U.S. patent application number 16/947738 was filed with the patent office on 2021-05-13 for methods of treating neurological diseases and disorders.
The applicant listed for this patent is Madrigal Pharmaceuticals, Inc.. Invention is credited to James Barsoum, Rongzhen Lu, Yumiko Wada.
Application Number | 20210140945 16/947738 |
Document ID | / |
Family ID | 1000005355779 |
Filed Date | 2021-05-13 |
United States Patent
Application |
20210140945 |
Kind Code |
A1 |
Lu; Rongzhen ; et
al. |
May 13, 2021 |
METHODS OF TREATING NEUROLOGICAL DISEASES AND DISORDERS
Abstract
The present invention is directed to compositions and methods
for modulating c-Rel-dependent cytokine production without
materially altering the level of expression of NF.kappa.B and/or
the amount of I.kappa.B. The present invention is also directed to
screening for modulators of c-Rel activity as determined by
assaying for altered subcellular localization of c-Rel but where
the level of expression of NF.kappa.B and/or the amount of
I.kappa.B is materially unaltered.
Inventors: |
Lu; Rongzhen; (Lincoln,
MA) ; Barsoum; James; (Lexington, MA) ; Wada;
Yumiko; (Billerica, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Madrigal Pharmaceuticals, Inc. |
West Conshohocken |
PA |
US |
|
|
Family ID: |
1000005355779 |
Appl. No.: |
16/947738 |
Filed: |
August 14, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15872331 |
Jan 16, 2018 |
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16947738 |
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14542453 |
Nov 14, 2014 |
9910031 |
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15872331 |
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13367825 |
Feb 7, 2012 |
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14542453 |
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11972592 |
Jan 10, 2008 |
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13367825 |
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10986553 |
Nov 10, 2004 |
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11972592 |
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60519040 |
Nov 11, 2003 |
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60519048 |
Nov 10, 2003 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/6872 20130101;
G01N 2333/4703 20130101; G01N 33/5035 20130101; G01N 2333/54
20130101; G01N 2500/10 20130101 |
International
Class: |
G01N 33/50 20060101
G01N033/50; G01N 33/68 20060101 G01N033/68 |
Claims
1-16. (canceled)
17. A method of treating dementia in a subject in need thereof, the
method comprising administering to the subject a pharmaceutical
composition comprising an amount of Compound 2:
N-(3-methyl-benzylidene)-N'-[6-morpholin-4-yl-2-(2-pyridin-2-yl-ethoxy)-p-
yrimidin-4-yl]-hydrazine, or a pharmaceutically acceptable salt
thereof.
18. The method of claim 17, wherein the dementia is selected from
the group consisting of AIDS dementia complex, senile dementia of
Lewy body type, and dementia pugilistica.
19. The method of claim 18, wherein the pharmaceutical composition
is formulated for a route of administration selected from
intravenous, intranasal, or oral.
20. The method of claim 19, wherein the pharmaceutical composition
is formulated for intravenous administration.
21. The method of claim 20, wherein the amount of Compound 2 in the
composition is from 1 to 50 mg per kilogram of the subject's body
weight.
22. The method of claim 19, wherein the pharmaceutical composition
is formulated for intranasal administration.
23. The method of claim 22, wherein the amount of Compound 2 in the
composition is from 0.1 to 50 mg per kilogram of the subject's body
weight.
24. The method of claim 19, wherein the pharmaceutical composition
is formulated for oral administration.
25. The method of claim 24, wherein the pharmaceutical composition
is a tablet, caplet, hard gelatin capsule, soft gelatin capsules,
troche, dragee, dispersion, suspension, or solution.
26. The method of claim 24, wherein the amount of Compound 2 in the
composition is from 1 microgram to 500 milligrams per kilogram of
the subject's body weight.
27. The method of claim 24, wherein the pharmaceutical composition
is a controlled release formulation.
28. The method of claim 17, wherein the subject is human.
29. The method of claim 18, wherein the subject is human.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 15/872,331, filed Jan. 16, 2018, which is a continuation of
U.S. application Ser. No. 14/542,453, filed Nov. 14, 2014, now U.S.
Pat. No. 9,910,031, which is a continuation of U.S. application
Ser. No. 13/367,825, filed Feb. 27, 2012 (abandoned), which is a
continuation of divisional U.S. patent application Ser. No.
11/972,592, filed Jan. 10, 2008 (abandoned), which is a divisional
application of U.S. patent application Ser. No. 10/986,553
(abandoned), filed Nov. 10, 2004, abandoned, which claims benefit
of U.S. Provisional Patent Application Nos. 60/519,048 (expired)
and 60/519,040 (expired) filed Nov. 10, 2003 and Nov. 11, 2003,
respectively. All of the foregoing applications are incorporated by
reference herein in their entirety.
FIELD OF THE INVENTION
[0002] The present invention is directed to compositions and
methods for modulating c-Rel-dependent cytokine production without
materially altering the level of expression of NF.kappa.B and/or
amount of I.kappa.B The present invention is also directed to
screening for modulators of c-Rel activity as determined by
assaying for altered subcellular localization of c-Rel but where
the level of expression of NF.kappa.B and/or amount of I.kappa.B is
materially unaltered.
BACKGROUND OF THE INVENTION
[0003] The role of cytokines in the development of autoimmune
diseases and inflammatory disorders is well known. Cytokines such
as interleukin-12 (IL-12) mediate the acute phase response to
inflammatory stimuli, enhance the microbicidal functions of
macrophages and other cells, and promote specific lymphocyte
responses. IL-12 plays a role in multiple-Thl dominant autoimmune
diseases including, but not limited to, multiple sclerosis,
myasthenia gravis, autoimmune neuropathies, Guillain-Barre
syndrome, autoimmune uveitis, autoimmune hemolytic anemia,
pernicious anemia, autoimmune thrombocytopenia, temporal arteritis,
anti-phospholipid syndrome, vasculitides, Wegener's granulomatosis,
Behcet's disease, psoriasis, dermatitis herpetiformis, pemphigus
vulgaris, vitiligo, Crohn's disease, ulcerative colitis, primary
biliary cirrhosis, autoimmune hepatitis, Type 1 or immune-mediated
diabetes mellitus, Grave's disease, Hashimoto's thyroiditis,
autoimmune oophoritis and orchitis, autoimmune disease of the
adrenal gland; rheumatoid arthritis, systemic lupus erythematosus,
scleroderma, polymyositis, dermatomyositis, spondyloarthropathies,
ankylosing spondylitis, Sjogren's syndrome and graft-versus-host
disease.
[0004] Interleukin-12 (IL-12) is a di-sulfide linked heterodimeric
cytokine (p70) composed of two independently regulated subunits,
p35 and p40. IL-12 is produced by phagocytic cells and antigen
presenting cells, in particular, macrophages and dendritic cells,
upon stimulation with bacteria, bacterial products such as
lipopolysaccharide (LPS), and intracellular parasites. The
well-documented biological functions of IL-12 are induction of
interferon-y expression from T and NK cells and differentiation
toward the Thl T lymphocyte type. IFN-.gamma., expression of which
is induced by IL-12, is a strong and selective enhancer of IL-12
production from monocytes and macrophages. The effect is evident
after extended treatment with IFN-.gamma. for at least 8 hours
prior to stimulation with LPS or Staphylococcus aureus Cowan I
(SAC), suggesting that, particularly in chronic diseases in which
there is ongoing production of IFN-.gamma., IL-12 production is
augmented by IFN-.gamma.. It is presumed that after an infective or
inflammatory stimulus that provokes IL-12 production, the powerful
feedback loop promotes IL-12-induced IFN-.gamma. to further augment
IL-12 production, leading to consequent excessive production of
pro-inflammatory cytokines. The cytokine IL-23 is a heterodimer
composed of a p19 subunit and the same p40 subunit of IL-12.
[0005] LPS stimulates the translocation of p50/c-Rel and p50/p65
heterodimers in macrophages from the cytoplasm to the nucleus. Both
of these heterodimers bind to the NF.kappa.B site in the promoter
of p40. However, only c-Rel has been shown to be important for the
LPS-induced signaling through Toll-like receptor-4 (TLR4) that
leads to the production of p40 in response to numerous
pro-inflammatory stimuli in vitro and in vivo.
[0006] Citation or identification of any reference in Section 2 or
in any other section of this application shall not be construed as
an admission that such reference is available as prior art to the
present invention.
SUMMARY OF THE INVENTION
[0007] The present invention provides methods of identifying a
molecule that selectively alters c-Rel-dependent transcription by
detecting alterations in the level of c-Rel molecules localized to
the nucleus of a cell (e.g., an immune cell) contacted with one or
more candidate molecules, without detecting any alterations in the
expression of NF.kappa.B and/or amount of I.kappa.B, relative to a
cell not contacted with a candidate molecule or contacted with a
negative control such as phosphate buffered saline (PBS), (e.g.,
assessing but detecting no material altering of the expression
levels of NF.kappa.B and or I.kappa.B). In one embodiment, the
invention provides a method of identifying a molecule that
selectively alters c-Rel-dependent transcription, comprising the
following steps in the order stated: (a) contacting a cell (e.g.,
an immune cell such as an natural killer cell, a T cell, a
macrophage, a dendritic cell, or a monocyte) with one or more
candidate molecules; and (b) detecting localization of c-Rel
molecules in the cell, wherein an increase or decrease in the
amount of c-Rel in the nucleus without materially altering the
level of expression of NF.kappa.B and/or amount of I.kappa.B
relative to said amount in a cell not so contacted with the one or
more candidate molecules or contacted with a negative control such
as PBS indicates that the candidate molecules alter c-Rel-dependent
transcription. In accordance with this embodiment, the cell may be
contacted with the candidate molecule(s) while concurrently being
stimulated with IFN-.gamma. and/or lipopolysaccharide (LPS).
Preferably, in accordance with this embodiment, the cell is
contacted with the candidate molecule(s) following stimulation with
IFN-.gamma. and/or lipopolysaccharide (LPS). In a specific
embodiment, the cell contacted with the candidate molecule is a
macrophage, monocyte or dendritic cell.
[0008] In another embodiment, the invention provides a method of
identifying a molecule that selectively alters c-Rel-dependent
transcription, comprising the following steps in the order stated:
(a) contacting a cell (e.g., an immune cell such as an natural
killer cell, a T cell, a macrophage, a dendritic cell, or a
monocyte) recombinantly expressing one or more candidate molecules;
and (b) detecting localization of c-Rel molecules in the cell,
wherein an increase or decrease in the amount of c-Rel in the
nucleus without materially altering the level of expression of
NF.kappa.B and/or amount of I.kappa.B relative to said amount in a
cell not expressing one or more candidate molecules indicates that
the candidate molecules alter c-Rel-dependent transcription. In
accordance with this embodiment, the cell may be stimulated with
IFN-.gamma. and/or lipopolysaccharide (LPS) prior to, concurrently
with or subsequent to the induction of the expression of the
candidate molecule(s). In a specific embodiment, the cell
expressing the candidate molecule(s) is a macrophage, monocyte or
dendritic cell.
[0009] Any method known in the art may be used to measure the level
of c-Rel localized to the nucleus of a cell. For example, the
localization of c-Rel in a cell may be detected by contacting the
cell with an antibody to c-Rel or a binding region of said
antibody, and a fluorescently labeled binding partner of said
antibody under conditions conducive to immunospecific binding.
Alternatively, the localization of c-Rel in a cell may be detected
by contacting the cell with a fluorescently labeled antibody to
c-Rel or a binding region of said antibody under conditions
conducive to immunospecific binding. The localization of c-Rel in a
cell may be detected also be detected by sequencing by mass
spectroscopy nuclear proteins isolated from the cell. Further, The
localization of c-Rel in a cell may be detected by measuring the
amount of c-Rel-dependent transcription, e.g., measuring p40
transcription, or total cellular p40 protein levels, or total
nuclear p40 protein levels.
[0010] Any method known in the art may be used to measure the level
of NF.kappa.B expression, including, but not limited to, measuring
the protein levels of NF.kappa.B family members p50, p65 and c-Rel
by immunospecific binding or measuring the levels of the encoding
mRNA. In a particular embodiment, expression of NF.kappa.B refers
to the expression of NF.kappa.B family members p50, p65 and c-Rel,
as measured, e.g., in a western blot using a whole cell protein
extract. Any method known in the art may be used to measure the
amount of I.kappa.B, including, but not limited to, measuring the
total amount of I.kappa.B protein or encoding mRNA in the cell, as
measured, e.g., in a western blot using either a whole cell or
cytoplasmic protein extract, or measuring the level of I.kappa.B
degradation by, e.g., measuring I.kappa.B protein levels in the
treated cells as compared to levels in the untreated cells.
[0011] In the context of NF.kappa.B and/or I.kappa.B (including
I.kappa.B.alpha. and I.kappa.B.beta.) expression and/or amount, the
term "materially altering" as used herein means a greater than 10%,
preferably greater than 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%
or 95% change in the level of expression of NF.kappa.B and/or
amount of I.kappa.B.
[0012] Any protein whose expression is dependent on c-Rel
transcription may be altered as result of altering c-Rel-dependent
transcription. Examples of such proteins include, but are not
limited to, IL-6, IL-10, IL-12, IL-15, IL-23, IFN-y, Bcl-xL, Mcl-1,
Jagged-1, ERF-4 and c-myc. Accordingly, the expression of one or
more of such proteins may be altered by a molecule identified in
accordance with the methods of the invention as selectively
altering c-Rel-dependent transcription. In a preferred embodiment,
the level of expression of IL-12 and/or IL-23 are altered by a
molecule identified in accordance with the methods of the invention
as selectively altering c-Rel-dependent transcription.
[0013] The present invention provides methods of identifying a
molecule that selectively alters c-Rel-dependent cytokine
production by detecting alterations in the level of c-Rel molecules
localized to the nucleus of a cell (e.g., an immune cell) of
contacted with one or more candidate molecules, without detecting
any alterations in the expression of NF.kappa.B and/or amount of
I.kappa.B, relative to a cell not contacted with a candidate
molecule or contacted with a negative control such phosphate
buffered saline (PBS) (e.g., assessing but detecting no material
altering of the expression levels of NF.kappa.B and or I.kappa.B).
Examples of cytokines dependent on c-Rel for production proteins
include, but are not limited to, IL-6, IL-10, IL-12, IL-15, IL-23,
and IFN-.gamma.. Accordingly, the expression of one or more of such
cytokines may be altered by a molecule identified in accordance
with the methods of the invention as selectively altering
c-Rel-dependent cytokine production. In a preferred embodiment, the
level of expression of IL-12 and/or IL-23 are altered by a molecule
identified in accordance with the methods of the invention as
selectively altering c-Rel-dependent cytokine production.
[0014] In one embodiment, the present invention provides a method
of identifying a molecule that selectively alters c-Rel-dependent
cytokine production in a cell comprising the following steps in the
order stated: (a) contacting the cell (preferably, after or
concurrently with IFN-.gamma. and/or LPS stimulation) with one or
more candidate molecules; and (b) detecting localization of c-Rel
molecules in the cell, wherein an increase or decrease in the
amount of c-Rel in the nucleus without materially altering the
level of expression of NF.kappa.B and/or amount of I.kappa.B
relative to said amount in a cell not so contacted with the one or
more candidate molecules or contacted with a negative control such
as PBS indicates that the candidate molecules alter c-Rel-dependent
cytokine production. In accordance with this embodiment, the cell
contacted with the candidate molecule(s) is preferably a
macrophage, monocyte or dendritic cell.
[0015] In another embodiment, the present invention provides a
method of identifying a molecule that selectively alters
c-Rel-dependent cytokine production in a cell comprising the
following steps in the order stated: (a) recombinantly expressing
within the cell one or more candidate molecules; and (b) detecting
localization of c-Rel molecules in the cell, wherein an increase or
decrease in the amount of c-Rel in the nucleus without materially
altering the level of expression of NF.kappa.B and/or amount of
I.kappa.B relative to said amount in a cell not expressing the one
or more candidate molecules indicates that the candidate molecules
alter c-Rel-dependent cytokine production. In accordance with this
embodiment, the cell may be stimulated with IFN-'y and/or
lipopolysaccharide (LPS) prior to, concurrently with or subsequent
to the induction of the expression of the candidate molecule(s). In
a specific embodiment, the cell expressing the candidate
molecule(s) is a macrophage, monocyte or dendritic cell.
[0016] In another embodiment, the present invention provides a
method of identifying a molecule that selectively alters
c-Rel-dependent cytokine production in a cell (after or
concurrently with IFN-.gamma. and/or LPS stimulation) comprising
the following steps in the order stated: (a) microinjecting into
the cell one or more candidate molecules; and (b) detecting
localization of c-Rel molecules in the cell, wherein an increase or
decrease in the amount of c-Rel in the nucleus without materially
altering the level of expression of NF.kappa.B and/or amount of
I.kappa.B relative to said amount in a cell not so microinjected
with the one or more candidate molecules or microinjected with a
negative control indicates that the candidate molecules alter
c-Rel-dependent cytokine production. In accordance with this
embodiment, the cell contacted with the candidate molecule(s) is
preferably a macrophage, monocyte or dendritic cell.
[0017] In one embodiment, a molecule identified in accordance with
the methods of the invention as selectively altering
c-Rel-dependent transcription and/or c-Rel-dependent cytokine
production in a cell does not alter the level of expression and/or
activity of one or more of the following proteins: PU-1, Jakl,
Jak2, STAT1, ERK, PICA, I.kappa.B and p38 kinases. In another
embodiment, a molecule identified in accordance with the methods of
the invention as selectively altering c-Rel-dependent transcription
and/or c-Rel-dependent cytokine production in a cell reduces the
level of ICSBP in the nucleus. In a particular embodiment, a
molecule identified in accordance with the methods of the invention
as selectively altering c-Rel-dependent transcription and/or
c-Rel-dependent cytokine production in a cell decreases in the
level of interferon consensus sequence binding protein (ICSBP) in
the nucleus of a cell by at least 10%, preferably, at least 15%, at
least 18%, at least 20%<at least 25%, at least 30%, at least
35%, at least 40% or at least 45%.
[0018] In another embodiment, a molecule identified in accordance
with the methods of the invention as selectively altering
c-Rel-dependent transcription and/or c-Rel-dependent cytokine
production in a cell reduces the level of p50 in the nucleus. In
another embodiment, a molecule identified in accordance with the
methods of the invention as selectively altering c-Rel-dependent
transcription and/or c-Rel-dependent cytokine production in a cell
increases the level of p65 in the nucleus. In a particular
embodiment, a molecule identified in accordance with the methods of
the invention as selectively altering c-Rel-dependent transcription
and/or c-Rel-dependent cytokine production in a cell increases the
level of p65 in the nucleus by at least 35%, preferably at least
40%, at least 45%, at least 50%, at least 55%, at least 59%, at
least 60%, at least 65%, at least 70%, at least 75% or at least
85%. In another embodiment, a molecule identified in accordance
with the methods of the invention as selectively altering
c-Rel-dependent transcription and/or c-Rel-dependent cytokine
production in a cell decreases p35 transcription. In yet another
embodiment, a molecule identified in accordance with the methods of
the invention as selectively altering c-Rel-dependent transcription
and/or c-Rel-dependent cytokine production in a cell results in two
or more of the following effects: a decrease in the level of ICSBP
in the nucleus, a decrease in the level of p50 in the nucleus, an
increase in the level of p65 in the nucleus, and a decrease in p35
transcription.
[0019] The present invention also provides a method for identifying
a drug target for mediating selective inhibition of c-Rel-dependent
transcription in a cell comprising the following steps in the order
stated: (a) labeling one or more agents that reduce the amount of
c-Rel in the nucleus of the cell without materially altering the
level of expression of NF.kappa.B or the amount of I.kappa.B; (b)
contacting the cell with the one or more labeled agents under
conditions that form a complex between the one or more labeled
agents and the drug target; (c) isolating the complex; and (d)
identifying the drug target from the complex. In another
embodiment, the present invention provides a method for identifying
a drug target that selectively inhibits c-Rel-dependent cytokine
production in a cell comprising the following steps in the order
stated: (a) labeling one or more agents that reduce the amount of
c-Rel in the nucleus of a cell without materially altering the
level of expression of NF.kappa.B or amount of I.kappa.B; (b)
contacting the cell with the one or more labeled agents under
conditions that form a complex between the one or more labeled
agents and the drug target; (c) isolating the complex; and (d)
identifying the drug target from the complex. The identified drug
target can then be used to identify compounds for altering cytokine
expression levels, in particular c-Rel-dependent cytokine
levels.
[0020] The present invention also is directed to a method for
identifying molecules whose expression or activity is altered due
to alteration of c-Rel subcellular localization, i.e., an increase
or decrease of c-Rel in the nucleus comprising altering the
subcellular localization of c-Rel by contacting a cell with a
molecule that alters c-Rel subcellular localization but does not
alter the level of expression of NF.kappa.B or amount of I.kappa.B,
and identifying molecules whose expression or activity is altered
in cells contacted with the molecule as compared to cells not
contacted with the molecule or contacted with a negative control
such as PBS.
[0021] In one embodiment of the present invention, methods are
provided for modulating the activity of c-Rel comprising contacting
a cell expressing c-Rel with a molecule that alters the subcellular
localization of c-Rel, particularly a decrease in the nucleus of
c-Rel, but does not materially alter the level of expression of
NF.kappa.B or amount of I.kappa.B in a cell. In an aspect of this
embodiment, the total level of c-Rel protein in the cell is
unchanged. In alternative aspect, the total level of c-Rel protein
in the cell is altered. In another embodiment the invention,
methods are provided for modulating c-Rel-dependent cytokine
production in a cell without materially altering the level of
NF.kappa.B expression or amount of I.kappa.B in the cell comprising
contacting the cell expressing c-Rel with a molecule that alters
the subcellular localization of c-Rel but does not materially alter
the level of NF.kappa.B expression or amount of I.kappa.B. In an
aspect of the above-embodiment, the molecule is identified by one
or more of the screening assays described above. In a particular
embodiment, the molecule is identified by a screening method
comprising contacting a cell with one or more candidate molecules;
and detecting localization of c-Rel molecules in the cell, such
that those molecules that cause a decrease in the amount of c-Rel
in the nucleus without materially altering the level of expression
of NF.kappa.B and/or amount of I.kappa.B relative to said amount in
a cell not so contacted or contacted with a negative control such
as PBS are identified.
[0022] In a specific embodiment, the present invention provides a
method for inhibiting c-Rel-dependent cytokine production in a cell
without materially altering the level of expression of NF.kappa.B
and/or amount of I.kappa.B comprising contacting a cell with a
molecule that reduces the amount of c-Rel in the nucleus but does
not materially alter the level of expression of NF.kappa.B and/or
amount of I.kappa.B, e.g., a molecule identified in a screening
assay described above. In one aspect of the embodiment, the method
comprises contacting a cell with a molecule that reduces the amount
of c-Rel in the nucleus but does not materially alter the level of
NF.kappa.B expression in the cell as measured by, e.g., the amount
of p50, p65 and c-Rel in a cell extract. In another aspect, the
method comprises contacting a cell with a molecule that reduces the
amount of c-Rel in the nucleus but does not materially alter the
amount of I.kappa.B in the cell as measured by, e.g., amount of
I.kappa.B in the cell.
[0023] The present invention also provides methods for diagnosing
or screening for the presence of or a predisposition for developing
a disease or disorder characterized by aberrant c-Rel subcellular
localization but with normal levels of expression of NF.kappa.B
and/or amount of I.kappa.B in a subject by measuring the level of
c-Rel localization to the nucleus in a sample derived from the
subject, in which a decrease or increase in the level of nuclear
localization of c-Rel relative to the level of localization in an
analogous sample not having the disease or disorder or a
predisposition for developing the disease or disorder indicates the
presence of the disease or disorder or the predisposition for
developing the disease or disorder.
[0024] The present invention also provides methods of treating a
disease or disorder associated with c-Rel-dependent cytokine
production in a subject in need thereof comprising administering to
the subject a molecule in an amount effective to reduce the amount
of c-Rel in the nucleus of a cell expressing a c-Rel dependent
cytokine but that does not materially alter the level of expression
of NF.kappa.B and/or amount of I.kappa.B in the cell. Non-limiting
examples of such molecules include those identified utilizing the
assays described herein. Preferably, the subject is human. In one
aspect of this embodiment, the disease or disorder is an IL-12
production-related disease. In another embodiment, the disease or
disorder is an autoimmune disease.
[0025] The present invention also provides a method of enhancing
the activity of a first agent that inhibits production of a first
cytokine in a subject in need thereof comprising administering to
the patient the first agent together with a second agent that
inhibits production of a second cytokine without materially
altering the level of expression of NF.kappa.B and/or amount of
I.kappa.B in a cell expressing the first and/or second cytokine, in
which said second cytokine is a c-Rel-dependent cytokine. In one
aspect of this embodiment, the first and second cytokine are the
same or are different. Preferably, the subject is human. In certain
aspects, the first agent is for treatment of
autoimmune/inflammatory diseases that does not alter c-Rel activity
or subcellular localization.
[0026] The present invention also provides a method for evaluating
the biological effect of an agent that reduces the amount of c-Rel
in the nucleus of a cell without materially altering the level of
expression of NF.kappa.B and/or amount of I.kappa.B in the cell
comprising contacting the cell with the agent and observing any
phenotypic effects in the cell. In another embodiment, the
invention is directed to a method for evaluating the biological
effect of an agent in a subject, which agent reduces the amount of
c-Rel in the nucleus of a cell without materially altering the
level of expression of NF.kappa.B and/or amount of I.kappa.B, said
method comprising administering the agent to the subject and
observing any phenotypic effects in the subject. The subject is a
mammal, e.g., mouse, rat, monkey, dog, pig, human, etc.
[0027] The present invention provides compositions comprising a
molecule that reduces the amount of c-Rel in the nucleus of a cell
without materially altering the level of expression of NF.kappa.B
and/or amount of I.kappa.B. The present invention further provides
methods of treating or preventing diseases or disorders associated
with aberrant c-Rel-dependent cytokine production, such as, e.g.,
autoimmune disorders, comprising administering to a subject in need
thereof such a composition. In certain embodiments the molecule
does not have a compound as described in U.S. Pat. No. 6,384,032;
U.S. patent application Ser. No. 09/594,362 filed May 7, 2002; U.S.
patent application Ser. No. 10/006,624 filed Nov. 30, 2001
(Publication No. 20020082259); U.S. patent application Ser. No.
10/000,742 filed Nov. 30, 2001 (Publication No. 20030139403); U.S.
patent application Ser. No. 10/192,347 filed Jul. 10, 2002
(Publication No. 20030114446); U.S. patent application Ser. No.
10/305,039 filed Nov. 26, 2002; International Patent Publication
No. WO 00/78757; International Patent Publication No. WO 03/04516;
International Patent Application PCT/US03/32546 filed Oct. 14,
2003; U.S. Provisional Patent Application Ser. No. 60/518,791 filed
Nov. 10, 2003; U.S. Provisional Patent Application Ser. No.
60/518,787 filed Nov. 10, 2003; U.S. Provisional Patent Application
Ser. No. 60/518,788 filed Nov. 10, 2003; U.S. patent application
Ser. No. 10/985,627, entitled, "Fused Heterocyclic Compounds,"
Mitsunori Ono et al, filed Nov. 10, 2004; U.S. patent application
Ser. No. 10/985,716, entitled, "Heteroaryl Hydrazone Compounds,"
Mitsunori Ono et al, filed Nov. 10, 2004; U.S. patent application
Ser. No. 10/985,696, entitled, "Pyridine Compounds," Mitsunori Ono,
et al, filed Nov. 10, 2004, each of which is incorporated by
reference herein in its entirety.
[0028] In certain embodiments the molecule does not have a compound
as described in U.S. Pat. No. 6,384,032; U.S. patent application
Ser. No. 09/594,362 filed May 7, 2002; U.S. patent application Ser.
