubilizing or dispersing
agents.
[0254] Should topical administration be desired, it can be
accomplished using any method commonly known to those skilled in
the art and includes but is not limited to incorporation of the
pharmaceutical composition into creams, ointments, or transdermal
patches.
[0255] Where the pharmaceutical compositions contain both agents
that act peripherally like HT1B antagonists or TPH1 inhibitors and
agents that act centrally like HT2C agonists, the compositions can
be formulated to increase delivery of the centrally acting
therapeutic agents to the central nervous system. If a compound
having therapeutic utility does not easily cross the blood brain
barrier, it can be modified using various methods in medicinal
chemistry known in the art that attach various side groups to
improve permeability through the blood brain barrier.
[0256] Serotonin receptor antagonists (e.g., HT1B receptor
antagonists) can be derivatized or otherwise designed to enhance
uptake by bone, using medicinal chemistry methods known in the
art.
[0257] The TPH1 inhibitors and HT1B antagonists of the present
invention can be derivatized by the formation of a reversible
linkage with one or more suitable groups to yield "pro-drugs,"
i.e., chemical derivatives that, after absorption by the host, are
converted into the parent compound. Liberation of the parent
compound may be by chemical hydrolysis or enzymatic attack. A
derivative or pro-drug can have enhanced permeability for the
target organ. In the case of TPH1 inhibitors, the target organ is
the duodenum where 95% of peripheral serotonin is made. HT1B
antagonists could be formulated to have enhanced penetration of
bone to reach the osteoblast target. The prodrug has an enhanced
permeability according to the present invention if, after
administration of the pro-drug or derivative thereof to a living
organism, a higher amount of the compound reaches the target organ,
resulting in a higher level of effective agent, as compared to
administration of the base compound without derivatization.
[0258] The amount of the therapeutic agents of the present
invention that may be combined with the carrier materials to
produce a pharmaceutical composition in a single dosage form will
vary depending upon the host treated and the particular mode of
administration. It should be understood that a specific dosage and
treatment regimen for any particular patient will depend upon a
variety of factors, including the activity of the specific compound
employed, the age, body weight, general health, sex, diet, time of
administration, rate of excretion, drug combination, and the
judgment of the treating physician as well as the severity of the
particular disease being treated. Despite their variety, accounting
for these factors in order to select an appropriate dosage or
treatment regimen would require no more than routine
experimentation.
[0259] Additional therapeutic agents, which are normally
administered to treat bone diseases associated with abnormally high
or abnormally low bone mass, may also be present in the
pharmaceutical compositions of this invention. As used herein,
additional therapeutic agents that are normally administered to
treat a particular disease, or condition, are known as "appropriate
for the disease, or condition, being treated." Examples of
appropriate agents for osteoporosis include FOSAMAX.RTM., other
bisphosphonates, FORTEO.RTM. (parathyroid hormone) and
beta-blockers. Those additional agents may be administered
separately from the therapeutic agents of the invention, as part of
a multiple dosage regimen. Alternatively, those agents may be part
of a single dosage form, mixed together with the therapeutic agents
of the invention in a single pharmaceutical composition. If
administered as part of a multiple dosage regime, the two active
agents may be administered simultaneously, sequentially or within a
period of time from one another. The amount of both the therapeutic
agent of the invention and the additional therapeutic agent (in
those compositions which comprise an additional therapeutic agent
as described above) that may be combined with the carrier materials
to produce a single dosage form will vary depending upon the host
treated and the particular mode of administration as well as on the
nature of the therapeutic agent of the invention and the additional
therapeutic agent.
[0260] TPH1 inhibitors and the other therapeutic agents described
herein (e.g., HT1B antagonists, HT2C agonists) may be proteins or
polypeptides, as well as any biologically active fragment, epitope,
modification, derivative or variant thereof. Biologically active
fragments of a protein or polypeptide are those fragments of the
protein or polypeptide exhibiting activity similar to, but not
necessarily identical to, an activity of the entire protein or
polypeptide. The biological activity of the fragments may include
an improved desired activity, or a decreased undesirable activity.
Variants include (i) substitutions of one or more of the amino acid
residues, where the substituted amino acid residues may or may not
be one encoded by the genetic code, or (ii) substitution with one
or more of amino acid residues having a substituent group, or (iii)
fusion of the mature polypeptide with another compound, such as a
compound to increase the stability and/or solubility of the
polypeptide (e.g., polyethylene glycol), or other molecule that
facilitates transport through stomach (if administered orally) or
through the endothelium (if administered intravenously), and (iv)
fusion of the polypeptide with additional amino acids, such as an
IgG Fc peptide, or a leader or secretary sequence, or a sequence
facilitating purification. Such variant polypeptides are deemed to
be within the scope of the present invention if they retain
therapeutic efficacy.
[0261] "Amino acid residue" refers to an amino acid which is part
of a polypeptide. The amino acid residues described herein are
preferably in the L isomeric form. However, residues in the D
isomeric form can be substituted for any L-amino acid residue, as
long as the desired functional property is retained by the
polypeptide. NH.sub.2 refers to the free amino group present at the
amino terminus of a polypeptide. COOH refers to the free carboxyl
group present at the carboxyl terminus of a polypeptide. "Amino
acid residue" is broadly defined to include the 20 amino acids
commonly found in natural proteins, as well as modified and unusual
amino acids, such as those referred to in 37 C.F.R. Sections
1.821-1.822, the entire contents of which are hereby incorporated
by reference as if fully set forth herein. In a polypeptide or
protein, suitable conservative substitutions of amino acids are
known to those of skill in this art and can be made generally
without altering the biological activity of the resulting molecule.
Those of skill in the art recognize that, in general, single amino
acid substitutions in non-essential regions of a polypeptide do not
substantially alter biological activity (see, e.g., Watson et al.
