U.S. patent application number 12/790099 was filed with the patent office on 2010-09-23 for method of modulating stress-activated protein kinase system.
Invention is credited to Leonid Beigelman, Lawrence M. Blatt, Karl Kossen, Ramachandran Radhakrishnan, Scott D. Seiwert, Vladimir Serebryany.
Application Number | 20100240704 12/790099 |
Document ID | / |
Family ID | 36954420 |
Filed Date | 2010-09-23 |
United States Patent
Application |
20100240704 |
Kind Code |
A1 |
Blatt; Lawrence M. ; et
al. |
September 23, 2010 |
METHOD OF MODULATING STRESS-ACTIVATED PROTEIN KINASE SYSTEM
Abstract
Disclosed are methods of modulating a stress activated protein
kinase (SAPK) system with an active compound, wherein the active
compound exhibits low potency for inhibition of at least one p38
MAPK; and wherein the contacting is conducted at a SAPK-modulating
concentration that is at a low percentage inhibitory concentration
for inhibition of the at least one p38 MAPK by the compound. Also
disclosed are derivatives of pirfenidone. These derivatives can
modulate a stress activated protein kinase (SAPK) system.
Inventors: |
Blatt; Lawrence M.; (San
Francisco, CA) ; Seiwert; Scott D.; (Pacifica,
CA) ; Beigelman; Leonid; (San Mateo, CA) ;
Radhakrishnan; Ramachandran; (Fremont, CA) ; Kossen;
Karl; (Brisbane, CA) ; Serebryany; Vladimir;
(Burlingame, CA) |
Correspondence
Address: |
Marshall, Gerstein & Borun LLP (Intermune)
233 South Wacker Drive, 6300 Willis Tower
Chicago
IL
60606
US
|
Family ID: |
36954420 |
Appl. No.: |
12/790099 |
Filed: |
May 28, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11431132 |
May 9, 2006 |
7728013 |
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12790099 |
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60679471 |
May 10, 2005 |
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60732230 |
Nov 1, 2005 |
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Current U.S.
Class: |
514/334 ; 506/9;
514/345; 514/350; 546/257; 546/290; 546/298; 546/301 |
Current CPC
Class: |
A61P 13/00 20180101;
A61P 25/16 20180101; C07D 213/63 20130101; A61P 11/00 20180101;
A61K 31/4704 20130101; A61P 11/16 20180101; A61K 31/4412 20130101;
A61P 25/28 20180101; A61K 31/44 20130101; Y02A 50/411 20180101;
A61P 1/04 20180101; A61P 11/06 20180101; Y02A 50/401 20180101; Y02A
50/409 20180101; A61P 17/00 20180101; A61P 31/00 20180101; A61P
31/08 20180101; A61P 35/00 20180101; A61P 9/00 20180101; A61P 9/08
20180101; A61P 31/12 20180101; A61P 31/22 20180101; A61K 31/7052
20130101; A61P 3/10 20180101; A61P 33/06 20180101; A61P 35/02
20180101; A61P 37/08 20180101; A61P 9/04 20180101; A61P 43/00
20180101; A61P 9/10 20180101; A61P 17/06 20180101; A61P 29/02
20180101; C07D 213/26 20130101; A61K 31/444 20130101; A61P 13/12
20180101; Y02A 50/475 20180101; A61K 31/45 20130101; A61K 47/58
20170801; A61P 21/04 20180101; A61P 31/04 20180101; C07D 213/16
20130101; A61P 25/00 20180101; C07D 213/69 20130101; C07H 15/26
20130101; C07K 1/1077 20130101; A61P 31/16 20180101; Y02A 50/30
20180101; A61P 7/04 20180101; A61P 1/00 20180101; A61P 7/02
20180101; A61P 29/00 20180101; C08F 289/00 20130101; A61P 31/18
20180101; C07D 215/227 20130101; A61K 47/56 20170801; A61P 21/00
20180101; A61P 37/02 20180101; A61P 19/02 20180101; A61P 19/08
20180101; C07D 213/64 20130101; A61P 19/06 20180101 |
Class at
Publication: |
514/334 ;
514/345; 514/350; 506/9; 546/257; 546/290; 546/298; 546/301 |
International
Class: |
A61K 31/444 20060101
A61K031/444; A61K 31/4418 20060101 A61K031/4418; C40B 30/04
20060101 C40B030/04; C07D 213/22 20060101 C07D213/22; C07D 213/63
20060101 C07D213/63; C07D 211/72 20060101 C07D211/72; A61P 25/28
20060101 A61P025/28; A61P 9/10 20060101 A61P009/10; A61P 31/18
20060101 A61P031/18; A61P 31/12 20060101 A61P031/12; A61P 31/22
20060101 A61P031/22; A61P 37/02 20060101 A61P037/02 |
Claims
1.-92. (canceled)
93. A method of modulating a stress activated protein kinase (SAPK)
system, comprising contacting a compound selected from the group
consisting of ##STR00054## ##STR00055## or a pharmaceutically
acceptable salt or ester thereof; with a p38 mitogen-activated
protein kinase (MAPK).
94. A method of treating or preventing an inflammatory or fibrotic
condition comprising administering to a subject in need thereof a
effective amount of at least one compound selected from the group
consisting of ##STR00056## ##STR00057## or a pharmaceutically
acceptable salt or ester thereof; wherein the effective amount
produces a blood or serum or other bodily fluid concentration that
is less than an EC.sub.30 for inhibition for at least one p38
MAPK.
95. The method of claim 94, wherein the anti-inflammatory or
fibrotic condition is selected from the group consisting of
fibrosis, chronic obstructive pulmonary disease, inflammatory
pulmonary fibrosis, idiopathic pulmonary fibrosis, bronchiolitis
obliterans syndrome, chronic allograft fibrosis, rheumatoid
arthritis, rheumatoid spondylitis, osteoarthritis, gout, sepsis,
septic shock; endotoxic shock, gram-negative sepsis, toxic shock
syndrome, myofacial pain syndrome (MPS), Shigellosis, asthma, adult
respiratory distress syndrome, inflammatory bowel disease, Crohn's
disease, psoriasis, eczema, ulcerative colitis, glomerular
nephritis, scleroderma, chronic thyroiditis, Grave's disease,
Ormond's disease, autoimmune gastritis, myasthenia gravis,
autoimmune hemolytic anemia, autoimmune neutropenia,
thrombocytopenia, pancreatic fibrosis, chronic active hepatitis,
hepatic fibrosis, renal disease, renal fibrosis, irritable bowel
syndrome, pyresis, restenosis, cerebral malaria, stroke and
ischemic injury, neural trauma, Alzheimer's disease, Huntington's
disease, Parkinson's disease, acute or chronic pain, an allergy,
cardiac hypertrophy, chronic heart failure, acute coronary
syndrome, cachexia, malaria, leprosy, leishmaniasis, Lyme disease,
Reiter's syndrome, acute synoviitis, muscle degeneration, bursitis,
tendonitis, tenosynoviitis, herniated, ruptured, or prolapsed
intervertebral disk syndrome, osteopetrosis, thrombosis, silicosis,
pulmonary sarcosis, bone resorption disease, cancer, Multiple
Sclerosis, lupus, fibromyalgia, AIDS, Herpes Zoster, Herpes
Simplex, influenza virus, Severe Acute Respiratory Syndrome (SARS),
cytomegalovirus, and diabetes mellitus.
96. The method of claim 94, wherein the effective amount is less
than 50% of an amount that causes an undesirable side effect in the
subject.
97. The method of claim 94, wherein the compound inhibits a kinase
in the SAPK signaling pathway.
98. The method of claim 94, wherein the administering of the
compound is on a schedule selected from the group consisting of
twice a day, once a day, once every two days, three times a week,
twice a week, and once a week.
99. The method of claim 95, wherein the administering of the
compound is on a schedule selected from the group consisting of
twice a day, once a day, once every two days, three times a week,
twice a week, and once a week.
100. The method of claim 95, wherein the anti-inflammatory or
fibrotic condition is bronchiolitis.
101. The method of claim 95, wherein the anti-inflammatory or
fibrotic condition is chronic allograft fibrosis.
102. The method of claim 95, wherein the anti-inflammatory or
fibrotic condition is idiopathic pulmonary fibrosis.
103. The method of claim 94, wherein the compound has a structure
##STR00058## or a pharmaceutically acceptable salt or ester
thereof.
104. The method of claim 94, wherein the compound has a structure
##STR00059## or a pharmaceutically acceptable salt or ester
thereof.
105. The method of claim 94, wherein the compound has a structure
##STR00060## or a pharmaceutically acceptable salt or ester
thereof.
106. The method of claim 94, wherein the compound has a structure
##STR00061## or a pharmaceutically acceptable salt or ester
thereof.
107. The method of claim 94, wherein the compound has a structure
##STR00062## or a pharmaceutically acceptable salt or ester
thereof.
108. The method of claim 94, wherein the compound has a structure
##STR00063## or a pharmaceutically acceptable salt or ester
thereof.
109. The method of claim 94, wherein the compound has a structure
##STR00064## or a pharmaceutically acceptable salt or ester
thereof.
110. The method of claim 94, wherein the compound has a structure
##STR00065## or a pharmaceutically acceptable salt or ester
thereof.
111. The method of claim 94, wherein the compound has a structure
##STR00066## or a pharmaceutically acceptable salt or ester
thereof.
112. The method of claim 94, wherein the compound has a structure
##STR00067## or a pharmaceutically acceptable salt or ester
thereof.
113. The method of claim 94, wherein the compound has a structure
##STR00068## or a pharmaceutically acceptable salt or ester
thereof.
114. The method of claim 94, wherein the compound has a structure
##STR00069## or a pharmaceutically acceptable salt or ester
thereof.
115. The method of claim 94, wherein the compound has a structure
##STR00070## or a pharmaceutically acceptable salt or ester
thereof.
116. The method of claim 94, wherein the compound has a structure
##STR00071## or a pharmaceutically acceptable salt or ester
thereof.
117. The method of claim 94, wherein the compound has a structure
##STR00072## or a pharmaceutically acceptable salt or ester
thereof.
118. A method of identifying a pharmaceutically active compound,
comprising: assaying a plurality of compounds from a library of
compounds for inhibition of at least one p38 MAPK; and selecting at
least one compound from the plurality of compounds, wherein the
selected compound exhibits an EC.sub.50 in the range of about 1
.mu.M to about 1000 .mu.M for inhibition of the at least one p38
MAPK.
119. A method of identifying a pharmaceutically active compound,
comprising: assaying a plurality of compounds from a library of
compounds for inhibition of TNF.alpha. secretion in a bodily fluid
in vivo; and selecting at least one compound from the plurality of
compounds, wherein the selected compound exhibits an EC.sub.50 in
the range of about 1 .mu.M to about 1000 .mu.M for inhibition of
TNF.alpha. secretion in a bodily fluid in vivo.
120. A compound having the formula of Subgenus III: ##STR00073##
wherein X.sub.3 is selected from the group consisting of H, F, and
OH; R.sub.2 is selected from the group consisting of H and
CF.sub.3; and wherein the compound exhibits an EC.sub.50 in the
range of about 1 .mu.M to about 1000 .mu.M for inhibition of p38
MAPK; or a pharmaceutically acceptable salt, ester, solvate or
prodrug of the compound.
121. A compound having the formula of Genus VII: ##STR00074##
wherein X.sub.3 is H, halogen, alkoxy, or OH; Y.sub.1, Y.sub.2,
Y.sub.3, and Y.sub.4 are independently selected from the group
consisting of H, C.sub.1-C.sub.10 alkyl, substituted
C.sub.1-C.sub.10 alkyl, C.sub.1-C.sub.10 alkenyl, C.sub.1-C.sub.10
haloalkyl, C.sub.1-C.sub.10 nitroalkyl, C.sub.1-C.sub.10 thioalkyl,
C.sub.1-C.sub.10 hydroxyalkyl, C.sub.1-C.sub.10 alkoxy, phenyl,
substituted phenyl, halogen, hydroxyl, C.sub.1-C.sub.10
alkoxyalkyl, C.sub.1-C.sub.10 carboxy, C.sub.1-C.sub.10
alkoxycarbonyl; R.sub.4 is H, halogen, or OH; and wherein the
compound exhibits an EC.sub.50 in the range of about 1 .mu.M to
about 1000 .mu.M for inhibition of p38 MAPK; or a pharmaceutically
acceptable salt, ester, solvate or prodrug of the compound.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 60/679,471 filed on May 10, 2005, and U.S.
Provisional Patent Application No. 60/732,230 filed on Nov. 1,
2005, both of which are hereby expressly incorporated by reference
in their entirety.
FIELD OF THE INVENTION
[0002] This invention relates to compounds and methods useful in
treating various inflammatory conditions and/or fibrotic
conditions, including those associated with enhanced activity of
kinase p38.
BACKGROUND OF THE INVENTION
[0003] A large number of chronic and acute conditions have been
recognized to be associated with perturbation of the inflammatory
response. A large number of cytokines participate in this response,
including IL-1, IL-6, IL-8 and TNF.alpha.. It appears that the
activity of these cytokines in the regulation of inflammation may
be associated with the activation of an enzyme of the cell
signaling pathway, a member of the MAP kinase family generally
known as p38 and also known as SAPK, CSBP and RK.
[0004] Several inhibitors of p38, such as NPC 31169, SB239063,
SB203580, FR-167653, and pirfenidone have been tested in vitro
and/or in vivo and found to be effective for modulating
inflammatory responses.
[0005] There continues to be a need for safe and effective drugs to
treat various inflammatory conditions and/or fibrotic conditions
such as inflammatory pulmonary fibrosis and/or idiopathic pulmonary
fibrosis.
SUMMARY OF THE INVENTION
[0006] One embodiment of the present invention is a method of
modulating a stress activated protein kinase (SAPK) system,
including contacting a compound with a p38 mitogen-activated
protein kinase (MAPK), wherein the compound exhibits an EC.sub.50
in the range of about 1 .mu.M to about 1000 .mu.M for inhibition of
at least one p38 MAPK; and wherein the contacting is conducted at a
SAPK-modulating concentration that is less than an EC.sub.30 for
inhibition of the at least one p38 MAPK by the compound.
[0007] Another embodiment of the present invention is a method of
treating or preventing a disease state in a subject, including,
identifying a subject at risk for or having a condition selected
from an inflammatory condition and a fibrotic condition;
administering a compound to the subject in an effective amount to
treat or prevent the condition; wherein the compound exhibits an
EC.sub.50 in the range of about 1 .mu.M to about 1000 .mu.M for
inhibition of at least one p38 MAPK; and wherein the effective
amount produces a blood or serum or another bodily fluid
concentration that is less than an EC.sub.30 for inhibition of the
at least one p38 MAPK.
[0008] Another embodiment of the present invention is a method of
identifying a pharmaceutically active compound, including:
providing a library of compounds; assaying a plurality of compounds
from the library for inhibition of at least one p38 MAPK; and
selecting at least one compound from the plurality of compounds,
wherein the selected compound exhibits an EC.sub.50 in the range of
about 1 .mu.M to about 1000 .mu.M for inhibition of the at least
one p38 MAPK.
[0009] Another embodiment of the present invention is a method of
identifying a pharmaceutically active compound, including:
providing a library of compounds; assaying a plurality of compounds
from the library for inhibition of TNF.alpha. secretion in a bodily
fluid in vivo; and selecting at least one compound from the
plurality of compounds, wherein the selected compound exhibits an
EC.sub.50 in the range of about 1 .mu.M to about 1000 .mu.M for
inhibition of TNF.alpha. secretion in a bodily fluid in vivo.
[0010] Another embodiment of the present invention is a method of
identifying a pharmaceutically active compound, including:
providing a library of compounds; assaying a plurality of compounds
from the library for inhibition of TNF.alpha. secretion by cultured
cells in vitro; and selecting at least one compound from the
plurality of compounds, wherein the selected compound exhibits an
EC.sub.50 in the range of about 1 .mu.M to about 1000 .mu.M for
inhibition of TNF.alpha. secretion by cultured cell in vitro.
[0011] Another embodiment of the present invention is a method of
identifying a pharmaceutically active compound, including:
providing a library of compounds; assaying a plurality of compounds
from the library for inhibition of TNF.alpha. secretion in a bodily
fluid in vivo; and selecting at least one compound from the
plurality of compounds, wherein the selected compound exhibits an
EC.sub.50 in the range of about 1 .mu.M to about 1000 .mu.M for
inhibition of TNF.alpha. secretion by cultured cells in vitro.
[0012] Another embodiment of the present invention is a method of
identifying a pharmaceutically active compound, including:
providing a library of compounds; assaying a plurality of compounds
from the library for inhibition of TNF.alpha. secretion by cultured
cells in vitro; and selecting at least one compound from the
plurality of compounds, wherein the selected compound exhibits an
EC.sub.50 in the range of about 1 .mu.M to about 1000 .mu.M for
inhibition of TNF.alpha. secretion in a bodily fluid in vivo.
[0013] Another embodiment of the present invention is a compound
having the formula of Subgenus III:
##STR00001##
[0014] Wherein X.sub.3 is selected from the group consisting of H,
F, and OH; and R.sub.2 is selected from the group consisting of H
and CF.sub.3; and wherein the compound exhibits an EC.sub.50 in the
range of about 1 .mu.M to about 1000 .mu.M for inhibition of p38
MAPK; or a pharmaceutically acceptable salt, ester, solvate or
prodrug of the compound.
[0015] Another embodiment of the present invention is a compound
having the formula of Genus VI:
##STR00002##
[0016] Wherein Ar is pyridinyl or phenyl; Z is O or S; X.sub.3 is
H, F, Cl, OH, or OCH.sub.3; R.sub.2 is methyl, C(.dbd.O)H,
C(.dbd.O)CH.sub.3, C(.dbd.O)O-glucosyl, fluoromethyl,
difluoromethyl, trifluoromethyl, methylmethoxyl, methylhydroxyl, or
phenyl; and R.sub.4 is H or hydroxyl; with the proviso that when
R.sub.2 is trifluoromethyl, Z is O, R.sub.4 is H and Ar is phenyl,
the phenyl is not solely substituted at the 4' position by H, F, or
OH; wherein the compound exhibits an EC.sub.50 in the range of
about 1 .mu.M to about 1000 .mu.M for inhibition of p38 MAPK; or a
pharmaceutically acceptable salt, ester, solvate or prodrug of the
compound.