No. 10/006,624 filed Nov. 30, 2001 (Publication No. 20020082259);
U.S. patent application Ser. No. 10/000,742 filed Nov. 30, 2001
(Publication No. 20030139403); U.S. patent application Ser. No.
10/192,347 filed Jul. 10, 2002 (Publication No. 20030114446);
International Patent Publication No. WO 00/78757; International
Patent Publication No. WO 03/04516, each of which is incorporated
by reference herein in its entirety.
[0029] In certain embodiments the molecule does not have a compound
as described in U.S. Pat. Nos. 6,680,315; 6,693,097; 6,660,733;
U.S. patent application Ser. No. 10/655,672; U.S. patent
application Ser. No. 10/656,671; U.S. patent application Ser. No.
10/305,039 filed Nov. 26, 2002; U.S. patent application Ser. No.
10/656,360 filed Sep. 5, 2003; International Patent Application
PCT/US03/32546 filed Oct. 14, 2003; U.S. Provisional Patent
Application Ser. No. 60/518,791 filed Nov. 10, 2003; U.S.
Provisional Patent Application Ser. No. 60/518,787 filed Nov. 10,
2003; U.S. Provisional Patent Application Ser. No. 60/518,788 filed
Nov. 10, 2003; U.S. patent application Ser. No. 10/985,627,
entitled, "Fused Heterocyclic Compounds," Mitsunori Ono et al,
filed Nov. 10, 2004; U.S. patent application Ser. No. 10/985,716,
entitled, "Heteroaryl Hydrazone Compounds," Mitsunori Ono et al,
filed Nov. 10, 2004; U.S. patent application Ser. No. 10/985,696,
entitled, "Pyridine Compounds," Mitsunori Ono, et al, filed Nov.
10, 2004, each of which is incorporated by reference herein in its
entirety.
[0030] In certain embodiments the molecule does not have a compound
as described in U.S. Pat. Nos. 6,680,315; 6,384,032; U.S. patent
application Ser. No. 10/656,360 filed Sep. 5, 2003; U.S. patent
application Ser. No. 09/594,362 filed May 7, 2002; U.S. patent
application Ser. No. 10/655,672; U.S. patent application Ser. No.
10/656,671; U.S. patent application Ser. No. 10/006,624 filed Nov.
30, 2001 (Publication No. 20020082259); U.S. patent application
Ser. No. 10/000,742 filed Nov. 30, 2001 (Publication No.
20030139403); U.S. patent application Ser. No. 10/192,347 filed
Jul. 10, 2002 (Publication No. 20030114446); International Patent
Publication No. WO 00/78757; International Patent Publication No.
WO 03/04516; International Patent Application PCT/US03/32546 filed
Oct. 14, 2003, each of which is incorporated by reference herein in
its entirety.
[0031] In certain embodiments the molecule does not have a compound
as described in U.S. Provisional Patent Application Ser. No.
60/518,791 filed Nov. 10, 2003; U.S. Provisional Patent Application
Ser. No. 60/518,787 filed Nov. 10, 2003; U.S. Provisional Patent
Application Ser. No. 60/518,788 filed Nov. 10, 2003; U.S. patent
application Ser. No. 10/985,627, entitled, "Fused Heterocyclic
Compounds," Mitsunori Ono et al, filed Nov. 10, 2004; U.S. patent
application Ser. No. 10/985,716, entitled, "Heteroaryl Hydrazone
Compounds," Mitsunori Ono et al, filed Nov. 10, 2004; U.S. patent
application Ser. No. 10/985,696, entitled, "Pyridine Compounds,"
Mitsunori Ono, et al, filed Nov. 10, 2004, each of which is
incorporated by reference herein in its entirety.
[0032] In other embodiments, the molecule does have a structure as
described in U.S. Pat. No. 6,384,032; U.S. patent application Ser.
No. 09/594,362 filed May 7, 2002; U.S. patent application Ser. No.
10/006,624 filed Nov. 30, 2001 (Publication No. 20020082259); U.S.
patent application Ser. No. 10/000,742 filed Nov. 30, 2001
(Publication No. 20030139403); U.S. patent application Ser. No.
10/192,347 filed Jul. 10, 2002 (Publication No. 20030114446); U.S.
patent application Ser. No. 10/305,039 filed Nov. 26, 2002;
International Patent Publication No. WO 00/78757; International
Patent Publication No. WO 03/04516; International Patent
Application PCT/US03/32546 filed Oct. 14, 2003; U.S. Provisional
Patent Application Ser. No. 60/518,791 filed Nov. 10, 2003; U.S.
Provisional Patent Application Ser. No. 60/518,787 filed Nov. 10,
2003; U.S. Provisional Patent Application Ser. No. 60/518,788 filed
Nov. 10, 2003; U.S. patent application Ser. No. 10/985,627,
entitled, "Fused Heterocyclic Compounds," Mitsunori Ono et al,
filed Nov. 10, 2004; U.S. patent application Ser. No. 10/985,716,
entitled, "Heteroaryl Hydrazone Compounds," Mitsunori Ono et al,
filed Nov. 10, 2004; U.S. patent application Ser. No. 10/985,696,
entitled, "Pyridine Compounds," Mitsunori Ono, et al, filed Nov.
10, 2004, each of which is incorporated by reference herein in its
entirety. In one aspect, the molecule is identified by any of the
screening methods disclosed herein. In another aspect, the molecule
is purified by techniques known in the art.
[0033] In other embodiments, the molecule does have a structure as
described in U.S. Pat. No. 6,384,032; U.S. patent application Ser.
No. 09/594,362 filed May 7, 2002; U.S. patent application Ser. No.
10/006,624 filed Nov. 30, 2001 (Publication No. 20020082259); U.S.
patent application Ser. No. 10/000,742 filed Nov. 30, 2001
(Publication No. 20030139403); U.S. patent application Ser. No.
10/192,347 filed Jul. 10, 2002 (Publication No. 20030114446);
International Patent Publication No. WO 00/78757; International
Patent Publication No. WO 03/04516, each of which is incorporated
by reference herein in its entirety. In one aspect, the molecule is
identified by any of the screening methods disclosed herein. In
another aspect, the molecule is purified by techniques known in the
art.
[0034] In other embodiments, the molecule does have a structure as
described in U.S. Pat. Nos. 6,680,315; 6,693,097; 6,660,733; U.S.
patent application Ser. No. 10/655,672; U.S. patent application
Ser. No. 10/656,671; U.S. patent application Ser. No. 10/305,039
filed Nov. 26, 2002; U.S. patent application Ser. No. 10/656,360
filed Sep. 5, 2003; International Patent Application PCT/US03/32546
filed Oct. 14, 2003; U.S. Provisional Patent Application Ser. No.
60/518,791 filed Nov. 10, 2003; U.S. Provisional Patent Application
Ser. No. 60/518,787. filed Nov. 10, 2003; U.S. Provisional Patent
Application Ser. No. 60/518,788 filed Nov. 10, 2003; U.S. patent
application Ser. No. 10/985,627, entitled, "Fused Heterocyclic
Compounds," Mitsunori Ono et al, filed Nov. 10, 2004; U.S. patent
application Ser. No. 10/985,716, entitled, "Heteroaryl Hydrazone
Compounds," Mitsunori Ono et al, filed Nov. 10, 2004; U.S. patent
application Ser. No. 10/985,696, entitled, "Pyridine Compounds,"
Mitsunori Ono, et al, filed Nov. 10, 2004, each of which is
incorporated by reference herein in its entirety. In one aspect,
the molecule is identified by any of the screening methods
disclosed herein. In another aspect, the molecule is purified by
techniques known in the art.
[0035] In other embodiments, the molecule does have a structure as
described in U.S. Pat. Nos. 6,680,315; 6,384,032; U.S. patent
application Ser. No. 10/656,360 filed Sep. 5, 2003; U.S. patent
application Ser. No. 09/594,362 filed May 7, 2002; U.S. patent
application Ser. No. 10/655,672; U.S. patent application Ser. No.
10/656,671; U.S. patent application Ser. No. 10/006,624 filed Nov.
30, 2001 (Publication No. 20020082259); U.S. patent application
Ser. No. 10/000,742 filed Nov. 30, 2001 (Publication No.
20030139403); U.S. patent application Ser. No. 10/192,347 filed
Jul. 10, 2002 (Publication No. 20030114446); International Patent
Publication No. WO 00/78757; International Patent Publication No.
WO 03/04516; International Patent Application PCT/US03/32546 filed
Oct. 14, 2003, each of which is incorporated by reference herein in
its entirety. In one aspect, the molecule is identified by any of
the screening methods disclosed herein. In another aspect, the
molecule is purified by techniques known in the art.
[0036] In other embodiments, the molecule does have a structure as
described in U.S. Provisional Patent Application Ser. No.
60/518,791 filed Nov. 10, 2003; U.S. Provisional Patent Application
Ser. No. 60/518,787 filed Nov. 10, 2003; U.S. Provisional Patent
Application Ser. No. 60/518,788 filed Nov. 10, 2003; U.S. patent
application Ser. No. 10/985,627, entitled, "Fused Heterocyclic
Compounds," Mitsunori Ono et al, filed Nov. 10, 2004; U.S. patent
application Ser. No. 10/985,716, entitled, "Heteroaryl Hydrazone
Compounds," Mitsunori Ono et al, filed Nov. 10, 2004; U.S. patent
application Ser. No. 10/985,696, entitled, "Pyridine Compounds,"
Mitsunori Ono, et al, filed Nov. 10, 2004, each of which is
incorporated by reference herein in its entirety. In one aspect,
the molecule is identified by any of the screening methods
disclosed herein. In another aspect, the molecule is purified by
techniques known in the art.
Other Embodiments
[0037] In other embodiments, the present invention is directed to a
method for inhibiting c-Rel-dependent cytokine production in a cell
without materially altering the level of NF.kappa.B transcription
factor expression and without materially altering the level of
I.kappa.B degradation comprising contacting a molecule to a cell,
which molecule reduces the amount of c-Rel in the nucleus of the
cell without materially altering the level of NF.kappa.B
transcription factor expression and without materially altering the
level of I.kappa.B degradation. In a particular embodiment, the
c-Rel-dependent cytokine is IL-12. In certain aspects of this
embodiment, IL-12 transcription is inhibited, or the amount of p65
in the nucleus is increased or c-Rel translocation to the nucleus
is inhibited, or c-Rel expression is not materially altered. In
other aspects, IL-12/IL-23 p40 expression is inhibited, or the
NF.kappa.B element of the IL-12/23 subunit p40 promoter is
inhibited, or activation of the Ets-2 element of the IL-12/IL-23
subunit p40 promoter is inhibited. In yet other aspects, IL-12
subunit p35 expression is inhibited, and/or the NF.kappa.B element
of IL-12 subunit p35 promoter is inhibited. In yet other aspects,
the amount of ICSBP in the nucleus is also reduced, or ICSBP
expression is inhibited. In certain aspects, the cell is selected
from the group consisting of macrophages, monocytes and dendritic
cells.
[0038] In yet another particular embodiment, the c-Rel-dependent
cytokine is IL-23. In certain aspects of this embodiment, the
amount of p65 in the nucleus is increased, or c-Rel translocation
to the nucleus is inhibited, or c-Rel expression is not materially
altered. In yet other aspects, IL-12/IL-23 subunit p40 expression
is inhibited, or the NF.kappa.B element of IL-12/23 subunit p40
promoter is inhibited, or activation of the Ets-2 element of the
IL-12/23 subunit p40 promoter is inhibited, or the amount of ICSBP
in the nucleus is also reduced. In yet other aspects, ICSBP
expression is inhibited. In certain aspects, the cell is selected
from the group consisting of macrophages, monocytes and dendritic
cells.
[0039] In other embodiments, the present invention is directed to a
method for identifying an agent (otherwise referred to as a
candidate molecule or compound) that selectively inhibits c-Rel
dependent cytokine production comprising contacting the cell with a
test agent; measuring the amount of c-Rel in the nucleus of the
cell; and selecting those agents that reduce the amount of c-Rel in
the nucleus of the cell without materially altering the level of
NF.kappa.B transcription factor expression and without materially
altering the level of I.kappa.B degradation. In certain aspects of
this embodiment, the amount of c-Rel in the nucleus is measured in
step using a luciferase assay. The c-Rel dependent cytokine can be
IL-12 or IL-23.
[0040] In yet another embodiment, the invention provides methods
for target discovery. In one aspect, a method for identifying an
agent that selectively inhibits c-Rel dependent cytokine production
is provided, which method comprises labeling an agent that reduces
the amount of c-Rel in the nucleus of a cell without materially
altering the level of NF.kappa.B transcription factor expression
and without materially altering the level of I.kappa.B degradation;
contacting the cell with the labeled agent under conditions that
form a complex between the labeled agent and the drug target;
isolating the complex; and identifying the drug target from the
complex. In this embodiment, the c-Rel dependent cytokine is IL-12
or IL-23.
[0041] The present invention is also directed to a method for
treating a disorder associated with c-Rel-dependent cytokine
production in a patient in need thereof comprising administering to
the patient an effective amount of an agent that reduces the amount
of c-Rel in the nucleus of a cell that produces the cytokine
without materially altering the level of NF.kappa.B transcription
factor expression and without materially altering the level of
I.kappa.B degradation. In one aspect, the disorder is an autoimuune
disease selected from the group consisting of: multiple sclerosis,
myasthenia gravis, autoimmune neuropathies, Guillain-Barre
syndrome, autoimmune uveitis, autoimmune hemolytic anemia,
pernicious anemia, autoimmune thrombocytopenia, temporal arteritis,
anti-phospholipid syndrome, vasculitides, Wegener's granulomatosis,
Behcet's disease, psoriasis, dermatitis herpetiformis, pemphigus
vulgaris, vitiligo, Cram's disease, ulcerative colitis, primary
biliary cirrhosis, autoimmune hepatitis, Type 1 or immune-mediated
diabetes mellitus, Grave's disease, Hashimoto's thyroiditis,
autoimmune oophoritis and orchitis, autoimmune disease of the
adrenal gland; rheumatoid arthritis, systemic lupus erythematosus,
sclerodenna, polymyositis, dermatomyositis, spondyloarthropathies,
ankylosing spondylitis, Sjogren's syndrome and graft-versus-host
disease.
[0042] In another embodiment, the present invention is also
directed to a method for treating a disorder associated with
c-Rel-dependent cytokine production in a subject identified as
being in need thereof. The subject may be identified as being in
need thereof by a health care professional or may be
self-diagnosed. The method comprises administering to the patient
an effective amount of an agent that reduces the amount of c-Rel in
the nucleus of a cell that produces the cytokine without materially
altering the level of NF.kappa.B transcription factor expression
and without materially altering the level of I.kappa.B degradation.
In one aspect, the disorder is an autoimuune disease selected from
the group consisting of: multiple sclerosis, myasthenia gravis,
autoimmune neuropathies, Guillain-Barre syndrome, autoimmune
uveitis, autoimmune hemolytic anemia, pernicious anemia, autoimmune
thrombocytopenia, temporal arteritis, anti-phospholipid syndrome,
vasculitides, Wegener's granulomatosis, Behcet's disease,
psoriasis, dermatitis herpetiformis, pemphigus vulgaris, vitiligo,
Crohn's disease, ulcerative colitis, primary biliary cirrhosis,
autoimmune hepatitis, Type 1 or immune-mediated diabetes mellitus.
Grave's disease, Hashimoto's thyroiditis, autoimmune oophoritis and
orchitis, autoimmune disease of the adrenal gland; rheumatoid
arthritis, systemic lupus erythematosus, scleroderma, polymyositis,
dermatomyositis, spondyloarthropathies, ankylosing spondylitis,
Sjogren's syndrome and graft-versus-host disease.
[0043] In another embodiment, a compound identified by a screening
method according to the invention is labeled with instructions for
use. The instructions may include directions for administration to
a subject identified to be in need thereof, dosages, dosage forms,
and duration of use. A subject may be a mammal, such as a human,
primate, dog, horse, pig, cow or cat.
[0044] In yet another embodiment, the invention is directed to a
method of enhancing the activity of a first agent that inhibits
production of a first cytokine in a patient in need thereof, said
method comprising administering to the patient the first agent
together with a second agent that inhibits production of a second
cytokine without materially altering the level of NF.kappa.B
transcription factor expression and without materially altering the
level of I.kappa.B degradation, wherein production of the second
cytokine is c-Rel-dependent. In certain aspects, the first cytokine
and the second cytokine are the same or the first cytokine and the
second cytokine are different. In other aspects, the second
cytokine is IL-12 or IL-23.
[0045] In yet another embodiment, the invention is directed to a
method for evaluating the biological effect of an agent that
reduces the amount of c-Rel in the nucleus of a cell without
materially altering the level of NF.kappa.B transcription factor
expression and without materially altering the level of I.kappa.B
degradation comprising contacting the cell with the agent and
observing any phenotypic effects in the cell. In yet another
embodiment, the invention is directed to a method for evaluating
the biological effect in a subject of an agent that reduces the
amount of c-Rel in the nucleus of a cell without materially
altering the level of NF.kappa.B transcription factor expression
and without materially altering the level of I.kappa.B degradation
comprising contacting the cell with the agent and observing any
phenotypic effects in the subject.
[0046] In yet another embodiment, the present invention is directed
to a compound that reduces the amount of c-Rel in the nucleus of a
cell without materially altering the level of NF.kappa.B
transcription factor expression and without materially altering the
level of I.kappa.B degradation, provided that the compound does not
have the structures described in U.S. Pat. No. 6,384,032; PCT
publication WO 00/78757.
BRIEF DESCRIPTION OF THE FIGURES
[0047] FIGS. 1A-1B recite the nucleotide and amino acid sequences
of human c-Rel (SEQ ID NOS:1 and 2, respectively).
[0048] FIGS. 2A and 2B are graphs showing the ability of test
molecules to inhibit IFN-.gamma. and IFN-.gamma./LPS induced p40
(FIG. 2A) and p35 (FIG. 2B) expression.
[0049] FIG. 3A is a schematic of the different test promoters used
and FIG. 3B is a graph demonstrating the ability of the various
test promoters to respond to IFN-.gamma./LPS stimulation.
[0050] FIG. 4 is a western blot analysis of THP-1 nuclear extracts
in stimulated and non-stimulated cells with regard to the presence
of NF.kappa.B family members c-Rel, p65 or p50; .alpha.-tubulin is
an internal control.
[0051] FIG. 5 is a western blot analysis of THP-1 nuclear extracts
with anti-ICSBP antibody in stimulated and non-stimulated
cells.
[0052] FIG. 6 is a western blot analysis of THP-1 nuclear extracts
with anti-PU-1 antibody in stimulated and non-stimulated cells.
[0053] FIG. 7 is an immunoblot that shows the effect of a test
molecule on NF-kB p50 nuclear translocation.
[0054] FIG. 8 graphically presents the results of a densitometry
showing the effect of a test molecule on p50 nuclear
translocation.
[0055] FIG. 9 depicts an immunoblot demonstrating the effect of a
test molecule on NF-kB p65 nuclear translocation.
[0056] FIG. 10 graphically presents the results of a densitometry
showing the effect of a test molecule on p65 nuclear
translocation.
[0057] FIG. 11 depicts an immunoblot demonstrating the effect of a
test molecule on nuclear translocation of NF-kB members, including
c rel.
DETAILED DESCRIPTION OF THE INVENTION
[0058] The present invention is based, in part, on the inventors'
discovery that the activity of c-Rel, particularly levels of c-Rel
present in the nucleus, can be increased or decreased without
materially altering the expression of NF.kappa.B or the amount of
I.kappa.B.
[0059] The present invention is directed to a method of identifying
a molecule that selectively alters c-Rel-dependent transcription in
a cell comprising the following steps in the order stated: (a)
contacting the cell with one or more candidate molecules; and (b)
detecting localization of c-Rel molecules in the cell, wherein an
increase or decrease in the amount of c-Rel in the nucleus without
materially altering the level of expression of NF.kappa.B and/or
amount of I.kappa.B relative to said amount in a cell not so
contacted with the one or more candidate molecules indicates that
the candidate molecules alter c-Rel-dependent transcription. In
another embodiment, the present invention is directed to a method
of identifying a molecule that selectively alters c-Rel-dependent
cytokine production in a cell comprising the following steps in the
order stated: (a) contacting the cell with one or more candidate
molecules; and (b) detecting localization of c-Rel molecules in the
cell, wherein an increase or decrease in the amount of c-Rel in the
nucleus without materially altering the level of expression of
NF.kappa.B and/or amount of I.kappa.B relative to said amount in a
cell not so contacted with the one or more candidate molecules
indicates that the candidate molecules alter c-Rel-dependent
cytokine production. In another embodiment, the present invention
is directed to a method of identifying a molecule that selectively
alters c-Rel-dependent cytokine production in a cell comprising the
following steps in the order stated: (a) recombinantly expressing
within the cell one or more candidate molecules; and (b) detecting
localization of c-Rel molecules in the cell, wherein an increase or
decrease in the amount of c-Rel in the nucleus without materially
altering the level of expression of NF.kappa.B and/or amount of
I.kappa.B relative to said amount in a cell not so contacted with
the one or more candidate molecules indicates that the candidate
molecules alter c-Rel-dependent cytokine production. In specific
embodiments, the c-Rel-dependent cytokine is IL-12 or IL-23.
[0060] The present invention is also directed to a method of
enhancing the activity of a first agent that inhibits production of
a first cytokine in a subject in need thereof comprising
administering to the patient the first agent together with a second
agent that inhibits production of a second cytokine without
materially altering the level of expression of NF.kappa.B and/or
amount of I.kappa.B in a cell expressing the first and/or second
cytokine, in which said second cytokine is a c-Rel-dependent
cytokine. In aspect of this embodiment, the first and second
cytokine are the same or are different. Preferably, the subject is
human.
Detection of c-Rel/KB Subcellular Localization
[0061] Any method known in the art for detecting the subcellular
localization of c-Rel, i.e., to the nucleus or cytoplasm, can be
used in the present invention. For example, and not by way of
limitation, one such method of detection is contacting a cell with
an antibody specific for c-Rel and then detecting whether the
antibody localizes to the nucleus. A particular method of detecting
c-Rel subcellular localization is to contact a labeled anti-c-Rel
antibody, e.g., labeled with a fluorescent dye, and a labeled
anti-DNA antibody, e.g., with a fluorescent dye different from the
anti-c-Rel antibody, to whole cells and then to detect cells having
both labels co-localized in the cell by, e.g., laser scanning
microscopy.
[0062] Thus, detection methods encompassed by the present invention
include immunofluorescence or immunoelectron microscopy, for in
situ detection of the c-Rel molecule. In situ detection may be
accomplished by contacting a cell endogenously or recombinantly
expressing a c-Rel molecule with a labeled molecule that binds to
c-Rel and detecting any binding that occurs and that is localized
to the nucleus. Alternatively, an unlabeled molecule may be used,
in combination with a labeled binding partner of the molecule.
Using such an assay, it is possible to determine not only the
presence of the c-Rel molecule, but also its subcellular
distribution, i.e., in the nucleus. Alternatively, c-Rel can be
expressed with a detectable moiety, such as a flag tag. An antibody
specific for the tag then allows for detection of the recombinant
c-Rel molecule.
[0063] Immunoassays for c-Rel will typically comprise incubating a
sample, such as a cell in vivo or in in vitro culture, in the
presence of a detectably labeled molecule specific for c-Rel, e.g.,
an antibody to c-Rel, and detecting the bound molecule by any of a
number of techniques known in the art.
[0064] In a specific embodiment, a biological sample, e.g., freshly
obtained cells, may be brought in contact with and immobilized onto
a solid phase support or carrier such as nitrocellulose, glass,
polystyrene, or other solid support, which is capable of
immobilizing cells. The support may then be washed with suitable
buffers followed by treatment with the detectably labeled molecule.
The solid phase support may then be washed with the buffer a second
time to remove unbound molecule. The amount of bound label on solid
support may then be detected by conventional means.
[0065] The binding activity of a given antibody to a c-Rel molecule
may be determined according to well-known methods. Those skilled in
the art will be able to determine operative and optimal assay
conditions for each determination by employing routine
experimentation.
[0066] One of the ways in which an antibody to c-Rel can be
detectably labeled is by linking the same to an enzyme and use in
an enzyme immunoassay (EIA) (Voller, A., "The Enzyme Linked
Immunosorbent Assay (ELISA)", 1978, Diagnostic Horizons 2:1-7,
Microbiological Associates Quarterly Publication, Walkersville,
Md.); Voller et al., 1978, J. Clin. Pathol. 31:507-520; Butler,
1981, Meth. Enzymol. 73:482-523; Maggio, E. (ed.), 1980, Enzyme
Immunoassay, CRC Press, Boca Raton, Fla.; Ishikawa et al., (eds.),
1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo)). The enzyme which is
bound to the antibody bound to a c-Rel molecule will react with an
appropriate substrate, preferably a chromogenic substrate, in such
a manner as to produce a chemical moiety which can be detected, for
example, by spectrophotometric, fluorimetric or by visual
means.
[0067] It is also possible to label the antibody with a fluorescent
or chemiluminescent or bioluminescent compound or with a
radioactive moiety or other label known in the art.
[0068] Another method of detecting and/or measuring c-Rel nuclear
localization is to isolate nuclear proteins by any method known in
the art and detect whether c-Rel is present in the pool of nuclear
proteins, preferably by mass spectroscopy analysis to identify the
proteins in the pool of nuclear proteins. Isolation of nuclear
proteins can be accomplished by any method know in the art. After
nuclear protein isolation, detection of c-Rel can be accomplished,
e.g., by immunoprecipitating c-Rel with an anti-c-Rel antibody or
binding to anti-c-Rel antibody on an immunoaffinity column or
immobilized on a plate or in a well, or visualizing the protein by
Western blotting. In another embodiment of the invention, c-Rel
localization to the nucleus can be detected and/or measured by
isolating and separating nuclear proteins on a SDS-PAGE gel,
eluting separated protein from the gel, and subjecting the eluted
protein to mass spectroscopy analysis to determine amino acid
sequence. Such mass spectroscopy analysis can be carried out by any
suitable method of mass spectroscopy known in the art, e.g., as
described in Neubauer et al., 1998, Nature Genetics 20:46-50;
Neubauer et al., 1997, Proc. Natl. Acad. Sci. USA 94:385-390; and
Wilm et al., 1996, Nature 379:466-469. By way of example but not
limitation, the eluted peptides are dissolved in a 5% methanol/5%
formic acid solution and desalted using a capillary column as
described in Wilm and Mann, 1996, Anal. Chem. 68:1-8. The peptides
are then diluted in one step in a 50% methanol/5% formic acid
solution (0.5-2 .mu.1) directly into the spraying needle of the
nanoelectrospray ion source. A mass spectrum of the peptides is
acquired. The peptides are then selected in turn in the first
quadrupole. This first part of the mass spectrometer is used as a
mass filter, allowing the transmission of a peptide ion species of
one m/z value at a time. Each peptide is then fragmented
individually by collision-induced dissociation with argon in the
collision cell. The resulting peptide fragment ions are separated
in the third quadrupole and detected. For tryptic peptides this
usually results in a `nested set` of peptide fragments containing
the carboxy-terminus. As the mass difference between two adjacent
fragments corresponds with the residue masses of the corresponding
amino acid, partial sequence of the peptide from its carboxy to
amino terminus can be determined.
[0069] The cell in which the localization of c-Rel is detected
and/or measured can be in vitro (e.g., isolated in cell culture) or
in vivo. The cell in which c-Rel subcellular localization is
detected can be any cell, e.g., one that endogenously or
recombinantly expresses c-Rel or a fragment or homolog thereof. The
cell can be vertebrate, insect (e.g., Drosophila), C. elegans,
mammalian, bovine, murine, rat, avian, fish, primate, human, etc.