Molecular Biology of the Gene, 4th Edition, 1987, The
Benjamin/Cummings Pub. Co., p. 224).
[0262] Examples of conservative substitutions are the replacements,
one for another, among the hydrophobic amino acids Ala, Val, Leu,
and Ile; interchange of the hydroxyl residues Ser and Thr; exchange
of the acidic residues Asp and Glu; substitution between the amide
residues Asn and Gln; exchange of the basic residues Lys, His and
Arg; replacements among the aromatic residues Phe, Trp and Tyr;
exchange of the polar residues Gln and Asn; and exchange of the
small residues Ala, Ser, Thr, Met, and Gly.
[0263] Acylation of the N-terminal amino group can be accomplished
using a hydrophilic compound, such as hydroorotic acid or the like,
or by reaction with a suitable isocyanate, such as methylisocyanate
or isopropylisocyanate, to create a urea moiety at the N-terminus.
Other agents can also be N-terminally linked that will increase the
duration of action of the variant as known in this art.
[0264] Reductive amination is the process by which ammonia is
condensed with aldehydes or ketones to form imines which are
subsequently reduced to amines. For therapeutic agents bearing one
or more amino groups, reductive amination is a potentially useful
method for conjugation to poly(ethylene glycol) (PEG). Covalent
linkage of PEG to drug molecules results in water-soluble
conjugates with altered bioavailability, pharmacokinetics,
immunogenic properties, and biological activities. For drugs
bearing one or more amino groups, reductive amination is a
potentially useful method for conjugation to PEG (Bentley et al.,
J. Pharm. Sci. 1998 November; 87(11):1446-1449).
[0265] As is also well known, polypeptides are not always entirely
linear. For instance, polypeptides may be branched as a result of
ubiquitination, and they may be circular, with or without
branching, generally as a result of post-translational events,
including natural processing events and events brought about by
human manipulation which do not occur naturally. Circular, branched
and branched circular polypeptides for use in the present invention
may be synthesized by non-translational natural processes and by
synthetic methods.
[0266] Modifications can occur anywhere in a protein or polypeptide
for use in the present invention, including the peptide backbone,
the amino acid side-chains and the amino or carboxyl termini.
Blockage of the amino or carboxyl group in a polypeptide, or both,
by a covalent modification, is common in naturally-occurring and
synthetic polypeptides. For instance, the amino-terminal residue of
polypeptides made in E. coli, prior to proteolytic processing,
almost invariably will be N-formylmethionine.
[0267] The modifications can be a function of how the protein is
made. For recombinant polypeptides used in the present invention,
for example, the modifications will be determined by the host cell
posttranslational modification capacity and the modification
signals in the polypeptide amino acid sequence. Accordingly, when
glycosylation is desired, a polypeptide should be expressed in a
glycosylating host, generally a eukaryotic cell. Insect cells often
carry out the same posttranslational glycosylations as mammalian
cells, and, for this reason, insect cell expression systems have
been developed to efficiently express mammalian proteins having
native patterns of glycosylation. Accordingly, the use of insect
systems in the present invention is contemplated. Similar
considerations apply to other modifications besides glycosylation.
The same type of modification may be present in the same or varying
degree at several sites in a given polypeptide. Also, a given
polypeptide may contain more than one type of modification.
[0268] The following table illustrates some of the common
modifications of proteins and polypeptides that may be used in the
present invention.
TABLE-US-00004 TABLE 4 Protein Modification Description Acetylation
Acetylation of N-terminus or .epsilon.-lysines. Introducing an
acetyl group into a protein, specifically, the substitution of an
acetyl group for an active hydrogen atom. A reaction involving the
replacement of the hydrogen atom of a hydroxyl group with an acetyl
group (CH.sub.3CO) yields a specific ester, the acetate. Acetic
anhydride is commonly used as an acetylating agent, which reacts
with free hydroxyl groups. Acylation may facilitate addition of
other functional groups. A common reaction is acylation of e.g.,
conserved lysine residues with a biotin appendage. ADP-ribosylation
Covalently linking proteins or other compounds via an
arginine-specific reaction. Alkylation Alkylation is the transfer
of an alkyl group from one molecule to another. The alkyl group may
be transferred as an alkyl carbocation, a free radical or a
carbanion (or their equivalents). Alkylation is accomplished by
using certain functional groups such as alkyl electrophiles, alkyl
nucleophiles or sometimes alkyl radicals or carbene acceptors. A
common example is methylation (usually at a lysine or arginine
residue). Amidation Reductive amidation of the N-terminus. Methods
for amidation are described in U.S. Pat. No. 4,489,159.
Carbamylation Adding a carbamoyl group to a protein or polypeptide.
Carboxylation Carboxylation typically occurs at the glutamate
residues of a protein. Carboxylation may be catalyzed by a
carboxylase enzyme (in the presence of Vitamin K - a cofactor).
Citrullination Citrullination involves the addition of citrulline
amino acids to the arginine residues of a protein, which is
catalyzed by peptidylarginine deaminase enzymes (PADs). This
generally converts a positively charged arginine into a neutral
citrulline residue, which may affect the hydrophobicity of the
protein (and can lead to unfolding). Condensation of amines with
Such reactions may be used, e.g., to attach a peptide to other
aspartate or glutamate proteins or to attach labels to proteins or
polypeptides. Covalent attachment of flavin Flavin mononucleotide
(FAD) may be covalently attached to serine and/or threonine
residues. May be used, e.g., as a light-activated tag. Covalent
attachment of heme A heme moiety is generally a prosthetic group
that consists moiety of an iron atom contained in the center of a
large heterocyclic organic ring, which is referred to as a
porphyrin. The heme moiety may be used, e.g., as a tag for the
peptide. Attachment of a nucleotide or May be used as a tag or as a
basis for further derivatising a nucleotide derivative peptide.