[0017] Another embodiment of the present invention is a compound
having the formula of Genus VII:
##STR00003##
[0018] Wherein X.sub.3 is H, halogen, C.sub.1-C.sub.10 alkoxy, or
OH; Y.sub.1, Y.sub.2, Y.sub.3, and Y.sub.4 are independently
selected from the group consisting of H, C.sub.1-C.sub.10 alkyl,
substituted C.sub.1-C.sub.10 alkyl, C.sub.1-C.sub.10 alkenyl,
C.sub.1-C.sub.10 haloalkyl, C.sub.1-C.sub.10 nitroalkyl,
C.sub.1-C.sub.10 thioalkyl, C.sub.1-C.sub.10 hydroxyalkyl,
C.sub.1-C.sub.10 alkoxy, phenyl, substituted phenyl, halogen,
hydroxyl, C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10 carboxy,
C.sub.1-C.sub.10 alkoxycarbonyl; R.sub.4 is H, halogen, or OH; and
wherein the compound exhibits an EC.sub.50 in the range of about 1
.mu.M to about 1000 .mu.M for inhibition of p38 MAPK; or a
pharmaceutically acceptable salt, ester, solvate or prodrug of the
compound.
[0019] A further embodiment of the present invention is a
pharmaceutical composition containing a compound having a formula
as described above and a pharmaceutically acceptable excipient.
[0020] These and other embodiments are described in greater detail
below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 is a schematic representation of the p38 MAPK
signalling cascade (prior art, FIG. 1 from Underwood et al. 2001
Prog Respir Res 31:342-345). This schematic shows activation of the
p38 signaling cascade by a variety of stimuli and the downstream
effects of p38 activation on an inflammatory response through
transcriptional activation and translation/mRNA stabilization.
[0022] FIG. 2 is a plot showing the fractional inhibition of
p38.gamma. by various pirfenidone metabolites and analogs as a
function of the concentration of the compounds. The EC50
concentrations of these compounds are shown to the right of the
plot. A detailed description of the assay can be found in Example
5.
[0023] FIG. 3 is a plot showing the fractional inhibition of
p38.alpha. by various pirfenidone metabolites and analogs as a
function of the concentration of the compounds. The EC.sub.50
concentrations of these compounds are shown to the right of the
plot. A detailed description of the assay can be found in Example
5.
[0024] FIG. 4 is a summary of the biochemical data for various
pirfenidone metabolites and analogs. The pirfenidone metabolites
and analogs referred to in FIGS. 2, 3, 5, and 6 are described by
the substitution pattern shown in FIG. 4. The summary shows the
effect of substitutions at three positions on the EC50
concentrations for inhibition of p38.alpha. and p38.gamma..
[0025] FIG. 5 is a plot showing the fractional activity (TNF.alpha.
release from macrophage in response to LPS) of various pirfenidone
metabolites and analogs as a function of the concentration of each
compound. A detailed description of the assay can be found in
Example 5.
[0026] FIG. 6 is a series of bar charts showing the cytotoxicity of
various pirfenidone metabolites and analogs at various
concentrations of the compounds. Cytotoxicity was determined by
measuring LDH release following incubation of cells in the presence
of the compound. The amount of LDH released is normalized to that
released upon treatment with Triton-X-100 and plotted versus
compound concentration.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0027] It has now been discovered that a high therapeutic effect in
treating various disorders associated with enhanced activity of
kinase p38 may be achieved by using a relatively low-potency p38
kinase inhibitor compound.
[0028] Therefore, in one embodiment there is provided a method of
modulating a stress-activated kinase (SAPK) system by contacting a
compound with a p38 mitogen-activated protein kinase (MAPK). A
preferred compound exhibits an EC.sub.50 in the range of about 1
.mu.M to about 1000 .mu.M, preferably about 50 .mu.M to about 650
.mu.M for the inhibition of at least one p38 MAPK. The
concentration at which the compound is contacted with the p38 MAPK
is generally less than EC.sub.30 for inhibition of the p38 by this
compound. Preferably, the concentration is less than EC.sub.20,
even more preferably, the concentration is less than EC.sub.10.
[0029] "Mitogen-activated protein kinases (MAPKs)" are
evolutionarily conserved serine/threonine kinases involved in the
regulation of many cellular events. Several MAPK groups have been
identified in mammalian cells, including extracellular
signal-regulated kinase (ERK), p38, and SAPK/JNK. It is believed
that MAPKs are activated by their specific MAPK kinases (MAPKKs):
ERK by MEK1 and MEK2, p38 by MKK3 and MKK6, and SAPK/JNK by SEK1
(also known as MKK4) and MKK7 (SEK2). These MAPKKs may also be
activated by various MAPKK kinases (MAPKKKs) such as Raf, MLK,
MEKK1, TAK1, and ASK1.
[0030] It is believed that the MAPK network involves at least
twelve cloned highly conserved, proline-directed serine-threonine
kinases which, when activated by cell stresses (oxidative stress,
DNA damage, heat or osmotic shock, ultraviolet irradiation,
ischemia-reperfusion), exogenous agents (anisomycin, Na arsenite,
lipopolysaccharide, LPS) or pro-inflammatory cytokines, TNF-.alpha.
and IL-.beta., can phosphorylate and activate other kinases or
nuclear proteins such as transcription factors in either the
cytoplasm or the nucleus (FIG. 1).
p38 MAPK
[0031] As used herein, "p38 MAPK" is a member of the
stress-activated protein kinase family, which includes at least 4
isoforms (.alpha., .beta., .gamma., .delta.), several of which are
considered important in processes critical to the inflammatory
response and tissue remodeling (Lee et al. 2000 Immunopharmacol.
47:185-201). The predominant kinases in monocytes and macrophages,
p38.alpha. and p38.beta., appear more widely expressed compared to
p38.gamma. (skeletal muscle) or p38.delta. (testes, pancreas,
prostate, small intestine, and in salivary, pituitary and adrenal
glands). The p38.gamma. isoform is expressed in myofibroblasts,
which have some phenotypic similarities to muscle cells including
expression of alpha-smooth muscle actin. A number of substrates of
p38 MAP kinase have been identified including other kinases (MAPKAP
K2/3, PRAK, MNK 1/2, MSK1/RLPK, RSK-B), transcription factors
(ATF2/6, myocyte enhancer factor 2, nuclear transcription
factor-.beta., CHOP/GADD153, Elk1 and SAP-1A1) and cytosolic
proteins (stathmin), many of which are important
physiologically.
[0032] Jiang, Y. et al. 1996 J Biol Chem 271:17920-17926 reported
characterization of p38.beta. as a 372-amino acid protein closely
related to p38-.alpha.. Both p38.alpha. and p38.beta. are activated
by proinflammatory cytokines and environmental stress, p38.beta. is
preferentially activated by MAP kinase kinase-6 (MKK6) and
preferentially phosphohorylate activated transcription factor 2
(ATF2). Kumar, S. et al. 1997 Biochem Biophys Res Comm 235:533-538
and Stein, B. et al. 1997 J Biol Chem 272:19509-19517 reported a
second isoform of p38.beta., p-38.beta.2, containing 364 amino
acids with 73% identity to p38.alpha.. It is believed that
p38.beta. is activated by proinflammatory cytokines and
environmental stress, although the second reported p38.beta.
isoform, p38.beta.2, appears to be preferentially expressed in the
central nervous system (CNS), heart and skeletal muscle, compared
to the more ubiquitous tissue expression of p38.alpha..
Furthermore, it is believed that activated transcription factor-2
(ATF-2) is a better substrate for p38.beta.2 than for
p38.alpha..
[0033] The identification of p38.gamma. was reported by Li, Z. et
al. 1996 Biochem Biophys Res Comm 228:334-340 and of p38.delta. by
Wang, X. et al. 1997 J Biol Chem 272:23668-23674 and by Kumar, S.
et al. 1997 Biochem Biophys Res Comm 235:533-538. These two p38
isoforms (.gamma. and .delta.) represent a unique subset of the
MAPK family based on their tissue expression patterns, substrate
utilization, response to direct and indirect stimuli, and
susceptibility to kinase inhibitors. Based upon primary sequence
conservation, p38.alpha. and .beta. are closely related, but
diverge from .gamma. and .delta., which are more closely related to
each other.
[0034] Typically the p38 MAP kinase pathway is directly or
indirectly activated by cell surface receptors, such as receptor
tyrosine kinases, chemokine or G protein-coupled receptors, which
have been activated by a specific ligand, e.g., cytokines,
chemokines or lipopolysaccharide (LPS) binding to a cognate
receptor. Subsequently, a p38 MAP kinase is activated by
phosphorylation on specific threonine and tyrosine residues. After
activation, p38 MAP kinase can phosphorylate other intracellular
proteins, including protein kinases, and can be translocated to the
cell nucleus, where it phosphorylates and activates transcription
factors leading to the expression of pro-inflammatory cytokines and
other proteins that contribute to the inflammatory response, cell
adhesion, and proteolytic degradation. For example, in cells of
myeloid lineage, such as macrophages and monocytes, both IL-1.beta.
and TNF.alpha. are transcribed in response to p38 activation.
Subsequent translation and secretion of these and other cytokines
initiates a local or systemic inflammatory response in adjacent
tissue and through infiltration of leukocytes. While this response
is a normal part of physiological responses to cellular stress,
acute or chronic cellular stress leads to the excess, unregulated,
or excess and unregulated expression of pro-inflammatory cytokines.
This, in turn, leads to tissue damage, often resulting in pain and
debilitation.
[0035] In alveolar macrophages, inhibition of p38 kinases with p38
inhibitor, SB203580, reduces cytokine gene products. It is believed
that inflammatory cytokines (TNF-.alpha., IFN-.gamma., IL-4, IL-5)
and chemokines (IL-8, RANTES, eotaxin) are capable of regulating or
supporting chronic airway inflammation. The production and action
of many of the potential mediators of airway inflammation appear to
be dependent upon the stress-activated MAP kinase system (SAPK) or
p38 kinase cascade (Underwood et al. 2001 Frog Respir Res
31:342-345). Activation of the p38 kinase pathway by numerous
environmental stimuli results in the elaboration of recognized
inflammatory mediators whose production is considered to be
translationally regulated. In addition, a variety of inflammatory
mediators activate p38 MAPK which may then activate downstream
targets of the MAPK system including other kinases or transcription
factors, thus creating the potential for an amplified inflammatory
process in the lung.
Downstream Substrates of P38 Group of Map Kinases
[0036] Protein kinase substrates of p38.alpha. or p38.beta.: MAP
kinase-activated protein kinase 2 (MAPKAPK2 or M2), MAP kinase
interaction protein kinase (MNK1), p38 regulated/activated kinase
(PRAK), mitogen- and stress-activated kinase (MSK: RSK-B or
RLPK).
[0037] Transcription factors activated by p38: activating
transcription factor (ATF)-1, 2 and 6, SRF accessory protein 1 (Sap
1), CHOP (growth arrest and DNA damage inducible gene 153, or
GADD153), p53, C/EBP.beta., myocyte enhance factor 2C (MEF2C),
MEF2A, MITF1, DDIT3, ELK1, NFAT, and high mobility group-box
protein (HBP1).
[0038] Other types of substrates for p38: cPLA2, Na.sup.+/H.sup.+
exchanger isoform-1, tau, keratin 8, and stathmin.
[0039] Genes regulated by the p38 pathway: c-jun, c-fos, junB,
IL-1, TNF, IL-6, IL-8, MCP-1, VCAM-1, iNOS, PPAR.gamma.,
cyclooxygenase (COX)-2, collagenase-1 (MMP-1), Collagenase-3
(MMP-13), HIV-LTR, Fgl-2, brain natriuretic peptide (BNP), CD23,
CCK, phosphoenolpyruvate carboxy-kinase-cytosolic, cyclin D1, LDL
receptor (Ono et al. 2000 Cellular Signalling 12:1-13).
Biological Consequences of p38 Activation
p38 and Inflammation
[0040] Acute and chronic inflammation are believed to be central to
the pathogenesis of many diseases such as rheumatoid arthritis,
asthma, chronic obstructive pulmonary disease (COPD) and acute
respiratory distress syndrome (ARDS). The activation of the p38
pathway may play an central role in: (1) production of
proflammatory cytokines such as IL-1.beta., TNF-.alpha. and IL-6;
(2) induction of enzymes such as COX-2, which controls connective
tissue remodeling in pathological condition; (3) expression of an
intracellular enzyme such as iNOS, which regulates oxidation; (4)
induction of adherent proteins such as VCAM-1 and many other
inflammatory related molecules. In addition to these, the p38
pathway may play a regulatory role in the proliferation and
differentiation of cells of the immune system. p38 may participate
in GM-CSF, CSF, EPO, and CD40-induced cell proliferation and/or
differentiation.
[0041] The role of the p38 pathway in inflammatory-related diseases
was studied in several animal models. Inhibition of p38 by SB203580
reduced mortality in a murine model of endotoxin-induced shock and
inhibited the development of mouse collagen-induced arthritis and
rat adjuvant arthritis. A recent study showed that SB220025, which
is a more potent p38 inhibitor, caused a significant dose-dependent
decrease in vascular density of the granuloma. These results
indicate that p38 or the components of the p38 pathway can be a
therapeutic target for inflammatory disease.
p38 and Apoptosis
[0042] It appears that concomitant activation of p38 and apoptosis
is induced by a variety of agents such as NGF withdrawal and Fas
ligation. Cysteine proteases (caspases) are central to the
apoptotic pathway and are expressed as inactive zymogens. Caspase
inhibitors may then block p38 activation through Fas cross-linking.
However, overexpression of dominant active MKK6b can also induce
caspase activity and cell death. The role of p38 in apoptosis is
cell type- and stimulus-dependent. While p38 signaling has been
shown to promote cell death in some cell lines, in different cell
lines p38 has been shown to enhance survival, cell growth, and
differentiation.
p38 in the Cell Cycle
[0043] Overexpression of p38.alpha. in yeast leads to significant
slowing of proliferation, indicating involvement of p38.alpha. in
cell growth. A slower proliferation of cultured mammalian cells was
observed when the cells were treated with p38.alpha./.beta.
inhibitor, SB203580.
p38 and Cardiomyocyte Hypertrophy
[0044] Activation and function of p38 in cardiomyocyte hypertrophy
has been studied. During progression of hypertrophy, both
p38.alpha. and p38.beta. levels were increased and constitutively
active MKK3 and MKK6-elicited hypertrophic responses enhanced by
sarcomeric organization and elevated atrial natriuretic factor
expression. Also, reduced signaling of p38 in the heart promotes
myocyte differentiation via a mechanism involving calcineurin-NFAT
signaling.
p38 and Development
[0045] Despite the non-viability of p38 knockout mice, evidence
exists regarding the differential role of p38 in development. p38
has been linked to placental angiogenesis but not cardiovascular
development in several studies. Furthermore, p38 has also been
linked to erythropoietin expression suggesting a role in
erythropoiesis. PRAK has recently been implicated in cell
development in murine implantation. PRAK mRNA, as well as p38
isoforms, were found to be expressed throughout blastocyst
development
p38 and Cell Differentiation
[0046] p38.alpha. and/or p38.beta. were found to play an important
role in cell differentiation for several different cell types. The
differentiation of 3T3-L1 cells into adipocytes and the
differentiation of PC12 cells into neurons both require p38.alpha.
and/or .beta.. The p38 pathway was found to be necessary and
sufficient for SKT6 differentiation into hemoglobinized cells as
well as C2C112 differentiation in myotubules.
p38 in Senescence and Tumor Suppression
[0047] p38 has a role in tumorigenesis and senescence. There have
been reports that activation of MKK6 and MKK3 led to a senescent
phenotype dependent upon p38 MAPK activity. Also, p38 MAPK activity
was shown responsible for senescence in response to telomere
shortening, H.sub.2O.sub.2 exposure, and chronic RAS oncogene
signaling. A common feature of tumor cells is a loss of senescence
and p38 is linked to tumorigenesis in certain cells. It has been
reported that p38 activation is reduced in tumors and that loss of
components of the p38 pathway such as MKK3 and MKK6 resulted in
increased proliferation and likelihood of tumorigenic conversion
regardless of the cell line or the tumor induction agent used in
these studies.
p38 MAP Kinase Inhibitors
[0048] A "p38 MAPK inhibitor" is a compound that inhibits the
activity of p38. The inhibitory effects of a compound on the
activity of p38 may be measured by various methods well-known to a
skilled artisan. For example, the inhibitory effects may be
measured by measuring the level of inhibition of lipopolysaccharide
(LPS)-stimulated cytokine production (Lee et al. 1988 Int J
Immunopharmacol 10:835-843; Lee et al. 1993 Ann NY Acad Sci
696:149-170; Lee et al. 1994 Nature 372:739-746; Lee et al. 1999
Pharmacol Ther 82:389-397).
[0049] Efforts to develop p38 MAPK inhibitors have focused on
increasing potency. SB203580 and other 2,4,5-triaryl imidazoles
were found to be potent p38 kinase inhibitors with EC.sub.50 values
in nanomolar range. For example, for SB203580 the EC.sub.50 was
found to be 48 nM. The pyridinylimidazoles SKF 86002 (P1) and
SB203582 (P2) shown below have been used as the template for the
majority of p38 inhibitors. Recent publications (Lee et al. 2000
Immunopharmacology 47:185-201) have disclosed the p38 inhibitors
(P3-P6) shown below. Notable among these inhibitors is the
relatively high potency and selectivity described for compound P4
(p38 EC.sub.50=0.19 nM) and the inhibition of inflammation driven
angiogenesis by SB 220025 (P6).
[0050] Two p38 inhibitors reported to be in clinical development
are HEP689 (P7) and VX-745 (P8). VX-745 is reportedly in Phase II
trials for rheumatoid arthritis. Potent topical anti-inflammatory
activity has been disclosed for HRP689, which has reportedly
entered clinical development to explore its potential as a topical
agent for the treatment of psoriasis and other skin disorders.
##STR00004## ##STR00005##
[0051] Further discussion of various p38 inhibitors can be found in
Boehm et al. 2000 Exp Opin Ther Pat 10:25-37; and Salituro et al.
1999 Curr Med Chem 6:807-823.
[0052] Preferred p38 inhibitors described herein are pirfenidone
derivatives and analogs that exhibit relatively low potency of p38
inhibition while, surprisingly, still having a relatively high
therapeutic effect (e.g., for modulating an SAPK system) as a
result of such inhibition. Preferably, the p38 inhibitors of the
embodiments exhibit EC.sub.50 in the range of about 1 .mu.M and
about 1000 .mu.M, preferably about 50 .mu.M to about 650 .mu.M for
the inhibition of the p38 MAPK.