The c-Rel which is expressed can be vertebrate, insect, C. elegans,
mammalian, bovine, murine, rat, avian, fish, primate, human, etc.
The cell can be a cell of primary tissue, a cell line, or of an
animal containing and expressing a c-Rel transgene. For example,
the transgenic animal can be a Drosophila (e.g., melanogaster) or a
C. elegans. In a preferred embodiment, the transgene encodes a
human c-Rel. Transgenic animals can be made by standard methods
well known in the art.
[0070] In specific embodiments of the invention, antibodies and
fragments containing the binding domain thereof, directed against
c-Rel are used to detect c-Rel in a specific embodiment of the
above methods. Accordingly, c-Rel proteins, fragments or analogs or
derivatives thereof, in particular, human c-Rel protein or
fragments thereof, may be used as immunogens to generate anti-c-Rel
protein antibodies. Such antibodies can be polyclonal, monoclonal,
chimeric, single chain, Fab fragments, or from an Fab expression
library. Methods for the production of such antibodies are well
known in the art, and some of which are described, infra.
[0071] The antibodies specific for c-Rel can be used in methods
known in the art, and those methods discussed above, relating to
the localization and/or quantification of c-Rel proteins of the
invention, e.g., for imaging these proteins, measuring levels
thereof in appropriate physiological samples, in diagnostic
methods, etc. This hold true also for a derivative, homolog, or
analog of a c-Rel protein.
[0072] The level of expression of NF.kappa.B or amount of I.kappa.B
can also be determined by using any method known in the art,
including the use of antibodies specific to NF.kappa.B family
members or any subunit thereof, e.g, p50, p65 or c-Rel or to
I.kappa.B. For example, using an antibody specific for I.kappa.B,
the amount of I.kappa.B can be determined, for example, by the
illustrative method taught in the Examples Section, infra. The
levels of expression of NF.kappa.B can be determined by measuring
the amount of p50, p65 or c-Rel.
[0073] Other methods for detection of whether c-Rel is located in
the nucleus can include measuring for the presence of proteins, or
their encoding mRNA molecules, that are dependent on c-Rel for
transcriptional activation and whether there is an increase
(increased c-Rel in nucleus) or a decrease in expression (decreased
c-Rel in the nucleus).
Antibody Production
[0074] Various procedures known in the art may be used for the
production of antibodies to c-Rel, NF.kappa.B family members or any
subunit thereof, or I.kappa.B, or a fragment, derivative, homolog
or analog of the protein. Antibodies of the invention include, but
are not limited to, synthetic antibodies, monoclonal antibodies,
recombinantly produced antibodies, intrabodies, multispecific
antibodies (including bi-specific antibodies), human antibodies,
humanized antibodies, chimeric antibodies, synthetic antibodies,
single-chain Fvs (scFv) (including bi-specific scFvs), single chain
antibodies Fab fragments, F(ab') fragments, disulfide-linked Fvs
(sdFv), and anti-idiotypic (anti-Id) antibodies, and
epitope-binding fragments of any of the above. In particular,
antibodies of the present invention include immunoglobulin
molecules and immunologically active portions of immunoglobulin
molecules, i.e., molecules that contain an antigen binding site
that immunospecifically binds to an antigen (e.g., one or more
complementarity determining regions (CDRs) of an antibody).
[0075] For production of the antibody, various host animals can be
immunized by injection with, e.g., a native c-Rel protein or a
synthetic version, or a derivative of the foregoing. Such host
animals include, but are not limited to, rabbits, mice, rats, etc.
Various adjuvants can be used to increase the immunological
response, depending on the host species, and include, but are not
limited to, Freund's (complete and incomplete), mineral gels such
as aluminum hydroxide, surface active substances such as
lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, dinitrophenol, and potentially useful human adjuvants
such as bacille Calmette-Guerin (BCG) and Corynebaeterium parvum.
Although the following refers specifically to c-Rel, any of the
methods described herein apply equally to c-Rel, NF.kappa.B family
members or subunits thereof, or I.kappa.B.
[0076] For preparation of monoclonal antibodies directed towards
c-Rel or a derivative, fragment, homolog or analog thereof, any
technique that provides for the production of antibody molecules by
continuous cell lines in culture may be used. Such techniques
include, but are not restricted to, the hybridoma technique
originally developed by Kohler and Milstein (1975, Nature
256:495-497), the trioma technique (Gustafsson et al., 1991, Hum.
Antibodies Hybridomas 2:26-32), the human B-cell hybridoma
technique (Kozbor et aL, 1983, Immunology Today 4:72), and the EBV
hybridoma technique to produce human monoclonal antibodies (Cole et
al., 1985, In: Monoclonal Antibodies and Cancer Therapy, Alan R.
Liss, Inc., pp. 77-96). In an additional embodiment of the
invention, monoclonal antibodies can be produced in germ-free
animals utilizing recent technology described in International
Patent Application PCT/US90/02545.
[0077] According to the present invention, human antibodies may be
used and can be obtained by using human hybridomas (Cote et al.,
1983, Proc. Natl. Acad. Sci. USA 80:2026-2030) or by transforming
human B cells with EBV virus in vitro (Cole et al., 1985, In:
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.
77-96). In fact, according to the invention, techniques developed
for the production of "chimeric antibodies" (Morrison et al., 1984,
Proc. Natl. Acad. Sci. USA 81:6851-6855; Neuberger et al, 1984,
Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) by
splicing the genes from a mouse antibody molecule specific for
c-Rel together with genes from a human antibody molecule of
appropriate biological activity can be used; such antibodies are
within the scope of this invention.
[0078] According to the present invention, techniques described for
the production of single chain antibodies (U.S. Pat. No. 4,946,778)
can be adapted to produce c-Rel-specific antibodies. An additional
embodiment of the invention utilizes the techniques described for
the construction of Fab expression libraries (Huse et al., 1989,
Science 246:1275-1281) to allow rapid and easy identification of
monoclonal Fab fragments with the desired specificity for c-Rel
proteins, derivatives, or analogs thereof. Non-human antibodies can
be "humanized" by known methods (e.g., U.S. Pat. No.
5,225,539).
[0079] Antibody fragments that contain the idiotypes of c-Rel can
be generated by techniques known in the art. For example, such
fragments include, but are not limited to, the F(ab')2 fragment
which can be produced by pepsin digestion of the antibody molecule;
the Fab' fragment that can be generated by reducing the disulfide
bridges of the F(ab')2 fragment; the Fab fragment that can be
generated by treating the antibody molecular with papain and a
reducing agent; and Fv fragments. Synthetic antibodies, e.g,
antibodies produced by chemical synthesis, are useful in the
present invention.
[0080] In the production of antibodies, screening for the desired
antibody can be accomplished by techniques known in the art, e.g.,
ELISA (enzyme-linked immunosorbent assay). To select antibodies
specific to a particular domain of c-Rel, or a derivative, homolog,
or analog thereof, one may assay generated hybridomas for a product
that binds to the fragment of the c-Rel protein, or a derivative,
homolog, or analog thereof, that contains such a domain.
Recombinant Expression
[0081] Methods for recombinant production of c-Rel and derivatives
or fragments or homologs thereof for use in the screening methods
of the present invention are well known to those skilled in the
art. Nucleic acids encoding c-Rel, derivatives, fragments, and
homologs thereof are known in the art. The nucleotide sequence
encoding an illustrative human c-Rel molecule is known and is
provided in FIG. 1 (SEQ ID NO:1). Nucleic acids encoding c-Rel can
be obtained by any method known in the art, e.g., by PCR
amplification using synthetic primers hybridizable to the 3' and 5'
ends of each sequence, and/or by cloning from a cDNA or genomic
library using an oligonucleotide specific for each nucleotide
sequence.
[0082] Homologs (e.g., nucleic acids encoding c-Rel of species
other than human) or other related sequences (e.g., paralogs) can
be obtained by low, moderate or high stringency hybridization with
all or a portion of the particular human sequence as a probe, using
methods well known in the art for nucleic acid hybridization and
cloning.
[0083] The encoded c-Rel protein, which is depicted in FIG. 1 (SEQ
ID NO:2) can be obtained by methods well known in the art for
protein purification and recombinant protein expression. For
recombinant expression of one or more of the proteins, the nucleic
acid containing all or a portion of the nucleotide sequence
encoding the protein can be inserted into an appropriate expression
vector, i.e., a vector that contains the necessary elements for the
transcription and translation of the inserted protein coding
sequence. The necessary transcriptional and translational signals
can also be supplied by the native promoter of the c-Rel gene,
and/or their flanking regions.
[0084] A variety of host-vector systems may be utilized to express
the protein coding sequence. These include but are not limited to
mammalian cell systems infected with virus (e.g., vaccinia virus,
adenovirus, etc.); insect cell systems infected with virus (e.g.,
baculovirus); microorganisms such as yeast containing yeast
vectors; or bacteria transformed with bacteriophage, DNA, plasmid
DNA, or cosmid DNA. The expression elements of vectors vary in
their strengths and specificities. Depending on the host-vector
system utilized, any one of a number of suitable transcription and
translation elements may be used.
[0085] In a preferred embodiment, human c-Rel is obtained by
expressing the human c-Rel coding sequence. In yet another
embodiment, a derivative, fragment or homolog of c-Rel is
recombinantly expressed. In one embodiment, the c-Rel protein is
expressed as chimeric or fusion protein in which an amino acid
sequence different from the c-Rel sequence is linked via a peptide
bond to the c-Rel sequence. The different amino acid sequence can
be a tag, such as a flag tag, for detection and isolation of the
expressed chimeric or fusion protein.
[0086] Any method available in the art can be used for the
insertion of DNA fragments into a vector to construct expression
vectors containing a chimeric gene consisting of appropriate
transcriptional/translational control signals and protein coding
sequences. These methods may include in vitro recombinant DNA and
synthetic techniques and in vivo recombinant techniques (genetic
recombination). Expression of nucleic acid sequences encoding
c-Rel, or a derivative, fragment or homolog thereof, may be
regulated by a second nucleic acid sequence so that the gene or
fragment thereof is expressed in a host transformed with the
recombinant DNA molecule(s). For example, expression of the
proteins may be controlled by any promoter/enhancer known in the
art. In a specific embodiment, the promoter is not native to the
gene for c-Rel. In another specific embodiment, the promoter is
active in immune cells, e.g., peripheral blood mononuclear cells,
dendritic cells or monocytes or splenocytes. Promoters that may be
used include but are not limited to the SV40 early promoter
(Bernoist and Chambon, 1981, Nature 290:304-310), the promoter
contained in the 3' long terminal repeat of Rous sarcoma virus
(Yamamoto et al., 1980, Cell 22:787-797), the herpes thymidine
kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA
78:1441-1445), the regulatory sequences of the metallothionein gene
(Brinster et al., 1982, Nature 296:39-42); prokaryotic expression
vectors such as the .beta.-lactamase promoter (Villa-Kamaroff et
al., 1978, Proc. Natl. Acad. Sci. USA 75:3727-3731) or the tac
promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25;
Gilbert et al., 1980, Scientific American 242:79-94); plant
expression vectors comprising the nopaline synthetase promoter
(Herrar-Estrella et al., 1984, Nature 303:209-213) or the
cauliflower mosaic virus 35S RNA promoter (Garder et al., 1981,
Nucleic Acids Res. 9:2871), and the promoter of the photosynthetic
enzyme ribulose bisphosphate carboxylase (Herrera-Estrella et al.,
1984, Nature 310:115-120); promoter elements from yeast and other
fungi such as the Gal4 promoter (Johnston et al., 1987, Microbiol.
Rev. 51:458-476), the alcohol dehydrogenase promoter (Schibler et
al., 1987, Annual Review Genetics 21:237-257), the phosphoglycerol
kinase promoter (Struhl et al., 1995, Annual Review Genetics
29:651-674-257; Guarente 1987, Annual Review Genetics 21:425-452),
the alkaline phosphatase promoter (Struhl et al., 1995, Annual
Review Genetics 29:651-674-257; Guarente 1987, Annual Review
Genetics 21:425-452), and the following animal transcriptional
control regions that exhibit tissue specificity and have been
utilized in transgenic animals: elastase I gene control region
which is active in pancreatic acinar cells (Swift et al., 1984,
Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp.
Quant. Biol. 50:399-409; MacDonald 1987, Hepatology 7:425-515);
insulin gene control region which is active in pancreatic beta
cells (Hanahan et al., 1985, Nature 315:115-122), immunoglobulin
gene control region which is active in lymphoid cells (Grosschedl
et al., 1984, Cell 38:647-658; Adams et al., 1985, Nature
318:533-538; Alexander et al., 1987, Mol. Cell Biol. 7:1436-1444),
mouse mammary tumor virus control region which is active in
testicular, breast, lymphoid and mast cells (Leder et al., 1986,
Cell 45:485-495), albumin gene control region which is active in
liver (Pinkert et al., 1987, Genes and Devel. 1:268-276),
alpha-fetoprotein gene control region which is active in liver
(Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et
al., 1987, Science 235:53-58), alpha-1 antitrypsin gene control
region which is active in liver (Kelsey et al., 1987, Genes and
Devel. 1:161-171), beta globin gene control region which is active
in myeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias
et al., 1986, Cell 46:89-94), myelin basic protein gene control
region which is active in oligodendrocyte cells of the brain
(Readhead et al., 1987, Cell 48:703-712), myosin light chain-2 gene
control region which is active in skeletal muscle (Sani 1985,
Nature 314:283-286), and gonadotrophic releasing hormone gene
control region which is active in gonadotrophs of the hypothalamus
(Mason et al., 1986, Science 234:1372-1378).
[0087] In a specific embodiment, a vector is used that comprises a
promoter operably linked to the nucleic acid sequence encoding
c-Rel, or a fragment, derivative or homo log thereof, one or more
origins of replication, and optionally, one or more selectable
markers (e.g., an antibiotic resistance gene).
[0088] In another specific embodiment, an expression vector
containing the coding sequence, or a portion thereof, of c-Rel is
made by subcloning the gene sequence into the EcoRI restriction
site of each of the three pGEX vectors (glutathione S-transferase
expression vectors; Smith and Johnson, 1988, Gene 7:31-40). This
allows for the expression of products in the correct reading
frame.
[0089] Expression vectors containing the sequences of interest can
be identified by three general approaches: (a) nucleic acid
hybridization, (b) presence or absence of "marker" gene function,
and (c) expression of the inserted sequences. In the first
approach, c-Rel sequences can be detected by nucleic acid
hybridization to probes comprising sequences homologous and
complementary to the inserted sequences. In the second approach,
the recombinant vector/host system can be identified and selected
based upon the presence or absence of certain "marker" functions
(e.g., resistance to antibiotics, occlusion body formation in
baculovirus, etc.) caused by insertion of the sequences of interest
in the vector. For example, if a c-Rel gene, or portion thereof, is
inserted within the marker gene sequence of the vector,
recombinants containing the c-Rel fragment will be identified by
the absence of the marker gene function (e.g., loss of
beta-galactosidase activity). In the third approach, recombinant
expression vectors can be identified by assaying for the c-Rel
expressed by the recombinant vector.
[0090] Once recombinant c-Rel molecules are identified and
isolated, several methods known in the art can be used to propagate
them. Using a suitable host system and growth conditions,
recombinant expression vectors can be propagated and amplified in
quantity. As previously described, the expression vectors or
derivatives which can be used include, but are not limited to,
human or animal viruses such as vaccinia virus or adenovirus;
insect viruses such as baculovirus, yeast vectors; bacteriophage
vectors such as lambda phage; and plasmid and cosmid vectors.
[0091] In addition, a host cell strain may be chosen that modulates
the expression of the inserted sequences, or modifies or processes
the expressed proteins in the specific fashion desired. Expression
from certain promoters can be elevated in the presence of certain
inducers; thus expression of the genetically-engineered c-Rel may
be controlled. Furthermore, different host cells have
characteristic and specific mechanisms for the translational and
post-translational processing and modification (e.g.,
glycosylation, phosphorylation, etc.) of proteins. Appropriate cell
lines or host systems can be chosen to ensure that the desired
modification and processing of the foreign protein is achieved. For
example, expression in a bacterial system can be used to produce an
unglycosylated core protein, while expression in mammalian cells
ensures "native" glycosylation of a heterologous protein.
Furthermore, different vector/host expression systems may effect
processing reactions to different extents.
[0092] In other specific embodiments, the c-Rel protein or a
fragment, homolog or derivative thereof, may be expressed as fusion
or chimeric protein products comprising the protein, fragment,
homolog, or derivative joined via a peptide bond to a heterologous
protein sequence of a different protein. Such chimeric products can
be made by ligating the appropriate nucleic acid sequences encoding
the desired amino acids to each other by methods known in the art,
in the proper coding frame, and expressing the chimeric products in
a suitable host by methods commonly known in the art.
Screening Methods for Identifying Modulators
[0093] In one embodiment of the invention, methods are provided for
the identification of modulators, e.g., inhibitors, antagonists, or
agonists, of c-Rel activity by detecting the ability of candidate
molecules to effect an alteration of c-Rel subcellular localization
(qualitatively and/or quantitatively), without materially altering
the level of expression of NF.kappa.B and/or amount of I.kappa.B,
and, thus, perhaps its activity in activating transcription of a
gene where c-Rel plays a role in creating the transcriptional
initiation complex by forming a sequence-specific DNA binding
complex at a NF.kappa.B binding site in the promoter and/or
enhancer of the gene. An illustrative example of such genes are
c-Rel-dependent cytokines, e.g., IL-12 and IL-23 whose subunits p40
and p35 each contain an NF.kappa.B site in its promoter. In one
aspect of this embodiment of the invention, the method for
identifying a modulator of c-Rel activity comprises providing a
cell with a candidate modulator molecule and detecting or measuring
the amount of c-Rel that co-purifies or co-localizes with the
nucleus without materially altering the levels of expression
NF.kappa.B or amount of I.kappa.B, wherein a difference in the
presence or amount of c-Rel co-purifying or co-localizing to the
nucleus compared to a cell not contacted with the candidate
molecule indicates that the candidate molecule modulates c-Rel
activity. Exemplary cells and cell lines useful in the screening
methods of the present invention include, but are not limited to,
macrophages, dendritic cells, monocytes, peripheral blood
mononuclear cells, which preferably are stimulated with IFN-.gamma.
and/or LPS prior to contacting with the candidate molecule.
[0094] A particular aspect of the present invention relates to
identifying molecules that inhibit or promote c-Rel localization to
the nucleus. In another particular aspect related to identifying
molecules that inhibit or promote c-Rel localization to the nucleus
without affecting the overall amount of c-Rel expressed in the
cell, either on the transcriptional or translational level. In a
preferred aspect, molecules are identified that reduce the amount
of c-Rel in the nucleus by, e.g., inhibiting translocation of c-Rel
into the nucleus or increasing the rate of degradation of c-Rel in
the nucleus. In other aspects, the amount of c-Rel in the nucleus
or c-Rel translocation to the nucleus is inhibited but the amount
of NF.kappa.B family member p65 increases in the nucleus. In yet
another aspect, c-Rel translocation to the nucleus is inhibited and
the amount of ICSBP is also reduced, with or without affecting the
levels of ICSBP mRNA or protein. In yet another aspect, c-Rel
translocation to the nucleus is reduced and the Ets-2 binding
domain in the promoter of p40 no longer has the ability to activate
transcription as compared to a cell where the amount of c-Rel in
the nucleus is not reduced.
[0095] Methods that can be used to carry out the foregoing are
commonly known in the art and/or those methods disclosed in Section
5.1, supra. The cells used in the methods of this embodiment of the
invention can either endogenously or recombinantly express c-Rel,
or a fragment, derivative or analog thereof. Recombinant expression
of c-Rel is carried out by introducing c-Rel encoding nucleic acids
into expression vectors and subsequently introducing the vectors
into a cell to express c-Rel or simply introducing c-Rel encoding
nucleic acids into a cell for expression, as described in Section
5.1.1 or using procedures well known in the art. In a specific
embodiment, c-Rel is expressed with a tag for ease of detection but
where the tag has no effect on c-Rel activity or subcellular
localization. Nucleic acids encoding c-Rel from a number of species
have been cloned and sequenced and their expression is well known
in the art. An illustrative example of a human c-Rel nucleotide and
amino acid sequence is set forth in FIG. 1 (SEQ ID NOS:1 and 2).
Expression can be from expression vectors or intrachromosomal. In a
specific embodiment, standard human cell lines, such as human
dendritic cell lines or the human monocyte cell line THP-1, or
human peripheral blood mononuclear cells, are employed in the
screening assays. In specific aspects, when immune cells are
employed, the immune cells are contacted with immunoactivating
compounds such as lipopolysaccharide (LPS) or interferon-.gamma.
(IFN-.gamma.), before, concurrently or after contacting with the
one or more candidate molecules.
[0096] Any method known to those of skill in the art for the
insertion of c-Rel-encoding DNA into a vector may be used to
construct expression vectors for expressing c-Rel, including those
methods described in Section 5.1, supra. In addition, a host cell
strain may be chosen which modulates the expression of c-Rel, or
modifies and processes the gene product in the specific fashion
desired. Expression from certain promoters can be elevated in the
presence of certain inducers; thus, expression of c-Rel protein may
be controlled. Furthermore, different host cells have
characteristic and specific mechanisms for the translational and
post-translational processing and modification (e.g.,
glycosylation, cleavage) of proteins. Appropriate cell lines or
host systems can be chosen to ensure the desired modification and
processing of the c-Rel protein expressed. illustrative cell lines
are those described in the Examples section, infra.
[0097] In another embodiment of the invention, methods are provided
for identifying a drug target for mediating selective inhibition of
c-Rel activity. One such illustrative method comprises the
following steps in the order stated: (a) labeling one or more
agents that reduce the amount of c-Rel in the nucleus of the cell
without materially altering the level of expression of NF.kappa.B
or amount of I.kappa.B; (b) contacting the cell with the one or
more labeled agents under conditions that form a complex between
the one or more labeled agents and the drug target; (c) isolating
the complex; and (d) identifying the drug target from the
complex.
Candidate Molecules
[0098] Any molecule known in the art can be tested for its ability
to modulate (increase or decrease) c-Rel activity as detected by a
change in the subcellular localization of c-ReI (or amount
thereof). By way of example, a change in the localization can be
detected by detecting a change in the amount of c-Rel that purifies
with or localizes to the nucleus before and after exposure to the
candidate molecules. For identifying a molecule that modulates
c-Rel activity, candidate molecules can be directly provided to a
cell expressing c-Rel, or, in the case of candidate proteins, can
be provided by providing their encoding nucleic acids under
conditions in which the nucleic acids are recombinantly expressed
to produce the candidate proteins within the c-Rel expressing
cell.
[0099] Preferred compounds that inhibit translocation of c-Rel to
the nucleus but do not materially alter expression of NF.kappa.B
and/or the amount of I.kappa.B include the following compounds:
[0100] Compound 1:
N-(1H-indol-3-ylmethylene)-N'-[4-morpholin-4-yl-6-(2-pyridin-2-yl-ethoxy)-
-[1,3,5]triazin-2-yl]-hydrazine; [0101] Compound 2:
N-(3-methyl-benzylidene)-N'-[6-morpholin-4-yl-2-(2-pyridin-2-yl-ethoxy)-p-
yrimidin-4-yl]-hydrazine; [0102] Compound 3:
N-(1H-indol-3-ylmethylene)-N'-[4-morpholin-4-yl-6-(2-morpholin-4-yl-ethox-
y)-pyridin-2-yl]-hydrazine [0103] Compound 4:
N-[3,5-Difluoro-2-morpholin-4-yl-6-(2-morpholin-4-yl-ethoxy)-pyridin-4-yl-
]-N'-(3-methyl-benzylidene)-hydrazine; [0104] Compound 5:
N-(3-methyl-benzylidene)-N'-[4-morpholin-4-yl-6-(2-morpholin-4-yl-ethoxy)-
-pyridin-2-yl]-hydrazine; [0105] Compound 6:
N-methyl-N'-(3-methyl-benzylidene)-N'-[4-morpholin-4-yl-6-(2-morpholin-4--
yl-ethoxy)-pyridin-2-yl]-hydrazine; [0106] Compound 7:
4-methyl-2-{[4-morpholin-4-yl-6-(2-morpholin-4-yl-ethoxy)-pyridin-2-yl]-h-
ydrazononomethyl}-phenylamine; [0107] Compound 8:
N-(6,7-dimethoxy-2-morpholin-4-yl-quinolin-4-yl)-N'-(3-methyl-benzylidene-
)-hydrazine; [0108] Compound 9:
N-(7-Chloro-2-morpholin-4-yl-quninazolin-4-yl)-N'-(3-methyl-benzylidene)--
hydrazine; [0109] Compound 10:
N-[7-methoxy-2-morpholin-4-yl-6-(2-phenoxy-ethoxy)-quinazolin-4-yl]-N'-(3-
-methyl-benzylidene)-hydrazine; [0110] Compound 11:
N-[6-Morpholin-4-yl-2-(2-pyridin-2-yl-ethoxy)-pyrimidin-4-ylmethylene]-N'-
-m-tolyl-hydrazine; [0111] Compound 12:
N-(3-Chloro-phenyl)-N'[6-morpholin-4-yl-2-(2-pyridin-2-yl-ethoxy)-pyrimid-
in-4-ylmethylene]-hydrazine; [0112] Compound 13:
N-(3-Methoxy-phenyl)-N'-[6-morpholin-4-yl-2-(2-pyridin-2-yl-ethoxy)-pyrim-
idin-4-ylmethylene]-hydrazine; and [0113] Compound 14:
N-(2,5-Dimethyl-phenyl)-N'-[6-morpholin-4-yl-2-(2-pyridin-2-yl-ethoxy)-py-
rimidin-4-ylmethylene]-hydrazine.
[0114] This embodiment of the invention is well suited to screen
chemical libraries for molecules which modulate, e.g., inhibit,
antagonize, or agonize, c-Rel activity by altering the amount of
c-Rel that purifies with or localizes to the nucleus. The chemical
libraries can be peptide libraries, peptidomimetic libraries,
chemically synthesized libraries, recombinant, e.g., phage display
libraries, and in vitro translation-based libraries, other
non-peptide synthetic organic libraries, etc.
[0115] Libraries screened using the methods of the present
invention can comprise a variety of types of compounds. Examples of
libraries that can be screened in accordance with the methods of
the invention include, but are not limited to, peptoids; random
biooligomers; diversomers such as hydantoins, benzodiazepines and
dipeptides; vinylogous polypeptides; nonpeptidal peptidomimetics;
oligocarbamates; peptidyl phosphonates; peptide nucleic acid
libraries; antibody libraries; carbohydrate libraries; and small
molecule libraries (preferably, small organic molecule libraries).
In some embodiments, the compounds in the libraries screened are
nucleic acid or peptide molecules. In a non-limiting example,
peptide molecules can exist in a phage display library. In other
embodiments, the types of compounds include, but are not limited
to, peptide analogs including peptides comprising non-naturally
occurring amino acids, e.g., D-amino acids, phosphorous analogs of
amino acids, such as .gamma.-amino phosphoric acids and
.gamma.-amino phosphoric acids, or amino acids having non-peptide
linkages, nucleic acid analogs such as phosphorothioates and PNAs,
hormones, antigens, synthetic or naturally occurring drugs,
opiates, dopamine, serotonin, catecholamines, thrombin,
acetylcholine, prostaglandins, organic molecules, pheromones,
adenosine, sucrose, glucose, lactose and galactose. Libraries of
polypeptides or proteins can also be used in the assays of the
invention.
