U.S. patent application number 12/765414 was filed with the patent office on 2010-08-19 for methods of using a g protein-coupled receptor to identify peptide yy (pyy) secretagogues and compounds useful in the treatment of conditions modulated by pyy.
This patent application is currently assigned to ARENA PHARMACEUTICALS, INC.. Invention is credited to Zhi-Liang Chu, Robert M. Jones, James N. Leonard.
Application Number | 20100210666 12/765414 |
Document ID | / |
Family ID | 40691322 |
Filed Date | 2010-08-19 |
United States Patent
Application |
20100210666 |
Kind Code |
A1 |
Chu; Zhi-Liang ; et
al. |
August 19, 2010 |
METHODS OF USING A G PROTEIN-COUPLED RECEPTOR TO IDENTIFY PEPTIDE
YY (PYY) SECRETAGOGUES AND COMPOUNDS USEFUL IN THE TREATMENT OF
CONDITIONS MODULATED BY PYY
Abstract
The present invention relates to methods of using GPR119
receptor to identify peptide YY (PYY) secretagogues and compounds
useful in the treatment of conditions modulated by PYY, such as
conditions modulated by stimulation of NPY Y2 receptor (Y2R).
Agonists of GPR119 receptor are useful as therapeutic agents for
treating or preventing a condition modulated by PYY, such as a
condition modulated by stimulation of Y2R. Conditions modulated by
PYY such as may be a condition modulated by stimulation of Y2R
include bone-related conditions, metabolic disorders,
angiogenesis-related conditions, ischemia-related conditions,
convulsive disorders, malabsorptive disorders, cancers, and
inflammatory disorders.
Inventors: |
Chu; Zhi-Liang; (San Diego,
CA) ; Leonard; James N.; (San Diego, CA) ;
Jones; Robert M.; (San Diego, CA) |
Correspondence
Address: |
FISH & RICHARDSON P.C.
P.O. BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Assignee: |
ARENA PHARMACEUTICALS, INC.
San Diego
CA
|
Family ID: |
40691322 |
Appl. No.: |
12/765414 |
Filed: |
April 22, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12391097 |
Feb 23, 2009 |
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12765414 |
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61123273 |
Apr 7, 2008 |
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Current U.S.
Class: |
514/256 ;
435/29 |
Current CPC
Class: |
G01N 2333/726 20130101;
A61P 13/00 20180101; A61P 43/00 20180101; G01N 2800/044 20130101;
A61P 19/08 20180101; A61P 3/04 20180101; A61P 3/10 20180101; A61P
9/10 20180101; A61P 19/10 20180101; G01N 2800/323 20130101; G01N
33/5044 20130101; G01N 2800/042 20130101; A61P 9/00 20180101; G01N
2800/065 20130101; A61P 35/00 20180101; G01N 33/68 20130101; A61P
1/04 20180101; G01N 2800/108 20130101; A61P 29/00 20180101; A61P
25/08 20180101; A61P 3/00 20180101; A61P 1/12 20180101 |
Class at
Publication: |
514/256 ;
435/29 |
International
Class: |
A61K 31/506 20060101
A61K031/506; C12Q 1/02 20060101 C12Q001/02; A61P 9/00 20060101
A61P009/00; A61P 35/00 20060101 A61P035/00; A61P 29/00 20060101
A61P029/00; A61P 3/00 20060101 A61P003/00 |
Claims
1.-109. (canceled)
110. A method of identifying a peptide YY (PYY) secretagogue,
comprising: (a) contacting a G protein coupled receptor 119
(GPR119) agonist in vitro with a vertebrate enteroendocrine cell or
with a cell capable of secreting PYY; and (b) determining whether
the GPR119 agonist stimulates PYY secretion from the vertebrate
enteroendocrine cell or the cell capable of secreting PYY, wherein
the ability of the GPR119 agonist to stimulate PYY secretion from
the vertebrate enteroendocrine cell or the cell capable of
secreting PYY is indicative of the agonist being a PYY
secretagogue.
111. A method of identifying a PYY secretagogue, comprising: (a)
administering a GPR119 agonist to a vertebrate; and (b) determining
whether the GPR119 agonist increases a PYY level in the vertebrate,
wherein the ability of the GPR119 agonist to increase a PYY level
in the vertebrate is indicative of the agonist being a PYY
secretagogue.
112. A method of identifying a PYY secretagogue, comprising
determining whether a GPR119 agonist increases a PYY level in a
sample, the sample having been obtained from a vertebrate
previously administered with the GPR119 agonist, wherein the
ability of the GPR119 agonist to increase a PYY level in the sample
is indicative of the agonist being a PYY secretagogue.
113. A method for identifying a PYY secretagogue, comprising: (a)
contacting a test compound with a host cell or with a membrane of a
host cell that expresses a G protein-coupled receptor (GPCR),
wherein the GPCR comprises an amino acid sequence selected from the
group consisting of: (i) amino acids 1-335 of SEQ ID NO:2; (ii)
amino acids 2-335 of SEQ ID NO:2; (iii) an amino acid sequence of a
GPCR encoded by a polynucleotide capable of hybridizing under
stringent conditions to the complement of SEQ ID NO:1; (iv) an
amino acid sequence of a GPCR having at least about 80% identity to
SEQ ID NO:2; (v) an amino acid sequence of a constitutively active
version of a GPCR having SEQ ID NO:2; and (vi) a biologically
active fragment of any one of (i) to (v); (b) determining the
ability of the test compound to stimulate functionality of the
GPCR; (c) contacting the test compound that stimulates
functionality of the GPCR in step (b) in vitro with a vertebrate
enteroendocrine cell or with a cell capable of secreting PYY; and
(d) determining whether the test compound stimulates PYY secretion
from the vertebrate enteroendocrine cell or the cell capable of
secreting PYY, wherein the ability of the test compound to
stimulate PYY secretion from the vertebrate enteroendocrine cell or
the cell capable of secreting PYY identifies the test compound as
the PYY secretagogue.
114. A method for identifying a PYY secretagogue, comprising: (a)
contacting a GPCR with a ligand to the GPCR in the presence of a
test compound, wherein the GPCR comprises an amino acid sequence
selected from the group consisting of: (i) amino acids 1-335 of SEQ
ID NO:2; (ii) amino acids 2-335 of SEQ ID NO:2; (iii) an amino acid
sequence of a GPCR encoded by a polynucleotide capable of
hybridizing under stringent conditions to the complement of SEQ ID
NO:1; (iv) an amino acid sequence of a GPCR having at least about
80% identity to SEQ ID NO:2; (v) an amino acid sequence of a
constitutively active version of a GPCR having SEQ ID NO:2; and
(vi) a biologically active fragment of any one of (i) to (v); (b)
detecting a complex between the GPCR and the ligand; (c)
determining whether less of the complex is formed in the presence
of the test compound than in the absence of the test compound; (d)
contacting the test compound in the presence of which less of the
complex is formed in step (c) in vitro with a vertebrate
enteroendocrine cell or with a cell capable of secreting PYY; and
(e) determining whether the test compound stimulates PYY secretion
from the vertebrate enteroendocrine cell or the cell capable of
secreting PYY, wherein the ability of the test compound to
stimulate PYY secretion from the vertebrate enteroendocrine cell or
the cell capable of secreting PYY identifies the test compound as a
PYY secretagogue.
115. A method for identifying a PYY secretagogue, comprising: (a)
contacting a test compound with a host cell or with a membrane of a
host cell that expresses a GPCR, wherein the GPCR comprises an
amino acid sequence selected from the group consisting of: (i)
amino acids 1-335 of SEQ ID NO:2; (ii) amino acids 2-335 of SEQ ID
NO:2; (iii) an amino acid sequence of a GPCR encoded by a
polynucleotide capable of hybridizing under stringent conditions to
the complement of SEQ ID NO:1; (iv) an amino acid sequence of a
GPCR having at least about 80% identity to SEQ ID NO:2; (v) an
amino acid sequence of a constitutively active version of a GPCR
having SEQ ID NO:2; and (vi) a biologically active fragment of any
one of (i) to (v); (b) determining the ability of the test compound
to stimulate functionality of the GPCR; (c) administering the test
compound that stimulates functionality of the GPCR in step (b) to a
vertebrate; and (d) determining whether the test compound increases
a PYY level in the vertebrate; wherein the ability of the test
compound to increase a PYY level in the vertebrate identifies the
test compound as a PYY secretagogue.
116. A method for identifying a PYY secretagogue, comprising: (a)
contacting a GPCR with a ligand to the GPCR in the presence of a
test compound, wherein the GPCR comprises an amino acid sequence
selected from the group consisting of: (i) amino acids 1-335 of SEQ
ID NO:2; (ii) amino acids 2-335 of SEQ ID NO:2; (iii) an amino acid
sequence of a GPCR encoded by a polynucleotide capable of
hybridizing under stringent conditions to the complement of SEQ ID
NO:1; (iv) an amino acid sequence of a GPCR having at least about
80% identity to SEQ ID NO:2; (v) an amino acid sequence of a
constitutively active version of a GPCR having SEQ ID NO:2; and
(vi) a biologically active fragment of any one of (i) to (v); (b)
detecting a complex between the GPCR and the ligand; (c)
determining whether less of the complex is formed in the presence
of the test compound than in the absence of the test compound; (d)
administering the test compound in the presence of which less of
the complex is formed in step (c) to a vertebrate; and (d)
determining whether the test compound increases a PYY level in the
vertebrate; wherein the ability of the test compound to increase a
PYY level in the vertebrate identifies the test compound as a PYY
secretagogue.
117. A method for identifying a PYY secretagogue, comprising: (a)
contacting a test compound with a host cell or with a membrane of a
host cell comprising a GPCR wherein the GPCR comprises an amino
acid sequence selected from the group consisting of: (i) amino
acids 1-335 of SEQ ID NO:2; (ii) amino acids 2-335 of SEQ ID NO:2;
(iii) an amino acid sequence of a GPCR encoded by a polynucleotide
capable of hybridizing under stringent conditions to the complement
of SEQ ID NO:1; (iv) an amino acid sequence of a GPCR having at
least about 80% identity to SEQ ID NO:2; (v) an amino acid sequence
of a constitutively active version of a GPCR having SEQ ID NO:2;
and (vi) a biologically active fragment of any one of (i) to (v);
(b) determining the ability of the test compound to stimulate
functionality of the GPCR; (c) determining whether the compound
that stimulates functionality of the GPCR in step (b) increases a
PYY level in a sample, the sample having been obtained from a
vertebrate previously administered with a compound which stimulates
functionality of the GPCR in step (b), wherein the ability of the
test compound to increase a PYY level in the vertebrate is
indicative of the test compound being a PYY secretagogue.
118. A method for identifying a PYY secretagogue, comprising: (a)
contacting a GPCR with a ligand to the GPCR in the presence of a
test compound, wherein the GPCR comprises an amino acid sequence
selected from the group consisting of: (i) amino acids 1-335 of SEQ
ID NO:2; (ii) amino acids 2-335 of SEQ ID NO:2; (iii) an amino acid
sequence of a GPCR encoded by a polynucleotide capable of
hybridizing under stringent conditions to the complement of SEQ ID
NO:1; (iv) an amino acid sequence of a GPCR having at least about
80% identity to SEQ ID NO:2; (v) an amino acid sequence of a
constitutively active version of a GPCR having SEQ ID NO:2; and
(vi) a biologically active fragment of any one of (i) to (v); (b)
detecting a complex between the GPCR and the ligand; (c)
determining whether less of the complex is formed in the presence
of the test compound than in the absence of the test compound; (d)
determining a PYY level in a sample obtained from a vertebrate, the
vertebrate having been administered with the test compound in the
presence of which less of the complex is formed in step (c),
wherein the ability of the test compound to increase a PYY level in
the sample obtained from the vertebrate identifies the test
compound as a PYY secretagogue.
119. A method for identifying compounds useful for treatment of a
condition modulated by PYY or compounds useful for a modulation of
a PYY-related activity in an individual, comprising: (a) contacting
a GPR119 agonist in vitro with a vertebrate enteroendocrine cell or
a cell capable of secreting PYY; and (b) determining whether the
GPR119 agonist stimulates PYY secretion from the vertebrate
enteroendocrine cell or the cell capable of secreting PYY; wherein
the ability of the GPR119 agonist to stimulate PYY secretion from
the vertebrate enteroendocrine cell or cell capable of secreting
PYY is indicative of the GPR119 agonist being a compound useful for
treatment or prevention of a condition modulated by PYY or a
compound useful for modulation of a PYY-related activity in an
individual.
120. A method for identifying compounds useful for treatment of a
condition modulated by PYY or compounds useful for a modulation of
a PYY-related activity in an individual, comprising: (a)
determining whether a GPR119 agonist increases a PYY level in a
vertebrate, wherein the ability of the GPR119 agonist to increase a
PYY level in the vertebrate is indicative of the GPR119 agonist
being a compound useful for treatment or prevention of a condition
modulated by PYY or a compound useful for a modulation of a
PYY-related activity in an individual.
121. A method for identifying compounds useful for treatment of a
condition modulated by PYY or compounds useful for a modulation of
a PYY-related activity in an individual, comprising: (a)
determining whether the compound increases a PYY level in a sample,
the sample having been obtained from a vertebrate previously
administered with a GPR119 agonist; wherein the ability of the
GPR119 agonist to increase a PYY level in the sample is indicative
of the GPR119 agonist being a compound useful for treatment or
prevention of a condition modulated by PYY or a compound useful for
a modulation of a PYY-related activity in an individual.
122. A method for identifying compounds useful for treatment of a
condition modulated by PYY or compounds useful for a modulation of
a PYY-related activity in an individual, comprising: (a) contacting
a test compound with a host cell or with a membrane of a host cell
comprising a GPCR, wherein the GPCR comprises an amino acid
sequence selected from the group consisting of: (i) amino acids
1-335 of SEQ ID NO:2; (ii) amino acids 2-335 of SEQ ID NO:2; (iii)
an amino acid sequence of a GPCR encoded by a polynucleotide
capable of hybridizing under stringent conditions to the complement
of SEQ ID NO:1; (iv) an amino acid sequence of a GPCR having at
least about 80% identity to SEQ ID NO:2; (v) an amino acid sequence
of a constitutively active version of a GPCR having SEQ ID NO:2;
and (vi) a biologically active fragment of any one of (i) to (v);
(b) determining the ability of the test compound to stimulate
functionality of the GPCR; (c) contacting a compound which
stimulates functionality of the GPCR in step (b) in vitro with a
vertebrate enteroendocrine cell or a cell capable of secreting PYY;
and (d) determining whether the compound stimulates PYY secretion
from the vertebrate enteroendocrine cell or the cell capable of
secreting PYY; wherein the ability of the test compound to
stimulate PYY secretion from the vertebrate enteroendocrine cell or
cell capable of secreting PYY is further indicative of the test
compound being a compound useful for treatment or prevention of a
condition modulated by PYY or a compound useful for a modulation of
a PYY-related activity in an individual.
123. A method for identifying compounds useful for treatment of a
condition modulated by PYY or compounds useful for a modulation of
a PYY-related activity in an individual, comprising the steps of:
(a) contacting a test compound with a host cell or with a membrane
of a host cell comprising a GPCR, wherein the GPCR comprises an
amino acid sequence selected from the group consisting of: (i)
amino acids 1-335 of SEQ ID NO:2; (ii) amino acids 2-335 of SEQ ID
NO:2; (iii) an amino acid sequence of a GPCR encoded by a
polynucleotide capable of hybridizing under stringent conditions to
the complement of SEQ ID NO:1; (iv) an amino acid sequence of a
GPCR having at least about 80% identity to SEQ ID NO:2; (v) an
amino acid sequence of a constitutively active version of a GPCR
having SEQ ID NO:2; and (vi) a biologically active fragment of any
one of (i) to (v); (b) determining the ability of the test compound
to stimulate functionality of the GPCR; (c) determining whether the
compound increases a PYY level in a sample, the sample being from a
vertebrate previously administered with a compound which stimulates
functionality of the GPCR in step (b); wherein the ability of the
test compound to increase a PYY level in the sample is indicative
of the test compound being a compound useful for treatment or
prevention of a condition modulated by PYY or a compound useful for
a modulation of a PYY-related activity in an individual.
124. A method for identifying compounds useful for treatment of a
condition modulated by PYY or compounds useful for a modulation of
a PYY-related activity in an individual, comprising the steps of:
(a) contacting a test compound with a host cell or with a membrane
of a host cell comprising a GPCR, wherein the GPCR comprises an
amino acid sequence selected from the group consisting of: (i)
amino acids 1-335 of SEQ ID NO:2; (ii) amino acids 2-335 of SEQ ID
NO:2; (iii) an amino acid sequence of a GPCR encoded by a
polynucleotide capable of hybridizing under stringent conditions to
the complement of SEQ ID NO:1; (iv) an amino acid sequence of a
GPCR having at least about 80% identity to SEQ ID NO:2; (v) an
amino acid sequence of a constitutively active version of a GPCR
having SEQ ID NO:2; and (vi) a biologically active fragment of any
one of (i) to (v); (b) determining the ability of the test compound
to stimulate functionality of the GPCR; (c) administering the test
compound that stimulates functionality of the GPCR in step (b) to a
vertebrate; and (d) determining whether the compound which
stimulates functionality of the GPCR in step (b) increases a PYY
level in the vertebrate, wherein the ability of the test compound
to increase a PYY level in the vertebrate is indicative of the test
compound being a compound useful for treatment or prevention of a
condition modulated by PYY or a compound useful for a modulation of
a PYY-related activity in an individual.
125. A method for identifying compounds useful for treatment of a
condition modulated by PYY or compounds useful for a modulation of
a PYY-related activity in an individual, comprising: (a) contacting
a GPCR with a ligand for the GPCR in the presence or absence of a
test compound, wherein the ligand for the GPCR is a ligand for
GPR119 and wherein the GPCR comprises an amino acid sequence
selected from the group consisting of: (i) amino acids 1-335 of SEQ
ID NO:2; (ii) amino acids 2-335 of SEQ ID NO:2; (iii) an amino acid
sequence of a GPCR encoded by a polynucleotide capable of
hybridizing under stringent conditions to the complement of SEQ ID
NO:1; (iv) an amino acid sequence of a GPCR having at least about
80% identity to SEQ ID NO:2; (v) an amino acid sequence of a
constitutively active version of a GPCR having SEQ ID NO:2; and
(vi) a biologically active fragment of any one of (i) to (v); (b)
detecting the complex between the ligand and the GPCR; and (c)
determining whether less of the complex is formed in the presence
of the test compound than in the absence of the test compound; (d)
contacting a compound in the presence of which less of the complex
is formed in step (c) in vitro with a vertebrate enteroendocrine
cell or a cell capable of secreting PYY; and (e) determining
whether the compound stimulates PYY secretion from the vertebrate
enteroendocrine cell or the cell capable of secreting PYY, wherein
the ability of the test compound to stimulate PYY secretion from
the vertebrate enteroendocrine cell or the cell capable of
secreting PYY is indicative of the test compound being a compound
useful for treatment or prevention of a condition modulated by PYY
or a compound useful for a modulation of a PYY-related activity in
an individual.
126. A method for identifying compounds useful for treatment of a
condition modulated by PYY or compounds useful for a modulation of
a PYY-related activity in an individual, comprising: (a) contacting
a GPCR with a ligand to the GPCR in the presence or absence of a
test compound, wherein the GPCR comprises an amino acid sequence
selected from the group consisting of: (i) amino acids 1-335 of SEQ
ID NO:2; (ii) amino acids 2-335 of SEQ ID NO:2; (iii) an amino acid
sequence of a GPCR encoded by a polynucleotide capable of
hybridizing under stringent conditions to the complement of SEQ ID
NO:1; (iv) an amino acid sequence of a GPCR having at least about
80% identity to SEQ ID NO:2; (v) an amino acid sequence of a
constitutively active version of a GPCR having SEQ ID NO:2; and
(vi) a biologically active fragment of any one of (i) to (v); (c)
determining whether less of the complex is formed in the presence
of the test compound than in the absence of the test compound; (d)
determining whether the compound increases a PYY level in a sample
from a vertebrate, the vertebrate having been administered with a
compound in the presence of which less of the complex is formed in
step (c); wherein the ability of the test compound to increase a
PYY level in the sample is indicative of the test compound being a
compound useful for treatment or prevention of a condition
modulated by PYY or a compound useful for a modulation of a
PYY-related activity in an individual.
127. A method for identifying compounds useful for treatment of a
condition modulated by PYY or compounds useful for a modulation of
a PYY-related activity in an individual, comprising: (a) contacting
a GPCR with a ligand for the GPCR in the presence or absence of a
test compound, wherein a ligand for the GPCR is a ligand for GPR119
and wherein the GPCR comprises an amino acid sequence selected from
the group consisting of: (i) amino acids 1-335 of SEQ ID NO:2; (ii)
amino acids 2-335 of SEQ ID NO:2; (iii) an amino acid sequence of a
GPCR encoded by a polynucleotide capable of hybridizing under
stringent conditions to the complement of SEQ ID NO:1; (iv) an
amino acid sequence of a GPCR having at least about 80% identity to
SEQ ID NO:2; (v) an amino acid sequence of a constitutively active
version of a GPCR having SEQ ID NO:2; and (vi) a biologically
active fragment of any one of (i) to (v); (b) detecting the complex
between the ligand and the GPCR; and (c) determining whether less
of the complex is formed in the presence of the test compound than
in the absence of the test compound; (d) determining whether a
compound in the presence of which less of the complex is formed in
step (c) increases a PYY level in a vertebrate; wherein the ability
of the test compound to increase a PYY level in the vertebrate is
indicative of the test compound being a compound useful for
treatment or prevention of a condition modulated by PYY or a
compound useful for a modulation of a PYY-related activity in an
individual.
128. A method of preparing a pharmaceutical composition comprising
a GPR119 agonist having the effect of a PYY secretagogue, the
method comprising: (a) contacting the GPR119 agonist in vitro with
a vertebrate enteroendocrine cell or with a cell capable of
secreting PYY; (b) determining whether the GPR119 agonist
stimulates PYY secretion from the vertebrate enteroendocrine cell
or the cell capable of secreting PYY, wherein the ability of the
GPR119 agonist to stimulate PYY secretion from the vertebrate
enteroendocrine cell or the cell capable of secreting PYY is
indicative of the agonist being the PYY secretagogue useful for
treating or preventing a condition ameliorated by increasing a PYY
level; and (c) admixing the GPR119 agonist having the effect of a
PYY secretagogue with a pharmaceutically acceptable carrier.
129. A method of preparing a pharmaceutical composition comprising
a GPR119 agonist having the effect of a PYY secretagogue, the
GPR119 agonist having been contacted in vitro with a vertebrate
enteroendocrine cell or with a cell capable of secreting PYY and
determined to stimulate PYY secretion from the vertebrate
enteroendocrine cell or the cell capable of secreting PYY, wherein
the ability of the GPR119 agonist to stimulate PYY secretion from
the vertebrate enteroendocrine cell or the cell capable of
secreting PYY is indicative of the agonist being a PYY
secretagogue, the method comprising admixing the GPR119 agonist
having the effect of a PYY secretagogue with a pharmaceutically
acceptable carrier.
130. A method of preparing a pharmaceutical composition comprising
a GPR119 agonist having the effect of a PYY secretagogue, the
method comprising: (a) determining a PYY level in a sample obtained
from a vertebrate, the vertebrate having been administered with the
GPR119 agonist, wherein the ability of the GPR119 agonist to
increase a PYY level in the sample obtained from the vertebrate is
indicative of the agonist being a PYY secretagogue useful for
treating or preventing a condition ameliorated by increasing a
blood PYY level; and (b) admixing the GPR119 agonist having the
effect of a PYY secretagogue with a pharmaceutically acceptable
carrier.
131. A method of preparing a pharmaceutical composition comprising
a GPR119 agonist having the effect of a PYY secretagogue, the
GPR119 agonist having been administered to a vertebrate and
determined to increase a PYY level in the vertebrate, wherein the
ability of GPR119 agonist to increase a PYY level is indicative of
the agonist being the PYY secretagogue, the method comprising
admixing the GPR119 agonist having the effect of a PYY secretagogue
with a pharmaceutically acceptable carrier.
132. A method of preparing a pharmaceutical composition comprising
a GPR119 agonist having the effect of a PYY secretagogue, the
method comprising: (a) administering a GPR119 agonist to a
vertebrate; (b) determining whether the GPR119 agonist increases a
PYY level in the vertebrate, wherein the ability of the GPR119
agonist to increase a PYY level in the vertebrate is indicative of
the agonist having the effect of a PYY secretagogue; and (b)
admixing the GPR119 agonist having the effect of a PYY secretagogue
with a pharmaceutically acceptable carrier.
133. A method of preparing a pharmaceutical composition comprising
a GPR119 agonist having the effect of a PYY secretagogue, wherein
the GPR119 agonist is determined to increase a PYY level in a
sample obtained from a vertebrate previously administered with the
GPR119 agonist, wherein the ability of GPR119 agonist to increase a
PYY level in the sample is indicative of the agonist being a PYY
secretagogue, the method comprising admixing the GPR119 agonist
having the effect of a PYY secretagogue with a pharmaceutically
acceptable carrier.
134. A method of treating a condition modulated by PYY in a subject
in need thereof, comprising administering to the subject a
therapeutically effective amount a GPR119 agonist, wherein the
condition modulated by PYY is selected from the group consisting of
angiogenesis-related condition, ischemia-related condition,
malabsorptive disorder, convulsive disorder, cancer, and
inflammatory disorder.
135. A method of modulating a PYY-related activity in a subject in
need thereof, comprising administering to the subject a
therapeutically effective amount a GPR119 agonist, wherein the
PYY-related activity is selected from the group consisting of
increasing plasma PYY, decreasing food uptake, decreasing body
weight, increasing satiety, increasing angiogenesis, decreasing
secretion in intestinal epithelium, increasing absorption in
intestinal epithelium, and increasing plasma adiponectin.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application No. 61/123,273, filed on Apr. 7, 2008, the disclosure
of which is incorporated by reference in its entirety in this
application.
FIELD OF THE INVENTION
[0002] The present invention relates to methods of using GPR119
receptor to identify peptide YY (PYY) secretagogues and compounds
useful in the treatment of conditions modulated by PYY, such as
conditions modulated by stimulation of NPY Y2 receptor (Y2R).
Agonists of GPR119 receptor are useful as therapeutic agents for
treating or preventing a condition modulated by PYY, such as a
condition modulated by stimulation of Y2R. Conditions modulated by
PYY such as may be a condition modulated by stimulation of Y2R
include bone-related conditions, metabolic disorders,
angiogenesis-related conditions, ischemia-related conditions,
convulsive disorders, malabsorptive disorders, cancers, and
inflammatory disorders.
BACKGROUND OF THE INVENTION
[0003] The following discussion is intended to facilitate the
understanding of the invention, but is not intended nor admitted to
be prior art to the invention.
[0004] A. Osteoporosis
[0005] Osteoporosis is a disabling disease characterized by the
loss of bone mass and microarchitectural deterioration of skeletal
structure leading to compromised bone strength, which predisposes a
patient to increased risk of fragility fractures. Osteoporosis
affects more than 75 million people in Europe, Japan and the United
States, and causes more than 2.3 million fractures in Europe and
the United States alone. In the United States, osteoporosis affects
at least 25% of all post-menopausal white women, and the proportion
rises to 70% in women older than 80 years. One in three women older
than 50 years will have an osteoporotic fracture that causes a
considerable social and financial burden on society. The disease is
not limited to women; older men also can be affected. By 2050, the
worldwide incidence of hip fracture in men is projected to increase
by 310% and 240% in women. The combined lifetime risk for hip,
forearm, and vertebral fractures presenting clinically is around
40%, equivalent to the risk for cardiovascular disease.
Osteoporotic fractures therefore cause substantial mortaility,
morbidity, and economic cost. With an ageing population, the number
of osteoporotic fractures and their costs will at least double in
the next 50 years unless effective preventive strategies are
developed. (See, e.g., Atik et al., Clin Orthop Relat Res (2006)
443:19-24; Raisz, J Clin Invest (2005) 115:3318-3325; and World
Health Organization Technical Report Series 921 (2003), Prevention
and Management of Osteoporosis.)
[0006] B. Obesity and Diabetes
[0007] Obesity is a major public health concern because of its
increasing prevalence and associated health risks. Moreover,
obesity may affect a person's quality of life through limited
mobility and decreased physical endurance as well as through
social, academic and job discrimination.
[0008] Obesity and being overweight are generally defined by body
mass index (BMI), which is correlated with total body fat and
serves as a measure of the risk of certain diseases. BMI is
calculated by weight in kilograms divided by height in meters
squared (kg/m.sup.2). Overweight is typically defined as a BMI of
25-29.9 kg/m.sup.2, and obesity is typically defined as a BMI of 30
kg/m.sup.2 or higher. See, e.g., National Heart, Lung, and Blood
Institute, Clinical Guidelines on the Identification, Evaluation,
and Treatment of Overweight and Obesity in Adults, The Evidence
Report, Washington, D.C.: U.S. Department of Health and Human
Services, NIH publication no. 98-4083 (1998).
[0009] Recent studies have found that obesity and its associated
health risks are not limited to adults, but also affect children
and adolescents to a startling degree. According to the Center for
Disease Control, the percentage of children and adolescents who are
defined as overweight has more than doubled since the early 1970s,
and about 15 percent of children and adolescents are now
overweight. Risk factors for heart disease, such as high
cholesterol and high blood pressure, occur with increased frequency
in overweight children and adolescents compared with normal-weight
subjects of similar age. Also, type 2 diabetes, previously
considered an adult disease, has increased dramatically in children
and adolescents. Overweight conditions and obesity are closely
linked to type 2 diabetes. It has recently been estimated that
overweight adolescents have a 70% chance of becoming overweight or
obese adults. The probability increases to about 80% if at least
one parent is overweight or obese. The most immediate consequence
of being overweight as perceived by children themselves is social
discrimination.
[0010] Overweight or obese individuals are at increased risk for
ailments such as hypertension, dyslipidemia, type 2 (non-insulin
dependent) diabetes, insulin resistance, glucose intolerance,
hyperinsulinemia, coronary heart disease, angine pectoris,
congestive heart failure, stroke, gallstones, cholescystitis,
cholelithiasis, gout, osteoarthritis, obstructive sleep apnea and
respiratory problems, gall bladder disease, certain forms of cancer
(e.g., endometrial, breast, prostate, and colon) and psychological
disorders (such as depression, eating disorders, distorted body
image and low self esteem). The negative health consequences of
obesity make it the second leading cause of preventable death in
the United States and impart a significant economic and
psychosocial effect on society. See, e.g., McGinnis et al, JAMA
(1993) 270:2207-2212.
[0011] Obesity is now recognized as a chronic disease that requires
treatment to reduce its associated health risks. Although weight
loss is an important treatment outcome, one of the main goals of
obesity management is to improve cardiovascular and metabolic
values to reduce obesity-related morbidity and mortality. It has
been shown that 5-10% loss of body weight can substantially improve
metabolic values, such as blood glucose, blood pressure, and lipid
concentrations. Hence, it is believed that a 5-10% reduction in
body weight may reduce morbidity and mortality.
[0012] Currently available prescription drugs for managing obesity
generally reduce weight by decreasing dietary fat absorption, as
with orlistat, or by creating an energy deficit be reducing food
intake and/or increasing energy expenditure, as seen with
sibutramine. The search for alternatives to presently available
antiobesity agents has taken several paths one of which has focused
on certain gut peptides that have been implicated in modulating
satiety such as peptide YY (PYY) (Chaudhri et al, Annu Rev Physiol
(2008) 70:239-255).
[0013] C. Atherosclerosis
[0014] Atherosclerosis is a complex disease characterized by
inflammation, lipid accumulation, cell death and fibrosis.
Atherosclerosis is characterized by cholesterol deposition and
monocyte infiltration into the subendothelial space, resulting in
foam cell formation. Thrombosis subsequent to atherosclerosis leads
to myocardial infarction and stroke. Atherosclerosis is the leading
cause of mortality in many countries, including the United States.
(See, e.g., Ruggeri, Nat Med (2002) 8:1227-1234; Arehart et al,
Circ Res, 2008, Mar. 6 Epub ahead of print.)
[0015] D. Inflammatory Bowel Disease (IBD)
[0016] Inflammatory bowel disease (IBD) is the general name for
diseases that cause inflammation in the intestines and includes,
e.g. Crohn's disease, ulcerative colitis, ulcerative proctitis.
U.S. medical costs of inflammatory bowel disease for 1990 have been
estimated to be $1.4 to $1.8 billion. Lost productivity has been
estimated to have added an additional $0.4 to $0.8 billion, making
the estimated cost of inflammatory bowel disease $1.8 to $2.6
billion. (See, e.g., Pearson, Nursing Times (2004) 100:86-90; Hay
et al, J Clin Gastroenterol (1992) 14:309-317; Keighley et al,
Ailment Pharmacol Ther (2003) 18:66-70.)
[0017] Enteritis refers to inflammation of the intestine,
especially the small intestine, a general condition that can have
any of numerous different causes. Enterocolitis refers to
inflammation of the small intestine and colon.
[0018] Crohn's disease (CD) is an inflammatory process that can
affect any portion of the digestive tract, but is most commonly
seen in the last part of the small intestine otherwise called the
(terminal) ileum and cecum. Altogether this area is also known as
the ileocecal region. Other cases may affect one or more of: the
colon only, the small bowel only (duodenum, jejunum and/or ileum),
the anus, stomach or esophagus. In contrast with ulcerative
colitis, CD usually does not affect the rectum, but frequently
affects the anus instead. The inflammation extends deep into the
lining of the affected organ. The inflammation can cause pain and
can make the intestines empty frequently, resulting in diarrhea.
Crohn's disease may also be called enteritis. Granulomatous colitis
is another name for Crohn's disease that affects the colon. Ileitis
is CD of the ileum which is the third part of the small intestine.
Crohn's colitis is CD affecting part or all of the colon.
[0019] Ulcerative colitis (UC) is an inflammatory disease of the
large intestine, commonly called the colon. UC causes inflammation
and ulceration of the inner lining of the colon and rectum. The
inflammation of UC is usually most severe in the rectal area with
severity diminishing (at a rate that varies from patient to
patient) toward the cecum, where the large and small intestine
join. Inflammation of the rectum is called proctitis. Inflammation
of the sigmoid colon (located just above the rectum) is called
sigmoiditis Inflammation involving the entire colon is termed
pancolitis. The inflammation causes the colon to empty frequently
resulting in diarrhea. As the lining of the colon is destroyed
ulcers form releasing mucus, pus and blood. Ulcerative proctitis is
a form of UC that affects only the rectum.
[0020] E. Peptide YY (PYY)
[0021] Peptide YY (PYY) is a 36 amino acid peptide originally
isolated in 1980 from porcine intestine (Tatemoto et al, Nature
(1980) 285:417-418). PYY is secreted from enteroendocrine L-cells
within both the large and small intestine. It has been shown that
in rat and human gut concentrations of immunoreactive PYY are low
in duodenum and jenunum, high in ileum and colon, and highest in
rectum (Lundberg et al, PNAS USA (1982) 79:4471-4475; Adrian et al,
Gastroenterol (1985) 89:1070-1077; Ekblad et al, Peptides (2002)
23:251-261; Ueno et al, Regul Pept (2008) 145:12-16). (PYY
expression in rat been reported to also extend to alpha cells of
the islets of Langerhans and to cells in the medulla oblongata
(Ekblad et al, Peptides (2002) 23:251-261; PYY is released into the
circulation as PYY.sub.1-36 and PYY.sub.3-36 (Eberlein et al,
Peptides (1989) 10:797-803). PYY.sub.3-36 is generated from
PYY.sub.1-36 by cleavage of the N-terminal Tyr and Pro residues by
dipeptidyl peptidase IV. PYY.sub.3-36 is the predominant form of
PYY in human postprandial plasma (Grandt et al, Regul Pept (1994)
51:151-159). PYY.sub.1-36 and PYY.sub.3-36 have been reported to
have comparable agonist activity at NPY Y2 receptor (Y2R), a G
protein-coupled receptor (Parker et al, Br J Pharmacol (2008)
153:420-431); however, PYY.sub.3-36 has been reported to be a
high-affinity Y2R selective agonist (Keire et al, Am J Physiol
Gastrointest Liver Physiol (2000) 279:G126-G131). PYY was
subsequently reported to reduce high-fat food intake in rats after
peripheral administration (Okada et al, Endocrinology Supplement
(1993) 180) and to cause weight loss in mice after peripheral
administration (Morley et al, Life Sciences (1987)
41:2157-2165).
[0022] Peripheral administration of PYY.sub.3-36 has been reported
to markedly reduce food intake and weight gain in rats, to decrease
appetite and food intake in humans, and to decrease food intake in
mice, but not in Y2R-null mice, which was said to suggest that the
food intake effect requires the Y2R. In human studies, infusion of
PYY.sub.3-36 was found to significantly decrease appetite and
reduce food intake by 33% over 24 hours. Infusion of PYY.sub.3-36
to reach the normal postprandial circulatory concentrations of the
peptide led to peak serum levels of PYY.sub.3-36 within 15 minutes,
followed by a rapid decline to basal levels within 30 minutes. It
was reported that there was significant inhibition of food intake
in the 12-hour period following the PYY.sub.3-36 infusion, but was
essentially no effect on food intake in the 12-hour to 24-hour
period. In a rat study, repeated administration of PYY.sub.3-36
intraperitoneally (injections twice daily for 7 days) reduced
cumulative food intake (Batterham et al, Nature (2002) 418:650-654;
Renshaw et al, Current Drug Targets (2005) 6:171-179).
[0023] Peripheral administration of PYY.sub.3-36 has been reported
to reduce food intake, body weight gain and glycemic indices in
diverse rodent models of metabolic diseases of both sexes (Pittner
et al, Int J Obes Relat Metab Disord (2004) 28:963-971). It has
been reported that blockade of Y2R with the specific antagonist
BIIE-246 attenuates the effect of peripherally administered
endogenous and exogenous PYY.sub.3-36 for reducing food intake
(Abbott et al, Brain Res (2005) 1043:139-144). It has been reported
that peripheral administration of a novel long-acting selective Y2R
polyethylene glycol-conjugated peptide agonist reduces food intake
and improves glucose metabolism (glucose disposal, plasma insulin
and plasma glucose) in rodents (Ortiz et al, JPET (2007)
323:692-700; Lamb et al, J Med Chem (2007) 50:2264-2268). It has
been reported that PYY ablation in mice leads to the development of
hyperinsulinemia and obesity (Boey et al, Diabetologia (2006)
49:1360-1370). It has been reported that peripheral administration
of a long-acting, potent and highly selective Y2R agonist inhibits
food intake and promotes fat metabolism in mice (Balasubramaniam et
al, Peptides (2007) 28:235-240).
[0024] There is evidence that agents which stimulate PYY synthesis
in vivo can confer protection against diet-induced and genetic
obesity and can improve glucose tolerance (Boey et al,
Neuropeptides (2008) 42:19-30).
[0025] It has been reported that Y2R agonists such as PYY.sub.1-36
and PYY.sub.3-36 can confer protection against epileptic seizures,
such as against kainate seizures (El Bahh et al, Eur J Neurosci
(2005) 22:1417-1430; Woldbye et al, Neurobiology of Disease (2005)
20:760-772). It has been reported that Y2R agonists such as
PYY.sub.1-36 and PYY.sub.3-36 act as proabsorbtive (or
anti-secretory) hormones, increasing upon intravenous
administration the absorption of both water and sodium in various
parts of the bowel (Bilchik et al, Gastroenterol (1993)
105:1441-1448; Liu et al, J Surg Res (1995) 58:6-11; Nightingale et
al, Gut (1996) 39:267-272; Liu et al, Am Surg (1996) 62:232-236;
Balasubramaniam et al, J Med Chem (2000) 43:3420-3427). It has been
reported that Y2R agonists such as PYY analogues inhibit secretion
and promote absorption and growth in the intestinal epithelium
(Balasubramaniam et al, J Med Chem (2000) 43:3420-3427). It has
been reported that PYY promotes intestinal growth in normal rats
(Gomez et al, Am J Physiol (1995) 268:G71-G81). It has been
reported that Y2R agonists such as PYY.sub.1-36 and PYY.sub.3-36
inhibit bowel motility and work to prevent diarrhea (EP1902730;
also see Cox, Peptides (2007) 28:345-351).
[0026] It has been reported that Y2R agonists such as PYY.sub.1-36
and PYY.sub.3-36 can confer protection against inflammatory bowel
disease such as ulcerative colitis and Crohn's disease (WO
03/105763). It has been reported that PYY-deficient mice exhibit an
osteopenic phenotype, i.e. that PYY can increase bone mass and/or
can confer protection against loss of bone mass (e.g., decreases
loss of bone mass) (Wortley et al, Gastroenterol (2007)
133:1534-1543). It has been reported that PYY.sub.3-36 can confer
protection in rodent models of pancreatitis (Vona-Davis et al,
Peptides (2007) 28:334-338).
[0027] It has been reported that angiogenesis is impaired in
Y2R-deficient mice (Lee et al, Peptides (2003) 24:99-106), i.e.
that agonists of Y2R such as PYY.sub.1-36 and PYY.sub.3-36 promote
angiogenesis. It has been reported that would healing is impaired
in Y2R-deficient mice (Ekstrand et al, PNAS USA (2003)
100:6033-6038), i.e. that agonists of Y2R such as PYY.sub.1-36 and
PYY.sub.3-36 promote wound healing. It has been reported that
ischemic angiogenesis is impaired in Y2R-deficient mice (Lee et al,
J Clin Invest (2003) 111:1853-1862), i.e. that agonists of Y2R such
as PYY.sub.1-36 and PYY.sub.3-36 promotes revascularization and
restoration of function of ischemic tissue. It has been reported
that agonists of Y2R such as PYY.sub.1-36 and PYY.sub.3-36 mediate
increases in collateral-dependent blood flow in a rat model of
peripheral arterial disease (Cruze et al, Peptides (2007)
28:269-280).
[0028] It has been reported that PYY and Y2R agonists such as
PYY.sub.3-36 can suppress tumor growth in the cases of, e.g.,
pancreatic cancer such as pancreatic ductal adenocarcinoma, breast
cancer such as breast infiltrative ductal adenocarcinoma, colon
cancer such as colon adenocarcinoma and Barrett's adenocarcinoma
(Liu et al, Surgery (1995) 118:229-236; Liu et al, J Surg Res
(1995) 58:707-712; Grise et al, J Surg Res (1999) 82:151-155; Tseng
et al, Peptides (2002) 23:389-395; McFadden et al, Am J Surg (2004)
188:516-519).
[0029] It has been reported that stimulation of Y2R such as by
PYY.sub.3-36 leads to an increase in plasma adiponectin (Ortiz et
al, JPET (2007) 323:692-700). Adiponectin is an adipokine with
potent anti-inflammatory properties (Ouchi et al, Clin Chim Acta
(2007) 380:24-30; Tilg et al, Nat Rev Immunol (2006) 6:772-783).
Adiponectin exerts anti-atherogenic effects by targeting vascular
endothelial cells and macrophages and insulin-sensitizing effects,
predominantly in muscle and liver (Kubota et al, J Biol Chem (2002)
277:25863-25866; Maeda et al, Nat Med (2002) 8:731-737). Low
adiponectin levels have been reported to be associated with
atherogenic lipoproteins in dyslipidemia (elevated triglycerides,
small dense LDL cholesterol, low HDL cholesterol) (Marso et al,
Diabetes Care (2008) February 5 Epub ahead of print). Adiponectin
has been implicated in high density lipoprotein (HDL) assembly (Oku
et al, FEBS Letters (2007) 581:5029-5033). Adiponectin has been
found to ameliorate the abnormalities of metabolic syndrome,
including insulin resistance, hyperglycemia, and dyslipidemia, in a
mouse model of obesity-linked metabolic syndrome associated with
decreased adiponectin levels (Hara et al, Diabetes Care (2006)
29:1357-1362). Adiponectin has been reported to stimulate
angiogenesis in response to tissue ischemia (Shibata et al, J Biol
Chem (2004) 279:28670-28674). Adiponectin has been reported to
prevent cerebral ischemic injury through endothelial nitric oxide
synthase-dependent mechanisms (Nishimura et al, Circulation (2008)
117:216-223). Adiponectin has been reported to confer protection
against myocardial ischemia-reperfusion injury (Shibata et al, Nat
Med (2005) 11:1096-1103; Tao et al, Circulation (2007)
115:1408-1416). Adiponectin has been reported to confer protection
against myocardial ischaemia-reperfusion injury via AMP-activated
protein kinase, Akt, and nitric oxide (Gonon et al, Cardiovasc Res
(2008) 78:116-122). Adiponectin has been reported to confer
protection against the development of systolic dysfunction
following myocardial infarction, through its abilities to suppress
cardiac hypertrophy and interstitial fibrosis, and proctect against
myocyte and capillary loss (Shibata et al, J Mol Cell Cardiol
(2007) 42:1065-1074). Adiponectin has been reported to confer
protection against inflammatory lung disease; adiponectin-deficient
mice exhibit an emphysema-like phenotype (Summer et al, Am J
Physiol Lung Cell Mol Physiol (Mar. 7, 2008)). Adiponectin has been
reported to confer protection against allergic airway inflammation
and airway hyperresponsiveness such as may be associated with
asthma (Shore et al, J Allergy Clin Immunol (2006) 118:389-395).
Adiponectin has been suggested to confer protection against
pulmonary arterial hypertension by virtue of its
insulin-sensitizing effects (Hansmann et al, Circulation (2007)
115:1275-1284). Adiponectin has been reported to ameliorate
obesity-related hypertension, with said amelioration of
hypertension being associated in part with upregulated prostacyclin
expression (Ohashi et al, Hypertension (2006) 47:1108-1116).
Adiponectin has been reported to decrease tumor necrosis factor
(TNF)-.alpha.-induced expression of the adhesion molecules VCAM-1,
E-selectin and ICAM-1 in human aortic endothelial cells (HAECs)
(Ouchi et al, Circulation (1999) 100:2473-2476) and to inhibit
production of TNF-a in macrophages (Yokota et al, Blood (2000)
96:1723-1732). Adiponectin has been reported to confer protection
against restenosis after vascular intervention (Matsuda et al, J
Biol Chem (2002) 277:37487-37491). The central role of TNF-.alpha.
in inflammation has been demonstrated by the ability of agents that
block the action of TNF-.alpha. to treat a range of inflammatory
conditions. TNF-.alpha.-mediated inflammatory conditions encompass
rheumatoid arthritis, inflammatory bowel disease such as Crohn's
disease, ankylosing spondylitis, psoriasis, ischemic brain injury,
cardiac allograft rejection, asthma, and the like (Bradley, J
Pathol (2008) 214:149-160). See, e.g., Yamamoto et al, Clinical
Science (2002) 103:137-142; Behre, Scand J Clin Lab Invest (2007)
67:449-458; Guerre-Millo, Diabetes & Metabolism (2008)
34:12-18; Parker et al, Br J Pharmacol (2008) 153:420-431.
[0030] F. Treatment of Conditions Modulated by PYY
[0031] An attractive alternative approach for treatment of
conditions modulated by PYY is to develop an orally active
composition for increasing an endogenous level of PYY in an
individual, such as an endogenous level of plasma PYY, such as by
stimulation of PYY secretion in gut enteroendocrine cells.
[0032] G. GPR119
[0033] GPR119 is a G protein-coupled receptor (GPR119; e.g., human
GPR119, GenBank.RTM. Accession No. AAP72125 and alleles thereof;
e.g., mouse GPR119, GenBank.RTM. Accession No. AY288423 and alleles
thereof). GPR119 stimulation as by an agonist leads to elevation of
the level of intracellular cAMP, consistent with GPR119 being
coupled to Gs. In the patent literature, GPR119 has been referred
to as RUP3 (e.g., WO 00/31258); GPR119 has also been referred to as
Glucose-Dependent Insulinotropic Receptor (GDIR).
