Note: Descriptions are shown in the official language in which they were submitted.
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MODULATORS OF CELLULAR PROLIFERATION AND ANGIOGENESIS,
METHODS FOR USE AND IDENTIFICATION THEREOF
FIELD OF THE INVENTION
The invention is directed to various "therapeutic uses of peptide and small
molecule compounds having
either hepatocyte growth factor/ scatter factor (HGF/SF), or the property of
inhibiting the activity of
HGF/SF. Such compounds have the potential for the treatment of conditions and
diseases in which
modulation of cellular proliferation, among other activities, is desired.
Methods for identifying such
compounds are also described.
BACKGROUND OF THE INVENTION
Scatter factor (SF; also known as hepatocyte growth factor [HGF], and
hereinafter referred to and
abbreviated as HGF/SF) is a pleiotropic growth factor that stimulates cell
growth, cell motility,
morphogenesis and angiogenesis. HGF/SF is produced as an inactive monomer (-
100 kDa) which is
proteolytically converted to its active form. Active HGF/SF is a heparin-
binding heterodimeric protein
composed of a 62 kDa a chain and a 34 kDa J3 chain. HGF/SF is a potent mitogen
for parenchymal liver,
epithelial and endothelial cells (Matsumoto, K, and Nakamura, T. , 1997,
Hepatocyte growth factor (HGF)
as a tissue organizer for organogenesis and regeneration. Biochein. Biophys.
Res. Commun. 239, 639-44;
Boros, P. and Miller, C.M., 1995, Hepatocyte growth factor: a multifunctional
cytokine. Lancet 345, 293-
5). It stimulates the growth of endothelial cells and also acts as a survival
factor against endothelial cell
death (Morishita, R, Nakamura, S, Nakamura, Y, Aoki, M, Moriguchi, A, Kida, I,
Yo, Y, Matsumoto, K,
Nakamura, T, Higaki, J, Ogihara, T, 1997, Potential role of an endothelium-
specific growth factor,
hepatocyte growth factor, on endothelial damage in diabetes. Diabetes 46:138-
42). HGF/SF synthesized
and secreted by vascular smooth muscle cells stimulate endothelial cells to
proliferate, migrate and
differentiate into capillary-like tubes in vitro (Grant, D.S, Kleinman, H.K.,
Goldberg, I.D., Bhargava,
M.M., Nickoloff, B.J., Kinsella, J.L., Polverini, P., Rosen, E.M., 1993,
Scatter factor induces blood vessel
formation in vivo. Proc. Natl. Acad. Sci. U S A 90:1937-41; Morishita, R.,
Nakamura, S., Hayashi, S.,
Taniyama, Y., Moriguchi, A., Nagano, T., Taiji, M., Noguchi, H., Takeshita,
S., Matsumoto, K.,
Nakamura, T., Higaki, J., Ogihara, T., 1999, Therapeutic angiogenesis induced
by human recombinant
hepatocyte growth factor in rabbit hind limb ischemia model as cytokine
supplement therapy.
Hypertension 33:1379-84). HGF/SF-containing implants in mouse subcutaneous
tissue and rat cornea
induce growth of new blood vessels from surrounding tissue. HGF/SF protein is
expressed at sites of
neovascularization including in tumors (Jeffers, M., Rong, S., Woude, G.F. ,
1996, Hepatocyte growth
factor/scatter factor-Met signaling in tumorigenicity and invasion/metastasis.
J. Mol. Med. 74:505-13;
Moriyama, T., Kataoka, H., Koono, M., Wakisaka, S., 1999, Expression of
hepatocyte growth
factor/scatter factor and its receptor c-met in brain tumors: evidence for a
role in progression of astrocytic
CA 02452445 2010-01-21
tumors Int. J. Mol. Med. 3: 531-6). These findings suggest that HGF/SF plays a
significant role in the formation and
repair of blood vessels under physiologic and pathologic conditions. Further
discussion of angiogenic proteins may
be found in U. S. Patents 6,011,009 and 5,997,868.
Modulation of cellular proliferation by exogenously-supplied therapeutic
agents has been offered as a new approach
for the prophylaxis and/or treatment of various conditions and diseases in
which limited cellular proliferation, or, in
contrast, excessive proliferation of cells, is responsible for pathology, or
at least for the prolongation of rebound
from a pathological state to homeostasis. For example, the duration of wound
healing, normalization of myocardial
perfusion as a consequence of chronic cardiac ischemia or myocardial
infarction, development or augmentation of
collateral vessel development after vascular occlusion or to ischemic tissues
or organs, and vascularization of
grafted or transplanted tissues, organs, or wound healing, may be accelerated
by promoting cellular proliferation,
particularly of vascular cells.
In other cases where abnormal or excessive cellular proliferation is the cause
of pathology, such as in
dysproliferative diseases including cancer, inflammatory joint and skin
diseases such as rheumatoid arthritis, and
neovascularization in the eye as a consequence of diabetic retinopathy,
suppression of cellular proliferation is a
desired goal in the treatment of these and other conditions. In either case,
therapy to promote or suppress
proliferation may be beneficial locally but not systemically, and for a
particular duration, and proliferation
modulating therapies must be appropriately applied.
Peptide mimetics with HGF/SF-like, proliferative activity and particularly
angiogenic activity, as well as
other agents, particularly peptide HGF/SF antagonists which inhibit cellular
proliferation and, in particular,
angiogenesis, were described, Such peptides have uses, for example, in the
treatment of inflammatory diseases,
cancer, neovascularization, cardiac ischemia, wound healing, and other
conditions in which modulation of cellular
proliferation including blood vessel growth is therapeutically beneficial, as
described above. Furthermore,
-various small-molecule compounds with HGF/SF-like activity or antagonistic
activity, with
the same aforementioned uses have been. described.
It is toward the identification of peptide and small organic molecules with
HGF/SF activity, or those that inhibit
HGF/SF activity, as well as methods for identifying and preparing such active
molecules, that the present invention
is directed.
The citation of any reference hertn should not be construed as an admission
that such reference is available as"Prior
Art"to the instant application.
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SUMMARY OF THE INVENTION
In one broad aspect, the present invention is directed to methods for the
modulation of hepatocyte growth
factor / scatter factor (HGF/SF) activities in a mammal for the treatment of
any of a number of conditions
or diseases in which either HGF/SF has a therapeutically useful role, or in
which the activity of
endogenous HGF/SF is desirably inhibited or abrogated. Such modulation is
achieved by the
administration to the mammal of a compound of the invention in an amount
effective to achieve the
desired outcome. In one embodiment, the compounds of the invention modulate
the activity of the
HGF/SF receptor, c-met. In a further embodiment, the compounds of the
invention bind to c-met.
In the instance where HGF/SF activity is desirable, certain compounds of the
invention have been found
to mimic or agonize the biological activities of HGF/SF, and thus are useful
in the treatment, for example,
of conditions or diseases in which enhanced cellular or vascular proliferation
is desirable, among other
desirable activities of HGF/SF. Such conditions or diseases include hepatic
disease, renal disease, bone
regeneration, hair growth, promoting wound or tissue healing, or augmenting or
restoring blood flow to
ischemic tissues such as the heart following myocardial infarction. Such
compounds may be administered
systemically or locally to particular tissues or organs, in order to achieve
the desired systemic or local
effect. Such desirable activities also include induction of proliferation of
endothelial cells, induction of
anti-apoptotic activity, induction of scatter activity, or any combination of
the foregoing activities. In a
preferred embodiment, any one of these activities is reduced or inhibited in
the presence of exogenous c-
met receptor by a compound of the invention.