[0116] In a preferred embodiment, the combinatorial libraries are
small organic molecule libraries including, but not limited to,
benzodiazepines, isoprenoids, thiazolidinones, metathiazanones,
pyrrolidines, morpholino compounds, and benzodiazepines. In another
embodiment, the combinatorial libraries comprise peptoids; random
bio-oligomers; benzodiazepines; diversomers such as hydantoins,
benzodiazepines and dipeptides; vinylogous polypeptides;
nonpeptidal peptidomimetics; oligocarbamates; peptidyl
phosphonates; peptide nucleic acid libraries; antibody libraries;
or carbohydrate libraries. Combinatorial libraries are themselves
commercially available (see, e.g., ComGenex, Princeton, N.J.;
Asinex, Moscow, Ru, Tripos, Inc., St. Louis, Mo.; ChemStar, Ltd,
Moscow, Russia; 3D Pharmaceuticals, Exton, Pa.; Martek Biosciences,
Columbia, Md.; etc.).
[0117] In a preferred embodiment, the library is preselected so
that the compounds of the library are more amenable for cellular
uptake. For example, compounds are selected based on specific
parameters such as, but not limited to, size, lipophilicity,
hydrophilicity, and hydrogen bonding, which enhance the likelihood
of compounds getting into the cells. In another embodiment, the
compounds are analyzed by three-dimensional or four-dimensional
computer computation programs.
[0118] The combinatorial compound library for use in accordance
with the methods of the present invention may be synthesized. There
is a great interest in synthetic methods directed toward the
creation of large collections of small organic compounds, or
libraries, which could be screened for pharmacological, biological
or other activity. The synthetic methods applied to create vast
combinatorial libraries are performed in solution or in the solid
phase, i.e., on a solid support. Solid-phase synthesis makes it
easier to conduct multi-step reactions and to drive reactions to
completion with high yields because excess reagents can be easily
added and washed away after each reaction step. Solid-phase
combinatorial synthesis also tends to improve isolation,
purification and screening. However, the more traditional solution
phase chemistry supports a wider variety of organic reactions than
solid-phase chemistry.
[0119] Combinatorial compound libraries of the present invention
may be synthesized using the apparatus described in U.S. Pat. No.
6,190,619 to Kilcoin et al., which is hereby incorporated by
reference in its entirety. U.S. Pat. No. 6,190,619 discloses a
synthesis apparatus capable of holding a plurality of reaction
vessels for parallel synthesis of multiple discrete compounds or
for combinatorial libraries of compounds.
[0120] In one embodiment, the combinatorial compound library can be
synthesized in solution. The method disclosed in U.S. Pat. No.
6,194,612 to Boger et al., which is hereby incorporated by
reference in its entirety, features compounds useful as templates
for solution phase synthesis of combinatorial libraries. The
template is designed to permit reaction products to be easily
purified from unreacted reactants using liquid/liquid or
solid/liquid extractions. The compounds produced by combinatorial
synthesis using the template will preferably be small organic
molecules. Some compounds in the library may mimic the effects of
non-peptides or peptides. In contrast to solid phase synthesize of
combinatorial compound libraries, liquid phase synthesis does not
require the use of specialized protocols for monitoring the
individual steps of a multistep solid phase synthesis (Egner et
al., 1995, J. Org. Chem. 60:2652; Anderson et al., 1995, J. Org.
Chem. 60:2650; Fitch et al., 1994, J. Org. Chem. 59:7955; Look et
al., 1994, J. Org. Chem. 49:7588; Metzger et al., 1993, Angew.
Chem., Int. Ed. Engl. 32:894; Youngquist et al., 1994, Rapid
Commun. Mass Spect. 8:77; Chu et al., 1995, J. Am. Chem. Soc.
117:5419; Brummel et aL, 1994, Science 264:399; and Stevanovic et
al., 1993, Bioorg. Med. Chem. Lett. 3:431).
[0121] Combinatorial compound libraries useful for the methods of
the present invention can be synthesized on solid supports. In one
embodiment, a split synthesis method, a protocol of separating and
mixing solid supports during the synthesis, is used to synthesize a
library of compounds on solid supports (see e.g., Lam et al., 1997,
Chem. Rev. 97:41-448; Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci.
USA 90:10922-10926 and references cited therein). Each solid
support in the final library has substantially one type of compound
attached to its surface. Other methods for synthesizing
combinatorial libraries on solid supports, wherein one product is
attached to each support, will be known to those of skill in the
art (see, e.g., Nefzi et al., 1997, Chem. Rev. 97:449-472).
[0122] As used herein, the term "solid support" is not limited to a
specific type of solid support. Rather a large number of supports
are available and are known to one skilled in the art. Solid
supports include silica gels, resins, derivatized plastic films,
glass beads, cotton, plastic beads, polystyrene beads, alumina
gels, and polysaccharides. A suitable solid support may be selected
on the basis of desired end use and suitability for various
synthetic protocols. For example, for peptide synthesis, a solid
support can be a resin such as p-methylbenzhydrylamine (pMBHA)
resin (Peptides International, Louisville, Ky.), polystyrenes
(e.g., PAM-resin obtained from Bachem Inc., Peninsula Laboratories,
etc.), including chloromethylpolystyrene, hydroxymethylpolystyrene
and aminomethylpolystyrene, poly(dimethylacrylamide)-grafted
styrene co-divinyl-benzene (e.g., POLYHIPE resin, obtained from
Aminotech, Canada), polyamide resin (obtained from Peninsula
Laboratories), polystyrene resin grafted with polyethylene glycol
(e.g., TENTAGEL or ARGOGEL, Bayer, Tubingen, Germany)
polydimethylacrylamide resin (obtained from MilligenlBiosearch,
California), or Sepharose (Pharmacia, Sweden).
[0123] In some embodiments of the present invention, compounds can
be attached to solid supports via linkers. Linkers can be integral
and part of the solid support, or they may be nonintegral that are
either synthesized on the solid support or attached thereto after
synthesis. Linkers are useful not only for providing points of
compound attachment to the solid support, but also for allowing
different groups of molecules to be cleaved from the solid support
under different conditions, depending on the nature of the linker.
For example, linkers can be, inter alia, electrophilically cleaved,
nucleophilically cleaved, photocleavable, enzymatically cleaved,
cleaved by metals, cleaved under reductive conditions or cleaved
under oxidative conditions. In a preferred embodiment, the
compounds are cleaved from the solid support prior to high
throughput screening of the compounds.
[0124] In certain embodiments of the invention, the compound is a
small molecule.
[0125] Exemplary libraries are commercially available from several
sources (ArQule, Tripos/PanLabs, ChemDesign, Pharmacopoeia). In
some cases, these chemical libraries are generated using
combinatorial strategies that encode the identity of each member of
the library on a substrate to which the member compound is
attached, thus allowing direct and immediate identification of a
molecule that is an effective modulator. Thus, in many
combinatorial approaches, the position on a plate of a compound
specifies that compound's composition. Also, in one example, a
single plate position may have from 1-20 chemicals that can be
screened by administration to a well containing the interactions of
interest. Thus, if modulation is detected, smaller and smaller
pools of interacting pairs can be assayed for the modulation
activity. By such methods, many candidate molecules can be
screened.
[0126] Many diversity libraries suitable for use are known in the
art and can be used to provide compounds to be tested according to
the present invention. Alternatively, libraries can be constructed
using standard methods. Chemical (synthetic) libraries, recombinant
expression libraries, or polysome-based libraries are exemplary
types of libraries that can be used.
[0127] The libraries can be constrained or semirigid (having some
degree of structural rigidity), or linear or nonconstrained. The
library can be a cDNA or genomic expression library, random peptide
expression library or a chemically synthesized random peptide
library, or non-peptide library. Expression libraries are
introduced into the cells in which the assay occurs, where the
nucleic acids of the library are expressed to produce their encoded
proteins.
[0128] In one embodiment, peptide libraries that can be used in the
present invention may be libraries that are chemically synthesized
in vitro. Examples of such libraries are given in Houghten et al.,
1991, Nature 354:84-86, which describes mixtures of free
hexapeptides in which the first and second residues in each peptide
were individually and specifically defined; Lam et al., 1991,
Nature 354:82-84, which describes a "one bead, one peptide"
approach in which a solid phase split synthesis scheme produced a
library of peptides in which each bead in the collection had
immobilized thereon a single, random sequence of amino acid
residues; Medynski, 1994, Bio/Technology 12:709-710, which
describes split synthesis and T-bag synthesis methods; and Gallop
et al., 1994, J. Medicinal Chemistry 37(9):1233-1251. Simply by way
of other examples, a combinatorial library may be prepared for use,
according to the methods of Ohlmeyer et at, 1993, Proc. Natl. Acad.
Sci. USA 90:10922-10926; Erb et al., 1994, Proc. Natl. Acad. Sci.
USA 91:11422-11426; Houghten et al., 1992, Biotechniques 13:412;
Jayawickreme et al., 1994, Proc. Natl. Acad. Sci. USA 91:1614-1618;
or Salmon et al., 1993, Proc. Natl. Acad. Sci. USA 90:11708-11712.
PCT Publication No. WO 93/20242 and Brenner and Lerner, 1992, Proc.
Natl. Acad. Sci. USA 89:5381-5383 describe "encoded combinatorial
chemical libraries," that contain oligonucleotide identifiers for
each chemical polymer library member.
[0129] In a preferred embodiment, the library screened is a
biological expression library that is a random peptide phage
display library, where the random peptides are constrained (e.g.,
by virtue of having disulfide bonding).
[0130] Further, more general, structurally constrained, organic
diversity (e.g., nonpeptide) libraries, can also be used. By way of
example, a benzodiazepine library (see e.g., Bunin et al., 1994,
Proc. Natl. Acad. Sci. USA 91:4708-4712) may be used.
[0131] Conformationally constrained libraries that can be used
include but are not limited to those containing invariant cysteine
residues which, in an oxidizing environment, cross-link by
disulfide bonds to form cystines, modified peptides (e.g.,
incorporating fluorine, metals, isotopic labels, are
phosphorylated, etc.), peptides containing one or more
non-naturally occurring amino acids, non-peptide structures, and
peptides containing a significant fraction of y-carboxyglutamic
acid.
[0132] Libraries of non-peptides, e.g., peptide derivatives (for
example, that contain one or more non-naturally occurring amino
acids) can also be used. One example of these is peptide libraries
(Simon et al., 1992, Proc. Natl. Acad. Sci. USA 89:9367-9371).
Peptoids are polymers of non-natural amino acids that have
naturally occurring side chains attached not to the alpha carbon
but to the backbone amino nitrogen. Since peptoids are not easily
degraded by human digestive enzymes, they are advantageously more
easily adaptable to drug use. Another example of a library that can
be used, in which the amide functionalities in peptides have been
premethylated to generate a chemically transformed combinatorial
library, is described by Ostresh et al., 1994, Proc. Natl. Acad.
Sci. USA 91:11138-11142).
[0133] The members of the peptide libraries that can be screened
according to the invention are not limited to containing the 20
naturally occurring amino acids. In particular, chemically
synthesized libraries and polysome based libraries allow the use of
amino acids in addition to the 20 naturally occurring amino acids
(by their inclusion in the precursor pool of amino acids used in
library production). In specific embodiments, the library members
contain one or more non-natural or non-classical amino acids or
cyclic peptides. Non-classical amino acids include but are not
limited to the D-isomers of the common amino acids, a-amino
isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid;
.gamma.-Abu, .epsilon.-Ahx, 6-amino hexanoic acid; Aib, 2-amino
isobutyric acid; 3-amino propionic acid; ornithine; norleucine;
norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid,
t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,
.beta.-alanine, designer amino acids such as .beta.-methyl amino
acids, C.alpha.-methyl amino acids, N.alpha.-methyl amino acids,
fluoro-amino acids and amino acid analogs in general. Furthermore,
the amino acid can be D (dextrorotary) or L (levorotary).
[0134] In a specific embodiment, fragments and/or analogs of c-Rel,
especially peptidomimetics, are screened for activity as
competitive or non-competitive inhibitors of c-Rel nuclear
localization or transport.
[0135] In another embodiment of the present invention,
combinatorial chemistry can be used to identify modulators of c-Rel
nuclear localization or transport. Combinatorial chemistry is
capable of creating libraries containing hundreds of thousands of
compounds, many of which may be structurally similar. While high
throughput screening programs are capable of screening these vast
libraries for affinity for known targets, new approaches have been
developed that achieve libraries of smaller dimension but which
provide maximum chemical diversity. (See e.g., Matter, 1997,
Journal of Medicinal Chemistry 40:1219-1229).
[0136] One method of combinatorial chemistry, affinity
fingerprinting, has previously been used to test a discrete library
of small molecules for binding affinities for a defined panel of
proteins. The fingerprints obtained by the screen are used to
predict the affinity of the individual library members for other
proteins or receptors of interest (in the instant invention,
c-Rel.) The fingerprints are compared with fingerprints obtained
from other compounds known to react with the protein of interest to
predict whether the library compound might similarly react. For
example, rather than testing every ligand in a large library for
interaction with c-Rel, those ligands having a fingerprint similar
to other compounds known to have that activity could be tested.
(See, e.g., Kauvar et al., 1995, Chemistry and Biology 2:107-118;
Kauvar, 1995, Affinity fingerprinting, Pharmaceutical Manufacturing
International. 8:25-28; and Kauvar, Toxic-Chemical Detection by
Pattern Recognition in New Frontiers in Agrochemical Immunoassay,
D. Kurtz. L. Stanker and J. H. Skerritt. Editors, 1995, AOAC:
Washington, D.C., 305-312).
[0137] Kay et al., 1993, Gene 128:59-65 (Kay) discloses a method of
constructing peptide libraries that encode peptides of totally
random sequence that are longer than those of any prior
conventional libraries. The libraries disclosed in Kay encode
totally synthetic random peptides of greater than about 20 amino
acids in length. Such libraries can be advantageously screened to
identify c-Rel modulators. (See also U.S. Pat. No. 5,498,538 dated
Mar. 12, 1996; and PCT Publication No. WO 94/18318 dated Aug. 18,
1994).
[0138] Other libraries can include antibody libraries and libraries
of intrabodies expressed in the cell.
[0139] If the library comprises arrays or microarrays of compounds,
wherein each compound has an address or identifier, the compound
can be deconvoluted, e.g., by cross-referencing the positive sample
to original compound list that was applied to the individual test
assays.
[0140] If the library is a peptide or nucleic acid library, the
sequence of the compound can be determined by direct sequencing of
the peptide or nucleic acid. Such methods are well known to one of
skill in the art.
[0141] A comprehensive review of various types of peptide libraries
can be found in Gallop et al., 1994, J. Med. Chem.
37:1233-1251.
Compounds Identified in Screening Assays
[0142] The present invention is further directed to the compounds
identified by the above-described screening assays and to processes
for producing such agents by use of these assays. The compounds can
include, but are not limited to, nucleic acids, antisense nucleic
acids, ribozyme, triple helix, antibody, and polypeptide molecules
and small inorganic or organic molecules. Accordingly, in one
embodiment, the present invention includes a compound obtained by a
method comprising the steps of any one of the aforementioned
screening assays. For example, the compound is obtained by a method
comprising contacting a cell with one or more candidate molecules;
and detecting localization of c-Rel molecules in the cell, wherein
an increase or decrease in the amount of c-Rel in the nucleus
without materially altering the level of expression of NF.kappa.B
and/or amount of I.kappa.B relative to said amount in a cell not so
contacted with the one or more candidate molecules.
[0143] Once a test compound has been identified as having an
appropriate activity according to the screening methods of the
present invention, the test compound can be subject to further
testing, for example, in animal models to confirm its activity as a
modulator of c-Rel activity or subcellular localization in the
animal, or for potential side effects. The test compound can also
be tested against known compounds that modulate c-Rel activity or
subcellular localization, in both cell based or animal assays, to
confirm its desired activity. The identified compound can also be
tested to determine its toxicity, or side effects that could be
associated with administration of such compound. Alternatively, a
compound identified as described herein can be used in an animal
model to determine the mechanism of action of such a compound.
[0144] Such exemplary evaluation methods for evaluating the
biological effect of an agent that reduces the amount of c-Rel in
the nucleus of a cell without materially altering the level of
expression of NF.kappa.B and/or amount of I.kappa.B in the cell
comprise contacting the cell with the agent and observing any
phenotypic effects in the cell. Another illustrative method
comprises administering the agent to a test subject/animal and
observing any phenotypic effects in the test subject/animal.
[0145] The present invention also pertains to uses of compounds
identified by the above-described screening assays for methods of
treatment as described herein. Accordingly, it is within the scope
of the present invention to use such compounds in the design,
formulation, synthesis, manufacture, and/or production of a drug or
pharmaceutical composition for use in diagnosis, prognosis, or
treatment, as described herein. For example, in one embodiment, the
present invention includes a method of synthesizing or producing a
drug or pharmaceutical composition by reference to the structure
and/or properties of a compound obtainable by one of the
above-described screening assays. For example, a drug or
pharmaceutical composition can be synthesized based on the
structure and/or properties of a compound obtained by the screening
methods described supra.
[0146] Furthermore, the identified compound, prior to formulation
for use in a method for treatment or prophylaxis can be modified
using methods known in the art to render the compound more stable,
i.e., increase its half-life in the subject, or render the compound
more readily absorbed into the tissues of the subject. Such
modifications include, but are not limited to, PEGylation,
multimerization. Such modifications are performed by a
pharmaceutical chemist to make the compound more suitable for
administration. Additionally, the identified compound can be
modified to allow for passage across the blood-brain barrier.
[0147] The compounds which display the desired biological activity
can be used as lead compounds for the development or design of
congeners or analogs having useful pharmacological activity. For
example, once a lead compound is identified, molecular modeling
techniques can be used to design variants of the compound that can
be more effective. Examples of molecular modeling systems are the
CHARM and QUANTA programs (Polygen Corporation, Waltham, Mass.).
CHARM performs the energy minimization and molecular dynamics
functions. QUANTA performs the construction, graphic modeling and
analysis of molecular structure. QUANTA allows interactive
construction, modification, visualization, and analysis of the
behavior of molecules with each other. Exemplary compounds that can
be used as lead compounds for the development or design of
congeners or analogs having useful pharmacological activity arc
described in U.S. Pat. No. 6,384,032; U.S. patent application Ser.
No. 09/594,362 filed May 7, 2002; U.S. patent application Ser. No.
10/006,624 filed Nov. 30, 2001 (Publication No. 20020082259); U.S.
patent application Ser. No. 10/000,742 filed Nov. 30, 2001
(Publication No. 20030139403); U.S. patent application Ser. No.
10/192,347 filed Jul. 10, 2002 (Publication No. 20030114446); U.S.
patent application Ser. No. 10/305,039 filed Nov. 26, 2002;
International Patent Publication No. WO 00/78757; International
Patent Publication No. WO 03/04516; International Patent
Application PCT/US03/32546 filed Oct. 14, 2003; U.S. Provisional
Patent Application Ser. No. 60/518,791 filed Nov. 10, 2003; U.S.
Provisional Patent Application Ser. No. 60/518,787 filed Nov. 10,
2003; U.S. Provisional Patent Application Ser. No. 60/518,788 filed
Nov. 10, 2003; U.S. patent application Ser. No. 10/985,627,
entitled, "Fused Heterocyclic Compounds," Mitsunori Ono et al,
filed Nov. 10, 2004; U.S. patent application Ser. No. 10/985,716,
entitled, "Heteroaryl Hydrazone Compounds," Mitsunori Ono et al,
filed Nov. 10, 2004; U.S. patent application Ser. No. 10/985,696,
entitled, "Pyridine Compounds," Mitsunori Ono, et al, filed Nov.
10, 2004, each of which is incorporated by reference herein in its
entirety.
[0148] A number of articles review computer modeling of drugs
interactive with specific proteins, such as Rotivinen et al., 1988,
Acta Pharmaceutical Fennica 97:159-166; Ripka, 1998, New Scientist
54-57; McKinaly & Rossmann, 1989, Annu. Rev. Pharmacol.
Toxiciol. 29:111-122; Perry & Davies, OSAR: Quantitative
Structure-Activity Relationships in Drug Design pp. 189-193 (Alan
R. Liss, Inc. 1989); Lewis & Dean, 1989, Proc. R. Soc. Lond.
236:125-140 and 141-162; Askew et al., 1989, J. Am. Chem. Soc.
111:1082-1090. Other computer programs that screen and graphically
depict chemicals are available from companies such as BioDesign,
Inc. (Pasadena, Calif.), Allelix, Inc. (Mississauga, Ontario,
Canada), and Hypercube, Inc. (Cambridge, Ontario). Although these
are primarily designed for application to drugs specific to
particular proteins, they can be adapted to design of drugs
specific to any identified region. Alternatively, lead compounds
with little or no biologic activity, as ascertained in the screen,
can also be used to design analogs and congeners of the compounds
that have biologic activity.
Pharmaceutical Compositions and Therapeutic/Prophylactic
Administration
[0149] The invention provides methods of treatment (and
prophylaxis) by administration to a subject of an effective amount
of a Therapeutic of the invention, i.e., a compound identified by
the screening methods of the present invention. In a preferred
aspect, the Therapeutic is substantially purified. The subject is
preferably an animal including, but not limited to animals such as
cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a
mammal, and most preferably human. In a specific embodiment, a
non-human mammal is the subject.
[0150] In a particular embodiment, the present invention provides a
method for treating a disease or disorder characterized by aberrant
subcellular localization of c-Rel without a material alteration of
the levels of expression of NF.kappa.B or amount of I.kappa.B
comprising administering to a subject having such disease or
disorder a composition comprising a molecule that reduces c-Rel
nuclear localization without materially altering the levels of
expression of NF.kappa.B or amount of I.kappa.B and a
pharmaceutically acceptable carrier. In another particular
embodiment, the invention provides a method for treating an IL-12
production-related disease or disorder comprising administering to
a subject having such a disease or disorder a composition
comprising a molecule that reduces c-Rel nuclear localization
without materially altering the levels of expression of NF.kappa.B
or amount of I.kappa.B and a pharmaceutically acceptable carrier.
In another particular embodiment, the invention provides a method
for treating a disease or disorder associated with c-Rel-dependent
cytokine production comprising administering to a subject having
such a disease or disorder a composition comprising a molecule that
reduces c-Rel nuclear localization without materially altering the
levels of expression of NF.kappa.B or amount of I.kappa.B and a
pharmaceutically acceptable carrier. In yet another particular
embodiment, the invention provides a method for treating an
autoimmune disease or disorder comprising administering to a
subject having such a disease or disorder a composition comprising
a molecule that reduces c-Rel nuclear localization without
materially altering the levels of expression of NF.kappa.B or
amount of I.kappa.B and a pharmaceutically acceptable carrier. The
molecule that reduces c-Rel nuclear localization without materially
altering the levels of expression of NF.kappa.B or amount of
I.kappa.B in the aforementioned methods can be those identified by
screening methods herein (e.g., those delineated in the detailed
description).
[0151] The compounds and compositions described herein are useful
to treat and prevent any IL-12 production-related disorders, e.g.,
inflammatory disorders, immune diseases, neurological disorders and
bone loss diseases. Methods of treatment and prevention are also
provided.
[0152] The term "inflammatory disorders" includes any inflammatory
disease, disorder or condition caused, exasperated or mediated by
IL-12 production. Such inflammatory disorders may include, without
limitation, asthma, adult respiratory distress syndrome, systemic
lupus erythematosus, inflammatory bowel disease (including Crohn's
disease and ulcerative colitis), multiple sclerosis,
insulin-dependent diabetes mellitus, autoimmune arthritis
(including rheumatoid arthritis, juvenile rheumatoid arthritis,
psoriatic arthritis), inflammatory pulmonary syndrome, pemphigus
vulgaris, idiopathic thrombocytopenic purpura, autoimmune
meningitis, myasthenia gravis, autoimmune thyroiditis, dermatitis
(including atopic dermatitis and eczematous dermatitis), psoriasis,
Sjogren's Syndrome (including keratoconjunctivitis sicca secondary
to Sjogren's Syndrome), alopecia areata, allergic responses due to
arthropod bite reactions, aphthous ulcer, iritis, conjunctivitis,
keratoconjunctivitis, cutaneous lupus erythematosus, scleroderma,
vaginitis, proctitis, drug eruptions (such as Stevens-Johnson
syndrome), leprosy reversal reactions, erythema nodosum leprosum,
autoimmune uveitis, allergic encephalomyelitis, aplastic anemia,
pure red cell anemia, idiopathic thrombocytopenia, polychondritis,
Wegener's granulomatosis, chronic active hepatitis, Graves
ophthalmopathy, primary biliary cirrhosis, uveitis posterior and
interstitial lung fibrosis.
[0153] "Inflammatory disorders" expressly include acute
inflammatory disorders. Examples of acute inflammatory disorders
include graft versus host disease, transplant rejection, septic
shock, endotoxemia, Lyme arthritis, infectious meningitis (e.g.,
viral, bacterial, Lyme disease-associated), an acute episode of
asthma and acute episodes of an autoimmune disease.
[0154] "Inflammatory disorders" expressly include chronic
inflammatory disorders. Nonlimiting examples of chronic
inflammatory disorder include asthma, rubella arthritis, and
chronic autoimmune diseases, such as systemic lupus erythematosus,
psoriasis, inflammatory bowel disease, including Crohn's disease
and ulcerative colitis, multiple sclerosis and rheumatoid
arthritis.
[0155] The term "immune diseases" includes any immune disease,
disorder or condition caused, exasperated or mediated by IL-12
production. Such immune diseases may include, without limitation,
rheumatoid arthritis, juvenile rheumatoid arthritis, systemic onset
juvenile rheumatoid arthritis, psoriatic arthritis, ankylosing
spondilitis, gastric ulcer, seronegative arthropathies,
osteoarthritis, inflammatory bowel disease, ulcerative colitis,
systemic lupus erythematosis, antiphospholipid syndrome,
iridocyclitis/uveitis/optic neuritis, idiopathic pulmonary
fibrosis, systemic vasculitis/wegener's granulomatosis,
sarcoidosis, orchitis/vasectomy reversal procedures,
allergic/atopic diseases, asthma, allergic rhinitis, eczema,
allergic contact dermatitis, allergic conjunctivitis,
hypersensitivity pneumonitis, transplants, organ transplant
rejection, graft-versus-host disease, systemic inflammatory
response syndrome, sepsis syndrome, gram positive sepsis, gram
negative sepsis, culture negative sepsis, fungal sepsis,
neutropenic fever, urosepsis, meningococcemia, trauma/hemorrhage,
burns, ionizing radiation exposure, acute pancreatitis, adult
respiratory distress syndrome, rheumatoid arthritis,
alcohol-induced hepatitis, chronic inflammatory pathologies,
sarcoidosis, Crohn's pathology, sickle cell anemia, diabetes,
nephrosis, atopic diseases, hypersensitity reactions, allergic
rhinitis, hay fever, perennial rhinitis, conjunctivitis,
endometriosis, asthma, urticaria, systemic anaphalaxis, dermatitis,
pernicious anemia, hemolytic disease, thrombocytopenia, graft
rejection of any organ or tissue, kidney transplant rejection,
heart transplant rejection, liver transplant rejection, pancreas
transplant rejection, lung transplant rejection, bone marrow
transplant (BMT) rejection, skin allograft rejection, cartilage
transplant rejection, bone graft rejection, small bowel transplant
rejection, fetal thymus implant rejection, parathyroid transplant
rejection, xenograft rejection of any organ or tissue, allograft
rejection, anti-receptor hypersensitivity reactions, Graves
disease, Raynoud's disease, type B insulin-resistant diabetes,
asthma, myasthenia gravis, antibody-meditated cytotoxicity, type
III hypersensitivity reactions, systemic lupus erythematosus, POEMS
syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal
gammopathy, and skin changes syndrome), polyneuropathy,
organomegaly, endocrinopathy, monoclonal gammopathy, skin changes
syndrome, antiphospholipid syndrome, pemphigus, scleroderma, mixed
connective tissue disease, idiopathic Addison's disease, diabetes
mellitus, chronic active hepatitis, primary biliary cirrhosis,
vitiligo, vasculitis, post-MI cardiotomy syndrome, type IV
hypersensitivity, contact dermatitis, hypersensitivity pneumonitis,
allograft rejection, granulomas due to intracellular organisms,
drug sensitivity, metabolic/idiopathic, Wilson's disease,
hemachromatosis, alpha-l-antitrypsin deficiency, diabetic
retinopathy, hashimoto's thyroiditis, osteoporosis,
hypothalamic-pituitary-adrenal axis evaluation, primary biliary
cirrhosis, thyroiditis, encephalomyelitis, cachexia, cystic
fibrosis, neonatal chronic lung disease, chronic obstructive
pulmonary disease (COPD), familial hematophagocytic
lymphohistiocytosis, dermatologic conditions, psoriasis, alopecia,
nephrotic syndrome, nephritis, glomerular nephritis, acute renal
failure, hemodialysis, uremia, toxicity, preeclampsia, okt3
therapy, anti-cd3 therapy, cytokine therapy, chemotherapy,
radiation therapy (e.g., including but not limited toasthenia,
anemia, cachexia, and the like), chronic salicylate intoxication,
and the like. See, e.g., the Merck Manual, 12th-17th Editions,
Merck & Company, Rahway, N.J. (1972, 1977, 1982, 1987, 1992,
1999), Pharmacotherapy Handbook, Wells et al., eds., Second
Edition, Appleton and Lange, Stamford, Conn. (1998, 2000), each
entirely incorporated by reference.