Pirfenidone Derivatives and Analogs
[0053] Pirfenidone (5-methyl-1-phenyl-2-(1H)-pyridone) itself is a
known compound and its pharmacological effects are disclosed, for
example, in Japanese Patent Application KOKAI (Laid-Open) Nos.
87677/1974 and 1284338/1976. U.S. Pat. No. 3,839,346, issued Oct.
1, 1974; U.S. Pat. No. 3,974,281, issued Aug. 10, 1976; U.S. Pat.
No. 4,042,699, issued Aug. 16, 1977; and U.S. Pat. No. 4,052,509,
issued Oct. 4, 1977, all of which are hereby incorporated by
reference in their entireties, describe methods of manufacture of
5-methyl-1-phenyl-2-(1H)-pyridone and its use as an
anti-inflammatory agent.
[0054] Pirfenidone and derivatives thereof are useful compounds for
modulating a stress activated protein kinase (SAPK) system.
[0055] The term "alkyl" used herein refers to a monovalent straight
or branched chain radical of from one to ten carbon atoms,
including, but not limited to, methyl, ethyl, n-propyl, isopropyl,
n-butyl, isobutyl, tert-butyl, n-hexyl, and the like.
[0056] The term "alkenyl" used herein refers to a monovalent
straight or branched chain radical of from two to ten carbon atoms
containing a carbon double bond including, but not limited to,
1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl,
and the like.
[0057] The term "halogen" used herein refers to fluorine, chlorine,
bromine, or iodine.
[0058] The term "haloalkyl" used herein refers to one or more
halogen groups appended to an alkyl radical.
[0059] The term "nitroalkyl" used herein refers to one or more
nitro groups appended to an alkyl radical.
[0060] The term "thioalkyl" used herein refers to one or more thio
groups appended to an alkyl radical.
[0061] The term "hydroxyalkyl" used herein refers to one or more
hydroxy groups appended to an alkyl radical.
[0062] The term "alkoxy" used herein refers to straight or branched
chain alkyl radical covalently bonded to the parent molecule
through an --O-- linkage. Examples of alkoxy groups include, but
are limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy,
n-butoxy, sec-butoxy, t-butoxy and the like.
[0063] The term "alkoxyalkyl" used herein refers to one or more
alkoxy groups appended to an alkyl radical.
[0064] The term "carboxy" used herein refers to --COOH optionally
appended to an alkyl group. Examples of carboxy groups include, but
are not limited to, --COOH, --CH.sub.2COOH, --CH.sub.2CH.sub.2COOH,
--CH(COOH)(CH.sub.3), and the like.
[0065] The term "alkoxycarbonyl" refers to --(CO)--O-alkyl.
Examples of alkoxycarbonyl groups include, but are limited to,
methoxycarbonyl group, ethoxycarbonyl group, propoxycarbonyl group,
and the like.
[0066] Carbohydrates are polyhydroxy aldehydes or ketones, or
substances that yield such compounds upon hydrolysis. Carbohydrates
comprise the elements carbon (C), hydrogen (H) and oxygen (O) with
a ratio of hydrogen twice that of carbon and oxygen.
[0067] In their basic form, carbohydrates are simple sugars or
monosaccharides. These simple sugars can combine with each other to
form more complex carbohydrates. The combination of two simple
sugars is a disaccharide. Carbohydrates consisting of two to ten
simple sugars are called oligosaccharides, and those with a larger
number are called polysaccharides.
[0068] The term "uronide" refers to a monosaccharide having a
carboxyl group (--COOH) on the carbon that is not part of the ring.
The uronide name retains the root of the monosaccharide, but the
-ose sugar suffix is changed to -uronide. For example, the
structure of glucuronide corresponds to glucose.
[0069] As used herein, a radical indicates species with a single,
unpaired electron such that the species containing the radical can
be covalently bonded to another species. Hence, in this context, a
radical is not necessarily a free radical. Rather, a radical
indicates a specific portion of a larger molecule. The term
"radical" can be used interchangeably with the term "group."
[0070] As used herein, a substituted group is derived from the
unsubstituted parent structure in which there has been an exchange
of one or more hydrogen atoms for another atom or group. When
substituted, the substituent group(s) is (are) one or more group(s)
individually and independently selected from C.sub.1-C.sub.10
alkyl, C.sub.1-C.sub.10 cycloalkyl, aryl, fused aryl, heterocyclyl,
heteroaryl, hydroxy, C.sub.1-C.sub.10 alkoxy, aryloxy, mercapto,
C.sub.1-C.sub.10 alkylthio, arylthio, cyano, halogen, carbonyl,
thiocarbonyl, C.sub.1-C.sub.10 alkoxycarbonyl, nitro, silyl,
trihalomethanesulfonyl, trifluoromethyl, and amino, including mono-
and di-substituted amino groups, and the protected derivatives
thereof. The protecting groups that can form the protective
derivatives of the above substituents are known to those of skill
in the art and can be found in references such as Greene and Wuts
Protective Groups in Organic Synthesis; John Wiley and Sons: New
York, 1999. Wherever a substituent is described as "optionally
substituted" that substituent can be substituted with the above
substituents.
[0071] The term "purified" refers to a compound which has been
separated from other compounds such that it comprises at least 95%
of the measured substance when assayed.
[0072] Asymmetric carbon atoms may be present in the compounds
described herein. All such isomers, including diastereomers and
enantiomers, as well as the mixtures thereof are intended to be
included in the scope of the recited compound. In certain cases,
compounds can exist in tautomeric forms. All tautomeric forms are
intended to be included in the scope of the recited compound.
Likewise, when compounds contain an alkenyl or alkenylene group,
there exists the possibility of cis- and trans-isomeric forms of
the compounds. Both cis- and trans-isomers, as well as the mixtures
of cis- and trans-isomers, are contemplated. Thus, reference herein
to a compound includes all of the aforementioned isomeric forms
unless the context clearly dictates otherwise.
[0073] Various forms are included in the embodiments, including
polymorphs, solvates, hydrates, conformers, salts, and prodrug
derivatives. A polymorph is a composition having the same chemical
formula, but a different structure. A solvate is a composition
formed by solvation (the combination of solvent molecules with
molecules or ions of the solute). A hydrate is a compound formed by
an incorporation of water. A conformer is a structure that is a
conformational isomer. Conformational isomerism is the phenomenon
of molecules with the same structural formula but different
conformations (conformers) of atoms about a rotating bond. Salts of
compounds can be prepared by methods known to those skilled in the
art. For example, salts of compounds can be prepared by reacting
the appropriate base or acid with a stoichiometric equivalent of
the compound. A prodrug is a compound that undergoes
biotransformation (chemical conversion) before exhibiting its
pharmacological effects. For example, a prodrug can thus be viewed
as a drug containing specialized protective groups used in a
transient manner to alter or to eliminate undesirable properties in
the parent molecule. Thus, reference herein to a compound includes
all of the aforementioned forms unless the context clearly dictates
otherwise.
[0074] The compounds described below are useful in the methods
described herein. In an embodiment, a compound as described below
exhibits an EC.sub.50 in the range of about 1 .mu.M to about 1000
.mu.M for inhibition of p38 MAPK.
[0075] An embodiment provides a family of compounds represented by
the following genus (Genus Ia):
##STR00006##
[0076] wherein
[0077] R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are independently
selected from the group consisting of H, C.sub.1-C.sub.10 alkyl,
substituted C.sub.1-C.sub.10 alkyl, C.sub.1-C.sub.10 alkenyl,
C.sub.1-C.sub.10 haloalkyl, C.sub.1-C.sub.10 nitroalkyl,
C.sub.1-C.sub.10 thioalkyl, C.sub.1-C.sub.10 hydroxyalkyl,
C.sub.1-C.sub.10 alkoxy, phenyl, substituted phenyl, halogen,
hydroxyl, C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10 carboxy,
C.sub.1-C.sub.10 alkoxycarbonyl, CO-uronide, CO-monosaccharide,
CO-oligosaccharide, and CO-polysaccharide; and
[0078] X.sub.1, X.sub.2, X.sub.3, X.sub.4, and X.sub.5 are
independently selected from the group consisting of H, halogen,
alkoxy, and hydroxy.
[0079] Another embodiment provides a family of compounds
represented by the following genus (Genus Ib):
##STR00007##
[0080] wherein
[0081] X.sub.3 is selected from the group consisting of H, halogen,
C.sub.1-C.sub.10 alkoxy, and OH;
[0082] R.sub.2 is selected from the group consisting of H, halogen,
C.sub.1-C.sub.10 alkyl, substituted C.sub.1-C.sub.10 alkyl,
C.sub.1-C.sub.10 hydroxyalkyl, C.sub.1-C.sub.10 alkoxyalkyl,
C.sub.1-C.sub.10 carboxy, C.sub.1-C.sub.10 alkoxycarbonyl,
CO-uronide, CO-monosaccharide, CO-oligosaccharide, and
CO-polysaccharide; and
[0083] R.sub.4 is selected from the group consisting of H, halogen,
and OH.
[0084] Another embodiment provides a family of compounds
represented by the following genus (Genus Ic):
##STR00008##
[0085] wherein
[0086] X.sub.3 is selected from the group consisting of H, F, OH,
and OCH.sub.3;
[0087] R.sub.2 is selected from the group consisting of H,
CF.sub.3, CHF.sub.2, CH.sub.2F, CH.sub.2OH, COOH, CO-Glucoronide,
Br, CH.sub.3, and CH.sub.2OCH.sub.3; and
[0088] R.sub.4 is selected from the group consisting of H and
OH;
[0089] with the proviso that when R.sub.4 and X.sub.3 are H,
R.sub.2 is not CH.sub.3
[0090] Another embodiment provides a family of compounds
represented by the following subgenus (Subgenus II):
##STR00009##
[0091] wherein
[0092] is selected from the group consisting of H, OH, and
OCH.sub.3;
[0093] R.sub.2 is selected from the group consisting of H,
CH.sub.2OH, COOH, CO-Glucoronide, Br, CH.sub.3, and
CH.sub.2OCH.sub.3; and
[0094] R.sub.4 is selected from the group consisting of H and OH,
with the proviso that when X.sub.3 is OH then R.sub.2 is not
CH.sub.3.
[0095] Another embodiment provides a family of compounds
represented by the following subgenus (Subgenus III):
##STR00010##
[0096] wherein
[0097] X.sub.3 is selected from the group consisting of H, F, and
OH; and
[0098] R.sub.2 is selected from the group consisting of H, Br,
CH.sub.2F, CHF.sub.2, and CF.sub.3.
[0099] Another embodiment provides a family of compounds
represented by the following subgenus (Subgenus IV):
##STR00011##
[0100] wherein X.sub.3 is selected from the group consisting of H,
halogen, C.sub.1-C.sub.10 alkoxy, OH, C.sub.1-C.sub.10 alkyl,
substituted C.sub.1-C.sub.10 alkyl, C.sub.1-C.sub.10 alkenyl,
C.sub.1-C.sub.10 haloalkyl, C.sub.1-C.sub.10 nitroalkyl,
C.sub.1-C.sub.10 thioalkyl, C.sub.1-C.sub.10 hydroxyalkyl, phenyl,
substituted phenyl, C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10
carboxy, C.sub.1-C.sub.10 alkoxycarbonyl, CO-uronide,
CO-monosaccharide, CO-oligosaccharide, and CO-polysaccharide.
[0101] Another embodiment provides a family of compounds
represented by the following subgenus (Subgenus V):
##STR00012##
[0102] wherein X.sub.3 is selected from the group consisting of H,
halogen, C.sub.1-C.sub.10 alkoxy, C.sub.1-C.sub.10 alkyl,
substituted C.sub.1-C.sub.10 alkyl, C.sub.1-C.sub.10 alkenyl,
C.sub.1-C.sub.10 haloalkyl, C.sub.1-C.sub.10 nitroalkyl,
C.sub.1-C.sub.10 thioalkyl, C.sub.1-C.sub.10 hydroxyalkyl, phenyl,
substituted phenyl, C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10
carboxy, C.sub.1-C.sub.10 alkoxycarbonyl, CO-uronide,
CO-monosaccharide, CO-oligosaccharide, and CO-polysaccharide.
[0103] Another embodiment provides a family of compounds
represented by the following genus (Genus VI):
##STR00013##
[0104] wherein
[0105] Ar is pyridinyl or phenyl;
[0106] Z is O or S;
[0107] X.sub.3 is H, F, Cl, OH, CH.sub.3, or OCH.sub.3;
[0108] R.sub.2 is methyl, C(.dbd.O)H, C(.dbd.O)CH.sub.3,
C(.dbd.O)OCH.sub.3, C(.dbd.O)O-glucosyl, fluoromethyl,
difluoromethyl, trifluoromethyl, bromo, methylmethoxyl,
methylhydroxyl, or phenyl; and
[0109] R.sub.4 is H or hydroxyl;
[0110] with the proviso that when R.sub.2 is trifluoromethyl, Z is
O, R.sub.4 is H and Ar is phenyl, the phenyl is not solely
substituted at the 4' position by H, F, or OH.
[0111] The Genus VI includes the families of compounds represented
by the Subgenus VIa and the Subgenus VIb:
##STR00014##
[0112] wherein Z, X.sub.3, R.sub.2 and R.sub.4 are defined as in
Genus VI. It will be recognized that the phenyl ring in the
structure represented by Subgenus VIa is substituted by X.sub.3 at
the 4' position.
[0113] Another embodiment provides a family of compounds
represented by the following genus (Genus VII):
##STR00015##
[0114] wherein
[0115] X.sub.3 is selected from the group consisting of H, halogen,
C.sub.1-C.sub.10 alkoxy, and OH;
[0116] Y.sub.1, Y.sub.2, Y.sub.3, and Y.sub.4 are independently
selected from the group consisting of H, C.sub.1-C.sub.10 alkyl,
substituted C.sub.1-C.sub.10 alkyl, C.sub.1-C.sub.10 alkenyl,
C.sub.1-C.sub.10 haloalkyl, C.sub.1-C.sub.10 nitroalkyl,
C.sub.1-C.sub.10 thioalkyl, C.sub.1-C.sub.10 hydroxyalkyl,
C.sub.1-C.sub.10 alkoxy, phenyl, substituted phenyl, halogen,
hydroxyl, C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10 carboxy,
C.sub.1-C.sub.10 alkoxycarbonyl; and
[0117] R.sub.4 is selected from the group consisting of H, halogen,
and OH.
[0118] It will be recognized that a particular compound described
herein may be a member of more than one of the various genera
described above. The compounds described herein are useful for
modulating a stress activated protein kinase (SAPK) system.
Exemplary compounds of Genera Ia-c, Subgenera II-V and Genera VI
and VII that are useful for modulating a stress activated protein
kinase (SAPK) system are set forth in Table 1 below. Compounds 1-6
are examples of compounds of Subgenus II. Compounds 7-12 are
examples of compounds of Subgenus III. Compound 13 is pirfenidone,
an example of a compound of Subgenus II. Compounds 14-32 are
examples of compounds of Genus VI. Compound 33 is an example of
Genus VII.
TABLE-US-00001 TABLE 1 Compound Number Compound 1 ##STR00016## 2
##STR00017## 3 ##STR00018## 4 ##STR00019## 5 ##STR00020## 6
##STR00021## 7 ##STR00022## 8 ##STR00023## 9 ##STR00024## 10
##STR00025## 11 ##STR00026## 12 ##STR00027## 13 ##STR00028## 14
##STR00029## 15 ##STR00030## 16 ##STR00031## 17 ##STR00032## 18
##STR00033## 19 ##STR00034## 20 ##STR00035## 21 ##STR00036## 22
##STR00037## 23 ##STR00038## 24 ##STR00039## 25 ##STR00040## 26
##STR00041## 27 ##STR00042## 28 ##STR00043## 29 ##STR00044## 30
##STR00045## 31 ##STR00046## 32 ##STR00047## 33 ##STR00048##
[0119] Another embodiment is directed to purified compounds
represented by Genera Ia-c, Subgenera II-V and/or Genera VI and
VII. The degree of purity may be expressed as a percentage as
described above. In preferred embodiments, purified compounds
represented by Genera Ia-c, Subgenera II-V and/or Genera VI and VII
have a purity of about 96% or greater, more preferably about 98% or
greater, by weight based on total weight of the composition that
comprises the purified compound. For example, an embodiment
provides purified Compound 3 (Table 1).
[0120] Compounds of Genera Ia-c, Subgenera II-V and/or Genera VI
and VII can be synthesized by using various reactions. Examples of
syntheses include the following, designated Synthetic Schemes 1, 2,
and 3.
##STR00049##
##STR00050##
[0121] Ullmann reaction: Chem. Pharm. Bull. 45(4) 719-721. Target
N-aryl-pyridine-2-ones were obtained via arylation of
2-hydroxypyridines. The Ullmann reaction is useful in the
preparation of disclosed compounds, except the 5-bromo analogs and
compound 33 which are afforded, for example, by synthetic scheme
3.
[0122] A mixture of 2-hydroxypyridine (1 mmol), aryl iodide or
bromide (2 mmol), CuI (0.1-0.5 mmol) and anhydrous potassium
carbonate (1 mmol) in DMF (3 ml) was stirred overnight at
135.degree. C. under argon atmosphere. Deep colored reaction
mixture was taken into ethyl acetate and 10% ammonium hydroxide.
Organic layer was washed with brine and dried over magnesium
sulfate. Column chromatography furnished target compounds as
off-white solids in 25-60% yield.
##STR00051##
[0123] Target N-aryl-2-pyridones may be obtained via arylation of
2-hydroxypyridines with alkylboronic acids (Tetrahedron Lett., 42
(2001) 3415-3418). The alkylboronic acid route is useful in the
preparation of disclosed compounds. A mixture of 2-hydroxypyridine
(5 mmol), arylboronic acid (10 mmol), cupper (1 l) acetate (0.5-1
mmol), pyridine (10 mmol) and molecular sieves 4A (0.5-1 g) in
dichloromethane (25 ml) was stirred for 24-48 hours at room
temperature opened to the air. Reaction mixture was washed with
saturated sodium bicarbonate with EDTA and organic phase was dried
over sodium sulfate. Target N-aryl-2-pyridones were isolated by
column chromatography as white solids in 85-100% yield.
[0124] As pirfenidone derivatives, compounds of Genera Ia-c,
Subgenera II-V and/or Genera VI and VII may also be synthesized by
any conventional reactions known in the art based on the known
synthetic schemes for pirfenidone, such as disclosed in U.S. Pat.