In another aspect, the present invention is directed to cellular proliferation
promoting agents and in
particular peptide agents characterized by the ability to bind to a monoclonal
or polyclonal antibody to
HGF/SF; and exhibit cellular proliferative activity in one or more in-vitro
and/or in-vivo assays. Such
agents may further exhibit the property of agonizing c-met, the HGF/SF
receptor. In one embodiment,
cellular proliferation comprises endothelial cell proliferation and
angiogenesis. By way of non-limiting
example, the agents may be small-molecule drugs, peptides, or proteins, such
as the peptides
TMGFTAPRFPHY (SEQ ID No:l) and KVWYHTTSIPSH (SEQ ID No:2), or their
conservatively-
substituted variants. The angiogenic peptides may further include a heparan
sulfate-binding peptide
conjugated thereto, such as KVWYHTTSIPSHCRPKAKAKAKAKDQTK (SEQ ID No:7) or
conservatively-substituted variants thereof. Non-limiting examples of such
conjugates include
TMGFTAPRFPHYKVWYHTTSIPSHCRPKAKAKAKAKDQTK (SEQ ID No:9),
KVWYHTTSIPSHKVWYHTTSIPSHCRPKAKAKAKAKDQTK (SEQ ID No:10), and conservatively-
substituted variants thereof. The invention is further directed to
pharmaceutical compositions comprising
the foregoing agents as well as polynucleotides comprising sequences encoding
the peptide agents.
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In a further aspect of the invention, a method is provided for promoting
cellular proliferation comprising
contacting cells or tissues with an effective proliferation promoting amount
of an agent characterized by
ability to bind to a monoclonal or polyclonal antibody to HGF/SF; and
exhibiting proliferation promoting
activity in one or more in-vitro and/or in-vivo assays. Such agents may
further exhibit the property of
agonizing c-met, the HGF/SF receptor. Such cells or tissues may further
exhibit the property of
expressing the c-met receptor. In one embodiment, the proliferation promoting
activity is angiogenesis.
By way of non-limiting example, the agents may be small-molecule drugs,
peptides, or proteins, such as
the peptides TMGFTAPRFPHY (SEQ ID No:1) and KVWYHTTSIPSH (SEQ ID No:2), or
their
conservatively-substituted variants. The proliferation-promoting peptides may
further include a heparan
sulfate-binding peptide conjugated thereto, such as
KVWYHTTSIPSHCRPKAKAKAKAKDQTK (SEQ
ID No:7) or conservatively-substituted variants thereof. Non-limiting examples
of such conjugates
include TMGFTAPRFPHYKVWYHTTSIPSHCRPKAKAKAKAKDQTK (SEQ ID No:9),
KVWYHTTSIPSHKVWYHTTSIPSHCRPKAKAKAKAKDQTK (SEQ ID No:10); and conservatively-
substituted variants thereof. The cells or tissue may be, for example, a
transplanted or grafted tissue or
organ such as skin, heart, vascular tissue or kidney, an ischemic organ, such
as a heart following
myocardial infarction or angina, a tissue or organ damaged by wounding,
surgical intervention, vascular
tissue, neural tissue, a wound, ulcer, etc. The cells may be, by way of non-
limiting example, epithelial
cells, endothelial cells, and smooth muscle cells, and tissues and organs
comprising such cells. Promotion
of growth and/or regeneration of neural tissue, teeth, and other tissues
comprising the c-met receptor are
embraced herein. Methods of treatment include application of an agent of the
invention, a pharmaceutical
composition comprising an agent of the invention, gene therapy in which
endogenous cells are transfected
to express a protein comprising an agent of the invention, or implanting cells
which secrete an agent of the
invention.
In another aspect of the invention, a method is provided for promoting
vascularization of a tissue
comprising contacting the tissue with an effective angiogenic amount of an
agent characterized by ability
to bind to a monoclonal or polyclonal antibody to HGF/SF; and exhibit
angiogenic activity in one or more
in-vitro and/or in-vivo assays. Such agents may further exhibit the property
of agonizing c-met, the
HGF/SF receptor. By way of non-limiting example, the agents may be small-
molecule drugs, peptides, or
proteins,suchasthepeptidesTMGFTAPRFPHY(SEQIDNo:l)andKVWYHTTSIPSH
(SEQ ID No:2), or their conservatively-substituted variants. The angiogenic
peptides may further include
a heparan sulfate-binding peptide conjugated thereto, such as K V W Y H T T S
I P S H C R P K A K A
K A K A K D Q T K (SEQ ID No:7) or conservatively-substituted variants
thereof. Non-limiting
examples of such conjugates includeTM GF YAP RFP HYKV WYHTTSIPSHCRPKAK
AKAKAKDQTK(SEQIDNo:9),KVWYHTTSIPSHKVWYHTTSIPSHCRPKA
K A K A K A K D Q T K(SEQ ID No:10); and conservatively-substituted variants
thereof. The cells or
tissue may be, for example, a transplanted or grafted tissue or organ such as
skin, heart, vascular tissue or
kidney, an ischemic organ, such as a heart following myocardial infarction or
angina, a tissue or organ
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damaged by wounding, surgical intervention, a wound, ulcer, etc. Means of
delivery of the agent are as
described hereinabove.
In a further embodiment, polynucleotides are described which comprise nucleic
acids encoding the
proliferation promoting peptides of the invention, including vectors encoding
the polynucleotides, as well
as microorganisms and cells comprising the vectors and expressing the
peptides. Therapeutic use of such
polynucleotide sequences, including gene therapy with naked DNA, viral
vectors, etc., and other means of
transfecting cells to express the agents of the invention, for all of the
above mentioned purposes, among
others, are embraced herein.
In yet another embodiment, the present invention is directed to methods for
identifying an proliferation
promoting agent, by carrying out the steps of a) providing a candidate agent;
b) measuring the ability of
the agent to bind to a monoclonal or polyclonal antibody to HGF/SF; and c)
measuring the ability of said
agent to exhibit cellular proliferation activity in one or more in-vitro
and/or in-vivo assays; wherein the
candidate agent with the ability to bind to a monoclonal or polyclonal
antibody to HGF/SF and ability to
exhibit proliferation promoting activity is an proliferation promoting agent.
The agent may further exhibit
the ability of agonizing the c-met receptor. In one embodiment, the agent is
an angiogenic agent. By way
of non-limiting example, the agent may be a small-molecule drug, a peptide, or
a protein. The ability of
the agent to bind to a monoclonal or polyclonal antibody to HGF/SF may be
determined by measurement
of binding of said agent to the antibody, or by measuring the ability of the
agent to compete with the
binding of the antibody with HGF/SF or a peptide mimetic thereof. In another
embodiment, a method of
preparing an proliferation promoting agent may be carried out by a)
identifying an proliferation promoting
peptide as described above; b) determining the three-dimensional structure of
the peptide; and c) modeling
a small-molecule drug on the three-dimensional structure of the peptide.
Another broad aspect of the invention is directed to antiproliferative agents
characterized by the ability to
bind to the extracellular domain of c-met; and inhibit HGF/SF-mediated
increase in cellular growth or
proliferation. In one embodiment, the inhibition of cellular growth or
proliferation is directed towards
cells expressing the c-met receptor. In another embodiment, the cells include
but are not limited to
epithelial cells, endothelial cells, fibroblasts and smooth muscle cells. By
way of non-limiting example,
the antiproliferative agent may be a small-molecule drug, a peptide or a
protein; examples of peptides
include but are not limited to A T W S H H L S S A G L (SEQ ID No:3); W P Q L
P P R P Y S T L (SEQ
IDNo:4); SNT S AGTPFTS L (SEQ IDNo:5); D STPKS TP WYYI(SEQIDNo:6);and
conservatively-substituted variants thereof. Pharmaceutical compositions
comprising the aforementioned
agents are embraced herein.