[0156] The term "neurological disorder" includes any neurological
disease, disorder or condition caused, exasperated or mediated by
IL-12 production. Such neurological disorders may include, without
limitation, neurodegenerative diseases, multiple sclerosis,
migraine headache, AIDS dementia complex, demyelinating diseases,
such as multiple sclerosis and acute transverse myelitis;
extrapyramidal and cerebellar disorders' such as lesions of the
corticospinal system; disorders of the basal ganglia or cerebellar
disorders; hyperkinetic movement disorders such as Huntington's
Chorea and senile chorea; drug-induced movement disorders, such as
those induced by drugs which block CNS dopamine receptors;
hypokinetic movement disorders, such as Parkinson's disease;
Progressive supranucleo Palsy; structural lesions of the
cerebellum; spinocerebellar degenerations, such as spinal ataxia,
Friedreich's ataxia, cerebellar cortical degenerations, multiple
systems degenerations (Mencel, Dejerine-Thomas, Shi-Drager, and
Machado-Joseph); systemic disorders (Refsum's disease,
abetalipoprotemia, ataxia telangiectasia, and mitochondrial
multi-system disorder); demyelinating core disorders, such as
multiple sclerosis, acute transverse myelitis; and disorders of the
motor unit` such as neurogenic muscular atrophies (anterior horn
cell degeneration, such as amyotrophic lateral sclerosis, infantile
spinal muscular atrophy and juvenile spinal muscular atrophy);
Alzheimer's disease; Down's Syndrome in middle age; Diffuse Lewy
body disease; Senile Dementia of Lewy body type; Wemicke-Korsakoff
syndrome; chronic alcoholism; Creutzfeldt-Jakob disease; Subacute
sclerosing panencephalitis, Hallerrorden-Spatz disease; and
Dementia pugilistica, and the like. Such a method can optionally
comprise administering an effective amount of a composition or
pharmaceutical composition comprising at least one TNF antibody or
specified portion or variant to a cell, tissue, organ, animal or
patient in need of such modulation, treatment or therapy. See,
e.g., the Merck Manual, 16, Edition, Merck & Company, Rahway,
N.J. (1992).
[0157] The term "bone loss disease" includes any bone loss disease,
disorder or condition caused, exasperated or mediated by IL-12
production e.g., periodontal disease, non-malignant bone disorders
(e.g., osteoporosis, Paget's disease of bone, osteogenesis
imperfecta, fibrous dysplasia, and primary hyperparathyroidism),
estrogen deficiency, inflammatory bone loss, bone malignancy,
arthritis, osteopetrosis, and certain cancer-related disorders
(e.g., hypercalcemia of malignancy (HCM), osteolytic bone lesions
of multiple myeloma and osteolytic bone metastases of breast cancer
and other metastatic cancers.
[0158] In the case of overlap in these definitions, the disease,
condition or disorder may be considered to be a member of any of
the above listed classes of IL-12 production-related disorders.
Specific IL-12 production related diseases include rheumatoid
arthritis, sepsis, Crohn's disease, multiple sclerosis, psoriasis,
or insulin-dependent diabetes mellitus).
[0159] Formulations and methods of administration that can be
employed when the Therapeutic comprises a modulating compound
identified by the assays described, supra; additional appropriate
formulations and routes of administration can be selected from
among those described herein below. Moreover, a Therapeutic of the
invention can be also be administered in conjunction with any known
drug to treat the disease or disorder of the invention.
[0160] Various delivery systems are known and can be used to
administer a Therapeutic of the invention, e.g., encapsulation in
liposomes, microparticles, and microcapsules, use of cells capable
of expressing the Therapeutic, use of receptor-mediated endocytosis
(e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432); construction
of a Therapeutic nucleic acid as part of a retroviral or other
vector, etc. Methods of introduction include but are not limited to
intradermal, intramuscular, intraperitoneal, intravenous,
subcutaneous, intranasal, epidural, and oral routes. The compounds
may be administered by any convenient route, for example by
infusion, by bolus injection, by absorption through epithelial or
mucocutaneous linings (e.g., oral, rectal and intestinal mucosa,
etc.), and may be administered together with other biologically
active agents. Administration can be systemic or local. In
addition, it may be desirable to introduce the pharmaceutical
compositions of the invention into the central nervous system by
any suitable route, including intraventricular and intrathecal
injection; intraventricular injection may be facilitated by an
intraventricular catheter, for example, attached to a reservoir,
such as an Ommaya reservoir. Pulmonary administration can also be
employed, e.g., by use of an inhaler or nebulizer, and formulation
with an aerosolizing agent.
[0161] In a preferred embodiment, the Therapeutic is formulated for
oral administration. These dosage forms include tablets (coated or
uncoated), caplets, hard gelatin capsules, soft gelatin capsules,
troches, dragees, dispersions, suspensions, solutions, and the
like, including sustained release formulations well known in the
art. See, e.g., Introduction to Pharmaceutical Dosage Forms, 1985,
Ansel, H. C., Lea and Febiger, Philadelphia, Pa.; Remington's
Pharmaceutical Sciences, 1995, Mack Publ. Co., Easton, Pa. Because
of their ease of administration, tablets and capsules are preferred
and represent the most advantageous oral dosage unit form, in which
case solid pharmaceutical excipients are employed. If desired,
tablets or caplets or capsules may be coated by standard aqueous or
non-aqueous techniques.
[0162] In a specific embodiment, it may be desirable to administer
the pharmaceutical compositions of the invention locally to the
area in need of treatment. This may be achieved by, for example,
and not by way of limitation, local infusion during surgery,
topical application, e.g., in conjunction with a wound dressing
after surgery, by injection, by means of a catheter, by means of a
suppository, or by means of an implant, said implant being of a
porous, nonporous, or gelatinous material, including membranes,
such as sialastic membranes, or fibers. In one embodiment,
administration can be by direct injection at the site (or former
site) of a malignant tumor or neoplastic or pre-neoplastic
tissue.
[0163] In another embodiment, the Therapeutic can be delivered in a
vesicle, in particular a liposome (Langer, 1990, Science
249:1527-1533; Treat et al., 1989, In: Liposomes in the Therapy of
Infectious Disease and Cancer, Lopez-Berestein and Fidler, eds.,
Liss, New York, pp. 353-365; Lopez-Berestein, ibid., pp. 317-327;
see generally ibid.)
[0164] In yet another embodiment, the Therapeutic can be delivered
via a controlled release system. In one embodiment, a pump may be
used (Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng.
14:201-240; Buchwald et al., 1980, Surgery 88:507-516; Saudek et
al., 1989, N. Engl. J. Med. 321:574-579). In another embodiment,
polymeric materials can be used (Medical Applications of Controlled
Release, Langer and Wise, eds., CRC Press, Boca Raton, Fla., 1974;
Controlled Drug Bioavailability, Drug Product Design and
Performance, Smolen and Ball, eds., Wiley, New York, 1984; Ranger
and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61; Levy
et al., 1985, Science 228:190-192; During et al., 1989, Ann.
Neurol. 25:351-356; Howard et al., 1989, J. Neurosurg. 71:858-863).
In yet another embodiment, a controlled release system can be
placed in proximity of the therapeutic target, i.e., the brain,
thus requiring only a fraction of the systemic dose (e.g., Goodson,
1984, In: Medical Applications of Controlled Release, supra, Vol.
2, pp. 115-138). Other controlled release systems are discussed in
the review by Langer (1990, Science 249:1527-1533).
[0165] The present invention also provides pharmaceutical
compositions. Such compositions comprise a therapeutically
effective amount of a Therapeutic, and a pharmaceutically
acceptable carrier. In a specific embodiment, the term
"pharmaceutically acceptable" means approved by a regulatory agency
of the Federal or a state government or listed in the U.S.
Pharmacopeia or other generally recognized pharmacopeia for use in
animals, and more particularly, in humans. The term "carrier"
refers to a diluent, adjuvant, excipient, or vehicle with which the
therapeutic is administered. Such pharmaceutical carriers can be
sterile liquids, such as water and oils, including those of
petroleum, animal, vegetable or synthetic origin, including but not
limited to peanut oil, soybean oil, mineral oil, sesame oil and the
like. Water can be a preferred carrier when the pharmaceutical
composition is administered orally. Saline and aqueous dextrose are
preferred carriers when the pharmaceutical composition is
administered intravenously. Saline solutions and aqueous dextrose
and glycerol solutions are preferably employed as liquid carriers
for injectable solutions. Suitable pharmaceutical excipients
include starch, glucose, lactose, sucrose, gelatin, malt, rice,
flour, chalk, silica gel, sodium stearate, glycerol monostearate,
talc, sodium chloride, dried skim milk, glycerol, propylene,
glycol, water, ethanol and the like. The composition, if desired,
can also contain minor amounts of wetting or emulsifying agents, or
pH buffering agents. These compositions can take the form of
solutions, suspensions, emulsions, tablets, pills, capsules,
powders, sustained-release formulations and the like. The
composition can be formulated as a suppository, with traditional
binders and carriers such as triglycerides. Oral formulation can
include standard carriers such as pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharine,
cellulose, magnesium carbonate, etc. Examples of suitable
pharmaceutical carriers are described in "Remington's
Pharmaceutical Sciences" by E. W. Martin. Such compositions will
contain a therapeutically effective amount of the Therapeutic,
preferably in purified form, together with a suitable amount of
carrier so as to provide the form for proper administration to the
patient. The formulation should suit the mode of
administration.
[0166] In a preferred embodiment, the composition is formulated, in
accordance with routine procedures, as a pharmaceutical composition
adapted for intravenous administration to human beings. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition
may also include a solubilizing agent and a local anesthetic such
as lidocaine to ease pain at the site of the injection. Generally,
the ingredients are supplied either separately or mixed together in
unit dosage form, for example, as a dry lyophilized powder or
water-free concentrate in a hermetically sealed container such as
an ampoule or sachette indicating the quantity of active agent.
Where the composition is to be administered by infusion, it can be
dispensed with an infusion bottle containing sterile pharmaceutical
grade water or saline. Where the composition is administered by
injection, an ampoule of sterile water or saline for injection can
be provided so that the ingredients may be mixed prior to
administration.
[0167] The Therapeutics of the invention can be formulated as
neutral or salt forms. Pharmaceutically acceptable salts include
those formed with free carboxyl groups such as those derived from
hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc.,
those formed with free amine groups such as those derived from
isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,
procaine, etc., and those derived from sodium, potassium, ammonium,
calcium, and ferric hydroxides, etc.
[0168] Preferred pharmaceutical compositions and dosage forms
comprise a Therapeutic of the invention, or a pharmaceutically
acceptable prodrug, salt, solvate, or clathrate thereof, optionally
in combination with one or more additional active agents.
[0169] The amount of the Therapeutic of the invention which will be
effective in the treatment of a particular disorder or condition
will depend on the nature of the disorder or condition, and can be
determined by standard clinical techniques. In addition, in vitro
assays may optionally be employed to help identify optimal dosage
ranges. The precise dose to be employed in the formulation will
also depend on the route of administration, and the seriousness of
the disease or disorder, and should be decided according to the
judgment of the practitioner and each patient's circumstances.
However, suitable dosage ranges for intravenous administration are
generally about 1-50 milligrams of active compound per kilogram
body weight. Suitable dosage ranges for intranasal administration
are generally about 0.1 mg/kg body weight to 50 mg/kg body weight.
Effective doses may be extrapolated from dose-response curves
derived from in vitro or animal model test systems.
[0170] Suppositories generally contain active ingredient in the
range of 0.5% to 10% by weight; oral formulations preferably
contain 10% to 95% active ingredient.
[0171] Exemplary doses of a small molecule include milligram or
microgram amounts of the small molecule per kilogram of subject or
sample weight (e.g., about 1 microgram per kilogram to about 500
milligrams per kilogram, about 100 micrograms per kilogram to about
5 milligrams per kilogram, or about 1 microgram per kilogram to
about 50 micrograms per kilogram).
[0172] For antibodies, proteins, polypeptides, peptides and fusion
proteins encompassed by the invention, the dosage administered to a
patient is typically 0.0001 mg/kg to 100 mg/kg of the patient's
body weight. Preferably, the dosage administered to a patient is
between 0.0001 mg/kg and 20 mg/kg, 0.0001 mg/kg and 10 mg/kg,
0.0001 mg/kg and 5 mg/kg, 0.0001 and 2 mg/kg, 0.0001 and 1 mg/kg,
0.0001 mg/kg and 0.75 mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001
mg/kg to 0.25 mg/kg, 0.0001 to 0.15 mg/kg, 0.0001 to 0.10 mg/kg,
0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kg or 0.01 to 0.10 mg/kg of the
patient's body weight. Generally, human antibodies have a longer
half-life within the human body than antibodies from other species
due to the immune response to the foreign polypeptides. Thus, lower
dosages of human antibodies and less frequent administration is
often possible. Further, the dosage and frequency of administration
of antibodies of the invention or fragments thereof may be reduced
by enhancing uptake and tissue penetration of the antibodies by
modifications such as, for example, lipidation.
[0173] Moreover, in certain embodiments, since IL-12 production can
be inhibited at a lower drug concentration that that needed to
inhibit IL-6 or 1FN-.gamma. production, appropriate dosages include
those that selectively inhibit IL-12 production but not other
cytokines.
[0174] The Therapeutics of the present invention may also be
administered by controlled release means or delivery devices that
are well known to those of ordinary skill in the art, such as those
described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809;
3,598,123; and 4,008,719, 5,674,533, 5,059,595, 5,591,767,
5,120,548, 5,073,543, 5,639,476, 5,354,556, and 5,733,566. These
controlled release compositions can be used to provide slow or
controlled-release of one or more of the active ingredients therein
using, for example, hydropropylmethyl cellulose, other polymer
matrices, gels, permeable membranes, osmotic systems, multilayer
coatings, microparticles, liposomes, microspheres, or the like, or
a combination thereof to provide the desired release profile in
varying proportions. Suitable controlled-release formulations known
to those of ordinary skill in the art may be readily selected for
use with the pharmaceutical compositions of the invention.
[0175] All controlled-release pharmaceutical products have a common
goal of improving drug therapy over that achieved by their
non-controlled counterparts. Ideally, the use of an optimally
designed controlled-release preparation in medical treatment is
characterized by a minimum of drug substance being employed to cure
or control the condition in a minimum amount of time. Advantages of
controlled-release formulations may include extended activity of
the drug, reduced dosage frequency, and/or increased patient
compliance.
[0176] Most controlled-release formulations are designed to
initially release an amount of the Therapeutic that promptly
produces the desired therapeutic effect, and gradually and
continually releases other amounts of the Therapeutic to maintain
the appropriate level of therapeutic effect over an extended period
of time. In order to maintain this constant level of Therapeutic in
the body, the Therapeutic must be released from the composition at
a rate that will replace the amount of Therapeutic being
metabolized and excreted from the body. The controlled-release of
the Therapeutic may be stimulated by various inducers, for example,
pH, temperature, enzymes, water, or other physiological conditions
or compounds. Such controlled-release components in the context of
the present invention include, but are not limited to, polymers,
polymer matrices, gels, permeable membranes, liposomes,
microspheres, or the like, or a combination thereof, that
facilitates the controlled-release of the active ingredient.
[0177] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Optionally associated with such container(s) can be a notice in the
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects approval by the agency of manufacture, use or
sale for human administration.
[0178] The methods for treating or preventing an IL-12 production
related disease or disorders, or those associated with aberrant
c-Rel subcellular localization or autoimmune disease or disorders
in a patient in need thereof can further comprise administering to
the patient being administered a compound of this invention, an
effective amount of one or more other therapeutic agents. Such
therapeutic agents may include other therapeutic agents such as
those conventionally used to prevent or treat disorders associated
with IL-12 production or aberrant c-Rel subcellular localization or
symptoms thereof. The other therapeutic agent can be a steroid or a
non-steroidal anti-inflammatory agent. Useful non-steroidal
anti-inflammatory agents, include, but are not limited to, aspirin,
ibuprofen, diclofenac, naproxen, benoxaprofen, flurbiprofen,
fenoprofen, flubufen, ketoprofen, indoprofen, piroprofen,
carprofen, oxaprozin, pramoprofen, muroprofen, trioxaprofen,
suprofen, aminoprofen, tiaprofenic acid, fluprofen, bucloxic acid,
indomethacin, sulindac, tolmetin, zomepirac, tiopinac, zidometacin,
acemetacin, fentiazac, clidanac, oxpinac, mefenamic acid,
meclofenamic acid, flufenamic acid, niflumic acid, tolfenamic acid,
diflurisal, flufenisal, piroxicam, sudoxicam, isoxicam; salicylic
acid derivatives, including aspirin, sodium salicylate, choline
magnesium trisalicylate, salsalate, diflunisal, salicylsalicylic
acid, sulfasalazine, and olsalazin; para-aminophennol derivatives
including acetaminophen and phenacetin; indole and indene acetic
acids, including indomethacin, sulindac, and etodolac; heteroaryl
acetic acids, including tolmetin, diclofenac, and ketorolac;
anthranilic acids (fenamates), including mefenamic acid, and
meclofenamic acid; enolic acids, including oxicams (piroxicam,
tenoxicam), and pyrazolidinediones (phenylbutazone,
oxyphenthartazone); and alkanones, including nabumetone and
pharmaceutically acceptable salts thereof and mixtures thereof. For
a more detailed description of the NSAIDs, see Paul A. Insel,
Analgesic-Antipyretic and Antiinflammatory Agents and Drugs
Employed in the Treatment of Gout, in Goodman & Gilman's The
Pharmacological Basis of Therapeutics 617-57 (Perry B. Molinhoff
and Raymond W. Ruddon eds., 9.sup.th ed 1996) and Glen R. Hanson,
Analgesic, Antipyretic and Anti-Inflammatory Drugs in Remington:
The Science and Practice of Pharmacy Vol II 1196-1221 (A. R.
Gennaro ed. 19th ed. 1995) which are hereby incorporated by
reference in their entireties.
[0179] Other Examples of prophylactic and therapeutic agents
include, but are not limited to, immunomodulatory agents,
anti-inflammatory agents (e.g., adrenocorticoids, corticosteroids
(e.g., beclomethasone, budesonide, flunisolide, fluticasone,
triamcinolone, methlyprednisolone, prednisolone, prednisone,
hydrocortisone), glucocorticoids, steroids, non-steriodal
anti-inflammatory drugs (e.g., aspirin, ibuprofen, diclofenac, and
COX-2 inhibitors), and leukotreine antagonists (e.g., montelukast,
methyl xanthines, zafirlukast, and zileuton), beta2-agonists (e.g.,
albuterol, biterol, fenoterol, isoetharie, metaproterenol,
pirbuterol, salbutamol, terbutalin formoterol, salmeterol, and
salbutamol terbutaline), anticholinergic agents (e.g., ipratropium
bromide and oxitropium bromide), sulphasalazine, penicillamine,
dapsone, antihistamines, anti-malarial agents (e.g.,
hydroxychloroquine), anti-viral agents, and antibiotics (e.g.,
dactinomycin (formerly actinomycin), bleomycin, erythomycin,
penicillin, mithramycin, and anthramycin (AMC)).
[0180] In combination therapy treatment, both the compounds of this
invention and the other drug agent(s) are administered to mammals
(e.g., humans, male or female) by conventional methods. The agents
may be administered in a single dosage form or in separate dosage
forms. Effective amounts of the other therapeutic agents are well
known to those skilled in the art. However, it is well within the
skilled artisan's purview to determine the other therapeutic
agent's optimal effective-amount range. In one embodiment of the
invention where another therapeutic agent is administered to an
animal, the effective amount of the compound of this invention is
less than its effective amount would be where the other therapeutic
agent is not administered. In another embodiment, the effective
amount of the conventional agent is less than its effective amount
would be where the compound of this invention is not administered.
In this way, undesired side effects associated with high doses of
either agent may be minimized. Other potential advantages
(including without limitation improved dosing regimens and/or
reduced drug cost) will be apparent to those of skill in the
art.
[0181] In various embodiments, the therapies (e.g., prophylactic or
therapeutic agents) are administered less than 5 minutes apart,
less than 30 minutes apart, 1 hour apart, at about 1 hour apart, at
about 1 to about 2 hours apart, at about 2 hours to about 3 hours
apart, at about 3 hours to about 4 hours apart, at about 4 hours to
about 5 hours apart, at about 5 hours to about 6 hours apart, at
about 6 hours to about 7 hours apart, at about 7 hours to about 8
hours apart, at about 8 hours to about 9 hours apart, at about 9
hours to about 10 hours apart, at about 10 hours to about 11 hours
apart, at about 11 hours to about 12 hours apart, at about 12 hours
to 18 hours apart, 18 hours to 24 hours apart, 24 hours to 36 hours
apart, 36 hours to 48 hours apart, 48 hours to 52 hours apart, 52
hours to 60 hours apart, 60 hours to 72 hours apart, 72 hours to 84
hours apart, 84 hours to 96 hours apart, or 96 hours to 120 hours
part. In preferred embodiments, two or more therapies are
administered within the same patent visit.
[0182] In certain embodiments, one or more compounds of the
invention and one or more other therapies (e.g., prophylactic or
therapeutic agents) are cyclically administered. Cycling therapy
involves the administration of a first therapy (e.g., a first
prophylactic or therapeutic agent) for a period of time, followed
by the administration of a second therapy (e.g., a second
prophylactic or therapeutic agent) for a period of time,
optionally, followed by the administration of a third therapy
(e.g., prophylactic or therapeutic agent) for a period of time and
so forth, and repeating this sequential administration, i.e., the
cycle in order to reduce the development of resistance to one of
the therapies, to avoid or reduce the side effects of one of the
therapies, and/or to improve the efficacy of the therapies.
[0183] In certain embodiments, the administration of the same
compounds of the invention may be repeated and the administrations
may be separated by at least 1 day, 2 days, 3 days, 5 days, 10
days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at
least 6 months. In other embodiments, the administration of the
same therapy (e.g., prophylactic or therapeutic agent) other than a
compound of the invention may be repeated and the administration
may be separated by at least at least 1 day, 2 days, 3 days, 5
days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3
months, or at least 6 months.
[0184] Any immunomodulatory agent well-known to one of skill in the
art may be used in the co-administration methods and compositions
of the invention. immunomodulatory agents can affect one or more or
all aspects of the immune response in a subject. Aspects of the
immune response include, but are not limited to, the inflammatory
response, the complement cascade, leukocyte and lymphocyte
differentiation, proliferation, and/or effector function, monocyte
and/or basophil counts, and the cellular communication among cells
of the immune system. In certain embodiments of the invention, an
immunomodulatory agent modulates one aspect of the immune response.
In other embodiments, an immunomodulatory agent modulates more than
one aspect of the immune response. In a preferred embodiment of the
invention, the administration of an immunomodulatory agent to a
subject inhibits or reduces one or more aspects of the subject's
immune response capabilities. In a specific embodiment of the
invention, the immunomodulatory agent inhibits or suppresses the
immune response in a subject. In accordance with the invention, an
immunomodulatory agent is not antibody that immunospecifically
binds to c-Rel. In certain embodiments, an immunomodulatory agent
is not an anti-inflammatory agent. In certain embodiments, an
immunomodulatory agent is'not an anti-angiogenic agent. In other
embodiments, an immunomodulatory agent is not an integrin
antagonist. In other embodiments, an immunomodulatory agent is not
a TNF-.alpha. antagonist. In certain embodiments, an
immunomodulatory agent is a chemotherapeutic agent. In certain
embodiments, an immunomodulatory agent is not a chemotherapeutic
agent.
[0185] Examples of immunomodulatory agents include, but are not
limited to, proteinaceous agents such as cytokines, peptide
mimetics, and antibodies (e.g., human, humanized, chimeric,
monoclonal, polyclonal, Fvs, ScFvs, Fab or F(ab)2 fragments or
epitope binding fragments), nucleic acid molecules (e.g., antisense
nucleic acid molecules and triple helices), small molecules,
organic compounds, and inorganic compounds. In particular,
immunomodulatory agents include, but are not limited to,
methotrexate, leflunomide, cyclophosphamide, cytoxan, Immuran,
cyclosporine A, minocycline, azathioprine, antibiotics (e.g., FK506
(tacrolimus)), methylprednisolone (MP), corticosteroids, steroids,
mycophenolate mofetil, rapamycin (sirolimus), mizoribine,
deoxyspergualin, brequinar, malononitriloamindes (e.g.,
leflunamide), T cell receptor modulators, cytokine receptor
modulators, and modulators mast cell modulators.
[0186] Examples of T cell receptor modulators include, but are not
limited to, anti-T cell receptor antibodies (e.g., anti-CD4
antibodies (e.g., cM-T412 (Boeringer), IDEC-CE9.1.RTM. (IDEC and
SKB), mAB 4162W94, Orthoclone and OKTcdr4a (Janssen-Cilag)),
anti-CD3 antibodies (e.g., Nuvion (Product Design Labs), OKT3
(Johnson & Johnson), or Rituxan (IDEC)), anti-CD5 antibodies
(e.g., an anti-CD5 ricin-linked immunoconjugate), anti-CD7
antibodies (e.g., CHH-380 (Novartis)), anti-CD8 antibodies,
anti-CD40 ligand monoclonal antibodies (e.g., IDEC-131 (IDEC)),
anti-CD52 antibodies (e.g., CAMPATH 1H (Ilex)), anti-CD2 antibodies
(e.g., MEDI-507 (Medlmmune, Inc., International Publication Nos. WO
02/098370 and WO 02/069904), anti-CD11a antibodies (e.g., Xanelim
(Genentech)), and anti-B7 antibodies (e.g., IDEC-114) (IDEC))),
CTLA4-immunoglobulin, and LFA-3TIP (Biogen, International
Publication No. WO 93/08656 and U.S. Pat. No. 6,162,432).
[0187] Examples of cytokine receptor modulators include, but are
not limited to, soluble cytokine receptors (e.g., the extracellular
domain of a TNF-.alpha. receptor or a fragment thereof, the
extracellular domain of an IL-10 receptor or a fragment thereof,
and the extracellular domain of an IL-6 receptor or a fragment
thereof), cytokines or fragments thereof (e.g., interleukin IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-13,
IL-15, IL-23, TNF-.alpha., TNF-.beta., interferon (IFN)-.alpha.,
IFN-.beta., IFN-.gamma., and GM-CSF), anti-cytokine receptor
antibodies (e.g., anti-IFN receptor antibodies, anti-IL-2 receptor
antibodies (e.g., Zenapax (Protein Design Labs)), anti-IL-3
receptor antibodies, anti-IL-4 receptor antibodies, anti-IL-6
receptor antibodies, anti-IL-10 receptor antibodies, anti-IL-12
receptor antibodies, anti-IL-13 receptor antibodies, anti-IL-15
receptor antibodies, and anti-IL-23 receptor antibodies),
anti-cytokine antibodies.