Nos. 3,839,346; 3,974,281; 4,042,699; and 4,052,509, all of which
are hereby expressly incorporated by reference in their
entirety.
[0125] Starting materials described herein are available
commercially, are known, or can be prepared by methods known in the
art. Additionally, starting materials not described herein are
available commercially, are known, or can be prepared by methods
known in the art.
[0126] Starting materials can have the appropriate substituents to
ultimately give desired products with the corresponding
substituents. Alternatively, substituents can be added at any point
of synthesis to ultimately give desired products with the
corresponding substituents.
[0127] Synthetic Schemes 1-3 show methods that can be used to
prepare compounds of Genera Ia-c, Subgenera II-V and/or Genera VI
and VII. One skilled in the art will appreciate that a number of
different synthetic reaction schemes can be used to synthesize the
compounds of Genera Ia-c, Subgenera II-V and/or Genera VI and VII.
Further, one skilled in the art will understand that a number of
different solvents, coupling agents, and reaction conditions can be
used in the syntheses reactions to yield comparable results.
[0128] One skilled in the art will appreciate variations in the
sequence and, further, will recognize variations in the appropriate
reaction conditions from the analogous reactions shown or otherwise
known which may be appropriately used in the processes above to
make the compounds of Genera Ia-c, Subgenera II-V and/or Genera VI
and VII.
[0129] In the processes described herein for the preparation of the
compounds of compounds of Genera Ia-c, Subgenera II-V and/or Genera
VI and VII, the use of protective groups is generally well
recognized by one skilled in the art of organic chemistry, and
accordingly the use of appropriate protecting groups may in some
cases be implied by the processes of the schemes herein, although
such groups may not be expressly illustrated. Introduction and
removal of such suitable protecting groups are well known in the
art of organic chemistry; see for example, T. W. Greene,
"Protective Groups in Organic Synthesis", Wiley (New York), 1999.
The products of the reactions described herein may be isolated by
conventional means such as extraction, distillation,
chromatography, and the like.
[0130] The salts, e.g., pharmaceutically acceptable salts, of the
compounds of Genera Ia-c, Subgenera II-V and/or Genera VI and VII
may be prepared by reacting the appropriate base or acid with a
stoichiometric equivalent of the compounds. Similarly,
pharmaceutically acceptable derivatives (e.g., esters),
metabolites, hydrates, solvates and prodrugs of the compounds of
Genera Ia-c, Subgenera II-V and/or Genera VI and VII may be
prepared by methods generally known to those skilled in the art.
Thus, another embodiment provides compounds that are prodrugs of an
active compound. In general, a prodrug is a compound which is
metabolized in vivo (e.g., by a metabolic transformation such as
deamination, dealkylation, de-esterification, and the like) to
provide an active compound. A "pharmaceutically acceptable prodrug"
means a compound which is, within the scope of sound medical
judgment, suitable for pharmaceutical use in a patient without
undue toxicity, irritation, allergic response, and the like, and
effective for the intended use, including a pharmaceutically
acceptable ester as well as a zwitterionic form, where possible, of
the compounds of the embodiments. Examples of
pharmaceutically-acceptable prodrug types are described in T.
Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14
of the A.C.S. Symposium Series, and in Edward B. Roche, ed.,
Bioreversible Carriers in Drug Design, American Pharmaceutical
Association and Pergamon Press, 1987, both of which are hereby
expressly incorporated by reference in their entirety.
[0131] The compounds and compositions described herein may also
include metabolites. As used herein, the term "metabolite" means a
product of metabolism of a compound of the embodiments or a
pharmaceutically acceptable salt, analog, or derivative thereof,
that exhibits a similar activity in vitro or in vivo to a compound
of the embodiments. The compounds and compositions described herein
may also include hydrates and solvates. As used herein, the term
"solvate" refers to a complex formed by a solute (herein, a
compound of Genera Ia-c, Subgenera II-V and/or Genera VI and VII)
and a solvent. Such solvents for the purpose of the embodiments
preferably should not interfere with the biological activity of the
solute. Solvents may be, by way of example, water, ethanol, or
acetic acid. In view of the foregoing, reference herein to a
particular compound or genus of compounds will be understood to
include the various forms described above, including
pharmaceutically acceptable salts, esters, prodrugs, metabolites
and solvates thereof unless stated otherwise.
Methods of Inhibiting p38 MAP Kinase
[0132] In an embodiment, methods are provided for modulating a SAPK
system, in vitro or in vivo. The methods include contacting a
SAPK-modulating concentration of a compound with at least one p38
MAPK (e.g., by contacting the compound with a cell or tissue
containing at least one p38 MAPK), where the compound has a
relatively low potency for inhibition of the at least one p38 MAPK,
corresponding to a relatively high inhibitory concentration for
inhibition of the at least one p38 MAPK by the compound.
[0133] "Contacting a cell" refers to a condition in which a
compound or other composition of matter is in direct contact with a
cell or tissue, or is close enough to induce a desired biological
effect in a cell or tissue. For example, contacting a cell or
tissue containing p38 MAPK with a compound may be conducted in any
manner that permits an interaction between p38 MAPK and the
compound, resulting in the desired biological effect in a cell.
Contacting a cell or tissue may be accomplished, for example, by
intermixing or administration of a compound (such as a compound of
Genera Ia-c, Subgenera II-V and/or Genera VI and VII and/or a salt,
ester, prodrug and/or intermediate thereof, and/or a pharmaceutical
composition comprising one or more of the foregoing).
[0134] Alternatively, contacting a cell or tissue may be
accomplished by introducing a compound in a manner such that the
compound will be targeted, directly or indirectly, to a cell or
tissue containing p38 MAPK. Contacting a cell or tissue may be
accomplished under conditions such that a compound binds to at
least one p38 MAPK. Such conditions may include proximity of the
compound and p38-containing cell or tissue, pH, temperature, or any
condition that affects the binding of a compound to p38 MAPK.
[0135] In certain embodiments, the cell is contacted with the
compound in vitro; in other embodiments, the cell is contacted with
the compound in vivo.
[0136] When the cell is contacted in vivo, the effective
concentration (EC) of a compound is a concentration that results in
a reduction of a specified endpoint by a target percentage (e.g.,
50%, 40%, 30%, 20%, 10%) relative to the maximal observable
reduction of the specified endpoint by that compound. Such an
endpoint may be a physiological response, for example, reduction in
blood or other bodily fluid concentration of TNF.alpha.. For
example, EC.sub.50, EC.sub.40, EC.sub.30, EC.sub.20 and EC.sub.10
are determined as concentrations that result in reductions in the
serum TNF.alpha. concentration by 50%, 40%, 30%, 20% and 10%,
respectively, relative to the maximal observable reduction on a
dose-response curve.
[0137] When the cell is contacted in vitro, except in a cell-based
assay, the effective concentration (EC) is a concentration that
results in a reduction in the activity of the specified target by a
given percentage (e.g., 50%, 40%, 30%, 20%, 10%). For example,
EC.sub.50, EC.sub.40, EC.sub.30, EC.sub.20 and EC.sub.10 are
determined as concentrations that result in reductions in the
activity of the specified target by 50%, 40%, 30%, 20% and 10%,
respectively, on a dose-response curve. When complete inhibition of
a specified target is not obtained, the effective concentration
(EC) of a compound is a concentration that results in a reduction
of a target activity by a given percentage (e.g., 50%, 40%, 30%,
20%, 10%) relative to the maximal reduction of the target activity
by that compound.
[0138] When the cell is contacted in vitro, in a cell-based assay,
the effective concentration (EC) of a compound is a concentration
that results in a reduction of a specified endpoint by a target
percentage (e.g., 50%, 40%, 30%, 20%, 10%) relative to the maximal
observable reduction of the specified endpoint by that compound.
Such an endpoint may be a cellular response, for example, reduction
in the secretion of TNF.alpha. as determined by the TNF.alpha.
concentration in cell medium. For example, EC.sub.50, EC.sub.40,
EC.sub.30, EC.sub.20 and EC.sub.10 are determined as concentrations
that result in reductions in the TNF.alpha. concentration by 50%,
40%, 30%, 20% and 10%, respectively, relative to the maximal
observable reduction on a dose-response curve.
[0139] The EC.sub.50 of the SAPK system-modulating compound is
preferably in the range of about 1 .mu.M to about 1000 .mu.M, more
preferably about 50 .mu.M to about 650 .mu.M for inhibition of at
least one p38 MAPK. Thus, for example, modulation of the SAPK
system may involve contacting a compound (e.g., a compound of
Genera Ia-c, Subgenera II-V and/or Genera VI and VII) with at least
one p38 MAPK at a concentration that is less than an EC.sub.40,
preferably less than EC.sub.30, more preferably less than
EC.sub.20, even preferably less than EC.sub.10 for inhibition of
the at least one p38 MAPK by the compound as determined on a
dose-response curve in vivo.
[0140] In certain embodiments, the compound is provided in the form
of a pharmaceutical composition, together with a pharmaceutically
acceptable carrier.
Screening a Library of Compounds for Low-Potency p38 Inhibitors
[0141] In another aspect, a method is provided for identifying a
pharmaceutically active compound, e.g., for determining whether a
compound is potentially useful as a therapeutic agent, e.g., for
the prevention or treatment of an inflammatory condition (such as
p38- or cytokine-associated condition). The method includes
assaying a plurality of compounds for inhibition of at least one
p38 MAPK and selecting a compound which exhibits a relatively low
potency for inhibiting p38 MAPK. Preferably, an EC.sub.50 of such a
low-potency p38 inhibitor compound is in the range of about 1 .mu.M
to about 1000 .mu.M, preferably about 50 .mu.M to about 650 .mu.M
for inhibition of the at least one p38 MAPK. The plurality of
compounds to be assayed is preferably selected from a library of
potential compounds. The assaying of the plurality of compounds
from the library may be conducted in various ways. For example, in
some embodiments, the methods further comprise contacting at least
one p38 MAPK with the plurality of compounds, and determining
whether the compounds inhibit the activity of cytokines. A p38 MAPK
is preferably selected from the group consisting of p38.alpha.,
p38.beta., p38.gamma., and p38.delta.. In preferred embodiments,
the contacting step takes place in vitro; in certain preferred
embodiments, the contacting step comprises contacting a cell
comprising p38 MAPK with the compound.
[0142] In yet another embodiment, methods are provided for
inhibiting the activity of p38 MAPK in a cell, in vitro or in vivo.
In general, such methods include contacting a cell containing at
least one p38 MAPK with an effective p38-inhibiting amount of a
compound (e.g., a compound of Genera Ia-c, Subgenera II-V and/or
Genera VI and VII), under conditions such that p38 activity in the
cell is inhibited. Examples of such methods are provided in the
EXAMPLES section below. The compound preferably exhibits an
EC.sub.50 in the range of about 1 .mu.M to about 1000 .mu.M,
preferably about 50 .mu.M to about 650 .mu.M for inhibition of the
at least one p38 MAPK. The contacting of at least one p38 MAPK with
the compound is preferably conducted at a SAPK system-modulating
concentration that is less than EC.sub.30, preferably less than
EC.sub.20, more preferably less than EC.sub.10 for inhibition of
the at least one p38 MAPK by the compound.
[0143] In vivo methods include for example, introducing into a
group of animals orally or by injection a compound of interest
(e.g., a compound of Genera Ia-c, Subgenera II-V and/or Genera VI
and VII) in various concentrations. Following the introduction of
the compound, lipopolysaccharide is administered intravenously.
Serum TNF.alpha. levels are measured and compared to that from
control animals. The preferred compounds inhibit the release of
TNF.alpha., thus reducing TNF.alpha. levels in the blood samples of
the tested animals. The compound preferably exhibits an EC.sub.50
in the range of about 1 .mu.M to about 1000 .mu.M, preferably about
50 .mu.M to about 650 .mu.M for inhibition of the release of
TNF.alpha..
[0144] The method of identifying a pharmaceutically active compound
may further include determining a mammalian toxicity of the
selected compound. Such methods are generally known to those
skilled in the art. The method of identifying a pharmaceutically
active compound may also include administering the selected
compound to a test subject, either in conjunction with the
deteintination of mammalian toxicity or for other reasons. In an
embodiment, the test subject test subject has or is at risk for
having an inflammatory condition. Preferably the test subject is a
mammal, and may be a human.
Methods of Treatment and/or Prevention
[0145] Another embodiment provides methods for treating or
preventing disease states, e.g., inflammatory condition(s) and/or
fibrotic condition(s). The methods include identifying a subject at
risk for or having at least one condition selected from an
inflammatory condition and a fibrotic condition and administering a
compound to the subject in an effective amount to treat or prevent
the inflammatory condition and/or fibrotic condition. In preferred
embodiments, the compound exhibits an EC.sub.50 in the range of
about 1 .mu.M to about 1000 .mu.M, preferably about 50 .mu.M to
about 650 .mu.M for inhibition of at least one p38 MAPK. In
preferred embodiments, the effective amount produces a blood or
serum or another bodily fluid concentration that is less than an
EC.sub.30 or, preferably, an EC.sub.20 or, more preferably, an
EC.sub.10 for inhibition of p38 MAPK by the compound. In preferred
embodiments, the compound exhibits an EC.sub.50 in the range of
about 1 .mu.M to about 1000 .mu.M, preferably about 50 .mu.M to
about 650 .mu.M for inhibition of the TNF.alpha. secretion. In
other preferred embodiments, the effective amount produces a blood
or serum or another bodily fluid concentration that is less than an
EC.sub.30 or, preferably, an EC.sub.20 or, more preferably, an
EC.sub.15 or, more preferably, an EC.sub.10 for inhibition of
LPS-stimulated TNF.alpha. release in a bodily fluid by the
compound. The effective amount is preferably about 70% or less,
more preferably less than about 50%, of an amount that causes an
undesirable side effect in the subject, such as, but not limited
to, drowsiness, nausea, cold symptom, gastrointestinal upset, and
photosensitivity rash. The compound used for the treatment or
prevention is preferably a compound of Genera Ia-c, Subgenera II-V
and/or Genera VI and VII.
[0146] Methods for identifying a subject at risk for or having an
inflammatory condition are known to those skilled in the art.
Examples of inflammatory conditions that may be treated or
prevented by the methods described herein include p-38 associated
conditions, e.g., conditions associated with altered cytokine
activity, conditions associated with modulation of an SAPK system,
autoimmune diseases, and diseases associated with acute and chronic
inflammation. The cytokine (or cytokines) is (are) preferably
selected from the group consisting of, but not limited to,
IL-1.beta., IL-6, IL-8, and TNF.alpha.. In an embodiment, the
compound used to treat or prevent the inflammatory condition is
compound that inhibits a kinase in the SAPK signaling pathway.
Examples of preferred compounds include compound of Genera Ia-c,
Subgenera II-V and/or Genera VI and VII.
[0147] The term "p38-associated condition" means a disease or other
deleterious condition in which the p38 MAP kinase signaling pathway
is implicated, whether directly or indirectly. Examples of
p38-associated conditions include conditions caused by IL-1.beta.,
TNF.alpha., IL-6 or IL-8 dysregulation or overexpression resulting
from sustained, prolonged, enhanced or elevated levels of p38
activity. Such conditions include, without limitation, inflammatory
diseases, autoimmune diseases, fibrotic diseases, destructive bone
disorders, proliferative disorders, infectious diseases,
neurodegenerative diseases, allergies, reperfusion ischemia in
stroke, heart attacks, angiogenic disorders, organ hypoxia,
vascular hyperplasia, cardiac hypertrophy, thrombin-induced
platelet aggregation, and conditions associated with the
prostaglandin or cyclooxygenase pathways, e.g., conditions
involving prostaglandin endoperoxide synthase. A p38-associated
condition can include any condition associated with or mediated by
an isoform of p38.
[0148] A "fibrotic condition," "fibroproliferative condition,"
"fibrotic disease," "fibroproliferative disease," "fibrotic
disorder," and "fibroproliferative disorder" are used
interchangeably to refer to a condition, disease or disorder that
is characterized by dysregulated proliferation or activity of
fibroblasts and/or pathologic or excessive accumulation of
collagenous tissue. Typically, any such disease, disorder or
condition is amenable to treatment by administration of a compound
having anti-fibrotic activity. Fibrotic disorders include, but are
not limited to, pulmonary fibrosis, including idiopathic pulmonary
fibrosis (IPF) and pulmonary fibrosis from a known etiology, liver
fibrosis, and renal fibrosis. Other exemplary fibrotic conditions
include musculoskeletal fibrosis, cardiac fibrosis, post-surgical
adhesions, scleroderma, glaucoma, and skin lesions such as
keloids.
[0149] The term "modulating SAPK system" means increasing or
decreasing activity of the stress-activated protein kinase system
activity, e.g., by inhibiting p38 activity, whether in vitro or in
vivo. In certain embodiments, the SAPK system is modulated when p38
activity in a cell is inhibited by about 50%, preferably by about
40%, more preferably by about 30%, even more preferably by about
20%, or yet even more preferably by about 10% compared to the p38
activity of an untreated control cell.
[0150] A condition associated with altered cytokine activity, as
used herein, refers to a condition in which cytokine activity is
altered compared to a non-diseased state. This includes, but is not
limited to, conditions caused by IL-1.beta., TNF.alpha., IL-6 or
IL-8 overproduction or dysregulation resulting in sustained,
prolonged, enhanced or elevated levels of cytokine activity, which
may be associated with p38 activity. Such conditions include,
without limitation, inflammatory diseases, autoimmune diseases,
fibrotic diseases, destructive bone disorders, proliferative
disorders, infectious diseases, neurodegenerative diseases,
allergies, reperfusion/ischemia in stroke, heart attacks,
angiogenic disorders, organ hypoxia, vascular hyperplasia, cardiac
hypertrophy, thrombin-induced platelet aggregation, and conditions
associated with the cyclooxygenase and lipoxygenase signaling
pathways, such as prostaglandin endoperoxide synthase. A
cytokine-associated condition can include any condition associated
with or mediated by IL-1 (particularly IL-1.beta.), TNF.alpha.,
IL-6 or IL-8, or any other cytokine which can be regulated by p38.
In preferred embodiments, the cytokine associated condition is a
condition associated with TNF.alpha..