[0034] H. G Protein-Coupled Receptors
[0035] Although a number of receptor classes exist in humans, by
far the most abundant and therapeutically relevant is represented
by the G protein-coupled receptor (GPCR) class. It is estimated
that there are some 30,000-40,000 genes within the human genome,
and of these, approximately 2% are estimated to code for GPCRs.
[0036] GPCRs represent an important area for the development of
pharmaceutical products. Drugs active at GPCRs have therapeutic
benefit across a broad spectrum of human diseases as diverse as
pain, cognitive dysfunction, hypertension, peptic ulcers, rhinitis,
and asthma. Of the approximately 500 clinically marketed drugs,
greater than 30% are modulators of GPCR function. These drugs exert
their activity at approximately 30 well-characterized GPCRs. (See,
e.g., Wise et al, Annu Rev Pharmacol Toxicol (2004) 44:43-66.)
[0037] GPCRs share a common structural motif, having seven
sequences of between 22 to 24 hydrophobic amino acids that form
seven alpha helices, each of which spans the membrane (each span is
identified by number, i.e., transmembrane-1 (TM-1), transmembrane-2
(TM-2), etc.). The transmembrane helices are joined by strands of
amino acids between transmembrane-2 and transmembrane-3,
transmembrane-4 and transmembrane-5, and transmembrane-6 and
transmembrane-7 on the exterior, or "extracellular" side, of the
cell membrane (these are referred to as "extracellular" regions 1,
2 and 3 (EC-1, EC-2 and EC-3), respectively). The transmembrane
helices are also joined by strands of amino acids between
transmembrane-1 and transmembrane-2, transmembrane-3 and
transmembrane-4, and transmembrane-5 and transmembrane-6 on the
interior, or "intracellular" side, of the cell membrane (these are
referred to as "intracellular" regions 1, 2 and 3 (IC-1, IC-2 and
IC-3), respectively). The "carboxy" ("C") terminus of the receptor
lies in the intracellular space within the cell, and the "amino"
("N") terminus of the receptor lies in the extracellular space
outside of the cell.
[0038] Generally, when a ligand binds with the receptor (often
referred to as "activation" of the receptor), there is a change in
the conformation of the receptor that facilitates coupling between
the intracellular region and an intracellular "G-protein." It has
been reported that GPCRs are "promiscuous" with respect to G
proteins, i.e., that a GPCR can interact with more than one G
protein. See, Kenakin, Life Sciences (1988) 43:1095-1101. Although
other G proteins exist, currently, Gq, Gs, Gi, Gz and Go are G
proteins that have been identified. Ligand-activated GPCR coupling
with the G-protein initiates a signaling cascade process (referred
to as "signal transduction"). Under normal conditions, signal
transduction ultimately results in cellular activation or cellular
inhibition. Although not wishing to be bound to theory, it is
thought that the IC-3 loop as well as the carboxy terminus of the
receptor interact with the G protein.
[0039] There are also promiscuous G proteins, which appear to
couple several classes of GPCRs to the phospholipase C pathway,
such as G15 or G16 (Offermanns & Simon, J Biol Chem (1995)
270:15175-80), or chimeric G proteins designed to couple a large
number of different GPCRs to the same pathway, e.g. phospholipase C
(Milligan & Rees, Trends in Pharmaceutical Sciences (1999)
20:118-24). Under physiological conditions, GPCRs exist in the cell
membrane in equilibrium between two different conformations: an
"inactive" state and an "active" state. A receptor in an inactive
state is unable to link to the intracellular signaling transduction
pathway to initiate signal transduction leading to a biological
response. Changing the receptor conformation to the active state
allows linkage to the transduction pathway (via the G-protein) and
produces a biological response.
[0040] A receptor may be stabilized in an active state by a ligand
or a compound such as a drug. Recent discoveries, including but not
exclusively limited to modifications to the amino acid sequence of
the receptor, provide means other than ligands or drugs to promote
and stabilize the receptor in the active state conformation. These
means effectively stabilize the receptor in an active state by
simulating the effect of a ligand binding to the receptor.
Stabilization by such ligand-independent means is termed
"constitutive receptor activation."
SUMMARY OF THE INVENTION
[0041] The present invention relates to the unexpected discovery by
Applicant that administration of a GPR119 agonist to an
individival, such as by oral administration, can act to increase
plasma total PYY in the individual. The present invention features
methods relating to GPR119 for identifying PYY secretagogues,
compounds useful for treatment or prevention of a condition
modulated by PYY, and compounds useful for a modulation of a
PYY-related activity in an individual. A GPR119 agonist is useful
for increasing plasma total PYY in an individual. In certain
embodiments, the individual is a human.
[0042] Nucleotide sequence encoding human GPR119 polypeptide is
given in SEQ ID NO: 1. The amino acid sequence of said encoded
human GPR119 polypeptide is given in SEQ ID NO: 2.
[0043] In a first aspect, the invention features a method for
identifying PYY secretagogues, compounds useful for treatment or
prevention of a condition modulated by PYY, or compounds useful for
a modulation of a PYY-related activity in an individual, comprising
the steps of: [0044] (a) contacting a test compound with a host
cell or with membrane of a host cell comprising a G protein-coupled
receptor, wherein the G protein-coupled receptor comprises an amino
acid sequence selected from the group consisting of: [0045] (i)
amino acids 1-335 of SEQ ID NO:2; [0046] (ii) amino acids 2-335 of
SEQ ID NO:2; [0047] (iii) an amino acid sequence comprising the
amino acid sequence of a polypeptide derived from (i) by
substitution, deletion or addition of one or several amino acids in
the amino acid sequence defined in (i); [0048] (iv) the amino acid
sequence of a G protein-coupled receptor encoded by a
polynucleotide hybridizing under stringent conditions to the
complement of SEQ ID NO:1; [0049] (v) a variant of SEQ ID NO: 2
when selected from the group consisting of: [0050] (a') the amino
acid sequence of a G protein-coupled receptor having at least about
80% identity to SEQ ID NO: 2; and [0051] (b') an amino acid
sequence of a G protein-coupled receptor comprising at least 20
contiguous amino acids of SEQ ID NO: 2; [0052] (vi) the amino acid
sequence of a constitutively active version of a G protein-coupled
receptor having SEQ ID NO: 2; and [0053] (vii) a biologically
active fragment of any one of (i) to (vi); and [0054] (b)
determining the ability of the test compound to stimulate
functionality of the G protein-coupled receptor; wherein the
ability of the test compound to stimulate functionality of the G
protein-coupled receptor is indicative of the test compound being a
PYY secretagogue, a compound useful for treatment or prevention of
a condition modulated by PYY, or a compound useful for a modulation
of a PYY-related activity in an individual.
[0055] In certain embodiments, the G protein-coupled receptor
comprises the amino acid sequence of a G protein-coupled receptor
having at least about 80% identity to SEQ ID NO: 2.
[0056] In certain embodiments, the G protein-coupled receptor
comprises the amino acid sequence of SEQ ID NO: 2.
[0057] In certain embodiments, the variant of SEQ ID NO: 2 is an
allele of SEQ ID NO: 2.
[0058] In certain embodiments, the variant of SEQ ID NO: 2 is an
ortholog of SEQ ID NO: 2. In certain embodiments, the variant of
SEQ ID NO: 2 is a mammalian ortholog of SEQ ID NO: 2.
[0059] In certain embodiments, the G protein-coupled receptor is
recombinant
[0060] In certain embodiments, the method is a method for
identifying PYY secretagogues.
[0061] In certain embodiments, the method comprises identifying an
agonist of the receptor.
[0062] In certain embodiments, the method comprises identifying a
partial agonist of the receptor.
[0063] In certain embodiments, the method is a method for
identifying compounds useful for treatment or prevention of a
condition modulated by PYY.
[0064] In certain embodiments, the method is a method for
identifying compounds useful for a modulation of a PYY-related
activity in an individual.
[0065] The invention additionally features a method for identifying
PYY secretagogues, compounds useful for treatment or prevention of
a condition modulated by PYY, or compounds useful for a modulation
of a PYY-related activity in an individual, comprising steps (a)
and (b) of this first aspect, and further comprising: [0066] (c)
optionally synthesizing a compound which stimulates the
functionality of the receptor in step (b); [0067] (d) contacting a
compound which stimulates functionality of the receptor in step (b)
in vitro with a vertebrate enteroendocrine cell or with a cell
capable of secreting PYY; and [0068] (e) determining whether the
compound stimulates PYY secretion from the vertebrate
enteroendocrine cell or from the cell capable of secreting PYY;
wherein the ability of the test compound to stimulate PYY secretion
from the vertebrate enteroendocrine cell or from the cell capable
of secreting PYY is further indicative of the test compound being a
PYY secretagogue, a compound useful for treatment or prevention of
a condition modulated by PYY, or a compound useful for a modulation
of a PYY-related activity in an individual.
[0069] In certain embodiments, the vertebrate enteroendocrine cell
is a mammalian enteroendocrine cell. In certain embodiments, the
enteroendocrine cell is an L cell. In certain embodiments, the
enteroendocrine cell is comprised by or derived from tissue
obtained from the small or large intestine. In certain embodiments,
the enteroendocrine cell is comprised by or derived from tissue
obtained from an L cell-containing region of small or large
intestine. In certain embodiments, the enteroendocrine cell is
comprised by or derived from duodenum, jejunum, ileum, colon or
rectum tissue (see, e.g., Lundberg et al, PNAS USA (1982)
79:4471-4475; Adrian et al, Gastroenterol (1985) 89:1070-1077;
Ekblad et al, Peptides (2002) 23:251-261; Ueno et al, Regul Pept
(2008) 145:12-16). In certain embodiments, the enteroendocrine cell
is an enteroendocrine cell line. In certain embodiments, the cell
capable of secreting PYY is a cell comprised by or derived from the
islets of Langerhans. In certain embodiments, the cell capable of
secreting PYY is a neuronal cell. In certain embodiments, the cell
capable of secreting PYY is a recombinant cell engineered to be
capable of secreting PYY.
[0070] The invention additionally features a method for identifying
PYY secretagogues, compounds useful for treatment or prevention of
a condition modulated by PYY, or compounds useful for a modulation
of a PYY-related activity in an individual, comprising steps (a)
and (b) of this first aspect, and further comprising: [0071] (c)
optionally synthesizing a compound which stimulates functionality
of the receptor in step (b); [0072] (d) administering a compound
which stimulates functionality of the receptor in step (b) to a
vertebrate; and [0073] (e) determining whether the compound
increases a PYY level in the vertebrate; wherein the ability of the
test compound to increase a PYY level in the vertebrate is further
indicative of the test compound being a PYY secretagogue, a
compound useful for treatment or prevention of a condition
modulated by PYY, or a compound useful for a modulation of a
PYY-related activity in an individual.
[0074] In certain embodiments, the PYY level is blood or plasma
concentration of total PYY. In certain embodiments, the PYY level
is blood or plasma concentration of PYY.sub.3-36.
[0075] In certain embodiments, the vertebrate is a mammal. In
certain embodiments, the vertebrate is a non-human vertebrate. In
certain embodiments, the vertebrate is a non-human mammal. In
certain embodiments, the mammal is a non-human mammal
[0076] In certain embodiments, the vertebrate or the mammal is a
human. In certain embodiments, the human is a participant in a
study reviewed by a governmental agency charged with marketing
approval for a drug. In certain embodiments, the study is a
clinical trial. In certain embodiments, the governmental agency is
the Food and Drug Administration of the United States.
[0077] In certain embodiments, the human is an individual or a
patient enrolled or undergoing treatment in a clinical trial.
[0078] The invention additionally features a method for identifying
PYY secretagogues, compounds useful for treatment or prevention of
a condition modulated by PYY, or compounds useful for a modulation
of a PYY-related activity in an individual, comprising steps (a)
and (b) of this first aspect, and further comprising: [0079] (c)
optionally synthesizing a compound which stimulates functionality
of the receptor in step (b); and [0080] (d) determining whether the
compound increases a PYY level in a sample, said sample having been
obtained from a vertebrate previously administered with a compound
which stimulates functionality of the receptor in step (b); wherein
the ability of the test compound to increase a PYY level in the
vertebrate is further indicative of the test compound being a PYY
secretagogue, a compound useful for treatment or prevention of a
condition modulated by PYY, or a compound useful for a modulation
of a PYY-related activity in an individual.
[0081] In certain embodiments, the vertebrate is a mammal. In
certain embodiments, the vertebrate is a non-human vertebrate. In
certain embodiments, the vertebrate is a non-human mammal. In
certain embodiments, the mammal is a non-human mammal
[0082] In certain embodiments, the vertebrate or the mammal is a
human. In certain embodiments, the human is a participant in a
study reviewed by a governmental agency charged with marketing
approval for a drug. In certain embodiments, the study is a
clinical trial. In certain embodiments, the governmental agency is
the Food and Drug Administration of the United States.
[0083] The invention additionally features a method for identifying
PYY secretagogues, compounds useful for treatment or prevention of
a condition modulated by PYY, or compounds useful for a modulation
of a PYY-related activity in an individual, comprising steps (a)
and (b) of this first aspect, and further comprising: [0084] (c)
optionally synthesizing a compound which stimulates functionality
of the receptor in step (b); [0085] (d) optionally providing a
compound which stimulates functionality of the receptor in step
(b); [0086] (e) contacting a compound which stimulates
functionality of the receptor in step (b) in vitro with a
vertebrate enteroendocrine cell or with a cell capable of secreting
PYY; and [0087] (f) determining whether the compound stimulates PYY
secretion from the vertebrate enteroendocrine cell or from the cell
capable of secreting PYY; wherein the ability of the test compound
to stimulate PYY secretion from the vertebrate enteroendocrine cell
or from the cell capable of secreting PYY is further indicative of
the test compound being a PYY secretagogue, a compound useful for
treatment or prevention of a condition modulated by PYY, or a
compound useful for a modulation of a PYY-related activity in an
individual.
[0088] In certain embodiments, the vertebrate enteroendocrine cell
is a mammalian enteroendocrine cell. In certain embodiments, the
enteroendocrine cell is an L cell. In certain embodiments, the
enteroendocrine cell is comprised by or derived from tissue
obtained from the small or large intestine. In certain embodiments,
the enteroendocrine cell is comprised by or derived from tissue
obtained from an L cell-containing region of small or large
intestine. In certain embodiments, the enteroendocrine cell is
comprised by or derived from duodenum, jejunum, ileum, colon or
rectum tissue (see, e.g., Lundberg et al, PNAS USA (1982)
79:4471-4475; Adrian et al, Gastroenterol (1985) 89:1070-1077;
Ekblad et al, Peptides (2002) 23:251-261; Ueno et al, Regul Pept
(2008) 145:12-16). In certain embodiments, the enteroendocrine cell
is an enteroendocrine cell line. In certain embodiments, the cell
capable of secreting PYY is a cell comprised by or derived from the
islets of Langerhans. In certain embodiments, the cell capable of
secreting PYY is a neuronal cell. In certain embodiments, the cell
capable of secreting PYY is a recombinant cell engineered to be
capable of secreting PYY.
[0089] The invention additionally features a method for identifying
PYY secretagogues, compounds useful for treatment or prevention of
a condition modulated by PYY, or compounds useful for a modulation
of a PYY-related activity, comprising steps (a) and (b) of this
first aspect, and further comprising: [0090] (c) optionally
synthesizing a compound which stimulates functionality of the
receptor in step (b); [0091] (d) optionally providing a compound
which stimulates functionality of the receptor in step (b); [0092]
(e) administering a compound which stimulates functionality of the
receptor in step (b) to a vertebrate; and [0093] (f) determining
whether the compound increases a PYY level in the vertebrate;
wherein the ability of the test compound to increase a PYY level in
the vertebrate is further indicative of the test compound being a
PYY secretagogue, a compound useful for treatment or prevention of
a condition modulated by PYY, or a compound useful for a modulation
of a PYY-related activity in an individual.
[0094] In certain embodiments, the PYY level is blood or plasma
concentration of total PYY. In certain embodiments, the PYY level
is blood or plasma concentration of PYY.sub.3-36.
[0095] In certain embodiments, the vertebrate is a mammal. In
certain embodiments, the vertebrate is a non-human vertebrate. In
certain embodiments, the vertebrate is a non-human mammal. In
certain embodiments, the mammal is a non-human mammal
[0096] In certain embodiments, the vertebrate or the mammal is a
human. In certain embodiments, the human is a participant in a
study reviewed by a governmental agency charged with marketing
approval for a drug. In certain embodiments, the study is a
clinical trial. In certain embodiments, the governmental agency is
the Food and Drug Administration of the United States.
[0097] The invention additionally features a method for identifying
PYY secretagogues, compounds useful for treatment or prevention of
a condition modulated by PYY, or compounds useful for a modulation
of a PYY-related activity, comprising steps (a) and (b) of this
first aspect, and further comprising: [0098] (c) optionally
synthesizing a compound which stimulates functionality of the
receptor in step (b); [0099] (d) optionally providing a compound
which stimulates functionality of the receptor in step (b); and
[0100] (e) determining whether the compound increases a PYY level
in a sample, said sample having been obtained from a vertebrate
previously administered with a compound which stimulates
functionality of the receptor in step (b); wherein the ability of
the test compound to increase a PYY level in the vertebrate is
further indicative of the test compound being a PYY secretagogue, a
compound useful for treatment or prevention of a condition
modulated by PYY, or a compound useful for a modulation of a
PYY-related activity in an individual.
[0101] In certain embodiments, the vertebrate is a mammal. In
certain embodiments, the vertebrate is a non-human vertebrate. In
certain embodiments, the vertebrate is a non-human mammal. In
certain embodiments, the mammal is a non-human mammal
[0102] In certain embodiments, the vertebrate or the mammal is a
human. In certain embodiments, the human is a participant in a
study reviewed by a governmental agency charged with marketing
approval for a drug. In certain embodiments, the study is a
clinical trial. In certain embodiments, the governmental agency is
the Food and Drug Administration of the United States.
[0103] In certain embodiments, the identified PYY secretagogue, or
the identified compound useful for treatment or prevention of a
condition modulated by PYY, or the identified compound useful for a
modulation of a PYY-related activity is an agonist of the receptor.
In some embodiments, the the identified PYY secretagogue, or the
identified compound useful for treatment or prevention of a
condition modulated by PYY, or the identified compound useful for a
modulation of a PYY-related activity is a partial agonist of the
receptor.
[0104] In certain embodiments, the G protein-coupled receptor is
coupled to a G protein. In certain embodiments, stimulation of the
G protein-coupled receptor increases a level of intracellular cAMP
accumulation. In certain embodiments, the G protein is Gs.
[0105] In some embodiments, a G protein-coupled receptor of the
invention is SEQ ID NO:2 or an allele thereof. In some embodiments,
a G protein-coupled receptor of the invention is an allele of SEQ
ID NO:2. In some embodiments, a G protein-coupled receptor of the
invention is an ortholog of SEQ ID NO:2.
[0106] In some embodiments, a G protein-coupled receptor of the
invention is a G protein-coupled receptor which specifically binds
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-l-yl]-5-nitro-pyrimidin-4-yl}-amine. In some
embodimen coupled receptor of the invention is a G protein-coupled
receptor for which
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadi-
azol-5-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine is a
ligand. In some embodiments, a G protein-coupled receptor of the
invention is a G protein-coupled receptor for which
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine is an agonist.
In some embodiments, a G protein-coupled receptor of the invention
exhibits a detectable level of constitutive activity. In some
embodiments, the constitutive activity is for increasing a level of
intracellular cAMP accumulation. In some embodiments, the
constitutive activity is for causing melanophore cells to undergo
pigment dispersion.
[0107] In certain embodiments, stringent hybridization conditions
comprise hybridization at 42.degree. C. in a solution comprising
50% formamide, 5.times.SSC (1.times.SSC=150mM NaCl, 15 mM trisodium
citrate), 50 mM sodium phosphate (pH 7.6), 5.times. Denhardt's
solution, 10% dextran sulfate, and 20 m/ml denatured, sheared
salmon sperm DNA, followed by washing at 65.degree. C. in a
solution comprising 0.1.times.SSC. Hybridization techniques are
well known to the skilled artisan.
[0108] In certain embodiments, the GPCR encoded by a polynucleotide
hybridizing under stringent conditions to the complement of SEQ ID
NO: 1 is SEQ ID NO:2 or an allele thereof. In certain embodiments,
the GPCR encoded by a polynucleotide hybridizing under stringent
conditions to the complement of SEQ ID NO: 1 is an allele of SEQ ID
NO:2. In certain embodiments, the GPCR encoded by a polynucleotide
hybridizing under stringent conditions to the complement of SEQ ID
NO: 1 is an ortholog of SEQ ID NO:2. In certain embodiments, the
GPCR encoded by a polynucleotide hybridizing under stringent
conditions to the complement of SEQ ID NO: 1 specifically binds
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-4-yl}-amine. In certain embodiments, the GPCR
encoded by a polynucleotide hybridizing under stringent conditions
to the complement of SEQ ID NO: 1 is a receptor for which
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine is an agonist.
In some embodiments, the GPCR encoded by a polynucleotide
hybridizing under stringent conditions to the complement of SEQ ID
NO: 1 exhibits a detectable level of constitutive activity. In some
embodiments, the constitutive activity is for increasing a level of
intracellular cAMP accumulation. In some embodiments, the
constitutive activity is for causing melanophore cells to undergo
pigment dispersion.
[0109] In certain embodiments, the variant of SEQ ID NO:2 is a G
protein-coupled receptor having at least about 80% identity to SEQ
ID NO:2. In certain embodiments, the variant of SEQ ID NO:2 is a G
protein-coupled receptor comprising at least 20 contiguous amino
acids of SEQ ID NO:2. In certain embodiments, the variant of SEQ ID
NO:2 which is a G protein-coupled receptor is an endogenous G
protein-coupled receptor. In certain embodiments, the variant of
SEQ ID NO:2 which is a G protein-coupled receptor is a
non-endogenous G protein-coupled receptor.
[0110] In certain embodiments, the G protein-coupled receptor which
has at least about 80% identity to SEQ ID NO: 2 is SEQ ID NO: 2 or
an allele thereof. In certain embodiments, the G protein-coupled
receptor which has at least about 80% identity to SEQ ID NO: 2 is
an allele of SEQ ID NO: 2. In certain embodiments, the G
protein-coupled receptor which has at least about 80% identity to
SEQ ID NO: 2 is an ortholog of SEQ ID NO: 2. In certain
embodiments, the G protein-coupled receptor which has at least
about 80% identity to SEQ ID NO: 2 specifically binds
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine. In certain
embodiments, the G protein-coupled receptor which has at least
about 80% identity to SEQ ID NO: 2 is a receptor for which
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine is an agonist.
In some embodiments, the the G protein-coupled receptor which has
at least about 80% identity to SEQ ID NO: 2 exhibits a detectable
level of constitutive activity. In some embodiments, the
constitutive activity is for increasing a level of intracellular
cAMP. In some embodiments, the constitutive activity is for causing
melanophore cells to undergo pigment dispersion.
[0111] In certain embodiments, the G protein-coupled receptor which
comprises at least 20 contiguous amino acids of SEQ ID NO: 2 is SEQ
ID NO: 2 or an allele of SEQ ID NO: 2. In certain embodiments, the
G protein-coupled receptor which comprises at least 20 contiguous
amino acids of SEQ ID NO: 2 is an allele of SEQ ID NO: 2. In
certain embodiments, the G protein-coupled receptor which comprises
at least 20 contiguous amino acids of SEQ ID NO: 2 is an ortholog
of SEQ ID NO: 2. In certain embodiments, the G protein-coupled
receptor which comprises at least 20 contiguous amino acids of SEQ
ID NO: 2 specifically binds
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadizol-5--
yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine. In certain
embodiments, the G protein-coupled receptor which comprises at
least 20 contiguous amino acids of SEQ ID NO: 2 is a receptor for
which
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl]-5-nitro-pyrimidin-4-yl}-amine is an agonist. In some
embodiments, the G protein-coupled receptor which comprises at
least 20 contiguous amino acids of SEQ ID NO: 2 exhibits a
detectable level of constitutive activity. In some embodiments, the
constitutive activity is for increasing a level of intracellular
cAMP. In some embodiments, the constitutive activity is for causing
melanophore cells to undergo pigment dispersion.
[0112] In some embodiments, the G protein-coupled receptor is part
of a fusion protein comprising a G protein. Techniques for making a
GPCR:G fusion construct are well known to the skilled artisan (see,
e.g., International Application WO 02/42461).
[0113] In certain embodiments, the host cell comprises an
expression vector, said expression vector comprising a
polynucleotide encoding the G protein-coupled receptor. An
exemplary suitable expression vector is pCMV. Other suitable
expression vectors will be readily apparent to those of ordinary
skill in the art, and a wide variety of expression vectors are
commercially available (e.g., from Clontech, Palo Alto, Calif.;
Stratagene, La Jolla, Calif.; and Invitrogen, Carlsbad,
Calif.).
[0114] In some embodiments, the host cell is a vertebrate cell. In
some embodiments, the host cell is a mammalian cell. In some
embodiments, the mammalian host cell is selected from the group
consisting of a 293 cell, a 293T cell, a CHO cell, an MCB3901 cell,
and a COS-7 cell. In some embodiments, the host cell is a yeast
cell. In some embodiments, the host cell is a melanophore cell.
Other suitable host cells will be readily apparent to those of
ordinary skill in the art, and a wide variety of cell lines are
available from the American Type Culture Collection, 10801
University Boulevard, Manassas, Va. 20110-2209.
[0115] In certain embodiments, said determining is consistent with
the G protein-coupled receptor being a Gs-coupled receptor.
[0116] In some embodiments, said determining is consistent with the
G protein-coupled receptor being coupled through a promiscuous G
protein, such as Ga 15 or Ga16, to the phopholipase C pathway.
Promiscuous G proteins are well known to the skilled artisan (see,
e.g., Offermanns et al., J Biol Chem (1995) 270:15175-15180). In
some embodiments, said determining is consistent with the G
protein-coupled receptor being coupled through a chimeric G
protein, e.g. to the phospholipase C pathway. Chimeric G proteins
are well known to the skilled artisan (see, e.g., Milligan et al.,
Trends in Pharmaceutical Sciences (1999) 20:118-124; and WO
02/42461).
[0117] In some embodiments, said determining is through the
measurement of a level of a second messenger.
[0118] In some embodiments, said determining is through the
measurement of a level of a second messenger selected from the
group consisting of cyclic AMP (cAMP), cyclic GMP (cGMP), inositol
1,4,5-triphosphate (IP.sub.3), diacylglycerol (DAG), MAP kinase
activity, MAPK/ERK kinase kinase-1 (MEKK1) activity, and Ca.sup.2+.
In some preferred embodiments, the second messenger is cAMP. In
certain embodiments, a level of intracellular cAMP is
increased.
[0119] In certain embodiments, said determining is carried out
using isolated membrane comprising the G protein-coupled
receptor.
[0120] In certain embodiments, said determining is through the use
of a melanophore assay. In certain embodiments, a level of pigment
dispersion is increased.
[0121] In some embodiments, said determining is through a reporter
assay. In some embodiments, said reporter assay is CRE-Luc reporter
assay.
[0122] In some embodiments, said determining is through the
measurement of an activity mediated by increasing a level of
intracellular cAMP.
[0123] In some embodiments, said determining is through CRE-Luc
reporter assay. In certain embodiments, a level of luciferase
activity is increased.
[0124] In some embodiments, said determining is through the
measurement of GTPyS binding to membrane comprising the G
protein-coupled receptor. In certain embodiments, said GTPyS is
labeled with [.sup.35S]. In certain embodiments, said GTPyS binding
to membrane comprising the GPCR is increased.
[0125] In some embodiments, the test compound is a small molecule.
In some embodiments, the test compound is a small molecule, with
the proviso that the small molecule is not a polypeptide. In some
embodiments, the test compound is a small molecule, with the
proviso that the small molecule is not an antibody or an
antigen-binding fragment thereof. In some embodiments, the test
compound is a small molecule, with the proviso that the small
molecule is not a lipid. In some embodiments, the test compound is
a small molecule, with the proviso that the small molecule is not a
polypeptide or a lipid. In some embodiments, the test compound is a
polypeptide. In some embodiments, the test compound is a
polypeptide, with the proviso that the polypeptide is not an
antibody or an antigen-binding fragment thereof. In some
embodiments, the test compound is a lipid. In some embodiments, the
test compound is not an antibody or an antigen-binding fragment
thereof. In some embodiments, the test compound is an antibody or
an antigen-binding fragment thereof.
[0126] In some embodiments, the method further comprises the step
of optionally determining the structure of the PYY secretagogue,
the compound useful for treatment or prevention of a condition
modulated by PYY, or the compound useful for a modulation of a
PYY-related activity in an individual.
[0127] In some embodiments, the method further comprises the step
of optionally providing the name or structure of the PYY
secretagogue, the compound useful for treatment or prevention of a
condition modulated by PYY, or the compound useful for a modulation
of a PYY-related activity in an individual.
[0128] In some embodiments, said method further comprises the step
of optionally producing or synthesizing the PYY secretagogue, the
compound useful for treatment or prevention of a condition
modulated by PYY, or the compound useful for a modulation of a
PYY-related activity in an individual.
[0129] In some embodiments, said method further comprises the step
of formulating the PYY secretagogue, the compound useful for
treatment or prevention of a condition modulated by PYY, or the
compound useful for a modulation of a PYY-related activity in an
individual as a pharmaceutical.
[0130] In some embodiments, said method further comprises
evaluating the bioavailability of the PYY secretagogue, the
compound useful for treatment or prevention of a condition
modulated by PYY, or the compound useful for a modulation of a
PYY-related activity.
[0131] In some embodiments, said method further comprises the step
of formulating the PYY secretagogue, the compound useful for
treatment or prevention of a condition modulated by PYY, or the
compound useful for a modulation of a PYY-related activity in an
individual into a pharmaceutical composition.
[0132] In certain embodiments, the condition modulated by PYY is a
bone-related condition.
[0133] In certain embodiments, the bone-related condition is a
condition characterized by low bone mass. In certain embodiments,
the condition characterized by low bone mass is selected from the
group consisting of osteopenia, osteoporosis, rheumatoid arthritis,
osteoarthritis, periodontal disease, alveolar bone loss, osteotomy
bone loss, childhood idiopathic bone loss, Paget's disease, bone
loss due to metastatic cancer, osteolytic lesions, curvature of the
spine, and loss of height.
[0134] In certain embodiments, the bone-related condition is a bone
fracture. In certain embodiments, the bone fracture is selected
from the group consisting of a traumatic bone fracture, a long-term
bone fracture, and an osteoporotic bone fracture.
[0135] In certain embodiments, the bone-related condition is a bone
disease. In certain embodiments, the bone disease is selected from
the group consisting of osteopenia, osteoporosis, rheumatoid
arthritis, osteoarthritis, periodontal disease, alveolar bone loss,
osteotomy bone loss, childhood idiopathic bone loss, Paget's
disease, bone loss due to metastatic cancer, osteolytic lesions,
curvature of the spine, and loss of height.
[0136] In certain embodiments, the bone-related condition is
enhanced bone healing or bone growth, wherein said enhanced bone
healing or bone growth is selected from the group consisting of
enhanced bone healing following facial reconstruction, maxillary
reconstruction, mandibular reconstruction, periodontal disease or
tooth extraction, enhanced long bone extension, enhanced prosthetic
ingrowth, and increased bone synostosis.
[0137] In certain embodiments, the condition modulated by PYY is a
metabolic disorder.
[0138] In certain embodiments, the metabolic disorder is selected
from the group consisting of overweight, obesity, hyperphagia,
diabetes (inclusive of type 1 diabetes and type 2 diabetes), type 2
diabetes, impaired glucose tolerance, insulin resistance,
hyperinsulinemia, dyslipidemia, hypertension and metabolic
syndrome.
[0139] In certain embodiments, the condition modulated by PYY is an
angiogenesis-related condition.
[0140] In certain embodiments, the angiogeneisis-related condition
is selected from the group consisting of peripheral arterial
disease, wound healing and ischemia.
[0141] In certain embodiments, the condition modulated by PYY is an
ischemia-related condition.
[0142] In certain embodiments, the ischemia-related condition is
selected from the group consisting of ischemic stroke, ischemic
heart disease, myocardial infarction, ischemia-reperfusion injury,
post-myocardial infarction remodeling, heart failure, and
congestive heart failure.
[0143] In certain embodiments, the condition modulated by PYY is a
malabsorptive disorder.
[0144] In certain embodiments, the malabsorptive disorder is
selected from the group consisting of short bowel syndrome,
enterocolitis, Crohn's disease, ulcerative colitis, irritable bowel
syndrome, cholera, and a disorder accompanied by diarrhea.
[0145] In certain embodiments, the condition modulated by PYY is a
convulsive disorder.
[0146] In certain embodiments, the convulsive disorder is
epilepsy.
[0147] In certain embodiments, the condition modulated by PYY is a
a cancer.
[0148] In certain embodiments, the cancer is selected from the
group consisting of pancreatic cancer, breast cancer, colon cancer,
and Barrett's adenocarcinoma.
[0149] In certain embodiments, the condition modulated by PYY is an
inflammatory disorder.
[0150] In certain embodiments, the inflammatory disorder is
selected from the group consisting of atherosclerosis,
atherothrombosis, insulin resistance, diabetes (inclusive of type 1
diabetes and type 2 diabetes), type 1 diabetes, ischemic stroke,
pancreatitis, restenosis, an inflammatory lung disease, and an
inflammatory bowel disease. In certain embodiments, the
inflammatory lung disease is selected from the group consisting of
asthma, emphysema, and pulmonary arterial hypertension. In certain
embodiments, the inflammatory bowel disease is selected from the
group consisting of enteritis, enterocolitis, Crohn's disease,
granulomatous colitis, ileitis, Crohn's colitis, ulcerative
colitis, sigmoiditis, pancolitis, and ulcerative proctitis.
[0151] In certain embodiments, the modulation of a PYY-related
activity is selected from the group consisting of: [0152] (a)
increasing plasma PYY; [0153] (b) increasing bone mass; [0154] (c)
decreasing loss of bone mass; [0155] (d) increasing bone formation;
[0156] (e) decreasing food uptake; [0157] (f) decreasing body
weight; [0158] (g) increasing satiety; [0159] (h) increasing
glucose disposal; [0160] (i) decreasing plasma glucose; [0161] (j)
increasing angiogenesis; [0162] (k) decreasing secretion in
intestinal epithelium; [0163] (l) increasing absorption in
intestinal epithelium; and [0164] (m) increasing plasma adiponectin
[0165] The invention additionally features a process comprising
performing a method according to this first aspect.
[0166] In a second aspect, the invention features a method for
identifying PYY secretagogues, compounds useful for treatment or
prevention of a condition modulated by PYY, or compounds useful for
a modulation of a PYY-related activity, comprising the steps of:
[0167] (a) contacting a compound in vitro with a vertebrate
enteroendocrine cell or with a cell capable of secreting PYY, said
compound having been identified by a method according to the first
aspect; and [0168] (b) determining whether the compound stimulates
PYY secretion from the vertebrate enteroendocrine cell or from the
cell capable of secreting PYY; wherein the ability of the test
compound to stimulate PYY secretion from the vertebrate
enteroendocrine cell or from the cell capable of secreting PYY is
further indicative of the test compound being a PYY secretagogue, a
compound useful for treatment or prevention of a PYY-related
condition, or a compound useful for a modulation of a PYY-related
activity.
[0169] In certain embodiments, the vertebrate enteroendocrine cell
is a mammalian enteroendocrine cell. In certain embodiments, the
enteroendocrine cell is an L cell. In certain embodiments, the
enteroendocrine cell is comprised by or derived from tissue
obtained from the small or large intestine. In certain embodiments,
the enteroendocrine cell is comprised by or derived from tissue
obtained from an L cell-containing region of small or large
intestine. In certain embodiments, the enteroendocrine cell is
comprised by or derived from duodenum, jejunum, ileum, colon or
rectum tissue (see, e.g., Lundberg et al, PNAS USA (1982)
79:4471-4475; Adrian et al, Gastroenterol (1985) 89:1070-1077;
Ekblad et al, Peptides (2002) 23:251-261; Ueno et al, Regul Pept
(2008) 145:12-16). In certain embodiments, the enteroendocrine cell
is an enteroendocrine cell line. In certain embodiments, the cell
capable of secreting PYY is a cell comprised by or derived from the
islets of Langerhans. In certain embodiments, the cell capable of
secreting PYY is a neuronal cell. In certain embodiments, the cell
capable of secreting PYY is a recombinant cell engineered to be
capable of secreting PYY.
[0170] The invention additionally features a method for identifying
PYY secretagogues, compounds useful for treatment or prevention of
a condition modulated by PYY, or compounds useful for a modulation
of a PYY-related activity in an individual, comprising the steps
of:
[0171] (a) administering a compound to a vertebrate, said compound
having been identified by a method according to the first aspect;
and
[0172] (b) determining whether the compound increases a PYY level
in the vertebrate;
wherein the ability of the test compound to increase a PYY level in
the vertebrate is further indicative of the test compound being a
PYY secretagogue, a compound useful for treatment or prevention of
a PYY-related condition, or a compound useful for a modulation of a
PYY-related activity in an individual.
[0173] In certain embodiments, the PYY level is blood or plasma
concentration of total PYY. In certain embodiments, the PYY level
is blood or plasma concentration of PYY.sub.3-36.
[0174] In certain embodiments, the vertebrate is a mammal. In
certain embodiments, the vertebrate is a non-human vertebrate. In
certain embodiments, the vertebrate is a non-human mammal. In
certain embodiments, the mammal is a non-human mammal.
[0175] In certain embodiments, the vertebrate or the mammal is a
human. In certain embodiments, the human is a participant in a
study reviewed by a governmental agency charged with marketing
approval for a drug. In certain embodiments, the study is a
clinical trial. In certain embodiments, the governmental agency is
the Food and Drug Administration of the United States.
[0176] The invention additionally features a method for identifying
PYY secretagogues, compounds useful for treatment or prevention of
a condition modulated by PYY, or compounds useful for a modulation
of a PYY-related activity in an individual, comprising the steps
of: [0177] (a) determining whether the compound increases a PYY
level in a sample, said sample having been obtained from a
vertebrate previously administered with a compound which stimulates
functionality of the receptor in step (b); wherein the ability of
the test compound to increase a PYY level in the vertebrate is
further indicative of the test compound being a PYY secretagogue, a
compound useful for treatment or prevention of a condition
modulated by PYY, or a compound useful for a modulation of a
PYY-related activity in an individual.
[0178] In certain embodiments, the PYY level is blood or plasma
concentration of total PYY. In certain embodiments, the PYY level
is blood or plasma concentration of PYY.sub.3-36.
[0179] In certain embodiments, the vertebrate is a mammal. In
certain embodiments, the vertebrate is a non-human vertebrate. In
certain embodiments, the vertebrate is a non-human mammal. In
certain embodiments, the mammal is a non-human mammal.
[0180] In certain embodiments, the vertebrate is a mammal. In
certain embodiments, the vertebrate is a non-human vertebrate. In
certain embodiments, the vertebrate is a non-human mammal.
[0181] In certain embodiments, the vertebrate or the mammal is a
human. In certain embodiments, the human is a participant in a
study reviewed by a governmental agency charged with marketing
approval for a drug. In certain embodiments, the study is a
clinical trial. In certain embodiments, the governmental agency is
the Food and Drug Administration of the United States.
[0182] In some embodiments, the test compound is a small molecule.
In some embodiments, the test compound is a small molecule, with
the proviso that the small molecule is not a polypeptide. In some
embodiments, the test compound is a small molecule, with the
proviso that the small molecule is not an antibody or an
antigen-binding fragment thereof. In some embodiments, the test
compound is a small molecule, with the proviso that the small
molecule is not a lipid. In some embodiments, the test compound is
a small molecule, with the proviso that the small molecule is not a
polypeptide or a lipid. In some embodiments, the test compound is a
polypeptide. In some embodiments, the test compound is a
polypeptide, with the proviso that the polypeptide is not an
antibody or an antigen-binding fragment thereof. In some
embodiments, the test compound is a lipid. In some embodiments, the
test compound is not an antibody or an antigen-binding fragment
thereof. In some embodiments, the test compound is an antibody or
an antigen-binding fragment thereof.
[0183] In some embodiments, the method further comprises the step
of optionally determining the structure of the PYY secretagogue,
the compound useful for treatment or prevention of a condition
modulated by PYY, or the compound useful for a modulation of a
PYY-related activity in an individual.
[0184] In some embodiments, the method further comprises the step
of optionally providing the name or structure of the PYY
secretagogue, the compound useful for treatment or prevention of a
condition modulated by PYY, or the compound useful for a modulation
of a PYY-related activity in an individual.
[0185] In some embodiments, said method further comprises the step
of optionally producing or synthesizing the PYY secretagogue, the
compound useful for treatment or prevention of a condition
modulated by PYY, or the compound useful for a modulation of a
PYY-related activity in an individual.
[0186] In some embodiments, said method further comprises the step
of formulating the PYY secretagogue, the compound useful for
treatment or prevention of a condition modulated by PYY, or the
compound useful for a modulation of a PYY-related activity in an
individual as a pharmaceutical.
[0187] In some embodiments, said method further comprises
evaluating the bioavailability of the PYY secretagogue, the
compound useful for treatment or prevention of a condition
modulated by PYY, or the compound useful for a modulation of a
PYY-related activity.
[0188] In some embodiments, said method further comprises the step
of formulating the PYY secretagogue, the compound useful for
treatment or prevention of a condition modulated by PYY, or the
compound useful for a modulation of a PYY-related activity in an
individual into a pharmaceutical composition.
[0189] The invention additionally features a process comprising
performing a method according to this second aspect.
[0190] In a third aspect, the invention features a method for
identifying PYY secretagogues, compounds useful for treatment or
prevention of a condition modulated by PYY, or compounds useful for
a modulation of a PYY-related activity in an individual, comprising
the steps of: [0191] (a) contacting a GPR119 agonist in vitro with
a vertebrate enteroendocrine cell or with a cell capable of
secreting PYY; and [0192] (b) determining whether the GPR119
agonist stimulates PYY secretion from the vertebrate
enteroendocrine cell or from the cell capable of secreting PYY;
wherein the ability of the GPR119 agonist to stimulate PYY
secretion from the vertebrate enteroendocrine cell or from the cell
capable of secreting PYY is indicative of the GPR119 agonist being
a PYY secretagogue, a compound useful for treatment or prevention
of a condition modulated by PYY, or a compound useful for a
modulation of a PYY-related activity in an individual.
[0193] In certain embodiments, the vertebrate enteroendocrine cell
is a mammalian enteroendocrine cell. In certain embodiments, the
enteroendocrine cell is an L cell. In certain embodiments, the
enteroendocrine cell is comprised by or derived from tissue
obtained from the small or large intestine. In certain embodiments,
the enteroendocrine cell is comprised by or derived from tissue
obtained from an L cell-containing region of small or large
intestine. In certain embodiments, the enteroendocrine cell is
comprised by or derived from duodenum, jejunum, ileum, colon or
rectum tissue (see, e.g., Lundberg et al, PNAS USA (1982)
79:4471-4475; Adrian et al, Gastroenterol (1985) 89:1070-1077;
Ekblad et al, Peptides (2002) 23:251-261; Ueno et al, Regul Pept
(2008) 145:12-16). In certain embodiments, the enteroendocrine cell
is an enteroendocrine cell line. In certain embodiments, the cell
capable of secreting PYY is a cell comprised by or derived from the
islets of Langerhans. In certain embodiments, the cell capable of
secreting PYY is a neuronal cell. In certain embodiments, the cell
capable of secreting PYY is a recombinant cell engineered to be
capable of secreting PYY.
[0194] The invention additionally features a method for identifying
PYY secretagogues, compounds useful for treatment or prevention of
a condition modulated by PYY, or compounds useful for a modulation
of a PYY-related activity in an individual, comprising the steps
of:
[0195] (a) administering a GPR119 agonist to a vertebrate; and
[0196] (b) determining whether the GPR119 agonist increases a PYY
level in the vertebrate;
wherein the ability of the GPR119 agonist to increase a PYY level
in the vertebrate is indicative of the GPR119 agonist being a PYY
secretagogue, a compound useful for treatment or prevention of a
condition modulated by PYY, or a compound useful for a modulation
of a PYY-related activity in an individual.
[0197] In certain embodiments, the PYY level is blood or plasma
concentration of total PYY. In certain embodiments, the PYY level
is blood or plasma concentration of PYY.sub.3-36.
[0198] In certain embodiments, the vertebrate is a mammal. In
certain embodiments, the vertebrate is a non-human vertebrate. In
certain embodiments, the vertebrate is a non-human mammal. In
certain embodiments, the mammal is a non-human mammal.
[0199] In certain embodiments, the vertebrate or the mammal is a
human. In certain embodiments, the human is a participant in a
study reviewed by a governmental agency charged with marketing
approval for a drug. In certain embodiments, the study is a
clinical trial. In certain embodiments, the governmental agency is
the Food and Drug Administration of the United States.
[0200] The invention additionally features a method for identifying
PYY secretagogues, compounds useful for treatment or prevention of
a condition modulated by PYY, or compounds useful for a modulation
of a PYY-related activity in an individual, comprising the steps
of: [0201] (a) determining whether the compound increases a PYY
level in a sample, said sample having been obtained from a
vertebrate previously administered with a GPR119 agonist;
administering a GPR119 agonist to a vertebrate; wherein the ability
of the GPR119 agonist to increase a PYY level in the vertebrate is
indicative of the GPR119 agonist being a PYY secretagogue, a
compound useful for treatment or prevention of a condition
modulated by PYY, or a compound useful for a modulation of a
PYY-related activity in an individual.
[0202] In certain embodiments, the PYY level is blood or plasma
concentration of total PYY. In certain embodiments, the PYY level
is blood or plasma concentration of PYY.sub.3-36.
[0203] In certain embodiments, the vertebrate is a mammal. In
certain embodiments, the vertebrate is a non-human vertebrate. In
certain embodiments, the vertebrate is a non-human mammal. In
certain embodiments, the mammal is a non-human mammal
[0204] In certain embodiments, the vertebrate or the mammal is a
human. In certain embodiments, the human is a participant in a
study reviewed by a governmental agency charged with marketing
approval for a drug. In certain embodiments, the study is a
clinical trial. In certain embodiments, the governmental agency is
the Food and Drug Administration of the United States.
[0205] In certain embodiments, the GPR119 agonist is an agonist of
an endogenous GPR119.
[0206] In certain embodiments, the GPR119 agonist is an agonist of
human GPR119.
[0207] In certain embodiments, the GPR119 agonist is a GPR119
partial agonist.
[0208] In certain embodiments, the GPR119 agonist is a selective
GPR119 agonist.
[0209] In certain embodiments, the GPR119 agonist is a small
molecule. In some embodiments, the small molecule is not a
polypeptide. In some embodiments, the small molecule is not an
antibody or an antigen-binding fragment thereof. In some
embodiments, the small molecule is not a lipid. In some
embodiments, the small molecule is not a polypeptide or a
lipid.