In a further aspect, the invention is directed to a method for inhibiting
cellular proliferation in a tissue or
organ by contacting the tissue or organ with an effective antiproliferative
amount of an agent
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characterized by the ability to bind to the extracellular domain of c-met; and
inhibit HGF/SF-mediated
increase in cell growth or proliferation. In one embodiment, the agent
inhibits HGF/SF-mediated increase
in endothelial, epithelial cell, fibroblast or smooth muscle cell growth or
proliferation. In a preferred
embodiment, the agent inhibits endothelial cell growth. By way of non-limiting
example, the agent may
be a small-molecule drug, a peptide or a protein; examples of peptides include
but are not limited to A T
W S H H L S S A G L (SEQ ID No:3); W P Q L P P R P Y S T L (SEQ ID No:4); S N
T S A G T P F T S
L (SEQ ID No:5); D S T P K S T P W Y Y I (SEQ ID No:6); and conservatively-
substituted variants
thereof The tissue or organ may be a dysproliferative tissue, such as a tumor
or metastasis, or psoriasis, a
tissue or organ involved in inflammatory diseases such as rheumatoid
arthritis, the eye involved in
neovascularization such as results from chronic diabetes, an abnormal growth
such as keloid formation
during wound healing, or an intentional disruption of cellular proliferation
such as to prevent the genesis
or maturation of a developing organ or tissue. Methods of treatment include
application of an agent of the
invention to the desired target site(s), a pharmaceutical composition
comprising an agent of the invention,
gene therapy in which endogenous cells are transfected to express a protein
comprising an agent of the
invention, or implanting cells which secrete an agent of the invention.
In a further aspect, the invention is directed to a method for inhibiting the
vascularization of a tissue or
organ by contacting the tissue or organ with an effective angiostatic amount
of an agent characterized by
the ability to bind to the extracellular domain of c-met; and inhibit HGF/SF-
mediated increase in
endothelial cell growth or proliferation. By way of non-limiting example, the
agent maybe a small-
molecule drug, a peptide or a protein; examples of peptides include but are
not limited to A T W S H H L
S S A G L (SEQ ID No:3); W P Q L P P R P Y S T L (SEQ ID No:4); S N T S A G T
P F T S L (SEQ ID
No:5); D S T P K S T P W Y Y I (SEQ ID No:6); and conservatively-substituted
variants thereof.
Examples of small-molecule compounds are described below. The tissue or organ
may be a
dysproliferative tissue, such as a tumor or metastasis, or psoriasis, a tissue
or organ involved in
inflammatory diseases such as rheumatoid arthritis, the eye involved in
neovascularization such as results
from chronic diabetes, an abnormal growth such as keloid formation during
wound healing, or an
intentional disruption of cellular proliferation such as to prevent the
genesis or maturation of a developing
organ or tissue. Means for application of the agent to the desired site(s) are
as described hereinabove.
The present invention is also directed to polynucleotide sequences comprising
nucleic acids encoding the
aforementioned antiproliferative peptide sequences, including vectors encoding
the polynucleotides, as
well as microorganisms and cells comprising the vectors and expressing the
antiproliferative peptides.
Therapeutic use of such polynucleotide sequences, including gene therapy with
naked DNA, viral vectors,
etc., and other means of transfecting cells to express the agents of the
invention are embraced herein. In a
preferred embodiment, the antiproliferative peptides and nucleic sequences
encoding them are
antiangiogenic peptides.
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In a further aspect, the invention is directed to a method for identifying an
agent capable of inhibiting
cellular proliferation comprising the steps of a) providing a candidate agent;
b) measuring the ability of
the agent to bind to the extracellular domain of C-met; and c) measuring the
ability of the agent to inhibit
the antiproliferative activity of HGF/SF; wherein the candidate agent with the
ability to bind to the
extracellular domain of C-met and inhibit the proliferative activity of
scatter factor is an antiproliferative
agent. By way of non-limiting example, the agent may be a small-molecule drug,
a peptide, or a protein.
In yet another aspect, a method of preparing an antiproliferative agent is
provided comprising the steps of
a) identifying an antiproliferative peptide as described above; b) determining
the three-dimensional
structure of said peptide; and c) modeling a small-molecule drug on the three-
dimensional structure of the
peptide.
In a further embodiment, the invention is directed to a method for identifying
an agent capable of
inhibiting angiogenesis comprising the steps of a) providing a candidate
agent; b) measuring the ability of
the agent to bind to the extracellular domain of C-met; and c) measuring the
ability of the agent to inhibit
the angiogenic activity of scatter factor; wherein the candidate agent with
the ability to bind to the
extracellular domain of C-met and inhibit the angiogenic activity of scatter
factor is an angiostatic agent.
By way of non-limiting example, the agent may be a small-molecule drug, a
peptide, or a protein. In yet
another aspect, a method of preparing an angiostatic agent is provided
comprising the steps of a)
identifying an angiostatic peptide as described above; b) determining the
three-dimensional structure of
said angiostatic peptide; and c) modeling a small-molecule drug on the three-
dimensional structure of the
angiostatic peptide.
The invention is also directed to small-molecule compounds that are agonists
or antagonists of HGF/SF
for all of the aforementioned uses of the agonist and antagonist peptides
described above. Such agonist
compounds of the invention are useful for mimicking or agonizing HGF/SF
activity and are characterized
by being non-peptide, non-protein organic molecules with one or more of the
activities of promoting
proliferation of endothelial cells in vitro or in vivo, promoting angiogenesis
in vitro or in vivo, increasing
angiogenesis in wounds in vivo, promoting the growth of tumor cells in vitro
or in vivo, promoting
scatter, promoting anti-apoptotic activity, or inducing gene expression of
angiogenic-cascade-related
genes such as but not limited to IL-8 and angiopoietin-2. Preferred are
compounds in which the
aforementioned activity is inhibited or competed in the presence of
exogenously-added c-met receptor.
The compounds may bind to c-met. The present invention embraces the use of all
such molecules for
treatment of various conditions or diseases in which increased or enhanced
HGF/SF activity is desirable.
In one embodiment, a compound of the invention has a molecular weight of under
1,000 Daltons,
preferably above about 200 Daltons to about 1,000 Daltons; more preferably
between about 300 Daltons
and about 750 Daltons, and most preferably between about 300 Daltons and about
500 Daltons.
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Thus, a method is provided for increasing hepatocyte growth factor / scatter
factor (HGF/SF) activities in
a mammal by administration to the mammal an effective amount of a compound
having a molecular
weight below about 1,000 Daltons, the compound exhibiting HGF/SF-like activity
in at least one of the
following HGF/SF activity assays:
induction of proliferation of endothelial cells in vitro or in vivo;
induction of angiogenesis in vitro or in vivo;
increasing angiogenesis in wounds in vivo;
promoting tumor growth;
inducing gene expression of angiogenic-cascade-related genes such as but not
limited to IL-8 and
angiopoietin-2;
inducing anti-apoptotic activity; or
inducing scatter activity.
In a preferred embodiment, the HGF/SF activity of the foregoing compound is
inhibited in the presence of
c-met. In another preferred embodiment, the compound binds to c-met.
A compound of the invention may exhibit HGF/SF-like activity in at least two
of the aforementioned
HGF/SF activity assays, or in at least three of the HGF/SF activity assays, or
in at least four said HGF/SF
activity assays, or in at least five of the HGF/SF activity assays, or in at
least six of the HGF/SF activity
assays or in all of the HGF/SF activity assays. The compound preferably has a
molecular weight between
about 200 Daltons and about 750 Daltons, more preferably between about 300
Daltons and about 500
Daltons.
In another embodiment, the invention is directed to a method for the
prophylaxis or treatment in a
mammal of hepatic disease, renal disease, bone regeneration, hair growth,
promoting wound or tissue
healing, promoting vascularization of a tissue, promoting vascularization of
an ischemic tissue, promoting
vascularization of a tissue susceptible to ischemia, or augmenting or
restoring blood flow to ischemic
tissues such as the heart following myocardial infarction comprising
administered systemically or locally
to particular tissues or organ in need thereof an effective amount of a
compound having a molecular
weight between below about 1,000 Daltons, the compound exhibiting HGF/SF-like
activity in at least one
HGF/SF activity assays:
induction of proliferation of endothelial cells in vitro or in vivo;
induction of angiogenesis in vitro or in vivo;
increasing angiogenesis in wounds in vivo;
promoting tumor growth;
inducing gene expression of angiogenic-cascade-related genes such as but not
limited to IL-8 and
angiopoietin-2;
inducing anti-apoptotic activity; or
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inducing scatter activity.