[0188] In a specific embodiment, a cytokine receptor modulator is
IL-3, IL-4, IL-10, or a fragment thereof. In another embodiment, a
cytokine receptor modulator is the extracellular domain of a
TNF-.alpha. receptor or a fragment thereof. In certain embodiments,
a cytokine receptor modulator is not a TNF-.alpha. antagonist.
[0189] In one embodiment, a cytokine receptor modulator is a mast
cell modulator. In an alternative embodiment, a cytokine receptor
modulator is not a mast cell modulator. Examples of mast cell
modulators include, but are not limited to stem cell factor (c-kit
receptor ligand) inhibitor (e.g., mAb 7H6, mAb 8H7a, pAb 1337,
FK506, CsA, dexamthasone, and fluconcinonide), c-kit receptor
inhibitor (e.g., STI 571 (formerly known as CGP 57148B)), mast cell
protease inhibitor (e.g., GW-45, GW-58, wortmannin, LY 294002,
calphostin C, cytochalasin D, genistein, KT5926, staurosproine, and
lactoferrin), relaxin ("RLX"), IgE antagonist (e.g., antibodies
rhuMAb-E25 omalizumab, HMK-12 and 6HD5, and mAB Hu-901), IL-3
antagonist, IL-4 antagonists, IL-10 antagonists, and TGF-beta.
[0190] An immunomodulatory agent may be selected to interfere with
the interactions between the T helper subsets (TH1 or TH2) and B
cells to inhibit neutralizing antibody formation. Antibodies that
interfere with or block the interactions necessary tier the
activation of B cells by TH (T helper) cells, and thus block the
production of neutralizing antibodies, are useful as
immunomodulatory agents in the methods of the invention. For
example, B cell activation by T cells requires certain interactions
to occur (Durie et aL, Immunol. Today, 15(9):406-410 (1994)), such
as the binding of CD40 ligand on the T helper cell to the CD40
antigen on the B cell, and the binding of the CD28 and/or CTLA4
ligands on the T cell to the B7 antigen on the B cell. Without both
interactions, the B cell cannot be activated to induce production
of the neutralizing antibody.
[0191] The CD40 ligand (CD40L)-CD40 interaction is a desirable
point to block the immune response because of its broad activity in
both T helper cell activation and function as well as the absence
of redundancy in its signaling pathway. Thus, in a specific
embodiment of the invention, the interaction of CD40L with CD40 is
transiently blocked at the time of administration of one or more of
the compounds of the invention and immunomodulatory agents. This
can be accomplished by treating with an agent which blocks the CD40
ligand on the TH cell and interferes with the normal binding of
CD40 ligand on the T helper cell with the CD40 antigen on the B
cell. An antibody to CD40 ligand (anti-CD40L) (available from
Bristol-Myers Squibb Co; see, e.g., European patent application
555,880, published Aug. 18, 1993) or a soluble CD40 molecule can be
selected and used as an immunomodulatory agent in accordance with
the methods of the invention.
[0192] An immunomodulatory agent may be selected to inhibit the
interaction between TH1 cells and cytotoxic T lymphocytes ("CTLs")
to reduce the occurrence of CTL-mediated killing. An
immunomodulatory agent may be selected to alter (e.g., inhibit or
suppress) the proliferation, differentiation, activity and/or
function of the CD4+ and/or CD8+ T cells. For example, antibodies
specific for T cells can be used as immunomodulatory agents to
deplete, or alter the proliferation, differentiation, activity
and/or function of CD4+ and/or CD8+ T cells.
[0193] In one embodiment of the invention, an immunomodulatory
agent that reduces or depletes T cells, preferably memory T cells,
is administered to a subject at risk of or with a disease or
disorder associated with or characterized by aberrant expression
and/or activity of an IL-9 polypeptide, a disease or disorder
associated with or characterized by aberrant subcellular
localization of c-Rel, an autoimmune disease, an inflammatory
disease, a proliferative disease, or an infection (preferably, a
respiratory infection) in accordance with the methods of the
invention. See, e.g., U.S. Pat. No. 4,658,019. in another
embodiment of the invention, an immunomodulatory agent that
inactivates CD8+ T cells is administered to a subject at risk of or
with a disease or disorder associated with or characterized by
aberrant subcellular localization of c-Rel, a disease or disorder
associated with or characterized by aberrant subcellular
localization of c-Rel, an autoimmune disease, an inflammatory
disease, a proliferative disease, or an infection (preferably, a
respiratory infection) in accordance with the methods of the
invention. In a specific embodiment, anti-CD8 antibodies are used
to reduce or deplete CD8+ T cells.
[0194] In another embodiment, an immunomodulatory agent which
reduces or inhibits one or more biological activities (e.g., the
differentiation, proliferation, and/or effector functions) of TH0,
TH1, and/or TH2 subsets of CD4+ T helper cells is administered to a
subject at risk of or with a disease or disorder associated with or
characterized by aberrant expression subcellular localization of
c-Rel, a disease or disorder associated with or characterized by
aberrant subcellular localization of c-Rel, an autoimmune disease,
an inflammatory disease, a proliferative disease, or an infection
(preferably, a respiratory infection) in accordance with the
methods of the invention. One example of such an immunomodulatory
agent is IL-4. IL-4 enhances antigen-specific activity of TH2 cells
at the expense of the TH1 cell function (see, e.g., Yokota et al,
1986 Proc. Natl. Acad. Sci., USA, 83:5894-5898; and U.S. Pat. No.
5,017,691). Other examples of immunomodulatory agents that affect
the biological activity (e.g., proliferation, differentiation,
and/or effector functions) of T-helper cells (in particular, TH1
and/or TH2 cells) include, but are not limited to, IL-2, IL-4,
IL-5, IL-6, IL-10, IL-13, IL-15, and interferon (IFN)-.gamma..
[0195] In another embodiment, an immunomodulatory agent
administered to a subject at risk of or with a disease or disorder
associated with or characterized by aberrant subcellular
localization of c-Rel, an autoimmune disease, an inflammatory
disease, a proliferative disease, or an infection (preferably, a
respiratory infection) in accordance with the methods of the
invention is a cytokine that prevents antigen presentation. In a
specific embodiment, an immunomodulatory agent used in the methods
of the invention is IL-10. IL-10 also reduces or inhibits
macrophage action which involves bacterial elimination.
[0196] An immunomodulatory agent may be selected to reduce or
inhibit the activation, degranulation, proliferation, and/or
infiltration of mast cells. In certain embodiments, the
immunomodulatory agent interferes with the interactions between
mast cells and mast cell activating agents, including, but not
limited to stem cell factors (c-kit ligands), IgE, IL-4,
environmental irritants, and infectious agents. In a specific
embodiment, the immunomodulatory agent reduces or inhibits the
response of mast cells to environmental irritants such as, but not
limited to pollen, dust mites, tobacco smoke, and/or pet dander. In
another specific embodiment, the immunomodulatory agent reduces or
inhibits the response of mast cells to infectious agents, such as
viruses, bacteria, and fungi. Examples of mast cell modulators that
reduce or inhibit the activation, degranulation, proliferation,
and/or infiltration of mast cells include, but are not limited to,
stem cell factor (c-kit receptor ligand) inhibitors (e.g., mAb 7H6,
mAb 8H7a, and pAb 1337 (see Mendiaz et al., 1996, Eur J Biochem
293(3):842-849), FK506 and CsA (Ito et al., 1999 Arch Dermatol Res
291(5):275-283), dexamthasone and fluconcinonide (see Finooto et
al. J Clin Invest 1997 99(7):1721-1728)), c-kit receptor inhibitors
(e.g., STI 571 (formerly known as CGP 57148B) (see Heinrich et al.,
2000 Blood 96(3):925-932)), mast cell protease inhibitors (e.g.,
GW-45 and GW-58 (see Temkin et al., 2002 J Immunol
169(5):2662-2669), wortmannin, LY 294002, calphostin C, and
cytochalasin D (see Vosseller et al., 1997, Mal Biol Cell
1997:909-922), genistein, KT5926, and staurosproine (see Nagai et
al. 1995, Biochem Biophys Res Commun 208(2):576-581), and
lactoferrin (see He et al., 2003 Biochem Pharmacol
65(6):1007-1015)), relaxin ("RLX") (see Bath et al., 2002 Int
Immunopharmacol 2(8):1195-1294)), IgE antagonists (e.g., antibodies
rhuMAb-E25 omalizumab (see Finn et al., 2003 J Allergy Clin Immuno
111(2):278-284; Corren et al., 2003 J Allergy Clin Immuno
111(1):87-90; Busse and Neaville, 2001 Curr Opin Allergy Clin
Immuno 1(1):105-108; and Tang and Powell, 2001, Eur J Pediatr
160(12): 696704), HMK-12 and 6HD5 (see Miyajima et al., 2202 Int
Arch Allergy Immuno 128(1):24-32), and mAB Hu-901 (see van Neerven
et al., 2001 Int Arch Allergy Immuno 124(1-3):400), IL-3
antagonist, IL-4 antagonists, IL-10 antagonists, and TGF-beta (see
Metcalfe et al., 1995, Exp Dennatol 4(4 Pt 2):227-230).
[0197] In a preferred embodiment, proteins, polypeptides or
peptides (including antibodies) that are utilized as
immunomodulatory agents are derived from the same species as the
recipient of the proteins, polypeptides or peptides so as to reduce
the likelihood of an immune response to those proteins,
polypeptides or peptides. In another preferred embodiment, when the
subject is a human, the proteins, polypeptides, or peptides that
are utilized as immunomodulatory agents are human or humanized.
[0198] In accordance with one embodiment of the invention, one or
more immunomodulatory agents are administered to a subject at risk
of or with a disease or disorder associated with or characterized
by aberrant subcellular localization of c-Rel, an autoimmune
disease, an inflammatory disease, a proliferative disease, or an
infection (preferably, a respiratory infection) prior to,
subsequent to, or concomitantly with a compound of the invention
that alters the subcellular localization of c-Rel and that does not
materially alter the expression of NF.kappa.B and/or the amount of
I.kappa.B. Preferably, one or more immunomodulatory agents are
administered in combination with a compound of the invention that
alters the subcellular localization of c-Rel and that does not
materially alter the expression of NF.kappa.B and/or the amount of
I.kappa.B to a subject at risk of or with a disease or disorder
associated with or characterized by aberrant subcellular
localization of c-Rel, an autoimmune disease, an inflammatory
disease, a proliferative disease, or an infection (preferably, a
respiratory infection) to reduce or inhibit one or more aspects of
the immune response as deemed necessary by one of skill in the art.
Any technique well-known to one skilled in the art can be used to
measure one or more aspects of the immune response in a particular
subject, and thereby determine when it is necessary to administer
an immunomodulatory agent to said subject. In a preferred
embodiment, a mean absolute lymphocyte count of approximately 500
cells/mm3, preferably 600 cells/mm3, 650 cells/mm3, 700 cells/mtn3,
750 cells/mm3, 800 cells/mm3, 900 cells/mm3, 1000 cells/mm3, 1100
cells/mm3, or 1200 cells/mm3 is maintained in a subject. In another
preferred embodiment, the subject is not administered a compound of
the invention if their absolute lymphocyte count is 500 cells/mm3
or less, 550 cells/mm3 or less, 600 cells/mm3 or less, 650
cells/mm3 or less, 700 cells/mm3 or less, 750 cells/mm3 or less, or
800 cells/mm3 or less.
[0199] In a preferred embodiment, one or more immunomodulatory
agents are administered in combination with a compound of the
invention so as to transiently reduce or inhibit one or more
aspects of the immune response. Such a transient inhibition or
reduction of one or more aspects of the immune system can last for
hours, days, weeks, or months. Preferably, the transient inhibition
or reduction in one or more aspects of the immune response lasts
for a few hours (e.g., 2 hours, 4 hours, 6 hours, 8 hours, 12
hours, 14 hours, 16 hours, 18 hours, 24 hours, 36 hours, or 48
hours), a few days (e.g., 3 days, 4 days, 5 days, 6 days, 7 days,
or 14 days), or a few weeks (e.g., 3 weeks, 4 weeks, 5 weeks or 6
weeks).
[0200] Any anti-inflammatory agent, including agents useful in
therapies for inflammatory disorders, well-known to one of skill in
the art can be used in the compositions and methods of the
invention. Non-limiting examples of anti-inflammatory agents
include non-steroidal anti-inflammatory drugs (NSAIDs), steroidal
anti-inflammatory drugs, anticholinergics (e.g., atropine sulfate,
atropine methylnitrate, and ipratropium bromide (ATROVENT.TM.)),
beta2-agonists (e.g., abuterol (VENTOLIN.TM. and PROVENTIL.TM.),
bitolterol (TORNALATE.TM.), levalbuterol (XOPONEX.TM.),
metaproterenol (ALUPENT.TM.), pirbuterol (MAXAIR.TM.), terbutlaine
(BRETHAIRE.TM. and BRETHINE.TM.), albuterol (PROVENTIL.TM.,
REPETABS.TM., and VOLMAX.TM.), formoterol (FORADIL AEROLIZER.TM.),
and salmeterol (SEREVENT.TM. and SEREVENT DISKUS.TM.)), and
methylxanthines (e.g., theophylline (UNIPHYL.TM., THEO-DUR.TM.,
SLO-BID.TM., AND TEHO-42.TM.)). Examples of NSAIDs include, but are
not limited to, aspirin, ibuprofen, celecoxib (CELEBREX.TM.),
diclofenac (VOLTAREN.TM.), etodolac (LODINE.TM.), fenoprofen
(NALFON.TM.), indomethacin (INDOCIN.TM.), ketoralac (TORADOL.TM.),
oxaprozin (DAYPRO.TM.), nabumentone (RELAFEN.TM.), sulindac
(CLINORIL.TM.), tolmentin (TOLECTIN.TM.), rofecoxib (VIOXX.TM.),
naproxen (ALEVE.TM. NAPROSYN.TM.), ketoprofen (ACTRON.TM.) and
nabumetone (RELAFEN.TM.). Such NSAIDs function by inhibiting a
cyclooxgenase enzyme (e.g., COX-1 and/or COX-2). Examples of
steroidal anti-inflammatory drugs include, but are not limited to,
glucocorticoids, dexamethasone (DECADRON.TM.), corticosteroids
(e.g., methylprednisolone (MEDROL.TM.)), cortisone, hydrocortisone,
prednisone (PREDNISONE.TM. and DELTASONE.TM.), prednisolone
(PRELONE.TM. and PEDIAPRED.TM.), triamcinolone, azulfidine, and
inhibitors of eicosanoids (e.g., prostaglandin, thromboxanes, and
leukotrienes). Anti-inflammatory therapies and their dosages,
routes of administration, and recommended usage are known in the
art and have been described in such literature as the Physician's
Desk Reference (57th ed., 2003).
[0201] For arthritis, inflammation-mediated bone loss and other
disorders that have an inflammatory component, preferred
conventional treatments for use in combination therapy with the
compounds and compositions of this invention include (without
limitation) naproxen sodium (Anaprox.RTM. and Anaprox.RTM. DS,
Roche), flurbiprofen (Ansaid.RTM.; Pharmacia), diclofenac
sodium+misoprostil (Arthrotec.RTM., Searle), valdecoxib
(Bextra.RTM., Pharmacia), diclofenac potassium (Cataflam.RTM. and
Voltaren.RTM., Novartis), celecoxib (Celebrex.RTM., Pharmacia),
sulindac (Clinoril.RTM., Merck), oxaprozin (Daypro.RTM.,
Pharmacia), salsalate (Disalcid.RTM., 3M), diflunisal
(Dolobid.RTM.), Merck), naproxen sodium (EC Naprosyn.RTM., Roche),
piroxicam (Feldene.RTM., Pfizer), indomethacin (Indocine.RTM. and
lndocin SR.RTM., Merck), etodolac (Lodine.RTM. and Lodine XL.RTM.,
Wyeth), meloxicam (Mobic.RTM., Boehringer Ingelheim), ibuprofen
(Motrin.RTM., Pharmacia), naproxen (Naprelan.RTM., Elan), naproxen
(Naprosyn.RTM., Roche), ketoprofen (Orudis.RTM. and Oruvail.RTM.,
Wyeth), nabumetone (Relafen.RTM., SmithKline), tolmetin sodium
(Tolectin.RTM., McNeil), choline magnesium trisalicylate
(Trilisate.RTM., Purdue Fredrick), and rofecoxib (Vioxx.RTM.,
Merck).
[0202] In any case where pain in a component of the target
disorder, the other therapeutic agent can be an analgesic. Useful
analgesics include, but are not limited to, phenacetin, butacetin,
acetaminophen, nefopam, acetoamidoquinone, and mixtures
thereof.
[0203] For use against osteoporosis, Paget's disease and other
disorders associated with bone deterioration, preferred
conventional agents that mayu be used in combination with compounds
and compositions of this invention include (without limitation)
bisphosphonates (such as etidronate (Didronel.RTM., Procter &
Gamble), pamidronate (Aredia.RTM., Novartis), and alendronate
(Fosamax.RTM., Merck)), tiludronate (Skelid.RTM.,
Sanofi-Synthelabo, Inc.), risedronate (Actonel.RTM., Procter &
Gamble/Aventis), calcitonin (Miacalcin.RTM.), estrogens
(Climara.RTM., Estrace.RTM., Estraderm.RTM., Estratab.RTM.,
Ogen.RTM., Ortho-Est.RTM., Premarin.RTM., and others) estrogens and
progestins (Activella.TM., FemHrt.RTM., Premphase.RTM.,
Prempro.RTM., and others), parathyroid hormone and portions
thereof, such as teriparatide (Forteo.RTM., Eli Lilly and Co.),
selective estrogen receptor modulators (SERMs) (such as raloxifene
(Evista.RTM.)) and treatments currently under investigation (such
as other parathyroid hormones, sodium fluoride, vitamin D
metabolites, and other bisphosphonates and selective estrogen
receptor modulators).
[0204] Any parathyroid hormone (PTH) may be used in combination
with the compound of this invention. The term parathyroid hormone
refers to parathyroid hormone, fragments or metabolites thereof and
structural analogs thereof which can stimulate bone formation and
increase bone mass. Also included are parathyroid hormone related
peptides and active fragments and analogs of parathyroid related
peptides (see PCT publication No. WO 94/01460). Such bone anabolic
functional activity is readily determined by those skilled in the
art of standard assays. A variety of these compounds are described
and referenced below. However, other parathyroid hormone will be
known to those skilled in the art. Exemplary parathyroid hormones
are disclosed in the following references. "Human Parathyroid
Peptide Treatment of Vertebral Osteoporosis", Osteoporosis Int., 3,
(Supp 1):199-203. "PTH 1-34 Treatment of Osteoporosis with Added
Hormone Replacement Therapy: Biochemical, Kinetic and Histological
Responses" Osteoporosis Int. 1: 162-170.
[0205] Any growth hormone or growth hormone secretagogue may be
used in combination with the compounds of this invention. The term
growth hormone secretagogue refers to a compound which stimulates
the release of growth hormone or mimics the action of growth
hormone (e.g., increases bone formation leading to increased bone
mass). Such actions are readily determined by those skilled in the
art of standard assays well known to those of skill in the art. A
variety of these compounds are disclosed in the following published
PCT patent applications: WO 95/14666; WO 95/13069; WO 94/19367; WO
94/13696; and WO 95/34311.
However, other growth hormones or growth hormone secretagogues will
be known to those skilled in the art. In particular, a preferred
growth hormone secretagogue is
N-[1(R)-[1,2-Dihydro-1-methanesulfonylspiro
[3H-indole-3,4'-piperidin]-l'-y1)carbonyl]-2-(phenylmethyloxy)ethyl]-2-am-
ino-2-methylpropanamide: MK-667. Other preferred growth hormone
secretagogues include
2-amino-N-(2-(3a-(R)-benzyl-2-methy1-3-oxo-2,3,3a,4,6,7-hexahydro-pyrazol-
o-[4,3-c]pyridin-5-y1)-1-(R)-benzyloxymethyl-2-oxo-ethyl)-isobutyramide
or its L-tartaric acid salt;
2-amino-N-(1-(R)-benzyloxymethyl-2-(3a-(R)-(4-fluoro-benzyl)-2-methyl-3-o-
xo-2,3,3a,4,6,7-hexahydro-pyrazolo[4,3-c]pyridin-5-yl)-2-oxo-ethyl)isobuty-
ramide;
2-amino-N-(2-(3a-(R)-benzyl-3-oxo-2,3,3a,4,6,7-hexahydro-pyrazolo[-
4,3-c]pyridin-5-yl)-1-(R)benzyloxymethyl-2-oxo-ethyl)isobutyramide,
and
2-amino-N-(1-(2,4-difluoro-benzyloxymethyl)-2-oxo-2-(3-oxo-3a-pyridin-2-y-
lmethy1-2-(2,2,2-trifluoro-ethyl)-2,3,3a,4,6,7-hexahydro-pyrazolo[4,3-c]py-
ridin-5-yl)-ethyl)-2-methyl-propionamide.
[0206] Any method of the present invention can comprise
administering an effective amount of a composition or
pharmaceutical composition comprising at least one compound of this
invention to a cell, tissue, organ, animal or patient in need of
such modulation, treatment or therapy. Such a method can optionally
further comprise co-administration or combination therapy for
treating an IL-12 production related disorder, wherein the
administering further comprises administering before, concurrently
with, and/or after the compound of this invention, at least one
additional active agent selected from a TNF antagonist (e.g., but
not limited to a TNF antibody or fragment, a soluble TNF receptor
or fragment, fusion proteins thereof, or a small molecule TNF
antagonist), an antirheumatic (e.g., methotrexate, auranofin,
aurothioglucose, azathioprine, etanercept, gold sodium thiomalate,
hydroxychloroquine sulfate, leflunomide, sulfasalzine), a muscle
relaxant, a narcotic, a non-steroid anti-inflammatory drug (NSAID),
an analgesic, an anesthetic, a sedative, a local anesthetic, a
neuromuscular blocker, an antimicrobial (e.g., aminoglycoside, an
antifungal, an antiparasitic, an antiviral, a carbapenem,
cephalosporin, a fluoroquinolone, a macrolide, a penicillin, a
sulfonamide, a tetracycline, another antimicrobial), an
antipsoriatic, a corticosteriod, an anabolic steroid, a diabetes
related agent, a mineral, a nutritional, a thyroid agent, a
vitamin, a calcium related hormone, an antidiarrheal, an
antitussive, an antiemetic, an antiulcer, a laxative, an
anticoagulant, an erythropieitin (e.g., epoetin alpha), a filgastim
(e.g., G-CSF, Neupogen), a sargramostim (GM-CSF, Leukine), an
immunization, an immunoglobulin, an immunosuppressive (e.g.,
basiliximab, cyclosporine, daclizumab), a growth hormone, a hormone
replacement drug, an estrogen receptor modulator, a mydriatic, a
cycloplegic, an alkylating agent, an antimetabolite, a mitotic
inhibitor, a radiopharmaceutical, an antidepressant, antimanic
agent, an antipsychotic, an anxiolytic, a hypnotic, a
sympathomimetic, a stimulant, donepezil, tacrine, an asthma
medication, a beta agonist, an inhaled steroid, a leukotriene
inhibitor, a methylxanthine, a cromolyn, an epinephrine or analog,
domase alpha (Pulmozyme), a cytokine or a cytokine antagonistm.
Suitable dosages are well known in the art. See, e.g., Wells et
al., eds., Pharmacotherapy Handbook, 2.sup.nd Edition, Appleton and
Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket
Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma
Linda, Calif. (2000), each of which references are entirely
incorporated herein by reference.
[0207] TNF antagonists suitable for compositions, combination
therapy, co-administration, devices and/or methods of the present
invention include, but are not limited to, anti-TNF antibodies
(such as, Remicade (Infliximab) or Humira (adalimumab)) for
example, or, antigen-binding fragments thereof, and receptor
molecules which bind specifically to TNF (such as, for example,
Enbrel (Etanercept)); compounds which prevent and/or inhibit TNF
synthesis, TNF release or its action on target cells, such as
thalidomide, tenidap, phosphodiesterase inhibitors (e.g,
pentoxifylline and rolipram), A2b adenosine receptor agonists and
A2b adenosine receptor enhancers; compounds which prevent and/or
inhibit TNF receptor signaling, such as mitogen activated protein
(MAP) kinase inhibitors; compounds which block and/or inhibit
membrane TNF cleavage, such as metalloproteinase inhibitors;
compounds which block and/or inhibit TNF activity, such as
angiotensin converting enzyme (ACE) inhibitors (e.g., captopril);
and compounds which block and/or inhibit TNF production and/or
synthesis, such as MAP kinase inhibitors.
[0208] For clarification, a "tumor necrosis factor antibody," "TNF
antibody," "TNF antibody," or fragment and the like decreases,
blocks, inhibits, abrogates or interferes with TNF activity in
vitro, in situ and/or preferably in vivo. For example, a suitable
TNF human antibody of the present invention can bind TNFa and
includes anti-TNF antibodies, antigen-binding fragments thereof,
and specified mutants or domains thereof that bind specifically to
TNFa. A suitable TNF antibody or fragment can also decrease block,
abrogate, interfere, prevent and/or inhibit TNF RNA, DNA or protein
synthesis, TNF release, TNF receptor signaling, membrane TNF
cleavage, TNF activity, TNF production and/or synthesis.
[0209] The foregoing and other useful combination therapies will be
understood and appreciated by those of skill in the art. Potential
advantages of such combination therapies include the ability to use
less of each of the individual active ingredients to minimize toxic
side effects, synergistic improvements in efficacy, improved ease
of administration or use and/or reduced overall expense of compound
preparation or formulation. The biological activities of a compound
of this invention can be evaluated by a number of cell-based
assays. One of such assays can be conducted using cells from human
peripheral blood mononuclear cells (PBMC) or human monocytic cell
line (THP-1). The cells are stimulated with a combination of human
interferon-y (IFN-.gamma.) and lipopolysaccharide or a combination
of IFN-.gamma. and Staphylococcus aureus Cowan I in the presence of
a test compound. The level of inhibition of IL-12 production can be
measured by, e.g., determining the amount of p70 by using a
sandwich ELISA assay with anti-human IL-12 antibodies. IC.sub.50 of
the test compound can then be determined. Specifically, PBMC or
THP-1 cells are incubated with the test compound. Cell viability
was assessed using the bioreduction of MTS
[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl-
)-2H-tetrazoliumj (Promega, Madison, Wis.).
[0210] The other therapeutic agent can include bone anti-resorptive
agents for example progestins, polyphosphonates, bisphosphonate(s),
estrogen agonists/antagonists, estrogen (such as Premarine.RTM.),
estrogenIprogestin combinations, and estrogen derivatives (such as
estrone, estriol or 17.alpha., 17.beta.-ethynyl estradiol).
Exemplary progestins are available from commercial sources and
include: algestone acetophenide, altrenogest, amadinone acetate,
anagestone acetate, chlorrnadinone acetate, cingestol, clogestone
acetate, clomegestone acetate, delmadinone acetate, desogestrel,
dimethisterone, dydrogesterone, ethynerone, dthynodiol diacetate,
etonogestrel, tlurogestone acetate, gestaclone, gestodene,
gestonorone caproate, gestrinone, haloprogesterone,
hydroxyprogesterone, caproate, levonorgestrel, lynestrenol,
medrogestone, medroxyprogesterone acetate, melengestrol acetate,
methynodiol diacetate, norethindrone, norethindrone acetate,
norethynodrel, norgestimate, norgestomet, norgestrel, oxogestone
phenpropionate, progesterone, quingestanol acetate, quingestrone,
and tigestol. Preferred progestins are medroxyprogestrone,
norethindrone and norethynodrel.