[0210] In certain embodiments, the GPR119 agonist is orally
available. In certain embodiments, the GPR119 agonist has an
EC.sub.50 value of less than about 10 .mu.M, less than about 1
.mu.M, less than about 100 nM, less than about 75 nM, less than
about 50 nM, less than about 25 nM, less than about 15 nM, less
than about 10 nM, less than about 5 nM, less than about 4 nM, less
than about 3 nM, less than about 2 nM, or less than about 1 nM. In
certain embodiments, the GPR119 agonist has an EC.sub.50 value of
less than about 10 .mu.M, less than about 1 .mu.M, less than about
100 nM, less than about 75 nM, less than about 50 nM, less than
about 25 nM, less than about 15 nM, less than about 10 nM, less
than about 5 nM, less than about 4 nM, less than about 3 nM, less
than about 2 nM, or less than about 1 nM at human GPR119 having SEQ
ID NO: 2. In certain embodiments, the GPR119 agonist has an
EC.sub.50 value of less than about 10 .mu.M, less than about 1
.mu.M, less than about 100 nM, less than 75 nM, less than about 50
nM, less than about 25 nM, less than about 15 nM, less than about
10 nM, less than about 5 nM, less than about 4 nM, less than about
3 nM, less than about 2 nM, or less than about 1 nM at human GPR119
having SEQ ID NO: 2 in adenylyl cyclase assay (exemplary adenylyl
cyclase assay is provided in Example 9, Example 10 and in Example
11, infra). In certain embodiments, the GPR119 agonist has an
EC.sub.50 value of less than about 10 .mu.M, less than about 1
.mu.M, less than about 100 nM, less than about 75 nM, less than
about 50 nM, less than about 25 nM, less than about 15 nM, less
than about 10 nM, less than about 5 nM, less than about 4 nM, less
than about 3 nM, less than about 2 nM, or less than about 1 nM at
human GPR119 having SEQ ID NO: 2 in melanophore assay (exemplary
melanophore assay is provided in Example 12, infra).
[0211] Exemplary GPR119 agonists are disclosed, e.g., in
International Application No. PCT/US2004/001267 (published as WO
04/065380); International Application No. PCT/US2004/005555
(published as WO 04/076413); International Application No.
PCT/US2004/022327 (published as WO 05/007647); International
Application No. PCT/US2004/022417 (published as WO 05/007658);
International Application No. PCT/US2005/019318 (published as WO
2005/121121); International Application No. PCT/GB2004/050046
(published as WO 2005/061489); International Application No.
PCT/US06/00567 (published as WO 2006/083491); International
Application No. PCT/GB2005/050264 (published as WO 2006/067531);
International Application No. PCT/GB2005/050265 (published as WO
2006/067532); International Application No. PCT/GB2005/050266
(published as WO 2006/070208); International Application No.
PCT/JP02/09350 (published as WO 03/026661); International
Application No. PCT/JP2005/018412 (published as WO 06/040966);
International Application No. PCT/JP2005/019000 (published as WO
2006/043490); International Application No. PCT/GB2006/050176
(published as WO 2007/003960); International Application No.
PCT/GB2006/050177 (published as WO 2007/003961); International
Application No. PCT/GB2006/050178 (published as WO 2007/003962);
International Application No. PCT/GB2006/050182 (published as WO
2007/003964); International Application No. PCT/JP02/09350
(published as WO 03/026661); International Application No.
PCT/GB2007/050183 (published as WO 2007/116229); International
Application No. PCT/GB2007/050190 (published as WO 2007/116230);
International Application No. PCT/US2007/073364 (published as WO
2008/008895); International Application No. PCT/EP2007/058991
(published as WO 2008/025798); International Application No.
PCT/EP2007/058993 (published as WO 2008/025799); and International
Application No. PCT/EP2007/058995 (published as WO
2008/025800).
[0212] In certain embodiments, the method comprises providing the
GPR119 agonist.
[0213] In certain embodiments, the GPR119 agonist is identifiable
by a method according to the first aspect.
[0214] In certain embodiments, the method comprises carrying out a
method according to the first aspect to identify the GPR119
agonist.
[0215] In certain embodiments, the method comprises having
identified the GPR119 agonist by a method according to the first
aspect.
[0216] In some embodiments, the method further comprises the step
of optionally determining the structure of the PYY secretagogue,
the compound useful for treatment or prevention of a condition
modulated by PYY, or the compound useful for a modulation of a
PYY-related activity in an individual.
[0217] In some embodiments, the method further comprises the step
of optionally providing the name or structure of the PYY
secretagogue, the compound useful for treatment or prevention of a
condition modulated by PYY, or the compound useful for a modulation
of a PYY-related activity in an individual.
[0218] In some embodiments, said method further comprises the step
of optionally producing or synthesizing the PYY secretagogue, the
compound useful for treatment or prevention of a condition
modulated by PYY, or the compound useful for a modulation of a
PYY-related activity in an individual.
[0219] In some embodiments, said method further comprises the step
of formulating the PYY secretagogue, the compound useful for
treatment or prevention of a condition modulated by PYY, or the
compound useful for a modulation of a PYY-related activity in an
individual as a pharmaceutical.
[0220] In some embodiments, said method further comprises
evaluating the bioavailability of the PYY secretagogue, the
compound useful for treatment or prevention of a condition
modulated by PYY, or the compound useful for a modulation of a
PYY-related activity.
[0221] In some embodiments, said method further comprises the step
of formulating the PYY secretagogue, the compound useful for
treatment or prevention of a condition modulated by PYY, or the
compound useful for a modulation of a PYY-related activity in an
individual into a pharmaceutical composition.
[0222] The invention additionally features a process comprising
performing a method according to this third aspect.
[0223] In a fourth aspect, the invention features a method for
identifying PYY secretagogues, compounds useful for treatment or
prevention of a condition modulated by PYY, or compounds useful for
a modulation of a PYY-related activity in an individual, comprising
the steps of: [0224] (a) contacting a G protein-coupled receptor
with an optionally labeled known ligand for the receptor in the
presence or absence of a test compound, wherein a ligand for the
receptor is a ligand for GPR119 and wherein the G protein-coupled
receptor comprises an amino acid sequence selected from the group
consisting of: [0225] (i) amino acids 1-335 of SEQ ID NO:2; [0226]
(ii) amino acids 2-335 of SEQ ID NO:2; [0227] (iii) an amino acid
sequence comprising the amino acid sequence of a polypeptide
derived from (i) by substitution, deletion or addition of one or
several amino acids in the amino acid sequence defined in (i);
[0228] (iv) the amino acid sequence of a G protein-coupled receptor
encoded by a polynucleotide hybridizing under stringent conditions
to the complement of SEQ ID NO:1; [0229] (v) a variant of SEQ ID
NO: 2 when selected from the group consisting of: [0230] (a') the
amino acid sequence of a G protein-coupled receptor having at least
about 80% identity to SEQ ID NO: 2; and [0231] (b') the amino acid
sequence of a G protein-coupled receptor comprising at least 20
contiguous amino acids of SEQ ID NO: 2; [0232] (vi) the amino acid
sequence of a constitutively active version of a G protein-coupled
receptor having SEQ ID NO: 2; and [0233] (vii) a biologically
active fragment of any one of (i) to (vi); and [0234] (b) detecting
the complex between said known ligand and said receptor; and [0235]
(c) determining whether less of said complex is formed in the
presence of the test compound than in the absence of the test
compound; wherein said determination is indicative of the test
compound being a PYY secretagogue, a compound useful for treatment
or prevention of a condition modulated by PYY, or a compound useful
for a modulation of a PYY-related activity in an individual.
[0236] In certain embodiments, the G protein-coupled receptor
comprises the amino acid sequence of a G protein-coupled receptor
having at least about 80% identity to SEQ ID NO: 2.
[0237] In certain embodiments, the receptor comprises the amino
acid sequence of SEQ ID NO: 2.
[0238] In certain embodiments, the variant of SEQ ID NO: 2 is an
allele of SEQ ID NO: 2.
[0239] In certain embodiments, the variant of SEQ ID NO: 2 is an
ortholog of SEQ ID NO: 2. In certain embodiments, the variant of
SEQ ID NO: 2 is a mammalian ortholog of SEQ ID NO: 2.
[0240] In certain embodiments, the G protein-coupled receptor is
recombinant.
[0241] In certain embodiments, the method is a method for
identifying PYY secretagogues.
[0242] In certain embodiments, the method is a method for
identifying compounds useful for treatment or prevention of a
condition modulated by PYY.
[0243] In certain embodiments, the method is a method for
identifying compounds useful for a modulation of a PYY-related
activity in an individual.
[0244] In certain embodiments, the ligand for GPR119 is a GPR119
agonist. In certain embodiments, the ligand for GPR119 or the
GPR119 agonist is a ligand or agonist of an endogenous vertebrate,
mammalian or human GPR119 receptor. In certain embodiments, the
ligand for GPR119 or the GPR119 agonist is a ligand or agonist of
an endogenous human GPR119 receptor. In certain embodiments, the
ligand for GPR119 or the GPR119 agonist is identical to a compound
disclosed in, e.g., in International Application No.
PCT/US2004/001267 (published as WO 04/065380); International
Application No. PCT/US2004/005555 (published as WO 04/076413);
International Application No. PCT/US2004/022327 (published as WO
05/007647); International Application No. PCT/US2004/022417
(published as WO 05/007658); International Application No.
PCT/US2005/019318 (published as WO 2005/121121); International
Application No. PCT/GB2004/050046 (published as WO 2005/061489);
International Application No. PCT/US06/00567 (published as WO
2006/083491); International Application No. PCT/GB2005/050264
(published as WO 2006/067531); International Application No.
PCT/GB2005/050265 (published as WO 2006/067532); International
Application No. PCT/GB2005/050266 (published as WO 2006/070208);
International Application No. PCT/JP02/09350 (published as WO
03/026661); International Application No. PCT/JP2005/018412
(published as WO 06/040966); International Application No.
PCT/JP2005/019000 (published as WO 2006/043490); International
Application No. PCT/GB2006/050176 (published as WO 2007/003960);
International Application No. PCT/GB2006/050177 (published as WO
2007/003961); International Application No. PCT/GB2006/050178
(published as WO 2007/003962); International Application No.
PCT/GB2006/050182 (published as WO 2007/003964); International
Application No. PCT/JP02/09350 (published as WO 03/026661);
International Application No. PCT/GB2007/050183 (published as WO
2007/116229); International Application No. PCT/GB2007/050190
(published as WO 2007/116230); International Application No.
PCT/US2007/073364 (published as WO 2008/008895); International
Application No. PCT/EP2007/058991 (published as WO 2008/025798);
International Application No. PCT/EP2007/058993 (published as WO
2008/025799); or International Application No. PCT/EP2007/058995
(published as WO 2008/025800). In certain embodiments, the ligand
for GPR119 or the GPR119 agonist is
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadizol-5--
yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine. In certain
embodiments, the ligand for GPR119 is an endogenous ligand of an
endogenous vertebrate, mammalian, or human GPR119 receptor.
[0245] In certain embodiments, the optionally labeled known ligand
is a labeled known ligand. In certain embodiments, the labeled
known ligand is a radiolabeled known ligand. Techniques for
radiolabeling a compound, such as for labeling a known ligand of a
G protein-coupled receptor of the invention, are well known to the
skilled artisan. See, e.g., International Application WO 04/065380.
Also see, e.g., Example 14, infra.
[0246] Techniques for detecting the complex between a G
protein-coupled receptor and a compound known to be a ligand for
the G protein-coupled receptor are well known to the skilled
artisan. See, e.g., International Application WO 04/065380. Also
see, e.g., Example 15, infra.
[0247] The invention additionally features a method for identifying
PYY secretagogues, compounds useful for treatment or prevention of
a condition modulated by PYY, or compounds useful for a modulation
of a PYY-related activity in an individual, comprising steps (a) to
(c) of this fourth aspect, and further comprising: [0248] (d)
optionally synthesizing a compound in the presence of which less of
said complex is formed in step (c); [0249] (e) contacting a
compound in the presence of which less of said complex is formed in
step (c) in vitro with a vertebrate enteroendocrine cell or with a
cell capable of secreting PYY; and [0250] (f) determining whether
the compound stimulates PYY secretion from the vertebrate
enteroendocrine cell or from the cell capable of secreting PYY;
wherein the ability of the test compound to stimulate PYY secretion
from the vertebrate enteroendocrine cell or from the cell capable
of secreting PYY is further indicative of the test compound being a
PYY secretagogue, a compound useful for treatment or prevention of
a condition modulated by PYY, or a compound useful for a modulation
of a PYY-related activity in an individual.
[0251] In certain embodiments, the vertebrate enteroendocrine cell
is a mammalian enteroendocrine cell. In certain embodiments, the
enteroendocrine cell is an L cell. In certain embodiments, the
enteroendocrine cell is comprised by or derived from tissue
obtained from the small or large intestine. In certain embodiments,
the enteroendocrine cell is comprised by or derived from tissue
obtained from an L cell-containing region of small or large
intestine. In certain embodiments, the enteroendocrine cell is
comprised by or derived from duodenum, jejunum, ileum, colon or
rectum tissue (see, e.g., Lundberg et al, PNAS USA (1982)
79:4471-4475; Adrian et al, Gastroenterol (1985) 89:1070-1077;
Ekblad et al, Peptides (2002) 23:251-261; Ueno et al, Regul Pept
(2008) 145:12-16). In certain embodiments, the enteroendocrine cell
is an enteroendocrine cell line. In certain embodiments, the cell
capable of secreting PYY is a cell comprised by or derived from the
islets of Langerhans. In certain embodiments, the cell capable of
secreting PYY is a neuronal cell. In certain embodiments, the cell
capable of secreting PYY is a recombinant cell engineered to be
capable of secreting PYY.
[0252] The invention additionally features a method for identifying
PYY secretagogues, compounds useful for treatment or prevention of
a condition modulated by PYY, or compounds useful for a modulation
of a PYY-related activity in an individual, comprising steps (a) to
(c) of this fourth aspect, and further comprising: [0253] (d)
optionally synthesizing a compound in the presence of which less of
said complex is formed in step (c); [0254] (e) administering a
compound in the presence of which less of said complex is formed in
step (c) to a vertebrate; and [0255] (f) determining whether the
compound increases a PYY level in the vertebrate; wherein the
ability of the test compound to increase a PYY level in the
vertebrate is further indicative of the test compound being a PYY
secretagogue, a compound useful for treatment or prevention of a
condition modulated by PYY, or a compound useful for a modulation
of a PYY-related activity in an individual.
[0256] In certain embodiments, the vertebrate is a mammal. In
certain embodiments, the vertebrate is a non-human vertebrate. In
certain embodiments, the vertebrate is a non-human mammal. In
certain embodiments, the mammal is a non-human mammal
[0257] In certain embodiments, the vertebrate or the mammal is a
human. In certain embodiments, the human is a participant in a
study reviewed by a governmental agency charged with marketing
approval for a drug. In certain embodiments, the study is a
clinical trial. In certain embodiments, the governmental agency is
the Food and Drug Administration of the United States.
[0258] The invention additionally features a method for identifying
PYY secretagogues, compounds useful for treatment or prevention of
a condition modulated by PYY, or compounds useful for a modulation
of a PYY-related activity in an individual, comprising steps (a) to
(c) of this fourth aspect, and further comprising: [0259] (d)
optionally synthesizing a compound in the presence of which less of
said complex is formed in step (c); and [0260] (e) determining
whether the compound increases a PYY level in a sample obtained
from a vertebrate, said vertebrate having been administered with a
compound in the presence of which less of said complex is formed in
step (c); wherein the ability of the test compound to increase a
PYY level in the vertebrate is indicative of the test compound
being PYY secretagogue, a compound useful for treatment or
prevention of a condition modulated by PYY, or a compound useful
for a modulation of a PYY-related activity in an individual.
[0261] In certain embodiments, the vertebrate is a mammal. In
certain embodiments, the vertebrate is a non-human vertebrate. In
certain embodiments, the vertebrate is a non-human mammal. In
certain embodiments, the mammal is a non-human mammal.
[0262] In certain embodiments, the vertebrate or the mammal is a
human. In certain embodiments, the human is a participant in a
study reviewed by a governmental agency charged with marketing
approval for a drug. In certain embodiments, the study is a
clinical trial. In certain embodiments, the governmental agency is
the Food and Drug Administration of the United States.
[0263] The invention additionally features a method for identifying
PYY secretagogues, compounds useful for treatment or prevention of
a condition modulated by PYY, or compounds useful for a modulation
of a PYY-related activity in an individual, comprising steps (a) to
(c) of this fourth aspect, and further comprising: [0264] (d)
optionally synthesizing a compound in the presence of which less of
said complex is formed according to step (c); [0265] (e) optionally
providing a compound in the presence of which less of said complex
is formed according to step (c); [0266] (f) contacting a compound
in the presence of which less of said complex is formed according
to step (c) in vitro with a vertebrate enteroendocrine cell or with
a cell capable of secreting PYY; and [0267] (g) determining whether
the compound stimulates PYY secretion from the vertebrate
enteroendocrine cell or from the cell capable of secreting PYY;
wherein the ability of the test compound to stimulate PYY secretion
from the vertebrate enteroendocrine cell or from the cell capable
of secreting PYY is further indicative of the test compound being a
PYY secretagogue, a compound useful for treatment or prevention of
a condition modulated by PYY, or a compound useful for a modulation
of a PYY-related activity in an individual.
[0268] In certain embodiments, the vertebrate enteroendocrine cell
is a mammalian enteroendocrine cell. In certain embodiments, the
enteroendocrine cell is an L cell. In certain embodiments, the
enteroendocrine cell is comprised by or derived from tissue
obtained from the small or large intestine. In certain embodiments,
the enteroendocrine cell is comprised by or derived from tissue
obtained from an L cell-containing region of small or large
intestine. In certain embodiments, the enteroendocrine cell is
comprised by or derived from duodenum, jejunum, ileum, colon or
rectum tissue (see, e.g., Lundberg et al, PNAS USA (1982)
79:4471-4475; Adrian et al, Gastroenterol (1985) 89:1070-1077;
Ekblad et al, Peptides (2002) 23:251-261; Ueno et al, Regul Pept
(2008) 145:12-16). In certain embodiments, the enteroendocrine cell
is an enteroendocrine cell line. In certain embodiments, the cell
capable of secreting PYY is a cell comprised by or derived from the
islets of Langerhans. In certain embodiments, the cell capable of
secreting PYY is a neuronal cell. In certain embodiments, the cell
capable of secreting PYY is a recombinant cell engineered to be
capable of secreting PYY.
[0269] The invention additionally features a method for identifying
PYY secretagogues, compounds useful for treatment or prevention of
a condition modulated by PYY, or compounds useful for a modulation
of a PYY-related activity in an individual, comprising steps (a) to
(c) of this fourth aspect, and further comprising: [0270] (d)
optionally synthesizing a compound in the presence of which less of
said complex is formed according to step (c); [0271] (e) optionally
providing a compound in the presence of which less of said complex
is formed according to step (c); [0272] (f) administering a
compound in the presence of which less of said complex is formed in
step (c) to a vertebrate; and [0273] (g) determining whether the
compound increases a PYY level in the vertebrate; wherein the
ability of the test compound to increase a PYY level in the
vertebrate is further indicative of the test compound being a PYY
secretagogue, a compound useful for treatment or prevention of a
condition modulated by PYY, or a compound useful for a modulation
of a PYY-related activity in an individual.
[0274] In certain embodiments, the vertebrate is a mammal. In
certain embodiments, the vertebrate is a non-human vertebrate. In
certain embodiments, the vertebrate is a non-human mammal. In
certain embodiments, the mammal is a non-human mammal
[0275] In certain embodiments, the vertebrate or the mammal is a
human. In certain embodiments, the human is a participant in a
study reviewed by a governmental agency charged with marketing
approval for a drug. In certain embodiments, the study is a
clinical trial. In certain embodiments, the governmental agency is
the Food and Drug Administration of the United States.
[0276] The invention additionally features a method for identifying
PYY secretagogues, compounds useful for treatment or prevention of
a condition modulated by PYY, or compounds useful for a modulation
of a PYY-related activity in an individual, comprising steps (a) to
(c) of this fourth aspect, and further comprising: [0277] (d)
optionally synthesizing a compound in the presence of which less of
said complex is formed according to step (c); [0278] (e) optionally
providing a compound in the presence of which less of said complex
is formed according to step (c); and [0279] (f) determining whether
the compound increases a PYY level in a sample obtained from a
vertebrate, said vertebrate having been administered with a
compound in the presence of which less of said complex is formed in
step (c); wherein the ability of the test compound to increase a
PYY level in the vertebrate is further indicative of the test
compound being a PYY secretagogue, a compound useful for treatment
or prevention of a condition modulated by PYY, or a compound useful
for a modulation of a PYY-related activity in an individual.
[0280] In certain embodiments, the vertebrate is a mammal. In
certain embodiments, the vertebrate is a non-human vertebrate. In
certain embodiments, the vertebrate is a non-human mammal. In
certain embodiments, the mammal is a non-human mammal
[0281] In certain embodiments, the vertebrate or the mammal is a
human. In certain embodiments, the human is a participant in a
study reviewed by a governmental agency charged with marketing
approval for a drug. In certain embodiments, the study is a
clinical trial. In certain embodiments, the governmental agency is
the Food and Drug Administration of the United States.
[0282] In some embodiments, a G protein-coupled receptor of the
invention is SEQ ID NO:2 or an allele thereof. In some embodiments,
a G protein-coupled receptor of the invention is an allele of SEQ
ID NO:2. In some embodiments, a G protein-coupled receptor of the
invention is an ortholog of SEQ ID NO:2.
[0283] In some embodiments, a G protein-coupled receptor of the
invention is a G protein-coupled receptor which specifically binds
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine. In some
embodiments, a G protein-coupled receptor of the invention is a G
protein-coupled receptor for which
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine is a ligand. In
some embodiments, a G protein-coupled receptor of the invention is
a G protein-coupled receptor for which
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine is an agonist.
In some embodiments, a G protein-coupled receptor of the invention
exhibits a detectable level of constitutive activity. In some
embodiments, the constitutive activity is for increasing a level of
intracellular cAMP accumulation. In some embodiments, the
constitutive activity is for causing melanophore cells to undergo
pigment dispersion.
[0284] In certain embodiments, stringent hybridization conditions
comprise hybridization at 42.degree. C. in a solution comprising
50% formamide, 5.times.SSC (1.times.SSC=150 mM NaCl, 15 mM
trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5.times.
Denhardt's solution, 10% dextran sulfate, and 20 m/ml denatured,
sheared salmon sperm DNA, followed by washing at 65.degree. C. in a
solution comprising 0.1.times.SSC. Hybridization techniques are
well known to the skilled artisan.
[0285] In certain embodiments, the GPCR encoded by a polynucleotide
hybridizing under stringent conditions to the complement of SEQ ID
NO: 1 is SEQ ID NO:2 or an allele thereof. In certain embodiments,
the GPCR encoded by a polynucleotide hybridizing under stringent
conditions to the complement of SEQ ID NO: 1 is an allele of SEQ ID
NO:2. In certain embodiments, the GPCR encoded by a polynucleotide
hybridizing under stringent conditions to the complement of SEQ ID
NO: 1 is an ortholog of SEQ ID NO:2. In certain embodiments, the
GPCR encoded by a polynucleotide hybridizing under stringent
conditions to the complement of SEQ ID NO: 1 specifically binds
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-yl}-amine. In certain embodiments, the GPCR encoded
by a polynucleotide hybridizing under stringent conditions to the
complement of SEQ ID NO: 1 is a receptor for which
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine is a ligand. In
certain embodiments, the GPCR encoded by a polynucleotide
hybridizing under stringent conditions to the complement of SEQ ID
NO: 1 is a receptor for which
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadi-
azol-5-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine is a
agonist. In some embodiments, the GPCR encoded by a polynucleotide
hybridizing under stringent conditions to the complement of SEQ ID
NO: 1 exhibits a detectable level of constitutive activity. In some
embodiments, the constitutive activity is for increasing a level of
intracellular cAMP. In some embodiments, the constitutive activity
is for causing melanophore cells to undergo pigment dispersion.
[0286] In certain embodiments, the variant of SEQ ID NO:2 is a G
protein-coupled receptor having at least about 80% identity to SEQ
ID NO:2. In certain embodiments, the variant of SEQ ID NO:2 is a G
protein-coupled receptor comprising at least 20 contiguous amino
acids of SEQ ID NO:2. In certain embodiments, the variant of SEQ ID
NO:2 which is a G protein-coupled receptor is an endogenous G
protein-coupled receptor. In certain embodiments, the variant of
SEQ ID NO:2 which is a G protein-coupled receptor is a
non-endogenous G protein-coupled receptor.
[0287] In certain embodiments, the G protein-coupled receptor which
has at least about 80% identity to SEQ ID NO: 2 is SEQ ID NO: 2 or
an allele thereof. In certain embodiments, the G protein-coupled
receptor which has at least about 80% identity to SEQ ID NO: 2 is
an allele of SEQ ID NO: 2. In certain embodiments, the G
protein-coupled receptor which has at least about 80% identity to
SEQ ID NO: 2 is an ortholog of SEQ ID NO: 2. In certain
embodiments, the G protein-coupled receptor which has at least
about 80% identity to SEQ ID NO: 2 specifically binds
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine. In certain
embodiments, the G protein-coupled receptor which has at least
about 80% identity to SEQ ID NO: 2 is a receptor for which
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine is an agonist. In
some embodiments, the the G protein-coupled receptor which has at
least about 80% identity to SEQ ID NO: 2 exhibits a detectable
level of constitutive activity. In some embodiments, the
constitutive activity is for increasing a level of intracellular
cAMP. In some embodiments, the constitutive activity is for causing
melanophore cells to undergo pigment dispersion.
[0288] In certain embodiments, the G protein-coupled receptor which
comprises at least 20 contiguous amino acids of SEQ ID NO: 2 is SEQ
ID NO: 2 or an allele of SEQ ID NO: 2. In certain embodiments, the
G protein-coupled receptor which comprises at least 20 contiguous
amino acids of SEQ ID NO: 2 is an allele of SEQ ID NO: 2. In
certain embodiments, the G protein-coupled receptor which comprises
at least 20 contiguous amino acids of SEQ ID NO: 2 is an ortholog
of SEQ ID NO: 2. In certain embodiments, the G protein-coupled
receptor which comprises at least 20 contiguous amino acids of SEQ
ID NO: 2 specifically binds
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadizol-5--
yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine. In certain
embodiments, the G protein-coupled receptor which comprises at
least 20 contiguous amino acids of SEQ ID NO: 2 is a receptor for
which
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl]-5-nitro-pyrimidin-4-yl}-amine is an agonist. In some
embodiments, the G protein-coupled receptor which comprises at
least 20 contiguous amino acids of SEQ ID NO: 2 exhibits a
detectable level of constitutive activity. In some embodiments, the
constitutive activity is for increasing a level of intracellular
cAMP. In some embodiments, the constitutive activity is for causing
melanophore cells to undergo pigment dispersion.
[0289] In some embodiments, the G protein-coupled receptor is part
of a fusion protein comprising a G protein. Techniques for making a
GPCR:G fusion construct are well known to the skilled artisan (see,
e.g., International Application WO 02/42461).
[0290] In certain embodiments, said determining is carried out
using a host cell comprising the G protein-coupled receptor. In
certain embodiments, the host cell comprises an expression vector,
said expression vector comprising a polynucleotide encoding the
GPCR. An exemplary suitable expression vector is pCMV. Other
suitable expression vectors will be readily apparent to those of
ordinary skill in the art, and a wide variety of expression vectors
are commercially available (e.g., from Clontech, Palo Alto, Calif.;
Stratagene, La Jolla, Calif.; and Invitrogen, Carlsbad,
Calif.).
[0291] In some embodiments, the host cell is a vertebrate cell. In
some embodiments, the host cell is a mammalian cell. In some
embodiments, the mammalian host cell is selected from the group
consisting of a 293 cell, a 293T cell, a CHO cell, a MCB3901 cell,
and a COS-7 cell. In some embodiments, the host cell is a yeast
cell. In some embodiments, the host cell is a melanophore cell.
Other suitable host cells will be readily apparent to those of
ordinary skill in the art, and a wide variety of cell lines are
available from the American Type Culture Collection, 10801
University Boulevard, Manassas, Va. 20110-2209.
[0292] In certain embodiments, said determining is carried out
using membrane comprising the G protein-coupled receptor.
[0293] In some embodiments, the test compound is a small molecule.
In some embodiments, the test compound is a small molecule, with
the proviso that the small molecule is not a polypeptide. In some
embodiments, the test compound is a small molecule, with the
proviso that the small molecule is not an antibody or an
antigen-binding fragment thereof. In some embodiments, the test
compound is a small molecule, with the proviso that the small
molecule is not a lipid. In some embodiments, the test compound is
a small molecule, with the proviso that the small molecule is not a
polypeptide or a lipid. In some embodiments, the test compound is a
polypeptide. In some embodiments, the test compound is a
polypeptide, with the proviso that the polypeptide is not an
antibody or an antigen-binding fragment thereof. In some
embodiments, the test compound is a lipid. In some embodiments, the
test compound is not an antibody or an antigen-binding fragment
thereof. In some embodiments, the test compound is an antibody or
an antigen-binding fragment thereof.
[0294] In some embodiments, the method further comprises the step
of optionally determining the structure of the PYY secretagogue,
the compound useful for treatment or prevention of a condition
modulated by PYY, or the compound useful for a modulation of a
PYY-related activity in an individual.
[0295] In some embodiments, the method further comprises the step
of optionally providing the name or structure of the PYY
secretagogue, the compound useful for treatment or prevention of a
condition modulated by PYY, or the compound useful for a modulation
of a PYY-related activity in an individual.
[0296] In some embodiments, said method further comprises the step
of optionally producing or synthesizing the PYY secretagogue, the
compound useful for treatment or prevention of a condition
modulated by PYY, or the compound useful for a modulation of a
PYY-related activity in an individual.
[0297] In some embodiments, the method further comprises the step
of optionally determining the structure of the PYY secretagogue,
the compound useful for treatment or prevention of a condition
modulated by PYY, or the compound useful for a modulation of a
PYY-related activity in an individual.
[0298] In some embodiments, the method further comprises the step
of optionally providing the name or structure of the PYY
secretagogue, the compound useful for treatment or prevention of a
condition modulated by PYY, or the compound useful for a modulation
of a PYY-related activity in an individual.
[0299] In some embodiments, said method further comprises the step
of optionally producing or synthesizing the PYY secretagogue, the
compound useful for treatment or prevention of a condition
modulated by PYY, or the compound useful for a modulation of a
PYY-related activity in an individual.
[0300] In some embodiments, said method further comprises the step
of formulating the PYY secretagogue, the compound useful for
treatment or prevention of a condition modulated by PYY, or the
compound useful for a modulation of a PYY-related activity in an
individual as a pharmaceutical.
[0301] In some embodiments, said method further comprises
evaluating the bioavailability of the PYY secretagogue, the
compound useful for treatment or prevention of a condition
modulated by PYY, or the compound useful for a modulation of a
PYY-related activity.
[0302] In some embodiments, said method further comprises the step
of formulating the PYY secretagogue, the compound useful for
treatment or prevention of a condition modulated by PYY, or the
compound useful for a modulation of a PYY-related activity in an
individual into a pharmaceutical composition.
[0303] The invention additionally features a process comprising
performing a method according to this fourth aspect.
[0304] In a fifth aspect, the invention features a method of
screening test compounds to identify a PYY secretagogue, a compound
for treatment or prevention of a condition modulated by PYY, or a
compound for a modulation of a PYY-related activity in an
individual, which is characterized by using a G protein-coupled
receptor comprising an amino acid sequence selected from the group
consisting of: [0305] (a) amino acids 1-335 of SEQ ID NO: 2; [0306]
(b) amino acids 2-335 of SEQ ID NO: 2; [0307] (c) an amino acid
sequence comprising the amino acid sequence of a polypeptide
derived from (a) by substitution, deletion or addition of one or
several amino acids in the amino acid sequence defined in (a);
[0308] (d) the amino acid sequence of a G protein-coupled receptor
encoded by a polynucleotide hybridizing under stringent conditions
to the complement of SEQ ID NO: 1; [0309] (e) a variant of SEQ ID
NO: 2 when selected from the group consisting of: [0310] (i) the
amino acid sequence of a G protein-coupled receptor having at least
about 80% identity to SEQ ID NO: 2; and [0311] (ii) the amino acid
sequence of a G protein-coupled receptor comprising at least 20
continguous amino acids of SEQ ID NO: 2; [0312] (f) the amino acid
sequence of a constitutively active version of a G protein-coupled
receptor having SEQ ID NO: 2; and
[0313] 1(g) a biologically active fragment of any one of any one of
(a) to (f).
[0314] In certain embodiments, the G protein-coupled receptor
comprises the amino acid sequence of a G protein-coupled receptor
having at least about 80% identity to SEQ ID NO: 2.
[0315] In certain embodiments, the receptor comprises the amino
acid sequence of SEQ ID NO: 2.
[0316] In certain embodiments, the variant of SEQ ID NO: 2 is an
allele of SEQ ID NO: 2.
[0317] In certain embodiments, the variant of SEQ ID NO: 2 is an
ortholog of SEQ ID NO: 2. In certain embodiments, the variant of
SEQ ID NO: 2 is a mammalian ortholog of SEQ ID NO: 2.
[0318] In certain embodiments, the G protein-coupled receptor is
recombinant
[0319] In certain embodiments, the method comprises identifying an
agonist of the receptor.
[0320] In certain embodiments, the method comprises identifying a
partial agonist of the receptor.
[0321] In some embodiments, said method further comprises the step
of formulating the PYY secretagogue, the compound useful for
treatment or prevention of a condition modulated by PYY, or the
compound useful for a modulation of a PYY-related activity in an
individual as a pharmaceutical.
[0322] In some embodiments, said method further comprises
evaluating the bioavailability of the PYY secretagogue, the
compound useful for treatment or prevention of a condition
modulated by PYY, or the compound useful for a modulation of a
PYY-related activity.
[0323] In some embodiments, said method further comprises the step
of formulating the PYY secretagogue, the compound useful for
treatment or prevention of a condition modulated by PYY, or the
compound useful for a modulation of a PYY-related activity in an
individual into a pharmaceutical composition.
[0324] The invention additionally features a process comprising
performing a method according to this fifth aspect.
[0325] In a sixth aspect, the invention features a method
comprising, having identified a PYY secretagogue, a compound for
treatment or prevention of a condition modulated by PYY, or a
compound for a modulation of a PYY-related activity in an
individual according to the first aspect, the second aspect, the
third aspect, the fourth aspect or the fifth aspect, formulating
said PYY secretagogue, said compound for treatment or prevention of
a condition modulated by PYY, or said compound for a modulation of
a PYY-related activity in an individual into a pharmaceutical
composition.
[0326] In a seventh aspect, the invention features use of a G
protein-coupled receptor to screen test compounds as PYY
secretagogues, compounds for treatment or prevention of a condition
modulated by PYY, or compounds for a modulation of a PYY-related
activity in an individual, wherein the G protein-coupled receptor
comprises an amino acid sequence selected from the group consisting
of: [0327] (a) amino acids 1-335 of SEQ ID NO: 2; [0328] (b) amino
acids 2-335 of SEQ ID NO: 2; [0329] (c) an amino acid sequence
comprising the amino acid sequence of a polypeptide derived from
(a) by substitution, deletion or addition of one or several amino
acids in the amino acid sequence defined in (a); [0330] (d) the
amino acid sequence of a G protein-coupled receptor encoded by a
polynucleotide hybridizing under stringent conditions to the
complement of SEQ ID NO: 1; [0331] (e) a variant of SEQ ID NO: 2
when selected from the group consisting of: [0332] (i) the amino
acid sequence of a G protein-coupled receptor having at least about
80% identity to SEQ ID NO: 2; and [0333] (ii) the amino acid
sequence of a G protein-coupled receptor comprising at least 20
contiguous amino acids of SEQ ID NO: 2; [0334] (f) the amino acid
sequence of a constitutively active version of a G protein-coupled
receptor having SEQ ID NO: 2; and [0335] (g) a biologically active
fragment of any one of any one of (a) to (f).
[0336] In certain embodiments, the G protein-coupled receptor
comprises the amino acid sequence of a G protein-coupled receptor
having at least about 80% identity to SEQ ID NO: 2.
[0337] In certain embodiments, the receptor comprises the amino
acid sequence of SEQ ID NO: 2.
[0338] In certain embodiments, the variant of SEQ ID NO: 2 is an
allele of SEQ ID NO: 2.
[0339] In certain embodiments, the variant of SEQ ID NO: 2 is an
ortholog of SEQ ID NO: 2. In certain embodiments, the variant of
SEQ ID NO: 2 is a mammalian ortholog of SEQ ID NO: 2.
[0340] In certain embodiments, the receptor is recombinant
[0341] In certain embodiments, the test compound is a small
molecule.
[0342] In certain embodiments, the test compound is a GPR119
agonist.
[0343] In certain embodiments, the GPR119 agonist is an agonist of
an endogenous GPR119.
[0344] In certain embodiments, the GPR119 agonist is an agonist of
human GPR119.
[0345] In certain embodiments, the GPR119 agonist is a GPR119
partial agonist.
[0346] In certain embodiments, the GPR119 agonist is a selective
GPR119 agonist.
[0347] In certain embodiments, the GPR119 agonist is a small
molecule.
[0348] In certain embodiments, the GPR119 agonist is orally
available.
[0349] In certain embodiments, the GPR119 agonist has an EC.sub.50
value of less than about 10 .mu.M, less than about 1 .mu.M, less
than about 100 nM, less than about 75 nM, less than about 50 nM,
less than about 25 nM, less than about 15 nM, less than about 10
nM, less than about 5 nM, less than about 4 nM, less than about 3
nM, less than about 2 nM, or less than about 1 nM. In certain
embodiments, the GPR119 agonist has an EC.sub.50 value of less than
about 10 .mu.M, less than about 1 .mu.M, less than about 100 nM,
less than about 75 nM, less than about 50 nM, less than about 25
nM, less than about 15 nM, less than about 10 nM, less than about 5
nM, less than about 4 nM, less than about 3 nM, less than about 2
nM, or less than about 1 nM at human GPR119 having SEQ ID NO: 2. In
certain embodiments, the GPR119 agonist has an EC.sub.50 value of
less than about 10 .mu.M, less than about 1 .mu.M, less than about
100 nM, less than 75 nM, less than about 50 nM, less than about 25
nM, less than about 15 nM, less than about 10 nM, less than about 5
nM, less than about 4 nM, less than about 3 nM, less than about 2
nM, or less than about 1 nM at human GPR119 having SEQ ID NO: 2 in
adenylyl cyclase assay (exemplary adenylyl cyclase assay is
provided in Example 9, Example 10 and in Example 11, infra). In
certain embodiments, the GPR119 agonist has an EC.sub.50 value of
less than about 10 .mu.M, less than about 1 .mu.M, less than about
nM, less than about 75 nM, less than about 50 nM, less than about
25 nM, less than about 15 nM, less than about 10 nM, less than
about 5 nM, less than about 4 nM, less than about 3 nM, less than
about 2 nM, or less than about 1 nM at human GPR119 having SEQ ID
NO: 2 in melanophore assay (exemplary melanophore assay is provided
in Example 12, infra).
[0350] Exemplary GPR119 agonists are disclosed, e.g., in
International Application No. PCT/US2004/001267 (published as WO
04/065380); International Application No. PCT/US2004/005555
(published as WO 04/076413); International Application No.
PCT/US2004/022327 (published as WO 05/007647); International
Application No. PCT/US2004/022417 (published as WO 05/007658);
International Application No. PCT/US2005/019318 (published as WO
2005/121121); International Application No. PCT/GB2004/050046
(published as WO 2005/061489); International Application No.
PCT/US06/00567 (published as WO 2006/083491); International
Application No. PCT/GB2005/050264 (published as WO 2006/067531);
International Application No. PCT/GB2005/050265 (published as WO
2006/067532); International Application No. PCT/GB2005/050266
(published as WO 2006/070208); International Application No.
PCT/JP02/09350 (published as WO 03/026661); International
Application No. PCT/JP2005/018412 (published as WO 06/040966);
International Application No. PCT/JP2005/019000 (published as WO
2006/043490); International Application No. PCT/GB2006/050176
(published as WO 2007/003960); International Application No.
PCT/GB2006/050177 (published as WO 2007/003961); International
Application No. PCT/GB2006/050178 (published as WO 2007/003962);
International Application No. PCT/GB2006/050182 (published as WO
2007/003964); International Application No. PCT/JP02/09350
(published as WO 03/026661); International Application No.
PCT/GB2007/050183 (published as WO 2007/116229); International
Application No. PCT/GB2007/050190 (published as WO 2007/116230);
International Application No. PCT/US2007/073364 (published as WO
2008/008895); International Application No. PCT/EP2007/058991
(published as WO 2008/025798); International Application No.
[0351] PCT/EP2007/058993 (published as WO 2008/025799); and
International Application No. PCT/EP2007/058995 (published as WO
2008/025800).
[0352] In an eighth aspect, the invention features a method for
identifying PYY secretagogues, compounds useful for treatment or
prevention of a condition modulated by PYY, or compounds useful for
a modulation of a PYY-related activity in an individual, comprising
the steps of: [0353] (a) contacting a compound which stimulates
functionality of a G protein-coupled receptor, wherein said G
protein-coupled receptor comprises an amino acid sequence selected
from the group consisting of: [0354] (i) amino acids 1-335 of SEQ
ID NO:2; [0355] (ii) amino acids 2-335 of SEQ ID NO:2; [0356] (iii)
an amino acid sequence comprising the amino acid sequence of a
polypeptide derived from (i) by substitution, deletion or addition
of one or several amino acids in the amino acid sequence defined in
(i); [0357] (iv) the amino acid sequence of a G protein-coupled
receptor encoded by a polynucleotide hybridizing under stringent
conditions to the complement of SEQ ID NO:1; [0358] (v) a variant
of SEQ ID NO: 2 when selected from the group consisting of: [0359]
(a') the amino acid sequence of a G protein-coupled receptor having
at least about 80% identity to SEQ ID NO: 2; and [0360] (b') an
amino acid sequence of a G protein-coupled receptor comprising at
least 20 contiguous amino acids of SEQ ID NO: 2; [0361] (vi) the
amino acid sequence of a constitutively active version of a G
protein-coupled receptor having SEQ ID NO: 2; and [0362] (vii) a
biologically active fragment of any one of (i) to (vi); [0363] in
vitro with a vertebrate enteroendocrine cell or with a cell capable
of secreting PYY, said compound having been determined or
identified by a method according to the first aspect; and [0364]
(b) determining whether the compound stimulates PYY secretion from
the vertebrate enteroendocrine cell or from the cell capable of
secreting PYY; wherein the ability of the test compound to
stimulate PYY secretion from the vertebrate enteroendocrine cell or
from the cell capable of secreting PYY is further indicative of the
test compound being a PYY secretagogue, a compound useful for
treatment or prevention of a condition modulated by PYY, or a
compound useful for a modulation of a PYY-related activity in an
individual.
[0365] In certain embodiments, the G protein-coupled receptor
comprises the amino acid sequence of a G protein-coupled receptor
having at least about 80% identity to SEQ ID NO: 2.
[0366] In certain embodiments, the receptor comprises the amino
acid sequence of SEQ ID NO: 2.
[0367] In certain embodiments, the variant of SEQ ID NO: 2 is an
allele of SEQ ID NO: 2.
[0368] In certain embodiments, the variant of SEQ ID NO: 2 is an
ortholog of SEQ ID NO: 2. In certain embodiments, the variant of
SEQ ID NO: 2 is a mammalian ortholog of SEQ ID NO: 2.
[0369] In certain embodiments, the G protein-coupled receptor is
recombinant
[0370] In certain embodiments, the vertebrate enteroendocrine cell
is a mammalian enteroendocrine cell. In certain embodiments, the
enteroendocrine cell is an L cell. In certain embodiments, the
enteroendocrine cell is comprised by or derived from tissue
obtained from the small or large intestine. In certain embodiments,
the enteroendocrine cell is comprised by or derived from tissue
obtained from an L cell-containing region of small or large
intestine. In certain embodiments, the enteroendocrine cell is
comprised by or derived from duodenum, jejunum, ileum, colon or
rectum tissue (see, e.g., Lundberg et al, PNAS USA (1982)
79:4471-4475; Adrian et al, Gastroenterol (1985) 89:1070-1077;
Ekblad et al, Peptides (2002) 23:251-261; Ueno et al, Regul Pept
(2008) 145:12-16). In certain embodiments, the enteroendocrine cell
is an enteroendocrine cell line. In certain embodiments, the cell
capable of secreting PYY is a cell comprised by or derived from the
islets of Langerhans. In certain embodiments, the cell capable of
secreting PYY is a neuronal cell. In certain embodiments, the cell
capable of secreting PYY is a recombinant cell engineered to be
capable of secreting PYY.
[0371] The invention additionally features a method for identifying
PYY secretagogues, compounds useful for treatment or prevention of
a condition modulated by PYY, or compounds useful for a modulation
of a PYY-related activity in an individual, comprising the steps
of: [0372] (a) administering to a vertebrate a compound which
stimulates functionality of a G protein-coupled receptor, wherein
said G protein-coupled receptor comprises an amino acid sequence
selected from the group consisting of: [0373] (i) amino acids 1-335
of SEQ ID NO:2; [0374] (ii) amino acids 2-335 of SEQ ID NO:2;
[0375] (iii) an amino acid sequence comprising the amino acid
sequence of a polypeptide derived from (i) by substitution,
deletion or addition of one or several amino acids in the amino
acid sequence defined in (i); [0376] (iv) the amino acid sequence
of a G protein-coupled receptor encoded by a polynucleotide
hybridizing under stringent conditions to the complement of SEQ ID
NO:1; [0377] (v) a variant of SEQ ID NO: 2 when selected from the
group consisting of: [0378] (a') the amino acid sequence of a G
protein-coupled receptor having at least about 80% identity to SEQ
ID NO: 2; and [0379] (b') an amino acid sequence of a G
protein-coupled receptor comprising at least 20 contiguous amino
acids of SEQ ID NO: 2; [0380] (vi) the amino acid sequence of a
constitutively active version of a G protein-coupled receptor
having SEQ ID NO: 2; and [0381] (vii) a biologically active
fragment of any one of (i) to (vi); [0382] said compound having
been determined or identified by a method according to the first
aspect; and [0383] (b) determining whether the compound increases a
PYY level in the vertebrate; wherein the ability of the test
compound to increase a PYY level in the vertebrate is further
indicative of the test compound being a PYY secretagogue, a
compound useful for treatment or prevention of a condition
modulated by PYY, or a compound useful for a modulation of a
PYY-related activity in an individual.
[0384] In certain embodiments, the G protein-coupled receptor
comprises the amino acid sequence of a G protein-coupled receptor
having at least about 80% identity to SEQ ID NO: 2.
[0385] In certain embodiments, the receptor comprises the amino
acid sequence of SEQ ID NO: 2.
[0386] In certain embodiments, the variant of SEQ ID NO: 2 is an
allele of SEQ ID NO: 2.
[0387] In certain embodiments, the variant of SEQ ID NO: 2 is an
ortholog of SEQ ID NO: 2. In certain embodiments, the variant of
SEQ ID NO: 2 is a mammalian ortholog of SEQ ID NO: 2.
[0388] In certain embodiments, the G protein-coupled receptor is
recombinant
[0389] In certain embodiments, the PYY level is blood or plasma
concentration of total PYY. In certain embodiments, the PYY level
is blood or plasma concentration of PYY.sub.3-36.
[0390] In certain embodiments, the vertebrate is a mammal. In
certain embodiments, the vertebrate is a non-human vertebrate. In
certain embodiments, the vertebrate is a non-human mammal. In
certain embodiments, the mammal is a non-human mammal.
[0391] In certain embodiments, the vertebrate or the mammal is a
human. In certain embodiments, the human is a participant in a
study reviewed by a governmental agency charged with marketing
approval for a drug. In certain embodiments, the study is a
clinical trial. In certain embodiments, the governmental agency is
the Food and Drug Administration of the United States.