In a preferred embodiment, the HGF/SF activity of the foregoing compound is
inhibited in the presence of
c-met.
A compound of the invention may exhibit HGF/SF-like activity in at least two
of the aforementioned
HGF/SF activity assays, or in at least three of the HGF/SF activity assays, in
at least five said HGF/SF
assays, in at least six said HGF/SF assays, or in at least four said HGF/SF
activity assays, or in all of the
HGF/SF activity assays. The compound preferably has a molecular weight between
about 200 Daltons
and about 750 Daltons, more preferably between about 300 Daltons and about 500
Daltons.
The invention is also directed to a method for inhibiting the activity of
hepatocyte growth factor / scatter
factor (HGF/SF) in a mammal comprising administering to the mammal an
effective amount of a
compound having a molecular weight below about 1,000 Daltons, the compound
exhibiting HGF/SF
inhibitory or antagonistic activity in at least one of the following HGF/SF
activity assays:
inhibiting proliferation of endothelial cells in vitro or in vivo;
inhibiting the growth of tumor cells in vitro or in vivo;
inhibiting scatter of normal or tumor cells; or
inhibiting anti-apoptotic activity.
In a preferred embodiment, the HGF/SF inhibitory activity of the foregoing
compound occurs in the
presence of exogenously added HGF/SF or in cells or tissues in which HGF/SF is
expressed or induced.
A compound of the invention may exhibit HGF/SF inhibitory activity in at least
two of the
aforementioned HGF/SF activity inhibition assays, or in at least three of the
HGF/SF activity inhibition
assays, or in all of the HGF/SF activity inhibition assays. The compound
preferably has a molecular
weight between about 200 Daltons and about 750 Daltons, more preferably
between about 300 Daltons
and about 500 Daltons.
In yet another embodiment, a method is provided for the prophylaxis or
treatment in a mammal of a
condition of disease selected from the group consisting of excessive cellular
proliferation, angiogenesis, a
dysproliferative disease, cancer, metastasis, inflammatory disease, diabetic
retinopathy, inflammatory
joint disease, and inflammatory skin disease comprising administering to a
mammal an effective amount
of a compound having a molecular weight below about 1,000 Daltons, said
compound exhibiting HGF/SF
inhibitory or antagonistic activity in at least one of the following HGFISF
activity inhibition assays:
inhibiting proliferation of endothelial cells in vitro or in vivo;
inhibiting the growth of tumor cells in vitro or in vivo;
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inhibiting scatter of normal or tumor cells; and
inhibiting anti-apoptotic activity.
In a preferred embodiment, the HGF/SF inhibitory activity of the foregoing
compound occurs in the
presence of exogenously added HGF/SF or in cells or tissues in which HGF/SF is
expressed or induced.
A compound of the invention may exhibit HGF/SF inhibitory activity in at least
two of the
aforementioned HGF/SF activity inhibition assays, or in at least three of the
HGF/SF activity inhibition
assays, or in all of the HGF/SF activity inhibition assays. The compound
preferably has a molecular
weight between about 200 Daltons and about 750 Daltons, more preferably
between about 300 Daltons
and about 500 Daltons.
In another embodiment, the invention is directed to a method for the use for
any of the aforementioned
purposes of compounds that modulate HGF/SF activity with the general formula
I:
R5
RI N R4
N
R3
Formula I
wherein
R3 and R5 are independently or together a straight-chain or branched C1-C6
alkyl optionally
substituted with a cyano or halogen, halogen, trifluoromethyl or
difluoromethyl groups;
Rl is hydrogen, methyl, CO-Aryl, S02-Aryl, CO-heteroaryl, or CO-alkyl; and
R4 is CH2-Aryl, halogen, arylcarbonylvinyl or S-heteroaryl.
Certain of the compounds of Formula I are novel, and the present invention is
also directed to all such
novel compounds with an activity as described herein.
The invention is also directed to a pharmaceutical composition comprising at
least one compound of
Formula I and a pharmaceutically-acceptable carrier, for any of the uses
described herein.
Non-limiting example of modulators of HGF/SF activity of Formula I include the
following compounds,
most of which, as will be seen in the examples below, exhibit HGF/SF agonist
activity.
3-(5-chloro-1,3-dimethyl-lH-pyrazol-4-yl)-1-(4-chlorophenyl)prop-2-en-l-one
[4-(2,6-dichlorobenzyl)-3,5-dimethyl-lH-pyrazol-1-yl][3-(2,6-dichlorophenyl)-5-
methylisoxazol-4-
yl]methanone
(4-(2-chloro-6-fluorobenzyl)-3,5-dimethyl-1H pyrazole-l-yl)(3-(2,6-
dicblorophenyl)-5-
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methylisoxazol-4-yl)methanone
4-(2-chloro-6-fluorobenzyl)-1-((3,4-dichlorophenyl)sulfonyl)-3,5-dimethyl-1H-
pyrazole
4-(2-chloro-6-fluorobenzyl)-1,3,5-timethyl-1H-pyrazole
4-(2-chloro-6-fluorobenzyl)-3,5-dimethyl-1H-pyrazole
(4-bromo-3,5-dimethyl-lH-pyrazol-l -yl)(3-(2,6-dichlorophenyl)isoxazole-4-
carbohydrazide)
3-(4-(2,6-dichlorobenzyl)-3,5-dimethyl-lH-pyrazol-l -yl)propanenitrile
3,5-di(tert-butyl)-4-(2-chloro-6-fluorobenzyl)-1H-pyrazole
(4-(2-chloro-6-fluorobenzyl)-3,5-dimethyl-1H-pyrazole-1-yl)(2,6-
dichlorophenyl)methanone
1-(4-(2-chloro-6-fluorobenzyl)-3,5-dimethyl-1H-pyrazole-1-yl)2,2-
dimethylpropan-l -one
(4-(2-chloro-6-fluorobenzyl)-3,5-dimethyl-1 H-pyrazole-l -yl)(4-
chlorophenyl)methanone
(4-(2-chloro-6-fluorobenzyl)-3,5-dimethyl-1H-pyrazole-1-yl)(2-
thienyl)methanone
(4-chlorophenyl)(3,5-dimethyl-4-((1-methyl-lH-imidazol-2-yl)thio)-1H-pyrazol-l-
yl)methanone
In another embodiment, the invention is directed to methods for the use for
the aforementioned purposes
of compounds that modulate HGF/SF activity with the general formula II:
R3 O
RI
0 N-N,
R2
R5
Formula II
wherein
R5 is a Cl to C6 branched or straight-chained alkyl group;
R3 is a substituted or unsubstituted Aryl group;
R1 is hydrogen or a Cl to C4 straight-chained, branched or cycloalkyl group;
R2 is COCH2ONCH-Aryl; heteroaryl, COCH2CH2Aryl; Aryl; COS-Aryl; CO-Heteroaryl;
Cl to
C4 straight-chained alkyl, branched alkyl, or cycloalkyl; or wherein RI and R2
form a cyclic group of 5 or
6 carbon atoms.
Certain of the compounds of Formula II are novel, and the present invention is
directed to all such novel
compounds with an activity as described herein.
The invention is also directed to a pharmaceutical composition comprising at
least one compound of
Formula II, in a pharmaceutically-acceptable carrier, for any of the uses
described herein.