[0211] Exemplary bone resorption inhibiting polyphosphonates
include polyphosphonates of the type disclosed in U.S. Pat. No.
3,683,080. Preferred polyphosphonates are geminal
dipolyphosphonates (also referred to as bis-phosphonates).
Tiludronate disodium is an especially preferred polyphosphonate.
Ibandronic acid is an especially preferred polyphosphonate.
Alendronate is an especially preferred polyphosphonate. Zoledronic
acid is an especially preferred polyphosphonate. Other preferred
polyphosphonates are 6-amino-1-hydroxy-hexylidene-biphosphonic acid
and 1-hydroxy-3(methylpentylamino)-propylidene-bisphosphonic acid.
The polyphosphonates may be administered in the form of the acid,
or of a soluble alkali metal salt or alkaline earth metal salt.
Hydrolyzable esters of the polyphosphonates are likewise included.
Specific examples include ethane-1-hydroxy 1,1-diphosphonic acid,
methane diphosphonic acid, pentane-1-hydroxy-1,1-diphosphonic acid,
methane dichloro diphosphonic acid, methane hydroxy diphosphonic
acid, ethane-1-amino-1,1-diphosphonic acid,
ethane-2-amino-1,1-diphosphonic acid,
propane-3-amino-1-hydroxy-1,1-diphosphonic acid,
propane-N,N-dimethyl-3-amino-1-hydroxy-1,1-diphosphonic acid,
propane-3,3-dimethyl-3-amino-l-hydroxy-1,1-diphosphonic acid,
phenyl amino methane diphosphonic acid, N,N-dimethylamino methane
diphosphonic acid, N(2-hydroxyethyl)amino methane diphosphonic
acid, butane-4-amino-1-hydroxy-1,1-diphosphonic acid,
pentane-5-amino-1-hydroxy-1,1-diphosphonic acid,
hexane-6-amino-1-hydroxy-1,1-diphosphonic acid and pharmaceutically
acceptable esters and salts thereof.
[0212] In particular, the compounds of this invention may be
combined with a mammalian estrogen agonist/antagonist. Any estrogen
agonist/antagonist may be used for this purpose. The term estrogen
agonist/antagonist refers to compounds which bind with the estrogen
receptor, inhibit bone turnover and/or prevent bone loss. In
particular, estrogen agonists are herein defined as chemical
compounds capable of binding to the estrogen receptor sites in
mammalian tissue, and mimicking the actions of estrogen in one or
more tissue. Estrogen antagonists are herein defined as chemical
compounds capable of binding to the estrogen receptor sites in
mammalian tissue; and blocking the actions of estrogen in one or
more tissues. Such activities are readily determined by those
skilled in the art of standard assays including estrogen receptor
binding assays, standard bone histomorphometric and densitometer
methods, and E. F Eriksen et al., Bone Histomorphometry, Raven
Press, New York, pp. 1-74 (1994); S. J. Grier et al., The Use of
Dual-Energy X-Ray Absorptiometry In Animals, Inv. Radiol. 31(1);
50-62 (1996); Wahner H. W. and Fogelman I., The Evaluation of
Osteoporosis: Dual Energy X-Ray Absorptiometry in Clinical
Practice, Martin Dunitz Ltd., London, pp. 1-296 (1994)). A variety
of these compounds are described and referenced below.
[0213] A preferred estrogen agonist/antagonist is droloxifene:
(phenol,
3-04442-(dimethylamino)ethoxy)phenyl)-2-phenyl-1-butenyl)-, (E)-)
and related compounds which are disclosed in U.S. Pat. No.
5,047,431. Another preferred estrogen agonist/antagonist is
3-(4-(1,2-diphenyl-but-1-enyl)-phenyl)-acrylic acid, which is
disclosed in Wilson et al., Endocrinology 138: 3901-11 (1997).
Another preferred estrogen agonist/antagonist is tamoxifen:
(ethanamine, 24-4-(1,2-diphenyl-1-butenyl)phenoxy)-N,N-dimethyl,
(Z)-2-, 2-hydroxy-1,2,3-propanetricarboxylate (1:1)) and related
compounds which are disclosed in U.S. Pat. No. 4,536,516. Another
related compound is 4-hydroxy tamoxifen which is disclosed in U.S.
Pat. No. 4,623,660.
[0214] A preferred estrogen agonist/antagonist is raloxifene:
(methanone,
(6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thien-3-yl)(4-(2-(1-piperidinyl)eth-
oxy)phenyl)hydrochloride) which is disclosed in U.S. Pat. No.
4,418,068. Another preferred estrogen agonist/antagonist is
toremifene: (ethanamine,
2-(4-(4-chloro-1,2-diphenyl-1-butenyl)phenoxy)-N,N-dimethyl-, (Z)-,
2-hydroxy-1,2,3-propanetricarboxylate (1:1) which is disclosed in
U.S. Pat. No. 4,996,225. Another preferred estrogen
agonist/antagonist is centchroman:
1-(24(44-methoxy-2,2,dimethyl-3-phenyl-chmman-4-yl)-phenoxy)-ethyl)-pyrro-
lidine, which is disclosed in U.S. Pat. No. 3,822,287. Also
preferred is levormeloxifene. Another preferred estrogen
agonist/antagonist is idoxifene:
(E)-1-(2-(4-(1-(4-iodo-phenyl)-2-phenyl-but-1-enyl)-phenoxy)-ethyl)-pyrro-
lidinone, which is disclosed in U.S. Pat. No. 4,839,155. Another
preferred estrogen agonist/antagonist is
2-(4-methoxy-phenyl)-3-[4-(2-piperidin-1-yl-ethoxy)-phenoxy]-benzo[b]thio-
phen-6-ol which is disclosed in U.S. Pat. No. 5,488,058. Another
preferred estrogen agonist/antagonist is
6-(4-hydroxy-phenyl)-5-(4-(2-piperidin-1-yl-ethoxy)-benzyl)-naphthalen-2--
ol which is disclosed in U.S. Pat. No. 5,484,795. Another preferred
estrogen agonist/antagonist is
(4-(2-(2-aza-bicyclo[2.2.1]hept-2-yl)-ethoxy)-phenyl)-(6-hydroxy-2-(4-hyd-
roxy-phenyl)-benzo[b]thiophen-3-yl)-methanone which is disclosed,
along with methods of preparation, in PCT publication no. WO
95/10513 assigned to Pfizer Inc. Other preferred estrogen
agonist/antagonists include compounds as described in U.S. Pat. No.
5,552,412. Especially preferred compounds described therein are:
cis-6-(4-fluoro-phenyl)-5-(4-(2-piperidin-I-yl-ethoxy)-phenyl)-5,6,7,8-te-
trahydro-naphthalene-2-ol;
(-)-cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy)-phenyl)-5,6,7,8-tetrahyd-
ro-naphthalene-2-ol;
cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy)-phenyl)-5,6,7,8-tetrahydro-n-
aphthalene-2-ol;
cis-1-(6'-pyrrolodinoethoxy-3'-pyridyl)-2-phenyl-6-hydroxy-1,2,3,4-tetrah-
ydronaphthalene;
1-(4'-pyrrolidinoethoxyphenyl)-2-(4''-fluorophenyI)-6-hydroxy-1,2,3,4-tet-
rahydroisoquinoline;
cis-6-(4-hydroxyphenyl)-5-(4-(2-piperidin-l-yl-ethoxy)-phenyl)-5,6,7,8-te-
trahydro-naphthalene-2-ol; and
1-(4'-pyrrolidinolethoxyphenyl)-2-phenyl-6-hydroxy-1,2,3,4-tetrahydroisoq-
uinoline. Other estrogen agonist/antagonists are described in U.S.
Pat. No. 4,133,814. U.S. Pat. No. 4,133,814 discloses derivatives
of 2-phenyl-3-aroyl-benzothiophene and
2-phenyl-3-aroylbenzothiophene-1-oxide.
[0215] Those skilled in the art will recognize that other bone
anabolic agents, also referred to as bone mass augmenting agents,
may be used in conjunction with the compounds of this invention. A
bone mass augmenting agent is a compound that augments bone mass to
a level which is above the bone fracture threshold as detailed in
the World Health Organization Study World Health Organization,
"Assessment of Fracture Risk and its Application to Screening for
Postmenopausal Osteoporosis (1994). Report of a WHO Study Group.
World Health Organization Technical Series 843." Any prostaglandin,
or prostaglandin agonist/antagonist may be used in combination with
the compounds of this invention. Those skilled in the art will
recognize that IGF-1, sodium fluoride, parathyroid hormone (PTH),
active fragments of parathyroid hormone, growth hormone or growth
hormone secretagogues may also be used. The following paragraphs
describes in greater detail exemplary compounds that may be
administered in combination with compounds of this invention.
[0216] Prostaglandins: The term prostaglandin refers to compounds
which are analogs of the natural prostaglandins PGD.sub.1,
PGD.sub.2, PGE.sub.2, PGE.sub.1 and PGF.sub.2 which are useful in
the treatment of osteoporosis and other disorders associated with
excessive osteoclastic bone resorption. These compounds bind to the
prostaglandins receptors. Such binding is readily determined by
those skilled in the art of standard assays (e.g., S. An et al.,
Cloning and Expression of the EP.sub.2 Subtype of Human Receptors
for Prostaglandin E.sub.2 Biochemical and Biophysical Research
Communications, 197(1): 263-270 (1993)).
[0217] Prostaglandins are alicyclic compounds related to the basic
compound prostanoic acid. The carbon atoms of the basic
prostaglandin are numbered sequentially from the carboxylic carbon
atom through the cyclopentyl ring to the terminal carbon atom on
the adjacent side chain. Normally the adjacent side chains are in
the trans orientation. The presence of an oxo group at C-9 of the
cyclopentyl moiety is indicative of a prostaglandin within the E
class while PGE.sub.2 contains a trans unsaturated double bond at
the C.sub.13-C.sub.14 and a cis double bond at the C.sub.5-C.sub.6
position.
[0218] A variety of prostaglandins are described and referenced
below. However, other prostaglandins will be known to those skilled
in the art. Exemplary prostaglandins are disclosed in U.S. Pat.
Nos. 4,171,331 and 3,927,197, Norrdin et al., The Role of
Prostaglandins in Bone in Vivo, Prostaglandins Leukotriene
Essential Fatty Acids 41: 139-150 (1990) is a review of bone
anabolic prostaglandins. Any prostaglandin agonist/antagonist may
be used in combination with the compounds of this invention. The
term prostaglandin agonist/antagonist refers to compounds which
bind to prostaglandin receptors (e.g., An S. et al., Cloning and
Expression of the EP.sub.2 Subtype of Human Receptors for
Prostaglandin E.sub.2, Biochemical and Biophysical Research
Communications 197(1): 263-70 (1993)) and mimic the action of
prostaglandin in vivo (e.g., stimulate bone formation and increase
bone mass). Such actions are readily determined by those skilled in
the art of standard assays. Eriksen E. F. et al., Bone
Histomorphometry, Raven Press, New York, 1994, pp. 1-74; S. J.
Grier et al., The Use of Dual-Energy X-Ray Absorptiometry In
Animals, Inv. Radio`. 31(1): 50-62 (1996); H. W. Warner and I.
Fogelman, The Evaluation of Osteoporosis: Dual Energy X-Ray
Absorptiometry in Clinical Practice, Martin Dunitz Ltd. London, pp.
1-296 (1994). A number of these compounds are described and
reference below. However, other prostaglandin agonists/antagonists
will be known to those skilled in the art. Exemplary prostaglandin
agonists/antagonists are disclosed as follows. U.S. Pat. No.
3,932,389 discloses
2-descarboxy-2-(tetrazol-5-yl)-11-desoxy-15-substituted-omega-pentanorpro-
s taglandins useful for bone formation activity. U.S. Pat. No.
4,018,892, discloses 16-aryl-13,14-dihydro-PGE.sub.2 p-biphenyl
esters useful for bone formation activity. U.S. Pat. No. 4,219,483,
discloses 2,3,6-substituted-4-pyrones useful for bone formation
activity. U.S. Pat. No. 4,132,847, discloses
2,3,6-substituted-4-pyrones useful for bone formation activity.
U.S. Pat. No. 4,000,309, discloses 16-aryl-13,14-dihydro-PGE.sub.2
p-biphenyl esters useful for bone formation activity. U.S. Pat. No.
3,982,016, discloses 16-aryl-13,14-dihydro-PGE.sub.2 p-biphenyl
esters useful for bone formation activity. U.S. Pat. No. 4,621,100,
discloses substituted cyclopentanes useful for bone formation
activity. U.S. Pat. No. 5,216,183, discloses cyclopentanones useful
for bone formation activity.
[0219] Sodium fluoride may be used in combination with the
compounds of this invention. The term sodium fluoride refers to
sodium fluoride in all its forms (e.g., slow release sodium
fluoride, sustained release sodium fluoride). Sustained release
sodium fluoride is disclosed in U.S. Pat. No. 4,904,478. The
activity of sodium fluoride is readily determined by those skilled
in the art of biological protocols.
[0220] Bone morphogenetic protein may be used in combination with
the compounds of this invention (e.g., see Ono et al., Promotion of
the Osteogenetic Activity of Recombinant Human Bone Morphogenetic
Protein by Prostaglandin E.sub.l, Bone 19(6): 581-588 (1996)).
Animal Models
[0221] Animal models for autoimmune disorders can be used to assess
the efficacy of the Therapeutics or pharmaceutical compositions of
invention. Animal models for autoimmune disorders such as type 1
diabetes, thyroid autoimmunity, systemic lupus eruthematosus, and
glomerulonephritis have been developed (Flanders et al., 1999,
Autoimmunity 29:235-246; Krogh et al., 1999, Biochimie 81:511-515;
Foster, 1999, Semin. Nephrol. 19:12-24).
[0222] The following series of examples are presented by way of
illustration and not by way of limitation on the scope of the
present invention.
Examples
I. Measuring the Level of IL-12 p40
[0223] Northern blot analysis was performed to examine the mRNA
levels of IL-12 p35 and p40. Human PBMC and the human monocyte cell
line THP-1 cells were stimulated with IFN-.gamma./SAC in the
presence or absence of Compound 1. Human PBMC were isolated by
centrifugation using Ficoll-Paque (Pharmacia Biotech, Uppsala,
Sweden) and prepared in RPMI medium supplemented with 10% fetal
calf serum (FCS), 100 U/ml penicillin, and 100 .mu.g/ml
streptomycin, in a 96-well plate with 5.times.10.sup.5 cells/well.
The cells were then primed with IFN-.gamma. (100 U/ml) and followed
by 0.01% SAC or 1 .mu.g/ml LPS, in the presence of different
concentrations of Compound 2 or other compounds. The test compounds
were prepared in DMSO and the final DMSO concentration was adjusted
to 0.25% in all cultures, including the compound-free control.
Cell-free supernatants were taken 18 h later for the measurement of
cytokines. The THP-1 cells were obtained from American Type Culture
Collection (Manassas, Va.) and were cultured in RPMI 1640 (ATCC,
Manassas, Va.), supplemented with 10% FCS (ATCC, Manassas, Va.),
and 1% penicillin/Streptomycin (Gibco-BRL, New York, N.Y.). Total
RNA was isolated and subjected to Northern blot analysis using
IL-12 p35 and p40 cDNA probes. We first examined the kinetics of
mRNA accumulation in cultures of hPBMC and THP-1 cells primed with
IFN-.gamma. followed by SAC stimulation in the presence or absence
of 1 .mu.M Compound 1.
[0224] In hPBMC, both IL-12 p35 and p40 mRNA were detectable by 4 h
and peaked at 6 h after the addition of SAC. The expression of p35
mRNA was completely inhibited by Compound 1 at all sampling times,
whereas the expression of the mRNA for the p40 subunit was reduced
significantly but incompletely. In THP-1 cells stimulated with
IFN-.gamma./SAC, IL-12 p35 mRNA was barely visible in compound-free
control and was undetectable in the presence of 1 .mu.M Compound I.
In contrast, IL-12 p40 mRNA was readily detectable by 4 h and
peaked at 6 h after the addition of SAC. Again, Compound I
significantly but incompletely reduced the expression of the p40
message.
[0225] We conducted a dose-response study of the inhibitory effects
of Compound 1 on IL-12 mRNA expression in
IFN-.gamma./SAC-stimulated hPBMC. Because both IL-12 p35 and p40
mRNA levels were maximal at 6 h after the addition of SAC, this
time point was selected for the dose-response analysis. The
induction of IL-12 p35 mRNA accumulation by IFN-.gamma./SAC was
completely reversed by 3 nM Compound 1, with an IC.sub.50 below 1
nM. In contrast, IL-12 p40 mRNA accumulation was barely inhibited
by 1 nM Compound 1, with maximum, though still incomplete
inhibition at 10 nM. This apparent weaker inhibition of p40
relative to p35 could be due to more effective inhibition of the
p35 promoter or it simply may be the product of the fact that p40
is produced in vast excess to p35 and its inhibition may require
greater concentrations of drug.
[0226] Thus, Compound 1 caused a decrease in both p35 and p40 mRNA
levels. Subsequent nuclear run-on experiments showed that this
effect was at the level of transcription initiation.
II. Effect of Compound 2 on IL-12 p35 and p40 Promoter Activity
[0227] As a result of the Northern blot findings, we undertook a
study of the p35 and p40 promoter activities. We transiently
transfected the murine macrophage cell line RAW264.7 with DNA
constructs in which the p35 and p40 promoters directed expression
of the luciferase reporter gene. The RAW264.7 cell line was
obtained from American Type Culture Collection (Manassas, Va.) and
was cultured in DMEM (ATCC, Manassas, Va.) supplemented with 10%
FCS (ATCC, Manassas, Va.), and 1% penicillin/Streptomycin
(Gibco-BRL, New York, N.Y.).
[0228] Both p35 and p40 promoter-driven luciferase production in
response to stimulation were determined in the presence or absence
of Compound 1 and Compound 2. To construct the human IL-12 p35 and
p40 promoter/luciferase reporter constructs, we generated p35 (-1.5
kb to +3 bp) and p40 (-1.3 kb to +56 bp) promoter fragments, which
contained several sequence motifs of the human IL-12 p35 and p40
genes. The fragments were generated by PCR from genomic DNA
obtained from human PBMC using primers as follows: IL-12 p35 1.5
kb-F: 5'-GCAGCATTAGAAGGGGCCTTAGAGA-3'(SEQ ID NO:3) and IL-12 p35
1.5 kb-R: TTTTATAATTGTCCCGAGGCGCG-3' (SEQ ID NO:4); IL-12 p40 -1.3
kb-F: 5'-ACGGCGAGGAAAGTTAGCCCG-3' (SEQ ID NO:5) and IL-12 p40 1.3
kb-R: 5'. TTGCTCTGGGCAGGACGGAG-3' (SEQ ID NO:6). The deletion in
the p40 promoter reporter constructs were generated by PCR with
primers as follows: IL-12 p40 -250 bp to +56 bp (p40/-250 bp) F:
5'-CACCCAAAAGTCATTTCCTC-3' (SEQ ID NO:7) and IL-12 p40 -250 bp to
+56 bp (p401-250 bp) R: 5'-TGCTCTGGGCAGGACGGAG-3' (SEQ ID NO:8);
IL-12 p40 -150 bp to +56 bp (p40/-150 bp) F:
5'-AGAGTTGTTTTCAATGTTGCAAC-3' (SEQ ID NO:9) and IL-12 p40 -150 bp
to +56 bp (p401-150 bp) R: 5'-TGCTCTGGGCAGGACGGAG-3' (SEQ ID
NO:10). The resulting PCR products were ligated upstream of the
luciferase gene in pGL3-Basic vector (Promega). All constructs were
verified by DNA sequencing.
[0229] RAW267.4 cells were transiently transfected and the cells
were then stimulated with murine recombinant IFN-.gamma. (100
ng/ml) for 10 h followed by LPS (1 .mu.g/ml) or SAC (0.025%) in the
presence or absence of Compound 1, Compound 2, or a negative
control (a structurally-related inactive compound) at different
concentrations for an additional 16 h. Transfection was
accomplished using SuperFect Transfection Reagent (Qiagen) by the
described protocol. Total amount of transfect DNA was kept constant
by including the respective control plasmids without
insertions.
[0230] Cells were co-transfected with the vector pCMV (BD
Biosciences Clontech) in which the constitutively active CMV
promoter directs .beta.-galactosidase expression for the monitoring
of transfection efficiency. Luciferase and .beta.-galactosidase
activity were determined in cell extracts prepared according to the
Luciferase assay system (Promega) and Luminescent .beta.-gal
Detection system (BD Biosciences Clontech). Luciferase activity was
then normalized using the .beta.-galactosidase value. The
luciferase activities were strongly induced in the case of the
IL-12 p40 and p35 promoter constructs in RAW264.7 cells after the
stimulation with IFN-.gamma./LPS or IFN-.gamma./SAC. This p40 and
p35 promoter-driven luciferase expression was suppressed in the
presence of Compound 1 and Compound 2, but not the inactive
negative control compound. The results are shown in FIGS. 2A-2B.
This result supports a mechanism in which Compound 2 inhibits IL-12
transcription. p35 promoter-driven luciferase expression stimulated
by IFN-.gamma./LPS was inhibited more effectively by Compound 2
than by Compound 1, while the negative control compound did not
suppress the promoter activity at all. The IC.sub.50s of Compound
1, Compound 2, and the negative control compound against IL-12
production in THP-1 cells were 40 nM, 10 nM, and greater than 1000
nM, respectively. These results are in agreement with the
inhibitory activity against IL-12 protein production evaluated by
ELISA, signifying that the inhibition of the p35 promoter activity
is a reflection of the inhibitory activity against IL-12. ELISA was
performed by the following method. Human IL-12 p70 (heterodimer)
was assayed using ELI-PAIR kit from Cell Sciences (Norwood, Mass.),
according to the manufacturer's instructions. Human IL-12 p40 was
assayed using ELISA kit from Cell Sciences (Norwood, Mass.)
according to the manufacturer's instructions.
[0231] Northern blot analysis to examine the mRNA levels of IL-12
p35 and p40 was used to elucidate the mechanism of action. Compound
1 caused a decrease in the levels of p35 and p40 mRNA. Nuclear
run-on experiments showed that this effect was at the level of
transcription initiation. When DNA expression plasmids in which the
p35 and p40 promoters directed the expression of the luciferase
reporter gene were transfected into cells, it was shown that
expression of luciferase could be inhibited by Compound 1 and
Compound 2. These results confirm a mechanism in which Compound 2
inhibits IL-12 transcription of the p35 and p40 genes.
[0232] We then set out to analyze the IL-12 transcriptional
promoter elements that played a role in this effect. We performed
deletion analyses using the p40 promoter. This promoter, rather
than the p35 promoter, was chosen because the transcriptional
elements are better defined in the p40 promoter. To identify the
IL-12 inhibitor responsive elements involved in the p40 gene
transcription activation, three different promoters were
constructed and transiently transfected into RAW264.7 cells. As
shown in FIGS. 3A-3B, the promoters that consisted of the
NF-.kappa.B through the AP1 element region showed diminished
promoter activation, while the promoter that contained the 5'
flanking region of p40 promoter but had a large proximal deletion
displayed significantly decreased promoter activity in response to
stimulation with IFN-.gamma./LPS. Only the promoter which contained
the Ets-2 element along with the PU-1, NF-.kappa.B and API elements
showed high activity of luciferase in response to IFN-.gamma./LPS
stimulation, suggesting that the Ets-2 element plays a role in the
regulation of IL-12 p40 promoter activity. This IL-12
promoter-driven luciferase activity was significantly suppressed in
the presence of Compound 1. These results suggest that the element
that is responsive in the suppression of promoter activity lies in
the region from the TATA box to -250 bp in the IL-12 p40
promoter.
[0233] To assess the role of individual p40 promoter transcription
elements in more detail, mutations within many of these elements
have been generated. The goal of this work is to assess the effect
of mutations which decrease but do not eliminate p40 promoter
activity on inhibition by Compound 2. All mutations in the Ets-2
element have completely eliminated the induction of reporter gene
expression, emphasizing the importance of this element.
Site-directed mutatgenesis of the NF.kappa.B element resulted in a
p40 promoter having reduced but clearly measurable induction by
IFN-.gamma./LPS. Site-directed mutagenesis was performed with the
GeneTailor Site-directed Mutagenesis System (Invitrogen, Carlsbad,
Calif.) according to the manufacturer's instructions. The IL-12 p40
Mutant primer sequences were as follows: IL-12 p40-Ets2 mut-F:
5'-TATTCCCCACCCAAAAGTCACTTAGTTCATT-3' (SEQ ID NO:11) and IL-12
p40-Ets2 mut-R: 5'-TGACITTTGGGTGGGGAATAAGGAAGGAGA-3' (SEQ ID
NO:12); IL-12 p40-AP-1 mut-F: 5'-TTGTTTTCAATGTTGCAACATTTCTAGTTTA-3'
(SEQ ID NO:13) and IL-12 p40-AP-1 mut-R:
5'-TGTTGCAACATTGAAAACAACTCTCAAAAC-3' (SEQ ID NO:14); IL-12
p40-NF.kappa.B mut-F: 5'-CAAACAAAAAAGGAACTTCTCAGAAGGT1TT-3' (SEQ ID
NO:15) and IL-12 p40-NF.kappa.B mut-R: 5'-AGAAGTTCC
TTTTTT'GTTTGTCTCTCTCTG-3' (SEQ ID NO:16); IL-12 p40-PU-1 mut-F:
5'-ACAGAGAGAGACAAACAAAACTTCTTGAAAT-3' (SEQ ID NO:17) and IL-12
p40-PU-1 mut-R: 5'-TTTTGTTTGTCTCTCTCTGTGTGTGTATCA-3' (SEQ ID
NO:18).
[0234] Interestingly, inhibition of expression by Compound 2 was
reduced in this mutant construct, indicating a role of NF.kappa.B.
Since the transcription factor NF.kappa.B has been shown to be
involved in the regulation of IL-12 p40 gene expression, we
examined whether STA-1856 alters the binding of NF.kappa.B to its
cognate site on the p40 promoter. Nuclear extracts were prepared
from IFN-.gamma.-primed THP-1 cells that had been treated with or
without SAC and incubated in the presence or absence of 1 .mu.M
Compound 1 or 10 mM ASA. Isolation of nuclear extracts was
accomplished by first suspending THP-1 cells in 20 volumes of
buffer A containing 10 mM KCl, 10 mM HEPES (pH 7.9), 1 mM MgC12, 1
mM dithiothreitol (DTT), 0.1% Nonidet p40 (NP-40), and 0.5 mM
phenylmethylsulfonyl fluoride (PMSF) and then homogenizing and
centrifuging at 10,000 rpm at 4.degree. C. for 5 min. Nuclear
pellets were then suspended in buffer C containing 400 mM NaCI, 20
mM HEPES pH 7.9), 15 mM MgC1.sub.2, 0.2 mM EDTA, 1 mM DTT, 25%
glycerol, 1 mM PMSF, and 10 .mu.g of leupeptin, 20 .mu.g of
pepstatin, and 10 .mu.g of antipain per ml, incubated for 30 min at
4.degree. C., and centrifuge at 14,000 rpm for 20 min. The
supernatants were dialyzed against buffer D containing 100 mM NaCl,
20 mM HEPES (pH 7.9), 20% glycerol, 1 mM PMSF, and 1 mM DTT.