Cross-linking Cross-linking is a method of covalently joining two
proteins. Cross-linkers contain reactive ends to specific
functional groups (primary amines, sulfhydryls, etc.) on proteins
or other molecules. Several chemical groups may be targets for
reactions in proteins and peptides. For example, Ethylene glycol
bis[succinimidylsuccinate, Bis[2-
(succinimidooxycarbonyloxy)ethyl]sulfone, and
Bis[sulfosuccinimidyl] suberate link amines to amines. Cyclization
For example, cyclization of amino acids to create optimized
delivery forms that are resistant to, e.g., aminopeptidases (e.g.,
formation of pyroglutamate, a cyclized form of glutamic acid).
Disulfide bond formation Disulfide bonds in proteins are formed by
thiol-disulfide exchange reactions, particularly between cysteine
residues (e.g., formation of cystine). Demethylation See, e.g.,
U.S. Pat. No. 4,250,088 (Process for demethylation). Formylation
The addition of a formyl group to, e.g., the N-terminus of a
protein. See, e.g., U.S. Pat. Nos. 4,059,589, 4,801,742, and
6,350,902. Glycosylation Glycosylation may be used to add
saccharides (or polysaccharides) to the hydroxy oxygen atoms of
serine and threonine side chains (which is also known as O-linked
Glycosylation). Glycosylation may also be used to add saccharides
(or polysaccharides) to the amide nitrogen of asparagine side
chains (which is also known as N-linked Glycosylation), e.g., via
oligosaccharyl transferase. GPI anchor formation The addition of
glycosylphosphatidylinositol to the C- terminus of a protein. GPI
anchor formation involves the addition of a hydrophobic
phosphatidylinositol group - linked through a carbohydrate
containing linker (e.g., glucosamine and mannose linked to
phosphoryl ethanolamine residue) - to the C-terminal amino acid of
a protein. Hydroxylation A chemical process that introduces one or
more hydroxyl groups (--OH) into a protein (or polypeptide).
Hydroxylation reactions are typically catalyzed by hydroxylases.
Proline is the principal residue to be hydroxylated in proteins,
which occurs at the C.sup..gamma. atom, forming hydroxyproline
(Hyp). In some cases, proline may be hydroxylated at its
C.sup..beta. atom. Lysine may also be hydroxylated on its
C.sup..delta. atom, forming hydroxylysine (Hyl). These three
reactions are catalyzed by large, multi-subunit enzymes known as
prolyl 4-hydroxylase, prolyl 3-hydroxylase and lysyl 5-hydroxylase,
respectively. These reactions require iron (as well as molecular
oxygen and .alpha.-ketoglutarate) to carry out the oxidation, and
use ascorbic acid to return the iron to its reduced state.
Iodination See, e.g., U.S. Pat. No. 6,303,326 for a disclosure of
an enzyme that is capable of iodinating proteins or polypeptides.
U.S. Pat. No. 4,448,764 discloses, e.g., a reagent that may be used
to iodinate proteins. ISGylation Covalently linking a peptide to
the ISG15 (Interferon- Stimulated Gene 15) protein, for, e.g.,
modulating immune response. Methylation Reductive methylation of
protein amino acids with formaldehyde and sodium cyanoborohydride
has been shown to provide up to 25% yield of N-cyanomethyl
(--CH.sub.2CN) product. The addition of metal ions, such as
Ni.sup.2+, which complex with free cyanide ions, improves reductive
methylation yields by suppressing by-product formation. The
N-cyanomethyl group itself, produced in good yield when cyanide ion
replaces cyanoborohydride, may have some value as a reversible
modifier of amino groups in proteins. Methylation may occur at the
arginine and lysine residues of a protein, as well as the N- and
C-terminus thereof. Myristoylation Myristoylation involves the
covalent attachment of a myristoyl group (a derivative of myristic
acid), via an amide bond, to the alpha-amino group of an N-terminal
glycine residue. This addition is catalyzed by the N-
myristoyltransferase enzyme. Oxidation Oxidation of cysteines.
Oxidation of N-terminal Serine or Threonine residues (followed by
hydrazine or aminooxy condensations). Oxidation of glycosylations
(followed by hydrazine or aminooxy condensations). Palmitoylation
Palmitoylation is the attachment of fatty acids, such as palmitic
acid, to cysteine residues of proteins. Palmitoylation increases
the hydrophobicity of a protein. (Poly)glutamylation
Polyglutamylation occurs at the glutamate residues of a protein.
Specifically, the gamma-carboxy group of a glutamate will form a
peptide-like bond with the amino group of a free glutamate whose
alpha-carboxy group may be extended into a polyglutamate chain. The
glutamylation reaction is catalyzed by a glutamylase enzyme (or
removed by a deglutamylase enzyme). Polyglutamylation has been
carried out at the C-terminus of proteins to add up to about six
glutamate residues. Phosphopantetheinylation The addition of a
4'-phosphopantetheinyl group. Phosphorylation A process for
phosphorylation of a protein or peptide by contacting a protein or
peptide with phosphoric acid in the presence of a non-aqueous
apolar organic solvent and contacting the resultant solution with a
dehydrating agent is disclosed e.g., in U.S. Pat. No. 4,534,894.