[0151] The methods described herein may also be used to treat
autoimmune diseases and diseases associated with acute and chronic
inflammation. These diseases include, but are not limited to:
chronic obstructive pulmonary disease (COPD), bronchiolitis
obliterans syndrome, chronic allograft fibrosis, inflammatory
pulmonary fibrosis (IPF), rheumatoid arthritis; rheumatoid
spondylitis; osteoarthritis; gout, other arthritic conditions;
sepsis; septic shock; endotoxic shock; gram-negative sepsis; toxic
shock syndrome; myofacial pain syndrome (MPS); Shigellosis; asthma;
adult respiratory distress syndrome; inflammatory bowel disease;
Crohn's disease; psoriasis; eczema; ulcerative colitis; glomerular
nephritis; scleroderma; chronic thyroiditis; Grave's disease;
Oiniond's disease; autoimmune gastritis; myasthenia gravis;
autoimmune hemolytic anemia; autoimmune neutropenia;
thrombocytopenia; pancreatic fibrosis; chronic active hepatitis
including hepatic fibrosis; acute and chronic renal disease; renal
fibrosis, irritable bowel syndrome; pyresis; restenosis; cerebral
malaria; stroke and ischemic injury; neural trauma; Alzheimer's
disease; Huntington's disease; Parkinson's disease; acute and
chronic pain; allergies, including allergic rhinitis and allergic
conjunctivitis; cardiac hypertrophy, chronic heart failure; acute
coronary syndrome; cachexia; malaria; leprosy; leishmaniasis; Lyme
disease; Reiter's syndrome; acute synoviitis; muscle degeneration,
bursitis; tendonitis; tenosynoviitis; herniated, ruptured, or
prolapsed intervertebral disk syndrome; osteopetrosis; thrombosis;
silicosis; pulmonary sarcosis; bone resorption diseases, such as
osteoporosis or multiple myeloma-related bone disorders; cancer,
including but not limited to metastatic breast carcinoma,
colorectal carcinoma, malignant melanoma, gastric cancer, and
non-small cell lung cancer; graft-versus-host reaction; and
auto-immune diseases, such as Multiple Sclerosis, lupus and
fibromyalgia; AIDS and other viral diseases such as Herpes Zoster,
Herpes Simplex I or II, influenza virus, Severe Acute Respiratory
Syndrome (SARS) and cytomegalovirus; and diabetes mellitus. In
addition, the methods of the embodiments can be used to treat
proliferative disorders (including both benign and malignant
hyperplasias), including acute myelogenous leukemia, chronic
myelogenous leukemia, Kaposi's sarcoma, metastatic melanoma,
multiple myeloma, breast cancer, including metastatic breast
carcinoma; colorectal carcinoma; malignant melanoma; gastric
cancer; non-small cell lung cancer (NSCLC); bone metastases, and
the like; pain disorders including neuromuscular pain, headache,
cancer pain, dental pain, and arthritis pain; angiogenic disorders
including solid tumor angiogenesis, ocular neovascularization, and
infantile hemangioma; conditions associated with the cyclooxygenase
and lipoxygenase signaling pathways, including conditions
associated with prostaglandin endoperoxide synthase-2 (including
edema, fever, analgesia, and pain); organ hypoxia; thrombin-induced
platelet aggregation. In addition, the methods described herein may
be useful for the treatment of protozoal diseases in animals,
including mammals.
[0152] A subject may include one or more cells or tissues, or
organisms. A preferred subject is a mammal. A mammal may include
any mammal. As a non-limiting example, preferred mammals include
cattle, pigs, sheep, goats, horses, camels, buffalo, cats, dogs,
rats, mice, and humans. A highly preferred subject mammal is a
human. The compound(s) may be administered to the subject via any
drug delivery route known in the art. Specific exemplary
administration routes include oral, ocular, rectal, buccal,
topical, nasal, ophthalmic, subcutaneous, intramuscular,
intravenous (bolus and infusion), intracerebral, transdermal, and
pulmonary.
[0153] The terms "therapeutically effective amount" and
"prophylactically effective amount", as used herein, refer to an
amount of a compound sufficient to treat, ameliorate, or prevent
the identified disease or condition, or to exhibit a detectable
therapeutic, prophylactic, or inhibitory effect. The effect can be
detected by, for example, the assays disclosed in the following
examples. The precise effective amount for a subject will depend
upon the subject's body weight, size, and health; the nature and
extent of the condition; and the therapeutic or combination of
therapeutics selected for administration. Therapeutically and
prophylactically effective amounts for a given situation can be
determined by routine experimentation that is within the skill and
judgment of the clinician. Preferably, the effective amount of the
compound of the embodiments produces a blood or serum or another
bodily fluid concentration that is less than an EC.sub.10,
EC.sub.20 or EC.sub.10 for inhibition of p38 MAP kinase.
[0154] For any compound, the therapeutically or prophylactically
effective amount can be estimated initially either in cell culture
assays, e.g., of neoplastic cells, or in animal models, usually
rats, mice, rabbits, dogs, or pigs. The animal model may also be
used to determine the appropriate concentration range and route of
administration. Such information can then be used to determine
useful doses and routes for administration in humans.
[0155] Therapeutic/prophylactic efficacy and toxicity may be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., ED.sub.50 (the dose therapeutically
effective in 50% of the population) and LD.sub.50 (the dose lethal
to 50% of the population). The dose ratio between therapeutic and
toxic effects is the therapeutic index, and it can be expressed as
the ratio, ED.sub.50/LD.sub.50. Pharmaceutical compositions that
exhibit large therapeutic indices are preferred. However, the
pharmaceutical compositions that exhibit narrow therapeutic indices
are also within the scope of the embodiments. The data obtained
from cell culture assays and animal studies may be used in
formulating a range of dosage for human use. The dosage contained
in such compositions is preferably within a range of circulating
concentrations that include an ED.sub.50 with little or no
toxicity. The dosage may vary within this range depending upon the
dosage form employed, sensitivity of the patient, and the route of
administration.
[0156] More specifically, the maximum plasma concentrations
(C.sub.max) may range from about 65 .mu.M to about 115 .mu.M, or
about 75 .mu.M to about 105 .mu.M, or about 85 .mu.M to about 95
.mu.M, or about 85 .mu.M to about 90 .mu.M depending upon the route
of administration. Guidance as to particular dosages and methods of
delivery is provided in the literature and is generally available
to practitioners in the art. In general the dose will be in the
range of about 100 mg/day to about 10 g/day, or about 200 mg to
about 5 g/day, or about 400 mg to about 3 g/day, or about 500 mg to
about 2 g/day, in single, divided, or continuous doses for a
patient weighing between about 40 to about 100 kg (which dose may
be adjusted for patients above or below this weight range,
particularly children under 40 kg). Generally the dose will be in
the range of about 25 mg/kg to about 300 mg/kg of body weight per
day.
[0157] The exact dosage will be determined by the practitioner, in
light of factors related to the subject that requires treatment.
Dosage and administration are adjusted to provide sufficient levels
of the active agent(s) or to maintain the desired effect. Factors
which may be taken into account include the severity of the disease
state, general health of the subject, age, weight, and gender of
the subject, diet, time and frequency of administration, drug
combination(s), reaction sensitivities, and tolerance/response to
therapy. Long-acting pharmaceutical compositions may be
administered twice a day, once a day, once very two days, three
times a week, twice a week, every 3 to 4 days, or every week
depending on half-life and clearance rate of the particular
formulation. For example, in an embodiment, a pharmaceutical
composition contains an amount of a compound as described herein
that is selected for administration to a patient on a schedule
selected from: twice a day, once a day, once very two days, three
times a week, twice a week, and once a week.
[0158] It will be appreciated that treatment as described herein
includes preventing a disease, ameliorating symptoms, slowing
disease progression, reversing damage, or curing a disease.
[0159] In one aspect, treating an inflammatory condition results in
an increase in average survival time of a population of treated
subjects in comparison to a population of untreated subjects.
Preferably, the average survival time is increased by more than
about 30 days; more preferably, by more than about 60 days; more
preferably, by more than about 90 days; and even more preferably by
more than about 120 days. An increase in survival time of a
population may be measured by any reproducible means. In a
preferred aspect, an increase in average survival time of a
population may be measured, for example, by calculating for a
population the average length of survival following initiation of
treatment with an active compound. In an another preferred aspect,
an increase in average survival time of a population may also be
measured, for example, by calculating for a population the average
length of survival following completion of a first round of
treatment with an active compound.
[0160] In another aspect, treating an inflammatory condition
results in a decrease in the mortality rate of a population of
treated subjects in comparison to a population of subjects
receiving carrier alone. In another aspect, treating an
inflammatory condition results in a decrease in the mortality rate
of a population of treated subjects in comparison to an untreated
population. In a further aspect, treating an inflammatory condition
results a decrease in the mortality rate of a population of treated
subjects in comparison to a population receiving monotherapy with a
drug that is not a compound of the embodiments, or a
pharmaceutically acceptable salt, metabolite, analog or derivative
thereof. Preferably, the mortality rate is decreased by more than
about 2%; more preferably, by more than about 5%; more preferably,
by more than about 10%; and most preferably, by more than about
25%. In a preferred aspect, a decrease in the mortality rate of a
population of treated subjects may be measured by any reproducible
means. In another preferred aspect, a decrease in the mortality
rate of a population may be measured, for example, by calculating
for a population the average number of disease-related deaths per
unit time following initiation of treatment with an active
compound. In another preferred aspect, a decrease in the mortality
rate of a population may also be measured, for example, by
calculating for a population the average number of disease related
deaths per unit time following completion of a first round of
treatment with an active compound.
[0161] In another aspect, treating an inflammatory condition
results in a decrease in growth rate of a tumor. Preferably, after
treatment, tumor growth rate is reduced by at least about 5%
relative to/number prior to treatment; more preferably, tumor
growth rate is reduced by at least about 10%; more preferably,
reduced by at least about 20%; more preferably, reduced by at least
about 30%; more preferably, reduced by at least about 40%; more
preferably, reduced by at least about 50%; even more preferably,
reduced by at least 60%; and most preferably, reduced by at least
about 75%. Tumor growth rate may be measured by any reproducible
means of measurement. In a preferred aspect, tumor growth rate is
measured according to a change in tumor diameter per unit time.
[0162] In another aspect, treating an inflammatory condition
results in a reduction in the rate of cellular proliferation.
Preferably, after treatment, the rate of cellular proliferation is
reduced by at least about 5%; more preferably, by at least about
10%; more preferably, by at least about 20%; more preferably, by at
least about 30%; more preferably, by at least about 40%; more
preferably, by at least about 50%; even more preferably, by at
least about 60%; and most preferably, by at least about 75%. The
rate of cellular proliferation may be measured by any reproducible
means of measurement. In a preferred aspect, the rate of cellular
proliferation is measured, for example, by measuring the number of
dividing cells in a tissue sample per unit time.
[0163] In another aspect, treating an inflammatory condition
results in a reduction in the proportion of proliferating cells.
Preferably, after treatment, the proportion of proliferating cells
is reduced by at least about 5%; more preferably, by at least about
10%; more preferably, by at least about 20%; more preferably, by at
least about 30%; more preferably, by at least about 40%; more
preferably, by at least about 50%; even more preferably, by at
least about 60%; and most preferably, by at least about 75%. The
proportion of proliferating cells may be measured by any
reproducible means of measurement. In a preferred aspect, the
proportion of proliferating cells is measured, for example, by
quantifying the number of dividing cells relative to the number of
nondividing cells in a tissue sample. In another preferred aspect,
the proportion of proliferating cells is equivalent to the mitotic
index.
[0164] In another aspect, treating an inflammatory condition
results in a decrease in size of an area or zone of cellular
proliferation. Preferably, after treatment, size of an area or zone
of cellular proliferation is reduced by at least 5% relative to its
size prior to treatment; more preferably, reduced by at least about
10%; more preferably, reduced by at least about 20%; more
preferably, reduced by at least about 30%; more preferably, reduced
by at least about 40%; more preferably, reduced by at least about
50%; even more preferably, reduced by at least about 60%; and most
preferably, reduced by at least about 75%. Size of an area or zone
of cellular proliferation may be measured by any reproducible means
of measurement. In a preferred aspect, size of an area or zone of
cellular proliferation may be measured as a diameter or width of an
area or zone of cellular proliferation.
[0165] The methods described herein may include identifying a
subject in need of treatment. In a preferred embodiment, the
methods include identifying a mammal in need of treatment. In a
highly preferred embodiment, the methods include identifying a
human in need of treatment. Identifying a subject in need of
treatment may be accomplished by any means that indicates a subject
who may benefit from treatment. For example, identifying a subject
in need of treatment may occur by clinical diagnosis, laboratory
testing, or any other means known to one of skill in the art,
including any combination of means for identification.
[0166] As described elsewhere herein, the compounds described
herein may be formulated in pharmaceutical compositions, if
desired, and can be administered by any route that permits
treatment of the disease or condition. A preferred route of
administration is oral administration. Administration may take the
form of single dose administration, or the compound of the
embodiments can be administered over a period of time, either in
divided doses or in a continuous-release formulation or
administration method (e.g., a pump). However the compounds of the
embodiments are administered to the subject, the amounts of
compound administered and the route of administration chosen should
be selected to permit efficacious treatment of the disease
condition.
[0167] The methods of the embodiments also include the use of a
compound or compounds as described herein together with one or more
additional therapeutic agents for the treatment of disease
conditions. Thus, for example, the combination of active
ingredients may be: (1) co-formulated and administered or delivered
simultaneously in a combined formulation; (2) delivered by
alternation or in parallel as separate formulations; or (3) by any
other combination therapy regimen known in the art. When delivered
in alternation therapy, the methods described herein may comprise
administering or delivering the active ingredients sequentially,
e.g., in separate solution, emulsion, suspension, tablets, pills or
capsules, or by different injections in separate syringes. In
general, during alternation therapy, an effective dosage of each
active ingredient is administered sequentially, i.e., serially,
whereas in simultaneous therapy, effective dosages of two or more
active ingredients are administered together. Various sequences of
intermittent combination therapy may also be used.
[0168] Diagnostic tests are contemplated as part of the methods
described herein. For example, a tissue biopsy sample may be taken
from a subject suffering from an inflammatory condition, e.g., a
p38-associated or cytokine-associated condition. The biopsy sample
can be tested to determine the level of p38 activity (or cytokine
levels) present in the sample; the sample can then be contacted
with a selected compound of the embodiments, and the p38 activity
(or cytokine levels) measured to determine whether the compound has
a desired effect (e.g., inhibition of p38 or cytokine activity with
an EC.sub.50 in the range of about 100 .mu.M and about 1000 .mu.M,
preferably about 50 .mu.M to about 650 .mu.M). Such a test may be
used to determine whether treatment with such a compound is likely
to be effective in that subject. Alternatively, the sample may be
contacted with a labeled compound (e.g., a fluorescently-labeled
compound, or a radioactivity-labeled compound) and the sample then
examined and the fluorescent or radioactive signal detected to
determine the distribution of p38 in the tissue sample. Repeated
biopsy samples taken during a course of treatment may also be used
to study the efficacy of the treatment. Other diagnostic tests
using the compounds described herein will be apparent to one of
ordinary skill in the art in light of the teachings of this
specification.
[0169] Thus, for example, an embodiment provides methods for
determining the presence, location, or quantity, or any combination
thereof of p38 protein in a cell or tissue sample. The methods
include: a) contacting the cell or tissue sample with a compound of
the embodiments under conditions such that the compound can bind to
at least one p38 MAPK; and b) determining the presence, location,
or quantity, or any combination thereof of the compound in the cell
or tissue sample, thereby determining the presence, location, or
quantity, or any combination thereof of the at least one p38 MAPK
in the cell or tissue sample. Determining the presence, location,
or quantity, or any combination thereof of the compound in the cell
or tissue sample may be conducted by any means that reveals the
presence, location, or quantity, or any combination thereof of the
compound in the cell or tissue. For example, as described
previously, radioactive or fluorescent labeling methods may be
used. Additional methods of determining the presence, location, or
quantity, or any combination thereof of the compound will be
apparent to a skilled artisan.
[0170] Another embodiment provides methods for determining: (1)
whether a compound will be a useful therapeutic agent for treatment
of a subject suffering from an inflammatory condition, or (2) the
severity of disease or (3) the course of disease during treatment
with a disease-modifying agent. The methods include: a) obtaining a
cell or tissue sample from the subject before, during and after
termination of treatment with a compound as described herein or
another disease-modifying agent; b) contacting the sample with the
compound; and c) determining the amount of the compound that binds
to the sample, wherein binding to p38 MAPK by the compound is
related to the amount of p38 MAPK in the sample.
Specific Examples of Diseases Contemplated to be Treated by the
Compounds and Methods Described Herein
COPD
[0171] Chronic obstructive pulmonary disease (COPD) is
characterized by a chronic inflammatory process in the lung that
includes (1) increased number of inflammatory cells (neutrophils,
macrophages and SD8.sup.+T cells) in the airways and parenchyma,
(2) increased inflammatory cytokine and chemokine expression, and
(3) increased number of proteases (elastases, cathepsins, and
matrix metalloproteinases, MMPs). The production and action of many
of potential mediators of airway inflammation are believed to be
dependent on the stress-induced MAPK or p38 kinase cascade. Several
reports support the association pf p38 kinase activation with as
plethora of pulmonary events: LPS- and TNF-.alpha.-induced
intercellular adhesion molecule-1 expression on pulmonary
microvascular endothelial cells, MMP-9 activation, hypoxia-induced
stimulation of pulmonary arterial cells, hyperosmolarity-induced
IL-8 expression in bronchial epithelial cells, and enhanced
eosinophil trafficking and survival.
[0172] Trifilieff et al. (2005 Brit J Pharmacol 144:1002-10)
reported that CGH2466, a combined adenosine receptor antagonist,
p38 MAPK and phosphodiesterase type 4 inhibitor showed potent in
vitro and in vivo anti-inflammatory activities in diseases such as
asthma and COPD. Underwood et al. (2000 Am J Physiol Lung Cell Mol
Physiol 279:L895-L902) demonstrated that the potent and selective
p38 MAPK inhibitor, SB239063, reduced proinflammatory cytokine
production, including IL-1.beta., TNF-.alpha., IL-6, and IL-8,
which have been linked to airway fibrosis because of their ability
to regulate fibroblast proliferation and matrix production; that
leads to diminished neutrophil trafficking and activation in the
lung. Earlier, the same compound was found capable of altering
responses associated with chronic fibrosis induced by bleomycin.
This inhibitory activity was selective for the .alpha. and .beta.
isoforms of the p38. The compounds and methods described herein are
useful in the treatment of COPD.
