U.S. patent application number 11/860933 was filed with the patent office on 2008-06-26 for methods relating to the treatment of fibrosis.
This patent application is currently assigned to UNIVERSITY OF WASHINGTON. Invention is credited to Emil Y. Chi, William R. HENDERSON, Michael Kahn.
Application Number | 20080153743 11/860933 |
Document ID | / |
Family ID | 36698775 |
Filed Date | 2008-06-26 |
United States Patent
Application |
20080153743 |
Kind Code |
A1 |
HENDERSON; William R. ; et
al. |
June 26, 2008 |
METHODS RELATING TO THE TREATMENT OF FIBROSIS
Abstract
The invention provides .alpha.-helix mimetic structures of
formula (I) with the definitions of A, B, D, E, G, W, R.sub.1 and
R.sub.2 as set out in the description and a chemical library
relating thereto. The compounds, pharmaceutical compositions
comprising the compounds, and methods of the invention using the
compounds, relate to the reversal of fibrotic conditions, such as
pulmonary fibrosis. ##STR00001##
Inventors: |
HENDERSON; William R.;
(Seattle, WA) ; Chi; Emil Y.; (Seattle, WA)
; Kahn; Michael; (Kirkland, WA) |
Correspondence
Address: |
NIXON PEABODY LLP - PATENT GROUP
1100 CLINTON SQUARE
ROCHESTER
NY
14604
US
|
Assignee: |
UNIVERSITY OF WASHINGTON
Seattle
WA
INSTITUTE FOR CHEMICAL GENOMICS
Kirkland
WA
|
Family ID: |
36698775 |
Appl. No.: |
11/860933 |
Filed: |
September 25, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/US2006/009191 |
Mar 15, 2006 |
|
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11860933 |
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60663499 |
Mar 18, 2005 |
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Current U.S.
Class: |
514/183 ;
514/15.4; 514/17.8; 514/18.6; 514/20.8; 514/6.9 |
Current CPC
Class: |
A61P 15/00 20180101;
A61P 27/06 20180101; A61P 7/04 20180101; C07K 7/56 20130101; A61P
29/00 20180101; A61P 1/16 20180101; A61P 17/00 20180101; A61P 27/02
20180101; A61P 25/28 20180101; A61P 11/00 20180101; A61P 3/00
20180101; A61P 43/00 20180101; A61P 13/12 20180101; A61P 19/00
20180101 |
Class at
Publication: |
514/10 |
International
Class: |
A61K 38/12 20060101
A61K038/12; A61P 3/00 20060101 A61P003/00; A61P 11/00 20060101
A61P011/00; A61P 19/00 20060101 A61P019/00; A61P 17/00 20060101
A61P017/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under grant
R01 HL073722 awarded by the National Institutes of Health. The
government has certain rights in the invention.
[0003] The subject matter of this application was made with support
from the United States Government under The National Institutes of
Health, Grant No. R01 HL073722. The U.S. Government may have
certain rights.
Claims
1. A method of reversing a fibrotic condition in a mammal, said
method comprising administering to a mammal in need of said
treatment at least one compound having the following general
formula (I) or a stereoisomer thereof: ##STR00014## wherein A is
--(CHR.sub.3)--(C.dbd.O)--; B is --(NR.sub.4)--; D is
--(CHR.sub.5)-- or --(C.dbd.O)--; E is -(ZR.sub.6)-- or
--(C.dbd.O)--, where Z is nitrogen or CH; G is
--(XR.sub.7).sub.n--, --(CHR.sub.7)--(NR.sub.8)--,
--(C.dbd.O)--(XR.sub.9)--, or --(C.dbd.O)--, where X is nitrogen or
CH and n=0 or 1; W is --(Y)--(C.dbd.O)--, --(C.dbd.O)--(NH)--,
--(SO.sub.2)--, or nothing, where Y is oxygen or sulfur; and
R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7,
R.sub.8 and R.sub.9 are the same or different and independently
selected from an amino acid side chain, a derivative of an amino
acid side chain, a linker facilitating linkage of the compound to
another moiety or compound, a linker joining the compound to a
solid support, and a solid support; under conditions effective to
reverse a fibrotic condition in the mammal.
2. The method according to claim 1, wherein the fibrotic condition
is at least one selected from the group consisting of
radiation-induced pulmonary fibrosis, chemotherapy-induced
pulmonary fibrosis, obliterative bronchiolitis, and silicosis
lesions.
3. The method according to claim 1, wherein the fibrotic condition
is at least one of kidney disease, polycystic kidney disease, renal
fibrotic disease, glomerular nephritis, liver cirrhosis, nephritis
associated with systemic lupus, peritoneal fibrosis, liver
fibrosis, polycystic ovarian syndrome, myocardial fibrosis, Grave's
opthalmopathy, glaucoma, scarring, skin lesions, diabetic
retinopathy, scleroderma, and Alzheimer's disease.
4. The method according to claim 1, wherein the fibrotic condition
is at least one selected from the group consisting of interstitial
lung disease, fibrotic lung disease, and pulmonary fibrosis.
5. The method of claim 1, wherein the compound is administered
orally, transdermally, intravenously, by inhalation, or
rectally.
6. The method of claim 5, wherein the compound is administered
orally.
7. The method of claim 4, wherein the compound is administered in a
form selected from the group consisting of capsules, tablets,
powders, granules, syrups, injectable fluids, creams, ointments,
hydrophilic ointments, inhalable fluids, eye drops, and
suppositories.
8. The method according to claim 1, The method according to claim
1, wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6,
R.sub.7, R.sub.8 and R.sub.9 are independently selected from the
group consisting of aminoC.sub.2-5alkyl, guanidinoC.sub.2-5alkyl,
C.sub.1-4alkylguanidinoC.sub.2-5alkyl,
diC.sub.1-4alkylguanidino-C.sub.2-5alkyl, amidinoC.sub.2-5alkyl,
C.sub.1-4alkylamidinoC.sub.2-5alkyl,
diC.sub.1-4alkylamidinoC.sub.2-5alkyl, C.sub.1-3alkoxy, Phenyl,
substituted phenyl (where the substituents are independently
selected from one or more of amino, amidino, guanidino, hydrazino,
amidrazonyl, C.sub.1-4alkylamino, C.sub.1-4-dialkylamino, halogen,
perfluoro C.sub.1-4alkyl, C.sub.1-4alkyl, C.sub.1-3alkoxy, nitro,
carboxy, cyano, sulfuryl, and hydroxyl), benzyl, substituted benzyl
(where the substituents on the benzyl are independently selected
from one or more of amino, amidino, guanidino, hydrazino,
amidrazonyl, C.sub.1-4alkylamino, C.sub.1-4dialkylamino, halogen,
perfluoro C.sub.1-4alkyl, C.sub.1-4alkyl, C.sub.1-3alkoxy, nitro,
carboxy, cyano, sulfuryl, and hydroxyl), naphthyl, substituted
naphthyl (where the substituents are independently selected from
one or more of amino, amidino, guanidino, hydrazino, amidrazonyl,
C.sub.1-4alkylamino, C.sub.1-4dialkylamino, halogen, perfluoro
C.sub.1-4alkyl, C.sub.1-4alkyl, C.sub.1-3alkoxy, nitro, carboxy,
cyano, sulfuryl, and hydroxyl), bis-phenyl methyl, substituted
bis-phenyl methyl (where the substituents are independently
selected from one or more of amino, amidino, guanidino, hydrazino,
amidrazonyl, C.sub.1-4alkylamino, C.sub.1-4dialkylamino, halogen,
perfluoro C.sub.1-4alkyl, C.sub.1-4alkyl, C.sub.1-3alkoxy, nitro,
carboxy, cyano, sulfuryl, and hydroxyl), pyridyl, substituted
pyridyl (where the substituents are independently selected from one
or more of amino, amidino, guanidino, hydrazino, amidrazonyl,
C.sub.1-4alkylamino, C.sub.1-4dialkylamino, halogen, perfluoro
C.sub.1-4alkyl, C.sub.1-4alkyl, C.sub.1-3alkoxy, nitro, carboxy,
cyano, sulfuryl, and hydroxyl), pyridylC.sub.1-4alkyl, substituted
pyridylC.sub.1-4alkyl (where the pyridine substituents are
independently selected from one or more of amino, amidino,
guanidino, hydrazino, amidrazonyl, C.sub.1-4alkylamino,
C.sub.1-4dialkylamino, halogen, perfluoro C.sub.1-4alkyl,
C.sub.1-4alkyl, C.sub.1-3alkoxy, nitro, carboxy, cyano, sulfuryl,