Most of the compounds of Formula II exhibit HGF/SF antagonist or inhibitory
activity, as will be seen in
the examples below. Non-limiting examples of compounds of Formula II include
N'4,5-dimethyl-N'4-(5-nitro-2-pyridyl)-3-(2,6-dichlorophenyl)isoxazole-4-
carbohydrazide
N'4-(2-(((2,4-dichlorobenzylidene)amino)oxy)acetyl)-3-(2,6-dichlorophenyl)-5-
methylisoxazole-4-
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carbohydrazide
N'4-(3-(3,4,5-trimethoxyphenyl)propanoyl)-3 -(2,6-dichlorophenyl)-5-
methylisoxazole-4-
carbohydrazide
2-nitrophenyl 2-((3-(2, 6-dichlorophenyl)-5-methylisoxazol-4-
yl)carbonyl)hydrazine- l -carbothioate
N'4-((2-methyl-1,3-thiazol-4-4y1)carbonyl)-3-(2,6-dichlorophenyl)-5-
methylisoxazole-4-
4carbohydrazide
N 1-((2-((3-(2,6-dichlorophenyl)-5-methylisoxazol-4-
yl)carbonyl)hydrazino)(methylthio)methylidene)benzene-l-sulfonamide
N'4-(2,4,6-trichlorophenyl)-3-3 (2,6-dichlorophenyl)-5-methylisoxazole-4-
carbohydrazide
N'4,3-di(2,6-dichlorophenyl)-5-methylisoxazole-4-carbohydrazide
N'4-(3,5-dichloro-4-pyridyl)-3-(2,6-dichlorophenyl)-5-methylisoxazole-4-
carbohydrazide
N'4-phenyl-3-(2,6-dichlorophenyl)-5-methylisoxazole-4-carbohydrazide
N'4,N'4,5-trimethyl-3-(2,6-dichlorophenyl)isoxazole-4-carbohydrazide
N4-azepan-1-y1-3-(2,6-dichlorophenyl)-5-methylisoxazole-4-carboxamide
N'4-(6-(trifluoromethyl)-2-pyridyl)-3-(2,6-dichlorophenyl)-5-methylisoxazole-4-
carbohydrazide
N'4-(3,3-diethoxypropanoyl)-3-(2,6-dichlorophenyl)-5-methylisoxazole-4-
carbohydrazide
In a further embodiment, the invention is directed to methods for the use for
any of the aforementioned
purposes of compounds that modulate HGF/SF activity with the general formula
III:
R1 ~N\\
N
R3
Formula III
wherein
RI is SO2Alkyl, SO,-Aryl, CO-t-Butyl, COAryl, CONHA1ky1; CONHAryl; and
R3 is CHCH-heteroaryl; phenoxyphenyl; heteroaryl; or Aryl substituted
heteroaryl.
Certain of the compounds of Formula III are novel, and the present invention
is directed to all such novel
compounds with an activity as described herein.
The invention is also directed to a pharmaceutical composition comprising at
least one compound of
Formula III, in a pharmaceutically-acceptable carrier, for any of the uses
described herein.
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These compounds generally exhibit HGF/SF stimulatory or agonist activity. Non-
limiting examples of
compounds of Formula III include
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone;
1-(methylsulfonyl)-3-(2-(2-thienyl)vi yl)-1H-pyrazole;
2,2-dimethyl-l-(3-(2-(2-thienyl)vinyl)-1H-pyrazole-1-yl)propan-1-one
N-methyl-3-(2-(2-thienyl)vinyl)-1 H-pyrazole-l -carboxamide
(4-chlorophenyl)(3-(3-phenylisoxazol-5-yl)-1 H-pyrazol- l -yl)methanone
(4-chlorophenyl)(3-(3-(4-chlorophenyl)-5-methylisoxazol-4-yl)-1H-pyrazol-l -
yl)methanone
(4-chlorophenyl)(3-(5-(2-thienyl)-2-thienyl)-1H-pyrazol-1-yl)methanone
(2,4-dichlorophenyl)(3-(5-(2,4-dichorophenyl)-2-furyl)-1H-pyrazol-l-
yl)lnethanone
N l -phenyl-3 -(2-(2-thienyl)vinyl)-1 H-pyrazole- l -carboxamide
(4-chlorophenyl)(3-(2-(5-(2-thienyl)-2-thienyl)-4-methyl-1,3-thiazol-5-yl)-1H-
pyrazol-l-
yl)methanone
(3-benzhydryl-1H-pyrazol-1-yl)(4-chlorophenyl)methanone
N 1-(4-chlorophenyl)-3-(2-(2-thienyl)vinyl)-1 H-pyrazole- l -carboxamide
(4-chlorophenyl)(3-(2-methylimidazo(1,2-a)pyridin-3-yl)-1H-pyrazol-1-
yl)methanone
2-chloro-6-(4-(1-(4-chlorobenzyl)-1H-pyrazol-3-yl)phenoxy)benzonitrile
1-((4-chlorophenyl) sulfonyl)-3 -(2-(2-thienyl)vinyl)-1 H-pyrazole
In a further embodiment, the invention is directed to methods for the use for
any of the aforementioned
purposes of compounds that modulate HGF/SF activity with the general formula
IV:
O
RI R2
Formula IV
Wherein
RI is Aryl or Heteroaiyl; and
R2 is one or more halogen, nitro, Cl to C4 straight-chained alkyl, branched
alkyl, or cycloalkyl,
or C l to C4 alkyloxy groups.
Certain of the compounds of Formula IV are novel, and the present invention is
directed to all such novel
compounds with an activity as described herein.
The invention is also directed to a pharmaceutical composition comprising at
least one compound of
Formula N, in a pharmaceutically-acceptable carrier, for any of the uses
described herein.
The compounds in this group may be HGF/SF agonists or antagonists. Non-
limiting examples of
modulators of Formula N include:
1-(4-chloro-3-methylphenyl)-3-(2,6-dichlorophenyl)-prop-2-en-l-one
1-(4-chloro-3-methylphenyl)-3-(2-chlorophenyl)prop-2-en-1-one
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3 -(2-chloro-6-fluorophenyl)-1-(4-chloro-3 -(4-chloro-3-methylpheiiyl)prop-2-
en-1 -one
3-(4-bromo-2-thienyl)-1-(3,4-dichlorophenyl)prop-2-en-l-one
3-(4-bromo-2-thienyl)-1-(4-chloro-3-methylphenyl)prop-2-en-l-one
3 -(4-bromo-2 -thienyl)-1-(4-fluorophenyl)prop-2-en- l -one
3-(4-bromo-2-thienyl)-1-(4-chlorophenyl)prop-2-en-l-one
1 -(4-chlorophenyl)-3 -(2,4-diclalorophenyl)prop-2-en-l-one
3-(1,3-benzodioxol-5-yl)-1-(4-bromophenyl)prop-2-en-l-one
3-(3-phenoxy-2-thienyl)-1-(2-thienyl)prop-2-en-1-one
3-(3-bromo-4-methoxyphenyl)-1-phenylprop-2-en-one
3-(3,4-dichlorophenyl)-1-(2-nitrophenyl)prop-2-en-l-one
1-(4-chlorophenyl)-3-(3,4-dichlorophenyl)prop-2-en- 1 -one
1-(4-chlorophenyl)-3-(3,5 -dichloro-2-hydroxyphenyl)prop-2-en-1-one
1-(2-chlorophenyl)-3-(3,5-dichloro-2-hydroxyphenyl)prop-2-en-1-one
3 -(4-chlorophenyl)-1-(2,6-dichlorophenyl)prop-2-en-1-one
1-(4-bromophenyl)-3-(4-chlorophenyl)prop-2-en-1-one
1-(2-chlorophenyl)-3-(2,6-dichlorophenyl)prop-2-en-1-one
1-(4-chlorophenyl)-3-(2,6-dichlorophenyl)prop-2-en-1-one
3 -(2, 6-dichlorophenyl)-1-(4-methoxyphenyl)prop-2-en-1-one
3 -(4-chloro- l -inethyl-1 H-pyrazol-3 -yl)-1-[4-(trifluoromethyl)phenyl]prop-
2-en-1-one
3-(2,4-dichlorophenyl)-1-(2-methylphenyl)prop-2-en- l-one
3 -(2, 6-dichlorophenyl)-1-(2-methylphenyl)prop-2-en- l -one
3-(3 ,4-dichlorophenyl)-1-(2-methylphenyl)prop-2-en- l -one
3-(5-bromo-2-hydroxyphenyl)- 1 -(3-methylphenyl)prop-2-en- 1 -one
3 -(5 -bromo-2 -hydroxyphenyl)-1-(4-methylphenyl)prop-2-en- l -one
3-(2,4-dichlorophenyl)-1-(3-methylphenyl)prop-2-en-l-one
3-(2,4-dichlorophenyl)-1-(4-methoxyphenyl)prop-2-en-l-one
1-[4-amino-2-(methylthio)-1,3-thiazol-5-yl]-3-(4-chlorophenyl)prop-2-en-l-one
1-(4-chlorophenyl)-3-[4-(trifluoromethyl)phenyl]prop-2-en-l -one
1-benzo[b]thiophen-3-yl-3-(4-chlorophenyl)prop-2-en-1-one
1,3-di(5-nitro-3-thienyl)prop-2-en-1-one
1-(4-bromophenyl)-3-(3,5-difluorophenyl)prop-2-en-I -one
3-(3,5-difluorophenyl)-1-(3-nitrophenyl)prop-2-en-1-one
The foregoing compounds also have antagonistic activity to other tyrosine
kinase receptor growth factors
including VEGF and FGF, and maybe used to inhibit such activities for the
treatment of various
conditions and diseases arising from the activities of these growth factors.