Pulmonary Fibrosis
[0173] Pulmonary fibrosis also called idiopathic pulmonary fibrosis
(IPF), interstitial diffuse pulmonary fibrosis, inflammatory
pulmonary fibrosis, or fibrosing alveolitis, is an inflammatory
lung disorder and a heterogeneous group of conditions characterized
by abnormal formation of fibrous tissue between alveoli caused by
alveolitis comprising an inflammatory cellular infiltration into
the alveolar septae with resulting fibrosis. The effects of IPF are
chronic, progressive, and often fatal. p38 MAPK activation has been
demonstrated in the lung of patients with pulmonary fibrosis. A
number of investigations about pulmonary fibrosis have indicated
that sustained and augmented expression of some cytokines in the
lung are relevant to recruitment of inflammatory cells and
accumulation of extracellular matrix components followed by
remodeling of the lung architecture. In particular, proinflammatory
cytokines such as TNF-.alpha. and interleukin IL-1.beta. were
demonstrated to play major roles in the formation of pneumonitis
and pulmonary fibrosis. In addition, profibrotic cytokines such as
TGF-.beta. and CTGF also play critical roles in the pathogenesis of
pulmonary fibrosis. Matsuoka et al. (2002 Am J Physiol Lung Cell
Mol Physiol 283:L103-L112) have demonstrated that a p38 inhibitor,
FR-167653, ameliorates murine bleomycin-induced pulmonary fibrosis.
Furthermore, pirfenidone (5-methyl-1-phenyl-2-(1H)-pyridone), a
compound with combined anti-inflammatory, antioxidant and
antifibrotic effects was found effective in experimental models of
pulmonary fibrosis as well as in clinical studies (Raghu et al.
1999 Am J Respir Crit Care Med 159:1061-1069; Nagai et al. 2002
Intern Med 41:1118-1123; Gahl et al. 2002 Mol Genet Metab
76:234-242; Azuma et al. 2002 Am J Respir Crit. Care Med 165:A729).
The compounds and methods described herein are useful in the
treatment of pulmonary fibrosis, such as IPF.
Bronchiolitis Obliterans and Bronchiolitis Obliterans Syndrome
[0174] Bronchiolitis obliterans, and its correlating clinical
condition bronchiolitis obliterans syndrome, are characterized by
obstruction of the pulmonary pathway via obliteration of pulmonary
small airways. In bronchiolitis obliterans, pathologic examination
characteristically finds lesions which obstruct or obliterate small
airways in the lung. These lesions are granular fibromyxoid tissue
and dense submucosal scar tissue. Lesions progress from prolonged,
abnormal, or aberrant inflammation of epithelial and
epithelial-localized structures of the small airways, mediated by
proinflammatory cytokines such as TNF-.alpha. and result in
excessive fibroproliferation. The obliteration of pulmonary small
airways progressively leads to airflow obstruction, characterized
by progressive decline in forced expiratory volume in one second
(FEV.sub.1), and is often accompanied by recurring infections of
the lower respiratory tract and colonization of pulmonary tissue by
pathogenic microorganisms.
[0175] Bronchiolitis obliterans syndrome affects 50-60% of patients
surviving five years after lung transplantation surgery, and five
year survival after onset of bronchiolitis obliterans syndrome is
only 30-40%. Lung transplantation patients experiencing
bronchiolitis obliterans syndrome often respond poorly to augmented
immunosuppression. In patients with idiopathic pulmonary fibrosis,
survival after bronchiolitis obliterans syndrome is shorter than in
patients with emphysema. The compounds and methods described herein
are useful in the treatment of bronchiolitis obliterans
syndrome.
Chronic Allograft Fibrosis
[0176] Allograft failure is a profound concern in transplantation
management. One of the primary causes of allograft failure is
chronic allograft dysfunction. Hallmarks of chronic allograft
dysfunction are chronic inflammation and chronic fibrosis, both of
which are associated with the production of inflammatory cytokines
and growth factors. Mediation of inflammatory cytokine and growth
factors, particularly resulting in interruption of collagen and TGF
production, is useful in the treatment of chronic allograft
fibrosis. The term "chronic allograft fibrosis" as used herein is
intended to encompass both chronic inflammation and chronic
fibrosis associated with chronic allograft fibrosis. The compounds
and methods described herein are useful in the treatment of chronic
allograft fibrosis.
Renal Fibrosis
[0177] Irrespective of the nature of the initial insult, renal
fibrosis is considered to be the common final pathway by which
kidney disease progresses to end-stage renal failure. Stambe et al.
(2004 J Am Soc Nephrol 15:370-379) tested an inhibitor of the
active (phosphorylated) form of p38, NPC 31169, developed by Scios
Inc. (San Francisco, Calif.) in a rat model of renal fibrosis, and
reported a significant reduction in renal fibrosis assessed by
interstitial volume, collagen IV deposition, and connective tissue
growth mRNA levels. The compounds and methods described herein are
useful in the treatment of renal fibrosis.
Leiomyoma
[0178] Uterine leiomyomas or fibroids are the most common pelvic
tumors in women with no known long-term effective drug therapies
available. Leiomyomas are characterized by increased cell
proliferation and tissue fibrosis. Pirfenidone was tested on cell
proliferation and collagen expression in cultured myometrial and
leiomyoma smooth muscle cells, and was found to be an effective
inhibitor of myometrial and leiomyoma cell proliferation (Lee et
al. 1998 J Clin Endocrinol Metab 83:219-223). The compounds and
methods described herein are useful in the treatment of
leiomyomas.
Endomyocardial Fibrosis
[0179] Endomyocardial fibrosis (EMF) is a disorder characterized by
the development of restrictive cardiomyopathy. EMF is sometimes
considered part of a spectrum of a single disease process that
includes Loffler endocarditis (nontropical eosinophilic
endomyocardial fibrosis or fibroplastic parietal endocarditis with
eosinophilia). In EMF, the underlying process produces patchy
fibrosis of the endocardial surface of the heart, leading to
reduced compliance and, ultimately, restrictive physiology as the
endomyocardial surface becomes more generally involved. Endocardial
fibrosis principally involves the inflow tracts of the right and
left ventricles and may affect the atrioventricular valves, leading
to tricuspid and mitral regurgitation. MAPK activation was shown to
contribute to arrhythmogenic atrial structural remodeling in EMF.
The compounds and methods described herein are useful in the
treatment and/or prevention of endomyocardial fibrosis.
Other Inflammatory Diseases
[0180] Many autoimmune diseases and diseases associated with
chronic inflammation, as well as acute responses, have been linked
to activation of p38 MAP kinase and overexpression or dysregulation
of inflammatory cytokines. These diseases include, but are not
limited to: rheumatoid arthritis; rheumatoid spondylitis;
osteoarthritis; gout, other arthritic conditions; sepsis; septic
shock; endotoxic shock; gram-negative sepsis; toxic shock syndrome;
asthma; adult respiratory distress syndrome; chronic obstructive
pulmonary disease; chronic pulmonary inflammation; inflammatory
bowel disease; Crohn's disease; psoriasis; eczema; ulcerative
colitis; pancreatic fibrosis; hepatic fibrosis; acute and chronic
renal disease; irritable bowel syndrome; pyresis; restenosis;
cerebral malaria; stroke and ischemic injury; neural trauma;
Alzheimer's disease; Huntington's disease; Parkinson's disease;
acute and chronic pain; allergic rhinitis; allergic conjunctivitis;
chronic heart failure; acute coronary syndrome; cachexia; malaria;
leprosy; leishmaniasis; Lyme disease; Reiter's syndrome; acute
synoviitis; muscle degeneration, bursitis; tendonitis;
tenosynovitis; herniated, ruptures, or prolapsed intervertebral
disk syndrome; osteopetrosis; thrombosis; cancer; restenosis;
silicosis; pulmonary sarcosis; bone resorption diseases, such as
osteoporosis; graft-versus-host reaction; and auto-immune diseases,
such as Multiple Sclerosis, lupus and fibromyalgia; AIDS and other
viral diseases such as Herpes Zoster, Herpes Simplex I or II,
influenza virus and cytomegalovirus; and diabetes mellitus.
[0181] Many studies have shown that reducing the activity of p38
MAP kinase, its upstream activators or its downstream effectors,
either through genetic or chemical means, blunts the inflammatory
response and prevents or minimizes tissue damage (see, e.g.,
English, et al. 2002 Trends Pharmacol Sci 23:40-45; and Dong et al.
2002 Annu Rev Immunol 20:55-72). Thus, inhibitors of p38 activity,
which also inhibit excess or unregulated cytokine production and
may inhibit more than a single pro-inflammatory cytokine, may be
useful as anti-inflammatory agents and therapeutics. Furthermore,
the large number of diseases associated with p38 MAP
kinase-associated inflammatory responses indicates that there is a
need for effective methods for treating these conditions.
[0182] Cardiovascular disease. Inflammation and leukocyte
activation/infiltration play a major role in the initiation and
progression of cardiovascular diseases including atherosclerosis
and heart failure. Acute p38 mitogen-activated protein kinase
(MAPK) pathway inhibition attenuates tissue damage and leukocyte
accumulation in myocardial ischemia/reperfusion injury. The
compounds and methods described herein are useful for treating
cardiovascular disease.
[0183] Multiple sclerosis. Inflammation in the central nervous
system occurs in diseases such as multiple sclerosis and leads to
axon dysfunction and destruction. Both in vitro and in vivo
observations have shown an important role for nitric oxide (NO) in
mediating inflammatory axonopathy. p38 MAP kinase is activated by
NO exposure and inhibition of p38 signalling was shown to lead to
neuronal and axonal survival effects. OCM and IGF-1 reduced p38
activation in NO-exposed cortical neurons and improved axon
survival in cultures exposed to NO, a process dependent on
mitogen-activated protein kinase/extracellular signal-related
kinase signalling. The compounds and methods described herein are
useful for treating multiple sclerosis.
[0184] Primary graft nonfunction. Nonspecific inflammation is
associated with primary graft nonfunction (PNF). Inflammatory islet
damage is mediated at least partially by pro-inflammatory
cytokines, such as interleukin-1.beta. (IL-1.beta.) and tumor
necrosis factor-.alpha. (TNF-.alpha.) produced by resident islet
macrophages. The p38 pathway is known to be involved in cytokine
production in the cells of the monocyte-macrophage lineage.
Inhibition of the p38 pathway by a chemical p38 inhibitor,
SB203580, suppresses IL-1.beta. and TNF-.alpha. production in human
islets exposed to lipopolysaccharide (LPS) and/or inflammatory
cytokines. Although IL-1.beta. is predominantly produced by
resident macrophages, ductal cells and islet vascular endothelial
cells were found to be another cellular source of IL-1.beta. in
isolated human islets. SB203580 also inhibited the expression of
inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2)
in the treated islets. Furthermore, human islets treated with
SB203580 for 1 h prior to transplantation showed significantly
improved graft function. The compounds and methods described herein
are useful for improving graft survival in clinical islet
transplantation.
[0185] Acute renal injury. Cisplatin is an important
chemotherapeutic agent but can cause acute renal injury. Part of
this acute renal injury is mediated through tumor necrosis
factor-.alpha. (TNF-.alpha.). Cisplatin activates p38 MAPK and
induces apoptosis in cancer cells. p38 MAPK activation leads to
increased production of TNF-.alpha. in ischemic injury and in
macrophages. In vitro, cisplatin caused a dose dependent activation
of p38 MAPK in proximal tubule cells. Inhibition of p38 MAPK
activation led to inhibition of TNF-.alpha. production. In vivo,
mice treated with a single dose of cisplatin developed severe renal
dysfunction, which was accompanied by an increase in kidney p38
MAPK activity and an increase in infiltrating leukocytes. However,
animals treated with a p38 MAPK inhibitor SKF86002 along with
cisplatin showed less renal dysfunction, less severe histologic
damage and fewer leukocytes compared with cisplatin+vehicle treated
animals. The compounds and methods described herein are useful for
preventing acute renal injury.
[0186] Periodontitis. The proinflammatory mediator bradykinin (BK)
stimulates interleukin-8 (IL-8) production in human gingival
fibroblasts in vitro and plays an important role in the
pathogenesis of various inflammatory diseases including
periodontitis. The specific p38 mitogen-activated protein kinase
(MAPK) inhibitor SB 203580 reduced IL-8 production stimulated by
the combination of BK and IL-1.beta. as well as the
IL-1.beta.-stimulated IL-8 production. The compounds and methods
described herein are useful for treating or preventing
periodontitis.
Pharmaceutical Compositions
[0187] While it is possible for the compounds described herein to
be administered alone, it may be preferable to formulate the
compounds as pharmaceutical compositions. As such, in yet another
aspect, pharmaceutical compositions useful in the methods of the
invention are provided. More particularly, the pharmaceutical
compositions described herein may be useful, inter alia, for
treating or preventing inflammatory conditions, e.g., conditions
associated with p38 activity or cytokine activity or any
combination thereof. A pharmaceutical composition is any
composition that may be administered in vitro or in vivo or both to
a subject in order to treat or ameliorate a condition. In a
preferred embodiment, a pharmaceutical composition may be
administered in vivo. A subject may include one or more cells or
tissues, or organisms. A preferred subject is a mammal. A mammal
includes any mammal, such as by way of non-limiting example,
cattle, pigs, sheep, goats, horses, camels, buffalo, cats, dogs,
rats, mice, and humans. A highly preferred subject mammal is a
human.
[0188] In an embodiment, the pharmaceutical compositions may be
formulated with pharmaceutically acceptable excipients such as
carriers, solvents, stabilizers, adjuvants, diluents, etc.,
depending upon the particular mode of administration and dosage
form. The pharmaceutical compositions should generally be
formulated to achieve a physiologically compatible pH, and may
range from a pH of about 3 to a pH of about 11, preferably about pH
3 to about pH 7, depending on the formulation and route of
administration. In alternative embodiments, it may be preferred
that the pH is adjusted to a range from about pH 5.0 to about pH 8.
More particularly, the pharmaceutical compositions may comprise a
therapeutically or prophylactically effective amount of at least
one compound as described herein, together with one or more
pharmaceutically acceptable excipients. Optionally, the
pharmaceutical compositions may comprise a combination of the
compounds described herein, or may include a second active
ingredient useful in the treatment or prevention of bacterial
infection (e.g., anti-bacterial or anti-microbial agents).
[0189] Formulations, e.g., for parenteral or oral administration,
are most typically solids, liquid solutions, emulsions or
suspensions, while inhalable formulations for pulmonary
administration are generally liquids or powders, with powder
formulations being generally preferred. A preferred pharmaceutical
composition may also be formulated as a lyophilized solid that is
reconstituted with a physiologically compatible solvent prior to
administration. Alternative pharmaceutical compositions may be
formulated as syrups, creams, ointments, tablets, and the like.
[0190] The term "pharmaceutically acceptable excipient" refers to
an excipient for administration of a pharmaceutical agent, such as
the compounds described herein. The term refers to any
pharmaceutical excipient that may be administered without undue
toxicity.
[0191] Pharmaceutically acceptable excipients are determined in
part by the particular composition being administered, as well as
by the particular method used to administer the composition.
Accordingly, there exists a wide variety of suitable formulations
of pharmaceutical compositions (see, e.g., Remington's
Pharmaceutical Sciences).
[0192] Suitable excipients may be carrier molecules that include
large, slowly metabolized macromolecules such as proteins,
polysaccharides, polylactic acids, polyglycolic acids, polymeric
amino acids, amino acid copolymers, and inactive virus particles.
Other exemplary excipients include antioxidants such as ascorbic
acid; chelating agents such as EDTA; carbohydrates such as dextrin,
hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid;
liquids such as oils, water, saline, glycerol and ethanol; wetting
or emulsifying agents; pH buffering substances; and the like.
Liposomes are also included within the definition of
pharmaceutically acceptable excipients.
[0193] The pharmaceutical compositions described herein may be
formulated in any form suitable for the intended method of
administration. When intended for oral use for example, tablets,
troches, lozenges, aqueous or oil suspensions, non-aqueous
solutions, dispersible powders or granules (including micronized
particles or nanoparticles), emulsions, hard or soft capsules,
syrups or elixirs may be prepared. Compositions intended for oral
use may be prepared according to any method known to the art for
the manufacture of pharmaceutical compositions, and such
compositions may contain one or more agents including sweetening
agents, flavoring agents, coloring agents and preserving agents, in
order to provide a palatable preparation.
[0194] Pharmaceutically acceptable excipients particularly suitable
for use in conjunction with tablets include, for example, inert
diluents, such as celluloses, calcium or sodium carbonate, lactose,
calcium or sodium phosphate; disintegrating agents, such as
cross-linked povidone, maize starch, or alginic acid; binding
agents, such as povidone, starch, gelatin or acacia; and
lubricating agents, such as magnesium stearate, stearic acid or
talc,
[0195] Tablets may be uncoated or may be coated by known techniques
including microencapsulation to delay disintegration and adsorption
in the gastrointestinal tract and thereby provide a sustained
action over a longer period. For example, a time delay material
such as glyceryl monostearate or glyceryl distearate alone or with
a wax may be employed.
[0196] Formulations for oral use may be also presented as hard
gelatin capsules where the active ingredient is mixed with an inert
solid diluent, for example celluloses, lactose, calcium phosphate
or kaolin, or as soft gelatin capsules wherein the active
ingredient is mixed with non-aqueous or oil medium, such as
glycerin, propylene glycol, polyethylene glycol, peanut oil, liquid
paraffin or olive oil.
[0197] In another embodiment, pharmaceutical compositions may be
formulated as suspensions comprising a compound of the embodiments
in admixture with at least one pharmaceutically acceptable
excipient suitable for the manufacture of a suspension.
[0198] In yet another embodiment, pharmaceutical compositions may
be formulated as dispersible powders and granules suitable for
preparation of a suspension by the addition of suitable
excipients.
[0199] Excipients suitable for use in connection with suspensions
include suspending agents, such as sodium carboxymethylcellulose,
methylcellulose, hydroxypropyl methylcellulose, sodium alginate,
polyvinylpyrrolidone, gum tragacanth, gum acacia, dispersing or
wetting agents such as a naturally occurring phosphatide (e.g.,
lecithin), a condensation product of an alkylene oxide with a fatty
acid (e.g., polyoxyethylene stearate), a condensation product of
ethylene oxide with a long chain aliphatic alcohol (e.g.,
heptadecaethyleneoxycetanol), a condensation product of ethylene
oxide with a partial ester derived from a fatty acid and a hexitol
anhydride (e.g., polyoxyethylene sorbitan monooleate);
polysaccharides and polysaccharide-like compounds (e.g. dextran
sulfate); glycosaminoglycans and glycosaminoglycan-like compounds
(e.g., hyaluronic acid); and thickening agents, such as carbomer,
beeswax, hard paraffin or cetyl alcohol. The suspensions may also
contain one or more preservatives such as acetic acid, methyl
and/or n-propyl p-hydroxy-benzoate; one or more coloring agents;
one or more flavoring agents; and one or more sweetening agents
such as sucrose or saccharin.