and hydroxyl), pyrimidylC.sub.1-4alkyl, substituted
pyrimidylC.sub.1-4alkyl (where the pyrimidine substituents are
independently selected from one or more of amino, amidino,
guanidino, hydrazino, amidrazonyl, C.sub.1-4alkylamino,
C.sub.1-4dialkylamino, halogen, perfluoro C.sub.1-4alkyl,
C.sub.1-4alkyl, C.sub.1-3alkoxy, nitro, carboxy, cyano, sulfuryl,
and hydroxyl), triazin-2-yl-C.sub.1-4alkyl, substituted
triazin-2-yl-C.sub.1-4alkyl (where the triazine substituents are
independently selected from one or more of amino, amidino,
guanidino, hydrazino, amidrazonyl, C.sub.1-4alkylamino,
C.sub.1-4dialkylamino, halogen, perfluoro C.sub.1-4alkyl,
C.sub.1-4alkyl, C.sub.1-3alkoxy, nitro, carboxy, cyano, sulfuryl,
and hydroxyl), imidazoC.sub.1-4alkyl, substituted imidazol
C.sub.1-4alkyl (where the imidazole substituents are independently
selected from one or more of amino, amidino, guanidino, hydrazino,
amidrazonyl, C.sub.1-4alkylamino, C.sub.1-4dialkylamino, halogen,
perfluoro C.sub.1-4alkyl, C.sub.1-4alkyl, C.sub.1-3alkoxy, nitro,
carboxy, cyano, sulfuryl, and hydroxyl),
imidazolinylC.sub.1-4alkyl, N-amidinopiperazinyl-N--C.sub.0-4alkyl,
hydroxyC.sub.2-5alkyl, C.sub.1-5alkylaminoC.sub.2-5alkyl,
hydroxyC.sub.2-5alkyl, C.sub.1-5alkylaminoC.sub.2-5alkyl,
C.sub.1-5dialkylaminoC.sub.2-5alkyl,
N-amidinopiperidinylC.sub.1-4alkyl, and
4-aminocyclohexylCO.sub.2alkyl.
9. The method according to claim 1, wherein the compound has the
following general formula (III): ##STR00015## with the proviso that
when Z is CH, X is nitrogen.
10. The method according to claim 8, wherein the compound has the
formula (IV): ##STR00016## wherein when Z is nitrogen, n is zero;
and when Z is CH, X is nitrogen and n is 1, or X(n) is zero.
11. A method of reversing a fibrotic condition in a mammal, said
method comprising administering to a mammal in need of said
treatment at least one compound having the following general
formula (V) or a stereoisomer thereof: ##STR00017## under
conditions effective to reverse a fibrotic condition in the mammal.
Description
CROSS REFERENCE TO RELATED APPLICATION(S)
[0001] The present application is a continuation of PCT
International Patent Application No. PCT/US2006/009191, filed Mar.
15, 2006, which claims priority from U.S. Provisional Application
No. 60/663,499, filed Mar. 18, 2005, both of which are hereby
incorporated by reference in their entirety.
TECHNICAL FIELD
[0004] The present invention relates generally to .alpha.-helix
mimetic structures and to a chemical library relating thereto. The
invention also relates to applications in the treatment of diseases
and pharmaceutical compositions comprising them.
BACKGROUND OF THE INVENTION
[0005] Fibrosis can occur in the lung, liver, kidney, eye, hear and
other major organs of the body. Fibrosis can be due to toxic or
infectious injury, such as cigarette smoke to the lungs or viral
hepatitis infection of the liver. The cause of some fibrotic
diseases is unknown, which is the case with idiopathic pulmonary
fibrosis.
[0006] Idiopathic pulmonary fibrosis (IPF) is a chronic and
insidious inflammatory disease of the lung that kills most of its
victims within five years after diagnosis. IPF afflicts 83,000
Americans and more than 31,000 new cases develop each year. It is
believed that death due to IPF is greatly underreported and the
considerable morbidity of IPF is not recognized. IPF represents
just one of the many fibrotic diseases that occurs as a result of
chronic inflammation. It is estimated by the United States
government that 45% of all deaths in the U.S. can be attributed to
fibrotic disorders, and therapeutic agents are needed for treating
this condition, especially fibrotic disease of the lungs.
[0007] Pulmonary fibrosis leads to progressive scarring and lung
destruction. Currently, there are five million people worldwide
that are affected by pulmonary fibrosis with 50% mortality at 5
years after diagnosis (Katzenstein A and Meyers Am. J. Respir.
Crit. Care Med. 1998, 157, 130-1-15 and American Thoracic Society,
Am. J, Respir. Care Med. 2000, 161, 646, 664.). Pulmonary fibrosis
is believed to be initiated by insult to the lung parenchyma
(either acute or chronic) and develop in patients unable to
effectively heal the damage (Gross T. J. N. Eng. J Med 345, 517,
2001). The fibrosis is refractory to corticosteroids and no
effective therapy currently exists.
[0008] Random screening of molecules for possible activity as
therapeutic agents has occurred for many years and resulted in a
number of important drug discoveries. While advances in molecular
biology and computational chemistry have led to increased interest
in what has been termed "rational drug design", such techniques
have not proven as fast or reliable as initially predicted. Thus,
in recent years there has been a renewed interest and return to
random drug screening. To this end, particular strides having been
made in new technologies based on the development of combinatorial
chemistry libraries, and the screening of such libraries in search
for biologically active members.
[0009] In general, combinatorial chemistry libraries are simply a
collection of molecules. Such libraries vary by the chemical
species within the library, as well as the methods employed to both
generate the library members and identify which members interact
with biological targets of interest. While this field is still
young, methods for generating and screening libraries have already
become quite diverse and sophisticated. For example, a recent
review of various combinatorial chemical libraries has identified a
number of such techniques (Dolle, J. Com. Chem., 2(3): 383-433,
2000), including the use of both tagged and untagged library
members (Janda, Proc. Natl. Acad. Sci. USA 91:10779-10785,
1994).
[0010] Initially, combinatorial chemistry libraries were generally
limited to members of peptide or nucleotide origin. To this end,
the techniques of Houghten et al. illustrate an example of what is
termed a "dual-defined iterative" method to assemble soluble
combinatorial peptide libraries via split synthesis techniques
(Nature (London) 354:84-86, 1991; Biotechniques 13:412-421, 1992;
Bioorg. Med. Chem. Lett. 3:405-412, 1993). By this technique,
soluble peptide libraries containing tens of millions of members
have been obtained. Such libraries have been shown to be effective
in the identification of opioid peptides, such as methionine- and
leucine-enkephalin (Dolley and Houghten, Life Sci. 52, 1509-1517,
1993), and N-acylated peptide library has been used to identify
acetalins, which are potent opioid antagonists (Dooley et al.,
Proc. Natl. Acad. Sci. USA 90:10811-10815, 1993). More recently, an
all D-amino acid opioid peptide library has been constructed and
screened for analgesic activity against the mu (".mu.") opioid
receptor (Dooley et al., Science 266:2019-2022, 1994).
[0011] While combinatorial libraries containing members of peptide
and nucleotide origin are of significant value, there is still a
need in the art for libraries containing members of different
origin. For example, traditional peptide libraries to a large
extent merely vary the amino acid sequence to generate library
members. While it is well recognized that the secondary structures
of peptides are important to biological activity, such peptide
libraries do not impart a constrained secondary structure to its
library members.