Typically, phosphorylation occurs at the serine, threonine, and
tyrosine residues of a protein. Prenylation Prenylation (or
isoprenylation or lipidation) is the addition of hydrophobic
molecules to a protein. Protein prenylation involves the transfer
of either a farnesyl (linear grouping of three isoprene units) or a
geranyl-geranyl moiety to C- terminal cysteine(s) of the target
protein or polypeptide. Proteolytic Processing Processing, e.g.,
cleavage of a protein at a peptide bond. Selenoylation The exchange
of, e.g., a sulfur atom in the peptide for selenium, using a
selenium donor, such as selenophosphate. Sulfation Processes for
sulfating hydroxyl moieties, particularly tertiary amines, are
described in, e.g., U.S. Pat. No. 6,452,035. A process for
sulfation of a protein or polypeptide by contacting the protein or
polypeptide with sulfuric acid in the presence of a non-aqueous
apolar organic solvent and contacting the resultant solution with a
dehydrating agent is disclosed. SUMOylation Covalently linking a
protein or polypeptide to a SUMO (small ubiquitin-related modifier)
protein, for, e.g., stabilizing the peptide. Transglutamination
Covalently linking other protein(s) or chemical groups (e.g., PEG)
via a bridge at glutamine residues tRNA-mediated addition of For
example, the site-specific modification (insertion) of an amino
acids (e.g., amino acid analog into a peptide. arginylation)
Ubiquitination The small peptide ubiquitin is covalently linked to,
e.g., lysine residues of a protein. The ubiquitin-proteasome system
can be used to carryout such reaction. See, e.g., U.S. Patent
Application Publication 2007/0059731.
Identifying Therapeutic Agents of the Present Invention
[0269] Inhibitors of TPH1 may be identified by any methods known in
the art. In particular, inhibitors of TPH1 may be identified by a
method comprising:
[0270] (a) providing a source of TPH1;
[0271] (b) exposing the source of TPH1 to L-tryptophan in the
absence of a candidate compound;
[0272] (c) measuring the amount of 5-hydroxytryptophan produced by
the source of TPH1 in the absence of the candidate compound;
[0273] (d) exposing the source of TPH1 to L-tryptophan in the
presence of the candidate compound;
[0274] (e) measuring the amount of 5-hydroxytryptophan produced by
the source of TPH1 in the presence of the candidate compound;
[0275] (f) where, if the amount of 5-hydroxytryptophan produced by
the source of TPH1 in the presence of the candidate compound is
less than the amount of 5-hydroxytryptophan produced by the source
of TPH1 in the absence of the candidate compound, the candidate
compound is a TPH1 inhibitor.
[0276] In certain embodiments, the method described above includes
the further step of administering the TPH1 inhibitor identified in
step (0 to a patient in need of therapy for a low bone mass
disease.
[0277] "Less than" for the purpose of the herein-described methods
of identifying therapeutic agents from a collection of candidate
compounds refers to an amount that would not be attributed by those
of skill in the art to normal variation seen in the method.
Preferably, "less than" is at least about 10%, at least about 20%,
at least about 50%, at least about 75%, or at least about 95% less
than the amount observed in the absence of the candidate
compound.
[0278] In certain embodiments, the source of TPH1 is an isolated
TPH1 enzyme, preferably human. Isolated TPH1 can be produced by in
vitro expression of TPH1, e.g., in a coupled in vitro
transcription/translation system. Alternatively, the source of TPH1
may be partially or highly purified preparations from cells
expressing TPH1. In other embodiments, the source of TPH1 is a
whole cell expressing TPH1, preferably human. In some embodiments,
the whole cell has been transfected with a expression vector
comprising TPH1 so that the cell expresses recombinant TPH1,
preferably human.
[0279] The mRNA and amino acid sequence of human TPH1 can be found
in GenBank, at accession no. X52836. The genomic sequence can be
found at AF057280. These nucleotide sequences can be used in
methods well-known in the art to construct suitable expression
vectors for expressing TPH1 recombinantly in cells, or in
vitro.
[0280] Activators of TPH2 may be identified by a method
comprising:
[0281] (a) providing a source of TPH2;
[0282] (b) exposing the source of TPH2 to L-tryptophan in the
absence of a candidate compound;
[0283] (c) measuring the amount of 5-hydroxytryptophan produced by
the source of TPH2 in the absence of the candidate compound;
[0284] (d) exposing the source of TPH2 to L-tryptophan in the
presence of the candidate compound;
[0285] (e) measuring the amount of 5-hydroxytryptophan produced by
the source of TPH2 in the presence of the candidate compound;
[0286] (f) where, if the amount of 5-hydroxytryptophan produced by
the source of TPH2 in the presence of the candidate compound is
greater than the amount of 5-hydroxytryptophan produced by the
source of TPH2 in the absence of the candidate compound, the
candidate compound is a TPH2 activator.
[0287] "Greater than" for the purpose of the herein-described
methods of identifying therapeutic agents from a collection of
candidate compounds refers to an amount that would not be
attributed by those of skill in the art to normal variation seen in
the method. Preferably, "greater than" is at least about 50%, at
least about 75%, at least about 100%, at least about 250%, or at
least about 500% more than the amount observed in the absence of
the candidate compound.
[0288] In certain embodiments, the method described above includes
the further step of administering the TPH2 activator identified in
step (f) to a patient I need of therapy for a low bone mass
disease.
[0289] In certain embodiments, the source of TPH2 is an isolated
TPH2 enzyme, preferably human. Isolated TPH2 can be produced by in
vitro expression of TPH1, e.g., in a coupled in vitro
transcription/translation system. Alternatively, the source of TPH2
may be partially or highly purified preparations from cells
expressing TPH2. In other embodiments, the source of TPH2 is a
whole cell expressing TPH2, preferably human. In some embodiments,
the whole cell has been transfected with a expression vector
comprising TPH2 so that the cell expresses recombinant TPH2,
preferably human.
[0290] The mRNA and amino acid sequence of human TPH2 can be found
in GenBank, at accession no. AY098914. The genomic sequence can be
found at AC090109. These nucleotide sequences can be used in
methods well-known in the art to construct suitable expression
vectors for expressing TPH2 recombinantly in cells, or in
vitro.