[0392] The invention additionally features a method for identifying
PYY secretagogues, compounds useful for treatment or prevention of
a condition modulated by PYY, or compounds useful for a modulation
of a PYY-related activity in an individual, comprising the steps
of: [0393] (a) determining whether the compound increases a PYY
level in a sample, said sample having been obtained from a
vertebrate previously administered with a compound which stimulates
functionality of a G protein-coupled receptor, wherein said G
protein-coupled receptor comprises an amino acid sequence selected
from the group consisting of: [0394] (i) amino acids 1-335 of SEQ
ID NO:2; [0395] (ii) amino acids 2-335 of SEQ ID NO:2; [0396] (iii)
an amino acid sequence comprising the amino acid sequence of a
polypeptide derived from (i) by substitution, deletion or addition
of one or several amino acids in the amino acid sequence defined in
(i); [0397] (iv) the amino acid sequence of a G protein-coupled
receptor encoded by a polynucleotide hybridizing under stringent
conditions to the complement of SEQ ID NO:1; [0398] (v) a variant
of SEQ ID NO: 2 when selected from the group consisting of: [0399]
(a') the amino acid sequence of a G protein-coupled receptor having
at least about 80% identity to SEQ ID NO: 2; and [0400] (b') an
amino acid sequence of a G protein-coupled receptor comprising at
least 20 contiguous amino acids of SEQ ID NO: 2; [0401] (vi) the
amino acid sequence of a constitutively active version of a G
protein-coupled receptor having SEQ ID NO: 2; and [0402] (vii) a
biologically active fragment of any one of (i) to (vi); [0403] said
compound having been determined or identified by a method according
to the first aspect; and wherein the ability of the test compound
to increase a PYY level in the vertebrate is further indicative of
the test compound being a PYY secretagogue, a compound useful for
treatment or prevention of a condition modulated by PYY, or a
compound useful for a modulation of a PYY-related activity in an
individual.
[0404] In certain embodiments, the G protein-coupled receptor
comprises the amino acid sequence of a G protein-coupled receptor
having at least about 80% identity to SEQ ID NO: 2.
[0405] In certain embodiments, the receptor comprises the amino
acid sequence of SEQ ID NO: 2.
[0406] In certain embodiments, the variant of SEQ ID NO: 2 is an
allele of SEQ ID NO: 2.
[0407] In certain embodiments, the variant of SEQ ID NO: 2 is an
ortholog of SEQ ID NO: 2. In certain embodiments, the variant of
SEQ ID NO: 2 is a mammalian ortholog of SEQ ID NO: 2.
[0408] In certain embodiments, the G protein-coupled receptor is
recombinant
[0409] In certain embodiments, the vertebrate is a mammal. In
certain embodiments, the vertebrate is a non-human vertebrate. In
certain embodiments, the vertebrate is a non-human mammal. In
certain embodiments, the mammal is a non-human mammal
[0410] In certain embodiments, the vertebrate or the mammal is a
human. In certain embodiments, the human is a participant in a
study reviewed by a governmental agency charged with marketing
approval for a drug. In certain embodiments, the study is a
clinical trial. In certain embodiments, the governmental agency is
the Food and Drug Administration of the United States.
[0411] In some embodiments, a G protein-coupled receptor of the
invention is SEQ ID NO:2 or an allele thereof. In some embodiments,
a G protein-coupled receptor of the invention is an allele of SEQ
ID NO:2. In some embodiments, a G protein-coupled receptor of the
invention is an ortholog of SEQ ID NO:2.
[0412] In some embodiments, a G protein-coupled receptor of the
invention is a G protein-coupled receptor which specifically binds
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine. In some
embodiments, a G protein-coupled receptor of the invention is a G
protein-coupled receptor for which
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine is a ligand. In
some embodiments, a G protein-coupled receptor of the invention is
a G protein-coupled receptor for which
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine is an agonist.
In some embodiments, a G protein-coupled receptor of the invention
exhibits a detectable level of constitutive activity. In some
embodiments, the constitutive activity is for increasing a level of
intracellular cAMP accumulation. In some embodiments, the
constitutive activity is for causing melanophore cells to undergo
pigment dispersion.
[0413] In certain embodiments, stringent hybridization conditions
comprise hybridization at 42.degree. C. in a solution comprising
50% formamide, 5.times.SSC (1.times.SSC=150 mM NaCl, 15 mM
trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5.times.
Denhardt's solution, 10% dextran sulfate, and 20 m/ml denatured,
sheared salmon sperm DNA, followed by washing at 65.degree. C. in a
solution comprising 0.1.times.SSC. Hybridization techniques are
well known to the skilled artisan.
[0414] In certain embodiments, the GPCR encoded by a polynucleotide
hybridizing under stringent conditions to the complement of SEQ ID
NO: 1 is SEQ ID NO:2 or an allele thereof. In certain embodiments,
the GPCR encoded by a polynucleotide hybridizing under stringent
conditions to the complement of SEQ ID NO: 1 is an allele of SEQ ID
NO:2. In certain embodiments, the GPCR encoded by a polynucleotide
hybridizing under stringent conditions to the complement of SEQ ID
NO: 1 is an ortholog of SEQ ID NO:2. In certain embodiments, the
GPCR encoded by a polynucleotide hybridizing under stringent
conditions to the complement of SEQ ID NO: 1 specifically binds
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine. In certain
embodiments, the GPCR encoded by a polynucleotide hybridizing under
stringent conditions to the complement of SEQ ID NO: 1 is a
receptor for which
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadi-
azol-5-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine is a
ligand. In certain embodiments, the GPCR encoded by a
polynucleotide hybridizing under stringent conditions to the
complement of SEQ ID NO: 1 is a receptor for which
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine is an agonist.
In some embodiments, the GPCR encoded by a polynucleotide
hybridizing under stringent conditions to the complement of SEQ ID
NO: 1 exhibits a detectable level of constitutive activity. In some
embodiments, the constitutive activity is for increasing a level of
intracellular cAMP. In some embodiments, the constitutive activity
is for causing melanophore cells to undergo pigment dispersion.
[0415] In certain embodiments, the variant of SEQ ID NO:2 is a G
protein-coupled receptor having at least about 80% identity to SEQ
ID NO:2. In certain embodiments, the variant of SEQ ID NO:2 is a G
protein-coupled receptor comprising at least 20 contiguous amino
acids of SEQ ID NO:2. In certain embodiments, the variant of SEQ ID
NO:2 which is a G protein-coupled receptor is an endogenous G
protein-coupled receptor. In certain embodiments, the variant of
SEQ ID NO:2 which is a G protein-coupled receptor is a
non-endogenous G protein-coupled receptor.
[0416] In certain embodiments, the G protein-coupled receptor which
has at least about 80% identity to SEQ ID NO: 2 is SEQ ID NO: 2 or
an allele thereof. In certain embodiments, the G protein-coupled
receptor which has at least about 80% identity to SEQ ID NO: 2 is
an allele of SEQ ID NO: 2. In certain embodiments, the G
protein-coupled receptor which has at least about 80% identity to
SEQ ID NO: 2 is an ortholog of SEQ ID NO: 2. In certain
embodiments, the G protein-coupled receptor which has at least
about 80% identity to SEQ ID NO: 2 specifically binds
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine. In certain
embodiments, the G protein-coupled receptor which has at least
about 80% identity to SEQ ID NO: 2 is a receptor for which
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine is an agonist. In
some embodiments, the the G protein-coupled receptor which has at
least about 80% identity to SEQ ID NO: 2 exhibits a detectable
level of constitutive activity. In some embodiments, the
constitutive activity is for increasing a level of intracellular
cAMP. In some embodiments, the constitutive activity is for causing
melanophore cells to undergo pigment dispersion.
[0417] In certain embodiments, the G protein-coupled receptor which
comprises at least 20 contiguous amino acids of SEQ ID NO: 2 is SEQ
ID NO: 2 or an allele of SEQ ID NO: 2. In certain embodiments, the
G protein-coupled receptor which comprises at least 20 contiguous
amino acids of SEQ ID NO: 2 is an allele of SEQ ID NO: 2. In
certain embodiments, the G protein-coupled receptor which comprises
at least 20 contiguous amino acids of SEQ ID NO: 2 is an ortholog
of SEQ ID NO: 2. In certain embodiments, the G protein-coupled
receptor which comprises at least 20 contiguous amino acids of SEQ
ID NO: 2 specifically binds
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine. In certain
embodiments, the G protein-coupled receptor which comprises at
least 20 contiguous amino acids of SEQ ID NO: 2 is a receptor for
which
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitropyrimidin-4-yl}-amine is an agonist. In
some embodiments, the G protein-coupled receptor which comprises at
least 20 contiguous amino acids of SEQ ID NO: 2 exhibits a
detectable level of constitutive activity. In some embodiments, the
constitutive activity is for increasing a level of intracellular
cAMP. In some embodiments, the constitutive activity is for causing
melanophore cells to undergo pigment dispersion.
[0418] In some embodiments, the G protein-coupled receptor is part
of a fusion protein comprising a G protein. Techniques for making a
GPCR:G fusion construct are well known to the skilled artisan (see,
e.g., International Application WO 02/42461).
[0419] In some embodiments, the compound which stimulates
functionality of a G protein-coupled receptor is a small molecule.
In some embodiments, the compound which stimulates functionality of
a G protein-coupled receptor is a small molecule, with the proviso
that the small molecule is not a polypeptide. In some embodiments,
the compound which stimulates functionality of a G protein-coupled
receptor is a small molecule, with the proviso that the small
molecule is not an antibody or an antigen-binding fragment thereof.
In some embodiments, the compound which stimulates functionality of
a G protein-coupled receptor is a small molecule, with the proviso
that the small molecule is not a lipid. In some embodiments, the
compound which stimulates functionality of a G protein-coupled
receptor is a small molecule, with the proviso that the small
molecule is not a polypeptide or a lipid. In some embodiments, the
compound which stimulates functionality of a G protein-coupled
receptor is a polypeptide. In some embodiments, the compound which
stimulates functionality of a G protein-coupled receptor is a
polypeptide, with the proviso that the polypeptide is not an
antibody or an antigen-binding fragment thereof. In some
embodiments, the compound which stimulates functionality of a G
protein-coupled receptor is a lipid. In some embodiments, the
compound which stimulates functionality of a G protein-coupled
receptor is not an antibody or an antigen-binding fragment thereof.
In some embodiments, the compound which stimulates functionality of
a G protein-coupled receptor is an antibody or an antigen-binding
fragment thereof.
[0420] In some embodiments, the method further comprises the step
of optionally determining the structure of the PYY secretagogue,
the compound useful for treatment or prevention of a condition
modulated by PYY, or the compound useful for a modulation of a
PYY-related activity in an individual.
[0421] In some embodiments, the method further comprises the step
of optionally providing the name or structure of the PYY
secretagogue, the compound useful for treatment or prevention of a
condition modulated by PYY, or the compound useful for a modulation
of a PYY-related activity in an individual.
[0422] In some embodiments, said method further comprises the step
of optionally producing or synthesizing the PYY secretagogue, the
compound useful for treatment or prevention of a condition
modulated by PYY, or the compound useful for a modulation of a
PYY-related activity in an individual.
[0423] In some embodiments, said method further comprises the step
of formulating the PYY secretagogue, the compound useful for
treatment or prevention of a condition modulated by PYY, or the
compound useful for a modulation of a PYY-related activity in an
individual as a pharmaceutical.
[0424] In some embodiments, said method further comprises
evaluating the bioavailability of the PYY secretagogue, the
compound useful for treatment or prevention of a condition
modulated by PYY, or the compound useful for a modulation of a
PYY-related activity.
[0425] In some embodiments, said method further comprises the step
of formulating the PYY secretagogue, the compound useful for
treatment or prevention of a condition modulated by PYY, or the
compound useful for a modulation of a PYY-related activity in an
individual into a pharmaceutical composition.
[0426] The invention additionally features a process comprising
performing a method according to this eighth aspect.
[0427] In a ninth aspect, the invention features a method for
identifying PYY secretagogues, compounds useful for treatment or
prevention of a condition modulated by PYY, or compounds useful for
a modulation of a PYY-related activity in an individual, comprising
the steps of: [0428] (a) contacting a compound in the presence of
which less of a complex between a G protein-coupled receptor and an
optionally labelled known ligand to the receptor is formed than in
the absence of the compound, wherein the G protein-coupled receptor
comprises an amino acid sequence selected from the group consisting
of: [0429] (i) amino acids 1-335 of SEQ ID NO:2; [0430] (ii) amino
acids 2-335 of SEQ ID NO:2; [0431] (iii) an amino acid sequence
comprising the amino acid sequence of a polypeptide derived from
(i) by substitution, deletion or addition of one or several amino
acids in the amino acid sequence defined in (i); [0432] (iv) the
amino acid sequence of a G protein-coupled receptor encoded by a
polynucleotide hybridizing under stringent conditions to the
complement of SEQ ID NO:1; [0433] (v) a variant of SEQ ID NO: 2
when selected from the group consisting of: [0434] (a') the amino
acid sequence of a G protein-coupled receptor having at least about
80% identity to SEQ ID NO: 2; and [0435] (b') the amino acid
sequence of a G protein-coupled receptor comprising at least 20
contiguous amino acids of SEQ ID NO: 2; [0436] (vi) the amino acid
sequence of a constitutively active version of a G protein-coupled
receptor having SEQ ID NO: 2; and [0437] (vii) a biologically
active fragment of any one of (i) to (vi); [0438] in vitro with a
vertebrate enteroendocrine cell or with a cell capable of secreting
PYY, said compound having been determined or identified by a method
according to the fourth aspect; and [0439] (b) determining whether
the compound stimulates PYY secretion from the vertebrate
enteroendocrine cell or from the cell capable of secreting PYY;
wherein the ability of the test compound to stimulate PYY secretion
from the vertebrate enteroendocrine cell or from the cell capable
of secreting PYY is further indicative of the test compound being a
PYY secretagogue, a compound useful for treatment or prevention of
a condition modulated by PYY, or a compound useful for a modulation
of a PYY-related activity in an individual.
[0440] In certain embodiments, the G protein-coupled receptor
comprises the amino acid sequence of a G protein-coupled receptor
having at least about 80% identity to SEQ ID NO: 2.
[0441] In certain embodiments, the receptor comprises the amino
acid sequence of SEQ ID NO: 2.
[0442] In certain embodiments, the variant of SEQ ID NO: 2 is an
allele of SEQ ID NO: 2.
[0443] In certain embodiments, the variant of SEQ ID NO: 2 is an
ortholog of SEQ ID NO: 2. In certain embodiments, the variant of
SEQ ID NO: 2 is a mammalian ortholog of SEQ ID NO: 2.
[0444] In certain embodiments, the G protein-coupled receptor is
recombinant.
[0445] In certain embodiments, the vertebrate enteroendocrine cell
is a mammalian enteroendocrine cell.
[0446] In certain embodiments, the enteroendocrine cell is an L
cell. In certain embodiments, the enteroendocrine cell is comprised
by or derived from tissue obtained from the small or large
intestine. In certain embodiments, the enteroendocrine cell is
comprised by or derived from tissue obtained from an L
cell-containing region of small or large intestine. In certain
embodiments, the enteroendocrine cell is comprised by or derived
from duodenum, jejunum, ileum, colon or rectum tissue (see, e.g.,
Lundberg et al, PNAS USA (1982) 79:4471-4475; Adrian et al,
Gastroenterol (1985) 89:1070-1077; Ekblad et al, Peptides (2002)
23:251-261; Ueno et al, Regul Pept (2008) 145:12-16). In certain
embodiments, the enteroendocrine cell is an enteroendocrine cell
line. In certain embodiments, the cell capable of secreting PYY is
a cell comprised by or derived from the islets of Langerhans. In
certain embodiments, the cell capable of secreting PYY is a
neuronal cell. In certain embodiments, the cell capable of
secreting PYY is a recombinant cell engineered to be capable of
secreting PYY.
[0447] The invention additionally features a method for identifying
PYY secretagogues, compounds useful for treatment or prevention of
a condition modulated by PYY, or compounds useful for a modulation
of a PYY-related activity in an individual, comprising the steps
of: [0448] (a) administering a compound to a vertebrate, wherein in
the presence of said compound less of a complex between a G
protein-coupled receptor and an optionally labelled known ligand to
the receptor is formed than in the absence of the compound, wherein
the G protein-coupled receptor comprises an amino acid sequence
selected from the group consisting of: [0449] (i) amino acids 1-335
of SEQ ID NO:2; [0450] (ii) amino acids 2-335 of SEQ ID NO:2;
[0451] (iii) an amino acid sequence comprising the amino acid
sequence of a polypeptide derived from (i) by substitution,
deletion or addition of one or several amino acids in the amino
acid sequence in (i); [0452] (iv) the amino acid sequence of a G
protein-coupled receptor encoded by a polynucleotide hybridizing
under stringent conditions to the complement of SEQ ID NO:1; [0453]
(v) a variant of SEQ ID NO: 2 when selected from the group
consisting of: [0454] (a') the amino acid sequence of a G
protein-coupled receptor having at least about 80% identity to SEQ
ID NO: 2; and [0455] (b') the amino acid sequence of a G
protein-coupled receptor comprising at least 20 contiguous amino
acids of SEQ ID NO: 2; [0456] (vi) the amino acid sequence of a
constitutively active version of a G protein-coupled receptor
having SEQ ID NO: 2; and [0457] (vii) a biologically active
fragment of any one of (i) to (vi); [0458] said compound having
been determined or identified by a method according to the fourth
aspect; and [0459] (b) determining whether the compound increases a
PYY level in the vertebrate; wherein the ability of the test
compound to increase a PYY level in the vertebrate is further
indicative of the test compound being a PYY secretagogue, a
compound useful for treatment or prevention of a condition
modulated by PYY, or a compound useful for a modulation of a
PYY-related activity in an individual.
[0460] In certain embodiments, the vertebrate is a mammal. In
certain embodiments, the vertebrate is a non-human vertebrate. In
certain embodiments, the vertebrate is a non-human mammal. In
certain embodiments, the mammal is a non-human mammal
[0461] In certain embodiments, the vertebrate or the mammal is a
human. In certain embodiments, the human is a participant in a
study reviewed by a governmental agency charged with marketing
approval for a drug. In certain embodiments, the study is a
clinical trial. In certain embodiments, the governmental agency is
the Food and Drug Administration of the United States.
[0462] The invention additionally features a method for identifying
PYY secretagogues, compounds useful for treatment or prevention of
a condition modulated by PYY, or compounds useful for a modulation
of a PYY-related activity in an individual, comprising the steps
of: [0463] (a) determining whether the compound increases a PYY
level in a sample, said sample having been obtained from a
vertebrate previous administered with a compound in the presence of
which less of a complex between a G protein-coupled receptor and an
optionally labelled known ligand to the receptor is formed than in
the absence of the compound, wherein the G protein-coupled receptor
comprises an amino acid sequence selected from the group consisting
of: [0464] (i) amino acids 1-335 of SEQ ID NO:2; [0465] (ii) amino
acids 2-335 of SEQ ID NO:2; [0466] (iii) an amino acid sequence
comprising the amino acid sequence of a polypeptide derived from
(i) by substitution, deletion or addition of one or several amino
acids in the amino acid sequence defined in (i); [0467] (iv) the
amino acid sequence of a G protein-coupled receptor encoded by a
polynucleotide hybridizing under stringent conditions stringency to
the complement of SEQ ID NO:1; [0468] (v) a variant of SEQ ID NO: 2
when selected from the group consisting of: [0469] (a') the amino
acid sequence of a G protein-coupled receptor having at least about
80% identity to SEQ ID NO: 2; and [0470] (b') the amino acid
sequence of a G protein-coupled receptor comprising at least 20
contiguous amino acids of SEQ ID NO: 2; [0471] (vi) the amino acid
sequence of a constitutively active version of a G protein-coupled
receptor having SEQ ID NO: 2; and [0472] (vii) a biologically
active fragment of any one of (i) to (vi); [0473] said compound
having been determined or identified by a method according to the
fourth aspect; and wherein the ability of the test compound to
increase a PYY level in the vertebrate is further indicative of the
test compound being a PYY secretagogue, a compound useful for
treatment or prevention of a condition modulated by PYY, or a
compound useful for a modulation of a PYY-related activity in an
individual.
[0474] In certain embodiments, the vertebrate is a mammal. In
certain embodiments, the vertebrate is a non-human vertebrate. In
certain embodiments, the vertebrate is a non-human mammal. In
certain embodiments, the mammal is a non-human mammal.
[0475] In certain embodiments, the vertebrate or the mammal is a
human. In certain embodiments, the human is a participant in a
study reviewed by a governmental agency charged with marketing
approval for a drug. In certain embodiments, the study is a
clinical trial. In certain embodiments, the governmental agency is
the Food and Drug Administration of the United States.
[0476] In some embodiments, a G protein-coupled receptor of the
invention is SEQ ID NO:2 or an allele thereof. In some embodiments,
a G protein-coupled receptor of the invention is an allele of SEQ
ID NO:2. In some embodiments, a G protein-coupled receptor of the
invention is an ortholog of SEQ ID NO:2.
[0477] In some embodiments, a G protein-coupled receptor of the
invention is a G protein-coupled receptor which specifically binds
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine. In some
embodiments, a G protein-coupled receptor of the invention is a G
protein-coupled receptor for which
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine is a ligand. In
some embodiments, a G protein-coupled receptor of the invention is
a G protein-coupled receptor for which
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine is an agonist.
In some embodiments, a G protein-coupled receptor of the invention
exhibits a detectable level of constitutive activity. In some
embodiments, the constitutive activity is for increasing a level of
intracellular cAMP accumulation. In some embodiments, the
constitutive activity is for causing melanophore cells to undergo
pigment dispersion.
[0478] In certain embodiments, stringent hybridization conditions
comprise hybridization at 42.degree. C. in a solution comprising
50% formamide, 5.times.SSC (1.times.SSC=150 mM NaCl, 15 mM
trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5.times.
Denhardt's solution, 10% dextran sulfate, and 20m/ml denatured,
sheared salmon sperm DNA, followed by washing at 65.degree. C. in a
solution comprising 0.1.times.SSC. Hybridization techniques are
well known to the skilled artisan.
[0479] In certain embodiments, the GPCR encoded by a polynucleotide
hybridizing under stringent conditions to the complement of SEQ ID
NO: 1 is SEQ ID NO:2 or an allele thereof. In certain embodiments,
the GPCR encoded by a polynucleotide hybridizing under stringent
conditions to the complement of SEQ ID NO: 1 is an allele of SEQ ID
NO:2. In certain embodiments, the GPCR encoded by a polynucleotide
hybridizing under stringent conditions to the complement of SEQ ID
NO: 1 is an ortholog of SEQ ID NO:2. In certain embodiments, the
GPCR encoded by a polynucleotide hybridizing under stringent
conditions to the complement of SEQ ID NO: 1 specifically binds
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-4-yl}-amine. In certain embodiments, the GPCR
encoded by a polynucleotide hybridizing under stringent conditions
to the complement of SEQ ID NO: 1 is a receptor for which
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine is a ligand. In
certain embodiments, the GPCR encoded by a polynucleotide
hybridizing under stringent conditions to the complement of SEQ ID
NO: 1 is a receptor for which
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadi-
azol-5-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine is an
agonist. In some embodiments, the GPCR encoded by a polynucleotide
hybridizing under stringent conditions to the complement of SEQ ID
NO: 1 exhibits a detectable level of constitutive activity. In some
embodiments, the constitutive activity is for increasing a level of
intracellular cAMP. In some embodiments, the constitutive activity
is for causing melanophore cells to undergo pigment dispersion.
[0480] In certain embodiments, the variant of SEQ ID NO:2 is a G
protein-coupled receptor having at least about 80% identity to SEQ
ID NO:2. In certain embodiments, the variant of SEQ ID NO:2 is a G
protein-coupled receptor comprising at least 20 contiguous amino
acids of SEQ ID NO:2. In certain embodiments, the variant of SEQ ID
NO:2 which is a G protein-coupled receptor is an endogenous G
protein-coupled receptor. In certain embodiments, the variant of
SEQ ID NO:2 which is a G protein-coupled receptor is a
non-endogenous G protein-coupled receptor.
[0481] In certain embodiments, the G protein-coupled receptor which
has at least about 80% identity to SEQ ID NO: 2 is SEQ ID NO: 2 or
an allele thereof. In certain embodiments, the G protein-coupled
receptor which has at least about 80% identity to SEQ ID NO: 2 is
an allele of SEQ ID NO: 2. In certain embodiments, the G
protein-coupled receptor which has at least about 80% identity to
SEQ ID NO: 2 is an ortholog of SEQ ID NO: 2. In certain
embodiments, the G protein-coupled receptor which has at least
about 80% identity to SEQ ID NO: 2 specifically binds
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine. In certain
embodiments, the G protein-coupled receptor which has at least
about 80% identity to SEQ ID NO: 2 is a receptor for which
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine is an agonist. In
some embodiments, the the G protein-coupled receptor which has at
least about 80% identity to SEQ ID NO: 2 exhibits a detectable
level of constitutive activity. In some embodiments, the
constitutive activity is for increasing a level of intracellular
cAMP. In some embodiments, the constitutive activity is for causing
melanophore cells to undergo pigment dispersion.
[0482] In certain embodiments, the G protein-coupled receptor which
comprises at least 20 contiguous amino acids of SEQ ID NO: 2 is SEQ
ID NO: 2 or an allele of SEQ ID NO: 2. In certain embodiments, the
G protein-coupled receptor which comprises at least 20 contiguous
amino acids of SEQ ID NO: 2 is an allele of SEQ ID NO: 2. In
certain embodiments, the G protein-coupled receptor which comprises
at least 20 contiguous amino acids of SEQ ID NO: 2 is an ortholog
of SEQ ID NO: 2. In certain embodiments, the G protein-coupled
receptor which comprises at least 20 contiguous amino acids of SEQ
ID NO: 2 specifically binds
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadizol-5--
yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine. In certain
embodiments, the G protein-coupled receptor which comprises at
least 20 contiguous amino acids of SEQ ID NO: 2 is a receptor for
which
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine is an agonist.
In some embodiments, the G protein-coupled receptor which comprises
at least 20 contiguous amino acids of SEQ ID NO: 2 exhibits a
detectable level of constitutive activity. In some embodiments, the
constitutive activity is for increasing a level of intracellular
cAMP. In some embodiments, the constitutive activity is for causing
melanophore cells to undergo pigment dispersion.
[0483] In some embodiments, the G protein-coupled receptor is part
of a fusion protein comprising a G protein. Techniques for making a
GPCR:G fusion construct are well known to the skilled artisan (see,
e.g., International Application WO 02/42461).
[0484] In some embodiments, the compound in the presence of which
less of a complex between a G protein-coupled receptor and an
optionally labelled known ligand to the receptor is formed than in
the absence of the compound is a small molecule. In some
embodiments, the compound in the presence of which less of a
complex between a G protein-coupled receptor and an optionally
labelled known ligand to the receptor is formed than in the absence
of the compound is a small molecule, with the proviso that the
small molecule is not a polypeptide. In some embodiments, the
compound in the presence of which less of a complex between a G
protein-coupled receptor and an optionally labelled known ligand to
the receptor is formed than in the absence of the compound is a
small molecule, with the proviso that the small molecule is not an
antibody or an antigen-binding fragment thereof. In some
embodiments, the compound in the presence of which less of a
complex between a G protein-coupled receptor and an optionally
labelled known ligand to the receptor is formed than in the absence
of the compound is a small molecule, with the proviso that the
small molecule is not a lipid. In some embodiments, the test
compound is a small molecule, with the proviso that the small
molecule is not a polypeptide or a lipid. In some embodiments, the
test compound is a polypeptide. In some embodiments, the compound
in the presence of which less of a complex between a G
protein-coupled receptor and an optionally labelled known ligand to
the receptor is formed than in the absence of the compound is a
polypeptide, with the proviso that the polypeptide is not an
antibody or an antigen-binding fragment thereof. In some
embodiments, the compound in the presence of which less of a
complex between a G protein-coupled receptor and an optionally
labelled known ligand to the receptor is formed than in the absence
of the compound is a lipid. In some embodiments, the compound in
the presence of which less of a complex between a G protein-coupled
receptor and an optionally labelled known ligand to the receptor is
formed than in the absence of the compound is not an antibody or an
antigen-binding fragment thereof. In some embodiments, the compound
in the presence of which less of a complex between a G
protein-coupled receptor and an optionally labelled known ligand to
the receptor is formed than in the absence of the compound is an
antibody or an antigen-binding fragment thereof.
[0485] In certain embodiments, the method is a method for
identifying PYY secretagogues.
[0486] In certain embodiments, the method is a method for
identifying compounds useful for treatment or prevention of a
condition modulated by PYY.
[0487] In certain embodiments, the method is a method for
identifying compounds useful for a modulation of a PYY-related
activity in an individual.
[0488] In certain embodiments, the ligand for GPR119 is a GPR119
agonist. In certain embodiments, the ligand for GPR119 or the
GPR119 agonist is a ligand or agonist of an endogenous vertebrate,
mammalian or human GPR119 receptor. In certain embodiments, the
ligand for GPR119 or the GPR119 agonist is a ligand or agonist of
an endogenous human GPR119 receptor. In certain embodiments, the
ligand for GPR119 or the GPR119 agonist is identical to a compound
disclosed in, e.g., in International Application No.
PCT/US2004/001267 (published as WO 04/065380); International
Application No. PCT/US2004/005555 (published as WO 04/076413);
International Application No. PCT/US2004/022327 (published as WO
05/007647); International Application No. PCT/US2004/022417
(published as WO 05/007658); International Application No.
PCT/US2005/019318 (published as WO 2005/121121); International
Application No. PCT/GB2004/050046 (published as WO 2005/061489);
International Application No. PCT/US06/00567 (published as WO
2006/083491); International Application No. PCT/GB2005/050264
(published as WO 2006/067531); International Application No.
PCT/GB2005/050265 (published as WO 2006/067532); International
Application No. PCT/GB2005/050266 (published as WO 2006/070208);
International Application No. PCT/JP02/09350 (published as WO
03/026661); International Application No. PCT/JP2005/018412
(published as WO 06/040966); International Application No.
PCT/JP2005/019000 (published as WO 2006/043490); International
Application No. PCT/GB2006/050176 (published as WO 2007/003960);
International Application No. PCT/GB2006/050177 (published as WO
2007/003961); International Application No. PCT/GB2006/050178
(published as WO 2007/003962); International Application No.
PCT/GB2006/050182 (published as WO 2007/003964); International
Application No. PCT/JP02/09350 (published as WO 03/026661);
International Application No. PCT/GB2007/050183 (published as WO
2007/116229); International Application No. PCT/GB2007/050190
(published as WO 2007/116230); International Application No.
PCT/US2007/073364 (published as WO 2008/008895); International
Application No. PCT/EP2007/058991 (published as WO 2008/025798);
International Application No. PCT/EP2007/058993 (published as WO
2008/025799); or International Application No. PCT/EP2007/058995
(published as WO 2008/025800). In certain embodiments, the ligand
for GPR119 or the GPR119 agonist is
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadizol-5--
yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine. In certain
embodiments, the ligand for GPR119 is an endogenous ligand of an
endogenous vertebrate, mammalian, or human GPR119 receptor.
[0489] In certain embodiments, the optionally labeled known ligand
is a labeled known ligand. In certain embodiments, the labeled
known ligand is a radiolabeled known ligand. Techniques for
radiolabeling a compound, such as for labeling a known ligand of a
G protein-coupled receptor of the invention, are well known to the
skilled artisan. See, e.g., International Application WO 04/065380.
Also see, e.g., Example 14, infra.
[0490] Techniques for detecting the complex between a G
protein-coupled receptor and a compound known to be a ligand of the
G protein-coupled receptor are well known to the skilled artisan.
See, e.g., International Application WO 04/065380. Also see, e.g.,
Example 15, infra.
[0491] In some embodiments, the method further comprises the step
of optionally determining the structure of the PYY secretagogue,
the compound useful for treatment or prevention of a condition
modulated by PYY, or the compound useful for a modulation of a
PYY-related activity in an individual.
[0492] In some embodiments, the method further comprises the step
of optionally providing the name or structure of the PYY
secretagogue, the compound useful for treatment or prevention of a
condition modulated by PYY, or the compound useful for a modulation
of a PYY-related activity in an individual.
[0493] In some embodiments, said method further comprises the step
of optionally producing or synthesizing the PYY secretagogue, the
compound useful for treatment or prevention of a condition
modulated by PYY, or the compound useful for a modulation of a
PYY-related activity in an individual.
[0494] In some embodiments, said method further comprises the step
of formulating the PYY secretagogue, the compound useful for
treatment or prevention of a condition modulated by PYY, or the
compound useful for a modulation of a PYY-related activity in an
individual as a pharmaceutical.
[0495] In some embodiments, said method further comprises
evaluating the bioavailability of the PYY secretagogue, the
compound useful for treatment or prevention of a condition
modulated by PYY, or the compound useful for a modulation of a
PYY-related activity.
[0496] In some embodiments, said method further comprises the step
of formulating the PYY secretagogue, the compound useful for
treatment or prevention of a condition modulated by PYY, or the
compound useful for a modulation of a PYY-related activity in an
individual into a pharmaceutical composition.
[0497] The invention additionally features a process comprising
performing a method according to this ninth aspect.
[0498] Applicant reserves the right to exclude any one or more
candidate compounds from any of the embodiments of the invention.
Applicant reserves the right to exclude any one or more modulators
from any of the embodiments of the invention. By way of example and
not limitation, Applicant reserves the right to exclude any one or
more agonists from any of the embodiments of the invention.
Applicant reserves the right to exclude any polynucleotide or
polypeptide from any of the embodiments of the invention. Applicant
additionally reserves the right to exclude any condition modulated
by PYY, any bone-related condition, any bone-related condition
wherein the bone-related condition is a condition characterized by
low bone mass, any bone-related condition wherein the bone-related
condition is a bone fracture, any bone-related condition wherein
the bone-related condition is a bone disease, any bone-related
condition wherein the bone-related condition is enhanced bone
healing or bone growth, any metabolic disorder, any
angiogenesis-related condition, any ischemia-related condition, any
malabsorptive disorder, any convulsive disorder, any cancer, any
inflammatory disorder, any inflammatory lung disease, any
inflammatory bowel disease, and any modulation of a PYY-related
activity in an individual from any of the embodiments of the
invention. It is also expressly contemplated that conditions
modulated by PYY, bone-related conditions, bone-related conditions
wherein the bone-related condition is a condition characterized by
low bone mass, bone-related conditions wherein the bone-related
condition is a bone fracture, bone-related conditions wherein the
bone-related condition is a bone disease, bone-related conditions
wherein the bone-related condition is enhanced bone healing or bone
growth, metabolic disorders, angiogenesis-related conditions,
ischemia-related conditions, malabsorptive disorders, convulsive
disorders, cancers, inflammatory disorders, inflammatory lung
diseases, inflammatory bowel diseases, and modulations of a
PYY-related activity in an individual of the invention can be
included in an embodiment either individually or in any
combination.
BRIEF DESCRIPTION OF THE DRAWINGS
[0499] The invention is illustrated in connection with the figures
appended hereto in which:
[0500] FIG. 1 shows that GPR119 receptor exhibits constitutive
activity for increasing cAMP accumulation in transfected HEK293
cells.
[0501] FIG. 2 shows that Compound 1 is an agonist of GPR119
receptor.
[0502] FIG. 3 shows an effect of administration of GPR119 agonist
on plasma total PYY in wild-type mice.
DETAILED DESCRIPTION OF THE INVENTION
[0503] The present invention is based, at least in part, on the
surprising discovery by Applicant that administration of a GPR119
agonist to an individual, such as by oral administration, can act
to increase a plasma total PYY in the individual. The present
invention features methods relating to GPR119 for identifying PYY
secretagogues, compounds useful for treatment or prevention of a
condition modulated by PYY, and compounds useful for a modulation
of a PYY-related activity in an individual. A GPR119 agonist is
useful for increasing plasma total PYY in an individual. In certain
embodiments, the individual is a human. GPR119 agonists are useful
as pharmaceutical agents for treatment or prevention of a condition
modulated by PYY and for a modulation of a PYY-related activity in
an individual.
[0504] The term "ligand", as used herein, shall mean a molecule
(e.g., test compound) that specifically binds to a polypeptide,
such as GPR119. A ligand may be, for example, a polypeptide, a
lipid, a small molecule, an antibody. Compound 1 is an exemplary
ligand of GPR119 receptor polypeptide (see, Table A, which sets
forth the chemical structure and chemical name of Compound 1).
Compound 1 is identical to a compound disclosed in International
Patent Application No. PCT/US2004/001267 (published as WO
2004/065380).
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadizol-5--
yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine (see, Table A) is
an exemplary ligand of GPR119 receptor polypeptide. An endogenous
ligand is a ligand that is an endogenous, natural ligand for a
native polypeptide, such as GPR119. A ligand may be an
"antagonist", "agonist", "partial agonist", or "inverse agonist",
or the like.
TABLE-US-00001 TABLE A ##STR00001## (2-Fluoro-4- methanesulfonyl-
phenyl)-{6-[4-(3- isopropyl- [1,2,4]oxadiazol-5-
yl)-piperidin-1-yl]- 5-nitro-pyrimidin-4- yl}-amine
[0505] The term "agonist", as used herein, shall mean an agent
(e.g., ligand, test compound) that by virtue of binding to a GPCR
activates (e.g., stimulates) the GPCR so as to elicit an
intracellular response mediated by the GPCR.
[0506] The term "partial agonist", as used herein, shall mean an
agent (e.g., ligand, test compound) that by virtue of binding to a
GPCR activates (e.g., stimulates) the GPCR so as to elicit an
intracellular response mediated by the GPCR, albeit to a lesser
exent or degree than does a full agonist.
[0507] The term "antagonist" shall mean an agent (e.g., ligand,
test compound) that binds, and preferably binds competitively, to a
GPCR at about the same site as an agonist or partial agonist but
which does not activate an intracellular response initiated by the
active form of the GPCR, and can thereby inhibit the intracellular
response by agonist or partial agonist. An anatagonist typically
does not diminish the baseline intracellular response in the
absence of an agonist or partial agonist.
[0508] The term "inverse agonist" shall mean an agent (e.g.,
ligand, test compound) which binds to a GPCR and which inhibits the
baseline intracellular response initiated by the active form of the
receptor below the normal base level activity which is observed in
the absence of an agonist or partial agonist.
[0509] The term "GPR119 agonist," as used herein, refers to a
compound that binds to GPR119 receptor and acts as an agonist.
Compound 1 is an exemplary GPR119 agonist (see, Table A, which sets
forth the chemical structure and chemical name of Compound 1).
Compound 1 is identical to a compound disclosed in International
Patent Application No. PCT/US2004/001267 (published as WO
2004/065380). (2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3
-isopropyl-[1,2,4]oxadiazol-5-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-
-amine is an exemplary GPR119 agonist.
[0510] The term "selective GPR119 agonist," as used herein, refers
to a GPR119 agonist having selectivity for GPR119 receptor over one
or more related receptors, such as corticotrophin-releasing
factor-1 (CRF-1) receptor. Compound 1 is an exemplary selective
GPR119 agonist (see, Table A, which sets forth the chemical
structure and chemical name of Compound 1). Compound 1 is identical
to a compound disclosed in International Patent Application No.
PCT/US2004/001267 (published as WO 2004/065380).
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3
-isopropyl-[1,2,4]oxadizol-5-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}--
amine is an exemplary selective GPR119 agonist.
[0511] The term "PYY secretagogue" shall mean an agent (e.g.,
ligand, test compound) that promotes (e.g., stimulates) secretion
of PYY in a cell, e.g. an enteroendocrine cell, or that increases a
level of total PYY, e.g. a level of blood or plasma total PYY, on
administration to an individual such as a vertebrate or a mammal.
In certain embodiments, a PYY secretagogue is a compound suitable
for increasing a level of total PYY in an individual, for example a
level of blood or plasma total PYY.
[0512] As used herein, the term "condition modulated by PYY" refers
to a condition modulated by PYY.sub.1-36 or by a fragment thereof
that is capable of modulating a G protein-coupled receptor. In
certain embodiments, the term "condition modulated by PYY" refers
to a condition modulated by PYY.sub.3-36. In certain embodiments,
the term "condition modulated by PYY" refers to a condition
modulated by an NPY Y2 receptor (Y2R) agonist. In certain
embodiments, the term "condition modulated by PYY" refers to a
condition modulated by stimulation of Y2R. In certain embodiments,
the term "condition modulated by PYY.sub.3-36" refers to a
condition modulated by a Y2R agonist. In certain embodiments, the
term "condition modulated by PYY.sub.3-36" refers to a condition
modulated by stimulation of Y2R.
[0513] As used herein, the term "PYY-related activity" refers to an
activity for modulating a level of PYY or to an activity associated
with PYY. In certain embodiments, the term "PYY-related activity"
refers to an activity for increasing a level of PYY or to an
activity associated with PYY.
[0514] The term "individual," as used herein, refers to a
vertebrate, including but not limited to fish (such as commercially
farmed fish, pet fish, etc.), amphibians (such as frogs, toads, pet
amphibians, etc.), reptiles (such as snakes, lizards, turtles, pet
reptiles, etc.), birds (such as chickens, turkeys, pet birds, etc.)
and mammals (such as mice, rats, hamsters, rabbits, pigs, dogs,
cats, horses, cows, sheep, goats, non-human primates, non-human
mammals, pet non-human mammals, humans, etc.). In certain
embodiments, the individual is a fish. In certain embodiments, the
individual is an amphibian. In certain embodiments, the individual
is a reptile. In certain embodiments, the individual is a bird. In
certain embodiments, the individual is a turkey. Over the past 25
yr, commercial selection pressure for turkeys with larger breast
muscle mass has placed increasing demands on skeletal integrity.
The increased breast muscle mass, however, has not been accompanied
by compensatory changes in the skeleton, with the result that the
turkey industry has experienced an increase in leg problems. Long
bone fracture in young adult male turkeys has been reported. (See,
e.g., Crespo et al, Poult Sci (2000) 79:602-608.) In certain
embodiments, the individual is a mammal. In certain embodiments,
the individual is a mouse, a rat, a hamster, a rabbit, a pig, a
dog, a cat, a horse, a cow, a sheep, a goat, a non-human primate or
a human (which may be included in embodiments of the invention
individually or in any combination). In certain embodiments, the
individual is a horse. Performance horses, which are horses
involved in activities such as racing, pacing and other competitive
events, are susceptible to bone fracture. In certain embodiments,
the individual is a dog or a cat. In certain embodiments, the
individual is a human companion animal (such as a dog, a cat,
etc.), a farm animal (such as a cow, a sheep, a goat, a pig, a
chicken, etc.), a sports animal (such as a horse, a dog, etc.), a
beast of burden (such as a mule, a camel, etc.) or an exotic animal
(such as an animal found in a zoo, etc.), which may be included in
embodiments of the invention individually or in any combination. In
certain embodiments, the individual is a non-human vertebrate. In
certain embodiments, the individual is a non-human mammal. In
certain embodiments, the individual is a non-human primate (such as
a rhesus monkey, a chimpanzee, etc.). In certain embodiments, the
individual is a human.
[0515] The term "in need of prevention or treatment" as used herein
refers to a judgement made by a caregiver (e.g. physician, nurse,
nurse practitioner in the case of humans; veterinarian in the case
of non-human vertebrates, and in particular embodiment non-human
mammals) that an individual requires or will benefit from
treatment.
[0516] The term "therapeutically effective amount" or
"therapeutically effective dose" as used herein refers to the
amount of active compound or pharmaceutical agent that elicits the
biological or medicinal response in a tissue, system, animal,
individual or human that is being sought by a researcher,
veterinarian, medical doctor or other clinician, which includes one
or more of the following: [0517] (1) Preventing the disease; for
example, preventing a disease, condition or disorder in an
individual that may be predisposed to the disease, condition or
disorder but does not yet experience or display the pathology or
symptomatology of the disease, [0518] (2) Inhibiting the disease;
for example, inhibiting a disease, condition or disorder in an
individual that is experiencing or displaying the pathology or
symptomatology of the disease, condition or disorder (i.e.,
arresting further development of the pathology and/or
symptomatology), and [0519] (3) Ameliorating the disease; for
example, ameliorating a disease, condition or disorder in an
individual that is experiencing or displaying the pathology or
symptomatology of the disease, condition or disorder (i.e.,
reversing the pathology and/or symptomatology).
[0520] The term "therapeutic efficacy" as used herein refers to
elicitation of the biological or medicinal response in a tissue,
system, animal, individual or human that is being sought by a
researcher, veterinarian, medical doctor or other clinician, which
includes one or more of the following: [0521] (1) Preventing the
disease; for example, preventing a disease, condition or disorder
in an individual that may be predisposed to the disease, condition
or disorder but does not yet experience or display the pathology or
symptomatology of the disease, [0522] (2) Inhibiting the disease;
for example, inhibiting a disease, condition or disorder in an
individual that is experiencing or displaying the pathology or
symptomatology of the disease, condition or disorder (i.e.,
arresting further development of the pathology and/or
symptomatology), and [0523] (3) Ameliorating the disease; for
example, ameliorating a disease, condition or disorder in an
individual that is experiencing or displaying the pathology or
symptomatology of the disease, condition or disorder (i.e.,
reversing the pathology and/or symptomatology).
[0524] The term "amount that is effective to prevent" refers to
that amount of drug that will prevent or reduce the risk of
occurrence of the biological or medical event that is sought to be
prevented. In many instances, the amount that is effective to
prevent is the same as the therapeutically effective amount.
[0525] The term "peripheral administration" shall mean
administration outside of the central nervous system. Peripheral
administration does not include direct administration to the brain.
Peripheral administration includes, but is not limited to,
intravascular, intramuscular, subcutaneous, inhalation, oral,
sublingual, enteral, rectal, transdermal, or intra-nasal
administration.
[0526] The term "composition" shall mean a material comprising at
least one component.
[0527] The term "active ingredient" shall mean any component that
provides pharmacological activity or other direct effect in the
diagnosis, cure, mitigation, treatment, or prevention of
disease.
[0528] The term "pharmaceutical composition" shall mean a
composition comprising at least one active ingredient, whereby the
composition is amenable to investigation and treatment in a
mammal
[0529] By "pharmaceutically acceptable" it is meant that the
carrier, vehicle, diluent, excipients, and/or salt must be
compatible with the other ingredients of the formulation, and not
deleterious to the recipient thereof.
[0530] The term "dosage form" shall mean the physical form in which
a drug is produced and dispensed, such as a tablet, capsule, or an
injectable.
[0531] By "bone" is intended the dense, semi-rigid, porous,
calcified connective tissue forming the major portion of the
skeleton of most vertebrates, comprising a dense organic matrix and
an inorganic, mineral component. Bone is any of numerous
anatomically distinct structures making up the skeleton of a
vertebrate. The terms "bone mass" and "bone mineral density (BMD)"
are used interchangeably herein.
[0532] BMD in humans is usually measured by a standard radiographic
technique, dual energy X-ray absorptiometry (DXA). Of the many
techniques developed to assess BMD, DXA is the most highly
developed technically and the most thoroughly validated
biologically. DXA technology, with suitably adapted software, can
also be used to reliably assess BMD in animal studies. DXA is used
in the diagnosis of osteoporosis, prognosis (fracture prediction),
monitoring the natural history of the disorder, and assessing
response to treatment.
[0533] The term "low bone mass" as used herein refers to any
decrease or reduction in bone mineral density (BMD) in an
individual, and includes both osteoporosis and osteopenia as
defined in proposals by the World Health Organization (WHO). The
WHO has defined normal as a value of BMD within one standard
deviation of the young adult reference mean (T-score.ltoreq.-1).
The WHO has defined osteopenia as a value of BMD more than 1
standard deviation below the young adult mean, but less than 2.5
standard deviations below this value (T-score<-1 and >-2.5).
The WHO has characterized osteoporosis as a more severe form of
osteopenia, and has defined it by value of BMD 2.5 standard
deviations or more blow the young adult mean (T-score.ltoreq.-2.5).
(See, e.g., World Health Organization Technical Report Series 921
(2003), Prevention and Management of Osteoporosis, the disclosure
of which is herein incorporated by reference in its entirety.) More
commonly, osteopenia is defined as a T-score of less than -1 and
greater than -2, and osteoporosis is defined as a T-score of less
than or equal to -2. In certain embodiments of the present
invention, the T-score is measured at the hip with DXA.
[0534] The term "osteoporosis" as used herein is defined by a value
of BMD 2 standard deviations or more below the young adult
reference mean (T-score.ltoreq.-2) or refers to a diagnosis made by
a caregiver (e.g. physician, nurse, nurse practitioner in the case
of humans; veterinarian in the case of non-human vertebrates).