[0012] To this end, some researchers have cyclized peptides with
disulfide bridges in an attempt to provide a more constrained
secondary structure (Tumelty et al., J. Chem. Soc. 1067-68, 1994;
Eichler et al., Peptide Res. 7:300-306, 1994). However, such
cyclized peptides are generally still quite flexible and are poorly
bioavailable, and thus have met with only limited success.
[0013] More recently, non-peptide compounds have been developed
which more closely mimic the secondary structure of reverse-turns
found in biologically active proteins or peptides. For example,
U.S. Pat. No. 5,440,013 to Kahn and published PCT WO94/03494, PCT
WO01/00210A1, and PCT WO01/16135A2 to Kahn disclose
conformationally constrained, non-peptidic compounds, which mimic
the three-dimensional structure of reverse-turns.
[0014] While significant advances have been made in the synthesis
and identification of conformationally constrained, reverse-turn
mimetics, there remains a need in the art for small molecules,
which mimic the secondary structure of peptides. There has been
also a need in the art for libraries containing such members, as
well as techniques for synthesizing and screening the library
members against targets of interest, particularly biological
targets, to identify bioactive library members. For example, U.S.
Pat. No. 5,929,237 and its continuation-in-part U.S. Pat. No.
6,013,458 to Kahn also discloses conformationally constrained
compounds which mimic the secondary structure of reverse-turn
regions of biologically active peptides and proteins. The synthesis
and identification of conformationally constrained .alpha.-helix
mimetics and their application to diseases are discussed in
Walensky, L. D. et al Science 305, 1466, 2004; Klein, C. Br. J.
Cancer. 91:1415, 2004.
[0015] Many models of pulmonary fibrosis have been developed,
however regardless of the nature of the initial insult the stages
of progression appear to be quite similar. A generally accepted
model involves damage to the endothelial and type I alveolar
epithelial cells followed by interstitial edema, deposition of
fibrous materials in the alveolus in areas of loss of type I
epithelial cells. It is believed that limited proliferation of the
type II cells and subsequent differentiation into type I and Clara
cells is critical to reestablishment of normal gas exchange.
[0016] Anti-inflammatory therapies (e.g. corticosteroids,
interferon-.gamma.) to treat pulmonary fibrosis have been
disappointing to date due to limited efficacy and severe adverse
side effects. An important unmet need exists to identify the key
molecular pathways involved in the development and progression of
pulmonary fibrotic diseases and to develop new therapeutic agents
to prevent the progression and reverse the disease process. No
drugs have been approved for the treatment of any fibrotic disease
in the United States. Research and development is desperately
needed to provide treatments to those afflicted with
fibroproliferative diseases. The present invention fulfills these
needs, and provides further related advantages by providing
conformationally constrained compounds which mimic the secondary
structure of .alpha.-helix regions of biologically active peptides
and proteins.
SUMMARY OF THE INVENTION
[0017] In brief, the present invention is directed to
conformationally constrained compounds, which mimic the secondary
structure of .alpha.-helix regions of biologically active peptides
and proteins and their use for treating fibrosis, such as pulmonary
fibrosis. This invention also discloses libraries containing such
compounds, as well as the synthesis and screening thereof.
[0018] The compounds of the present invention have the following
general formula (I):
##STR00002##
Wherein A is --(C.dbd.O)--CHR.sup.3--, B is N--R.sub.4--, D is
--(C.dbd.O)--(CHR.sub.5)-- or --(C.dbd.O)--, E is -(ZR.sub.6)-- or
(C.dbd.O), G is (XR.sub.7).sub.n--, --(CHR.sub.7)--(NR.sub.8)--,
--(C.dbd.O)--(XR.sub.9)--, or --(C.dbd.O)--, W is --Y(C.dbd.O)--,
--(C.dbd.O)NH--, --(SO.sub.2)-- or nothing, Y is oxygen or sulfur,
X and Z is independently nitrogen or CH, n=0 or 1; and R.sub.1,
R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, and
R.sub.9 are the same or different and independently selected from
an amino acid side chain moiety or derivative thereof, the
remainder of the molecule, a linker and a solid support, and
stereoisomers thereof.
[0019] In the embodiment wherein A is --(C.dbd.O)--CHR.sup.3--, B
is --(NR.sub.4)--, D is --(C.dbd.O)--, E is -(ZR.sub.6)--, and G is
--(C.dbd.O)--(XR.sub.9)--, the compounds of this invention have the
following formula (III):
##STR00003##
Wherein W, Y and n are as defined above, Z is nitrogen or CH (when
Z is CH, then X is nitrogen), and R.sub.1, R.sub.2, R.sub.3
R.sub.4, R.sub.6, and R.sub.9 are as defined in the following
detailed description.
[0020] In the embodiment wherein A is --(C.dbd.O)--(CHR.sub.3), B
is --(CHR.sub.4)--, D is --(C.dbd.O)--, E is -(ZR.sub.6)--, and G
is (XR.sub.7).sub.n--, the compounds of this invention have the
following general formula (IV):
##STR00004##
Wherein W, Y and n are as defined above, Z is nitrogen or CH (when
Z is nitrogen, then n is zero, and when Z is CH, then X is nitrogen
and n is not zero), and R.sub.1, R.sub.2, R.sub.4, R.sub.6, and
R.sub.7, are as defined in the following detailed description.
[0021] The present invention is also directed to libraries
containing compounds of formula (I) above, as well as methods for
synthesizing such libraries and methods for screening the same to
identify biologically active compounds. Compositions containing a
compound of this invention in combination with a pharmaceutically
acceptable carrier or diluent are also disclosed.
[0022] Especially, the present invention relates pharmaceutical
compositions containing compounds of formula (I) for treating
disorders including fibrosis of the lung. It further relates to
methods for treating disorders including fibrosis of the lung which
are associated with TGF-.beta. signaling pathway.
[0023] The compound V (ICG-001) is useful for treating fibrosis as
described in Example 1.
[0024] These and other aspects of this invention will be apparent
upon reference to the attached figures and following detailed
description. To this end, various references are set forth herein,
which describe in more detail certain procedures, compounds and/or
compositions, and are incorporated by reference in their
entirety.
BRIEF DESCRIPTION OF THE FIGURES
[0025] FIG. 1 depicts lung sections taken from Bat-Gal transgenic
mice given intratracheal saline or bleomycin and either treated
with ICG-001 (5 mgs/Kg/day subcutaneously) or saline as vehicle
control. The lungs were sectioned and stained with X-Gal (blue
color.) FIG. 1A) intratracheal bleo+saline; FIG. 1B) intratracheal
bleo+ICG-001; FIG. 1 C) saline+saline.
[0026] FIG. 2 depicts lung sections taken from C57/B16 mice treated
with intratracheal bleomeycin (lower left) or saline (upper left)
for 5 days and stained with trichrome (red color) to stain
collagen.
[0027] FIG. 3 shows RT-PCR data for S100A4 and collagen1A2, which
are increased in the bleomycin treated mice (treated with saline
control). Message is reduced essentially to negative control (i.e.
saline/saline mice) levels by ICG-001 treatment (5 mgs/Kg/day
s.c.)
[0028] FIG. 4 shows the results of IPF patient fibroblasts after
culture in RPMI1640+10% FBS for 2 days and treatment with ICG-001.
Western blots for S100A4 (also known as FSP-1 or fibroblast
specific protein-1) and E-Cadherin were performed on whole cell
lysates. ICG-001 decreased S100A4 expression and increased
E-cadherin expression.
DETAILED DESCRIPTION OF THE INVENTION
[0029] Transforming growth factor .beta. (TGF.beta.), a key
mediator in the development of fibrosis, is important in cell
proliferation and differentiation, apoptosis, and deposition of
extracellular matrix (ECM). TGF.beta. signaling activates both the
Smad and AP-1 transcription pathways. TGF.beta. in the airways of
patients with pulmonary fibrosis (PF) may function initially as a
"healing molecule" involved in the diminution of initial airway
inflammation and in tissue repair. However, with continued
inflammatory response such as may occur in PF, the balance may be
shifted, to excessive ECM deposition and development of airway
fibrosis.