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The foregoing general and specific structures of compounds with HGF/SF
activity are merely illustrative
of compounds of the invention with the desired activities, and are in no way
limiting.
In a further embodiment, the invention is directed to pharmaceutical
compositions comprising any one or
a combination of the foregoing compounds, together with a pharmaceutically-
acceptable carrier, for use in
any of the aforementioned purposes.
In a further embodiment, the aforementioned compounds with activities of
promotion of cellular
proliferation or angiogenesis are useful for promoting vascularization of a
tissue, particularly of an
ischernic tissue or a tissue susceptible to ischemia.
Prophylaxis or treatment maybe provided by contacting the tissue with an
effective angiogenic amount of
an agent of the invention. Contact may be provided by any appropriate means to
deliver an effective
amount of the agent for a duration to achieve the desired results. By way of
non-limiting example, topical
application may be applied to the desired target, or by infusion, bathing, or
implantation of a sustained
delivery device. For systemic administration, oral or parenteral routes may be
employed. The target cells
or tissue may be, for example, a transplanted or grafted tissue or organ such
as skin, heart, vascular tissue
or kidney, an ischemic organ, such as a heart following myocardial infarction
or angina, a tissue or organ
damaged by wounding, surgical intervention, vascular tissue, neural tissue, a
wound, ulcer, etc. The cells
may be, by way of non-limiting example, epithelial cells, endothelial cells,
and smooth muscle cells, and
tissues and organs comprising such cells. Promotion of growth and/or
regeneration of neural tissue, teeth,
and other tissues are embraced herein. Preferred cells, organs and tissues
comprise the c-met receptor.
The aforementioned compounds with HGF/SF activity are also desirably useful
for the treatment of
various hepatic diseases including cirrhosis and liver failure; various renal
diseases including renal failure.
The compounds are also useful for inducing bone regeneration.
In another broad aspect of the invention, undesirable activities of HGF/SF in
vivo may be therapeutically
inhibited by the administration to a mammal of an effective amount of certain
compounds of the invention
for the treatment of various conditions and diseases generally involved in
cellular proliferation and
angiogenesis, among others. Inhibition of HGF/SF is desired, for example, in
the treatment of
dysproliferative diseases such as cancer and metastases, as well as various
inflammatory diseases such as
inflammatory joint and skin diseases. Other activities include but are not
limited to inhibition of
endothelial cell proliferation, inhibition of angiogenesis, angiostasis,
tumoricidal activity, and any
combination of the foregoing. In a further embodiment, the agents inhibit
activity in the presence of
exogenously-added or in cells in which activity is present or induced.
Abnormal vascular proliferation
such as occurs in diabetic retinopathy is also treatable by the methods of the
invention.
CA 02452445 2010-01-21
The compounds of the invention useful for inhibiting HGF/SF activity are
characterized by being small organic molecules or
peptides with one or more of the activities of inhibiting proliferation of
endothelial cells in vitro or in vivo, inhibiting the growth,
scatter or metastasis of tumor cells in vitro or in vivo, inhibiting scatter,
or inhibiting anti-apoptotic activity. Preferred are
compounds in which such activities are exhibitable in the presence of
exogenously-added HGF/SF. The present invention
embraces the use of all such molecules for treatment of various conditions or
diseases in which decreased or inhibited HGF/SF
activity is desirable.
In addition, certain compounds of the invention have been found to have either
antagonistic (activating) or antagonistic
(inhibitory) activities directed not only to HGF/SF but also to VEGF and FGF,
as will be seen in the examples below. The
invention is also directed to use of these compounds to agonize or antagonize
the activities of these growth factors, as well as to
other factors which are tyrosine kinase receptors. In particular, the
compounds 3,3-dibromo-l-phenyl-1,2,3,4-
tetrahydroquinoline-2,4-dione and 4- (4-chlorophenyl)-6- (dimetlrylamino)-2-
plienyl-5-pyrimidinecarbonitrile have VEGF-like
activity and these compounds and structurally-related VEGF agonists or mimics
are embraced herein for the treatment of various
conditions and diseases for which VEGF would be useful for therapy in a
mammal, preferably a human, such as but not limited to
acceleration of wound healing, and in particular, diabetic wound healing. The
compounds are generally useful for promoting
proliferation of vascular endothelial cells and promoting vascularization, for
such other uses as restenosis for treatment of
coronary artery disease, angina and other ischemic diseases, including stroke.
These and other aspects of the present invention will be better appreciated by
reference to the following drawings and Detailed
Description.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts the ability of angiogenic peptides P3 (TMGFTAPRFPHY; SEQ ID
NO: 1) and P4 (KVWYHTTSIPSH; SEQ ID
NO:2) of the invention to stimulate endothelial cell proliferation, as
compared to HGFISF, measured by the uptake of
radiolabeled thymidine.
Figure 2 shows the effects of angiogenic peptides P3 (TMGFTAPRFPHY; SEQ ID NO:
I) and P4 (KVWYHTTSIPSH; SEQ ID
NO:2) and HGF/SF on angiogenesis in a rat aortic ring vascular sprouting
assay.
Figure 3 shows the effects of angiogenic peptides P3 (TMGFTAPRFPHY; SEQ ID
NO:1) and P4 (KVWYHTTSIPSH; SEQ ID
NO:2) of the invention on angiogenesis in an in-vivo assay using basement
membrane matrix implanted subcutaneously in mice.
Figures 4A-48 depict the promotion of angiogenic activity of a peptide of the
invention when conjugated to a lysine-rich,
heparan-sulfate-binding sequence or a control sequence. Figure 4A shows the
effect on endothelial cell proliferation compared to
that of the conjugated peptide measured by radiolabeled thymidine
incorporation; P4 (KV WYHTTSIPSH; SEQ ID NO:2), NS-P4
(KVWYHTTSIPSHCQKAKTRAKAAKPDKK; SEQ ID NO:8), HS-P4
(KVWYHTTSIPSHCRPKAKAKAKAKDQTK; SEQ
ID NO: 7); Figure 4B compared the activity to growth factors HGF/SF and bFGF.
Figure 5 shows the ability of angiostatic peptides of the invention to inhibit
HGF/SF-mediated increase in endothelial cell
proliferation, measured by incorporation of radiolabeled thymidine. Met-I
(ATWSHHLSSAGL; SEQ ID NO:3); Met-2
(WPQLPPRPYSTL; SEQ ID NO:4); Met-3 (SNTSAGTPFTSL; SEQ ID NO:5); and Met-4
(DSTPKSTPWYYI; SEQ ID NO:6).
16
CA 02452445 2010-01-21
Figures 6A-6B illustrate the angiostatic activity of peptides of the invention
Met-1 (ATWSHHLSSAGL; SEQ ID NO:3); Met-2
(WPQLPPRPYSTL; SEQ ID NO:4); Met-3 (SNTSAGTPFTSL; SEQ ID NO:5); Met-4
(DSTPKSTPWYYI; SEQ ID NO:6) and
Met-5 (AKTYAGSSYQFG; SEQ ID NO:11) in a human glioblastoma cell line (U87,
Figure 6A) and in a human glioma cell line
(Hs 683, Figure 6B), the extent of tumor growth measured by incorporation of
radiolabeled thymidine.