[0291] Antagonists of a serotonin receptor may be identified by a
method comprising:
[0292] (a) providing a cell expressing the serotonin receptor;
[0293] (b) exposing the cell expressing the serotonin receptor to
serotonin or a serotonin analogue in the absence of a candidate
compound;
[0294] (c) measuring the activation of the serotonin receptor in
the absence of the candidate compound;
[0295] (d) exposing the cell expressing the serotonin receptor to
serotonin or a serotonin analogue in the presence of a candidate
compound;
[0296] (e) measuring the activation of the serotonin receptor in
the presence of the candidate compound;
[0297] (f) where, if the amount of activation of the serotonin
receptor in the presence of the candidate compound is less than the
amount of activation of the serotonin receptor in the absence of
the candidate compound, the candidate compound is a serotonin
receptor antagonist.
[0298] Antagonists of a serotonin receptor may also be identified
by a method comprising:
[0299] (a) providing a cell expressing the serotonin receptor;
[0300] (b) exposing the cell expressing the serotonin receptor to
serotonin or a serotonin analogue in the absence of a candidate
compound;
[0301] (c) measuring the binding of the serotonin or the serotonin
analogue to the serotonin receptor in the absence of the candidate
compound;
[0302] (d) exposing the cell expressing the serotonin receptor to
serotonin or a serotonin analogue in the presence of a candidate
compound;
[0303] (e) measuring the binding of the serotonin or the serotonin
analogue to the serotonin receptor in the presence of the candidate
compound;
[0304] (f) where, if the binding of the serotonin or the serotonin
analogue to the serotonin receptor in the presence of the candidate
compound is less than the binding of the serotonin or the serotonin
analogue to the serotonin receptor in the absence of the candidate
compound, the candidate compound is a serotonin receptor
antagonist.
[0305] By "serotonin analogue" is meant a substance that binds to a
serotonin receptor with binding characteristics similar to those of
serotonin and/or activates a serotonin receptor in a manner similar
to that of serotonin.
[0306] In certain embodiments, the present invention provides a
method of lowering serum serotonin levels in a patient known or
suspected to be in need of lowering of serum serotonin levels
comprising:
[0307] (a) providing a plurality of candidate compounds;
[0308] (b) determining that one of the plurality of candidate
compounds is an inhibitor of TPH1;
[0309] (c) administering to the patient known or suspected to be in
need of lowering of serum serotonin levels a therapeutically
effective amount of the candidate compound determined to be a TPH1
inhibitor in step (b).
[0310] In certain embodiments, the present invention provides a
method of lowering serum serotonin levels in a patient known or
suspected to be in need of lowering of serum serotonin levels
comprising:
[0311] (a) providing a plurality of candidate compounds;
[0312] (b) determining that one of the plurality of candidate
compounds is a serotonin receptor antagonist;
[0313] (c) administering to the patient known or suspected to be in
need of lowering of serum serotonin levels a therapeutically
effective amount of the candidate compound determined to be a
serotonin receptor antagonist in step (b).
[0314] In the present specification, the invention has been
described with reference to specific embodiments thereof. It will,
however, be evident that various modifications and changes may be
made thereto without departing from the broader spirit and scope of
the invention. The specification and drawings are, accordingly, to
be regarded in an illustrative rather than a restrictive sense.
EXAMPLES
Example 1
Assessment of Effect of Catechol-3,6-Bis Methyleneiminodiacetic
Acid (CBMIDA) on Peripheral Serotonin Production in Mice
Animals
[0315] One month old C57Bl/6 inbred male mice, weighing 15-16 g
were used in the experiments. Animals were housed under 12 h
light/12 h dark conditions in a room with controlled temperature
(22.degree. C.) and humidity (60%). Mice had ad libitum access to
food and water, and were used after a minimum of 4 days of
acclimatization to the housing conditions. All experiments were
conducted following Columbia University Guidelines for the Animal
Use and Care of laboratory mice.
Experimental Protocol
[0316] Before the experiments, animals were separated into
individual cages one day prior to the experiment. Compounds were
fed orally (gavage) to the mouse, calculated according to the
weight of the mouse, twice a day at 1700 h and at 1100 h. Oral
feeding was selected over intravenous or intraperitoneal infusion
of the compound for better inhibition of Tryptophan hydroxylase-1
(TPH1) present in the gut vs TPH2 that synthesizes serotonin and is
present in the brain. This route created two potential barriers for
the compound to reach the brain. First, the intestinal blood
barrier that has poor permeability to the EDTA-based compounds (as
is the case with CBMIDA), hence does not allow all the amount given
orally to be absorbed in the circulation (only 5-10% is transported
to blood). The second barrier is the blood-brain barrier that shows
poor permeability to a large number of compounds including EDTA
compounds. Control animals received the same volume of vehicle.
Blood was collected through heart puncture on
isofluorane-anaesthesized animals and allowed to clot for 5 minutes
on ice. The serum was separated, snap chilled in liquid nitrogen
and frozen at -80.degree. C. till analyzed. Brainstems from all the
animals were collected and processed for brain serotonin
measurement through HPLC. Mice were observed for any physical or
behavioral abnormality during the course of investigation.
Serotonin Measurements in Serum
[0317] The Serotonin ELISA kit obtained from the Fitzgerald company
was used to measure derivatized serotonin from serum.
Derivatization is part of the sample preparation. Serotonin present
in the serum was first quantitatively acylated into N-Acylserotonin
using the acylation reagent. The principle of the assay is based on
competitive ELISA, wherein serotonin that is bound to the solid
phase of the plate and the N-acylserotonin compete for the fixed
number of antiserum binding sites. When the reaction is in
equilibrium, free antigen and free antigen-antiserum complexes are
removed by washing.