[0535] Osteoporosis can be classified as either primary or
secondary. (See, e.g., World Health Organization Technical Report
Series 921 (2003), Prevention and Management of Osteoporosis.) As
used herein, the term "osteoporosis" encompasses primary
osteoporosis and secondary osteoporosis. In certain embodiments,
osteoporosis is primary osteoporosis. In certain embodiments,
osteoporosis is secondary osteoporosis.
[0536] "Primary osteoporosis" as used herein is associated with
menopause (natural, premature, or surgical), aging, or both. It
shall be understood that in the present invention, primary
osteoporosis associated with menopause (natural, premature, or
surgical), primary osteoporosis associated with aging, and primary
osteoporosis associated with menopause and aging can be included in
embodiments individually or in any combination.
[0537] "Secondary osteoporosis" as used herein refers to
osteoporosis which is associated not with menopause or aging but
rather with medical conditions or with the use of medications or
drugs. An increased risk of osteoporosis is associated with a host
of medical conditions, including but not limited to endocrine and
metabolic disorders, and malignant disease, and with the use of
certain medications and drugs, examples of which are well known to
those skilled in the art (see, e.g., World Health Organization
Technical Report Series 921 (2003), Prevention and Management of
Osteoporosis; Williams Textbook of Endocrinology, 10.sup.th
Edition; the disclosure of which is herein incorporated by
reference in its entirety.) Secondary osteoporosis can also be
associated with immobilization. A diagnosis of osteoporosis
secondary to a medical condition, to use of a medication or drug,
or to immobilization can be made by a caregiver (e.g. physician,
nurse, nurse practitioner in the case of humans; veterinarian in
the case of non-human vertebrates).
[0538] By "bone fracture" is intended a complete or incomplete
break, rupture or crack of a bone. Diagnosis of fractures normally
depends upon clinical examination and radiological findings. In the
invention, bone fractures include, but are not limited to,
traumatic fractures, long-term fractures, and pathological
fractures.
[0539] "Traumatic fracture" as used herein shall refer to an
immediate fracture which involves a supraliminal trauma with a
degree of local violence that exceeds the natural elasticity of the
bone. It can be accompanied by simultaneous injury to the soft
tissues and very often the skin. A traumatic fracture can be closed
(the adjacent soft tissue can be injured but the covering soft
parts are largely preserved). A traumatic fracture can be open (the
broken ends of the bone are freed by extensive soft tissue injury
so that pathogens from outside can enter the wound directly).
[0540] "Long-term fracture" as used herein shall refer to a chronic
fracture, fatigue fracture, stress fracture or spontaneous fracture
type I.
[0541] "Pathological fracture" as used herein shall refer to a
spontaneous fracture type II. A pathological fracture arises
spontaneously, without adequate trauma to account for it. The bone
may have been previously damaged, either by a systemic disease
(e.g., osteoporosis, osteodystrophy, or Paget's osteitis deformans)
or by a local bone lesion (e.g., metastasis, radioosteonecrosis, or
bone tumor). See, Adler, Claus-Peter, BONE DISEASES, p. 114
(Springer-Verlag, Germany 2000).
[0542] Fractures also include, but are not limited no, oblique
torsion fracture, transverse fracture, comminuted fracture,
compression fracture, rib fractures, creeping fracture, and
fractured femoral neck (Adler, Claus-Peter, BONE DISEASES,
Springer-Verlag, Germany (2000)).
[0543] As used herein, "bone-related condition" refers to a
condition that derives benefit from treatment according to the
invention, by increasing bone mass or bone growth or by decreasing
loss of bone mass, and includes but is not limited to a condition
characterized by low bone mass, a bone fracture, a bone disease,
and enhancing bone healing or bone growth. It shall be understood
that in the present invention, bone-related conditions that can be
treated according to the invention can be included in embodiments
individually or in any combination.
[0544] As used herein, the term "condition characterized by low
bone mass" includes but is not limited to osteopenia, osteoporosis,
rheumatoid arthritis, osteoarthritis, periodontal disease, alveolar
bone loss, osteotomy bone loss, childhood idiopathic bone loss,
curvature of the spine and loss of height. In certain embodiments,
osteoporosis is primary osteoporosis. In certain embodiments,
osteoporosis is secondary osteoporosis. In certain embodiments,
secondary osteoporosis is associated with a medical condition. In
certain embodiments, secondary osteoporosis is associated with the
use of a medication or drug. In certain embodiments, secondary
osteoporosis is associated with immobilization. Conditions
characterized by low bone mass also include but are not limited to
Paget's disease, bone loss due to metastatic cancer, and osteolytic
lesions such as those caused by neoplastic disease, radiotherapy,
or chemotherapy. Conditions characterized by low bone mass also
include but are not limited to long-term complications of
osteoporosis such as curvature of the spine, loss of height and
prosthetic surgery. It shall be understood that in the present
invention, conditions characterized by low bone mass can be
included in embodiments individually or in any combination. (See,
e.g., World Health Organization Technical Report Series 921 (2003),
Prevention and Management of Osteoporosis; Williams Textbook of
Endocrinology, 10.sup.th Edition, Larsen et al, Eds. (2002), W.B.
Saunders Company; and Endocrinology and Metabolism, 4.sup.th
Edition, Felig et al, Eds. (2001), McGraw-Hill Book Company; the
disclosure of each of which is herein incorporated by reference in
its entirety.)
[0545] As used herein, "bone disease" refers to a disorder or
condition relating to abnormality of the bone. Bone diseases that
can be treated according to the invention, by increasing bone mass
or bone growth or decreasing loss of bone mass, include but are not
limited to osteopenia, osteoporosis, rheumatoid arthritis,
osteoarthritis, periodontal disease, alveolar bone loss, osteotomy
bone loss, childhood idiopathic bone loss, curvature of the spine,
and loss of height. In certain embodiments, osteoporosis is primary
osteoporosis. In certain embodiments, osteoporosis is secondary
osteoporosis. In certain embodiments, secondary osteoporosis is
associated with a medical conditions. In certain embodiments,
secondary osteoporosis is associated with the use of a medication
or drug. In certain embodiments, secondary osteoporosis is
associated with immobilization. Bone diseases that can be treated
according to the invention, by increasing bone mass or bone growth,
also include but are not limited to Paget's disease and bone loss
due to metastatic cancer. Destructive bone disorders that can be
treated according to the invention, by increasing bone mass or
growth, include but are not limited to osteoporosis,
osteoarthritis, and osteolytic lesions such as those caused by
neoplastic disease, radiotherapy, or chemotherapy. It shall be
understood that in the present invention, bone diseases that can be
treated according to the invention, by increasing bone mass or
growth, can be included in embodiments individually or in any
combination. (See, e.g., World Health Organization Technical Report
Series 921 (2003), Prevention and Management of Osteoporosis;
Williams Textbook of Endocrinology, 10.sup.th Edition, Larsen et
al, Eds. (2002), W.B. Saunders Company; and Endocrinology and
Metabolism, 4.sup.th Edition, Felig et al, Eds. (2001), McGraw-Hill
Book Company; the disclosure of each of which is herein
incorporated by reference in its entirety.)
[0546] The present invention also relates to the other conditions
that derive benefit from treatment according to the invention, by
increasing bone mass or bone growth or decreasing loss of bone
mass, including but not limited to enhanced bone healing following
facial reconstruction, maxillary reconstruction, mandibular
reconstruction, periodontal disease or tooth extraction, enhanced
long bone extension, enhanced prosthetic ingrowth and increased
bone synostosis.
[0547] As used herein, "metabolic disorder" refers to a disorder
relating to abnormality of metabolism. Metabolic disorders that can
be treated according to the invention include but are not limited
to overweight, obesity, hyperphagia, diabetes (inclusive of type 1
diabetes and type 2 diabetes), type 2 diabetes, impaired glucose
tolerance, insulin resistance, hyperinsulinemia, dyslipidemia,
hypertension and metabolic syndrome. It shall be understood that in
the present invention, metabolic disorders that can be treated
according to the invention can be included in embodiments
individually or in any combination.
[0548] The term "metabolic syndrome" as defined herein, and
according to the Adult Treatment Panel III (ATP III; National
Institutes of Health: Third Report of the National Cholesterol
Education Program Expert Panel on Detection, Evaluation, and
Treatment of High Blood Cholesterol in Adults (Adult Treatment
Panel III), Executive Summary; Bethesda, Md., National Institutes
of Health, National Heart, Lung and Blood Institute, 2001 (NIH pub.
No 01-3670), occurs when a person meets three or more of five
criteria related to obesity, hypertriglyceridemia, low HDL
cholesterol, high blood pressure, and high fasting glucose.
[0549] The term "obesity," as used herein, is defined as a body
mass index (BMI) of 30.0 or greater, in accordance with the WHO
classifications of weight [Kopelman, Nature (2000) 404:635-643; the
disclosure of which is herein incorporated by reference in its
entirety].
[0550] "Impaired glucose tolerance" (IGT) as used herein is
intended to indicate that condition associated with
insulin-resistance that is intermediate between frank, type 2
diabetes and normal glucose tolerance
[0551] (NGT). IGT is diagnosed by a procedure wherein an affected
person's postprandial glucose response is determined to be abnormal
as assessed by 2-hour postprandial plasma glucose levels. In this
test, a measured amount of glucose is given to the patient and
blood glucose levels are measured at regular intervals, usually
every half hour for the first two hours and every hour thereafter.
In a "normal" or non-IGT individual, glucose levels rise during the
first two hours to a level less than 140 mg/dl and then drop
rapidly. In an IGT individual, the blood glucose levels are higher
and the drop-off level is at a slower rate.
[0552] "Insulin resistance" as used herein is intended to encompass
the usual diagnosis of insulin resistance made by any of a number
of methods, including but not restricted to: the intravenous
glucose tolerance test or measurement of the fasting insulin level.
It is well known that there is an excellent correlation between the
height of the fasting insulin level and the degree of insulin
resistance. Therefore, one could use elevated fasting insulin
levels as a surrogate marker for insulin resistance for the purpose
of identifying which normal glucose tolerance (NGT) individuals
have insulin resistance. A diagnosis of insulin resistance can also
be made using the euglycemic glucose clamp test.
[0553] "Dyslipidemia" is intended herein to encompass disorders
comprising any one of elevated level of plasma free fatty acids,
elevated level of plasma cholesterol, elevated level of
LDL-cholesterol, reduced level of HDL-cholesterol, elevated ratio
of total cholesterol to HDL cholesterol, and elevated level of
plasma triglycerides.
[0554] As used herein, "angiogenesis-related condition" refers to a
condition that derives benefit from increased angiogenesis.
Angiogenesis-related conditions that can be treated according to
the invention, by increasing angiogenesis, include but are not
limited to peripheral arterial disease, wound healing, and
ischemia. It shall be understood that in the present invention,
angiogenesis-related conditions that can be treated according to
the invention can be included in embodiments individually or in any
combination.
[0555] As used herein, "ischemia" refers to a state of tissue
oxygen deprivation or insufficiency.
[0556] As used herein, "ischemia-related condition" refers to a
condition which relates to ischemia. Ischemia-related conditions
that can be treated according to the invention include but are not
limited to cerebral ischemia, stroke, ischemic heart disease,
myocardial infarction, ischemia-reperfusion injury, post-myocardial
infarction remodeling, heart failure, and congestive heart failure.
It shall be understood that in the present invention,
ischemia-related conditions that can be treated according to the
invention can be included in embodiments individually or in any
combination.
[0557] "Stroke" is a cardiovascular disease that affects the blood
vessels supplying blood to the brain and is intended herein to
include cerebral thrombosis, the most common type of stroke.
Cerebral thrombosis occurs when a blood clot (thrombus) forms and
blocks blood flow in an artery bring blood to part of the brain.
Blood clots usually form in arteries damaged by
atherosclerosis.
[0558] As used herein, "ischemic heart disease" refers to a
disorder caused by lack of oxygen to the tissues of the heart, in
which muscles of the heart are affected and the heart cannot pump
properly. Ischemic heart disease is the most common cardiomyopathy
in the United States.
[0559] As used here, "myocardial infarction" refers to the damage
or death of an area of heart muscle because of an inadequate supply
of oxygen to that are. Myocardial infarctions are often caused by a
blood clot that blocks one of the coronary arteries (the blood
vessels that bring oxygen to heart muscle). The clot prevents blood
and oxygen from reaching that area of the heart, leading to the
death of heart cells in that area.
[0560] The loss of myocardial tissue due to myocardial infarction
results in a sustained excessive hemodynamic burden placed on the
ventricle. Ventricular hypertrophy constitutes one of the principle
mechanisms by which the heart compensates for an increased load.
However, the capacity for this adaptation to sustain cardiac
performance in the face of hemodynamic overload is finite and, when
chronically maintained, becomes maladaptive. Gradually, the
adaptive hypertrophic phenotype transitions to overt heart failure
as the enlarged ventricles progressively dilate and contractile
function weakens. The natural history of the adaptive and
maladaptive response to myocardial infarction in the heart is
referred to as "pos-myocardial infaraction remodeling".
[0561] "Congestive heart failure" refers to a disorder in which the
heart loses its ability to pump blood efficiently. Congestive heart
failure becomes more prevalent with advancing age. Ischemic heart
disease is the most common cause of congestive heart failure,
accounting for 60-70% of all cases. An increased venous pressure
greater than 12 mmHg is one of the major Framingham criteria for
congestive heart failure, as is a reduction in cardiac output
equivalent to a circulation time great than 25 seconds.
[0562] As used herein, "malabsorptive disorder" refers to a
disorder relating to abnormality of secretion and/or absorption in
the intenstinal epithelium. Malabsorptive disorders that can be
treated according to the invention include but are not limited to
short bowel syndrome, enterocolitis, Crohn's disease, ulcerative
colitis, irritable bowel syndrome, cholera and a disorder
accompanied by diarrhea. As used herein, the "disorder accompanied
by diarrhea" is not particularly limited as long as it is a disease
or a symptom accompanied by diarrhea, and it does not matter
whether it is an acute disease or a chronic disease. Examples of
such a disease or symptom include diarrhea (caused by a parasite, a
bacterium, a virus, physical stimulation, dyspepsia, mental
disorder, gastrointestinal inflammation or allergy, etc.),
irritable bowel syndrome, ulcerative colitis and the like. It is
expressly contemplated that each disorder accompanied by diarrhea
is a separate embodiment within the scope of the present invention.
It shall be understood that in the present invention, malabsorptive
disorders that can be treated according to the invention can be
included in embodiments individually or in any combination.
[0563] As used herein, "convulsive disorder" refers to a disorder
of an individual in which the subject suffers from convulsion,
e.g., convulsions due to epileptic seizure. Convulsive disorders
that can be treated according to the invention include epilepsy and
non-epileptic seizures, e.g., convulsions due to administration of
a convulsant agent to the individual. It shall be understood that
in the present invention, convulsive disorders that can be treated
according to the invention can be included in embodiments
individually or in any combination.
[0564] As used herein, "cancer" refers to a malignant neoplasm.
Cancers that can be treated according to the invention include but
are not limited to pancreatic cancer, breast cancer, colon cancer,
and Barrett's adenocarcinoma. It shall be understood that in the
present invention, cancers that can be treated according to the
invention can be included in embodiments individually or in any
combination.
[0565] As used herein, "inflammatory disorder" refers to a disorder
relating to abnormality of inflammation. Inflammatory disorders
that can be treated according to the invention include but are not
limited to atherosclerosis, atherothrombosis, insulin resistance,
pancreatitis, restenosis, an inflammatory lung disease, and an
inflammatory bowel disease. It shall be understood that in the
present invention, inflammatory disorders that can be treated
according to the invention can be included in embodiments
individually or in any combination.
[0566] As used herein, "atherothrombosis" refers to thrombus
formation in an atherosclerotic vessel.
[0567] As used herein, "inflammatory lung disease" refers to a
condition relating to abnormality of inflammation in the lung.
Inflammatory lung diseases that can be treated according to the
invention include but are not limited to asthma, emphysema and
pulmonary arterial hypertension (PAH). PAH is a life-threatening
disease characterized by a progressive pulmonary vasculopathy
leading to right ventricular hypertrophy; right heart failure
occurs if left untreated. PAH shall be understood to encompass the
following forms of pulmonary arterial hypertension described in the
2003 World Health Organization (WHO) clinical classification of
pulmonary arterial hypertension: idiopathic PAH, familial PAH, PAH
associated with other condition, and PAH associated with
significant venous or capillary involvement. It shall be understood
that in the present invention, inflammatory lung diseases that can
be treated according to the invention can be included in
embodiments individually or in any combination.
[0568] As used herein, the "inflammatory bowel disease" refers to a
condition relating to abnormality of inflammation in the bowel.
Inflammatory bowel diseases that can be treated according to the
invention include but are not limited to Crohn's disease as may be
enteritis, enterocolitis, Crohn's disease, granulomatous colitis,
ileitis or Crohn's colitis and ulcerative colitis as may be
ulcerative colitis, sigmoiditis, pancolitis and ulcerative
proctitis. It shall be understood that in the present invention,
inflammatory bowel diseases that can be treated according to the
invention can be included in embodiments individually or in any
combination.
[0569] The term "atherosclerosis" as used herein refers to a form
of vascular disease characterized by the deposition of atheromatous
plaques containing cholesterol and lipids on the innermost layer of
the walls of large and medium-sized arteries.
[0570] "Alzheimer's disease" is a progressive neurodegenerative
disease characterized by significant loss of function in more than
one cognitive domain and often accompanied by changes in behavior
or personality. Alzheimer's disease is the most common cause of
dementia. The beta-amyloid peptide (A.beta.) is believed to play a
central role in the pathology of Alzheimer's disease. "Mild
cognitive impairment" (MCI) is a state of cognitive impairment,
most often of memory only, that is not severe enough to
significantly impact occupational or social functioning. However,
many people with MCI progress to Alzheimer's disease, at a rate of
10-15% per year and up to 60% within five years. Growing evidence
shows that Alzheimer's disease pathology begins much earlier than
the development of symptoms severe enough to allow a diagnosis of
Alzheimer's criteria. In 2001, the American Academy of Neurology
(AAN) published clinical practice parameters for the detection of
MCI that will increase awareness, diagnosis, and treatment of both
MCI and mild forms of Alzheimer's disease.
[0571] As used herein, "prion-associated disease" is intended to
include but not be limited to bovine spongiform encephalopathy,
Creutzfeldt-Jakob disease, kuru, Gerstmann-Straussler-Scheinker
syndrome, and fatal familial insomnia (Collins et al, Lancet (2004)
363:51-61).
[0572] As used herein, "excitotoxic injury" refers to neuronal cell
damage or neuronal cell death caused resulting from
substance-induced paroxysmal overactivity, e.g. glutamate-induced
paroxysmal activity.
[0573] "Parkinson's disease" is a chronic, progressive
neurodegenerative disorder characterized by motor symptoms such as
tremor, bradykinesia, muscle rigidity, gait dysfunction, and
postural instability. Researchers have identified degeneration of
dopaminergic neurons in the substantia nigra as the primary
pathophysiological mechanism of Parkinson's disease.
[0574] "Adiponectin" is a plasma protein secreted by adipocytes and
comprised of an N-terminally disposed collagen-like region and a
C-terminal globular region. Reduced levels of adiponectin have been
associated with a number of metabolic-related disorders, including
atherosclerosis, coronary heart disease, stroke, insulin resistance
and type 2 diabetes. The serum adiponectin level for women has been
reported to be higher than that for men, for example 13.5 .mu.g/m1
versus 7.2 .mu.g/m1 in one study (Yamamoto et al, Clin Sci (London)
(2002) 103:137-142).
[0575] The term "endogenous" shall mean a material that an
individual (for example, and not limitation, a human) naturally
produces. By contrast, "non-endogenous" shall mean that which is
not naturally produced by an individual (for example, and not
limitation, a human).
[0576] The term "biologically active fragment" of a G
protein-coupled receptor shall mean a fragment of the GPCR having
structural and biochemical functions of a naturally occurring GPCR.
In certain embodiments, the biologically active fragment couples to
a G protein. In certain embodiments, the biologically active
fragment binds to a known ligand of the GPCR.
[0577] The term "primer" is used herein to denote a specific
nucleotide sequence which is complementary to a target nucleotide
sequence and used to hybridize to the target nucleotide sequence. A
primer servesas an initiation point for nucleotide polymerization
catalyzed by DNA polymerase, RNA polymerase, or reverse
transcriptase.
[0578] The term "expression vector" shall mean a DNA sequence that
is required for the transcription of cloned DNA and translation of
the transcribed mRNA in an appropriate host cell recombinant for
the expression vector. An appropriately contructed expression
vector should contain an origin of replication for autonomous
replication in host cells, selectable markers, a limited number of
useful restriction enzymne sites, a potential for high copy number,
and active promoters. The cloned DNA to be transcribed is operably
linked to a constitutively or conditionally active promoter within
the expression vector.
[0579] The term "host cell" shall mean a cell capable of having a
vector incorporated therein. In the present context, the vector
will typically contain nucleic acid encoding a GPCR or GPCR fusion
protein in operable conncection with a suitable promoter sequence
to permit expression of the GPCR or GPCR fusion protein to occur.
In particular embodiment, the host cell is a eukaryotic host cell.
In certain embodiments, the eukaryotic host cell is a mammalian
host cell. In certain embodiments, the eukaryotic host cell is a
yeast host cell. In certain embodiments, the eukaryotic host cell
is a melanophore host cell.
[0580] The term "contact" or "contacting" shall mean bringing at
least two moieties together.
[0581] The terms "modulate" or "modify" shall be taken to refer to
an increase or decrease in the amount, quality, or effect of a
particular activity, function or molecule or, when in reference to
a condition, shall be taken to refer to a change in the status of
said condition. By way of illustration and not limitation,
agonists, partial agonists, inverse agonists, and antagonists of a
G protein-coupled receptor are modulators of the receptor.
[0582] The term "small molecule" shall be taken to mean a compound
having a molecular weight of less than about 10,000 grams per mole,
including a peptide, peptidomimetic, amino acid, amino acid
analogue, polynucleotide, polynucleotide analogue, nucleotide,
nucleotide analogue, organic compound or inorganic compound (i.e.
including a heterorganic compound or organometallic compound), and
salts, esters and other pharmaceutically acceptable forms thereof.
In certain embodiments, small molecules are organic or inorganic
compounds having a molecular weight of less than about 5,000 grams
per mole. In certain embodiments, small molecules are organic or
inorganic compounds having molecular weight of less than about
1,000 grams per mole. In certain embodiments, small molecules are
organic or inorganic compounds having a molecular weight of less
than about 800 grams per mole. In certain embodiments, small
molecules are organic or inorganic compounds having a molecular
weight of less than about 600 grams per mole. In certain
embodiments, small molecules are organic or inorganic compounds
having a molecular weight of less than about 500 grams per
mole.
[0583] Amino acid abbreviations used herein are set out in Table
B:
TABLE-US-00002 TABLE B ALANINE ALA A ARGININE ARG R ASPARAGINE ASN
N ASPARTIC ACID ASP D CYSTEINE CYS C GLUTAMIC ACID GLU E GLUTAMINE
GLN Q GLYCINE GLY G HISTIDINE HIS H ISOLEUCINE ILE I LEUCINE LEU L
LYSINE LYS K METHIONINE MET M PHENYLALANINE PHE F PROLINE PRO P
SERINE SER S THREONINE THR T TRYPTOPHAN TRP W TYROSINE TYR Y VALINE
VAL V
[0584] The term "polypeptide" shall refer to a polymer of amino
acids without regard to the length of the polymer. Thus, peptides,
oligopeptides, and proteins are included within the definition of
polypeptide. This term also does not specify or exclude
post-expression modifications of polypeptides. For example,
polypeptides that include the covalent attachment of glycosyl
groups, acetyl groups, phosphate groups, lipid groups and the like
are expressly encompassed by the term polypeptide.
[0585] The term "polynucleotide" shall refer to RNA, DNA, or
RNA/DNA hybrid sequence of more than one nucleotide in either
single chain or duplex form. The polynucleotides of the invention
may be prepared by any known method, including synthetic,
recombinant, ex vivo generation, or a combination thereof, as well
as utilizing any purification methods known in the art.
[0586] The term "antibody" is intended herein to encompass
monoclonal antibody and polyclonal antibody.
[0587] The term "second messenger" shall mean an intracellular
response produced as a result of receptor activation. A second
messenger can include, for example, inositol 1,4,5-triphosphate
(IP.sub.3), diacylglycerol (DAG), cyclic AMP (cAMP), cyclic GMP
(cGMP), MAP kinase activity, MAPK/ERK kinase kinase-1 (MEKK1)
activity, and Ca.sup.2-. Second messenger response can be measured
for a determination of receptor activation.
[0588] The term "receptor functionality" shall refer to the normal
operation of a receptor to receive a stimulus and moderate an
effect in the cell, including, but not limited to regulating gene
transcription, regulating the influx or efflux of ions, effecting a
catalytic reaction, and/or modulating activity through G-proteins,
such as eliciting a second messenger response.
[0589] The term "stimulate" or "stimulating," in relationship to
the term "response" or "functionality of the receptor" shall mean
that a response or a functionality of the receptor is increased in
the presence of a compound as opposed to in the absence of the
compound.
[0590] The term "inhibit" or "inhibiting," in relationship to the
term "response" or "functionality of the receptor" shall mean that
a response a functionality of the receptor is decreased or
prevented in the presence of a compound as opposed to in the
absence of the compound.
[0591] The term "compound efficacy" shall mean a measurement of the
ability of a compound to inhibit or stimulate receptor
functionality, as opposed to receptor binding affinity.
[0592] The term "test compound," used interchangeably herein with
"candidate compound," shall mean a molecule (for example, and not
limitation, a chemical compound) which is amenable to a screening
technique.
[0593] The term "constitutively active" in relationship to a G
protein-coupled receptor shall mean that the G protein-coupled
receptor exhibits agonist-independent activity.
[0594] The term "directly identifying" or "directly identified", in
relationship to the phrase "test compound," shall mean the
screening of a compound against a G protein-coupled receptor in the
absence of a known ligand (e.g., a known agonist) to the G
protein-coupled receptor.
[0595] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the lower limit unless the
context clearly indicates otherwise, between the upper and lower
limit of that range and any other stated or intervening value in
that stated range, is encompassed within the invention. The upper
and lower limits of these smaller ranges may independently be
included in the smaller ranges, and are also encompassed within the
invention, subject to any specifically excluded limit in the stated
range. Where the stated range includes one or both of the limits,
ranges excluding either or both of those included limits are also
included in the invention.
A. Introduction
[0596] The order of the following sections is set forth for
presentational efficiency and is not intended, nor should be
construed, as a limitation on the disclosure or the claims to
follow.
B. Receptor Expression
[0597] 1. GPCR Polypeptides of Interest
[0598] A GPCR of the invention may comprise an amino acid sequence
selected from the group consisting of: [0599] (a) amino acids 1-335
of SEQ ID NO: 2; [0600] (b) amino acids 2-335 of SEQ ID NO: 2;
[0601] (c) an amino acid sequence comprising the amino acid
sequence of a polypeptide derived from (a) by substitution,
deletion or addition of one or several amino acids in the amino
acid sequence defined in (a); [0602] (d) the amino acid sequence of
a G protein-coupled receptor encoded by a polynucleotide
hybridizing under stringent conditions to the complement of SEQ ID
NO: 1; [0603] (e) a variant of SEQ ID NO: 2 when selected from the
group consisting of: [0604] (i) the amino acid sequence of a G
protein-coupled receptor having at least about 80% identity to SEQ
ID NO: 2; and [0605] (ii) the amino acid sequence of a G
protein-coupled receptor comprising at least 20 contiguous amino
acids of SEQ ID NO: 2; [0606] (f) the amino acid sequence of a
constitutively active version of a G protein-coupled receptor
having SEQ ID NO: 2; and [0607] (g) a biologically active fragment
of any one of any one of (a) to (f).
[0608] In certain embodiments, a GPCR of the invention comprises
the amino acid sequence of SEQ ID
[0609] NO: 2. In some embodiments, a GPCR of the invention is an
allele of SEQ ID NO:2. In some embodiments, a GPCR of the invention
is an ortholog of SEQ ID NO:2.
[0610] In some embodiments, a GPCR of the invention is recombinant
In some embodiments, the recombinant GPCR is recombinant human
GPR119.
[0611] In certain embodiments, a GPCR that may be used in the
subject methods exhibits a detectable level of constitutive
activity.
[0612] In some embodiments, a GPCR of the invention is endogenous.
In some embodiments, a GPCR of the invention is a mammalian GPR119.
In some embodiments, a GPCR of the invention that is endogenous is
a mammalian GPR119.
[0613] By way of illustration and not limitation, deletion of an
N-terminal methionine residue is envisioned to provide a
biologically active fragment that may be used in the subject
invention. In certain embodiments, a biologically active fragment
of the invention is a fragment optionally fused at its N-terminus
to a peptide comprising an N-terminal methionine residue and an HA
epitope tag (from hemagglutinin influenza virus) that specifically
binds Compound 1. In certain embodiments, a biologically active
fragment of the invention is a fragment optionally fused at its
N-terminus to a peptide comprising an N-terminal methionine residue
and an HA epitope tag (from hemagglutinin influenza virus) that
specifically binds
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine In certain
embodiments, a biologically active fragment of the invention is a
fragment optionally fused at its N-terminus to a peptide comprising
an N-terminal methionine residue and an HA epitope tag that
specifically binds
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4yl}-amine with an IC.sub.50
value in receptor binding assay according to Example 15, infra, of
less than about 50 .mu.M, less than about 25 .mu.M, less than about
10 .mu.M, less than about 5 .mu.M, less than about 1 .mu.M, less
than about 500 nM, less than about 100 nM, or less than about 50
nM. In certain embodiments, a biologically active fragment of the
invention is a fragment optionally fused at its N-terminus to a
peptide comprising an N-terminal methionine residue and an HA
epitope tag that specifically binds
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine with an
IC.sub.50 value in receptor binding assay according to Example 15,
infra, of less than about 10 .mu.M, less than about 5 .mu.M, less
than about 1 .mu.M, less than about 500 nM, less than about 100 nM,
or less than about 50 nM. In certain embodiments, a biologically
active fragment of the invention is a fragment optionally fused at
its N-terminus to a peptide comprising an N-terminal methionine
residue and an HA epitope tag for which
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine is an agonist
having an EC.sub.50 value at said fragment optionally fused at its
N-terminus to said peptide in whole cell adenylyl cyclase assay
according to Example 10 or in HTRF.RTM. assay according to Example
11, infra, of less than about 5 .mu.M, less than about 1 .mu.M,
less than about 100 nM, less than about 50 nM, less than about 25
nM, less than about 10 nM, less than about 5 nM, or less than about
1 nM. In certain embodiments, a biologically active fragment of the
invention is a fragment optionally fused at its N-terminus to a
peptide comprising an N-terminal methionine residue and an HA
epitope tag for which
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine is an agonist
having an EC.sub.50 value at said fragment optionally fused at its
N-terminus to said peptide in whole cell adenylyl cyclase assay
according to Example 10 or in HTRF.RTM. assay according to Example
11, infra, of less than about 100 nM, less than about 50 nM, less
than about 25 nM, less than about 10 nM, less than about 5 nM, or
less than about 1 nM. In some embodiments, a biologically active
fragment of the invention is a fragment optionally fused at its
N-terminus to a peptide comprising an N-terminal methionine residue
and an HA epitope tag that exhibits a detectable level of
constitutive activity. In some embodiments, the constitutive
activity is for increasing a level of intracellular cAMP. In some
embodiments, the constitutive activity is for causing melanophore
cells to undergo pigment dispersion. In certain embodiments, the
fragment is fused at its N-terminus to a peptide consisting
essentially of an N-terminal methionine residue and an HA epitope
tag. Techniques for fusing a peptide comprising or consisting
essentially of an N-terminal methionine residue and an HA epitope
tag to the N-terminus of a polypeptide fragment are well known in
the art and can be obtained commercially (e.g., Clontech, Mountain
View, Calif.).
[0614] An allelic variant of human GPR119 of SEQ ID NO: 2 is
envisioned to be within the scope of the invention. Human GPR119 is
envisioned to be within the scope of the invention.
[0615] A variant which is a vertebrate ortholog of human GPR119 of
SEQ ID NO: 2 is envisioned to be within the scope of the invention.
A variant which is a mammalian ortholog of human GPR119 of SEQ ID
NO: 2 is envisioned to be within the scope of the invention. By way
of illustration and not limitation, mouse GPR119 (e.g.,
GenBank.RTM. Accession No. AY288423), rat GPR119 (GenBank.RTM.
Accession No. AAN95195), hamster GPR119, dog GPR119, and non-human
primate GPR119 are envisioned to be within the scope of the
invention.
[0616] In certain embodiments, the variant of SEQ ID NO: 2 is a
GPCR.
[0617] A variant of SEQ ID NO: 2 having at least about 80%, at
least about 85%, at least about 90%, at least about 95%, at least
about 96%, at least about 97%, at least about 98%, or at least
about 99% identity to SEQ ID NO: 2 is envisioned to be within the
scope of the invention. In certain embodiments, the variant of SEQ
ID NO: 2 having at least about 80%, at least about 85%, at least
about 90%, at least about 95%, at least about 96%, at least about
97%, at least about 98%, or at least about 99% identity to SEQ
ID
[0618] NO: 2 is a GPCR. In some embodiments, the variant of SEQ ID
NO: 2 is an endogenous GPCR. In some embodiments, the variant of
SEQ ID NO: 2 is a non-endogenous GPCR. In some embodiments, the
variant of SEQ ID NO: 2 that is an endogenous GPCR is a mammalian
GPCR. In certain embodiments, the variant of SEQ ID NO: 2
specifically binds Compound 1. In certain embodiments, the variant
of SEQ ID NO: 2 specifically binds
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine. In certain
embodiments, the variant of SEQ ID NO: 2 specifically binds
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine with an
IC.sub.50 value in receptor binding assay according to Example 15,
infra, of less than about 50 .mu.M, less than about 25 .mu.M, less
than about 10 .mu.M, less than about 5 .mu.M, less than about 1
.mu.M, less than about 500 nM, less than about 100 nM, or less than
about 50 nM. In certain embodiments, the variant of SEQ ID NO: 2
specifically binds
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine with an
IC.sub.50 value in receptor binding assay according to Example 15,
infra, of less than about 10 .mu.M, less than about 5 .mu.M, less
than about 1 .mu.M, less than about 500 nM, less than about 100 nM,
or less than about 50 nM. In certain embodiments, the variant of
SEQ ID NO: 2 is a receptor for which
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine is an agonist
having an EC.sub.50 value at said receptor in whole cell adenylyl
cyclase assay according to Example 10 or in HTRF.RTM. assay
according to Example 11, infra, of less than about 5 .mu.M, less
than about 1 .mu.M, less than about 100 nM, less than about 50 nM,
less than about 25 nM, less than about 10 nM, less than about 5 nM,
or less than about 1 nM. In certain embodiments, the variant of SEQ
ID NO: 2 is a receptor for which
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine is an agonist
having an EC.sub.50 value at said receptor in whole cell adenylyl
cyclase assay according to Example 10 or in HTRF.RTM. assay
according to Example 11, infra, of less than about 100 nM, less
than about 50 nM, less than about 25 nM, less than about 10 nM,
less than about 5 nM, or less than about 1 nM. In some embodiments,
the variant of SEQ ID NO: 2 exhibits a detectable level of
constitutive activity. In some embodiments, the constitutive
activity is for increasing a level of intracellular cAMP. In some
embodiments, the constitutive activity is for causing melanophore
cells to undergo pigment dispersion. Percent identity can be
determined conventionally using known computer programs.
[0619] In certain embodiments, a variant GPCR that may be used in
the subject methods has an amino acid sequence having at least
about 80%, at least about 85%, at least about 90%, at least about
95%, of at least about 96%, at least about 97%, at least about 98%,
or at least about 99% identity to SEQ ID NO: 2. By a variant GPCR
having, for example, 95% "identity" to SEQ ID NO: 2 is meant that
the amino acid sequence of the variant is identical to amino acids
1-335 of SEQ ID NO: 2 except that it may include up to five amino
acid alterations per each 100 amino acids of SEQ ID NO: 2. Thus, to
obtain for example an amino acid sequence having at least 95%
identity to the amino acid sequence of SEQ ID NO: 2, up to 5% (5 of
100) of the amino acid residues in the sequence may be inserted,
deleted, or substituted with another amino acid compared with amino
acids 1-335 of SEQ ID NO: 2. These alternations may occur at the
amino or carboxy termini or anywhere between those terminal
positions, interspersed either subjectly among residues in the
sequence or in one or more contiguous groups within the
sequence.
[0620] In certain embodiments, a variant G protein-coupled receptor
that may be used in the subject methods is a G protein-coupled
receptor having an amino acid sequence derived from SEQ ID NO: 2 by
deletion, substitution, and/or addition of one or several amino
acids. In certain embodiments, a variant G protein-coupled receptor
that may be used in the subject methods is a G protein-coupled
receptor having an amino acid sequence derived from SEQ ID NO: 2 by
no more than 10 conservative amino acid substitutions and/or no
more than 3 non-conservative amino acid substitutions in the amino
acid sequence of SEQ ID NO: 2. In certain embodiments, arginine,
lysine and histidine may conservatively substitute for each other;
glutamic acid and aspartic acid may conservatively substitute for
each other; glutamine and asparagine may conservatively substitute
for each other; leucine, isoleucine and valine may conservatively
substitute for each other; phenylalanine, tryptophan and tyrosine
may conservatively substitute for each other; and glycine, alanine,
serine, threonine and methionine may conservatively substitute for
each other. The amino acid substitutions, amino acid deletions, and
amino acid additions may be at any position (e.g., the C- or
N-terminus, or at internal positions). In some embodiments, the
variant is an endogenous G protein-coupled receptor. In some
embodiments, the variant is an endogenous vertebrate G
protein-coupled receptor. In some embodiments, the variant is an
endogenous mammalian G protein-coupled receptor. In some
embodiments, the variant is an endogenous human G protein-coupled
receptor. In some embodiments, the variant is a non-endogenous G
protein-coupled receptor. In some embodiments, the variant exhibits
a detectable level of constitutive activity. In some embodiments,
the constitutive activity is for increasing intracellular cAMP. In
some embodiments, the constitutive activity is for causing
melanophore cells to undergo pigment dispersion. In certain
embodiments, said G protein-coupled receptor having an amino acid
sequence derived from SEQ ID NO: 2 is a G protein-coupled receptor
for which Compound 1 is a ligand. In certain embodiments, said G
protein-coupled receptor having an amino acid sequence derived from
SEQ ID NO: 2 is a G protein-coupled receptor for which
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine is a ligand
having an IC.sub.50 value in receptor binding assay according to
Example 15, infra, of less than about 50 .mu.M, less than about 25
.mu.M, less than about 10 .mu.M, less than about 5 .mu.M, less than
about 1 .mu.M, less than about 500 nM, less than about 100 nM, or
less than about 50 nM. In certain embodiments, said G
protein-coupled receptor having an amino acid sequence derived from
SEQ ID NO: 2 is a G protein-coupled receptor for which Compound 1
is an agonist. In certain embodiments, said G protein-coupled
receptor having an amino acid sequence derived from SEQ ID NO: 2 is
a G protein-coupled receptor for which
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine is an agonist
having an EC.sub.50 value in in whole cell adenylyl cyclase assay
according to Example 10 or in HTRF.RTM. assay according to Example
11, infra, of less than about 5 .mu.M, less than about 1 .mu.M,
less than about 100 nM, less than about 50 nM, less than about 25
nM, less than about 10 nM, less than about 5 nM, or less than about
1 nM. A variant of SEQ ID NO: 2 that is a G protein-coupled
receptor comprising at least 20, at least 30, at least 40, at least
50, at least 75, or at least 100 contiguous amino acids of SEQ ID
NO: 2 is envisioned to be within the scope of the invention. In
certain embodiments, the variant of SEQ ID NO: 2 comprising at
least 20, at least 30, at least 40, at least 50, at least 75, or at
least 100 contiguous amino acids of SEQ ID NO: 2 is a GPCR. In some
embodiments, the variant of SEQ ID NO: 2 is an endogenous GPCR. In
some embodiments, the variant of SEQ ID NO: 2 is a non-endogenous
GPCR. In some embodiments, the variant of SEQ ID NO: 2 that is an
endogenous GPCR is a mammalian GPCR. In certain embodiments, the
variant of SEQ ID NO: 2 specifically binds Compound 1. In certain
embodiments, the variant of SEQ ID NO: 2 specifically binds
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine In certain
embodiments, the variant of SEQ ID NO: 2 specifically binds
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine with an
IC.sub.50 value in receptor binding assay according to Example 15,
infra, of less than about 50 .mu.M, less than about 25 .mu.M, less
than about 10 .mu.M, less than about 5 .mu.M, less than about 1
.mu.M, less than about 500 nM, less than about 100 nM, or less than
about 50 nM. In certain embodiments, the variant of SEQ ID NO: 2
specifically binds
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine with an
IC.sub.50 value in receptor binding assay according to Example 15,
infra, of less than about 10 .mu.M, less than about 5 .mu.M, less
than about 1 .mu.M, less than about 500 nM, less than about 100 nM,
or less than about 50 nM. In certain embodiments, the variant of
SEQ ID NO: 2 is a receptor for which
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine is an agonist
having an EC.sub.50 value at said receptor in whole cell adenylyl
cyclase assay according to Example 10 or in HTRF.RTM. assay
according to Example 11, infra, of less than about 5 .mu.M, less
than about 1 .mu.M, less than about 100 nM, less than about 50 nM,
less than about 25 nM, less than about 10 nM, less than about 5 nM,
or less than about 1 nM. In certain embodiments, the variant of SEQ
ID NO: 2 is a receptor for which
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine is an agonist
having an EC.sub.50 value at said receptor in whole cell adenylyl
cyclase assay according to Example 10 or in HTRF.RTM. assay
according to Example 11, infra, of less than about 100 nM, less
than about 50 nM, less than about 25 nM, less than about 10 nM,
less than about 5 nM, or less than about 1 nM. In some embodiments,
the variant of SEQ ID NO: 2 exhibits a detectable level of
constitutive activity. In some embodiments, the constitutive
activity is for increasing a level of intracellular cAMP. In some
embodiments, the constitutive activity is for causing melanophore
cells to undergo pigment dispersion. In some embodiments, the G
protein-coupled receptor comprising at least 20, at least 30, at
least 40, at least 50, at least 75, or at least 100 contiguous
amino acids of SEQ ID NO: 2 exhibits a detectable level of
constitutive activity. In some embodiments, the constitutive
activity is for increasing a level of intracellular cAMP. In some
embodiments, the constitutive activity is for causing melanophore
cells to undergo pigment dispersion.
[0621] In some embodiments, a variant GPCR that may be used in the
subject methods is a GPCR encoded by a polynucleotide hybridizing
under stringent conditions to the complement of SEQ ID NO: 1. In
some embodiments, the GPCR encoded by a polynucleotide hybridizing
under stringent conditions to the complement of SEQ ID NO: 1 is an
endogenous GPCR. In some embodiments, the GPCR encoded by a
polynucleotide hybridizing under stringent conditions to the
complement of SEQ ID NO: 1 is a non-endogenous GPCR. In some
embodiments, the GPCR encoded by a polynucleotide hybridizing under
stringent conditions to the complement of SEQ ID NO: 1 and that is
an endogenous GPCR is a mammalian endogenous GPCR. In certain
embodiments, the GPCR encoded by a polynucleotide hybridizing under
stringent conditions to the complement of SEQ ID NO: 1 is SEQ ID
NO:2 or an allele thereof. In certain embodiments, the GPCR encoded
by a polynucleotide hybridizing under stringent conditions to the
complement of SEQ ID NO: 1 is an allele of SEQ ID NO:2. In certain
embodiments, the GPCR encoded by a polynucleotide hybridizing under
stringent conditions to the complement of SEQ ID NO: 1 is an
ortholog of SEQ ID NO:2. In certain embodiments, the GPCR encoded
by a polynucleotide hybridizing under stringent conditions to the
complement of SEQ ID NO: 1 specifically binds Compound 1. In
certain embodiments, the GPCR encoded by a polynucleotide
hybridizing under stringent conditions to the complement of SEQ ID
NO: 1 specifically binds
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine. In certain
embodiments, the GPCR encoded by a polynucleotide hybridizing under
stringent conditions to the complement of SEQ ID NO: 1 specifically
binds
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine with an
IC.sub.50 value in receptor binding assay according to Example 15,
infra, of less than about 50 .mu.M, less than about 25 .mu.M, less
than about 10 .mu.M, less than about 5 .mu.M, less than about 1
.mu.M, less than about 500 nM, less than about 100 nM, or less than
about 50 nM. In certain embodiments, the GPCR encoded by a
polynucleotide hybridizing under stringent conditions to the
complement of SEQ ID NO: 1 specifically binds
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine with an
IC.sub.50 value in receptor binding assay according to Example 15,
infra, of less than about 10 .mu.M, less than about 5 .mu.M, less
than about 1 .mu.M, less than about 500 nM, less than about 100 nM,
or less than about 50 nM. In certain embodiments, the GPCR encoded
by a polynucleotide hybridizing under stringent conditions to the
complement of SEQ ID NO: 1 is a receptor for which
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine is an agonist
having an EC.sub.50 value at said receptor in whole cell adenylyl
cyclase assay according to Example 10 or in HTRF.RTM. assay
according to Example 11, infra, of less than about 5 .mu.M, less
than about 1 .mu.M, less than about 100 nM, less than about 50 nM,
less than about 25 nM, less than about 10 nM, less than about 5 nM,
or less than about 1 nM. In certain embodiments, the GPCR encoded
by a polynucleotide hybridizing under stringent conditions to the
complement of SEQ ID NO: 1 is a receptor for which
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadi-
azol-5-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine is an
agonist having an EC.sub.50 value at said receptor in whole cell
adenylyl cyclase assay according to Example 10 or in HTRF.RTM.
assay according to Example 11, infra, of less than about 100 nM,
less than about 50 nM, less than about 25 nM, less than about 10
nM, less than about 5 nM, or less than about 1 nM. In some
embodiments, the GPCR encoded by a polynucleotide hybridizing under
stringent conditions to the complement of SEQ ID NO: 1 exhibits a
detectable level of constitutive activity. In some embodiments, the
constitutive activity is for increasing a level of intracellular
cAMP. In some embodiments, the constitutive activity is for causing
melanophore cells to undergo pigment dispersion.
[0622] Hybridization techniques are well known to the skilled
artisan. In some embodiments, stringent hybridization conditions
include overnight incubation at 42.degree. C. in a solution
comprising: 50% formamide, 5.times.SSC (1.times.SSC=150 mM NaCl, 15
mM trisodium citrate), 50mM sodium phosphate (pH 7.6), 5.times.
Denhardt's solution, 10% dextran sulfate, and 20 .mu.g/ml
denatured, sheared salmon sperm DNA; followed by washing the filter
in 0.1.times.SSC at about 65.degree. C. In some embodiments,
stringent hybridization conditions include overnight incubation at
42.degree. C. in a solution comprising: 50% formamide, 5.times.SSC
(1.times.SSC=150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium
phosphate (pH 7.6), 5.times. Denhardt's solution, 10% dextran
sulfate, and 20 .mu.g/ml denatured, sheared salmon sperm DNA;
followed by a wash in 0.1.times.SSC/0.1% SDS (sodium dodecyl
sulfate) or in 0.2.times.SSC/0.1% SDS at about 50.degree. C., at
about 55.degree. C., at about 60.degree. C. or at about 65.degree.
C. In some embodiments, said stringent conditions comprise washing
at 65.degree. C. with 0.1.times.SSC. In some embodiments, said
stringent conditions comprise washing at about 50.degree. C., at
about 55.degree. C., at about 60.degree. C., or at about 65.degree.
with 0.1.times.SSC/0.1% SDS or with 0.2.times.SSC/0.1% SDS.