[0030] Fibroproliferative diseases are generally caused by the
activation of resident stellate cells which are found in most
organs. This activation of stellate cells leads to their conversion
to myofibroblasts which display characteristics of muscle and
non-muscle cells. Activated stellate cells initiate inflammatory
signals, principally mediated through TGF-.beta.. Inflammatory
cytokines and mediators in addition to TGF-.beta., lead to
proliferation of myofibroblasts. Stellate-derived myofibroblasts
proliferate and replace healthy, functional organ cells with
extra-cellular matrix that exhibit muscle and connective tissue
traits. Ultimately, organ failure results when the nonfunctional
fibrotic honeycomb matrix replaces a critical number of healthy
cells.
[0031] The initial cause of fibrosis is believed to be the result
of injury or insult to organ tissues. This cellular injury to organ
tissues can often be traced to toxic or infectious agents.
Pulmonary fibrosis, or interstitial lung disease, is often the
result of smoking, chronic asthma, chronic obstructive pulmonary
disease (COPD) or pneumonia.
[0032] Pulmonary fibrosis destroys the lung's ability to transport
oxygen and other gases into or out of the blood. This disease
modifies the delicate and elastic tissues of the lung, changing
these tissues into thicker, stiff fibrous tissue. This change or
replacement of the original tissue is similar to the permanent
scarring that can occur to other damaged tissues. Scarring of the
lung reduces the lung's ability to allow gases to pass into or out
of the blood (i.e. oxygen, carbon dioxide). Gradually, the air sacs
of the lungs become replaced by fibrotic tissue. When the scar
forms, the tissue becomes thicker causing an irreversible loss of
the tissue's ability to transfer oxygen into the bloodstream.
Symptoms include shortness of breath, particularly with exertion;
chronic dry, hacking cough; fatigue and weakness; discomfort in the
chest; loss of appetite; and rapid weight loss.
[0033] Several causes of pulmonary fibrosis are known and they
include occupational and environmental exposures. Many jobs,
particularly those that involve mining or that expose workers to
asbestos or metal dusts, can cause pulmonary fibrosis. Workers
doing these kinds of jobs may inhale small particles (like silica
dusts or asbestos fibers) that can damage the lungs, especially the
small airways and air sacs, and cause the scarring associated with
fibrosis. Agricultural workers also can be affected. Some organic
substances, such as moldy hay, cause an allergic reaction in the
lung. This reaction is called Farmer's Lung and can cause pulmonary
fibrosis. Other fumes found on farms are directly toxic to the
lungs.
[0034] Another cause is Sarcoidosis, a disease characterized by the
formation of granulomas (areas of inflammatory cells), which can
attack any area of the body but most frequently affects the lungs.
Certain medicines may have the undesirable side effect of causing
pulmonary fibrosis, as can radiation, such as treatment for breast
cancer. Connective tissue or collagen diseases such as rheumatoid
arthritis and systemic sclerosis are also associated with pulmonary
fibrosis. Although genetic and familial factors may be involved,
this cause is not as common as the other causes discussed above. In
Chronic Obstructive Pulmonary Disease (COPD), connective tissue
proliferation and fibrosis can characterize severe COPD. COPD can
develop as a result of smoking or chronic asthma.
[0035] When all known causes of interstitial lung disease have been
ruled out, the condition is called "idiopathic" (of unknown origin)
pulmonary fibrosis (IPF). Over 83,000 Americans are living with
IPF, and more than 31,000 new cases develop each year. This
debilitating condition involves scarring of the lungs. The lungs'
air sacs develop scar, or fibrotic tissue, which gradually
interferes with the body's ability to transfer the oxygen into the
bloodstream, preventing vital organs and tissue from obtaining
enough oxygen to function normally.
[0036] There are several theories as to what may cause IPF,
including viral illness and allergic or environmental exposure
(including tobacco smoke). These theories are still being
researched. Bacteria and other microorganisms are not thought to be
the cause of IPF. There is also a familial form of the disease,
known as familial idiopathic pulmonary fibrosis. Additional
research is being done to determine whether there is a genetic
tendency to develop the disease, as well as to determine other
causes of EPF.
[0037] Patients with IPF suffer similar symptoms to those with
pulmonary fibrosis when their lungs lose the ability to transfer
oxygen into the bloodstream. The symptoms include shortness of
breath, particularly during or after physical activity; spasmodic,
dry cough; gradual, unintended weight loss; fatigue and weakness;
chest discomfort; clubbing, or enlargement of the ends of the
fingers (or sometimes the toes) due to a buildup of tissue. These
symptoms can greatly reduce IPF patients' quality of life.
Pulmonary rehabilitation, and oxygen therapy can reduce the
lifestyle-altering effects of IPF, but do not provide a cure.
[0038] Other mammalian fibrotic diseases that are amenable to
treatment according to the invention include kidney disease,
polycystic kidney disease, renal fibrotic disease, glomerular
nephritis, liver cirrhosis, nephritis associated with systemic
lupus, peritoneal fibrosis, liver fibrosis, polycystic ovarian
syndrome, myocardial fibrosis, Grave's opthalmopathy, glaucoma,
scarring, skin lesions, diabetic retinopathy, scleroderma, and
Alzheimer's disease.
[0039] In order to develop a treatment for fibrotic disease, it is
important to focus on the common pathway to the ultimate pathology
that is shared by the disease states, regardless of cause or of
tissue in which it is manifested. .beta.-catenin plays a role in
the development of fibrosis, and compounds that modulate this
pathway are useful for treating fibrosis.
[0040] Wnt signaling plays an essential role in both the
development and maintenance of multiple organ systems including the
brain, intestines, skin and lung. A number of Wnt genes including
Wnt2, Wnt5a, Wnt7b, Wnt11 and Wnt13 are expressed both in the
developing and adult lung (Morrisey E. 2003, Am. J. Pathology, 162,
1393-7). In both epithelial (type 2 pneumocytes) and mesenchymal
(myofibroblasts) cells, accumulation of nuclear .beta.-catenin, a
hallmark of activated Wnt signaling has been observed (Chilosi et
al 2003, Am J. Pathology 162, 1495-1502). Importantly, increased
proliferation of type 2 cells in IPF has been observed (Kawanami O
et al. Lab Invest 1982, 46, 39-53 and Kasper M et al. Histol.
Histopathol 1996, 11, 463-83). Furthermore, activation of Wnt
signaling in the adjacent mesenchyme may further prevent the proper
differentiation of the alveolar epithelium.
[0041] The well established bleomycin induced model of pulmonary
fibrosis in transgenic Bat-Gal mice was used herein to demonstrate
that aberrant activation of Wnt signaling in the lungs is induced
after insult. Furthermore, utilizing a specific inhibitor of
Wnt/.beta.-catenin/CBP driven transcription (ICG-001, 5 mg/Kg/day
s.c.) Wnt/.beta.-catenin was inhibited by >95% as judged by
.beta.-galactogidase activity. ICG-001 is among the structures
described in detail below.
[0042] The Wnt/.beta.-catenin pathway initiates a signaling cascade
critical in normal development of many organ systems including the
lung (Morrisey E 2003 Am J Pathology, 162, 1393-7). The hallmark of
this pathway is that it activates the transcriptional role of the
multifunctional protein .beta.-catenin. Canonical Wnt signaling
inactivates GSK-3.beta., preventing .beta.-catenin phosphorylation.
This leads to accumulation of .beta.-catenin in the cytoplasm and
subsequent translocation to the nucleus (Behrens J, 2000, Ann. NY
Acad. Sci. 910, 21-33.). A key step in the activation of target
genes is the formation of a complex between .beta.-catenin and
members of the T-cell factor (TCF)/lymphoid enhancer factor (LEF-1)
family of transcription factors. To generate a transcriptionally
active complex, .beta.-catenin recruits the transcriptional
coactivators, Creb-Binding Protein (CBP) or its closely related
homolog, p300 as well as other components of the basal
transcription machinery.