[0318] The antibody bound to the solid phase serotonin is then
detected using antirabbit/peroxidase. The substrate TMB/Peroxidase
reaction is read at 450 nm. The amount of antibody bound to the
solid phase serotonin is inversely proportional to the
concentration of serotonin in the sample.
Drugs Used in the Study
[0319] Catechol-3,6-bis methyleneiminodiacetic acid (CBMIDA) (basic
structure is an EDTA-like compound and the catechol ring is at the
centre) synthesized at the Columbia University Chemistry division,
and para-chlorophenylalanine (pCPA) obtained from Sigma Aldrich
Corp. were used. Each compound was dissolved with twice molar
solution of NaHCO.sub.3 in water and given to mouse orally at 250
and 500 mg/kg/dose.
Results
[0320] As can be seen in FIG. 11, oral administration of CBMIDA
decreased serotonin serum levels to 80% below normal at a dose of
500 mg/kg twice daily. Lowering this dose to 250 mg/kg produced the
effect but to a lesser extent. In fact when one compares the two
doses versus control animals, a dose response curve is produced.
While pCPA, a well known inhibitor of tryptophan hydroxylase used
as a control, decreased the serum serotonin levels as expected to
>60% below normal range.
Example 2
Synthesis of catechol TPH1 inhibitors
Synthesis of
2,2',2'',2'''-(2,3-dihydroxy-1,4-phenylene)bis(methylene)bis(azanetriyl)t-
etraacetic acid
##STR00032##
[0322] Catechol (5.5 g, 0.05 mol) and iminodiacetic acid (11.3 g,
0.1 mol) were suspended in a mixture of acetic acid (20 ml) and
water (40 ml). Formaldehyde (37%, 10 ml) was added slowly. The
reaction was stirred at 60.degree. C. for 1 hour. The solution was
cooled and white precipitate was filtered, washed with water and
ethanol, dried. 15.0 g of product was obtained (yield: 75%).
Synthesis of
2,2'-(2,3-dihydroxy-1,4-phenylene)bis(methylene)bis((2-amino-2-oxoethyl)a-
zanediyl)diacetic acid
##STR00033##
[0324]
2,2',2'',2'''-(2,3-dihydroxy-1,4-phenylene)bis(methylene)bis(azanet-
riyl)tetraacetic acid (5 g, 12.5 mmol) was added to acetyl
anhydride (10 ml) followed by pyridine (3 ml). The reaction was
heated at 70.degree. C. for 5 h. The excess anhydride and pyridine
were removed under reduced pressure and the residue was
recrystallized in acetyl anhydride. 2.8 g of
3,6-bis((2,6-dioxomorpholino)methyl)-1,2-phenylene diacetate was
obtained as white solid (yield: 51%).
[0325] 3,6-bis((2,6-dioxomorpholino)methyl)-1,2-phenylene diacetate
(2.24 g, 10 mmol) was added to the solution of ammonia in methanol
(7N, 20 ml). The reaction was stirred at room temperature for 1
hour. After removing methanol, the residue was recrystallized in
methanol and acetone. 3.5 g of product was obtained as white solid
(yield: 88%).
Synthesis of
2,2'-((11,12-dihydroxy-6-methyl-4,5,6,8-tetrahydropyrido[3,2,1-de]phenant-
hridin-10-yl)methylazanediyl)diacetic acid
##STR00034##
[0327] Catechol (5.5 g, 0.05 mol) and iminodiacetic acid (11.3 g,
0.1 mol) were suspended in the mixture of acetic acid (20 ml) and
water (40 ml). Formaldehyde (37%, 10 ml) was added slowly. The
reaction was stirred at 60.degree. C. for 1 hour. The solution was
cooled and the white precipitate was filtered, washed with water
and ethanol, and dried. 15.0 g of product was obtained (yield:
75%).
[0328]
6-methyl-4,5,6,8-tetrahydropyrido[3,2,1-de]phenanthridine-11,12-dio-
l (1.33 g, 5 mmol) and iminodiacetic acid (1.13 g, 10 mmol) were
suspended in a mixture of acetic acid (2 ml) and water (4 ml).
Formaldehyde (37%, 1 ml) was added slowly. The reaction was stirred
at 60.degree. C. for 3 hours. The solvent was removed under reduced
pressure and the residue was recrystallized in methanol and water.
1.8 g of product was obtained as a white solid (yield: 87%).
Example 3
Measurement of Serum Serotonin
[0329] Two possible methods of measuring serum serotonin levels are
as follows:
[0330] (1) Initial steps are performed at room temperature using
polypropylene tubes and pipettes. Establishing free flow by
venipuncture, blood is collected from an antecubital vein with a
19-gauge, thin-walled butterfly needle into EDTA-containing vacuum
tubes. The tubes are centrifuged (Sorvall GLC-2B) at 800 rpm
(100.times.g) for 15 minutes at room temperature. The upper layer
of platelet-rich plasma (PRP), about 0.3 cm from the interface
layer (buffy coat), is removed with a plastic pipette and
transferred to a new polypropylene test tube. The tube containing
the platelet-poor plasma (PRP) is iced for 10 min before being
centrifuged at 11,000 rpm (14,500.times.g) in a Sorvall SS-34 rotor
for 6 min at 4.degree. C. to yield the platelet pellet and platelet
poor plasma (PPP). The supernate containing PPP is removed and
placed into a new polypropylene test tube in a volume of 500
microliters in Eppendorf tubes. The platelet-rich pellets are
resuspended in 1 ml saline. Mixing or vortexing, before transfer to
an Eppendorf tube, is sometimes required to maintain a homogenous
suspension without clumps. The aliquoted plasma supernate (PPP) and
the resuspended pellet (PRP) are kept at -20. For the serotonin
assay, the samples are resuspended in saline. The `hormonal`
element of serotonin that is of most interest is the circulating
level in PPP but the PRP fraction will also be measured. The method
is an ELISA obtained from Fitzgerald Industries International
(Concord, Mass.). It measures the derivatized serotonin from serum
or plasma samples or urine samples. Derivatization is part of the
sample preparation. Serotonin present in the biological fluids
(e.g., serum) is first quantitatively acylated using the acylation
reagent into N-acylserotonin. The assay is based on the competitive
ELISA principle wherein serotonin that is bound to the solid phase
of the plate and the N-acylserotonin competes for the fixed number
of antiserum binding sites. When the reaction is in equilibrium,
free antigen and free antigen-antiserum complexes are removed by
washing. The antibody bound to the solid phase serotonin is then
detected by the anti-rabbit/peroxidase. The substrate
TMB/Peroxidase reaction is read at 450 nm. The amount of antibody
bound to the solid phase serotonin is inversely proportional to the
concentration of serotonin in the sample. Although the ELISA assay
is useful, we will have the opportunity to apply an even more
precise assay namely HPLC coupled with electrochemical
detection.