[0623] In some embodiments, a GPCR that may be used in the subject
methods is a non-endogenous, constitutively activated receptor
comprising the amino acid sequence of SEQ ID NO: 2, wherein the
leucine at amino acid position 224 of SEQ ID NO: 2 is substituted
with an amino acid other than leucine. In some embodiments, the
amino acid other than leucine is lysine. In some embodiments, the
amino acid other than leucine is alanine. In some embodiments, the
amino acid other than leucine is arginine. In some embodiments, the
amino acid other than leucine is histidine. In some embodiments,
the constitutive activity is for increasing a level of
intracellular cAMP. In some embodiments, the constitutive activity
is for causing melanophore cells to undergo pigment dispersion.
[0624] In certain embodiments, a GPCR of the invention comprises a
constitutively active version of a G protein-coupled receptor
having SEQ ID NO: 2. In some embodiments, the constitutively active
version of the receptor is an endogenous constitutively active
version having SEQ ID NO: 2. In some embodiments, the
constitutively active version of the receptor is a non-endogenous
constitutively active version having a mutation positioned at amino
acid position 224 of SEQ ID NO: 2. In some embodiments, the mutated
residue has been mutated to a residue other than leucine. In some
embodiments, the mutated residue has been mutated to a lysine
residue. In some embodiments, the mutated residue has been mutated
to an alanine residue. In some embodiments, the mutated residue has
been mutated to an arginine residue. In some embodiments, the
mutated residue has been mutated to a histidine residue. In some
embodiments, the constitutive activity is for increasing a level of
intracellular cAMP. In some embodiments, the constitutive activity
is for causing melanophore cells to undergo pigment dispersion.
[0625] In certain embodiments, a GPCR of the invention forms part
of a fusion protein with a G protein.
[0626] a. Sequence Identity
[0627] In certain embodiments, percent identity is evaluated using
the Basic Local Alignment Search Tool ("BLAST"), which is well
known in the art [See, e.g., Karlin and Altschul, Proc Natl Acad
Sci USA (1990) 87:2264-2268; Altschul et al., J Mol Biol (1990)
215:403-410; Altschul et all, Nature Genetics (1993) 3:266-272; and
Altschul et al., Nucleic Acids Res (1997) 25:3389-3402; the
disclosure of each of which is herein incorporated by reference in
its entirety]. The BLAST programs may be used with the default
parameters or with modified parameters provided by the user.
Preferably, the parameters are default parameters.
[0628] In certain embodiments, a preferred method for determining
the best overall match between a query sequence (e.g., the amino
acid sequence of SEQ ID NO:2) and a sequence to be interrogated,
also referred to as a global sequence alignment, can be determined
using the FASTDB computer program based on the algorithm of Brutlag
et al. (Comp App Biosci (1990) 6:237-245; the disclosure of which
is herein incorporated by reference in its entirety). In a sequence
alignment the query and interrogated sequences are both amino acid
sequences. The results of said global sequence alignment is in
percent identity. Preferred parameters used in a FASTDB amino acid
alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining
Penalty=20, Randomization Group=25, Length=0, Cutoff Score=1,
Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05,
Window Size=247 or the length of the interrogated amino acid
sequence, whichever is shorter.
[0629] If the interrogated sequence is shorter than the query
sequence due to N- or C-terminal deletions, not because of internal
deletions, the results, in percent identity, must be manually
corrected because the FASTDB program does not account for N- and
C-terminal truncations of the interrogated sequence when
calculating global percent identity. For interrogated sequences
truncated at the N- and C-termini, relative to the query sequence,
the percent identity is corrected by calculating the number of
residues of the query sequence that are N- and C-terminal of the
interrogated sequence, that are not matched/aligned with a
corresponding interrogated sequence residue, as a percent of the
total bases of the query sequence.
[0630] Whether a residue is matched/aligned is determined by
results of the FASTDB sequence alignment. This percentage is then
subtracted from the percent identity, calculated by the above
FASTDB program using the specified parameters, to arrive at a final
percent identity score. This final percent identity score is what
is used for the purposes of the present invention. Only residues to
the N- and C-termini of the interrogated sequence, which are not
matched/aligned with the query sequence, are considered for the
purposes of manually adjusting the percent identity score. That is,
only querey amino acid residues outside the farthest N- and
C-terminal residues of the interrogated sequence.
[0631] For example, a 90 amino acid residue interrogated sequence
is aligned with a 100-residue query sequence to determine percent
identity. The deletion occurs at the N-terminus of the interrogated
sequence and therefore, the FASTDB alignment does not match/align
with the first residues at the N-terminus The 10 unpaired residues
represent 10% of the sequence (number of residues at the N- and
C-termini not matched/total number of residues in the query
sequence) so 10% is subtracted from the percent identity score
calculated by the FASTDB program. If the remaining 90 residues were
perfectly matched, the final percent identity would be 90%.
[0632] In another example, a 90-residue interrogated sequence is
compared with a 100-residue query sequence. This time the deletions
are internal so there are no residues at the N- or C-termini of the
interrogated sequence, which are not matched/aligned with the
query. In this case, the percent identity calculated by FASTDB is
not manually corrected. Once again, only residue positions outside
the N-and C-terminal ends of the subject sequence, as displayed in
the FASTDB alignment, which are not matched/aligned with the query
sequence are manually corrected. No other corrections are made for
the purposes of the present invention.
[0633] b. Fusion Proteins
[0634] In certain embodiments, a polypeptide of interest is a
fusion protein, and may contain, for example, an affinity tag
domain or a reporter domain. Suitable affinity tags include any
amino acid sequence that may be specifically bound to another
moiety, usually another polypeptide, most usually an antibody.
Suitable affinity tags include epitope tags, for example, the the
V5 tag, the FLAG tag, the HA tag (from hemagglutinin influenza
virus), the myc tag, and the like, as is known in the art. Suitable
affinity tags also include domains for which, binding substrates
are known, e.g., HIS, GST and MBP tags, as is known in the art, and
domains from other proteins for which specific binding partners,
e.g., antibodies, particularly monoclonal antibodies, are
available. Suitable affinity tags also include any protein-protein
interaction domain, such as a IgG Fc region, which may be
specifically bound and detected using a suitable binding partner,
e.g. the IgG Fc receptor. It is expressly contemplated that such a
fusion protein may contain a heterologous N-terminal domain (e.g.,
an epitope tag) fused in-frame with a GPCR that has had its
N-terminal methionine residue either deleted or substituted with an
alternative amino acid.
[0635] Suitable reporter domains include any domain that can report
the presence of a polypeptide. While it is recognized that an
affinity tag may be used to report the presence of a polypeptide
using, e.g., a labeled antibody that specifically binds to the tag,
light emitting reporter domains are more usually used. Suitable
light emitting reporter domains include luciferase (from, e.g.,
firefly, Vargula, Renilla reniformis or Renilla muelleri), or light
emitting variants thereof. Other suitable reporter domains include
fluorescent proteins, (from e.g., jellyfish, corals and other
coelenterates as such those from Aequoria, Renilla, Ptilosarcus,
Stylatula species), or light emitting variants thereof. Light
emitting variants of these reporter proteins are very well known in
the art and may be brighter, dimmer, or have different excitation
and/or emission spectra, as compared to a native reporter protein.
For example, some variants are altered such that they no longer
appear green, and may appear blue, cyan, yellow, enhanced yellow
red (termed BFP, CFP, YFP eYFP and RFP, respectively) or have other
emission spectra, as is known in the art. Other suitable reporter
domains include domains that can report the presence of a
polypeptide through a biochemical or color change, such as
.beta.-galactosidase, .beta.-glucuronidase, chloramphenicol acetyl
transferase, and secreted embryonic alkaline phosphatase.
[0636] Also as is known in the art, an affinity tags or a reporter
domain may be present at any position in a polypeptide of interest.
However, in most embodiments, they are present at the C- or
N-terminal end of a polypeptide of interest.
[0637] 2. Nucleic Acids Encoding GPCR Polypeptides of Interest
[0638] Since the genetic code and recombinant techniques for
manipulating nucleic acid are known, and the amino acid sequences
of GPCR polypeptides of interest described as above, the design and
production of nucleic acids encoding a GPCR polypeptide of interest
is well within the skill of an artisan. In certain embodiments,
standard recombinant DNA technology (Ausubel, et al, Short
Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995;
Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second
Edition, (1989) Cold Spring Harbor, N.Y.) methods are used. For
example, GPCR coding sequences may be isolated from a library of
GPCR coding sequence using any one or a combination of a variety of
recombinant methods that do not need to be described herein.
Subsequent substitution, deletion, and/or addition of nucleotides
in the nucleic acid sequence encoding a protein may also be done
using standard recombinant DNA techniques.
[0639] For example, site directed mutagenesis and subcloning may be
used to introduce/delete/substitute nucleic acid residues in a
polynucleotide encoding a polypeptide of interest. In other
embodiments, PCR may be used. Nucleic acids encoding a polypeptide
of interest may also be made by chemical synthesis entirely from
oligonucleotides (e.g., Cello et al., Science (2002)
297:1016-8).
[0640] In some embodiments, the codons of the nucleic acids
encoding polypeptides of interest are optimized for expression in
cells of a particular species, particularly a mammalian, e.g.,
mouse, rat, hamster, non-human primate, or human, species. In some
embodiments, the codons of the nucleic acids encoding polypeptides
of interest are optimized for expression in cells of a particular
species, particularly an amphibian species.
[0641] a. Vectors
[0642] The invention further provides vectors (also referred to as
"constructs") comprising a subject nucleic acid. In many
embodiments of the invention, the subject nucleic acid sequences
will be expressed in a host after the sequences have been operably
linked to an expression control sequence, including, e.g. a
promoter. The subject nucleic acids are also typically placed in an
expression vector that can replicate in a host cell either as an
episome or as an integral part of the host chromosomal DNA.
Commonly, expression vectors will contain selection markers, e.g.,
tetracycline or neomycin, to permit detection of those cells
transformed with the desired DNA sequences (see, e.g., U.S. Pat.
No. 4,704,362, which is incorporated herein by reference). Vectors,
including single and dual expression cassette vectors are well
known in the art (Ausubel, et al, Short Protocols in Molecular
Biology, 3rd ed., Wiley & Sons, 1995; Sambrook, et al.,
Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold
Spring Harbor, N.Y.). Suitable vectors include viral vectors,
plasmids, cosmids, artificial chromosomes (human artificial
chromosomes, bacterial artificial chromosomes, yeast artificial
chromosomes, etc), mini-chromosomes, and the like. Retroviral,
adenoviral and adeno-associated viral vectors may be used.
[0643] A variety of expression vectors are available to those in
the art for purposes of producing a polypeptide of interest in a
cell and include expression vectors which are commercially
available (e.g., from Invitrogen, Carlsbad, Calif.; Clontech,
Mountain View, Calif.; Stratagene, La Jolla, Calif.). Commercially
available expression vectors include, by way of non-limiting
example, CMV promoter-based vectors. One suitable expression vector
is pCMV. The expression vector may be adenoviral. An exemplary
adenoviral vector may be purchased as AdEasy.TM. from Qbiogene
(Carlsbad, Calif.) (He T C et al, Proc Natl Acad Sci USA (1998)
95:2509-2514; and U.S. Pat. No. 5,922,576; the disclosure of each
of which is herein incorporated by reference in its entirety).
Other suitable expression vectors will be readily apparent to those
of ordinary skill in the art.
[0644] The subject nucleic acids usually comprise an single open
reading frame encoding a subject polypeptide of interest, however,
in certain embodiments, since the host cell for expression of the
polypeptide of interest may be a eukaryotic cell, e.g., a mammalian
cell, such as a human cell, the open reading frame may be
interrupted by introns. Subject nucleic acid are typically part of
a transcriptional unit which may contain, in addition to the
subject nucleic acid 3' and 5' untranslated regions (UTRs) which
may direct RNA stability, translational efficiency, etc. The
subject nucleic acid may also be part of an expression cassette
which contains, in addition to the subject nucleic acid a promoter,
which directs the transcription and expression of a polypeptide of
interest, and a transcriptional terminator.
[0645] Eukaryotic promoters can be any promoter that is functional
in a eukaryotic host cell, including viral promoters and promoters
derived from eukaryotic genes. Exemplary eukaryotic promoters
include, but are not limited to, the following: the promoter of the
mouse metallothionein I gene sequence (Hamer et al., J. Mol. Appl.
Gen. 1:273-288, 1982); the TK promoter of Herpes virus (McKnight,
Cell 31:355-365, 1982); the SV40 early promoter (Benoist et al.,
Nature (London) 290:304-310, 1981); the yeast gall gene sequence
promoter (Johnston et al., Proc. Natl. Acad. Sci. (USA)
79:6971-6975, 1982); Silver et al., Proc. Natl. Acad. Sci. (USA)
81:5951-59SS, 1984), the CMV promoter, the EF-1 promoter,
Ecdysone-responsive promoter(s), tetracycline-responsive promoter,
and the like. Viral promoters may be of particular interest as they
are generally particularly strong promoters. In certain
embodiments, a promoter is used that is a promoter of the target
pathogen. Promoters for use in the present invention are selected
such that they are functional in the cell type (and/or animal) into
which they are being introduced. In certain embodiments, the
promoter is a CMV promoter.
[0646] In certain embodiments, a subject vector may also provide
for expression of a selectable marker. Suitable vectors and
selectable markers are well known in the art and discussed in
Ausubel, et al, (Short Protocols in Molecular Biology, 3rd ed.,
Wiley & Sons, 1995) and Sambrook, et al, (Molecular Cloning: A
Laboratory Manual, Third Edition, (2001) Cold Spring Harbor, N.Y.).
A variety of different genes have been employed as selectable
markers, and the particular gene employed in the subject vectors as
a selectable marker is chosen primarily as a matter of convenience.
Known selectable marker genes include: the thymidine kinase gene,
the dihydrofolate reductase gene, the xanthine-guanine
phosphoribosyl transferase gene, CAD, the adenosine deaminase gene,
the asparagine synthetase gene, the antibiotic resistance genes,
e.g. tetr, ampr, Cmr or cat, kanr or neor (aminoglycoside
phosphotransferase genes), the hygromycin B phosphotransferase
gene, and the like.
[0647] As mentioned above, polypeptides of interest may be fusion
proteins that contain an affinity domain and/or a reporter domain.
Methods for making fusions between a reporter or tag and a GPCR,
for example, at the C- or N-terminus of the GPCR, are well within
the skill of one of skill in the art (e.g. McLean et al, Mol.
Pharma. Mol Pharmacol. 1999 56:1182-91; Ramsay et al., Br. J.
Pharmacology, 2001, 315-323) and will not be described any further.
It is expressly contemplated that such a fusion protein may contain
a heterologous N-terminal domain (e.g., an epitope tag) fused
in-frame with a GPCR that has had its N-terminal methionine residue
either deleted or substituted with an alternative amino acid. It is
appreciated that a polypeptide of interest may first be made from a
native polypeptide and then operably linked to a suitable
reporter/tag as described above.
[0648] The subject nucleic acids may also contain restriction
sites, multiple cloning sites, primer binding sites, ligatable
ends, recombination sites etc., usually in order to facilitate the
construction of a nucleic acid encoding a polypeptide of
interest.
[0649] b. Host Cells
[0650] The invention further provides host cells comprising a
vector comprising a subject nucleic acid. Suitable host cells
include prokaryotic, e.g., bacterial cells (for example E. coli),
as well as eukaryotic cells e.g. an animal cell (for example an
insect, mammal, fish, amphibian, bird or reptile cell), a plant
cell (for example a maize or Arabidopsis cell), or a fungal cell
(for example a S. cerevisiae cell). In certain embodiments, any
cell suitable for expression of a polypeptide of interest-encoding
nucleic acid may be used as a host cell. Usually, an animal host
cell line is used, examples of which are as follows: monkey kidney
cells (COS cells), monkey kidney CVI cells transformed by SV40
(COS-7, ATCC CRL 165 1); human embryonic kidney cells (HEK-293
["293"], Graham et al. J. Gen Virol. 36:59 (1977)); HEK-293T
["293T"] cells; baby hamster kidney cells (BHK, ATCC CCL 10);
chinese hamster ovary-cells (CHO, Urlaub and Chasin, Proc. Natl.
Acad. Sci. (USA) 77:4216, (1980); Syrian golden hamster cells
MCB3901 (ATCC CRL-9595); mouse sertoli cells (TM4, Mather, Biol.
Reprod. 23:243-251 (1980)); monkey kidney cells (CVI ATCC CCL 70);
african green monkey kidney cells (VERO-76, ATCC CRL-1587); human
cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells
(MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL
1442); human lung cells (W138, ATCC CCL 75); human liver cells (hep
G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL 51); TRI
cells (Mather et al., Annals N. Y. Acad. Sci 383:44-68 (1982));
NIH/3T3 cells (ATCC CRL-1658); and mouse L cells (ATCC CCL-1).
[0651] In certain embodiments, melanophores are used. Melanophores
are skin cells found in lower vertebrates. Relevant materials and
methods will be followed according to the disclosure of U.S. Pat.
No. 5,462,856 and U.S. Pat. No. 6,051,386. These patent disclosures
are herein incorporated by reference in their entirety.
[0652] Additional cell lines will become apparent to those of
ordinary skill in the art, and a wide variety of cell lines are
available from the American Type Culture Collection, 10801
University Boulevard, Manassas, Va. 20110-2209.
C. Screening of Candidate Compounds
[0653] 1. Generic GPCR Screening Assay Techniques
[0654] When a G protein receptor becomes active, it binds to a G
protein (e.g., Gq, Gs, Gi, Gz, Go) and stimulates the binding of
GTP to the G protein. The G protein then acts as a GTPase and
slowly hydrolyzes the GTP to GDP, whereby the receptor, under
normal conditions, becomes deactivated. However, activated
receptors continue to exchange GDP to GTP. A non-hydrolyzable
analog of GTP, [.sup.35S]GTP.gamma.S, can be used to monitor
enhanced binding to membranes which express activated receptors. It
is reported that [.sup.35S]GTP.gamma.S can be used to monitor G
protein coupling to membranes in the absence and presence of
ligand. An example of this monitoring, among other examples
well-known and available to those in the art, was reported by
Traynor and Nahorski in 1995. A preferred use of this assay system
is for initial screening of candidate compounds because the system
is generically applicable to all G protein-coupled receptors
regardless of the particular G protein that interacts with the
intracellular domain of the receptor.
[0655] 2. Specific GPCR Screening Assay Techniques
[0656] Once candidate compounds are identified using the "generic"
G protein-coupled receptor assay (i.e., an assay to select
compounds that are agonists or inverse agonists), in some
embodiments further screening to confirm that the compounds have
interacted at the receptor site is preferred. For example, a
compound identified by the "generic" assay may not bind to the
receptor, but may instead merely "uncouple" the G protein from the
intracellular domain.
[0657] a. Gs, Gz and Gi.
[0658] Gs stimulates the enzyme adenylyl cyclase. Gi (and Gz and
Go), on the other hand, inhibit adenylyl cyclase. Adenylyl cyclase
catalyzes the conversion of ATP to cAMP; thus, activated GPCRs that
couple the Gs protein are associated with increased cellular levels
of cAMP. On the other hand, activated GPCRs that couple Gi (or Gz,
Go) protein are associated with decreased cellular levels of cAMP.
See, generally, "Indirect Mechanisms of Synaptic Transmission,"
Chpt. 8, From Neuron To Brain (3.sup.rd Ed.) Nichols, J. G. et al
eds. Sinauer Associates, Inc. (1992). Thus, assays that detect cAMP
can be utilized to determine if a candidate compound is, e.g., an
inverse agonist to the receptor (i.e., such a compound would
decrease the levels of cAMP). A variety of approaches known in the
art for measuring cAMP can be utilized; in some embodiments a
preferred approach relies upon the use of anti-cAMP antibodies in
an ELISA-based format. Another type of assay that can be utilized
is a whole cell second messenger reporter system assay. Promoters
on genes drive the expression of the proteins that a particular
gene encodes. Cyclic AMP drives gene expression by promoting the
binding of a cAMP-responsive DNA binding protein or transcription
factor (CREB) that then binds to the promoter at specific sites
called cAMP response elements and drives the expression of the
gene. Reporter systems can be constructed which have a promoter
containing multiple cAMP response elements before the reporter
gene, e.g., .beta.-galactosidase or luciferase. Thus, an activated
Gs-linked receptor causes the accumulation of cAMP that then
activates the gene and expression of the reporter protein. The
reporter protein such as .beta.-galactosidase or luciferase can
then be detected using standard biochemical assays (Chen et al.
1995).
[0659] b. Go and Gq.
[0660] Gq and Go are associated with activation of the enzyme
phospholipase C, which in turn hydrolyzes the phospholipid
PIP.sub.2, releasing two intracellular messengers: diacyclglycerol
(DAG) and inositol 1,4,5-triphosphate (IP.sub.3). Increased
accumulation of IP.sub.3 is associated with activation of Gq- and
Go-associated receptors. See, generally, "Indirect Mechanisms of
Synaptic Transmission," Chpt. 8, From Neuron To Brain (3.sup.rd
Ed.) Nichols, J. G. et al eds. Sinauer Associates, Inc. (1992).
Assays that detect IP.sub.3 accumulation can be utilized to
determine if a candidate compound is, e.g., an inverse agonist to a
Gq- or Go-associated receptor (i.e., such a compound would decrease
the levels of IP.sub.3). Gq-associated receptors can also been
examined using an AP1 reporter assay in that Gq-dependent
phospholipase C causes activation of genes containing AP1 elements;
thus, activated Gq-associated receptors will evidence an increase
in the expression of such genes, whereby inverse agonists thereto
will evidence a decrease in such expression, and agonists will
evidence an increase in such expression. Commercially available
assays for such detection are available.
[0661] 3. GPCR Fusion Protein
[0662] The use of an endogenous, constitutively active GPCR or a
non-endogenous, constitutively activated GPCR, for use in screening
of candidate compounds for the direct identification of inverse
agonists or agonists provides an interesting screening challenge in
that, by definition, the receptor is active even in the absence of
an endogenous ligand bound thereto. Thus, in order to differentiate
between, e.g., the non-endogenous receptor in the presence of a
candidate compound and the non-endogenous receptor in the absence
of that compound, with an aim of such a differentiation to allow
for an understanding as to whether such compound may be an inverse
agonist or agonist or have no affect on such a receptor, in some
embodiments it is preferred that an approach be utilized that can
enhance such differentiation. In some embodiments, a preferred
approach is the use of a GPCR Fusion Protein.
[0663] Generally, once it is determined that a non-endogenous GPCR
has been constitutively activated using the assay techniques set
forth above (as well as others known to the art-skilled), it is
possible to determine the predominant G protein that couples with
the endogenous GPCR. Coupling of the G protein to the GPCR provides
a signaling pathway that can be assessed. In some embodiments it is
preferred that screening take place using a mammalian or a
melanophore expression system, as such a system will be expected to
have endogenous G protein therein. Thus, by definition, in such a
system, the non-endogenous, constitutively activated GPCR will
continuously signal. In some embodiments it is preferred that this
signal be enhanced such that in the presence of, e.g., an inverse
agonist to the receptor, it is more likely that it will be able to
more readily differentiate, particularly in the context of
screening, between the receptor when it is contacted with the
inverse agonist.
[0664] The GPCR Fusion Protein is intended to enhance the efficacy
of G protein coupling with the GPCR. The GPCR Fusion Protein may be
preferred for screening with either an endogenous, constitutively
active GPCR or a non-endogenous, constitutively activated GPCR
because such an approach increases the signal that is generated in
such screening techniques. This is important in facilitating a
significant "signal to noise" ratio; such a significant ratio is
preferred for the screening of candidate compounds as disclosed
herein.
[0665] The construction of a construct useful for expression of a
GPCR Fusion Protein is within the purview of those having ordinary
skill in the art. Commercially available expression vectors and
systems offer a variety of approaches that can fit the particular
needs of an investigator. Important criteria in the construction of
such a GPCR Fusion Protein construct include but are not limited
to, that the GPCR sequence and the G protein sequence both be
in-frame (preferably, the sequence for the endogenous GPCR is
upstream of the G protein sequence), and that the "stop" codon of
the GPCR be deleted or replaced such that upon expression of the
GPCR, the G protein can also be expressed. The GPCR can be linked
directly to the G protein, or there can be spacer residues between
the two (preferably, no more than about 12, although this number
can be readily ascertained by one of ordinary skill in the art).
Based upon convenience, it is preferred to use a spacer. In some
embodiments, it is preferred that the G protein that couples to the
non-endogenous GPCR will have been identified prior to the creation
of the GPCR Fusion Protein construct.
[0666] As noted above, activated GPCRs that couple to Gi, Gz and Go
are expected to inhibit the formation of cAMP making assays based
upon these types of GPCRs challenging (i.e., the cAMP signal
decreases upon activation, thus making the direct identification
of, e.g., agonists (which would further decrease this signal)
challenging). As will be disclosed herein, it has been ascertained
that for these types of receptors, it is possible to create a GPCR
Fusion Protein that is not based upon the GPCR's endogenous G
protein, in an effort to establish a viable cyclase-based assay.
Thus, for example, an endogenous Gi coupled receptor can be fused
to a Gs protein--such a fusion construct, upon expression, "drives"
or "forces" the endogenous GPCR to couple with, e.g., Gs rather
than the "natural" Gi protein, such that a cyclase-based assay can
be established. Thus, for Gi, Gz and Go coupled receptors, in some
embodiments it is preferred that when a GPCR Fusion Protein is used
and the assay is based upon detection of adenylyl cyclase activity,
that the fusion construct be established with Gs (or an equivalent
G protein that stimulates the formation of the enzyme adenylyl
cyclase).
TABLE-US-00003 TABLE C Effect of cAMP Effect of IP.sub.3 Production
upon Accumulation Effect of cAMP Activation of upon Activation
Production GPCR (i.e., of GPCR (i.e., upon contact Effect on
IP.sub.3 constitutive constitutive with an Accumulation upon G
activation or activation or Inverse contact with an protein agonist
binding) agonist binding) Agonist Inverse Agonist Gs Increase N/A
Decrease N/A Gi Decrease N/A Increase N/A Gz Decrease N/A Increase
N/A Go Decrease Increase Increase Decrease Gq N/A Increase N/A
Decrease
[0667] Equally effective is a G Protein Fusion construct that
utilizes a Gq Protein fused with a Gs, Gi, Gz or Go Protein. In
some embodiments a preferred fusion construct can be accomplished
with a Gq Protein wherein the first six (6) amino acids of the
G-protein .alpha.-subunit ("G.alpha.q") is deleted and the last
five (5) amino acids at the C-terminal end of G.alpha.q is replaced
with the corresponding amino acids of the G.alpha. of the G protein
of interest. For example, a fusion construct can have a Gq (6 amino
acid deletion) fused with a Gi Protein, resulting in a "Gq/Gi
Fusion Construct". This fusion construct will force the endogenous
Gi coupled receptor to couple to its non-endogenous G protein, Gq,
such that the second messenger, for example, inositol triphosphate
or diacylgycerol, can be measured in lieu of cAMP production.
[0668] 4. Co-Transfection of a Target Gi Coupled GPCR with a
Signal-Enhancer Gs Coupled GPCR (cAMP Based Assays)
[0669] A Gi coupled receptor is known to inhibit adenylyl cyclase,
and, therefore, decreases the level of cAMP production, which can
make the assessment of cAMP levels challenging. In certain
embodiments, an effective technique in measuring the decrease in
production of cAMP as an indication of activation of a receptor
that predominantly couples Gi upon activation can be accomplished
by co-transfecting a signal enhancer, e.g., a non-endogenous,
constitutively activated receptor that predominantly couples with
Gs upon activation (e.g., TSHR-A623I; see infra), with the Gi
linked GPCR. As is apparent, activation of a Gs coupled receptor
can be determined based upon an increase in production of cAMP.
Activation of a Gi coupled receptor leads to a decrease in
production cAMP. Thus, the co-transfection approach is intended to
advantageously exploit these "opposite" affects. For example,
co-transfection of a non-endogenous, constitutively activated Gs
coupled receptor (the "signal enhancer") with expression vector
alone provides a baseline cAMP signal (i.e., although the Gi
coupled receptor will decrease cAMP levels, this "decrease" will be
relative to the substantial increase in cAMP levels established by
constitutively activated Gs coupled signal enhancer). By then
co-transfecting the signal enhancer with the "target receptor", an
inverse agonist of the Gi coupled target receptor will increase the
measured cAMP signal, while an agonist of the Gi coupled target
receptor will decrease this signal.
[0670] Candidate compounds that are directly identified using this
approach should be assessed independently to ensure that these do
not target the signal enhancing receptor (this can be done prior to
or after screening against the co-transfected receptors).
Composition/Formulation and Methods of Treatment
[0671] A compound of the invention, e.g. a GPR119 agonist, a PYY
secretagogue, a compound useful for treatment or prevention of a
condition modulated by PYY, or a compound useful for a modulation
of a PYY-related activity in an individual, can be formulated into
pharmaceutical compositions and medicaments for use in accordance
with the present invention using techniques well known in the art.
Proper formulation is dependent on the route of administration
chosen. In certain embodiments, said administration is to a
non-human vertebrate or to a non-human mammal
[0672] As relates to therapies of the present invention, namely
therapies relating to a GPR119 agonist, the compounds according to
the invention can be administered in any suitable way. Suitable
routes of administration include oral, nasal, rectal, transmucosal,
transdermal, or intestinal administration, parenteral delivery,
including intramuscular, subcutaneous, intramedullary injections,
as well as intrathecal, direct intraventricular, intravenous,
intraperitoneal, intranasal, intrapulmonary (inhaled) or
intraocular injections using methods known in the art. Other
suitable routes of administration are aerosol and depot
formulation. Sustained release formulations, particularly depot, of
the invented medicaments are expressly contemplated. In certain
preferred embodiments, the compounds according to the present
invention are administered orally. The compounds according to the
present invention can be made up in solid or liquid form, such as
tablets, capsules, powders, syrups, elixirs and the like, aerosols,
sterile solutions, suspensions or emulsions, and the like. In
certain embodiments, the GPR119 agonist is administered orally.
[0673] In certain preferred embodiments, the compounds according to
the present invention, e.g. GPR119 agonists, are administered
orally. In certain preferred embodiments, the compounds according
to the present invention, e.g. GPR119 agonists, are administered in
a manner that allows for stimulation of PYY secretion in gut
enteroendocrine cells. In certain preferred embodiments, the
compounds according to the present invention, e.g. GPR119 agonists,
are administered orally in a manner that allows for stimulation of
PYY secretion in gut enteroendocrine cells.
[0674] In certain preferred embodiments, the compounds according to
the present invention, e.g. PYY secretagogues, compounds useful for
treatment or prevention of a condition modulated by PYY, or
compounds useful for a modulation of a PYY-related activity in an
individual, are administered orally. In certain preferred
embodiments, the compounds according to the present invention, e.g.
GPR119 agonists, are administered in a manner that allows for
stimulation of PYY secretion in gut enteroendocrine cells. In
certain preferred embodiments, the compounds according to the
present invention, e.g. GPR119 agonists, are administered orally in
a manner that allows for stimulation of PYY secretion in gut
enteroendocrine cells.
[0675] Formulations for oral administration may be in the form of
aqueous solutions and suspensions, in addition to solid tablet and
capsule formulations. The aqueous solutions and suspensions may be
prepared from sterile powders or granules. The compounds may be
dissolved in water, polyethylene glycol, propylene glycol, ethanol,
corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol,
sodium chloride, and/or various buffers. Other adjuvants are well
and widely known in the art.
[0676] Pharmaceutical compositions of the GPR119 agonist may be
prepared by methods well known in the art, e.g., by means of
conventional mixing, dissolving, granulation, dragee-making,
levigating, emulsifying, encapsulating, entrapping, lyophilizing
processes or spray drying.
[0677] Pharmaceutical compositions for use in accordance with the
present invention may be formulated in conventional manner using
one or more physiologically acceptable carriers comprising
excipients and auxiliaries which facilitate processing of the
active compounds into preparations which can be used
pharmaceutically. Suitable pharmaceutically acceptable carriers are
available to those in the art (see, e.g., Remington: The Science
and Practice of Pharmacy, (Gennaro et al., eds.), 20.sup.th
Edition, 2000, Lippincott Williams & Wilkins; and Handbook of
Pharmaceutical Excipients (Rowe et al., eds), 4.sup.th Edition,
2003, Pharmaceutical Press; the disclosure of each of which is
herein incorporated by reference in its entirety). Proper
formulation is dependent upon the route of administration chosen.
The term "carrier" material or "excipient" material herein means
any substance, not itself a therapeutic agent, used as a carrier
and/or dilutent and/or adjuvant, or vehicle for delivery of a
therapeutic agent to a subject or added to a pharmaceutical
composition to improve its handling or storage properties or to
permit or facilitate formation of a dose unit of the composition
into a discrete article such as a capsule or tablet suitable for
oral administration. Excipients can include, by way of illustration
and not limitation, diluents, disintegrants, binding agents,
adhesives, wetting agents, polymers, lubricants, glidants,
substances added to mask or counteract a disagreeable taste or
odor, flavors, dyes, fragrances, and substances added to improved
appearance of the composition. Acceptable excipients include
stearic acid, magnesium stearate, magnesium oxide, sodium and
calcium salts of phosphoric and sulfuric acids, magnesium
carbonate, talc, gelatin, acacia gum, sodium alginate, pectin,
dextrin, mannitol, sorbitol, lactose, sucrose, starches, gelatin,
cellulosic materials, such as cellulose esters of alkanoic acids
and cellulose alkyl esters, low melting wax cocoa butter or powder,
polymers, such as polyvinyl-pyrrolidone, polyvinyl alcohol, and
polytheylene glycols, and other pharmaceutically acceptable
materials. The components of the pharmaceutical composition can be
encapsulated or tableted for convenient administration.
[0678] Pharmaceutically acceptable refers to those properties
and/or substances which are acceptable to the patient from a
pharmacological/toxicological point of view and to the
manufacturing pharmaceutical chemist from a physical/chemical point
of view regarding composition, formulation, stability, patient
acceptance and bioavailability.
[0679] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used which may
optionally contain gum Arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0680] Pharmaceutical compositions which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with a filler such as lactose, a binder such as starch,
and/or a lubricant such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, liquid polyethylene glycols, cremophor, capmul,
medium or long chain mono-, di- or triglycerides. Stabilizers may
be added in these formulations, also.
[0681] Additionally, a GPR119 agonist may be delivered using a
sustained-release system. Various sustained-release materials have
been established and are well known to those skilled in the art.
Sustained-release tablets or capsules are particularly preferred.
For example, a time delay material such as glyceryl monostearate or
glyceryl distearate may be employed. The dosage form may also be
coated by the techniques described in the U.S. Pat. Nos. 4,256,108,
4,166,452, and 4,265,874 to form osmotic therapeutic tablets for
controlled release.
[0682] It is expressly contemplated that therapies of the present
invention, namely therapies relating to a GPR119 agonist, may be
administered or provided alone or in combination with one or more
other pharmaceutically or physiologically acceptable compound. In
one aspect of the present invention, the other pharmaceutically or
physiologically acceptable compound is not a GPR119 agonist. In one
aspect of the present invention, the other pharmaceutically or
physiologically acceptable compound is a pharmaceutical agent
selected from the group consisting of calcium, vitamin D, estrogen,
tibolone, selective estrogen receptor modulator (SERM; e.g.,
raloxifene, tamoxifen), biphosphonate (e.g., etidronate,
alendronate, risedronate), calcitonin, 1.alpha.-hydroxylated
metabolite of vitamin D, fluoride, thiazide, anabolic steroid,
ipriflavone, vitamin K, parathyroid hormone (PTH), strontium,
statin, osteoprotererin, EP4 receptor selective agonist,
cannabinoid receptor type 2 (CB2) selective agonist, and p38 MAP
kinase inhibitor. (See, e.g., World Health Organization Technical
Report Series 921 (2003), Prevention and Management of
Osteoporosis.)
[0683] In one aspect, the present invention features a composition
comprising or consisting essentially of an amount of a GPR119
agonist according to the present invention. In one aspect, the
present invention features a pharmaceutical composition comprising
or consisting essentially of an amount of a GPR119 agonist
according to the present invention and at least one
pharmaceutically acceptable carrier.
[0684] In one aspect, the present invention features a composition
comprising or consisting essentially of an amount of a GPR119
agonist according to the present invention. In one aspect, the
present invention features a pharmaceutical composition comprising
or consisting essentially of an amount of a GPR119 agonist
according to the present invention and at least one
pharmaceutically acceptable carrier. The present invention also
relates to a dosage form of the composition or of the
pharmaceutical composition wherein the GPR119 agonist, the PYY
secretagogue, the compound useful for treatment or prevention of a
condition modulated by PYY, or the compound useful for a modulation
of a PYY-related activity in an individual is in an amount
sufficient to increase a PYY level in an individual, such as a
plasma total PYY level or a plasma PYY.sub.3-36 level. The present
invention also relates to a dosage form of the composition or of
the pharmaceutical composition wherein the GPR119 agonist is in an
amount sufficient to give an effect in treating or preventing a
condition characterized by low bone mass, such as osteoporosis,
and/or in increasing bone mass in an individual.
[0685] In one aspect, the present invention features a composition
comprising or consisting essentially of an amount of a GPR119
agonist according to the present invention. In one aspect, the
present invention features a pharmaceutical composition comprising
or consisting essentially of an amount of a GPR119 agonist
according to the present invention and at least one
pharmaceutically acceptable carrier. The present invention also
relates to a dosage form of the composition or of the
pharmaceutical composition wherein the GPR119 agonist is in an
amount sufficient to give an effect in increasing a PYY level in an
individual. The present invention also relates to a dosage form of
the composition or of the pharmaceutical composition wherein the
GPR119 agonist is in an amount sufficient to give an effect in
increasing a total PYY (inclusive of PYY.sub.1-36 and PYY.sub.3-36)
level in an individual. The present invention also relates to a
dosage form of the composition or of the pharmaceutical composition
wherein the GPR119 agonist is in an amount sufficient to give an
effect in increasing a PYY.sub.3-36 level in an individual.
[0686] Pharmaceutical compositions suitable for use in the present
invention include compositions wherein the active ingredients are
contained in an amount to achieve their intended purpose. In some
embodiments, a pharmaceutical composition of the present invention
is understood to be useful for treatment or prevention of a
condition modulated by PYY. Conditions modulated by PYY are
according to the present invention. In some embodiments, a
pharmaceutical composition of the present invention is understood
to be useful for increasing a PYY level in an individual. As
relates to the present invention, determination of the amount of a
GPR119 agonist sufficient to achieve an intended purpose according
to the invention is well within the capability of those skilled in
the art, especially in light of the detailed disclosure provided
herein.
[0687] The data obtained from animal studies, including but not
limited to studies using mice, rats, rabbits, pigs, and non-human
primates, can be used in formulating a range of dosage for use in
humans. In general, one skilled in the art understands how to
extrapolate in vivo data obtained in an animal model system to
another, such as a human. In some circumstances, these
extrapolations may merely be based on the weight of the animal
model in comparison to another, such as a human; in other
circumstances, these extrapolations are not simply based on weights
but rather incorporate a variety of factors. Representative factors
include the type, age, weight, sex, diet and medical condition of
the patient, the severity of the disease, the route of
administration, pharmacological considerations such as the
activity, efficacy, pharmacokinetic and toxicology profiles of the
particular compound employed, whether a drug delivery system is
utilized, on whether an acute or chronic disease state is being
treated or prophylaxis is conducted or on whether further active
compounds are administered in addition to the compounds of the
present invention and as part of a drug combination. The dosage
regimen for treating a disease condition with the compounds and/or
compositions of this invention is selected in accordance with a
variety factors as cited above. Thus, the actual dosage regimen
employed may vary widely and therefore may deviate from a preferred
dosage regimen and one skilled in the art will recognize that
dosage and dosage regimen outside these typical ranges can be
tested and, where appropriate, may be used in the methods of this
invention.
[0688] An exemplary animal model system is the rat ovariectomy
(OVX) bone loss model. The ovariectomized rat is an excellent
preclinical animal model that correctly emulates the important
clinical feature of the estrogen depleted human skeleton and the
response of therapeutic agents. In this model, a therapeutic
efficacy is achieved when the bone loss associated with ovariectomy
is partially or completely prevented. (See, e.g., Bollag et al, Mol
Cell Endocrinol (2001) 177:35-41; and Jee et al, J Musculoskel
Neuron Interact (2001) 1:193-207.) In certain embodiments,
therapeutic efficacy is achieved when the bone loss associated with
ovariectomy is at least about 10% prevented, at least about 20%
prevented, at least about 30% prevented, at least about 40%
prevented, at least about 50% prevented, at least about 60%
prevented, at least about 70% prevented, at least about 75%
prevented, at least about 80% prevented, at least about 85%
prevented, at least about 90% prevented, at least about 95%
prevented, or 100% prevented.
[0689] An additional exemplary animal model system is increase of a
blood PYY level after glucose challenge in mice. In certain
embodiments, the blood PYY level is a plasma PYY level. In certain
embodiments, the PYY level is a glucose-independent PYY level. In
certain embodiments, the PYY level is a glucose-dependent PYY
level. In certain embodiments, the PYY is total PYY, inclusive of
PYY.sub.1-36 and PYY.sub.3-36. In certain embodiments, the PYY is
PYY.sub.3-36. Kits for measuring total PYY or PYY.sub.3-36 in
rodents or humans are commercially available, such as from Phoenix
Pharmaceuticals, Inc. (Burlingame, Calif.). Also, PYY.sub.1-36 and
PYY.sub.3-36 can be independently measured by biochemical means
(Eberlein et al, Peptides (1989) 10:797-803; Grandt et al, Regul
Pept (1994) 51:151-159; Cruze et al, Peptides (2007) 28:269-280).
In certain embodiments, a PYY-related activity of the invention is
a PYY.sub.3-36-related activity. In certain embodiments,
therapeutic efficacy is achieved when a blood or plasma PYY level
is increased by at least about 10%, at least about 25%, at least
about 50%, at least about 100%, at least about 150%, at least about
200%, at least about 300%, at least about 400%, at least about
500%, at least about 600%, at least about 700%, at least about
800%, at least about 900%, or at least about 1000%.
[0690] It is expressly contemplated that a GPR119 agonist can be
administered on a daily or regular basis to achieve an increased
level of PYY in an individual. In certain embodiments, a GPR119
agonist is administered on a daily or regular basis to achieve an
increased blood (e.g., plasma) level of PYY in an individual. In
certain embodiments, a GPR119 agonist is administered on a daily or
regular basis to achieve an increased blood (e.g., plasma) level of
PYY in an individual that is at least about 10%, at least about
25%, at least about 50%, at least about 100%, at least about 150%,
at least about 200%, at least about 300%, at least about 400%, at
least about 500%, at least about 600%, at least about 700%, at
least about 800%, at least about 900%, or at least about 1000% a
normal blood (e.g., plasma) level of PYY (e.g., a normal pre-meal
plasma PYY level (e.g., plasma total PYY level) or a plasma PYY
level (e.g., a plasma total PYY level) between the normal pre-meal
and post-meal plasma PYY levels) in an individual or the blood
(e.g., plasma) level of PYY (e.g., plasma total PYY) in the
individual prior to treatment. It is noted that these ranges of
increased blood (e.g., plasma) level of PYY (e.g., plasma total
PYY) are exemplary ranges and are not meant to be limiting to the
invention.
[0691] It is expressly contemplated that a GPR119 agonist can be
administered on a daily or regular basis to achieve an increased
level of PYY in an individual. In certain embodiments, a GPR119
agonist is administered on a daily or regular basis to achieve an
increased blood (e.g., plasma) level of PYY in an individual. In
certain embodiments, a GPR119 agonist is administered on a daily or
regular basis to achieve an increased blood (e.g., plasma) level of
PYY in an individual within a concentration range of 0.1 ng/ml to
10 ng/ml or 100 ng/ml, 0.2 ng/ml to 10 ng/ml or 100 ng/ml, 0.3
ng/ml to 10 ng/ml or 100 ng/ml, 0.4 ng/ml to 10 ng/ml or 100 ng/ml,
0.5 ng/ml to 10 ng/ml or 100 ng/ml, 0.6 ng/ml to 10 ng/ml or 100
ng/ml, 0.7 ng/ml to 10 ng/ml or 100 ng/ml, 0.8 ng/ml to 10 ng/ml or
100 ng/ml, 0.9 ng/ml to 10 ng/ml or 100 ng/ml, 1 ng/ml to 10 ng/ml
or 100 ng/ml, 2 ng/ml to 10 ng/ml or 100 ng/ml, 3 ng/ml to 10 ng/ml
or 100 ng/ml, or 5 ng/ml to 10 ng/ml or 100 ng/ml. It is noted that
these ranges of blood (e.g., plasma) PYY concentration (e.g.,
plasma total PYY concentration) are exemplary ranges and are not
meant to be limiting to the invention.
[0692] Dosage amount and interval may be adjusted in order to
provide an intended therapeutic effect. It will be appreciated that
the exact dosage of a GPR119 agonist in accordance with the present
invention will vary depending on the GPR119 agonist, its potency,
the mode of administration, the age and weight of the patient and
the severity of the condition to be treated. The exact formulation,
route of administration and dosage can be chosen by the individual
physician in view of the patient's condition. By way of
illustration and not limitation, an amount of a GPR119 agonist in
accordance with the present invention is an amount less than about
0.001 mg/kg body weight, less than about 0.005 mg/kg body weight,
less than about 0.01 mg/kg body weight, less than about 0.05 mg/kg
body weight, less than about 0.1 mg/kg body weight, less than about
0.5 mg/kg body weight, less than about 1 mg/kg body weight, less
than about 5 mg/kg body weight, less than about 10 mg/kg body
weight, less than about 50 mg/kg body weight, or less than about
100 mg/kg body weight.
[0693] A preferred dosage range for the amount of a compound of the
invention (e.g. a GPR119 agonist, a PYY secretagogue, a compound
useful for treatment or prevention of a condition modulated by PYY,
or a compound useful for a modulation of a PYY-related activity in
an individual), which can be administered on a daily or regular
basis to achieve desired results is 0.1-100 mg/kg body mass. Other
preferred dosage range is 0.1-30 mg/kg body mass. Other preferred
dosage range is 0.1-10 mg/kg body mass. Other preferred dosage
range is 0.1-3.0 mg/kg body mass. Exemplary preferred dosage is
selected from the group consisting of 0.1 mg/kg, 0.3 mg/kg, 1.0
mg/kg, 3.0 mg/kg, 10 mg/kg, 30 mg/kg and 100 mg/kg. Of course,
these daily dosages can be delivered or administered in small
amounts periodically during the course of a day. It is noted that
these dosage ranges are only preferred ranges and are not meant to
be limiting to the invention. It is noted that these dosages are
only preferred dosages and are not meant to be limiting to the
invention.
[0694] Dosage amount and interval may be adjusted individually to
provide plasma levels of a GPR119 agonist according to the present
invention which achieve an intended therapeutic effect. Dosage
intervals can also be determined using the value for a selected
range of GPR119 agonist concentration so as to achieve the intended
therapeutic effect. A GPR119 agonist should be administered using a
regimen that maintains plasma levels within the selected range of
GPR119 agonist concentration for 10-90% of the time, preferably
between 30-99% of the time, and most preferably between 50-90% of
the time. In cases of local administration or selective uptake, the
range of GPR119 agonist concentration providing the intended
therapeutic effect may not be related to plasma concentration.
[0695] The amount of composition administered will, of course, be
dependent on the individual being treated, on the individual's
weight, the severity of the affliction, the manner of
administration, and the judgement of the prescribing physician.
[0696] In one aspect, the present invention accordingly features a
method of treating or preventing a condition characterized by low
bone mass, such as osteoporosis, or of increasing bone mass
comprising administering to an individual in need thereof a
therapeutically effective amount of a composition comprising or
consisting essentially of an amount of a GPR119 agonist according
to the present invention. In certain embodiments, the composition
is a pharmaceutical composition.