[0200] The pharmaceutical compositions may also be in the form of
oil-in water emulsions. The oily phase may be a vegetable oil, such
as olive oil or arachis oil, a mineral oil, such as liquid
paraffin, or a mixture of these. Suitable emulsifying agents
include naturally-occurring gums, such as gum acacia and gum
tragacanth; naturally occurring phosphatides, such as soybean
lecithin, esters or partial esters derived from fatty acids;
hexitol anhydrides, such as sorbitan monooleate; and condensation
products of these partial esters with ethylene oxide, such as
polyoxyethylene sorbitan monooleate. The emulsion may also contain
sweetening and flavoring agents. Syrups and elixirs may be
formulated with sweetening agents, such as glycerol, sorbitol or
sucrose. Such formulations may also contain a demulcent, a
preservative, a flavoring or a coloring agent.
[0201] Additionally, the pharmaceutical compositions may be in the
form of a sterile injectable preparation, such as a sterile
injectable aqueous emulsion or oleaginous suspension. This emulsion
or suspension may be formulated according to the known art using
those suitable dispersing or wetting agents and suspending agents
which have been mentioned above. The sterile injectable preparation
may also be a sterile injectable solution or suspension in a
non-toxic parenterally acceptable diluent or solvent, such as a
solution in 1,2-propane-diol.
[0202] The sterile injectable preparation may also be prepared as a
lyophilized powder. Among the acceptable vehicles and solvents that
may be employed are water, Ringer's solution, and isotonic sodium
chloride solution. In addition, sterile fixed oils may be employed
as a solvent or suspending medium. For this purpose any bland fixed
oil may be employed including synthetic mono- or diglycerides. In
addition, fatty acids such as oleic acid may likewise be used in
the preparation of injectables.
[0203] To obtain a stable water-soluble dose form of a
pharmaceutical composition, a pharmaceutically acceptable salt of a
compound described herein may be dissolved in an aqueous solution
of an organic or inorganic acid, such as 0.3 M solution of succinic
acid, or more preferably, citric acid. If a soluble salt form is
not available, the compound may be dissolved in a suitable
co-solvent or combination of co-solvents. Examples of suitable
co-solvents include alcohol, propylene glycol, polyethylene glycol
300, polysorbate 80, glycerin and the like in concentrations
ranging from about 0 to about 60% of the total volume. In one
embodiment, the active compound is dissolved in DMSO and diluted
with water.
[0204] The pharmaceutical composition may also be in the form of a
solution of a salt form of the active ingredient in an appropriate
aqueous vehicle, such as water or isotonic saline or dextrose
solution. Also contemplated are compounds which have been modified
by substitutions or additions of chemical or biochemical moieties
which make them more suitable for delivery (e.g., increase
solubility, bioactivity, palatability, decrease adverse reactions,
etc.), for example by esterification, glycosylation, PEGylation,
etc.
[0205] In a preferred embodiment, the compounds described herein
may be formulated for oral administration in a lipid-based
formulation suitable for low solubility compounds. Lipid-based
formulations can generally enhance the oral bioavailability of such
compounds.
[0206] As such, a preferred pharmaceutical composition comprises a
therapeutically or prophylactically effective amount of a compound
described herein, together with at least one pharmaceutically
acceptable excipient selected from the group consisting of--medium
chain fatty acids or propylene glycol esters thereof (e.g.,
propylene glycol esters of edible fatty acids such as caprylic and
capric fatty acids) and pharmaceutically acceptable surfactants
such as polyoxyl 40 hydrogenated castor oil.
[0207] In an alternative preferred embodiment, cyclodextrins may be
added as aqueous solubility enhancers. Preferred cyclodextrins
include hydroxypropyl, hydroxyethyl, glucosyl, maltosyl and
maltotriosyl derivatives of .alpha.-, .beta.-, and
.gamma.-cyclodextrin. A particularly preferred cyclodextrin
solubility enhancer is hydroxypropyl-o-cyclodextrin (BPBC), which
may be added to any of the above-described compositions to further
improve the aqueous solubility characteristics of the compounds of
the embodiments. In one embodiment, the composition comprises about
0.1% to about 20% hydroxypropyl-o-cyclodextrin, more preferably
about 1% to about 15% hydroxypropyl-o-cyclodextrin, and even more
preferably from about 2.5% to about 10%
hydroxypropyl-o-cyclodextrin. The amount of solubility enhancer
employed will depend on the amount of the compound of the
embodiments in the composition.
[0208] A pharmaceutical composition contains a total amount of the
active ingredient(s) sufficient to achieve an intended therapeutic
effect. More specifically, in some embodiments, the pharmaceutical
composition contains a therapeutically effective amount (e.g., an
amount of an SAPK-modulating compound that is effective in the
prevention or treatment of the symptoms of an inflammatory disease
or condition, wherein the compound exhibits an EC.sub.50 in the
range of about 1 .mu.M to about 1000 .mu.M, preferably about 50
.mu.M to about 650 .mu.M, for inhibition of at least one p38 MAPK).
The total amounts of the compound that may be combined with the
carrier materials to produce a unitary dosing form will vary
depending upon the host treated and the particular mode of
administration. Preferably, the compositions are formulated so that
a dose of between 0.01 to 100 mg/kg body weight/day of an
SAPK-modulating compound is administered to a patient receiving the
compositions.
Example 1
[0209] Compounds are screened for the ability to inhibit ATF2
phosphorylation by p38 MAP kinase in vitro. The ability of
compounds to inhibit ATF2 phosphorylation in this in vitro assay is
correlated with the inhibition of p38 MAP kinase and TNF.alpha.
expression in vivo, and is therefore an indicator of potential in
vivo therapeutic activity (Raingeaud, J. et al. 1995 J. Biol. Chem.
270:7420-7426; Brinkman, M. N., et al. 1999 J. Biol. Chem.
274:30882-30886; and Fuchs, S. Y. et al. J. Biol. Chem.
275:12560-12564, 2000).
[0210] All kinases and the substrate ATF2 are acquired from Upstate
(Charlottesville, Va.). p38 MAP Kinases are recombinant human
full-length proteins with an amino-terminal GST fusion, expressed
in and purified from E. coli. ATF2 is a GST fusion protein
containing amino acids 19-96 of human ATF2 expressed in E. coli.
All proteins are aliquoted and stored at -80.degree. C.
[0211] p38 MAP kinase assays are performed using an assay buffer
containing 25 mM HEPES, pH 7.5, 10 mM MgCl.sub.2, 2 mM DTT, 20 mM
.beta.-glycerophosphate, 0.1 mM Na.sub.3VO.sub.4, 40 .mu.M ATP and
1.25 .mu.M of ATF2, together with 6 ng of p38.alpha. protein, 12 ng
p38.beta. protein, 1.5 ng p38.gamma., or 0.4 ng JNK2.alpha.2.
Compounds are serially diluted in DMSO and 2 .mu.L of test compound
at various concentrations is used. The vehicle control receives
DMSO only.
[0212] Test compounds are pre-incubated with 20 .mu.l of enzyme in
kinase buffer (25 mM HEPES, pH 7.5, 10 mM MgCl.sub.2, 2 mM DTT, 20
mM .beta.-glycerophosphate and 0.1 mM Na.sub.3VO.sub.4) at room
temperature for 15 minutes. Reactions are initiated by addition of
30 .mu.l substrate solution to yield a final concentration of 40
.mu.M ATP and 1.25 .mu.M ATF2 in kinase buffer. The reactions are
incubated for 30 minutes at 37.degree. C. and terminated by the
addition of 18 .mu.l of 200 mM EDTA. An ELISA method is used to
measure the phosphorylation of ATF2 at Thr 69. High binding 96-well
plates are coated with 50 .mu.l of kinase reaction for 1 hour at
37.degree. C. The coated plates are washed with 200 .mu.l washing
buffer (25 mM Tris HCl, pH 8.3, 192 mM glycine, 0.1% SDS and 0.05%
Tween-20) three times. The plates are then washed three times with
SuperBlock in TBS (Pierce, 37535). After blocking, plates are
incubated with 50 .mu.l of rabbit anti-phospho-ATF2 antibody (Cell
Signaling, 9221L, 1:500) for 30 minutes at 37.degree. C.
[0213] Plates are washed three times with washing buffer prior to
incubation with 50 .mu.l HRP-conjugated goat anti-rabbit antibody
(Cell Signaling, 7074, 1:500) for 30 minutes at 37.degree. C.
Plates are then washed three times with washing buffer before
incubation with 50 .mu.l of Ultra TMB-ELISA (Pierce, 34028) for 8
minutes at room temperature. Finally, 50 .mu.l of phosphoric acid
(1 M) is added to stop reactions and plate absorbance is read at
450 nm on a SpectraMax 250 plate reader.
[0214] The compounds inhibit the phosphorylation of ATF2 in this in
vitro assay. Preferred compounds exhibit EC.sub.50 values of
between about 1 .mu.M and about 1000 .mu.M, preferably about 50
.mu.M to about 650 .mu.M.
Example 2
[0215] Compounds are screened for the ability to inhibit TNF.alpha.
release from THP-1 cells stimulated with lipopolysaccharide (LPS)
in vitro. The ability of compounds to inhibit TNF.alpha. release in
this in vitro assay is correlated with the inhibition of p38
activity and TNF.alpha., and is therefore an indicator of potential
in vivo therapeutic activity (Lee J. C. et al. 1993 Ann. NY. Acad.
Sci. 696:149-170; and 1994 Nature 372:739-746).
[0216] THP-I cells from ATCC (TIB202) are maintained at 37.degree.
C., 5% CO.sub.2 in RPMI 1640 media (MediaTech, Herndon, Va.)
containing 4.5 g/L glucose, supplemented with 10% fetal bovine
serum, 1% penicillin/streptomycin and 50 .mu.M
.beta.-mercaptoethanol.
[0217] Test compounds are initially dissolved in RPMI media with 1%
DMSO (v/v). Compounds are then serially diluted in RPMI media for
all subsequent dilutions. The assay is performed under sterile
conditions. THP-1 cells at a culture density of 6-8.times.10.sup.5
cells/ml are collected and resuspended in the RPMI media at
10.sup.6 cells/ml. 100 .mu.l of resuspended cells are added to each
well, which contain 100 .mu.l of a test compound. Test compounds
are prepared at twice the final concentration. Final DMSO
concentration is no more than 0.5% (v/v). Cells are preincubated
with compound for 60 minutes at 37.degree. C., 5% CO.sub.2 prior to
stimulation with lipopolysaccharide (LPS) (Sigma L-2880, 4 mg/ml
stock in PBS). The final LPS concentration in each well is 10 or 30
.mu.g/ml for TNF.alpha. and IL-1.beta. release, respectively.
Unstimulated control cell suspensions receive PBS vehicle only.
Cell mixtures are incubated for 18 or 48 hours for TNF.alpha. and
IL-1.beta. release, respectively. 150 .mu.l of supernatants are
taken and transferred to a fresh plate and stored at -20.degree. C.
until farther analysis. TNF.alpha. and IL-1.beta. levels are
measured using ELISA kits. A Luminescence is used as the plate
reader. Analysis is performed by non-linear regression to generate
a dose response curve. The calculated EC.sub.50 value is the
concentration of the test compound that causes a 50% decrease in
TNF.alpha. or IL-1.beta. levels.
[0218] Compounds inhibit the release of TNF.alpha., IL-1.beta. or
both TNF.alpha., and IL-1.beta. in this in vitro assay. Preferred
compounds exhibit EC.sub.50 values for TNF.alpha. and/or IL-1.beta.
of between about 1 .mu.M and about 1000 .mu.M, preferably about 50
.mu.M to about 650 .mu.M. Data are provided in Table 2 below.
TABLE-US-00002 TABLE 2 ##STR00052## TNF No..sup.1 X.sub.3 R.sub.2
R.sub.4 Z EC.sub.50 (.mu.M).sup.2 Toxicity.sup.3 13 --H --CH.sub.3
--H O A .gtoreq.1 mM 7 --H --H --H O B D 9 --OH --H --H O C D 11
--F --H --H O B D 8 --H --CF.sub.3 --H O B D 12 --F --CF.sub.3 --H
O C D 5 --OH --CH.sub.3 --H O A .gtoreq.1 mM 1 --H --CH.sub.2OH --H
O B D 2 --H --COOH --H O C D 3 --H -glucuronide --H O C D 6 --H
--CH.sub.2OCH.sub.3 --H O A .gtoreq.1 mM 4 --H --CH.sub.3 --OH O A
.gtoreq.1 mM 14 --F --CH.sub.3 --H O B D 15 --OCH.sub.3 --CF.sub.3
--H O C D 16 --COCH.sub.3 --H --H O C D 10 --OH --CF.sub.3 --H O A
D 17 --H -phenyl --H O C D 18 --H --CH.sub.3 --H S C D 25 See note
CH.sub.3 --H O B B 4 26 --H --Br --H O A B 27 --OCH.sub.3 --Br --H
O A A 28 --OH --CH.sub.2F --H O C D 29 --OH --CHF.sub.2 --H O C D
30 --OH --Br --H O A A 31 --OCH.sub.3 --CH.sub.2F --H O A A 32
--OCH.sub.3 --CHF.sub.2 --H O A A 33 --H See note 5 --H O C D 24
--H CO.sub.2CH.sub.3 --H O C D .sup.1Compound number as shown in
Table 1 .sup.2A: .ltoreq.2,000; B: >2,000; C: Inconclusive
(e.g., no data or no activity observed) .sup.3D: Inconclusive
(e.g., no data or no toxicity observed) .sup.4Compound 25 is as
depicted in Table 1, namely the aryl group attached to a 2-pyridone
nitrogen is an N-methylpyridinium moiety .sup.5Compound 33 is as
depicted in Table 1, namely a bromoaryl group fused to the 5 and 6
positions of a 2-pyridone ring
Example 3
[0219] Compounds are screened for the ability to inhibit TNF.alpha.
release from primary human peripheral blood mononuclear cells
(PBMC) stimulated with lipopolysaccharide (LPS) in vitro. The
ability of compounds to inhibit TNF.alpha. release in this in vitro
assay is correlated with the inhibition of p38 activity and is
therefore an indicator of potential in vivo therapeutic activity
(2002 Osteoarthritis & Cartilage 10:961-967; and Laufer, S. A.
and Wagner, G. K. 2002 J. Med. Chem. 45: 2733-2740).
[0220] Human peripheral blood mononuclear cells (PBMC) are isolated
by differential centrifugation through a Ficoll-HyPaque density
gradient from pooled serum of 3-8 individual blood donors. Isolated
PBMC contain approximately 10% CD-14 positive monocytes, 90%
lymphocytes and <1% granulocytes and platelets. PBMC
(10.sup.6/ml) are cultured in polystyrene plates and stimulated
with lipopolysaccharide (LPS; 50 ng/ml; Sigma, St. Louis, Mo.) in
the presence and absence of the test compound in serial dilutions,
in duplicate, for 24 hr at 37.degree. C. in GIBCO.TM. RPM1 medium
(Invitrogen, Carlsbad, Calif.) without serum. The TNF.alpha. level
in cell supernatants is determined by ELISA using a commercially
available kit (MDS Panlabs #309700).
[0221] Preferred compounds inhibit the release of TNF.alpha. in
this assay with an EC.sub.50 value of between about 1 .mu.M and
about 1000 .mu.M, preferably about 50 .mu.M to about 650 .mu.M.
Example 4
[0222] Compounds are screened for the ability to inhibit the
release of TNF.alpha. in an in vivo animal model (See, e.g.,
Griswold D. E. et al. 1993 Drugs Exp. Clin. Res. 19:243-248;
Badger, A. M. et al. 1996 J. Pharmacol. Exp. Ther. 279:1453-1461;
Dong, C. et al. 2002 Annu. Rev. Immunol. 20:55-72 (and references
cited therein); Ono, K. and Han, J. 2000 Cellular Signalling
12:1-13 (and references cited therein); and Griffiths, J. B. et al.
1999 Curr. Rheumatol. Rep. 1:139-148).
[0223] Without being bound by any particular theory, it is believed
that inhibition of TNF.alpha. in this model is due to inhibition of
p38 MAP kinase by the compound.
[0224] Male Sprague-Dawley rats (0.2-0.35 kg) are randomly divided
into groups of six or more and are dosed intravenously by infusion
or bolus injection, or are dosed orally with test compounds in a
suitable formulation in each case. Thirty minutes following end of
infusion or bolus injection, and 1-2 hr following oral
administration, lipopolysaccharide E. coli/0127:B8 (0.8 mg/kg) is
administered IV. Blood samples are collected 1.5 hours
post-treatment with LPS. Serum TNF.alpha. levels are determined
using the ELISA kit from Biosource (KRC3011C) and compared to that
from vehicle-treated control.
[0225] Preferred compounds inhibit the release of TNF.alpha. in
this in vivo assay. Preferred compounds exhibit an ED.sub.50 value
of less than 500 mg/kg, preferably less than 400 mg/kg, preferably
less than 200 mg/kg, preferably less than 100 mg/kg, more
preferably, less than 50 mg/kg, more preferably, less than 40
mg/kg, more preferably, less than 30 mg/kg, more preferably, less
than 20 mg/kg, more preferably, less than 10 mg/kg.
[0226] The methods of determining the EC.sub.50 of the inhibition
of p38 by a compound include any methods known in the art that
allow the quantitative detection of any of the downstream
substrates of p38 MAPK as described above. Therefore, these methods
additionally include but limited to detection of expression of
genes known to be regulated by p38 either individually, or by gene
arrays.
Example 5
[0227] The following methods can be used for (1) a kinase assay for
determination of EC.sub.50, (2) a non-radiometric kinase assay for
determination of EC.sub.50, (3) modulation of induction of
TNF.alpha. expression, (4) a test for cell toxicity, and (5) an
assay to test the effect of compounds on collagen production.