[0331] (2) Another method relies on HPLC coupled with
electrochemical detection. Samples obtained in the manner described
above are precipitated with 1N HClO.sub.4 (1:1), diluted and
aliquoted into HPLC vials containing 32.5 .mu.l of 0.02 M acetic
acid. The fractions are injected via a Gilson 223 XL autoinjector
onto the column. 20 .mu.l of the microdialysis sample are injected
onto a 100.times.2 mm C18 Hypersil 3 .mu.m column and separated
with a mobile phase consisting of 4.1 g/l sodium acetate, 500 mg/l
Na2-EDTA, 50 mg/l heptane sulfonic acid, 4.5% methanol v/v, and 30
.mu.l/l of triethylamine, pH 4.75 at a flow rate of 0.4 ml/min
using a Shimadzu LC-10 AD pump. Serotonin is detected
amperometrically at a glassy carbon electrode at 500 mV vs Ag/AgCl.
The detection limit, 0.5 fmol serotonin per 20 .mu.l sample or 10
pM, is well within the circulating concentrations of serotonin.
Since serotonin measured in PPP is not bound to any appreciable
degree by plasma proteins, these measurements can be regarded as
equivalent to free serotonin levels.
Example 4
Synthesis Schemes for Additional TPH1 Inhibitors
[0332] TPH1 inhibitors having the following structures:
##STR00035##
can be synthesized by the following methods: for n=1,
##STR00036##
for n=2,
##STR00037##
for n=3,
##STR00038##
[0333] TPH1 inhibitors having the following structures:
##STR00039##
can be synthesized by the following methods: for n=1,
##STR00040##
for n=2,
##STR00041##
for n=3,
##STR00042##
Example 5
Generation of Mutant Animals and Animal Treatments
[0334] Generation of Lrp5-/- (Kato et al., 2002, J. Cell Biol.
157:303-314) 0-cateninfloxed/floxed (Glass et al., 2005, Dev. Cell
8:751-764), .alpha.1(I)collagen-cre transgenic (Dacquin et al.,
2002, Dev. Dyn. 224:245-251) and Htt-/- (Ansorge et al., 2008, J.
Neurosci. 28:199-207) mice were as described previously. Lrp5+/-;
Htt+/- double heterozygous mice were generated by crossing Lrp5+/-
and Htt+/- mice. Three week-old Wt or Lrp5-/- mice were
administered pCPA on alternate days for 9 weeks by i.p. All animal
protocols were approved by the Animal Care Committees of Columbia
University.
Example 6
Morphometric Measurements
[0335] Static histomorphometry measurements were performed as
previously described in accordance with standard nomenclature,
using the Osteomeasure Analysis System (Osteometrics, Inc) (Ducy et
al., 2000, Cell 100:197-207). Four to 9 animals were assigned per
group.
Example 7
Cell Cultures
[0336] Calvaria osteoblasts were extracted by triple
collagenase/trypsine digestion from 4 day-old CD1 pups and
differentiated with ascorbic acid as previously described (Ducy et
al., 2000, Cell 100:197-207).
Example 8
Gene Expression Studies
[0337] Osteoblasts were treated in serum-free medium with vehicle
or Serotonin (50 to 100 .mu.M, Sigma) for 24 hr. Total RNA were
extracted with Trizol (Invitrogen). cDNA were generated using the
ABI Reverse transcriptase system and random hexanucleotide primers.
Real-time PCR was performed using superarray primers on a
Stratagene real time PCR cycler and Actin expression was used as
endogenous control. Chromatin immunoprecipitation assays (ChIP)
were performed by standard procedures using primary osteoblasts.
Microarray analysis was performed as described previously (Glass et
al., 2005, Dev. Cell 8:751-764).
Example 9
Biochemical Studies
[0338] Osteoblasts were treated in serum-free medium with vehicle
or Serotonin (50 to 100 .mu.M, Sigma) for 24 hr. Lysates from
primary osteoblasts or crushed frozen bones were prepared in RIPA
buffer in the presence of protease and phosphatase inhibitors.
Twenty to 60 .mu.g of proteins were separated by SDS-PAGE in
reducing conditions and transferred on nitrocellulose membrane
using standard protocols. Membranes were incubated with primary
antibodies including total or anti-Phospho CREB (Cell Signaling
Technology).
Example 10
Hormone Measurements
[0339] Serotonin serum levels were quantified using immunoassay
kits from Fitzgerald (Serotonin) and serotonin levels in the
different regions of the brain were quantified by HPLC method as
described previously (Mann et al., 1992, Arch. Gen. Psychiatry
49:442-446).