[0043] Previously, aberrant Wnt/.beta.-catenin signaling has been
demonstrated in lung samples from patients with idiopathic
pulmonary fibrosis (IPF) (Chilosi M. et al 2003 Am. J. Pathol. 162,
1495-1502) with increased nuclear .beta.-catenin immunoreactivity
and increased expression of two TCF/.beta.-catenin regulated genes
i.e. cyclin D1 and matrilysin (MMP7). An important role for
increased MMP7 activity in pulmonary fibrotic disease is expected
as MMP7 (-/-) mice are protected from bleomycin-induced pulmonary
fibrosis (Zuo F et al PNAS 99, 6292, 2002).
[0044] ICG-001, a small molecule (FW 548) that selectively inhibits
TCF/.beta.-catenin transcription in a CBP-dependent fashion, was
recently identified (Emami et al. Proc. Natl. Acad. Sci. USA 2004,
101, 12682-7 and McMillan and Kahn Drug Discovery Today 2005, 10,
1467-74). ICG-001 selectively blocks the .beta.-catenin/CBP
interaction without interfering with the highly homologous
.beta.-catenin/p300 interaction. Using a well established murine
model of pulmonary fibrosis in transgenic Bat-Gal mice, we now
demonstrate that ICG-001 (5 mg/Kg/day) blocks >95% of
bleomycin-induced TCF/.beta.-catenin transcription. Furthermore,
ICG-001 at this dose not only halts but reverses disease
progression, as judged by reduced mortality, histopathology and
endogenous gene expression. Given the fact that currently no
effective treatments for pulmonary fibrotic disease exist
inhibition of Wnt/.beta.catenin/CBP dependent transcription
according to the invention appears to offer a novel therapeutic
approach and provides industrial applicability.
[0045] Canonical Wnt signaling has been shown to promote
self-renewal in a variety of tissue stem cells, including neuronal
stem cells and hematopoeitic stem cells. However, activation of the
canonical Wnt pathway can promote or inhibit differentiation
depending on the experimental circumstances.
[0046] The present invention therefore is directed to
conformationally constrained compounds which mimic the secondary
structure of .alpha.-helix regions of biological peptide and
proteins (also referred to herein as ".alpha.-helix mimetics" and
chemical libraries relating thereto. Such compounds find use in
treating fibrosis, including pulmonary fibrosis.
[0047] The .alpha.-helix mimetic structures of the present
invention are useful as bioactive agents, including (but not
limited to) use as diagnostic, prophylactic and/or therapeutic
agents. The .alpha.-helix mimetic structure libraries of this
invention are useful in the identification of such bioactive
agents. In the practice of the present invention, the libraries may
contain from tens to hundreds to thousands (or greater) of
individual .alpha.-helix structures (also referred to herein as
"members").
[0048] In one aspect of the present invention, a .alpha.-helix
mimetic structure is disclosed having the following formula
(I):
##STR00005##
wherein A is --(C.dbd.O)--(CHR.sub.3)--, B is --N--R.sub.4--, D is
--(CHR.sub.5)-- or --(C.dbd.O), E is -(ZR.sub.6)-- or
--(C.dbd.O)--, G is --(XR.sub.7).sub.n--,
--(CHR.sub.7)--(NR.sub.8)--, --(C.dbd.O)--(XR.sub.9)--, or
--(C.dbd.O), W is --Y(C.dbd.O)--, --(C.dbd.O)NH--, --(SO.sub.2)--
or nothing, Y is oxygen or sulfur, X and Z is independently
nitrogen or CH, n=0 or 1; and R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6, R.sub.7, R.sub.8 and R.sub.9, are the same or
different and independently selected from an amino acid side chain
moiety or derivative thereof, the remainder of the molecule, a
linker and a solid support, and stereoisomers thereof.
[0049] More specifically, R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6, R.sub.7, R.sub.8 and R.sub.9, are independently
selected from the group consisting of aminoC.sub.2-5alkyl,
guanidineC.sub.2-5alkyl, C.sub.1-4alkylguanidinoC.sub.2-5alkyl,
diC.sub.1-4alkylguanidino-C.sub.2-5alkyl, amidinoC.sub.2-5alkyl
C.sub.1-4alkylamidino C.sub.2-5alkyl,
diC.sub.1-4alkylamidinoC.sub.2-5alkyl, C.sub.1-3alkoxy, Phenyl,
substituted phenyl (where the substituents are independently
selected from one or more of amino, amidino, guanidino, hydrazino,
amidrazonyl, C.sub.1-4alkylamino, C.sub.1-4dialkylamino, halogen,
perfluoro C.sub.1-4alkyl, C.sub.1-4alkyl, C.sub.1-3alkoxy, nitro,
carboxy, cyano, sulfuryl or hydroxyl), benzyl, substituted benzyl
(where the substituents on the benzyl are independently selected
from one or more of amino, amidino, guanidino, hydrazino,
amidrazonyl, C.sub.1-4alkylamino, C.sub.1-4dialkylamino, halogen,
perfluoro C.sub.1-4alkyl, C.sub.1-3alkyl, nitro, carboxy, cyano,
sulfuryl or hydroxyl), naphthyl, substituted naphthyl (where the
substituents are independently selected from one or more of amino,
amidino, guanidino, hydrazino, amidrazonyl, C.sub.1-4alkylamino,
C.sub.1-4dialkylamino, halogen, perfluoro C.sub.1-4alkyl,
C.sub.1-4alkyl, C.sub.1-3alkoxy, nitro, carboxy, cyano, sulfuryl or
hydroxyl), bisphenyl methyl, substituted bis-phenyl methyl (where
the substituents are independently selected from one or more of
amino, amidino, guanidino, hydrazino, amidrazonyl,
C.sub.1-4alkylamino, C.sub.1-4dialkylamino, halogen, perfluoro
C.sub.1-4alkyl, C.sub.1-4alkyl, C.sub.1-3alkoxy, nitro, carboxy,
cyano, sulfuryl or hydroxyl), pyridyl, substituted pyridyl, (where
the substituents are independently selected from one or more of
amino, amidino, guanidino, hydrazino, amidrazonyl,
C.sub.1-4alkylamino, C.sub.1-4dialkylamino, halogen, perfluoro
C.sub.1-4alkyl, C.sub.1-4alkyl, C.sub.1-3alkoxy, nitro, carboxy,
cyano, sulfuryl or hydroxyl), pyridylC.sub.1-4alkyl, substituted
pyridylC1-.sub.4-alkyl (where the pyridine substituents are
independently selected from one or more of amino, amidino,
guanidino, hydrazino, amidrazonyl, C.sub.1-4alkylamino,
C.sub.1-4dialkylamino, halogen, perfluoro C.sub.1-4alkyl,
C.sub.1-4alkyl, C.sub.1-3alkoxy, nitro, carboxy, cyano, sulfuryl or
hydroxyl), pyrimidylC.sub.1-4alkyl, substituted
pyrimidylC.sub.1-4alkyl (where the pyrimidine substituents are
independently selected from one or more of amino, amidino,
guanidino, hydrazino, amidrazonyl, C.sub.1-4alkylamino,
C.sub.1-4dialkylamino, halogen, perfluoro C.sub.1-4alkyl,
C.sub.1-4alkyl, C.sub.1-3alkoxy, nitro, carboxy, cyano, sulfuryl or
hydroxyl), triazin-2-yl-C.sub.1-4alkyl, substituted
triazin-2-yl-C.sub.1-4alkyl (where the triazine substituents are
independently selected from one or more of amino, amidino,
guanidino, hydrazino, amidrazonyl, C.sub.1-4alkylamino,
C.sub.1-4dialkylamino, halogen, perfluoro C.sub.1-4alkyl,
C.sub.1-4alkyl, C.sub.1-3alkoxy, nitro, carboxy, cyano, sulfuryl or
hydroxyl), imidazoC.sub.1-4alkyl, substituted imidazol
C.sub.1-4alkyl (where the imidazole substituents are independently
selected from one or more of amino, amidino, guanidino, hydrazino,
amidrazonyl, C.sub.1-4alkylamino, C.sub.1-4dialkylamino, halogen,
perfluoro C.sub.1-4alkyl, C.sub.1-4alkyl, C.sub.1-3alkoxy, nitro,
carboxy, cyano, sulfuryl or hydroxyl), imidazolinylCalkyl,
N-amidinopiperazinyl-N--C.sub.0-4alkyl, hydroxyC.sub.2-5alkyl,
C.sub.1-5alkylaminoC.sub.2-5alkyl, hydroxyC.sub.2-5alkyl,
C.sub.1-5alkylaminOC.sub.2-5alkyl,
C.sub.1-5dialkylaminoC.sub.2-5alkyl,
N-amidinopiperidinylC.sub.1-4alkyl and
4-aminocyclohexylC.sub.0-2alkyl.