[0697] In one aspect, the present invention relates to a method of
treating or preventing a condition characterized by low bone mass,
such as osteoporosis, or of increasing bone mass comprising
administering to an individual in need thereof a therapeutically
effective amount of a composition comprising or consisting
essentially of an amount of a GPR119 agonist according to the
present invention. In a related aspect, the present invention
features said method wherein the GPR119 agonist is administered in
an amount sufficient to give an effect in increasing a PYY level in
the individual. In certain embodiments, the composition is a
pharmaceutical composition.
[0698] Therapies of the present invention, such as therapies
relating to a GPR119 agonist, are useful in treatment or prevention
of a condition modulated by PYY.
[0699] In certain embodiments, the condition modulated by PYY is a
bone-related condition.
[0700] In certain embodiments, the bone-related condition is a
condition characterized by low bone mass. In certain embodiments,
the condition characterized by low bone mass is selected from the
group consisting of osteopenia, osteoporosis, rheumatoid arthritis,
osteoarthritis, periodontal disease, alveolar bone loss, osteotomy
bone loss, childhood idiopathic bone loss, Paget's disease, bone
loss due to metastatic cancer, osteolytic lesions, curvature of the
spine, and loss of height.
[0701] In certain embodiments, the bone-related condition is a bone
fracture. In certain embodiments, the bone fracture is selected
from the group consisting of a traumatic bone fracture, a long-term
bone fracture, and an osteoporotic bone fracture.
[0702] In certain embodiments, the bone-related condition is a bone
disease. In certain embodiments, the bone disease is selected from
the group consisting of osteopenia, osteoporosis, rheumatoid
arthritis, osteoarthritis, periodontal disease, alveolar bone loss,
osteotomy bone loss, childhood idiopathic bone loss, Paget's
disease, bone loss due to metastatic cancer, osteolytic lesions,
curvature of the spine, and loss of height.
[0703] In certain embodiments, the bone-related condition is
enhanced bone healing or bone growth, wherein said enhanced bone
healing or bone growth is selected from the group consisting of
enhanced bone healing following facial reconstruction, maxillary
reconstruction, mandibular reconstruction, periodontal disease or
tooth extraction, enhanced long bone extension, enhanced prosthetic
ingrowth, and increased bone synostosis.
[0704] In certain embodiments, the condition modulated by PYY is a
metabolic disorder.
[0705] In certain embodiments, the metabolic disorder is selected
from the group consisting of overweight, obesity, hyperphagia,
diabetes (inclusive of type 1 diabetes and type 2 diabetes), type 1
diabetes, type 2 diabetes, impaired glucose tolerance, insulin
resistance, hyperinsulinemia, dyslipidemia, hypertension and
metabolic syndrome.
[0706] In certain embodiments, the condition modulated by PYY is an
angiogenesis-related condition.
[0707] In certain embodiments, the angiogeneisis-related condition
is selected from the group consisting of peripheral arterial
disease, wound healing and ischemia.
[0708] In certain embodiments, the condition modulated by PYY is an
ischemia-related condition.
[0709] In certain embodiments, the ischemia-related condition is
selected from the group consisting of cerebral ischemia, stroke,
ischemic heart disease, myocardial infarction, ischemia-reperfusion
injury, post-myocardial infarction remodeling, heart failure, and
congestive heart failure.
[0710] In certain embodiments, the condition modulated by PYY is a
malabsorptive disorder.
[0711] In certain embodiments, the malabsorptive disorder is
selected from the group consisting of short bowel syndrome,
enterocolitis, Crohn's disease, ulcerative colitis, irritable bowel
syndrome, cholera, and a disorder accompanied by diarrhea.
[0712] In certain embodiments, the condition modulated by PYY is a
convulsive disorder.
[0713] In certain embodiments, the convulsive disorder is
epilepsy.
[0714] In certain embodiments, the condition modulated by PYY is a
a cancer.
[0715] In certain embodiments, the cancer is selected from the
group consisting of pancreatic cancer, breast cancer, colon cancer,
and Barrett's adenocarcinoma.
[0716] In certain embodiments, the condition modulated by PYY is an
inflammatory disorder.
[0717] In certain embodiments, the inflammatory disorder is
selected from the group consisting of atherosclerosis,
atherothrombosis, psoriasis, psoriatic arthritis, rheumatoid
arthritis, osteoarthritis, insulin resistance, diabetes (inclusive
of type 1 diabetes and type 2 diabetes), type 1 diabetes, type 2
diabetes, metabolic syndrome, obesity, lower than normal
HDL-cholesterol, hypertension, hyperlipidemia, ischemic heart
disease, myocardial infarction, congestive heart failure,
osteoporosis, higher than normal osteoclastogenesis, pancreatitis,
interstitial nephritis, acute transplant rejection, chronic
hepatitis, hepatic steatosis, multiple sclerosis, systemic lupus
erythematosus (SLE), restenosis, ischemia-reperfusion injury,
allergic inflammation, Parkinson's disease, prion-associated
disease, excitotoxic injury, Alzheimer's disease, mild cognitive
impairment (MCI), disseminated intravascular coagulation, septic
shock, an inflammatory lung disease, and an inflammatory bowel
disease. In certain embodiments, the inflammatory lung disease is
selected from the group consisting of asthma, emphysema,
interstitial lung disease, idiopathic pulmonary fibrosis,
bronchiolitis obliterans syndrome, chronic obstructive pulmonary
disease (COPD), and pulmonary arterial hypertension. In certain
embodiments, the inflammatory bowel disease is selected from the
group consisting of enteritis, enterocolitis, Crohn's disease,
granulomatous colitis, ileitis, Crohn's colitis, ulcerative
colitis, sigmoiditis, pancolitis, and ulcerative proctitis.
[0718] In certain embodiments, the inflammatory disorder is
selected from the group consisting of atherosclerosis,
atherothrombosis, insulin resistance, pancreatitis, restenosis, an
inflammatory lung disease, and an inflammatory bowel disease. In
certain embodiments, the inflammatory lung disease is selected from
the group consisting of asthma, emphysema, and pulmonary arterial
hypertension. In certain embodiments, the inflammatory bowel
disease is selected from the group consisting of enteritis,
enterocolitis, Crohn's disease, granulomatous colitis, ileitis,
Crohn's colitis, ulcerative colitis, sigmoiditis, pancolitis, and
ulcerative proctitis.
[0719] Conditions characterized by low bone mass include but are
not limited to osteopenia, osteoporosis, rheumatoid arthritis,
osteoarthritis, periodontal disease, alveolar bone loss, osteotomy
bone loss, childhood idiopathic bone loss, Paget's disease, bone
loss due to metastatic cancer, osteolytic lesions, curvature of the
spine, and loss of height. In certain embodiments, the condition
characterized by low bone mass is osteoporosis. In certain
embodiments, the condition characterized by low bone mass is
osteoporosis. In certain embodiments, osteoporosis is primary
osteoporosis. In certain embodiments, osteoporosis is secondary
osteoporosis. Conditions characterized by low bone mass also
include but are not limited to long-term complications of
osteoporosis such as curvature of the spine, loss of height and
prosthetic surgery. It is understood that conditions characterized
by low bone mass can be included in embodiments individually or in
any combination. In certain embodiments, the condition
characterized by low bone mass is primary osteoporosis.
[0720] In certain embodiments, the individual in need of increased
bone mass has a bone mineral density (BMD) of greater than 1
(T-score<-1), greater than or equal to 1.5
(T-score.ltoreq.-1.5), greater than or equal to 2
(T-score.ltoreq.-2) or greater than or equal to 2.5
(T-score.ltoreq.-2.5) standard deviations below the young adult
reference mean. In certain embodiments, the individual in need of
increased bone mass is in need of treatment of bone fracture. In
certain embodiments, the individual in need of treatment of a bone
fracture has a traumatic bone fracture, a long-term bone fracture,
or an osteoporotic bone fracture. In certain embodiments, the
individual is in need of treatment for a bone disease. In certain
embodiments, the individual in need of treatment for a bone disease
has osteopenia, osteoporosis, rheumatoid arthritis, osteoarthritis,
periodontal disease, alveolar bone loss, osteotomy bone loss,
childhood idiopathic bone loss, Paget's disease, bone loss due to
metastatic cancer, osteolytic lesions, curvature of the spine, or
loss of height. In certain embodiments, the individual in need of
treatment for a bone disease has osteoporosis. In certain
embodiments, osteoporosis is primary osteoporosis. In certain
embodiments, osteoporosis is secondary osteoporosis. Destructive
bone disorders that can be treated according to the invention
include but are not limited to osteoporosis, osteoarthritis, and
osteolytic lesions such as those caused by neoplastic disease,
radiotherapy, or chemotherapy. In certain embodiments, osteoporosis
is primary osteoporosis. In certain embodiments, osteoporosis is
secondary osteoporosis.
[0721] Therapies of the present invention, such as therapies
relating to a GPR119 agonist are additionally useful in enhancing
bone healing following facial reconstruction, maxillary
reconstruction, mandibular reconstruction, periodontal disease or
tooth extraction, enhancing long bone extension, enhancing
prosthetic ingrowth or increasing bone synostosis in an
individual.
[0722] Therapies of the present invention, such as therapies
relating to a GPR119 agonist, are useful in a modulation of a
PYY-related activity in an individual. In certain embodiments, the
modulation of a PYY-related activity is selected from the group
consisting of:
[0723] (a) increasing plasma PYY;
[0724] (b) increasing bone mass;
[0725] (c) decreasing loss of bone mass;
[0726] (d) increasing bone formation;
[0727] (e) decreasing food uptake;
[0728] (f) decreasing body weight;
[0729] (g) increasing satiety;
[0730] (h) increasing glucose disposal;
[0731] (i) decreasing plasma glucose;
[0732] (j) increasing angiogenesis;
[0733] (k) decreasing secretion in intestinal epithelium;
[0734] (l) increasing absorption in intestinal epithelium; and
[0735] (m) increasing plasma adiponectin
[0736] In certain embodiments, the individual is a vertebrate. In
certain embodiments, the individual that is a vertebrate is a fish,
an amphibian, a reptile, a bird or a mammal In certain embodiments,
the individual or vertebrate is a mammal In certain embodiments,
the individual or vertebrate that is a mammal is a mouse, a rat, a
hamster, a rabbit, a pig, a dog, a cat, a horse, a cow, a sheep, a
goat, a non-human mammal, a non-human primate or a human. In
certain embodiments, the individual is a human. In certain
embodiments, the human is a post-menopausal woman or a man over the
age of 50.
[0737] Without further elaboration, it is believed that one skilled
in the art can, using the preceding description, practice the
present invention to its fullest extent. The foregoing detailed
description is given for clearness of understanding only, and no
unnecessary limitation should be understood therefrom, as
modifications within the scope of the invention may become apparent
to those skilled in the art.
[0738] Throughout this application, various publications, patents
and patent applications are cited. The disclosures of these
publications, patents and patent applications referenced in this
application are herein incorporated by reference in their entirety
into the present disclosure. Citation herein by Applicant of a
publication, patent, or patent application is not an admission by
Applicant of said publication, patent, or patent application as
prior art.
Examples
[0739] Without further elaboration, it is believed that one skilled
in the art can, using the preceding description, practice the
present invention to its fullest extent. The following detailed
examples are to be construed as merely illustrative, and not
limitations of the preceding disclosure in any way whatsoever.
Those skilled in the art will promptly recognize appropriate
variations from the procedures.
Example 1
[0740] GPR119 Receptor Exhibits Constitutive Activity for Incresing
cAMP Accumulation in Transfected HEK293 Cells
[0741] GPR119 receptor was shown to exhibit constitutive activity
in cAMP assay. cAMP measurements were done with a Flash Plate
adenylyl cyclase kit (NEN Life Science Products, Boston, Ma.)
according to the supplier's protocol. Briefly, HEK293 cells were
transfected with either empty vector DNA ("Vector") or human GPR119
receptor expression plasmid DNA ("GPR119") using Lipofectamine
(Invitrogen, Carlsbad, Calif.). After 24 h, transfected cells were
harvested in GIBCO (Gaithersburg, Md.) cell dissociation buffer
(Catalog no. 13151-014) and resuspended in assay buffer (50%
1.times. PBS/50% stimulation buffer). Incubation was carried out
with 10.sup.5 cells per well for 60 min at room temperature. After
another 2-h incubation with tracer in detection buffer, plates were
counted in a Wallac (Gaithersburg, Md.) MicroBeta scintillation
counter. Values of cAMP per well were extrapolated from a standard
cAMP curve that was included on each assay plate. Results are
presented in FIG. 1. From the results shown in FIG. 1, it is
apparent that GPR119 receptor exhibits constitutive activity for
increasing cAMP accumulation in transfected HEK293 cells.
Example 2
[0742] Compound 1 is an Agonist of GPR119 Receptor as Evidenced,
e.g., by Stimulation of cAMP Accumulation in GPR119-Transfected
HEK293 Cells
[0743] Compound 1
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine) was shown to be
an agonist of GPR119 receptor in cAMP assay. cAMP measurements were
done with a Flash Plate adenylyl cyclase kit (NEN Life Science
Products, Boston, Mass.) according to the supplier's protocol.
Briefly, HEK293 cells were transfected with either empty vector DNA
("Vector") or human GPR119 receptor expression plasmid DNA
("GPR119") using Lipofectamine (Invitrogen, Carlsbad, Calif.).
After 24 h, transfected cells were harvested in GIBCO
(Gaithersburg, Md.) cell dissociation buffer (Catalog no.
13151-014) and resuspended in assay buffer (50% 1.times. PBS/50%
stimulation buffer). Compound 1 was incubated with 10.sup.5 cells
per well for 60 min at room temperature. After another 2-h
incubation with tracer in detection buffer, plates were counted in
a Wallac (Gaithersburg, Md.) MicroBeta scintillation counter.
Values of cAMP per well were extrapolated from a standard cAMP
curve that was included on each assay plate. Results are presented
in FIG. 2. From the results shown in FIG. 2, it is apparent that
Compound 1 is an agonist of GPR119 receptor.
Example 3
Effect of Administration of GPR119 Agonist on Plasma PYY in
Wild-Type Mice
[0744] C57BL/6 male mice (approximately 8 weeks of age; obtained
from Harlan, Indianapolis, Ind.) were fasted for 18 hours, and
randomly assigned into eight groups with n=6 for each group. Mice
were administered per orally with vehicle (80% polyethylene glycol
400/10% Tween 80/10% ethanol) or with a GPR119 agonist (Compound 1;
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine) at 10 mg/kg, as
indicated in FIG. 3. Thirty minutes after treatment, a glucose
bolus at 3g/kg was delivered per orally, and blood was collected at
0 minute (no glucose bolus), 2 minutes, 5 minutes, and 10 minutes
after glucose bolus. Plasma total PYY (inclusive of PYY.sub.1-36
and PYY.sub.3-36) levels were determined by using a PYY EIA kit
purchased from Phoenix Pharmaceuticals, Inc. (Peptide YY (PYY)
(Rat, Mouse, Porcine, Canine) EIA kit, Catalog No. EK-059-03)
according to instructions provided in the kit. From the results
shown in FIG. 3, it is apparent that administration of the GPR119
agonist (Compound 1) increased plasma total PYY in the the mice.
From the results shown in FIG. 3, it is apparent that the GPR119
agonist (Compound 1) stimulated plasma total PYY in the mice.
Compound 1 is identical to a compound disclosed in International
Patent Application No. PCT/US2004/001267 (published as WO
2004/065380).
Example 4
Effect of Administration of GPR119 Agonist on Plasma PYY in
GPR119-Deficient (Knockout) Mice Compared to Wild-Type Mice
[0745] GPR119-deficient male mice (Chu et al, Endocrinology (2007)
148:2601-2609) and wild-type littermates are fasted for 18 hours.
Mice are administered per orally with vehicle (80% polyethylene
glycol 400/10% ethano1110% Tween 80) or with a GPR119 agonist in
accordance with the present invention (Compound 1;
(2-Fluoro-4-methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-
-yl)-piperidin-1-yl]-5-nitro-pyrimidin-4-yl}-amine) at 20 mg/kg.
Thirty minutes after treatment, blood (100 microliter) is collected
via retro orbital vein of the eye (time 0 minutes) followed by a
glucose bolus at 3g/kg (per orally). Five minutes after delivering
glucose, another blood sample (100 microliter) is collected (time 5
minutes). Plasma are collected after centrifugation and plasma
total PYY levels are determined by using PYY EIA kit purchased from
Phoenix Pharmaceuticals, Inc. (Peptide YY (PYY) (Rat, Mouse,
Porcine, Canine) EIA kit, Catalog No. EK-059-03) according to
instructions provided in the kit. It can be further shown that
functional GPR119 receptor is necessary for the administered GPR119
agonist to increase plasma total PYY in the mice. Compound 1 can be
shown to stimulate plasma total PYY in the wild-type mice but not
in the GPR119-deficient mice. Compound 1 is identical to a compound
disclosed in International Patent Application No. PCT/US2004/001267
(published as WO 2004/065380).
Example 5
Effect of Administration of GPR119 Agonist on Bone Mass in
Ovariectomized Rats
[0746] A GPR119 agonist in accordance with the present invention
can be shown to be effective in treating or preventing a condition
characterized by low bone mass, such as osteoporosis, and/or in
increasing bone mass in an individual using the in vivo
ovariectomized (OVX) rat model described below (see, e.g., Bollag
et al, Mol Cell Endocrinol (2001) 177:35-41).
[0747] Twenty virgin female OVX and 20 virgin non-OVX
Sprague-Dawley rats (150-175 g), age 8 weeks, are purchased from
Harlan Sprague-Dawley, Inc. (Indianapolis, Ind.). Animals are fed
ad libitum on a normal commercial pellet diet, Teklab Rodent diet
(1.46% calcium), with free access to water. The rats are randomly
divided into four weight-matched experimental groups and selected
to receive per orally vehicle or a GPR119 agonist in accordance
with the present invention. Treatment is continued on a daily basis
for 6 weeks.
[0748] 1. Control. Ten non-OVX rats are administered per orally
vehicle.
[0749] 2. Control+Treatment. Ten non-OVX rats are administered per
orally GPR119 agonist.
[0750] 3. OVX. Ten OVX rats are administered per orally
vehicle.
[0751] 4. OVX+Treatment. Ten OVX rats are administered per orally
GPR119 agonist.
The rats are weighed daily and length measured at baseline and
again at 6 weeks. Dual energy X-ray absorptiometry (DXA) using a
Hologic QDR 1000/W (Waltham, Mass.) is performed on all animals
prior to initiation of treatment and at 6 weeks, and data is
analyzed using the software Rat Whole Body version 5.53. Bone
mineral density (BMD) is determined at the spine.
[0752] The percent change in vertebral bone density after 6 weeks
of treatment is determined It is shown that administration of a
GPR119 agonist attenuates the negative effects of ovariectomy on
vertebral bone density. Attenuation of the negative effects of
ovariectomy on vertebral bone density is indicative of the
treatment having efficacy in treating or preventing a condition
characterized by low bone mass, such as osteoporosis, and/or in
increasing bone mass in an individual.
Example 6
Effect of Administration of GPR119 Agonist on Bone Fracture
Healing
[0753] A GPR119 agonist in accordance with the present invention
can be shown to be effective in treatment of bone fracture using
the in vivo assay described below.
Fracture Technique
[0754] Sprague-Dawley rats at 3 months of age are anesthetized with
Ketamine. A 1 cm incision is made on the anteromedial aspect of the
proximal part of the right tibia or femur. The following describes
the tibial surgical technique. The incision is carried through to
the bone, and a 1 mm hole is drilled 4 mm proximal to the distal
aspect of the tibial tuberosity 2 mm medial to the anterior ridge.
Intramedullary nailing is performed with a 0.8 mm stainless steel
tube (maximum load 36.3 N, maximum stiffness 61.8 N/mm, tested
under the same conditions as the bones). No reaming of the
medullary canal is performed. A standardized closed fracture is
produced 2 mm above the tibiofibular junction by three-point
bending using specially designed adjustable forceps with blunt
jaws. To minimize soft tissue damage, care is taken not to displace
the fracture. The skin is closed with monofilament nylon sutures.
The operation is performed under sterile conditions. Radiographs of
all fractures are taken immediately after nailing, and rats with
fractures outside the specified diaphyseal area or with displaced
nails are excluded. The remaining animals are divided randomly into
the following groups with 10-12 animals per each subgroup per time
point for testing the fracture healing. The rats are administered
on a daily basis per orally with vehicle or with a GPR119 agonist.
The GPR119 agonist is used at an amount between 0.001 mg/kg body
weight and 100 mg/kg body weight. Treatment is continued for 10,
20, 40 and 80 days.
[0755] At 10, 20, 40 and 80 days, 10-12 rats from each group are
anesthetized with Ketamine and sacrificed by exsanguination. Both
tibiofibular bones are removed by dissection and all soft tissue is
stripped. Bones from 5-6 rats for each group are stored in 70%
ethanol for histological analysis, and bones from another 5-6 rats
for each group are stored in a buffered Ringer's solution
(+4.degree. C., pH 7.4) for radiographs and biomechanical testing
which is performed.
Histological Analysis
[0756] The methods for histological analysis of fractured bone have
been previously published by
[0757] Mosekilde and Bak (Bone (1993) 14:19-27). Briefly, the
fracture site is sawed 8mm to each side of the fracture line,
embedded undecalcified in methymethacrylate, and cut frontals
sections on a Reichert-Jung Polycut microtome in 8 .mu.m thick.
Masson-Trichome stained mid-frontal sections (including both tibia
and fibula) are used for visualization of the cellular and tissue
response to fracture healing with and without treatment. Sirius red
stained sections are used to demonstrate the characteristics of the
callus structure and to differentiate between woven bone and
lamellar bone at the fracture site. The following measurements are
performed: (1) fracture gap--measured as the shortest distance
between the cortical bone ends in the fracture, (2) callus length
and callus diameter, (3) total bone volume area of callus, (4) bony
tissue per tissue area inside the callus area, (5) fibrous tissue
in the callus, and (6) cartilage area in the callus.
Biomechanical analysis
[0758] The methods for biomechanical analysis have been previously
published by Bak and Andreassen (Calcif Tissue Int (1989)
45:292-297). Briefly, radiographs of all fractures are taken prior
to the biomechanical test. The mechanical properties of the healing
fractures are analyzed by a destructive three- or four-point
bending procedure. Maximum load, stiffness, energy at maximum load,
deflection at maximum load, and maximum stress are determined
Example 7
Full-Length Cloning of Endogenous Human GPR119
[0759] Polynucleotide encoding endogenous human GPR119 was cloned
by PCR using the GPR119 specific primers
TABLE-US-00004 (SEQ ID NO: 3; sense, ATG as initiation codon)
5'-GTCCTGCCACTTCGAGACATGG-3' (SEQ ID NO: 4; antisense, 3' of stop
codon) 5'-GAAACTTCTCTGCCCTTACCGTC-3'
and human genomic DNA as template. TaqPlus Precision.TM. DNA
polymerase (Stratagene) was used for amplification by the following
cycle with step 2 to step 4 repeated 35 times: 94.degree. C., 3
minutes; 94.degree. C., 1 minute; 58.degree. C., 1 minute;
72.degree. C., 2 minutes; 72.degree. C., 10 minutes. A 1.0 Kb PCR
fragment of predicted size was isolated and cloned into the
pCRII-TOPO.TM. vector (Invitrogen) and completely sequenced using
the T7 DNA sequenase kit (Amersham). See, SEQ ID NO:1 for nucleic
acid sequence and SEQ ID NO:2 for the deduced amino acid
sequence.
Example 8
Receptor Expression
[0760] Although a variety of cells are available to the art for the
expression of G protein-coupled receptors, it is most preferred
that eukaryotic cells be utilized. In certain embodiments,
mammalian cells or melanophores are utilized. The following are
illustrative; those of ordinary skill in the art are credited with
the ability to determine those techniques that are preferentially
beneficial for the needs of the artisan. See, e.g., Example 12,
infra, as it relates to melanophores.
[0761] a. Transient Transfection
[0762] On day one, 6.times.10.sup.6/10 cm dish of 293 cells are
plated out. On day two, two reaction tubes are prepared (the
proportions to follow for each tube are per plate): tube A is
prepared by mixing 4 .mu.g DNA (e.g., pCMV vector; pCMV vector with
receptor cDNA, etc.) in 0.5 ml serum free DMEM (Gibco BRL); tube B
is prepared by mixing 24 .mu.l lipofectamine (Gibco BRL) in 0.5m1
serum free DMEM. Tubes A and B are admixed by inversions (several
times), followed by incubation at room temperature for 30-45 min.
The admixture is referred to as the "transfection mixture". Plated
293 cells are washed with 1.times. PBS, followed by addition of 5
ml serum free DMEM. 1 ml of the transfection mixture is added to
the cells, followed by incubation for 4hrs at 37.degree. C./5%
CO.sub.2. The transfection mixture is removed by aspiration,
followed by the addition of 10 ml of DMEM/10% Fetal Bovine Serum.
Cells are incubated at 37.degree. C./5% CO.sub.2. After 48 hr
incubation, cells are harvested and utilized for analysis.
[0763] b. Stable Cell Lines
[0764] Approximately 12.times.10.sup.6 293 cells are plated on a 15
cm tissue culture plate. Grown in DME High Glucose Medium
containing ten percent fetal bovine serum and one percent sodium
pyruvate, L-glutamine, and antibiotics. Twenty-four hours following
plating of 293 cells (or to .about.80% confluency), the cells are
transfected using 12 .mu.g of DNA (e.g., pCMV-neo.sup.r vector with
receptor cDNA). The 12 .mu.g of DNA is combined with 60 .mu.l of
lipofectamine and 2 ml of DME High Glucose Medium without serum.
The medium is aspirated from the plates and the cells are washed
once with medium without serum. The DNA, lipofectamine, and medium
mixture are added to the plate along with 10 ml of medium without
serum. Following incubation at 37.degree. C. for four to five
hours, the medium is aspirated and 25 ml of medium containing serum
is added. Twenty-four hours following transfection, the medium is
aspirated again, and fresh medium with serum is added. Forty-eight
hours following transfection, the medium is aspirated and medium
with serum is added containing geneticin (G418 drug) at a final
concentration of approximately 12.times.10.sup.6 293 cells are
plated on a 15 cm tissue culture plate. Grown in DME High Glucose
Medium containing ten percent fetal bovine serum and one percent
sodium pyruvate, L-glutamine, and antibiotics. Twenty-four hours
following plating of 293 cells (or to .about.80% confluency), the
cells are transfected using 12 .mu.g of DNA (e.g., pCMV vector with
receptor cDNA). The 12 .mu.g of DNA is combined with 60 .mu.l of
lipofectamine and 2m1 of DME High Glucose Medium without serum. The
medium is aspirated from the plates and the cells are washed once
with medium without serum. The DNA, lipofectamine, and medium
mixture are added to the plate along with 10 ml of medium without
serum. Following incubation at 37.degree. C. for four to five
hours, the medium is aspirated and 25m1 of medium containing serum
is added. Twenty-four hours following transfection, the medium is
aspirated again, and fresh medium with serum is added. Forty-eight
hours following transfection, the medium is aspirated and medium
with serum is added containing geneticin (G418 drug) at a final
concentration of 500 .mu.g/ml. The transfected cells now undergo
selection for positively transfected cells containing the G418
resistance gene. The medium is replaced every four to five days as
selection occurs. During selection, cells are grown to create
stable pools, or split for stable clonal selection.
Example 9
Assays for Screening Candidate Compounds as, e.g., GPR119
Agonist
[0765] A variety of approaches are available for screening
candidate compounds as, e.g., GPR119 agonists. The following are
illustrative; those of ordinary skill in the art are credited with
the ability to determine those techniques that are preferentially
beneficial for the needs of the artisan. Assays for screening
compounds as agonists of a G protein-coupled receptor are well
known to the skilled artisan (see, e.g., International Application
WO 02/42461).
[0766] 1. Membrane Binding Assays: [.sup.35S]GTP.gamma.S Assay
[0767] When a G protein-coupled receptor is in its active state,
either as a result of ligand binding or constitutive activation,
the receptor couples to a G protein and stimulates the release of
GDP and subsequent binding of GTP to the G protein. The alpha
subunit of the G protein-receptor complex acts as a GTPase and
slowly hydrolyzes the GTP to GDP, at which point the receptor
normally is deactivated. Activated receptors continue to exchange
GDP for GTP. The non-hydrolyzable GTP analog,
[.sup.35S]GTP.gamma.S, can be utilized to demonstrate enhanced
binding of [.sup.35S]GTP.gamma.S to membranes expressing activated
receptors. The advantage of using [.sup.35S]GTP.gamma.S binding to
measure activation is that: (a) it is generically applicable to all
G protein-coupled receptors; (b) it is proximal at the membrane
surface making it less likely to pick-up molecules which affect the
intracellular cascade.
[0768] The assay utilizes the ability of G protein coupled
receptors to stimulate [.sup.35S]GTP.gamma.S binding to membranes
expressing the relevant receptors. The assay is generic and has
application to drug discovery at all G protein-coupled
receptors.
Membrane Preparation
[0769] In some embodiments, membranes comprising a G
protein-coupled receptor of the invention and for use in the
identification of candidate compounds as, e.g.,. agonists of the
receptor, are preferably prepared as follows:
[0770] a. Materials
[0771] "Membrane Scrape Buffer" is comprised of 20mM HEPES and 10mM
EDTA, pH 7.4; "Membrane Wash Buffer" is comprised of 20 mM HEPES
and 0.1 mM EDTA, pH 7.4; "Binding Buffer" is comprised of 20 mM
HEPES, 100 mM NaCl, and 10 mM MgCl.sub.2, pH 7.4.
[0772] b. Procedure
[0773] All materials will be kept on ice throughout the procedure.
Firstly, the media will be aspirated from a confluent monolayer of
cells, followed by rinse with 10m1 cold PBS, followed by
aspiration. Thereafter, 5 ml of Membrane Scrape Buffer will be
added to scrape cells; this will be followed by transfer of
cellular extract into 50m1 centrifuge tubes (centrifuged at 20,000
rpm for 17 minutes at 4.degree. C.). Thereafter, the supernatant
will be aspirated and the pellet will be resuspended in 30 ml
Membrane Wash Buffer followed by centrifuge at 20,000 rpm for 17
minutes at 4.degree. C. The supernatant will then be aspirated and
the pellet resuspended in Binding Buffer. This will then be
homogenized using a Brinkman Polytron.TM. homogenizer (15-20 second
bursts until the all material is in suspension). This is referred
to herein as "Membrane Protein".
Bradford Protein Assay
[0774] Following the homogenization, protein concentration of the
membranes will be determined using the Bradford Protein Assay
(protein can be diluted to about 1.5mg/ml, aliquoted and frozen
(-80.degree. C.) for later use; when frozen, protocol for use will
be as follows: on the day of the assay, frozen Membrane Protein is
thawed at room temperature, followed by vortex and then homogenized
with a Polytron at about 12.times.1,000 rpm for about 5-10 seconds;
it is noted that for multiple preparations, the homogenizer should
be thoroughly cleaned between homogenization of different
preparations).
[0775] a. Materials
[0776] Binding Buffer (as per above); Bradford Dye Reagent;
Bradford Protein Standard will be utilized, following manufacturer
instructions (Biorad, cat. no. 500-0006).
[0777] b. Procedure
[0778] Duplicate tubes will be prepared, one including the
membrane, and one as a control "blank". Each contained 800 .mu.l
Binding Buffer. Thereafter, 10 .mu.l of Bradford Protein Standard
(1 mg/ml) will be added to each tube, and 10 .mu.l of membrane
Protein will then be added to just one tube (not the blank).
Thereafter, 200 .mu.l of Bradford Dye Reagent will be added to each
tube, followed by vortex of each. After five (5) minutes, the tubes
will be re-vortexed and the material therein will be transferred to
cuvettes. The cuvettes will then be read using a CECIL 3041
spectrophotometer, at wavelength 595.
Identification Assay
[0779] a. Materials
[0780] GDP Buffer consists of 37.5 ml Binding Buffer and 2mg GDP
(Sigma, cat. no. G-7127), followed by a series of dilutions in
Binding Buffer to obtain 0.2 .mu.M GDP (final concentration of GDP
in each well was 0.1 .mu.M GDP); each well comprising a candidate
compound, has a final volume of 200 .mu.l consisting of 100 .mu.l
GDP Buffer (final concentration, 0.1 .mu.M GDP), 50 .mu.l Membrane
Protein in Binding Buffer, and 50 .mu.l [.sup.35S]GTP.gamma.S (0.6
nM) in Binding Buffer (2.5 .mu.l [.sup.35S]GTP.gamma.S per 10 ml
Binding Buffer).
[0781] b. Procedure
[0782] Candidate compounds will be preferably screened using a
96-well plate format (these can be frozen at -80.degree. C.).
Membrane Protein (or membranes with expression vector excluding the
Target GPCR, as control), will be homogenized briefly until in
suspension. Protein concentration will then be determined using the
Bradford Protein Assay set forth above. Membrane Protein (and
control) will then be diluted to 0.25 mg/ml in Binding Buffer
(final assay concentration, 12.5 .mu.g/well). Thereafter, 100 .mu.l
GDP Buffer was added to each well of a Wallac Scintistrip.TM.
(Wallac). A 5 ul pin-tool will then be used to transfer 5 .mu.l of
a candidate compound into such well (i.e., 5 .mu.l in total assay
volume of 200 .mu.l is a 1:40 ratio such that the final screening
concentration of the candidate compound is 10 .mu.M). Again, to
avoid contamination, after each transfer step the pin tool should
be rinsed in three reservoirs comprising water (1.times.), ethanol
(1.times.) and water (2.times.)--excess liquid should be shaken
from the tool after each rinse and dried with paper and kimwipes.
Thereafter, 50 .mu.l of Membrane Protein will be added to each well
(a control well comprising membranes without the Target GPCR was
also utilized), and pre-incubated for 5-10 minutes at room
temperature. Thereafter, 50 .mu.l of [.sup.35S]GTP.gamma.S (0.6 nM)
in Binding Buffer will be added to each well, followed by
incubation on a shaker for 60 minutes at room temperature (again,
in this example, plates were covered with foil). The assay will
then be stopped by spinning of the plates at 4000 RPM for 15
minutes at 22.degree. C. The plates will then be aspirated with an
8 channel manifold and sealed with plate covers. The plates will
then be read on a Wallac 1450 using setting "Prot. #37" (as per
manufacturer's instructions).
[0783] 2. Adenylyl Cyclase Assay
[0784] A Flash Plate.TM. Adenylyl Cyclase kit (New England Nuclear;
Cat. No. SMP004A) designed for cell-based assays can be modified
for use with crude plasma membranes. The Flash Plate wells can
contain a scintillant coating which also contains a specific
antibody recognizing cAMP. The cAMP generated in the wells can be
quantitated by a direct competition for binding of radioactive cAMP
tracer to the cAMP antibody. The following serves as a brief
protocol for the measurement of changes in cAMP levels in cells
that express the receptors.
[0785] In certain embodiments, a modified Flash Plate.TM. Adenylyl
Cyclase kit (New England Nuclear; Cat. No. SMP004A) is utilized for
identification of candidate compounds as, e.g., GPR119 agonists in
accordance with the following protocol.
[0786] Cells transfected with a G protein-coupled receptor of the
invention are harvested approximately three days after
transfection. Membranes are prepared by homogenization of suspended
cells in buffer containing 20 mM HEPES, pH 7.4 and 10 mM
MgCl.sub.2. Homogenization is performed on ice using a Brinkman
Polytron.TM. for approximately 10 seconds. The resulting homogenate
is centrifuged at 49,000.times.g for 15 minutes at 4.degree. C. The
resulting pellet is then resuspended in buffer containing 20 mM
HEPES, pH 7.4 and 0.1 mM EDTA, homogenized for 10 seconds, followed
by centrifugation at 49,000.times.g for 15 minutes at 4.degree. C.
The resulting pellet is then stored at -80.degree. C. until
utilized. On the day of direct identification screening, the
membrane pellet is slowly thawed at room temperature, resuspended
in buffer containing 20 mM HEPES, pH 7.4 and 10 mM MgCl.sub.2, to
yield a final protein concentration of 0.60 mg/ml (the resuspended
membranes are placed on ice until use).
[0787] cAMP standards and Detection Buffer (comprising 2 .mu.Ci of
tracer ([.sup.125I]cAMP (100 .mu.l) to 11 ml Detection Buffer)) are
prepared and maintained in accordance with the manufacturer's
instructions. Assay Buffer was prepared fresh for screening and
contained 20 mM HEPES, pH 7.4, 10 mM MgCl.sub.2, 20 mM
phospocreatine (Sigma), 0.1 units/ml creatine phosphokinase
(Sigma), 50 .mu.M GTP (Sigma), and 0.2 mM ATP (Sigma); Assay Buffer
was then stored on ice until utilized.
[0788] Candidate compounds are added, preferably, to e.g. 96-well
plate wells (3 .mu.l/well; 12 .mu.M final assay concentration),
together with 40 .mu.l Membrane Protein (30 .mu.g/well) and 50
.mu.l of Assay Buffer. This admixture was then incubated for 30
minutes at room temperature, with gentle shaking.
[0789] Following the incubation, 100 .mu.l of Detection Buffer is
added to each well, followed by incubation for 2-24 hours. Plates
are then counted in a Wallac MicroBeta.TM. plate reader using
"Prot. #31" (as per manufacturer's instructions).
[0790] 3. CRE-Luc Reporter Assay
[0791] 293 and 293T cells are plated-out on 96 well plates at a
density of 2.times.10.sup.4 cells per well and were transfected
using Lipofectamine Reagent (BRL) the following day according to
manufacturer instructions. A DNA/lipid mixture is prepared for each
6-well transfection as follows: 260 ng of plasmid DNA in 100 .mu.l
of DMEM is gently mixed with 2 .mu.l of lipid in 100 .mu.l of DMEM
(the 260 ng of plasmid DNA consists of 200 ng of a 8.times.CRE-Luc
reporter plasmid, 50 ng of pCMV comprising a G protein-coupled
receptor of the invention or pCMV alone, and 10 ng of a GPRS
expression plasmid (GPRS in pcDNA3 (Invitrogen)). The
8.times.CRE-Luc reporter plasmid was prepared as follows: vector
SRIF-.beta.-gal was obtained by cloning the rat somatostatin
promoter (-71/+51) at BglV-HindIII site in the p.beta.gal-Basic
Vector (Clontech). Eight (8) copies of cAMP response element were
obtained by PCR from an adenovirus template AdpCF126CCRE8 [see,
Suzuki et al., Hum Gene Ther (1996) 7:1883-1893; the disclosure of
which is herein incorporated by reference in its entirety) and
cloned into the SRIF-.beta.-gal vector at the Kpn-BglV site,
resulting in the 8.times.CRE-.beta.-gal reporter vector. The
8.times.CRE-Luc reporter plasmid was generated by replacing the
beta-galactosidase gene in the 8.times.CRE-.beta.-gal reporter
vector with the luciferase gene obtained from the pGL3-basic vector
(Promega) at the HindIII-BamHI site. Following 30 min. incubation
at room temperature, the DNA/lipid mixture is diluted with 400
.mu.l of DMEM and 100 .mu.l of the diluted mixture is added to each
well. 100 .mu.l of DMEM with 10% FCS are added to each well after a
4 hr incubation in a cell culture incubator. The following day the
transfected cells are changed with 200 .mu.l/well of DMEM with 10%
FCS. Eight (8) hours later, the wells are changed to 100 .mu.l/well
of DMEM without phenol red, after one wash with PBS. Luciferase
activity is measured the next day using the LucLite.TM. reporter
gene assay kit (Packard) following manufacturer instructions and
read on a 1450 MicroBeta.TM. scintillation and luminescence counter
(Wallac).
Example 10
[0792] Whole Cell Adenylyl Cyclase Assay for, e.g., GPR119 Agonist
Activity
[0793] Cyclic AMP measurements are done with a Flash Plate.TM.
Adenylyl Cyclase kit (New England Nuclear) according to the
supplier's protocol. HEK293 cells are plated in 15-cm tissue
culture dish at 12.times.10.sup.6 cells per dish in regular growth
medium (DMEM/10% FBS). On the next day, 10 .mu.g of either empty
vector DNA or expression plasmid DNA are transfected into cells
with lipofectamine (Invitrogen, Carlsbad, Calif.) according to
manufacturer's protocol. After 24 hours in culture, transfected
cells are harvested in GIBCO cell dissociation buffer (Cat
#13151-014), pelleted by centrifugation for 5 minutes at 1,100 rpm,
and carefully re-suspended into an appropriate volume of Assay
Buffer (50% 1.times. PBS and 50% Stimulation Buffer) to give a
final cell count at 2.times.10.sup.6 cells/ml. Test compounds are
prepared in 50 .mu.l Assay Buffer at desired assay concentration
where indicated, and pipetted into wells of the 96-well Flash
Plate. The cell suspension prepared above was then added (50 .mu.l
per well). After an incubation time of 60 minutes at room
temperature, 100 .mu.l of Detection Mix containing tracer
[.sup.125I]-cAMP is then added to the wells. Plates are incubated
for additional 2 hours followed by counting in a Wallac MicroBeta
scintillation counter. Values of cAMP/well are extrapolated from a
standard cAMP curve which is included on each assay plate.
[0794] An increase in cAMP level in GPR119-transfected HEK293 cells
over that in HEK293 cells transfected with empty vector is
indicative of a test compound being a compound that stimulates
GPR119 receptor functionality.
Example 11
[0795] Homogeneous Time-Resolved Fluorescence (HTRF.RTM.) Assay for
Direct cAMP Measurement
[0796] Compounds are screened for compounds which stimulate GPR119
receptor, e.g. for agonists of GPR119 receptor, using HTRF.RTM.
assay for direct cAMP measurement (Gabriel et al, ASSAY and Drug
Development Technologies, 1:291-303, 2003) and recombinant CHO-K1
cells stably transfected with GPR119 receptor. Compounds are
screened for agonists of GPR119 receptor using HTRF.RTM. assay for
direct cAMP measurement (Gabriel et al, ASSAY and Drug Development
Technologies, 1:291-303, 2003) and recombinant CHO-K1 cells stably
transfected with GPR119 receptor. CHO-K1 cells are obtained from
ATCC.RTM. (Manassas, Va.; Catalog # CCL-61). An agonist of GPR119
receptor is detected in HTRF.RTM. assay for direct cAMP measurement
as a compound which increases cAMP concentration. HTRF.RTM. assay
also is used to determine EC.sub.50 values for GPR119 receptor
agonists.
[0797] Principle of the assay: HTRF.RTM. assay kit is purchased
from Cisbio-US, Inc. (Bedford, Mass.; Catalog #62AM4PEC). The
HTRF.RTM. assay supported by the kit is a competitive immunoassay
between endogenous cAMP produced by the CHO-K1 cells and tracer
cAMP labeled with the dye d2. The tracer binding is visualized by a
monoclonal anti-cAMP antibody labeled with Cryptate. The specific
signal (i.e., fluorescence resonance energy transfer, FRET) is
inversely proportional to the concentration of unlabeled cAMP in
the standard or sample.
[0798] Standard curve: The fluorescence ratio (665 nm/620 nm) of
the standards (0.17 to 712 nM cAMP) included in the assay is
calculated and used to generate a cAMP standard curve according to
the kit manufacturer's instructions. The fluorescence ratio of the
samples (test compound or compound buffer) is calculated and used
to deduce respective cAMP concentrations by reference to the cAMP
standard curve.
[0799] Setup of the assay: HTRF.RTM. assay is carried out using a
two-step protocol essentially according to the kit manufacturer's
instructions, in 20 .mu.l total volume per well in 384-well plate
format (ProxiPlates; PerkinElmer, Fremont, Calif.; catalog
#6008280). To each of the experimental wells is transferred 3000
recombinant CHO-K1 cells in 5 .mu.l assay buffer (phosphate
buffered saline containing calcium chloride and magnesium chloride
(Invitrogen, Carlsbad, Calif.; catalog #14040) supplemented with
IBMX (100 .mu.M) and rolipram (10 .mu.M) (phosphodiesterase
inhibitors; Sigma-Aldrich, St. Louis, MO; catalog #I5879 and
catalog #R6520, respectively) and 0.1% bovine serum albumin (BSA)
fraction V (Sigma-Aldrich; catalog #A3059)), followed by test
compound in 5 .mu.l assay buffer or 5 .mu.l assay buffer. The plate
is then incubated at room temperature for 1 hour. To each well is
then added 5 .mu.l cAMP-d2 conjugate in lysis buffer and 5 .mu.l
Cryptate conjugate in lysis buffer according to the kit
manufacturer's instructions. The plate is then further incubated at
room temperature for 1 hour, after which the assay plate is
read.
[0800] Assay readout: HTRF.RTM. readout is accomplished using a
PHERAstar (BMG LABTECH Inc., Durham, N.C.) or EnVision.TM.
(PerkinElmer, Fremont Calif.) microplate reader.
Example 12
[0801] Melanophore Assay for, e.g., GPR119 Agonist Activity
[0802] Melanophores are maintained in culture as reported by
Potenza et al [Pigment Cell Research (1992) 5:372-378] and
transfected with an expression vector encoding a GPR119 receptor
(GPR119; e.g., human GPR119, GenBank.RTM. Accession No. AAP72125
and alleles thereof) using electroporation. Following
electroporation, the transfected cells are plated into 96 well
plates for the assay. The cells are then allowed to grow for 48
hours in order to both recover from the electroporation procedure
and attain maximal receptor expression levels.
[0803] On the assay day, the growth medium on the cells is replaced
with serum-free buffer containing 10 nM melatonin. The melatonin
acts via an endogenous Gi-coupled GPCR in the melanophores to lower
intracellular cAMP levels. In response to lowered cAMP levels, the
melanophores translocate their pigment to the center of the cell.
The net effect of this is a significant decrease in the absorbance
reading of the cell monolayer in the well, measured at 600-650
nM.
[0804] After a 1-hour incubation in melatonin, the cells become
completely pigment-aggregated. At this point a baseline absorbance
reading is collected. Serial dilutions of test compounds are then
added to the plate, and compounds having GPR119 agonist activity
produce increases in intracellular cAMP levels. In response to
these increased cAMP levels, the melanophores translocate their
pigment back into the cell periphery. After one hour, stimulated
cells are fully pigment-dispersed. The cell monolayer in the
dispersed state absorbs much more light in the 600-650 nm range.
The measured increase in absorbance compared to the baseline
reading allows one to quantitate the degree of receptor stimulation
and plot a dose-response curve.
[0805] Materials and methods relating to melanophore assay are
found in U.S. Pat. Nos. 5,462,856 and 6,051,386, the disclosure of
each of which is herein incorporated by reference in its
entirety.
[0806] An increase in pigment dispersion in GPR119-transfected
melanophores over over that in melanophores transfected with empty
vector is indicative of a test compound being a compound that
stimulates GPR119 receptor functionality.
[0807] Other assays for identifying a compound as a GPR119 agonist
will be readily apparent to the skilled artisan (see, e.g., Example
9, supra).
Example 13
[0808] Yeast Reporter Assay for, e.g., GPR119 Agonist Activity
[0809] The yeast cell-based reporter assays have previously been
described in the literature (e.g., see Miret et al, J Biol Chem
(2002) 277:6881-6887; Campbell et al, Bioorg Med Chem Left (1999)
9:2413-2418; King et al, Science (1990) 250:121-123; WO 99/14344;
WO 00/12704; and U.S. Pat. No. 6,100,042). Briefly, yeast cells
have been engineered such that the endogenous yeast G-alpha (GPA1)
has been deleted and replaced with G-protein chimeras constructed
using multiple techniques. Additionally, the endogenous yeast
alpha-cell GPCR, Ste3 has been deleted to allow for a homologous
expression of a mammalian GPCR of choice. In the yeast, elements of
the pheromone signaling transduction pathway, which are conserved
in eukaryotic cells (for example, the mitogen-activated protein
kinase pathway), drive the expression of Fus1. By placing
.beta.-galactosidase (LacZ) under the control of the Fus1 promoter
(Fus1p), a system has been developed whereby receptor activation
leads to an enzymatic readout.