Figure 7 depicts the stimulation of endothelial cell proliferation by C6 ((4-
chlorophenyl) [3-(2-(2-thienyl)vinyl)-lH-pyrazol-l-
yl]methanone), a compound of the invention with HGF/SF-like activity, and the
inhibition of the observed stimulation by
inclusion of c-met.
Figure 8 A-B show the induction of scatter of MDCK cells by (4-chlorophenyl)[3-
(2-(2-thienyl)vinyl)-1H-pyrazol-l-yl] methanone.
Figure 9 shows the protection of MDCK cells from adriamycin-induced apoptosis
by C6 ((4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-
pyrazol- l -yl ]methanone).
Figure 10 shows a dose-response curve of the stimulation of endothelial cell
proliferation by C6 ((4-chlorophenyl)[3-(2-(2-
thienyl)vinyl)-I H-pyrazol- l -yl]methanone).
Figure 11 shows the ability of the compound C2 (4-(2,6-dichlorobenzyl)-3,5-
dimethyl-IH-pyrazol-l-yl)-3-(2,6-dichlorophenyl)-
5-methanone) on HGF/SF-mediated endothelial cell proliferation. First bar:
control; second bar: HGF/SF alone; third bar; C2
alone; fourth bar, HGF/SF + C2.
Figure 12 A-B shows the results from a Matrigel in-vivo assay using 1-(4-
chloro-3-methylphenyl)-3-(2, 6-dichlorophenyl)-prop-
2-en-l-one and (4-(2-chloro-6-fluorobenzyl)-3,5-dimetliyl-IH-pyrazole-l-yl) (3-
(2,6-dichlorophenyl)-5-methylisoxazol-4-yl)
methanone, respectively.
Figure 13 shows the results of a clonogenic assay using DU145 cells and C2A
((4-(2-chloro-6-fluorobenzyl)-3,5-dimethyl-1H-pyrazole-l-
yl) (3-(2, 6-dichlorophenyl)-5-methylisoxazol-4-yl)methanone).
Figure 14 shows the results of a clonogenic assay using DU145 cells and Met-8
(1-(4-chloro-3-methylphenyl)-3-(2,6-dichlorophenyl)-prop-
2-en-l-one).
Figure 15 shows improved blood flow in mice following removal of the femoral
artery after treatment with C6 ((4-
chlorophenyl)[3-(2-(2-thienyl) vinyl)-IH-pyrazol-l-yl] methanone).
Figure 16 A-B shows the effect of the compound M8 (1-(4-chloro-3-methylphenyl)-
3- (2, 6-dichlorophenyl)-prop-2-en-I -one) on
inhibition of endothelial cell proliferation induced by the growth factors
HGF/SF, VEOF and FGF at 1.5 micromolar (A) and 3.0
micromolar (B).
Figure 17 shows the VEGF-like activity of two compounds of the invention vs
control (C): 3,3-dibromo-l-phenyl-1,2,3,4-
tetrahydroquinoline-2,4-dione (VC8) and 4-(4- chlorophenyl)-6- (dimethylamino)-
2-phenyl-5-pyrimidinecarbonitrile (VC14).
17
CA 02452445 2010-01-21
Figure 18 shows the stimulation of 3H-thymidine incorporation into HUVEC by C6
((4-chlorophenyl)[3-(2-(2-
thienyl)vinyl)-IH-pyrazol-1-yl] methanone).
Figure 19 depicts the phosphorylation of Erk by HGF/SF and (4-chlorophenyl)[3-
(2-(2-thienyl)vinyl)-1H-
pyrazol-l -yl] methanone.
Figure 20 demonstrated the efficacy of C6 ((4-chlorophenyl)[3-(2-(2-
thienyl)vinyl)-1H-pyrazol-l-
yljmethanone) in a pig wound healing model.
Figure 21 shows the ability of C6 ((4-chlorophenyl) [3-(2-(2-thienyl)vinyl)-1H-
pyrazol-l-yl]methanone) to
increase capillary number in the ischemic mouse hindlimb.
Figure 22 depicts the dose-dependent phosphorylation of HUVECs and MDCK cells
by C6 ((4-
chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-l-yl] methanone).
Figure 23 A-B shows the increased survival time in mice by infra-tumor
injection of M8 (1-chloro-3-
methylphenyl)-3-(2, 6-dichlorophenyl)-prop-2-en- l -one).
DETAILED DESCRIPTION OF THE INVENTION
The agents and method of the invention are directed to modulation of cellular
proliferation to provide new
and effective agents and methods for the prophylaxis and/or treatment of
various conditions and diseases
in which limited cellular proliferation, or, in contrast, excessive
proliferation of cells, is responsible for
pathology, or at least for the prolongation of rebound from a pathological
state to homeostasis. Using new
identification methods, the inventors herein have found surprising and
unexpected activity of various
peptides as well as of small-molecule compounds, some of which promote, and
others inhibit, cellular
proliferation. The methods may be used to identify further agents with the
aforementioned activities. By
way of non-limiting examples, certain of such agents have been found to
promote angiogenesis in vitro
and in vivo ; others inhibit angiogenesis in vitro and in vivo, and inhibit
proliferation of dysproliferative
tissues as evaluated in two cancer models. Additional activities are also
seen. Moreover, and not to be
bound by theory, the proliferation promoting and antiproliferative peptides
identified herein are believed
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to exert their effects by agonizing and antagonizing, respectively, the c-met
receptor present on numerous
cell types within the body, comprising various tissues and organs, such cells
including but not limited to
epithelial cells, endothelial cells, fibroblasts, neuronal cells, and smooth
muscle cells. Tissues and organs
comprising such cell types are targets for the various activities described
herein. As the effects of the
agents herein on a single cell type comprising a tissue or organ may account
for the therapeutic goal
described, the agents herein may have profound effects on tissues or organs
whose cells expressing c-met
comprise only a small fraction. The extent of expression of the target
receptor does not detract from the
utility of the agents and methods herein. The foregoing activities display
agonist or antagonist activities
of HGF/SF, and as such the peptides, small-molecule compounds and other
compounds embraced herein
may be considered to be HGF/SF agonists or antagonists. Furthermore, as will
be seen below, the
compounds of the invention also act as agonists or antagonists of other
tyrosine kinase receptors,
including but not limited to VEGF and FGF, and the present invention and
identification of small-
molecule agonists and antagonists extends to these receptors generally.
For example, the duration of wound healing, vascularization of a damaged
and/or ischemic organs,
transplants or grafts, normalization of myocardial perfusion as a consequence
of chronic cardiac ischemia
or myocardial infarction, development or augmentation of collateral vessel
development after vascular
occlusion or to ischemic tissues or organs, and vascularization of grafted or
transplanted tissues, organs,
or wound healing, may be accelerated by promoting cellular proliferation,
particularly of vascular cells.
Further utility is in the promotion of endothelial growth in vascular grafts
and transplants.
In other cases where abnormal or excessive cellular proliferation is the cause
of pathology, such as in
dysproliferative diseases including cancer and psoriasis, various inflammatory
diseases characterized by
proliferation of cells such as atherosclerosis and rheumatoid arthritis, and
neovascularization in the eye as
a consequence of diabetic retinopathy, suppression cellular proliferation is a
desired goal in the treatment
of these and other conditions. As the antiproliferative agents of the
invention have been found to possess
antiproliferative activity on cells, as well as antiangiogenic activity, both
activities may be beneficial in
the treatment of, for example, solid tumors, in which both the
dysproliferative cells and the enhanced
tumor vasculature elicited thereby are targets for inhibition by the agents of
the invention. In either case,
therapy to promote or suppress proliferation may be beneficial locally but not
systemically, and for a
particular duration, and proliferation modulating therapies must be
appropriately applied. The invention
embraces localized delivery of such agents to the affected tissues and organs,
to achieve a particular
effect.