Example 11
Changes in Serotonin Levels Upon Oral Deeding of CBMIDA and Other
Compounds
[0340] Serotonin serum levels relative to vehicle were measured
following the oral feeding to 4 week old mice of the following
compounds:
##STR00043##
[0341] The feeding protocol is shown in FIG. 13. Results are shown
in FIG. 14. Experimental details were similar to those of Example
12, below.
Example 12
Assessment of Effect of Novel Inhibitors of Tryptophan Hydroxylase
in Protection from Ovariectomy-Induced Bone Loss
Animals
[0342] 6-week old C57Bl/6 inbred female mice, weighing 12-14 g,
were used in the experiments. Animals were housed under 12 h
light/12 h dark conditions in a room with controlled temperature
(22.degree. C.) and humidity (60%). Mice had ad libitum access to
food and water, and were used after a minimum of 4 days of
acclimatization to the housing conditions. All experiments were
conducted following Columbia University Guidelines for the Animal
Use and Care of laboratory mice.
Experimental Protocol
[0343] Animals were separated into different groups one day prior
to the experiments. Mice were ovariectomized (OVX) under
anaesthesia (Avertin). Compounds were fed orally (Gavage) twice a
day at 1700 h and at 1100 h for the pilot study while for the long
term study compounds were given at 1700 h once a day. Oral feeding
was selected over intravenous or intraperitoneal infusion of the
compound for better inhibition of Tryptophan hydroxylase-1 (Tph1)
present in the gut and to avoid affecting Tph2 function in the
brain. This route creates two potential barriers for the compound
to reach the brain. First, the intestinal blood barrier that has
poor permeability to the EDTA-based compounds (as is the case with
Compound 1), hence does not allow all the amount given orally to be
absorbed (only 5-10% is transported to blood) in the general
circulation; second, the blood-brain barrier itself shows poor
permeability to a large number of compounds including EDTA
compounds. Control animals received the same volume of vehicle.
Blood was collected through heart puncture on
isofluorane-anaesthesized animals, allowed to clot for 5 minutes on
ice and then serum was separated, snap chilled in liquid nitrogen
and frozen at -80.degree. C. till analyzed. Mice were observed
daily for any physical or behavioral abnormality during the course
of investigation.
Experiment I
Pilot Study to Determine Efficacy of the Compounds to be Tested
Group 1: Vehicle
Group 2: Compound 1
Group 3: Compound 2
Group 4: Compound 3
Group 5: Compound 4 (LP-533401)
Experiment II
Effect of Compounds 1 and 4 on Protection from OVX-Induced Bone
Loss
[0344] Protocol 1: Gavage Feeding of the Compounds Will be Started
1 Day after OVX for 6 Weeks Group 1: Sham treatment
Group 2: OVX
[0345] Group 3: OVX+Compound 1 (250 mg/kg) Group 4: OVX+Compound 1
(500 mg/kg) Group 5: OVX+Compound 4 (250 mg/kg) Protocol 2: Gavage
Feeding of the Compounds Will be Started 2 Weeks after OVX for 6
Weeks Group 1: Sham treatment
Group 2: OVX
[0346] Group 3: OVX+Compound 1 (250 mg/kg) Group 4: OVX+Compound 1
(500 mg/kg) Group 5: OVX+Compound 4 (250 mg/kg) Protocol 3: Gavage
Feeding of the Compounds Will be Started 4 Weeks after OVX for 6
Weeks Group 1: Sham treatment
Group 2: OVX
[0347] Group 3: OVX+Compound 1 (250 mg/kg) Group 4: OVX+Compound 1
(500 mg/kg) Group 2: OVX+Compound 4 (250 mg/kg)
Serotonin Measurement in Serum
[0348] The Serotonin ELISA kit obtained from the Fitzgerald company
was used to measure derivatized serotonin from serum.
Derivatization is part of the sample preparation. Serotonin present
in the serum was first quantitatively acylated using the acylation
reagent into N-Acylserotonin. The principle of the assay is based
on competitive ELISA, wherein serotonin that is bound to the solid
phase of the plate and the N-acylserotonin compete for the fixed
number of antiserum binding sites. When the reaction is in
equilibrium, free antigen and free antigen-antiserum complexes are
removed by washing. The antibody bound to the solid phase serotonin
is then detected by anti-rabbit/peroxidase. The substrate
TMB/Peroxidase reaction is read at 450 nm. The amount of antibody
bound to the solid phase serotonin is inversely proportional to the
concentration of serotonin in the sample.
Drugs Used in the Study
[0349] We used Catechol-3,6-bis methyleneiminodiacetic acid
[(CBMIDA) Compound 1: basic structure is a EDTA-like compound and
the catechol ring is at the center)] synthesized at the Columbia
University Chemistry division and para-Chlorophenylalanine (pCPA)
obtained from Sigma Aldrich Corp. for our experiments. Compounds
were dissolved with twice molar solution of NaHCO.sub.3 in water
and given to mice orally at 250 and 500 mg/kg/dose. Compounds 2 and
3 were dissolved in water.
Results
[0350] We tested the effect of the four compounds mentioned above
for their effect on peripheral serotonin production in mice. As
seen in FIG. 15, oral administration of Compound 1 (CBMIDA)
decreased serotonin serum levels to 80% below normal at a dose of
500 mg/kg twice daily. Lowering this dose to 250 mg/kg produced the
effect but to a lesser extent while Compound 2 had minimal effect.
Although Compound 3 decreased the serotonin levels dramatically, it
was toxic to the animals, as they looked very lethargic and
sick.
[0351] We also tested the effect of different doses of Compound 1
on serotonin levels. As can be seen in FIG. 15, Compound 1 produced
a dose response curve when compared to the control animals. While
pCPA, a well known inhibitor of Tryptophan hydroxylase used as a
control, decreased serotonin levels as expected to 60% below normal
range.
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