[0050] In one embodiment, R.sub.1, R.sub.2, R.sub.6 of E, and
R.sub.7, R.sub.8 and R.sub.9 of G are the same or different and
represent the remainder of the compound, and R.sub.3 or A, R.sub.4
of B or R.sub.5 of D is selected from an amino acid side chain
moiety or derivative thereof. As used herein, the term "remainder
of the compound" means any moiety, agent, compound, support,
molecule, linker, amino acid, peptide or protein covalently
attached to the .alpha.-helix mimetic structure at R.sub.1,
R.sub.2, R.sub.5, R.sub.6, R.sub.7, R.sub.8 and/or R.sub.9
positions. This term also includes amino acid side chain moieties
and derivatives thereof.
[0051] As used herein, the term "amino acid side chain moiety"
represents any amino acid side chain moiety present in naturally
occurring proteins including (but not limited to) the naturally
occurring amino acid side chain moieties identified in Table 1.
Other naturally occurring amino acid side chain moieties of this
invention include (but are not limited to) the side chain moieties
of 3,5-dibromotyrosine, 3,5-diiodotyrosine, hydroxylysine,
.gamma.-carboxyglutamate, phosphotyrosine and phosphoserine. In
addition, glycosylated amino acid side chains may also be used in
the practice of this invention, including (but not limited to)
glycosylated threonine, serine and asparagine.
TABLE-US-00001 TABLE 1 Amino Acid Side Chain Moieties Amino Acid
Side Chain Moiety Amino Acid --H Glycine --CH.sub.3 Alanine
--CH(CH.sub.3).sub.2 Valine --CH.sub.2CH(CH.sub.3).sub.2 Leucine
--CH(CH.sub.3)CH.sub.2CH.sub.3 Isoleucine
--(CH.sub.2).sub.4NH.sub.3.sup.+ Lysine
--(CH.sub.2).sub.3NHC(NH.sub.2)NH.sub.2.sup.+ Arginine Histidine
--CH.sub.2COO.sup.- Aspartic acid --CH.sub.2CH.sub.2COO.sup.-
Glutamic acid --CH.sub.2CONH.sub.2 Asparagine
--CH.sub.2CH.sub.2CONH.sub.2 Glutamine Phenylalanine Tyrosine
Tryptophan --CH.sub.2SH Cysteine --CH.sub.2CH.sub.2SCH.sub.3
Methionine --CH.sub.2OH Serine --CH(OH)CH.sub.3 Threonine Proline
Hydroxyproline
[0052] In addition to naturally occurring amino acid side chain
moieties, the amino acid side chain moieties of the present
invention also include various derivatives thereof. As used herein,
a "derivative" of an amino acid side chain moiety includes
modifications and/or variations to naturally occurring amino acid
side chain moieties. For example, the amino acid side chain
moieties of alanine, valine, leucine, isoleucine and pheylalanine
may generally be classified as lower chain alkyl, aryl, or
arylalkyl moieties. Derivatives of amino acid side chain moieties
include other straight chain or branched, cyclic or noncyclic,
substitutes or unsubstituted, saturated or unsaturated lower chain
alkyl, aryl or arylalkyl moieties.
[0053] As used herein, "lower chain alkyl moieties" contain from
1-12 carbon atoms, "lower chain aryl moieties" contain from 6-12
carbon atoms and "lower chain aralkyl moieties" contain from 7-12
carbon atoms. Thus, in one embodiment, the amino acid side chain
derivative is selected from a C.sub.1-12 alkyl a C.sub.6-12 aryl
and a C.sub.7-12 arylalkyl, and in a more preferred embodiment,
from a C.sub.1-7 alkyl, a C.sub.6-10 aryl and a C.sub.7-11
arylalkyl.
[0054] Amino side chain derivatives of this invention further
include substituted derivatives of lower chain alkyl, aryl, and
arylalkyl moieties, wherein the substituents is selected from (but
are not limited to) one or more of the following chemical moieties:
--OH, --OR, --COOH, --COOR, --CONH.sub.2, --NH.sub.2, --NHR, --NRR,
--SR, --SR, --SO.sub.2R, --SO.sub.2H, --SOR and halogen (including
F, Cl, Br and I), wherein each occurrence of R is independently
selected from straight chain or branched, cyclic or noncyclic,
substituted or unsubstituted, saturated or unsaturated lower chain
alkyl, aryl, and aralkyl moieties. Moreover, cyclic lower chain
alkyl, aryl and arylalkyl moieties of this invention include
naphthalene, as well as heterocyclic compounds such as thiophene,
pyrrole, furan, imidazole, oxazole, thiazole, pyrazole,
3-pyrroline, pyrrolidine, pyridine, pyrimidine, purine, quinoline,
isoquinoline and carbazole. Amino acid side chain derivatives fuher
include heteroalkyl derivatives of the alkyl portion of the lower
chain alkyl and aralkyl moieties, including (but not limited to)
alkyl and aralkyl phosphonates and silanes.
[0055] Representative R.sub.1, R.sub.2, R.sub.5, R.sub.6, R.sub.7,
R.sub.8 and R.sub.9 moieties specifically include (but are not
limited to) --OH, --OR, --COR, --COOR, --CONH.sub.2, --CONR,
--CONRR, --NH.sub.2, --NHR, --NRR, --SO.sub.2R and --COSR, wherein
each occurrence of R is as defined above.
[0056] In a further embodiment, and in addition to being an amino
acid side chain moiety or derivative thereof (or the remainder of
the compound in the case of R.sub.1, R.sub.2, R.sub.5, R.sub.6,
R.sub.7, R.sub.8 and R.sub.9), R.sub.1, R.sub.2, R.sub.5, R.sub.6,
R.sub.7, R.sub.8 or R.sub.9 may be a linker facilitating the
linkage of the compound to another moiety or compound. For example,
the compounds of this invention may be linked to one or more known
compounds, such as biotin, for use in diagnostic or screening
assay. Furthermore, R.sub.1, R.sub.2, R.sub.5, R.sub.6, R.sub.7,
R.sub.8 or R.sub.9 may be a linker joining the compound to a solid
support (such as a support used in solid phase peptide synthesis)
or alternatively, may be the support itself. In this embodiment,
linkage to another moiety or compound, or to a solid support is
preferable at the R.sub.1, R.sub.2, R.sub.7 or R.sub.8 position,
and more preferably at the R.sub.1 or R.sub.2 position.
[0057] In the embodiment wherein A is --(C.dbd.O)--CHR.sub.3--, B
is --N--R.sub.4, D is --(C.dbd.O)--, E is -(ZR)--, G is
--(C.dbd.O)--(XR.sub.9), the .alpha.-helix mimetic compounds of
this invention have the following general formula (III):
##STR00006##
wherein R.sub.1, R.sub.2, R.sub.4, R.sub.6, R.sub.9, W and X are as
defined above, Z is nitrogen or CH (when Z is CH, then X is
nitrogen). In a preferred embodiment, R.sub.1, R.sub.2, R.sub.6,
and R.sub.9 represent the remainder of the compound, and R.sub.4 is
selected from an amino acid side chain moiety. In a more specific
embodiment wherein A is --O--CHR.sub.3--, B is --NR.sub.4--, D is
--(C.dbd.O), E is -(ZR.sub.6)--, Gi is (XR.sub.7).sub.n--, the
.alpha.-helix mimetic compounds of this invention have the
following formula (IV):
##STR00007##
wherein R.sub.1, R.sub.2, R.sub.4, R.sub.6, R.sub.7, W, X and n are
as defined above, and Z is nitrogen or CH (when Z is nitrogen, then
n is zero, and when Z is CH, then X is nitrogen and n is not zero).