Kinase Assay
[0228] The activity of the P38 kinase isoforms P38.gamma. and
P38.alpha. is determined by phosphorylation of ATF-2 in presence of
.sup.32P-.gamma.-ATP. The incorporation of .sup.32P into ATF-2 in
the presence or absence of inhibitors is determined. Pirfenidone
and its different derivatives are tested for inhibition of
P38.gamma. and P38.alpha. kinase activity in this biochemical
assay. The compounds are solubilized in water or DMSO and tested at
different concentrations from 0 to 10 mM using the appropriate
solvent for dilutions and as vehicle control. The enzymes
P38.gamma. and P38.alpha. are obtained as activated and purified
recombinant protein (Upstate, Charlottesville, Va.). The activated
enzyme is used at 24.8 nM in the final reaction. The enzymes are
diluted prior to the reaction in the following buffer (1M HEPES, pH
7.4, 500 mM DTT, 1% Triton X-100 and 10 mg/ml BSA). The reaction is
performed in the following solution that is prepared as a two fold
stock solution (1M HEPES, pH 7.4, 500 mM DTT and 1% Triton X-100)
and non-radioactive ATP is present in the reaction at 6.25 .mu.M
ATP (Cell Signaling, Beverly, Mass.). To determine the
phosphorylation of ATF-2, .gamma.-[.sup.32P]-ATP 3000 Ci/mmol is
added to each reaction at a concentration of 7.5 .mu.M. ATF-2 (Cell
Signaling, Beverly, Mass.) as a kinase substrate is used at 3
.mu.M. As a first step in assembling the enzyme reaction, activated
kinase and inhibitor or the appropriate vehicle control are added
to reaction buffer and incubated for 30 min at room temperature.
The kinase reaction is initiated by the addition of ATF-2 and ATP
mixture. The final volume for each reaction is 20 .mu.A and
performed at room temperature for 30 minutes. After the 30 minutes
of incubation 80 ul of Laemmlie buffer is added. Subsequently 20%
of the reaction is separated by SDS-Page (BioRad, Hercules, Calif.)
under reducing conditions. After electrophoresis, the gel is
exposed to a phosphorimager plate and analyzed using a
phosphoimager (Storm System, Amersham Biosciences, Piscataway,
N.J.). The signal obtained is quantified after background
correction and calculated as percent inhibition using the
uninhibited kinase activity with the vehicle control as 0%
inhibition. The kinase activity, in the presence of different
inhibitor concentrations, is plotted using Kaleidagraph (Synergy
Software, Reading, Pa.) to determine the EC.sub.50 for each
compound and tested P38 kinase.
Non-Radiometric Kinase Assay
[0229] An alternate, non-radiometric kinase assay was also employed
to define the EC50 for inhibition of P38. In this assay, p38 kinase
transfers a phosphate from ATP to an EGF-R peptide substrate,
resulting in the formation of phosphorylated EGF-R peptide with the
concomitant conversion of ATP to ADP. In an uncoupled reaction, p38
also hydrolyzes ATP at a slower rate in the absence of peptide
substrate (Fox et al, FEBS Lett 1999), which contributes slightly
to ATP consumption. Thus, the amount of ATP consumed is directly
proportional to p38 activity. At the end of a kinase reaction, the
amount of ATP remaining is determined using Kinase-Glo Plus
Luminescent Kinase Assay (Promega, Inc., Madison, Wis.). These
reagents use residual ATP to support the ATP-dependent enzymatic
conversion of beetle luciferin to oxyluciferin with the concomitant
production of light, which is detected by a luminometer.
[0230] Kinase reactions are conducted by mixing compound (diluted
in DMSO and assay buffer) with either p38.alpha. or p38.gamma., and
EGF-R peptide substrate (AnaSpec, Inc., San Jose, Calif.) in assay
buffer. Reactions are then initiated by the addition of ATP and
allowed to run for 45 minutes at room temperature. Final buffer
conditions are: 20 mM HEPES (pH 7.4), 2 mM DTT, 0.1% Triton-X-100,
10 mM MgCl.sub.2, 10% glycerol, 12.5 mM p38.alpha. or p38.gamma.
(Upstate, Charlottesville, Va.), 50 .mu.M EGF-R peptide substrate,
and 10 .mu.M ATP. The final assay volume is 10 .mu.L. A control
reaction is performed in the absence of compound. Additional
control reactions that omit p38 are performed at every compound
concentration. All reactions are performed in triplicate.
[0231] Forty-five minutes after initiation of the kinase reaction,
the reaction is quenched by the addition of 10 .mu.L of Kinase-Glo
Plus assay reagent. The luciferase reaction is allowed to
equilibrate for 15 minutes prior to being read on an Envision
Multilabel Plate Reader (Perkin Elmer Life and Analytical Sciences,
Boston, Mass.). Data are plotted as luminescent signal versus log
compound concentration in KaleidaGraph (Synergy Software, Reading,
Pa.). EC.sub.50 values are determined by fitting the data to
4-paramater binding equation using a fixed upper bound that is
determined from control reactions in the absence of p38 (as
luminescence is inversely related to kinase activity).
Inhibition of TNF.alpha. Induction
[0232] THP-1 (ATCC, Rockville, Md.) is grown under regular tissue
culture conditions as recommended by ATCC. 18 hours prior to the
experiment, cells were plated in a 96 well format in regular
culture media containing 1% serum and 0.25 ml culture volume at a
density of 500,000 cells per well. The compound is added to each
well in triplicates and the appropriate solvent control is included
in each assay. The p38 inhibitor SB203850 at 1 mM/ml (Upstate,
Waltham, Mass.) is included as a positive control in each assay.
For the induction of TNF.alpha. expression, 1 .mu.g/ml LPS is added
to each well 30 minutes post compound addition. Following a 4 hour
incubation under tissue culture conditions the cells are sedimented
by centrifugation (10 min, 1000 rpm, Beckman table top centrifuge)
and a fraction of the cell free supernatant is collected and used
in a tenfold dilution for the quantification in the TNF.alpha.
specific ELISA (R&D Systems, Minneapolis, Minn.). The
TNF.alpha. ELISA is performed according to the directions provided
by the manufacturer. The TNF.alpha. is detected in pg/ml and
plotted as fractional activity normalized to the TNF.alpha.
expression in the solvent control.
Compound Toxicity Testing in a Cell Based Assay
[0233] The release of LDH as result of a disrupted cell membrane is
applied as a measure of cell toxicity. LDH is detected by its
enzymatic activity using a commercially available diagnostic kit
(Roche Diagnostics, Cat# 1 644 793). THP-1 cells are used for
determination of cell toxicity for consistency with the induced
TNF.alpha. expression in the previous experiment. As previously
described for testing of inhibition of TNF.alpha. induction, cells
are cultured in a 96 well format under 1% serum and regular tissue
culture conditions. The compounds are added at different
concentrations in triplicate. The appropriate solvent control is
used in each assay. After compound addition the cells are cultured
18 hours under regular tissue culture conditions. After this
incubation period, the positive control is initiated by adding
Triton-X-100 (2% v/v) to untreated cells and incubated for an
additional 10 minutes for complete cell lysis. Subsequently the
cells are sedimented by centrifugation and a fraction of the
supernatant removed and analyzed for LDH enzyme activity according
to the manufacturer's instructions. The data is typically reported
as % cell toxicity normalized to the Triton-X-100 lysed cells as
100% cell toxicity.
[0234] Toxicity data are also obtained using a commercially
available ATP assay (Molecular Probes' ATP Determination Kit
A22066, available from Invitrogen) and/or using a MTT assay. Both
the ATP and the MTT assays measure metabolic competence of the
cell. The MTT assay measures the ability of the cell to reduce a
marker substrate, which is related to metabolic competence (i.e.
viability). The ATP assay measures the cellular ATP concentration
in the presence and absence of compound. Toxic compounds lead to
reduced metabolic activity which leads to a reduction in ATP
concentration.
Assay for Effect of Compounds on Collagen Production
[0235] HFL-1 Cells (ATCC, Rockville, Md.) were grown under regular
tissue culture conditions in complete media containing 10% fetal
bovine serum (FBS; Mediatech, Inc., Herndon, Va.). Cells in early
passage were plated in 6 well plates. When the cells reached
confluence, the media was removed, cells washed with PBS, and the
cells were kept overnight in complete media containing 0.1% FBS.
The media was then replaced with fresh media plus 0.1% FCS, 10
.mu.M L-Proline (EMD Chemicals, Gibbstown, N.J.), 20 .mu.g/mL
ascorbic acid (EMD Chemicals, Gibbstown, N.J.). Compounds were
added to triplicate wells to a final concentration of 1 mM from
100.times. stock solutions in DMSO. One hour after the addition of
compound, the cells were treated with TGF-.beta.1 (Sigma-Aldrich,
St. Louis, Mo.) to a final concentration of 10 ng/mL (25 ng total).
Three days after addition of TGF-.beta., the media was removed,
cells were washed with PBS and then lysed. The total collagen
content of lysed cells was assessed with a dye-based collagen assay
(Sircol Collagen Assay, Newtownabbey, Northern Ireland) and a
.mu.Quant plate-based spectrophotometer (BioTek Instruments, Inc.,
Winooski, Vt.) with appropriate standard curves. The dynamic range
of the assay is defined by cells that were mock treated (1% DMSO
without compound) in the presence and absence of TGF-.beta.. Data
are reported in Table 3 as the percent inhibition of
TGF-.beta.-induced collagen as determined in the following
equation:
% inhibition=100*[(collagen, mock/+TGF-.beta.)-(collagen,
treated/+TGF-.beta.)]/[(collagen, mock/+TGF-.beta.)-(collagen,
mock/-TGF-.beta.)]
TABLE-US-00003 TABLE 3 % Inhibition of TGF-.beta. Stimulated
Compound No. Collagen Synthesis 8 48 14 23 15 49 26 58 30 69
Example 6
[0236] Preparation of
1-(4-hydroxyphenyl)-5-(trifloromethyl)-2-pyridone (Compound 10): A
mixture of 5-(trifloromethyl)-2(1H)-pyridone (815.5 mg, 5 mmol),
4-iodoanisole (2.34 g, 10 mmol), CuI (952 mg, 5 mmol),
K.sub.2CO.sub.3 (691 mg, 5 mmol) and DMF (5 ml) was heated at
135.degree. C. overnight. The reaction mixture was diluted with 10%
ammonia (15 ml) and extracted with ethyl acetate. The organic
extract was washed with saturated sodium chloride, dried over
magnesium sulfate and evaporated. Column chromatography
purification (30% ethyl acetate-hexane) afforded 526 mg (39.2%) of
1-(4-methoxyphenyl)-5-(trifloromethyl)-2-pyridone. This compound
(268.2 mg, 1 mmol) was treated with 1M BBr.sub.3 solution in
dichloromethane (DCM, 2 ml) in DCM (5 ml) for 2 hours at 0.degree.
C. Reaction mixture was diluted with DCM and washed 3 times with
water. Organic phase was dried over sodium sulfate and evaporated.
The residue was separated by column chromatography (20% ethyl
acetate-DCM) to afford the title compound as a off-white solid, 226
mg (89%). The .sup.1H NMR spectra was consistent with the structure
of Compound 10.
Example 7
[0237] Preparation of 1-phenyl-5-acetyl-2-pyridone (Compound 16):
2-methoxy-5-acetyl pyridine (1.51 g, 10 mmol) was treated with 6N
HCl at 100.degree. C. for 5 hours. The reaction mixture was
neutralized with sodium hydroxide to pH 7 and then extracted
several times with DCM. Organic layer was dried over sodium
sulfate, evaporated and the residue was crystallized from ethyl
acetate to give 5-acetyl-2(1H)-pyridone as a white solid, 1.06 g
(78%). This compound (685.7 mg, 5 mmol) was reacted with
iodobenzene (0.84 ml, 7.5 mmol) in the presence of CuI (95 mg, 0.5
mmol) and K.sub.2CO.sub.3 (691 mg, 5 mmol) in DMF (5 ml) at
135.degree. C. overnight. The reaction mixture was diluted with 10%
ammonia (15 ml) and extracted with ethyl acetate. The organic
extract was washed with saturated sodium chloride, dried over
magnesium sulfate and evaporated. Column chromatography (10% ethyl
acetate-DCM) afforded 407 mg (38%) of the target compound as a
white solid. The .sup.1H NMR spectra was consistent with the
structure of Compound 16.
Example 8
[0238] Preparation of 1-(4-pyridinyl)-5-methyl-2-pyridone (Compound
22): Compound 22 was synthesized by condensation of
5-methyl-2(1H)-pyridone (327.4 mg, 3 mmol) with 4-bromopyridine
hydrochloride (778 mg, 4 mmol) in the presence of CuI (60 mg, 0.3
mmol) and K.sub.2CO.sub.3 (1.36 g, 10 mmol) in DMF (3 ml) at
135.degree. C. overnight. The reaction mixture was diluted with 10%
ammonia (15 ml) and extracted with ethyl acetate. Organic extract
was washed with saturated sodium chloride, dried over magnesium
sulfate and evaporated. Column chromatography (5% MeOH-DCM)
afforded 197 mg (35%) of the target compound as a yellowish solid.
The .sup.1H NMR spectra was consistent with the structure of
Compound 22.
Example 9
[0239] Preparation of 1-phenyl-5-methyl-2-pyridinethione (Compound
18): 1-phenyl-5-methyl-2-pyridinone (555.7 mg, 3 mmol) was reacted
with Lawesson's reagent (606.7 mg, 1.5 mmol) in toluene (5 ml) at
90.degree. C. Reaction mixture was evaporated and the target
compound was isolated by column chromatography (20-30% ethyl
acetate-hexane) followed by crystallization from methyl-tert-butyl
ether. Yield 403 mg (67%), yellow solid. The .sup.1H NMR spectra
was consistent with the structure of Compound 18.
Example 10
[0240] Compound 33 was prepared according to the following
synthetic scheme:
##STR00053##
[0241] A mixture of commercially available ethyl
3,3-diethoxypropionate (9.7 ml, 50 mmol), sodium hydroxide (10M, 6
ml, 60 mmol) and water (15 ml) was refluxed until homogenous
(approximately 30 min). After cooling to 0.degree. C. 6N
hydrochloric acid was added to bring pH of the solution to 2-3
(.about.10 ml). The mixture was extracted with dichloromethane,
organic phase was washed with water, dried over sodium sulfate, and
the solvent was removed under vacuum to give acid 1 which was used
without any additional purification.
[0242] To a solution of crude acid 1 (approximately 50 mmol) in DCM
(100 ml) at 0.degree. C. was sequentially added 4-bromoaniline
(10.3 g, 60 mmol), HOST (675 mg, 5 mmol) and finally DCC (12.4 g,
60 mmol). The reaction mixture was stirred at 0.degree. C. for 1
hour and then refluxed for another 4 hours. The solid was filtered
off, filtrate was washed with saturated sodium bicarbonate, dried
over sodium sulfate, and the solvent was removed under vacuum to
give amide 2 as slightly yellow solid. This solid was dissolved in
sulfuric acid (96%, 50 ml) at 0.degree. C. The solution was kept at
the same temperature for another 3 h and then poured into ice-water
(500 ml). The solid was filtered off, washed with water, and
stirred with hot acetonitrile (100 ml). Quinolinone 3 was filtered
off and dried under vacuum. The yield was 10 g (91%).
[0243] Mixture of compound 3 (309 mg, 1.38 mmol), phenylboronic
acid (336 mg, 2.76 mmol), Cu(OAc).sub.2 (36 mg, 0.2 mmol),
molecular sieves 4a (0.3 g), pyridine (0.24 ml) and DCM (10 ml) was
stirred at room temperature for 2 days. Reaction mixture was
filtered trough Celite, washed with saturated sodium bicarbonate
with EDTA and organic phase was dried over sodium sulfate. Compound
33 was isolated by chromatography (50%
ethylacetate-hexane-ethylacetate). The yield was 352 mg (85%).
Example 11
[0244] The pharmacokinetic (PK) properties of pirfenidone,
pirfenidone analogs and derivatives were assessed in dual canulated
(right jugular/left carotid) Sprague Dawley rats (Charles River
Laboratories, Inc., Wilmington, Mass.). Male rats weighing
approximately 275-300 g were administered an intravenous (5 mg/Kg)
or oral (50 mg/Kg via gavage) dose of compound in an appropriate
formulation. Plasma samples were collected via intra-arterial
canula at desired times in the 24 hours after dosing using EDTA as
an anticoagulant. Three animals were used for each compound. All
experiments were conducted by trained personnel in accordance with
guidelines of the appropriate Institutional Animal Care and Use
Committees (IACUC).
[0245] Compound concentrations were assessed by LC-MS using a MDS
SCIEX API 3000 mass spectrometer (Applied Biosystems, Foster City,
Calif.) coupled to a Shimadzu VP HPLC (Shimadzu Corp., Kyoto,
Japan) outfitted with a Duragel G C.sub.18 guard cartridge (Peeke
Scientific, Redwood City, Calif.). Calibration samples were
prepared by mixing known amounts of pirfenidone, or a pirfenidone
analog or derivative, with rat plasma. A standard curve was created
by serial dilution of the calibration sample in the same matrix.
Both standard and analytical samples were prepared for injection to
the HPLC by mixing an aliquot of plasma sample with 3 volumes of
ice cold acetonitrile containing internal standard. Samples were
then centrifuged. An aliquot of the resulting supernatant was then
mixed with five volumes of 0.2% formic acid in water, injected to
the HPLC, and resolved in a methanol gradient (containing 0.18-0.2%
formic acid). The integrated analyte signal was corrected for that
of the internal standard and compared to the appropriate standard
curve in order to define the analyte concentration.
[0246] The pharmacokinetic parameters shown in Table 4 were derived
using the WinNonlin Software package (Pharsight Corp, Mountain
View, Calif.).
TABLE-US-00004 TABLE 4 AUClast Route of Dose MRTinf Clobs ng-hr/mL
.times. Admin. mg/kg Compound T.sub.1/2 hr. hr mL/hr/kg 10.sup.6 E
% IV 5 8 13.9 20.3 0.117 30.2 19 2.96 6.26 0.142 35 26 1.62 2.4
0.691 10.3 Oral 50 8 122.4 40.5 19 191.7 54.8 26 65.8 63.9
[0247] While the present invention has been described in some
detail for purposes of clarity and understanding, one skilled in
the art will appreciate that various changes in form and detail can
be made without departing from the true scope of the invention. All
figures, tables, appendices, patents, patent applications and
publications, referred to above, are hereby incorporated by
reference in their entirety.
* * * * *