[0810] Yeast cells are transformed by an adaptation of the lithium
acetate method described by Agatep et al (Agatep et al, 1998,
Transformation of Saccharomyces cerevisiae by the lithium
acetate/single-stranded carrier DNA/polyethylene glycol
(LiAc/ss-DNA/PEG) protocol. Technical Tips Online, Trends Journals,
Elsevier). Briefly, yeast cells are grown overnight on yeast
tryptone plates (YT). Carrier single-stranded DNA (10 .mu.g), 2
.mu.g of each of two Fus1p-LacZ reporter plasmids (one with URA
selection marker and one with TRP), 2 .mu.g of GPR119 (e.g., human
receptor) in yeast expression vector (2 .mu.g origin of
replication) and a lithium acetate/polyethylene glycol/TE buffer is
pipetted into an Eppendorf tube. The yeast expression plasmid
containing the receptor/no receptor control has a LEU marker. Yeast
cells are inoculated into this mixture and the reaction proceeds at
30.degree. C. for 60 min. The yeast cells are then heat-shocked at
42.degree. C. for 15 min. The cells are then washed and spread on
selection plates. The selection plates are synthetic defined yeast
media minus LEU, URA and TRP (SD-LUT). After incubating at
30.degree. C. for 2-3 days, colonies that grow on the selection
plates are then tested in the LacZ assay.
[0811] In order to perform fluorimetric enzyme assays for
.beta.-galactosidase, yeast cells carrying the subject GPR119
receptor are grown overnight in liquid SD-LUT medium to an
unsaturated concentration (i.e. the cells are still dividing and
have not yet reached stationary phase). They are diluted in fresh
medium to an optimal assay concentration and 90 .mu.l of yeast
cells are added to 96-well black polystyrene plates (Costar). Test
compounds, dissolved in DMSO and diluted in a 10% DMSO solution to
10.times. concentration, are added to the plates and the plates
placed at 30.degree. C. for 4h. After 4 h, the substrate for the
.beta.-galactosidase is added to each well. In these experiments,
Fluorescein di(.beta.-D-galactopyranoside) is used (FDG), a
substrate for the enzyme that releases fluorescein, allowing a
fluorimetric read-out. 20 .mu.l per well of 500 .mu.M FDG/2.5%
Triton X100 is added (the detergent is necessary to render the
cells permeable). After incubation of the cells with the substrate
for 60 min, 20 .mu.l per well of 1M sodium carbonate is added to
terminate the reaction and enhance the fluorescent signal. The
plates are then read in a fluorimeter at 485/535 nm.
[0812] An increase in fluorescent signal in GPR119-transformed
yeast cells over that in yeast cells transformed with empty vector
is indicative of a test compound being a compound that stimulates
GPR119 receptor functionality (e.g., a compound that is an agonist
or partial agonist of GPR119). In certain embodiments, compounds of
the invention give an increase in fluorescent signal above that of
the background signal (the signal obtained in the presence of
vehicle alone).
Example 14
Radiolabeled Compound
[0813] In certain embodiments, a compound known to be a ligand of a
G protein-coupled receptor of the invention is radiolabeled. A
radiolabeled compound as described herein can be used in a
screening assay to identify/evaluate compounds. In general terms, a
newly synthesized or identified compound (i.e., test compound) can
be evaluated for its ability to reduce binding of the radiolabeled
known ligand to the receptor, by its ability to reduce formation of
the complex between the radiolabeled known ligand and the receptor.
Suitable radionuclides that may be incorporated in compounds of the
present invention include but are not limited to .sup.3H (also
written as T), .sup.11C, .sup.14C, .sup.18F, .sup.125I, .sup.82Br,
.sup.123I, .sup.124I, .sup.125, .sup.131I, .sup.75Br, .sup.76Br,
.sup.15O, .sup.13N, .sup.35S or .sup.82Br. Compounds that
incorporate .sup.3H, .sup.14C, .sup.125I, .sup.131I, .sup.35S or
.sup.82Br will generally be most useful.
[0814] It is understood that a "radiolabelled" compound" is a
compound that has incorporated at least one radionuclide. In some
embodiments, the radionuclide is selected from the group consisting
of .sup.3H, .sup.14C, .sup.125, .sup.35S and .sup.82Br. In some
embodiments, the radionuclide .sup.3H or .sup.14C. Moreover, it
should be understood that all of the atoms represented in the
compounds known to be ligands of a G protein-coupled receptor of
the invention can be either the most commonly occurring isotope of
such atoms or the more scarce radioisotope or nonradioactive
isotope.
[0815] Synthetic methods for incorporating radioisotopes into
organic compounds including those applicable to those compounds
known to be ligands of a G protein-coupled receptor of the
invention are well known in the art and include incorporating
activity levels of tritium into target molecules include: A.
Catalytic Reduction with Tritium Gas--This procedure normally
yields high specific activity products and requires halogenated or
unsaturated precursors. B. Reduction with Sodium Borohydride
[.sup.3H]--This procedure is rather inexpensive and requires
precursors containing reducible functional groups such as
aldehydes, ketones, lactones, esters, and the like. C. Reduction
with Lithium Aluminum Hydride [.sup.3H ]--This procedure offers
products at almost theoretical specific activities. It also
requires precursors containing reducible functional groups such as
aldehydes, ketones, lactones, esters, and the like. D. Tritium Gas
Exposure Labeling--This procedure involves exposing precursors
containing exchangeable protons to tritium gas in the presence of a
suitable catalyst. E. N-Methylation using Methyl Iodide
[.sup.3H]--This procedure is usually employed to prepare O-methyl
or N-methyl (.sup.3H) products by treating appropriate precursors
with high specific activity methyl iodide (.sup.3H). This method in
general allows for high specific activity, such as about 80-87
Ci/mmol
[0816] Synthetic methods for incorporating activity levels of
.sup.1251 into target molecules include: A. Sandmeyer and like
reactions--This procedure transforms an aryl or heteroaryl amine
into a diazonium salt, such as a tetrafluoroborate salt, and
subsequently to .sup.125I labelled compound using Na.sup.125I. A
represented procedure was reported by Zhu, D.-G. and co-workers in
J. Org. Chem. 2002, 67, 943-948. B. Ortho .sup.125Iodination of
phenols--This procedure allows for the incorporation of .sup.125I
at the ortho position of a phenol as reported by Collier, T. L. and
co-workers in J. Labelled Compd Radiopharm. 1999, 42, S264-S266. C.
Aryl and heteroaryl bromide exchange with .sup.125I--This method is
generally a two step process. The first step is the conversion of
the aryl or heteroaryl bromide to the corresponding tri-alkyltin
intermediate using for example, a Pd catalyzed reaction (i.e.
Pd(Ph.sub.3P).sub.4] or through an aryl or heteroaryl lithium, in
the presence of a tri-alkyltinhalide or hexaalkylditin [e.g.,
(CH.sub.3).sub.3SnSn(CH.sub.3).sub.3). A represented procedure was
reported by Bas, M.-D. and co-workers in J. Labelled Compd
Radiopharm. 2001, 44, S280-S282.
[0817] The foregoing techniques are intended to be illustrative and
not limiting. Other techniques for radiolabeling a compound known
to be a ligand of a G protein-coupled receptor of the invention are
well known to the skilled artisan.
Example 15
Receptor Binding Assay
[0818] A test compound can be evaluated for its ability to reduce
formation of the complex between a compound known to be a ligand of
a G protein-coupled receptor of the invention and the receptor. In
certain embodiments, the known ligand is radiolabeled. The
radiolabeled known ligand can be used in a screening assay to
identify/evaluate compounds. In general terms, a newly synthesized
or identified compound (i.e., test compound) can be evaluated for
its ability to reduce binding of the radiolabeled known ligand to
the receptor, by its ability to reduce formation of the complex
between the radiolabeled known ligand and the receptor.
[0819] A level of specific binding of the radiolabled known ligand
in the presence of the test compound less than a level of specific
binding of the radiolabeled known ligand in the absence of the test
compound is indicative of less of the complex between said
radiolabeled known ligand and said receptor being formed in the
presence of the test compound than in the absence of the test
compound.
[0820] Assay Protocol for Detecting the Complex Between a Compound
Known to be a Ligand of a G Protein-Coupled Receptor of the
Invention and the Receptor
[0821] A. Preparation of the Receptor
[0822] 293 cells are transiently transfected with 10 ug expression
vector comprising a polynucleotide encoding a G protein-coupled
receptor of the invention using 60 ul Lipofectamine (per 15-cm
dish). The transiently transfected cells are grown in the dish for
24 hours (75% confluency) with a media change and removed with 10
ml/dish of Hepes-EDTA buffer (20 mM Hepes+10 mM EDTA, pH 7.4). The
cells are then centrifuged in a Beckman Coulter centrifuge for 20
minutes, 17,000 rpm (JA-25.50 rotor). Subsequently, the pellet is
resuspended in 20 mM Hepes+1 mM EDTA, pH 7.4 and homogenized with a
50-ml Dounce homogenizer and again centrifuged. After removing the
supernatant, the pellets are stored at -80.degree. C., until used
in binding assay. When used in the assay, membranes are thawed on
ice for 20 minutes and then 10 mL of incubation buffer (20 mM
Hepes, 1 mM MgCl.sub.2, 100 mM NaCl, pH 7.4) added. The membranes
are then vortexed to resuspend the crude membrane pellet and
homogenized with a Brinkmann PT-3100 Polytron homogenizer for 15
seconds at setting 6. The concentration of membrane protein is
determined using the BRL Bradford protein assay.
[0823] B. Binding Assay
[0824] For total binding, a total volume of 50 ul of appropriately
diluted membranes (diluted in assay buffer containing 50 mM Tris
HCl (pH 7.4), 10 mM MgCl.sub.2, and 1 mM EDTA; 5-50 ug protein) is
added to 96-well polyproylene microtiter plates followed by
addition of 100 ul of assay buffer and 50 ul of a radiolabeled
known ligand. For nonspecific binding, 50 ul of assay buffer is
added instead of 100 ul and an additional 50 ul of 10 uM said known
ligand which is not radiolabeled is added before 50 ul of said
radiolabeled known ligand is added. Plates are then incubated at
room temperature for 60-120 minutes. The binding reaction is
terminated by filtering assay plates through a Microplate Devices
GF/C Unifilter filtration plate with a Brandell 96-well plate
harvestor followed by washing with cold 50 mM Tris HCl, pH 7.4
containing 0.9% NaCl. Then, the bottom of the filtration plate are
sealed, 50u1 of Optiphase Supermix is added to each well, the top
of the plates are sealed, and plates are counted in a Trilux
MicroBeta scintillation counter. For determining whether less of
the complex between said radiolabeled known ligand and said
receptor is formed in the presence of a test compound, instead of
adding 100 ul of assay buffer, 100 ul of appropriately diluted said
test compound is added to appropriate wells followed by addition of
50 ul of said radiolabled known ligand.
[0825] C. Calculations
[0826] The test compounds are initially assayed at 10, 1 and 0.1
.mu.M and then at a range of concentrations chosen such that the
middle dose would cause about 50% inhibition of binding of the
radiolabeled known ligand (i.e., IC.sub.50). Specific binding in
the absence of test compound (B.sub.O) is the difference of total
binding (B.sub.T) minus non-specific binding (NSB) and similarly
specific binding (in the presence of test compound) (B) is the
difference of displacement binding (B.sub.D) minus non-specific
binding (NSB). IC.sub.50 is determined from an inhibition response
curve, logit-log plot of % B/B.sub.O vs concentration of test
compound.
[0827] K.sub.i is calculated by the Cheng and Prustoff
transformation:
K.sub.i=IC.sub.50/(1+[L]/K.sub.D)
[0828] where [L] is the concentration of radiolabled known ligand
used in the assay and K.sub.D is the dissociation constant of the
radiolabeled known ligand determined independently under the same
binding conditions.
Example 16
[0829] Effect of GPR119 Agonist on PYY Secretion in Enteroendocrine
Cell or in Cells in Tissue Derived from an L Cell-Containing Region
of Iintestine or in Cells Capable of Secreting PYY
[0830] A GPR119 agonist in accordance with the present invention
can be shown to stimulate PYY secretion in an enteroendocrine cell,
e.g. an enteroendocrine cell line, or in cells such as
enteroendocrine cells in tissue derived from an L cell-containing
region of intestine [e.g., duodenum, jejunum, ileum, colon or
rectum tissue; see, e.g., Lundberg et al, PNAS USA (1982)
79:4471-4475; Adrian et al, Gastroenterol (1985) 89:1070-1077;
Ekblad et al, Peptides (2002) 23:251-261; Ueno et al, Regul Pept
(2008) 145:12-16)] or in cells capable of secreting PYY using the
in vitro assay described here. Enteroendocrine cells or cells such
as enteroendocrine cells in tissue derived from an L
cell-containing region of intestine are plated in 24-well plates on
day one in complete culture medium (DMEM/10% FBS). On day two the
culture medium is replaced with a low glucose medium (DMEM/3 mM
Glucose/10% FBS). On day three cells are washed twice with 1.times.
PBS. The washed cells are stimulated with vehicle or with GPR119
agonist at various concentrations (e.g., in the range of 1 nM to 20
uM) or with forskolin (1 uM) as a positive control in serum free
DMEM with 15 mM glucose for one hour at 37.degree. C. and 5%
CO.sub.2 in a tissue culture incubator. The supernatants are then
collected and clarified by centrifugation at 500 g and 4.degree. C.
for 5 minutes. Total PYY released into the supernatant is
determined using a PYY EIA kit purchased from Phoenix
Pharmaceuticals, Inc. (Peptide YY (PYY) (Rat, Mouse, Porcine,
Canine) EIA kit, Catalog No. EK-059-03) according to instructions
provided in the kit.
[0831] It is expressly contemplated that cells capable of secreting
PYY include but are not limited to an enteroendocrine cell, as may
be an enteroendorine cell line, a cell such as may be an
enteroendocrine cell in tissue derived from an L cell-containing
region of intestine, an alpha cell of the islets of Langerhans, a
cell line derived from the islets of Langerhans such as may be
RINm5F (Bargsten, Arch Histol Cytol (2004) 67:79-94), a cell such
as a neuronal cell as may be in tissue from the medulla oblongata,
a neuronal cell line, and a recombinant cell.
Example 17
[0832] In vivo Effects of a GPR119 Agonist on Atherogenesis in
Mice
[0833] Male apoE.sup.-/- mice (Jackson Laboratory, Bar Harbor, Me.)
are fed a normal chow. The apolipoprotein E-deficient
(apoE.sup.-/-) mouse is an established animal model of
atherosclerosis, developing extensive atherosclerotic lesions on a
chow diet [Zhang et al, Science (1992) 258:468-471]. At the age of
12 weeks, a GPR119 agonist or vehicle alone is administered per
daily oral administration. A preferred dose of the GPR119 agonist
is 0.1-100 mg/kg. Other preferred dose is selected from the group
consisting of 0.1 mg/kg, 0.3 mg/kg, 1.0 mg/kg, 3.0 mg/kg, 10 mg/kg,
30 mg/kg and 100 mg/kg.
[0834] The mice are anesthetized with an intraperitoneal injection
of pentobarbital (50 mg/kg), and the hearts, which contain the
aoritic sinus and aortic arch, are harvested 14 days after the
administration of the GPR119 agonist. Hearts are also harvested
from unmanipulated mice at the start of the experiment. Ten mice
are used for each of the three experimental groups.
[0835] The frozen cross-sections (10 .mu.M thick) of aortic sinus
embedded in Optimal Cutting Temperature (OCT; Sakura Finetechnical
Co., Ltd) compound after overnight fixation in 10% formalin are
mounted on slides. For the analysis of plaque size, 3 sections (100
.mu.M apart) from each mouse were stained with Oil Red O. The
lesion size and the diameter of lipid droplets in the lesions are
quantified with an image analyzing computer software, and the mean
values are determined. The mean value for the group administered
with the GPR119 agonist is compared with the mean value for the
group administered with vehicle alone.
[0836] Data are presented as means.+-.SEM and are analyzed by
Student's t test or the Mann-Whitney U test, depending on their
distribution pattern. A value of P<0.05 is considered
statistically significant. See, e.g., Okamoto et al, Circulation
(2002) 106:2767-2770.
[0837] These results can demonstrate that the GPR119 agonist is an
inhibitor of atherogenesis.
[0838] It is expressly contemplated that in other embodiment a
GPR119 agonist can be shown to be an inhibitor of atherogenesis in
the ApoE.sup.-/- mice using a non-invasive in vivo technique [see,
e.g., Fayad et al, Circulation (1998) 98:1541-1547]. It is
expressly contemplated that in other embodiment, administration of
the agonist is other than oral, for example that administration of
the agonist is intraperitoneal or intravenous. It is expressly
contemplated that in other embodiment, treatment begins other than
at 12 weeks of age, either earlier or later than at 12 weeks of
age. It is expressly contemplated that in other embodiment
treatment continues for less than or more than 14 days. It is
expressly contemplated that administration may be other than
daily.
Example 18
[0839] In vivo Effects of a GPR119 Agonist on Chronic Inflammatory
Arthritis in Mice
[0840] Both male and female MRL-lpr mice (Jackson Laboratory, Bar
Harbor, Me.) are used at 13-14 weeks of age. MRL-lpr mice
spontaneously develop a chronic inflammatory arthritis with similar
characteristics to human rheumatoid arthritis including cell
infiltration, pannus formation, bone and cartilage breakdown, and
the presence of serum rheumatoid factor. The disease normally
develops towards the end of the animal's life span; however,
injection with complete Freund's adjuvant (CFA) initiates early
onset and increases the severity of arthritis [Rakay et al, J
Immunol (1993) 151:5081-5087].
[0841] On Day 0 of each experiment, all groups of mice are injected
with CFA intradermally into a thoracic and an inguinal site with
0.05 ml CFA supplemented to 10 mg/ml with heat inactivated
Mycobacterium tuberculosis H37 Ra (Difco, Detroit, Mich.).
Immediately, a GPR119 agonist or vehicle alone is administered per
daily oral administration. A preferred dose of the GPR119 agonist
is 0.1-100 mg/kg. Other preferred dose is selected from the group
consisting of 0.1 mg/kg, 0.3 mg/kg, 1.0 mg/kg, 3.0 mg/kg, 10 mg/kg,
30 mg/kg and 100 mg/kg.
[0842] Treatment is continued for 30 days. For quantifying
swelling, ankle widths are measured with a micrometer. The
statistical comparison of paired sets of ankle width measurements
is carried out using the Student's t test.
[0843] Histopathological Analysis
[0844] At day 30 after CFA priming, the hind paws are fixed in
buffered formalin. After decalcification in 10% formic acid for 48
hours, the tissues are processed for paraffin embedding. Serial
sections of the tarso-metatarsal joints are cut to a thickness of 5
mm and stained with hematoxylin and eosin. Sections are examined by
an individual without knowledge of the experimental protocol. A
minimum of 10 sections/joint are assessed and scored to provide a
semiquantitative measure of subsynovial inflammation (0, normal; 1
focal inflammatory infiltrates; 2, inflammatory infiltrate that
dominates the cellular histology), synovial hyperplasia (0, normal;
1, a continuous, minimum three-layer thick, synovial lining seen in
one joint; 2, minimum three-layer thick, synovial lining detetected
in several joints), pannus formation and cartilage erosion (0,
normal; 1, pannus partially covered cartilage surfaces without
evident cartilage loss; 2, pannus connected to evident cartilage
loss), bone destruction (0, normal; 1, detectable destruction of
bone by the pannus or osteoclast activity; 2, the pannus or
osteoclast activity has destroyed a significant part of the bone),
and finally, overall pathology is the overall assessment derived by
the summation of the values for these criteria [see, e.g., Gong et
al, J Exp Med (1997) 186:131-137]. Statistical analysis of the
histopathology indices is done using the Student's t test.
[0845] These results can demonstrate that the GPR119 agonist is an
inhibitor of a chronic inflammatory arthritis, for example that the
GPR119 agonist is an inhibitor of rheumatoid arthritis.
[0846] It is expressly contemplated that in other embodiment,
administration of the agonist is other than oral, for example that
administration of the agonist is intraperitoneal or intravenous. It
is expressly contemplated that in other embodiment, treatment
begins other than at 13-14 weeks of age, either earlier or later
than at 13-14 weeks of age. It is expressly contemplated that in
other embodiment treatment continues for less than or more than 30
days. It is expressly contemplated that administration may be other
than daily.
Example 19
Reduction of Colon Injury of Animal Model for Inflammatory Bowel
Disease
[0847] A GPR119 agonist can be shown to confer protection from
inflammatory bowel disease using the animal model essentially as
described in WO 03/105763.
[0848] Methods
[0849] Animal Preparation:
[0850] 10-11 weeks old male HSD rats, ranging 250-300 grams, are
housed in a 12:12 light:dark cycle, and allowed ad libitum access
to a standard rodent diet (Taklad L M 485, Madison, Wis.) and
water. The animals are fasted for 24 hours before the
experiment.
[0851] Procedure:
[0852] A simple and reproducible rat model of chronic colonic
inflammation has been previously described by Morris et al
(Gastroenterology (1989) 96:795-803). It exhibits a relatively long
duration of inflammation and ulceration, affording an opportunity
to study the pathophysiology of colonic inflammatory disease in a
specifically controlled fasion, and to evaluate new treatments
potentially applicable to inflammatory bowel disease in humans.
[0853] Rats are anesthetized with 3% isofluorane and placed on a
regulated heating pad set at 37.degree. C. A gavage needle is
inserted rectally into the colon 7 cm. The hapten
trinitrobenzenesulfonic acid (TNBS) dissolved in 50% ethanol (v/v)
is delivered into the lumen of the colon through the gavage needle
at a dose of 30 mg/kg, in a total volume of 0.4-0.6 mL. Control
groups receive saline solution (NaCl 0.9%) intracolonically.
[0854] Four days after induction of colitis, the colon is resected
from anesthetized rats, which are then euthanized by decapitation.
Weights of excised colon and spleen are measured, and the colon
photographed for scoring of gross morphologic damage according to
the a rating scale (0=no damage, 1=localized hyperemia but no
ulcers, 2=linear ulcers with no significant inflammation, 3=linear
ulcer with inflammation at one site, 4=two or more sites of
ulceration and/or inflammation, 5=two or more major sites of
inflammation and ulceration or one major site of inflammation and
ulceration extending >1 cm along the length of the colon) before
division into 3 parts. The segment of colon 1 cm from the cecum is
fixed in 10% formalin. Other segments are immediately frozen on dry
ice.
[0855] Inflammation is defined as regions of hyperemia and bowel
wall thickening. Colons are observed independently by several
observers blinded to experimental treatment.
[0856] For this assay, 5 treatment groups are set up as
follows:
[0857] Group A (n=3). intracolonic saline with oral treatment of
saline;
[0858] Group B (n=8). oral treatment of saline prior to induction
of colitis by 30 mg/kg TNBS;
[0859] Group C (n=10). Experimental group--oral treatment of GPR119
agonist (at a dose of, e.g., 30 mg/kg) twice daily for 2 days
before, 2 days after, and 30 min before induction of colitis by 30
mg/kg TNBS.
[0860] Group D (n=9). Positive control--intramuscular injection of
Depo-Medrol 600 .mu.g/kg 60 min prior to induction of colitis by 30
mg/kg TNBS;
[0861] Group E (n=10). Positive control, 5-amino salicylic acid
(Sigma) suspension at 200 mg/kg intracolonic administration in 0.5
mL saline, 60 min before induction of colitis and daily
thereafter.
[0862] A preferred dose of the GPR119 agonist is 0.1-100 mg/kg.
Other preferred dose is selected from the group consisting of 0.1
mg/kg, 0.3 mg/kg, 1.0 mg/kg, 3.0 mg/kg, 10 mg/kg, 30 mg/kg and 100
mg/kg. It is expressly contemplated that in other embodiment,
administration of the agonist is other than oral, for example that
administration of the agonist is subcutaneous or intravenous.
Example 20
Effect of GPR119 Agonist on Kainate Seizures in Mice
[0863] A GPR119 agonist can be shown to confer protection from
kainate seizures in mice using the animal model essentially as
described in Woldbye et al (Neurobiology of Disease (2005)
20:760-772).
[0864] Male NMRI mice (Taconic M&B, DK; 23-30 g) are
administered per orally with GPR119 agonist at doses of, e.g., 0.2,
1, 10, 20 or 30 mg/kg (n=7-8) in vehicle containing 0.05% bovine
serum albumin in 10 mM phosphate-buffered saline (PBS, 0.13 M NaCl,
7 mM Na.sub.2HPO.sub.4, 3 mM NaH.sub.2PO.sub.4). A control group
receives only vehicle by oral administration (n =28). Five minutes
later, all animals receive a subcutaneous injection of kainate
dissolved in isotonic saline and adjusted to pH 7.4 (30 mg/kg;
#2020, Ocean Produce International, Canada); seizure severity is
scored according to a modified rating scale of Marsh et al (PNAS
USA (1999) 96:13518-13523): 0 =no seizure activity, 1=staring or
facial movements, 2=head nodding or isolated twitches, 3=motor
seizure with forelimb colonus, 4=motor seizure with rearing,
5=motor seizure with loss of posture or status epilepticus (at
least 10 min of continuous motor seizure activity), 6=death. The
seizure score and latencies to first motor seizure (when scored as
at least 3) and motor seizure with loss of posture are determined
for each treatment group by an observed blinded to the identity of
the mice.
[0865] A preferred dose of the GPR119 agonist is 0.1-100 mg/kg.
Other preferred dose is selected from the group consisting of 0.1
mg/kg, 0.3 mg/kg, 1.0 mg/kg, 3.0 mg/kg, 10 mg/kg, 30 mg/kg and 100
mg/kg.
[0866] It is expressly contemplated that in other embodiment,
administration of the agonist is other than oral, for example that
administration of the agonist is intraperitoneal or intravenous. It
is expressly contemplated that the subcutaneous injection of
kainate may alternatively be carried out at a time after
administration of the GPR119 agonist which is greater than 5
minutes.
Example 21
Effect of GPR119 Agonist on Food Intake in Lean Mice
[0867] A GPR119 agonist can be shown to decrease food intake using
lean fasted-refed mice essentially as described in Ortiz et al
(JPET (2007) 323:692-700).
[0868] Lean C57BL/6 mice (Taconic Farms, Germantown, N.Y.) are
acclimated for a minimum of a week with controlled temperature and
humidity on a 12-h light/dark cycle. Mice are housed in pairs in
cages with a grid floor with food and water (standard chow pellet
diet) continuously available. A study includes 20 mice per
treatment group. The mice are fasted overnight (18 h) with water
available during the dark phase, and they are dosed orally with
GPR119 agonist (at a dose of, e.g., 30 mg/kg) in USP saline or only
vehicle. Preweighed food is provided 30 min after dosing, and
cumulative food intake is measured over 72 h (e.g., at 1 h, at 2h,
at 4 h, at 24 h, at 48 h, and at 72 h).
[0869] A preferred dose of the GPR119 agonist is 0.1-100 mg/kg.
Other preferred dose is selected from the group consisting of 0.1
mg/kg, 0.3 mg/kg, 1.0 mg/kg, 3.0 mg/kg, 10 mg/kg, 30 mg/kg and 100
mg/kg.
[0870] It is expressly contemplated that in other embodiment,
administration of the agonist is other than oral, for example that
administration of the agonist is intraperitoneal or
intravenous.
Example 22
Effect of GPR119 Agonist on Body Weight and Plasma Adiponectin in
Diet-Induced Obese (DIO) Mice
[0871] A GPR119 agonist can be shown to provide a reduction in body
weight and an increase in plasma adiponectin using the diet-induced
obese (DIO) mouse essentially as described in Ortiz et al (JPET
(2007) 323:692-700).
[0872] Male C57BL/6 mice (Taconic Farms) are fed a high-fat diet
containing 45% calories from fat (rodent diet D12451; Research
Diets, Inc., New Brunswick, N.J.) for 20 to 22 weeks, and they have
an average body weight over about 5 SDs greater than mice fed a
standard chow pellet diet (5% calories from fat). A study includes
10 mice per treatment group. Mice are kept in standard animal rooms
under controlled temperature and humidity and a 12/12-h light dark
cycle. Water and food are continuously available Animals are
adapted to the grid floors for 4 days, and they ar dosed with
vehicle (USP saline) for a further 4 days before recording of 2
days of baseline body weight and 24-h food and water consumption.
Mice are assigned to treatment groups based upon their body weight
such that the intial mean and standard error of the meand of body
weight is similar Animals are administered GPR119 agonist (at a
dose of, e.g., 30 mg/kg) in USP saline or only vehicle orally every
day before the dark phase, and body weight and food and water
consumption are measured. Animals are dosed daily for 40 days.
[0873] Blood is collected on day 40 by retro-orbital bleeding for
adiponectin analysis; plasma adiponectin is measured using a Mouse
Adiponectin ELISA kit (Catalog No. EZMADP-60K; Millipore Corp.,
Billerica, Mass.) according to manufacturer's instructions.
[0874] A preferred dose of the GPR119 agonist is 0.1-100 mg/kg.
Other preferred dose is selected from the group consisting of 0.1
mg/kg, 0.3 mg/kg, 1.0 mg/kg, 3.0 mg/kg, 10 mg/kg, 30 mg/kg and 100
mg/kg.
[0875] It is expressly contemplated that in other embodiment,
administration of the agonist is other than oral, for example that
administration of the agonist is subcutaneous or intravenous.
Example 23
Effect of GPR119 Agonist on Plasma Glucose, Plasma Indulin, and
Glucose Disposal in Diet-Induced Obese (DIO) Mice
[0876] A GPR119 agonist can be shown to provide favorable
improvements in metabolic parameters of diet-induced obese (DIO)
mice associated with risk for type 2 diabetes, such as glucose
disposal (to increase glucose disposal in fasted mice, e.g. glucose
disposal following an OGTT), plasma glucose level (to decrease
blood glucose in fed mice), and plasma insulin level (to increase
plasma insulin in fasted mice), essentially as described in Ortiz
et al (JPET (2007) 323:692-700).
[0877] Male C57BL/6 mice (Taconic Farms) are fed a high-fat diet
containing 45% calories from fat (rodent diet D12451; Research
Diets, Inc., New Brunswick, N.J.) for 20 to 22 weeks, and they have
an average body weight over 5 SDs greater than mice fed a standard
chow pellet diet (5% calories from fat). A study includes 10 mice
per treatment group. Mice are kept in standard animal rooms under
controlled temperature and humidity and a 12/12-h light dark cycle.
Water and food are continuously available. Animals are adapted to
the grid floors for 4 days, and they ar dosed with vehicle (USP
saline) for a further 4 days before recording of 2 days of baseline
body weight and 24-h food and water consumption. Mice are assigned
to treatment groups based upon their body weight such that the
intial mean and standard error of the mean of body weight is
similar.
[0878] Animals are orally administered GPR119 agonist (at a dose
of, e.g., 30 mg/kg) in USP saline or only vehicle every day before
the dark phase. Animals are dosed daily for 11 days. Blood samples
are collected from the tail of fed mice and analyzed with an
Ascensia glucose meter (Bayer Healthcare, Tarrytown, N.Y.) on day
10 of treatment. Food is removed 5 h into the feeding phase, and
the animals are fasted for 14 h before blood collection using
retro-orbital bleeding for insulin analysis. Oral glucose tolerance
test (OGTT) is performed 2 h after retro-orbital bleeding following
oral administration of 2 g/kg glucose, and tail blood glucose is
measured at 30-min intervals for 3 h. Insulin is measured by
enzyme-linked immunosorbent assay (EIA) (Alpco Diagnostics,
Windham, N.H.).
[0879] A preferred dose of the GPR119 agonist is 0.1-100 mg/kg.
Other preferred dose is selected from the group consisting of 0.1
mg/kg, 0.3 mg/kg, 1.0 mg/kg, 3.0 mg/kg, 10 mg/kg, 30 mg/kg and 100
mg/kg.
[0880] It is expressly contemplated that in other embodiment,
administration of the agonist is other than oral, for example that
administration of the agonist is subcutaneous or intravenous. It is
expressly contemplated in other embodiment, that intraperitoneal
glucose tolerance test (IPGTT) is performed 2 h after retro-orbital
bleeding following intraperitoneal administration of 2 g/kg
glucose.
Example 24
Effect of GPR119 Agonist on Collateral-Dependent Blood Flow in a
Rat Model of Peripheral Arterial Insufficiency
[0881] A GPR119 agonist can be shown to increase
collateral-dependent blood flow using a rat model of acute onset
peripheral arterial insufficiency that is characteristic of the
large vessel occlusive disease that often presents with symptoms of
intermittent claudication, essentially as described in Cruze et al
(Peptides (2007) 28:269-280).
[0882] Peripheral arterial insufficiency is established in adult
male Sprague Dawley rats (325-350 g; Taconic Farms, Inc.) with
bilateral occlusion of the femoral arteries distal to the inguinal
ligament. In the same surgical procedure, a catheter connected to a
14 days osmotic pump (Alzet.RTM.) is inserted into the iliac artery
for infusion near the site of collateral expansion for systemic
delivery of vascular growth factor-A (residues 1-165)
(VEGF.sub.165) at a dose of 15 .mu.g/kg/day; GPR119 agonist (at a
dose of, e.g., 30 mg/kg) and vehicle alone (phosphate buffered
saline, 10% sodium citrate, 1.6% glycerol, 0.008% Tween 20) are
administered per daily oral administration. All agents are supplied
and administered in a manner blinded to the investigators. Blood
flow is measured on day 16 to eliminate any acute effects of GPR119
agonist or VEGF.sub.165 on blood flow. Muscle blood flow is
determined in a blinded manner, utilizing radiolabeled microspheres
during treadmill running, as described in Yang et al (Am J Physiol
Heart Circ Phys (2000) 278:H1966-H1973). The dose of VEGF.sub.165
used (15 .mu.g/kg/day) gelds a near-maximal increase in
collateral-dependent blood flow in this model (Cruze et al,
Peptides (2007) 28:269-280).
[0883] A preferred dose of the GPR119 agonist is 0.1-100 mg/kg.
Other preferred dose is selected from the group consisting of 0.1
mg/kg, 0.3 mg/kg, 1.0 mg/kg, 3.0 mg/kg, 10 mg/kg, 30 mg/kg and 100
mg/kg.
[0884] It is expressly contemplated that in other embodiment,
administration of the agonist is other than oral, for example that
administration of the agonist is by intravenous infusion at a dose
of, e.g., 30 mg/kg/day. A preferred infusion dose of the GPR119
agonist is 0.1-100 mg/kg/day. Other preferred infusion dose is
selected from the group consisting of 0.1 mg/kg/day, 0.3 mg/kg/day,
1.0 mg/kg/day, 3.0 mg/kg/day, 10 mg/kg/day, 30 mg/kg/day and 100
mg/kg/day.
Example 25
[0885] Effect of GPR119 Agonist on Vasoactive Intestinal Peptide
(VIP)-Induced Jejunal Secretions in in vivo Rat Model
[0886] A GPR119 agonist can be shown to decrease secretion in
intestinal epithelium using an in vivo rat model of vasoactive
intestinal peptide (VIP)-induced jejunal secretions, essentially as
described by Balasubramaniam et al (J Med Chem (2000) 43:3420-3427)
and Souli et al (Peptides (1997) 18:551-557). Balasubramaniam et al
(J Med Chem (2000) 43:3420-3427) also describe analogues of PYY
having activity in this assay.
[0887] Male Wistar rats (Charles River Laboratories, Inc.,
Wilmington, Mass.) are anesthetized, and a saphenous vein is
cannulated to infuse drugs. The abdomen is opened, a a jejunal loop
(.about.20 cm long) is delimited by two ligatures. At time zero, 2
mL of 0.9% saline prewarmed to 37.degree. C. is instilled into the
jejunal loop. The loop is then returned into the abdomen and
closed. VIP (30 .mu.g/kg/hour) is infused through the saphenous
vein at the rate of 2.5 mL/hour for 30 minutes starting at time
zero. GPR119 agonist (at a dose of, e.g., 30 mg/kg) or saline
(controls) are orally administered as bolus doses 15 min before
starting VIP infusions. At the end of the experiment rats are
sacrificed, and the jejunal loop excised. The loop is then measured
and weighed before and after removing the instilled fluid. The net
water flux expressed as .mu.L/cm/30 min is calculated using the
formula (F-E-2000)/L, where F and E are the weights (mg) of the
loop before and after emptying the remaining fluid, L is the length
of the loop in cm, and 2000 is the volume in .mu.L of the fluid
instilled initially. Net absorption is indicated by a negative
value and net secretion by a positive value. Each dose is
investigated in 6-8 animals.
[0888] A preferred dose of the GPR119 agonist is 0.1-100 mg/kg.
Other preferred dose is selected from the group consisting of 0.1
mg/kg, 0.3 mg/kg, 1.0 mg/kg, 3.0 mg/kg, 10 mg/kg, 30 mg/kg and 100
mg/kg.
[0889] It is expressly contemplated that in other embodiment,
administration of the agonist is other than oral, for example that
administration of the agonist is subcutaneous or intravenous.
Example 26
[0890] Effect of GPR119 Agonist on Intestinal Absorption in in vivo
Dog Model
[0891] A GPR119 agonist can be shown to increase absorption in
intestinal epithelium using an in vivo dog model, essentially as
described by Balasubramaniam et al (J Med Chem (2000)
43:3420-3427). Balasubramaniam et al (J Med Chem (2000)
43:3420-3427) also describe analogues of PYY having activity in
this assay.
[0892] In vivo effects of GPR119 agonist on water and electrolyte
absorption by the jejunum, ileu, and colon under basal conditions
are investigated in awake dogs with jejunal, ileal and/or colonal
Thiry-Vella loops essentially as described in greater detail in
Bilchik et al (Gastroenterology (1993) 105:1441-1448) and Bilchik
et al (Am J Surg (1994) 167:570-574). Each experiment consists of a
90-min basal period followed by a 150-min experimental period. The
small bowel segment is perfused through the proximal cannula at 2
mL/min using a roller pump. The perfusate, pH 7.4 maintained at
37.degree. C., contained (in nmol/L) 140 Na.sup.+, 5.2 K.sup.-,
119.8Cl.sup.-, 25 HCO.sub.3, 1.2 Ca.sup.2+, 1.2 Mg.sup.2+, 2.4
HPO.sub.2.sup.4-, 0.4 H.sub.2PO.sub.4.sub.-, 10 glucose and 10
.mu.C [.sup.14C]poly(ethylene glycol) in 5 g/L poly(ethylene
glycol). After a 20-min washout period, perfusate is collected
every 15 min for determination of Na.sup.+, Cl.sup.- and water
contents.
[0893] GPR119 agonist is administered per orally at a dose of,
e.g., 30 mg/kg at time zero.
[0894] A preferred dose of the GPR119 agonist is 0.1-100 mg/kg.
Other preferred dose is selected from the group consisting of 0.1
mg/kg, 0.3 mg/kg, 1.0 mg/kg, 3.0 mg/kg, 10 mg/kg, 30 mg/kg and 100
mg/kg.
[0895] It is expressly contemplated that in other embodiment,
administration of the agonist is other than oral, for example that
administration of the agonist is by intraluminal infusion at a dose
of, e.g., 30 mg/kg/hour starting at time zero. A preferred infusion
dose of the GPR119 agonist is 0.1-100 mg/kg/hour. Other preferred
infusion dose is selected from the group consisting of 0.1
mg/kg/hour, 0.3 mg/kg/hour, 1.0 mg/kg/hour, 3.0 mg/kg/hour, 10
mg/kg/hour, 30 mg/kg/hour and 100 mg/kg/hour.
Example 27
Inhibition of Diarrhea of Castor Oil-Induced Defection and
Diarrheal Sympton in Mice
[0896] A GPR119 agonist can be shown to inhibit diarrheal symptom
using a castor oil-induced defecation and diarrheal symptom mouse
model essentially as described in EP 1902730.
[0897] C57BL/6 mice are used. Mice are individually housed in cages
with a floor mat in an animal room with a controlled environment
(temperature: 23.degree. C., relative humidity: 55%, light hours:
from 7:00 till 19:00) with ad libitum access to rodent chow (CE-2,
Nippon CLEA) and drinking water.
[0898] GPR119 agonist (at a dose of, e.g., 30 mg/kg), conventional
antidiarrheal agent loperamide (1 mg/kg), conventional
antidiarrheal agent atropine (10 mg/kg), or only vehicle are
administered per oral administration, intraperitoneal
administration, intraperitoneal administration, and oral
administration, respectively, to the mice, and at 15 minutes after
the administration, castor oil (0.3 ml/animal) is orally
administered to the mice. The fecal weight and diarrhea score
(normal: 0, soft feces: 1, watery feces: 2) is measured in a
time-dependent manner (1, 2, and 4 hours after administration). At
the same time, the same measurement is carried out for mice to
which only a vehicle is administered and castor oil is not
administered.
[0899] A preferred dose of the GPR119 agonist is 0.1-100 mg/kg.
Other preferred dose is selected from the group consisting of 0.1
mg/kg, 0.3 mg/kg, 1.0 mg/kg, 3.0 mg/kg, 10 mg/kg, 30 mg/kg and 100
mg/kg. It is expressly contemplated that in other embodiment,
administration of the agonist is other than oral, for example that
administration of the agonist is intraperitoneal or
intravenous.
Example 28
Oral Bioavailability
[0900] Physicochemico analytical approaches for directly assessing
oral bioavailability of a compound of the invention, such as a
GPR119 agonist, a PYY secretagogue, a compound useful for treatment
or prevention of a condition modulated by PYY, or a compound useful
for a modulation of a PYY-related activity, are well known to those
of ordinary skill in the art and may be used (see, e.g., without
limitation: Wong P C et al., Cardiovasc Drug Rev (2002) 20:137-52;
and Buchan P et al., Headache (2002) Suppl 2:S54-62; the disclosure
of each of which is hereby incorporated by reference in its
entirety). By way of further illustration and not limitation, said
alternative analytical approaches may comprise liquid
chromatography-tandem mass spectrometry (Chavez-Eng C M et al., J
ChromatogrB Analyt Technol Biomed Life Sci (2002) 767:117-29;
Jetter A et al., Clin Pharmacol Ther (2002) 71:21-9; Zimmerman J J
et al., J Clin Pharmacol (1999) 39:1155-61; and Banish A et al.,
Rapid Commun Mass Spectrom (1996) 10:1033-7; the disclosure of each
of which is hereby incorporated by reference in its entirety).
Recently, positron emission tomography (PET) has been successfully
used to obtain direct measurements of drug distribution, including
oral bioavailability, in the mammalian body following oral
administration of the drug (Gulyas et al., Eur J Nucl Med Mol
Imaging (2002) 29:1031-8; the disclosure of which is hereby
incorporated by reference in its entirety).
[0901] While the foregoing specification teaches the principles of
the present invention, with examples provided for the purpose of
illustration, it will be understood that the practice of the
invention encompasses all of the usual variations, adaptions, or
modifications, as come within the scope of the following claims and
its equivalents.
Sequence CWU 1
1
411008DNAHomo sapien 1atggaatcat ctttctcatt tggagtgatc cttgctgtcc
tggcctccct catcattgct 60actaacacac tagtggctgt ggctgtgctg ctgttgatcc
acaagaatga tggtgtcagt 120ctctgcttca ccttgaatct ggctgtggct
gacaccttga ttggtgtggc catctctggc 180ctactcacag accagctctc
cagcccttct cggcccacac agaagaccct gtgcagcctg 240cggatggcat
ttgtcacttc ctccgcagct gcctctgtcc tcacggtcat gctgatcacc
300tttgacaggt accttgccat caagcagccc ttccgctact tgaagatcat
gagtgggttc 360gtggccgggg cctgcattgc cgggctgtgg ttagtgtctt
acctcattgg cttcctccca 420ctcggaatcc ccatgttcca gcagactgcc
tacaaagggc agtgcagctt ctttgctgta 480tttcaccctc acttcgtgct
gaccctctcc tgcgttggct tcttcccagc catgctcctc 540tttgtcttct
tctactgcga catgctcaag attgcctcca tgcacagcca gcagattcga
600aagatggaac atgcaggagc catggctgga ggttatcgat ccccacggac
tcccagcgac 660ttcaaagctc tccgtactgt gtctgttctc attgggagct
ttgctctatc ctggaccccc 720ttccttatca ctggcattgt gcaggtggcc
tgccaggagt gtcacctcta cctagtgctg 780gaacggtacc tgtggctgct
cggcgtgggc aactccctgc tcaacccact catctatgcc 840tattggcaga
aggaggtgcg actgcagctc taccacatgg ccctaggagt gaagaaggtg
900ctcacctcat tcctcctctt tctctcggcc aggaattgtg gcccagagag
gcccagggaa 960agttcctgtc acatcgtcac tatctccagc tcagagtttg atggctaa
10082335PRTHomo sapien 2Met Glu Ser Ser Phe Ser Phe Gly Val Ile Leu
Ala Val Leu Ala Ser1 5 10 15Leu Ile Ile Ala Thr Asn Thr Leu Val Ala
Val Ala Val Leu Leu Leu 20 25 30Ile His Lys Asn Asp Gly Val Ser Leu
Cys Phe Thr Leu Asn Leu Ala 35 40 45Val Ala Asp Thr Leu Ile Gly Val
Ala Ile Ser Gly Leu Leu Thr Asp 50 55 60Gln Leu Ser Ser Pro Ser Arg
Pro Thr Gln Lys Thr Leu Cys Ser Leu65 70 75 80Arg Met Ala Phe Val
Thr Ser Ser Ala Ala Ala Ser Val Leu Thr Val 85 90 95Met Leu Ile Thr
Phe Asp Arg Tyr Leu Ala Ile Lys Gln Pro Phe Arg 100 105 110Tyr Leu
Lys Ile Met Ser Gly Phe Val Ala Gly Ala Cys Ile Ala Gly 115 120
125Leu Trp Leu Val Ser Tyr Leu Ile Gly Phe Leu Pro Leu Gly Ile Pro
130 135 140Met Phe Gln Gln Thr Ala Tyr Lys Gly Gln Cys Ser Phe Phe
Ala Val145 150 155 160Phe His Pro His Phe Val Leu Thr Leu Ser Cys
Val Gly Phe Phe Pro 165 170 175Ala Met Leu Leu Phe Val Phe Phe Tyr
Cys Asp Met Leu Lys Ile Ala 180 185 190Ser Met His Ser Gln Gln Ile
Arg Lys Met Glu His Ala Gly Ala Met 195 200 205Ala Gly Gly Tyr Arg
Ser Pro Arg Thr Pro Ser Asp Phe Lys Ala Leu 210 215 220Arg Thr Val
Ser Val Leu Ile Gly Ser Phe Ala Leu Ser Trp Thr Pro225 230 235
240Phe Leu Ile Thr Gly Ile Val Gln Val Ala Cys Gln Glu Cys His Leu
245 250 255Tyr Leu Val Leu Glu Arg Tyr Leu Trp Leu Leu Gly Val Gly
Asn Ser 260 265 270Leu Leu Asn Pro Leu Ile Tyr Ala Tyr Trp Gln Lys
Glu Val Arg Leu 275 280 285Gln Leu Tyr His Met Ala Leu Gly Val Lys
Lys Val Leu Thr Ser Phe 290 295 300Leu Leu Phe Leu Ser Ala Arg Asn
Cys Gly Pro Glu Arg Pro Arg Glu305 310 315 320Ser Ser Cys His Ile
Val Thr Ile Ser Ser Ser Glu Phe Asp Gly 325 330 335322DNAArtificial
SequencePrimer 3gtcctgccac ttcgagacat gg 22423DNAArtificial
SequencePrimer 4gaaacttctc tgcccttacc gtc 23
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