In a preferred embodiment R.sub.1, R.sub.2, R.sub.6, and R.sub.7
represent the remainder of the compound, and R.sub.4 is selected
from an amino acid side chain moiety. In this case, R.sub.6 or
R.sub.7 may be selected from an amino acid side chain moiety when Z
and X are CH, respectively.
[0058] The .alpha.-helix mimetic structures of the present
invention may be prepared by utilizing appropriate starting
component molecules (hereinafter referred to as "component
pieces"). Briefly, in the synthesis of .alpha.-helix mimetic
structures having formula (II), first and second component pieces
are coupled to form a combined first-second intermediate, if
necessary, third and/or fourth component pieces are coupled to form
a combined third-fourth intermediate (or, if commercially
available, a single third intermediate may be used), the combined
first-second intermediate and third-fourth intermediate (or third
intermediate) are then coupled to provide a
first-second-third-fourth intermediate (or first-second-third
intermediate) which is cyclized to yield the .alpha.-helix mimetic
structures of this invention. Alternatively, the .alpha.-helix
mimetic structures of formula (II) may be prepared by sequential
coupling of the individual component pieces either stepwise in
solution or by solid phase synthesis as commonly practiced in solid
phase peptide synthesis.
[0059] Within the context of the present invention, a "first
component piece" has the following formula S1
##STR00008##
Wherein R.sub.2 as defined above, and R is a protective group
suitable for use in peptide synthesis. Suitable R groups include
alkyl groups and, in a preferred embodiment, R is a methyl group.
Such first component pieces may be readily synthesized by reductive
amination or substitution reaction by displacement of
H.sub.2N--R.sub.2 from CH(OR).sub.2--CHO or
CH(OR).sub.2--CH.sub.2-Hal (wherein Hal means a halogen atom).
[0060] A "second component piece" of this invention has the
following formula S2:
##STR00009##
Where L.sub.1 is carboxyl-activation group such as halogen atom,
R.sub.3, R.sub.4 is as defined above, and P is an amino protective
group suitable for use in peptide synthesis. Preferred protective
groups include t-butyl dimethylsilyl (TBDMS), t-Butyloxycarbonyl
(BOC), Methylosycarbonyl (MOC), 9H-Fluorenylmethyloxycarbonyl
(FMOC), and allyloxycarbonyl (Alloc). When L is --C(O)NHR; --NHR
may be a carboxyl protective group. N-hydrazino amino acids can be
readily prepared according to the procedures of Vidal et al.
(Tetrahedron Letters 39:8845-8848, 1998). The conversion of these
compounds to the second component pieces of this invention may be
readily achieved by activation of the carboxylic acid group of the
N-protected hydrazine-amino acid. Suitable activated carboxylic
acid groups include acid halides where X is a halide such as
chloride or bromide, acid anhydrides where X is an acyl group such
as acetyl, reactive esters such as an N-hydroxysuccinimide esters
and pentafluorophenyl esters, and other activated intermediates
such as the active intermediate formed in a coupling reaction using
a carbodiimide such as dicyclohexylcarbodiimide (DCC).
[0061] A "third component piece" of this invention has the
following formula S3:
##STR00010##
where G, E, and L.sub.1 are as defined above. Suitable third
component pieces are commercially available from a variety of
sources or can be prepared by known methods in organic
chemistry.
[0062] More specifically, the .alpha.-helix mimetic structures of
this invention of formula (II) are synthesized by reacting a first
component piece with a second component piece to yield a combined
first-second intermediate, followed by either reacting the combined
first-second intermediate with third component pieces sequentially
to provide a combined first-second-third-fourth intermediate, and
the cyclizing this intermediate to yield the .alpha.-helix mimetic
structure.
[0063] The general synthesis of a .alpha.-helix having structure I'
may be synthesized by the following technique. A first component
piece 1 is coupled with a second component piece 2 by using
coupling reagent such as phosgene to yield, after N-deprotection, a
combined first-second intermediate 1-2 as illustrated below:
##STR00011##
wherein R.sub.1, R.sub.2, R.sub.4, R.sub.7 Fmoc, Moc and X are as
defined above, and Pol represents a polymeric support.
[0064] The synthesis of representative component pieces of this
invention are described in Preparation Examples and Working
Examples.
[0065] The .alpha.-helix mimetic structures of formula (II) and
(IV) may be made by techniques analogous to the modular component
synthesis disclosed above, but with appropriate modifications to
the component pieces.
[0066] As mentioned above, the reverse-turn mimetics of U.S. Pat.
No. 6,013,458 to Kahn, et al. are useful as bioactive agents, such
as diagnostic, prophylactic, and therapeutic agents. The opiate
receptor binding activity of representative reverse-turn mimetics
is presented in Example 9 of said U.S. Pat. No. 6,013,458, wherein
the reverse-turn mimetics of this invention were found to
effectively inhibit the binding of a radiolabeled enkephalin
derivative to the .delta. and .mu. opiate receptors, of which data
demonstrates the utility of these reverse-turn mimetics as receptor
agonists and as potential analgesic agents.
[0067] The .alpha.-helix mimetic structures of the present
invention will be useful as bioactive agents, such as diagnostic,
prophylactic, and therapeutic agents.
[0068] Therefore, since the compounds according to the present
invention are of .alpha.-helix mimetic structures, it may be useful
for modulating a cell signaling transcription factor related
peptides in a warm-blooded animal, comprising administering to the
animal an effective amount of the compound of formula (I).
[0069] A particular compound, referred to as ICG-001, is shown
below as compound V:
##STR00012##
[0070] Further, the .alpha.-helix mimetic structures of the present
invention may also be effective for inhibiting transcription
factor/coactivator and transcription factor corepressor
interactions.
[0071] Therefore, it is an object of the present invention to
provide a pharmaceutical composition comprising a safe and
effective amount of the compound having general formula (VI) and
pharmaceutically acceptable carrier, which can be used for
treatment of fibrotic disorders modulated by TGF-.beta. signaling
pathway.
[0072] In another aspect of this invention, libraries containing
.alpha.-helix mimetic structures of the present invention are
disclosed. Once assembled, the libraries of the present invention
may be screened to identify individual members having bioactivity.
Such screening of the libraries for bioactive members may involve;
for example, evaluating the binding activity of the members of the
library or evaluating the effect the library members have on a
functional assay. Screening is normally accomplished by contacting
the library members (or a subset of library members) with a target
of interest, such as, for example, an antibody, enzyme, receptor or
cell line. Library members, which are capable of interacting with
the target of interest, are referred to herein as "bioactive
library members" or "bioactive mimetics". For example, a bioactive
mimetic may be a library member which is capable of binding to an
antibody or receptor, which is capable of inhibiting an enzyme, or
which is capable of eliciting or antagonizing a functional response
associated, for example, with a cell line. In other words, the
screening of the libraries of the present invention determines
which library members are capable of interacting with one or more
biological targets of interest. Furthermore, when interaction does
occur, the bioactive mimetic (or mimetics) may then be identified
from the library members. The identification of a single (or
limited number) of bioactive mimetic(s) from the library yields
.alpha.-helix mimetic structures which are themselves biologically
active, and thus useful as diagnostic, prophylactic or therapeutic
agents, and may further be used to significantly advance
identification of lead compounds in these fields.
[0073] In another aspect of this invention, methods for
constructing the libraries are disclosed Traditional combinatorial
chemistry techniques (see, e.g., Gallop et al., J. Med. Chem.
37:1233-1251, 1994) permit a vast number of compounds to be rapidly
prepared by the sequential combination of reagents to a basic
molecular scaffold. Combinatorial techniques have been used to
construct peptide libraries derived from the naturally occurring
amino acids. For example, by taking 20 mixtures of 20 suitably
protected and different amino acids and coupling each with one of
the 20 amino acids, a library of 400 (i.e., 20.sup.2) dipeptides is
created. Repeating the procedure seven times results in the
preparation of a peptide library comprised of about 26 billion
(i.e., 20.sup.8) octapeptides.