[0235] The extracts obtained from this process were used in
gel-shift assays using oligonucleotides containing the NF.kappa.B
target sequence corresponding to the region -121 to -102 from the
transcription initiation site of IL-12 p40 or a mutated NF.kappa.B
binding site. The binding of NF.kappa.B to the probe comprising its
cognate sequence from the p40 promoter was strongly induced in
IFN-.gamma./SAC-stimulated THP-1 cells. This interaction was
specific as it was competed away by an excess of unlabeled probe,
but not by a mutated oligonucleotide in which two base-pairs were
substituted. Compound 1 did not show any influence on NF.kappa.B
binding. In contrast, ASA reduced the binding significantly,
despite the fact that the percent inhibition of production of IL-12
p70 protein elicited by 1 .mu.M Compound 1 and 10 mM ASA were 97%
and 45%, respectively. Combined with the lack of any effect of
Compound 2 on I.kappa.B, these results show that the strong
inhibitory activity of Compound 1/Compound 2 on IL-12 production is
not due to a gross reduction in total NF.kappa.B binding activity.
This is expected since a compound that potently blocks NF.kappa.B
have a far broader cytokine inhibitory profile than Compound
1/Compound 2.
[0236] To understand the action of Compound 2 in NF-.kappa.B
binding, several NF-.kappa.B family members, p50, c-Rel and p65
were investigated using an ELISA based transcriptional factor-DNA
binding activity assay system. DNA-transcription factor binding
activities assays were performed with EZ-detect transcription
Factor kit-NF.kappa.B p50 or p65 (Pierce, Rockford, Ill.), and BD
Mercury TransFactor Kits-NF.kappa.B (BD Biosciences Clontech, Palo
Alto, Calif.) according to the manufacturer's instruction.
[0237] The binding activities of p50, c-Rel and p65 were
significantly increased in nuclear extracts from THP-1 cells 3 hrs
after IFN-.gamma./LPS stimulation. The binding activity of c-Rel
was significantly decreased, and p50 was slightly decreased in the
presence of Compound 2 (500 nm) for 3 hrs. In the case of p65, the
increased binding activity was observed in the presence of Compound
2 in response to the IFN-.gamma./LPS stimulation. This is a
consequence of the lack of binding competition as a result of a
decrease in p50 and c-Rel.
III. NF-kB Proteins Translocation:
[0238] Our DNA-protein interaction study showed that the binding
activity of c-Rel and p50, which form functional active
heterodimers in IL-12, were decreased, and the binding activity of
p65 was increased in response to Compound 2 treatment. In order to
understand this changes, the subcellular localization of
NF-.kappa.B proteins was investigated using western blot analysis.
The amount of c-Rel and p50 protein in nuclear were found to be
decreased, and the amount of p65 in nuclear was found significantly
increased in cells treated with Compound 2 (500 nM) for 3 hr
relative to untreated cells. This finding is in agreement with our
DNA-protein interaction study, and indicated that the impaired
activity of p50/c-Rel and increased p65 binding activity could
cause the unbalance of the NF-.kappa.B proteins in nucleus and
effect the binding activity of p50/c-Rel.
IV. Effect of Compound 2 on c-Rel and ICSBP (Measuring the Level of
Both in the Nucleus)
[0239] Of the transcription factors that have been analyzed, two
factors, ICSBP and c-Rel, seem to be affected by Compound
1/Compound 2 treatment. ICSBP binds indirectly to the Ets-2 site.
The primary NF.kappa.B trans-activator for IL-12 is the c-Rel/p50
heterodimer. Other dimers (p65/p50 and p50/p50) either lack
activity or have inhibitory functions. Thus, c-Rel plays a role in
IL-12 transcription as a result of both activation through
NF.kappa.B and its interaction with ICSBP. Both Western blot
analysis and DNA binding studies showed a decrease in nuclear c-Rel
levels following Compound 2 treatment. As seen in FIG. 4, a western
blot assay of THP1 nuclear c-Rel, p50 and p65 proteins was carried
out by the following method: 10% SDS polyacrylamide gels
(Invitrogen) were transferred to a Pure nitrocellulose membrane
(BioRed, Hercules, Calif.). The membranes were blocked with 5% milk
in TBST buffer and then incubated with anti-c-Rel, anti-p65,
anti-p50, anti-ICSBP or anti-PU-1 antibody (all the antibodies were
purchased from Santa Cruz) at a dilution of 1:500 for 1 h at room
temperature or overnight at 4.degree. C. The membranes were washed
and incubated with Horseradish Peroxidase-conjugated anti-rabbit
IgG or anti-mouse IgG (Amersharm, England) at a dilution of 1:2000
at room temperature for 1 h.
[0240] Both IFN-.gamma. plus LPS and IFN-.gamma. plus SAC treatment
strongly increased the amount of nuclear c-Rel, p65 and p50.
Compound 2 treatment significantly reduced the levels of c-Rel,
with the post-treatment nuclear c-Rel level being equal to or below
the non-stimulated level. In contrast, nuclear p65 protein
increased following Compound 2 treatment. p50 levels decreased
slightly following Compound 2 treatment, but remained above the
non-stimulated levels. Thus, it is shown that Compound 2 treatment
causes a reduction in the amount of nuclear c-Rel/p50, the primary
IL-12 activating NF.kappa.B dimer.
[0241] ICSBP, whose expression was reduced by Compound 2, was
over-expressed using co-transfection with the IL-12 promoter-Luc
report system. The over-expression construct of ICSBP was generated
by PCR from cDNA of human PBMC using primers as follow:
ICSBP-exp-F: 5'-CCGGAATTCAGGATGTGTGACCGGAATGG-3' (SEQ ID NO:19) and
ICSBP-exp-R: 5'-ATATCTAGAATGGATGCAGGACGCAGAC-3' (SEQ ID NO:20), the
resulting PCR products was ligated to pCI vector (Promega). ICSBP
over-expression increased the level of p40 expression and decreased
the inhibition by Compound 2.
V. Effect of Compound 2 on I.kappa.B
[0242] I.kappa.B degradation is one of the steps in the signaling
pathway of NF.kappa.B dependent genes. The activity of Compound 2
in inducible degradation of I.kappa.B.alpha. and I.kappa.B.beta.
was investigated in THP-1 cells using Western blot and FACS
analysis. The amount of I.kappa.B.alpha. and I.kappa.B.beta. in the
cytoplasm of THP-1 and RAW267.4 cells was significantly reduced at
30 min in response to induction by IFN-.gamma./LPS or
IFN-.gamma./SAC. However, there was no significant difference
observed between the samples which were treated with or without
Compound 2 (500 nM) at 30 min and 2 hrs. Similar results were
observed from the Compound 2 pre-treatment samples in which
Compound 2 was added 30 min before stimulation. These results show
that Compound 2 does not induce the degradation of I.kappa.B.alpha.
and I.kappa.B.beta. to allow free NF.kappa.B to translocate into
the nucleus where it can act as a transcription factor.
VI. Measuring the Level of Ets2 in the Nucleus
[0243] The transcription factor ICSBP binds to the Ets-2 element
indirectly through binding to PU-1. Nuclear extracts were bound to
Ets-2 DNA element beads, the beads isolated to separate bound from
free protein, and the proteins analyzed on Western blots using
antibodies to either ICSBP or PU-1. Conjugation of Ets-2 DNA to
beads was accomplished by the following method. Biotinylated DNA
fragment encompassing the IL-12 p40 Ets-2 site (-292 to -196) were
synthesized from the 1.3 kb wild-type human IL-12 p40 reporter by
PCR using a biotinylated primer as detailed in The Journal of
Immunology, 2000, vol 165. pages 271-279. PCR products were
purified by the Qiaquick Kit (Qiagen, Chatsworth, Calif.). Two ttg
of biotinylated DNA were conjugated to 100 .mu.l of
streptavidin-bound magnetic beads (Dynabeads, M280, Dynal, Lake
Success, N.Y.) in buffer containing 10 mM Tris-HCl, pH 8.0, 1 mM
EDTA, 0.1 M NaCl. Ten .mu.l of beads conjugated to 2 .mu.g of DNA
were equilibrated with TGEDN buffer (120 mM Tris-HCL, pH 8.0, 1 mM
EDTA, 0.1 M NaCl, 1 mM DTT, 0.1% Triton X100, 10% glycerol) and
incubated with 500 .mu.g of THP-1 cell nuclear extracts and 20
.mu.g of Herring sperm DNA (GibCo) at 4.degree. C. for 2 h. Beads
were washed in TGEDN buffer, and bound materials were eluted in 20
.mu.l of the same buffer supplemented with 0.5% SDS and 1 M NaCl.
Eluted materials were separated by 10% SDS-PAGE and detected by
immunoblot analysis using anti-ICSBP or anti-PU-1 antibody.
[0244] Western blot analysis showed a significant reduction in the
amount of ICSBP protein in nuclear extracts of THP-1 cells treated
with Compound 1, see FIG. 5. In contrast, the levels of PU-1 were
unaffected by Compound 1 treatment, see FIG. 6.
[0245] Of particular interest is the finding that both ICSBP and
c-Rel were reduced in the nuclei of Compound 2 treated cells. Since
these two transcription factors interact with each other, a
decrease in the levels of both factors would be expected to have a
profound effect. Compound 2 selectively inhibits expression of
genes which are dependent upon the ICSBP-c-Rel interaction for
trans-activation.
[0246] Although c-Rel has a role in the expression of both p35 and
p40 in monocytes and macrophages as well as p35 in dendritic cells
(DCs), p40 expression in dendritic cells is c-Rel-independent
(Grumont et al. J. Exp. Med. 2001; 194:1021-1031). If Compound 2 is
acting through c-Rel, this drug should inhibit both p40 and p70
production by PBMCs. However, Compound 2 should inhibit the
production of p70 (through inhibition of p35) in DCs, but should
not inhibit p40 in DCs. This was tested by generating
monocyte-derived dendritic cells according to the following method.
Human PBMC at 1.times.10.sup.7 cells/ml were suspended in
serum-free DMEM and incubated for 2 hrs at 37.degree. C. under 5%
CO.sub.2. The non-adherent cells then were removed by washing with
PBS. The adherent cells were cultured in RPMI-1640 medium
containing rhIL-4 (100 11/m1) and rhGM-CSF (1000 U/ml) for 6-7
days. The half volume fresh media and full-volume fresh cytokines
were added every other day. The cells were then primed with
IFN-.gamma. (100 U/ml) and followed by 0.01% SAC or 1 .mu.g/ml LPS,
in the presence of different concentrations of Compound 2 or other
compounds. The test compounds were prepared in DMSO and the final
DMSO concentration was adjusted to 0.25% in all cultures, including
the compound-free control. Cell-free supernatants were taken 18 h
later for the measurement of cytokines.
[0247] Compound 2 did, in fact, inhibit p70 but not p40 production
in human DCs (no inhibition at up to 10 .mu.M Compound 2). Also,
the induction of IL-12 p40 production following Toxoplasma antigen
(STAg) stimulus has been shown to be c-Rel independent. Therefore,
if Compound 2 is acting through c-Re1, we should not observe potent
inhibition of IL-12 p40 following STAg stimulation. Preliminary
results showed that the IC.sub.50 of Compound 2-mediated p40
inhibition was approximately 1000 times higher with STAg induction
relative to IF N-.gamma./SAC stimulus, while the IC50 of
dexamethasone was the same in the case of both SAC and STAg
stimuli. These two results further confirm that the inhibition of
IL-12 production by Compound 2 is via the c-Rel pathway.
VII. Kinetics of the Members of NF-.kappa.B Nuclear Translocation
in Compound 2-Treated Cells:
[0248] Compound 2 impairs nuclear translocation of c-Rel and p50.
We examined the nuclear translocation kinetics of NF-.kappa.B
family members in LPS stimulated cells treated with Compound 2.
THP1 cells were stimulated with LPS in either the presence or
absence of 100 nM Compound 2, and the distribution of the
NF-.kappa.B Rel family members are determined by immunoblotting
nuclear (n.p.) extracts collected at 5 min, 15 min, 30 min, 1 h, 3
h and 6 h post-treatment. In response to LPS stimulation, p50
translocated into the nucleus as early as 5 minutes
post-stimulation and accumulates as time goes on (FIG. 7,
immunoblots and FIG. 8 densitometry). Treatment of LPS-stimulated
cells with Compound 2 had no effect on the kinetics of p50 nuclear
entry at 5 minutes to 1 hr post-stimulation, and showed a small
decrease in nuclear protein levels at 3 hours. The experiment
examining p65 nuclear translocation is shown in FIG. 9
(immunoblots) and FIG. 10 (densitometry). In LPS stimulated cells,
p65 translocated into the nucleus as early as 5 minutes
post-stimulation and accumulated to maximum levels at 15-30 minutes
post-stimulation. Treatment of LPS-stimulated cells with Compound 2
had no effect on the kinetics of p65 nuclear entry. The level of
nuclear p65 at later times (6 hours) showed a small increase in
Compound 2 treated cells relative to untreated cells.
[0249] Without wishing to be bound by theory, Compound 2 does not
affect the kinetics of p50 and p65 nuclear translocation in
response to LPS stimulation. At later times, Compound 2 impairs
nuclear translocation of p50 (at3 h time point), and enhances
nuclear translocation of p65 (at 6 h time point), indicating a
selective effect on the NF-.kappa.B family.
VIII. The Effects of Compound 2 on Nuclear Translocation of p52 and
Rel-B:
[0250] Rel B and p52 are two members of Rel family, which are
preferentially complexed with each other. Like p50 and p65, p52 is
found in virtually all cell types, whereas c-Rel and Rel B have
only been detected in lymphoid tissues. To determine the effect of
Compound 2 on p52 and Rel-B nuclear translocation, THP1 cells were
stimulated with IFNg+LPS in either the presence or absence of 100
nM Compound 2, and the distribution of p52 and Rel-B was determined
by immunoblotting of nuclear at 6 h post-treatment. As shown in
FIG. 11, the nuclear Rel-B was slightly increased in the presence
of Compound 2. No significant difference was found in p52. This
result indicates that Compound 2 specifically inhibits c-Rel and
p50 nuclear translocation, but not other NF-.kappa.B p52 and Rel-B
nuclear translocation.
[0251] Cell Lines and Culture Conditions:
[0252] THP-1 cell line were obtained from American Type Culture
Collection (Manassas, Va.). The THP-1 cells were cultured in RPMI
1640 (ATCC, Manassas, Va.), supplemented with 10% FCS (ATCC,
Manassas, Va.), and 1% penicillin/Streptomycin (Gibco-BRL, New
York, N.Y.). The cells were primed with IFNg (100 U/ml) followed by
1 ug/ml LPS in the presence of different concentrations of Compound
2. Compound 2 was prepared in DMSO and the final DMSO concentration
was adjusted to 0.25% in the cultures, including the compound-free
control. Cell-free supernatants were taken 18 h later for the
measurement of cytokines.
[0253] Isolation of Nuclear Extracts:
[0254] THP-1 cells were suspended in 20 volumes of buffer A
containing 10 mM KCl, 10 mM HEPES (pH 7.9), 1 mM MgCl.sub.2, 1 mM
dithiothreitol (DTT), 0.1% Nonidet p40 (NP-40), and 0.5 mM
phenylmethylsulfonyl fluoride (PMSF) and homogenized and
centrifuged at 10,000 rpm at 4 C afor 5 min. Nuclear pellets were
then suspended in buffer C containing 400 mM NaCl, 20 mM HEPES 9
(pH 7.9), 15 mM MgCl.sub.2, 0.2 mM EDTA, 1 mM DTT, 25% glycerol, 1
mM PMSF, and 10 ug of leupeptin, 20 ug of pepstatin, and 10 ug/m1
antipain, incubated for 30 min at 4 C, and centrifuged at 14,000
rpm for 20 min. The supernatants were dialyzed against buffer D
containing 100 mM NaCl, 20 mM HEPES (pH 7.9), 20% glycerol, 1 mM
PMSF, and 1 mM DTT.
[0255] Western Blot:
[0256] The 10% SDS Polyacrylamide gels (Invitrogen) were
transferred to Pure Nitrocellulose membrane (BioRed, Hercules,
Calif.). The membranes were blocked with 5% milk in TBST buffer and
incubated with anti-c-Rel, anti-p65, anti-p50, anti-ICSBP or
anti-PU-1 antibody (all the antibodies were purchased from Santa
Cruz) at a dilution of 1:500 for 1 h at room temperature or
overnight at 4 C. The membranes were washed and incubated with
Horseradish Peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG
(Amersham, England) at a dilution of 1:2000 at room temperature for
1 h.
[0257] Compounds 3-14 are expected to have similar activity as
Compounds 1 and 2 in the procedures described in Examples I through
VIII.
[0258] Many modifications and variations of this invention can be
made without departing from its spirit and scope, as will be
apparent to those skilled in the art. The specific embodiments
described herein are offered by way of example only, and the
invention is to be limited only by the terms of the appended
claims, along with the full scope of equivalents to which such
claims are entitled. Such modifications are intended to fall within
the scope of the appended claims.
[0259] All references, patent and non-patent, cited herein are
incorporated herein by reference in their entirety and for all
purposes to the same extent as if each individual publication or
patent or patent application was specifically and individually
indicated to be incorporated by reference in its entirety for all
purposes.
[0260] All of the features, specific embodiments and particular
substituents disclosed herein may be combined in any combination.
Each feature, embodiment or substituent disclosed in this
specification may be replaced by an alternative feature, embodiment
or substituent serving the same, equivalent, or similar purpose. In
the case of chemical compounds, specific values can be combined in
any combination resulting in a stable structure. Furthermore,
specific values (whether preferred or not) for substituents in one
type of chemical structure may be combined with values for other
substituents (whether preferred or not) in the same or different
type of chemical structure. Thus, unless expressly stated
otherwise, each feature, embodiment or substituent disclosed is
only an example of a generic series of equivalent or similar
features feature, embodiments or substituents.
Sequence CWU 1
1
2012337DNAHomo sapiensmodified_base(61)..(61)a, c, t, g, unknown or
othermodified_base(2277)..(2277)a, c, t, g, unknown or other
1cggaaggtgt gagccgcaaa cccagcggag ggcgggaaga aggaggaggc ctctagggtg
60ntcgggggac tgggggcccc gccggcagag gtccctcggc ctcctgactg actgactgcg
120gccgcctccg gccaggacgc tgggagctgc ctgcgggaag gtgcggggag
cggagccatg 180gcctccggtg cgtataaccc gtatatagag ataattgaac
aacccaggca gaggggaatg 240cgttttagat acaaatgtga agggcgatca
gcaggcagca ttccagggga gcacagcaca 300gacaacaacc gaacataccc
ttctatccag attatgaact attatggaaa aggaaaagtg 360agaattacat
tagtaacaaa gaatgaccca tataaacctc atcctcatga tttagttgga
420aaagactgca gagacggcta ctatgaagca gaatttggac aagaacgcag
acctttgttt 480ttccaaaatt tgggtattcg atgtgtgaag aaaaaagaag
taaaagaagc tattattaca 540agaataaagg caggaatcaa tccattcaat
gtccctgaaa aacagctgaa tgatattgaa 600gattgtgacc tcaatgtggt
gagactgtgt tttcaagttt ttctccctga tgaacatggt 660aatttgacga
ctgctcttcc tcctgttgtc tcgaacccaa tttatgacaa ccgtgctcca
720aatactgcag aattaaggat ttgtcgtgta aacaagaatt gtggaagtgt
cagaggagga 780gatgaaatat ttctactttg tgacaaagtt cagaaagatg
acatagaagt tcgttttgtg 840ttgaacgatt gggaagcaaa aggcatcttt
tcacaagctg atgtacaccg tcaagtagcc 900attgttttca aaactccacc
atattgcaaa gctatcacag aacccgtaac agtaaaaatg 960cagttgcgga
gaccttctga ccaggaagtt agtgaatcta tggattttag atatctgcca
1020gatgaaaaag atacttacgg caataaagca aagaaacaaa agacaactct
gcttttccag 1080aaactgtgcc aggatcacgt agaaacaggg tttcgccatg
ttgaccagga tggtcttgaa 1140ctcctgacat caggtgatcc acccaccttg
gcctcccaaa gtgctgggat tacagttaat 1200tttcctgaga gaccaagacc
tggtctcctc ggttcaattg gagaaggaag atacttcaaa 1260aaagaaccaa
acttgttttc tcatgatgca gttgtgagag aaatgcctac aggggtttca
1320agtcaagcag aatcctacta tccctcacct gggcccatct caagtggatt
gtcacatcat 1380gcctcaatgg cacctctgcc ttcttcaagc tggtcatcag
tggcccaccc caccccacgc 1440tcaggcaata caaacccact gagtagtttt
tcaacaagga cacttccttc taattcgcaa 1500ggtatcccac cattcctgag
aatacctgtt gggaatgatt taaatgcttc taatgcttgc 1560atttacaaca
atgccgatga catagtcgga atggaagcgt catccatgcc atcagcagat
1620ttatatggta tttctgatcc caacatgctg tctaattgtt ctgtgaatat
gatgacaacc 1680agcagtgaca gcatgggaga gactgataat ccaagacttc
tgagcatgaa tcttgaaaac 1740ccctcatgta attcagtgtt agacccaaga
gacttgagac agctccatca gatgtcctct 1800tccagtatgt cagcaggcgc
caattccaat actactgttt ttgtttcaca atcagatgca 1860tttgagggat
ctgacttcag ttgtgcagat aacagcatga taaatgagtc gggaccatca
1920aacagtacta atccaaacag tcatggtttt gttcaagata gtcagtattc
aggtattggc 1980agtatgcaaa atgagcaatt gagtgactcc tttccatatg
aattttttca agtataactt 2040gcaagattta aatcctttta aatcttgata
ccacctatat agatgcagca ttttgtattt 2100gtctaactgg ggatataata
ctatatttat actgtatata taatactgac tgagaatata 2160atactgtatt
tgagaatata aaaaactttt ttcagggaag aagcatacaa ctttggacat
2220agcgaataca aaattggaag ctgtcataaa aagacaactc agaggccagg
cgcaggngct 2280cacacctgta atcctagcac tttgggaggc caaggcgggt
ggatcacttg agaccag 23372619PRTHomo sapiens 2Met Ala Ser Gly Ala Tyr
Asn Pro Tyr Ile Glu Ile Ile Glu Gln Pro1 5 10 15Arg Gln Arg Gly Met
Arg Phe Arg Tyr Lys Cys Glu Gly Arg Ser Ala 20 25 30Gly Ser Ile Pro
Gly Glu His Ser Thr Asp Asn Asn Arg Thr Tyr Pro 35 40 45Ser Ile Gln
Ile Met Asn Tyr Tyr Gly Lys Gly Lys Val Arg Ile Thr 50 55 60Leu Val
Thr Lys Asn Asp Pro Tyr Lys Pro His Pro His Asp Leu Val65 70 75
80Gly Lys Asp Cys Arg Asp Gly Tyr Tyr Glu Ala Glu Phe Gly Gln Glu
85 90 95Arg Arg Pro Leu Phe Phe Gln Asn Leu Gly Ile Arg Cys Val Lys
Lys 100 105 110Lys Glu Val Lys Glu Ala Ile Ile Thr Arg Ile Lys Ala
Gly Ile Asn 115 120 125Pro Phe Asn Val Pro Glu Lys Gln Leu Asn Asp
Ile Glu Asp Cys Asp 130 135 140Leu Asn Val Val Arg Leu Cys Phe Gln
Val Phe Leu Pro Asp Glu His145 150 155 160Gly Asn Leu Thr Thr Ala
Leu Pro Pro Val Val Ser Asn Pro Ile Tyr 165 170 175Asp Asn Arg Ala
Pro Asn Thr Ala Glu Leu Arg Ile Cys Arg Val Asn 180 185 190Lys Asn
Cys Gly Ser Val Arg Gly Gly Asp Glu Ile Phe Leu Leu Cys 195 200
205Asp Lys Val Gln Lys Asp Asp Ile Glu Val Arg Phe Val Leu Asn Asp
210 215 220Trp Glu Ala Lys Gly Ile Phe Ser Gln Ala Asp Val His Arg
Gln Val225 230 235 240Ala Ile Val Phe Lys Thr Pro Pro Tyr Cys Lys
Ala Ile Thr Glu Pro 245 250 255Val Thr Val Lys Met Gln Leu Arg Arg
Pro Ser Asp Gln Glu Val Ser 260 265 270Glu Ser Met Asp Phe Arg Tyr
Leu Pro Asp Glu Lys Asp Thr Tyr Gly 275 280 285Asn Lys Ala Lys Lys
Gln Lys Thr Thr Leu Leu Phe Gln Lys Leu Cys 290 295 300Gln Asp His
Val Glu Thr Gly Phe Arg His Val Asp Gln Asp Gly Leu305 310 315
320Glu Leu Leu Thr Ser Gly Asp Pro Pro Thr Leu Ala Ser Gln Ser Ala
325 330 335Gly Ile Thr Val Asn Phe Pro Glu Arg Pro Arg Pro Gly Leu
Leu Gly 340 345 350Ser Ile Gly Glu Gly Arg Tyr Phe Lys Lys Glu Pro
Asn Leu Phe Ser 355 360 365His Asp Ala Val Val Arg Glu Met Pro Thr
Gly Val Ser Ser Gln Ala 370 375 380Glu Ser Tyr Tyr Pro Ser Pro Gly
Pro Ile Ser Ser Gly Leu Ser His385 390 395 400His Ala Ser Met Ala
Pro Leu Pro Ser Ser Ser Trp Ser Ser Val Ala 405 410 415His Pro Thr
Pro Arg Ser Gly Asn Thr Asn Pro Leu Ser Ser Phe Ser 420 425 430Thr
Arg Thr Leu Pro Ser Asn Ser Gln Gly Ile Pro Pro Phe Leu Arg 435 440
445Ile Pro Val Gly Asn Asp Leu Asn Ala Ser Asn Ala Cys Ile Tyr Asn
450 455 460Asn Ala Asp Asp Ile Val Gly Met Glu Ala Ser Ser Met Pro
Ser Ala465 470 475 480Asp Leu Tyr Gly Ile Ser Asp Pro Asn Met Leu
Ser Asn Cys Ser Val 485 490 495Asn Met Met Thr Thr Ser Ser Asp Ser
Met Gly Glu Thr Asp Asn Pro 500 505 510Arg Leu Leu Ser Met Asn Leu
Glu Asn Pro Ser Cys Asn Ser Val Leu 515 520 525Asp Pro Arg Asp Leu
Arg Gln Leu His Gln Met Ser Ser Ser Ser Met 530 535 540Ser Ala Gly
Ala Asn Ser Asn Thr Thr Val Phe Val Ser Gln Ser Asp545 550 555
560Ala Phe Glu Gly Ser Asp Phe Ser Cys Ala Asp Asn Ser Met Ile Asn
565 570 575Glu Ser Gly Pro Ser Asn Ser Thr Asn Pro Asn Ser His Gly
Phe Val 580 585 590Gln Asp Ser Gln Tyr Ser Gly Ile Gly Ser Met Gln
Asn Glu Gln Leu 595 600 605Ser Asp Ser Phe Pro Tyr Glu Phe Phe Gln
Val 610 615325DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 3gcagcattag aaggggcctt agaga
25423DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 4ttttataatt gtcccgaggc gcg 23521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
5acggcgagga aagttagccc g 21620DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 6ttgctctggg caggacggag
20720DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 7cacccaaaag tcatttcctc 20819DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
8tgctctgggc aggacggag 19923DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 9agagttgttt tcaatgttgc aac
231019DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 10tgctctgggc aggacggag 191131DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
11tattccccac ccaaaagtca cttagttcat t 311230DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
12tgacttttgg gtggggaata aggaaggaga 301331DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
13ttgttttcaa tgttgcaaca tttctagttt a 311430DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
14tgttgcaaca ttgaaaacaa ctctcaaaac 301531DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
15caaacaaaaa aggaacttct cagaaggttt t 311630DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
16agaagttcct tttttgtttg tctctctctg 301731DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
17acagagagag acaaacaaaa cttcttgaaa t 311830DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
18ttttgtttgt ctctctctgt gtgtgtatca 301929DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
19ccggaattca ggatgtgtga ccggaatgg 292028DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
20atatctagaa tggatgcagg acgcagac 28
* * * * *