[0074] Specifically, synthesis of the peptide mimetics of the
library of the present invention may be accomplished using known
peptide synthesis techniques, for example, the General Scheme of
[4,4,0] .alpha.-helix Mimetic Library as follows:
##STR00013##
[0075] Synthesis of the peptide mimetics of the libraries of the
present invention was accomplished using a FlexChem Reactor Block
which has 96 well plates by known techniques. In the above scheme
`Pol` represents a bromoacetal resin (Advanced ChemTech) and
detailed procedure is illustrated below.
Step 1
[0076] A bromoacetal resin (37 mg, 0.98 mmol/g) and a solution of
R.sub.2-amine in DMSO (1.4 mL) were placed in a Robbins block
(FlexChem) having 96 well plates. The reaction mixture was shaken
at 60.degree. C. using a rotating oven [Robbins Scientific] for 12
hours. The resin was washed with DMF, MeOH, and then DCM
Step 2
[0077] A solution of available Fmoc hydrazine Amino Acids (4
equiv.), PyBop (4 equiv.), HOAt (4 equiv.), and DIEA (12 equiv.) in
DMF was added to the resin. After the reaction mixture was shaken
for 12 hours at room temperature, the resin was washed with DMF,
MeOH, and them DCM.
Step 3
[0078] To the resin swollen by DMF before reaction was added 25%
piperidine in DMF and the reaction mixture was shaken for 30 min at
room temperature. This deprotection step was repeated again and the
resin was washed with DMF, Methanol, and then DCM. A solution of
hydrazine acid (4 equiv.), HOBt (4 equiv.), and DIC (4 equiv.) in
DMF was added to the resin and the reaction mixture was shaken for
12 hours at room temperature. The resin was washed with DMF, MeOK,
and then DCM.
Step 4a (Where Hydrazine Acid is MOC Carbamate)
[0079] The resin obtained in Step 3 was treated with formic acid
(1.2 mL each well) for 18 hours at room temperature. After the
resin was removed by filtration, the filtrate was condensed under a
reduced pressure using SpeedVac [SAVANT] to give the product as
oil. The product was diluted with 50% water/acetonitrile and then
lyophilized after freezing.
Step 4b (Where Fmoc Hydrazine Acid is Used to Make Urea Through
Isocynate)
[0080] To the resin swollen by DMF before reaction was added 25%
piperidine in DMF and the reaction mixture was shaken for 30 min at
room temperature. This deprotection step was repeated again and the
resin was washed with DMF, Methanol, then DCM. To the resin swollen
by DCM before reaction was added isocyanate (5 equiv.) in DCM.
After the reaction mixture was shaken for 12 hours at room
temperature the resin was washed with DMF, MeOH then DCM. The resin
was treated with formic acid (1.2 mL each well) for 18 hours at
room temperature. After the resin was removed by filtration, the
filtrate was condensed under a reduced pressure using SpeedVac
[SAVANI] to give the product as oil. The product was diluted with
50% water/acetonitrile and then lyophilized after freezing.
Step 4c (Where Fmoc-Hydrazine Acid is Used to Make Urea Through
Active Carbamate)
[0081] To the resin swollen by DMF before reaction was added 25%
piperidine in DMF and the reaction mixture was shaken for 30 min at
room temperature. This deprotection step was repeated again and the
resin was washed with DMF, MeOH, and then DCM. To the resin swollen
by DCM before reaction was added p-nitrophenyl chloroformate (5
equiv.) and diisopropyl ethylamine (5 equiv.) in DCM. After the
reaction mixture was shaken for 12 hours at room temperature, the
resin was washed with DMF, MeOH, and then DCM. To the resin was
added primary amines in DCM for 12 hours at room temperature and
the resin was washed with DMF, MeOH, and then DCM. After reaction
the resin was treated with formic acid (1.2 mL each well) for 18
hours at room temperature. After the resin was removed by
filtration, the filtrate was condensed under a reduced pressure
using SpeedVac [SAVANT] to give the product as oil. The product was
diluted with 50% water/acetonitrile and then lyophilized after
freezing.
[0082] It should be understood that the examples and embodiments
described herein are for illustrative purposes only and that
various modifications or changes in light thereof will be suggested
to persons skilled in the art and are to be included within the
spirit and purview of this application.
EXAMPLE 1
Effect of ICG-001 on Pulmonary Fibrosis
[0083] Murine models of bleomycin induced fibrosis have been
developed in order to study fibrotic disease progression. Bleomycin
induced murine fibrosis has been shown to lead to aberrant alveolar
epithelial repair, with increased metaplastic alveolar cells that
apparently do not properly differentiate to a type I phenotype
(Adamson and Bowden 1979 Am J Pathol. 1979 August; 96(2):531-44.).
Utilizing this model it is demonstrated in this Example that the
Wnt/.beta.-catenin pathway plays a critical role in the development
of pulmonary fibrosis and validates that the inhibition of this
pathway with ICG-001 represents a therapy for the treatment of
pulmonary fibrotic disease.
[0084] Using this murine model of pulmonary fibrosis in transgenic
Bat-al mice, ICG-001 (5 mg/Kg/day) blocked >95% of
bleomycin-induced TCF/.beta.catenin transcription. Furthermore,
ICG-001 at this dose not only halted but reversed disease
progression, as judged by reduced mortality, histopathology and
endogenous gene expression.
[0085] FIG. 1 shows lung sections taken from Bat-Gal transgenic
mice. These mice have a Beta-Galactosidase transgene driven by a
TCF/Catenin driven promoter (i.e. a read out for activated
Wnt/catenin signaling). The mice were given intratracheal saline or
bleomycin and either treated with ICG-001 (5 mgs/Kg/day
subcutaneously) or saline as vehicle control. The mice were
sacrificed and the lungs sectioned and stained with X-Gal (blue
color) A) intratracheal bleo+saline. B) intracheal bleo+ICG-001 C)
saline+saline.
[0086] The dose was selected because ICG-001 reduces
TCF/.beta.Catenin driven O-Galactosidase expression >95% at 5
mgs/Kg/day.
[0087] FIG. 2 shows lung sections taken from C57/B16 mice treated
with intratracheal bleomeycin (lower left) or saline (upper left)
for 5 days and stained with trichrome (red color) to stain
collagen. There is an absence of airway epithelium in lower left
compared to upper left (see arrow heads) and extensive collagen
deposition (lower left). On the sixth day, either saline (upper
right) or ICG-001 (5 mgs/Kg/day) was administered for 10 days after
which the mice were sacrificed and sectioned. Of interest is the
upper right (saline treatment) showing lack of normal airway
epithelialization, extensive collagen deposition and intra-airway
hypercellularity (fibroblasts and inflammatory influx). After
treatment with ICG-001, the airway looks essentially normal
(compare to untreated (saline) control) (upper left), with normal
collagen levels. The mice also regained normal body weight and
survived (untreated controls did not).
[0088] FIG. 3 shows RT-PCR data for S100A4 and collagen1A2, which
are increased in the bleomycin treated mice (treated with saline
control). Message is reduced essentially to negative control (i.e.
saline/saline mice) levels by ICG-001 treatment (5 mgs/Kg/day
s.c.)
[0089] As shown in FIG. 4, IPF patient fibroblasts were cultured in
RPMI1640+10% FBS for 2 days and treated with ICG-001. Western blots
for S100A4 (also know as FSP-1 or fibroblast specific protein-1)
and E-Cadherin were performed on whole cell lysates. ICG-001
decreased S100A4 expression and increased E-cadherin expression
(this is also true at the mRNA level). These data demonstrate that
ICG-001 mediates a mesenchymal to epithelial transition that is
essential for normal healing, re-epithelialization and
ameliorization of fibrosis.
[0090] All patents, patent applications, provisional applications,
and publications referred to or cited herein are incorporated by
reference in their entirety, including all figures and tables, to
the extent they are not inconsistent with the explicit teachings of
this specification.
* * * * *