As noted above, modulating cellular proliferation, either by promoting the
growth or new cells and/or
formation of new blood vessels, or by inhibiting growth of cells and/or
inducing destruction of existing
vasculature, is a therapeutically-desirable goal for the prophylaxis or
treatment of numerous conditions
and diseases, including such major pathologies as myocardial ischemia, cancer,
inflammatory joint and
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CA 02452445 2010-01-21
skin diseases, diabetic retinopathy, and wound healing, as well as adjunctive
therapy to increase the success rate of,
for example, organ transplants and skin grafts. The examples provided herein
below are merely illustrative of the
range of utilities or proliferation promoting and antiproliferative agents,
which include but are not limited to
angiogenic and angiostatic agents; such uses are known to the skilled artisan;
moreover, various citations referred to
herein offer guides to certain of the uses mentioned here. For example,
inhibition of
cellular growth is desirable in the treatment of dysproliferative diseases
including cancers, and the additional
antiangiogenic activity especially of solid tumors is desirable, as such
tumors not only require an enhanced blood
supply to feed the tumor, but produce factors that stimulate tumor
vascularization. Vascularization of the vitreous
humor of the eye as a consequence of diabetic retinopathy is a major cause of
blindness, and inhibition of such
vascularization is desirable. Other conditions in which vascularization is
undesirable include certain chronic
inflammatory diseases, in particular inflammatory joint and skin disease, but
also other inflammatory diseases in
which a proliferative response occurs and is responsible for part of all of
the pathology. For example, psoriasis is a
common inflammatory skin disease characterized by prominent epidermal
hyperplasia and neovascularization in the
dermal papillae. Proliferation of smooth muscle cells, perhaps as a
consequence of growth factors, is a factor in the
narrowing and occlusion of the macro vase ulature in atherosclerosis,
responsible for myocardial ischemia, angina,
myocardial infarction, and stroke, to name a few examples. Peripheral vascular
disease and arteriosclerosis
obliterans comprise an inflammatory component. Numerous diabetic complications
such as atherosclerosis, and
particularly diabetic nephropathy, characterized by basement membrane
thickening and mesangial cell proliferation,
are believed to have a component of cellular proliferation attendant to
excessive production of growth factors as a
consequence of chronic hyperglycemia. These examples of proliferative diseases
are given by way of illustration
only, and the theoretical basis for their etiology as proliferative processes
is not intended to be limiting to the
invention, and applicants have no duty to disclose or be bound by such
disclosure.
Moreover, localized ablation of tissues or even organs using antiproliferative
or antiangiogenic agents may find use
in treatment of certain central nervous system diseases or conditions which
otherwise may require dangerous
invasive procedures; removal of cosmetically undesirable cutaneous lesions are
further targets for the
antiproliferative agents of the invention. In reproductive biology, such
antiproliferative agents may be used as
abortifacients or for non-surgical castration, particularly for use in
livestock and domesticated animals. These are
also merely illustrative of the uses of the instant agents.
On the other hand, poorly perfused tissues and organs, such as the heart as a
sequela of myocardial infarction, as
well as to promote wound healing, organ transplantation, acceleration of
endothelial cell growth and vascularization
of vascular grafts in order to promote integration of the graft, prevent graft
failure due to reocclusion, and to enhance
skin grafting, are desirable targets for increasing vascularization and uses
of the angiogenic agents herein. Enhanced
vascularization of a chronically ischemic organ is a therapeutically
beneficial goal.
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WO 02/02593 PCT/US01/20849
The term "angiogenesis," as used herein, refers to the formation of blood
vessels. Specifically,
angiogenesis is a multistep process in which endothelial cells focally degrade
and invade through their
own basement membrane, migrate through interstitial stroina toward an
angiogenic stimulus, proliferate
proximal to the migrating tip, organize into blood vessels, and reattach to
newly synthesized basement
membrane (see Folkman et al., Adv. Cancer Res., Vol. 43, pp. 175-203 (1985)).
These processes are
controlled by soluble factors and by the extracellular matrix (see Ingber et
al., Cell, Vol. 58, pp. 803-805
(1985)).
Accordingly, an aspect of the present invention extends to methods for
identifying agents with cellular
proliferation promoting or antiproliferative activity, including but not
limited to angiogenic or angiostatic
(antiangiogenic) activity. Such agents are not limited to any particular
structural or chemical class. As
will be seen in the Examples below, the methods described herein were used to
identify peptides as well
as small-molecule compounds with agonist or antagonist activities, but such
methods may be used to
identify active agents in other structural classes. Furthennore, once such
agents are identified, analysis of
the three-dimensional structure may be used to model other small-molecule
compounds with similar or
increased activities, also as described below. Such methods, which often
employ structural determination
of the interactions between biomolecules and their ligands, such as by nuclear
magnetic resonance
spectroscopy or x-ray crystallographic methods, are known to the skilled
artisan and may be applied to the
identification of the sites of interaction between c-met and the peptides of
the invention for the
development of further compounds, which may be peptides or small-molecule,
organic drugs, which
mimic the interactions and activity of the peptides described herein. The
present invention embraces
methods for developing and screening small-molecule and other mimics of the
instant compounds based
upon the properties hereindescribed for the peptides of the invention.
The methods for identifying an proliferation promoting agent are based upon
the ability of certain active
agents, particularly peptides, to 1) bind to a monoclonal or polyclonal
antibody to HGF/SF, and 2) exhibit
proliferation promoting activity, such as angiogenic activity, in one or more
in-vitro and/or in-vivo assays.
Using these methods, which will be elaborated upon below, several peptides
have been identified with
potent proliferative and angiogenic activity.
Furthermore, antiproliferative agents, such as but not limited to angiostatic
agents, may be identified by
ability to 1) bind to the HGF/SF receptor, c-met, and 2) exhibit
antiproliferative activity, such as but not
limited to angiostatic activity, in one or more in-vitro or in-vivo assays.
Using these methods, which will
be elaborated upon below, several peptides have been identified with potent
antiproliferative activity.
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Before describing the particular aspects of the invention in more detail, the
following discussion is
applicable to all of the various aspects of the invention described herein, in
particular the peptide and
small-molecule compounds with agonist or antagonist activity.
The agents of the invention may be administered to the desired site in the
body or target tissue or organ by
any means that achieves the desired therapeutic effect. By way of non-limiting
example, proliferation
promoting agents including angiogenic agents maybe administered locally, such
as by injection or
deposition in a target tissue or organ, or by the implantation of a controlled
release delivery device or
matrix containing the agent, to achieve local effects. Such sites may be
accessed surgically, or via
transcutaneous catheterization to gain access to a tissue or organ through the
major vasculature. For
example, enhancing the perfusion of the ischemic heart may be achieved by use
of a transcutaneous
catheter that may be positioned to release the angiogenic agent of the
invention into the coronary
vasculature.
For antiproliferative agents including antiangiogenic (angiostatic) agents,
local administration of an agent
at the desired site of activity, such as a tumor or the vitreous humor, may be
carried out, or implantation of
a controlled release delivery device containing an agent of the invention in
the tumor or eye, may be
desirable to achieve local effects. Surgical or transcutaneous methods may
also be used. These and other
means for contacting the agents of the invention with the desired target
cells, tissue or organs will be
readily apparent to the skilled artisan.
In yet another aspect of the present invention, provided are pharmaceutical
compositions of the above
agents. As noted above, the application and duration of application of the
compounds of the invention
may require particular local placement or delivery, for example, exposure of
the antiproliferative
compounds to a solid tumor or within the vitreous humor; and avoidance, for
example, of systemic
exposure. Exposure of proliferation promoting agents such as angiogenic
compounds to a transplanted or
ischemic tissue or organ is desirable without exposing such agents to other
sites in the body. Such
considerations, depending on the target cells, tissues or organs, whether the
therapy is to promote or
suppress proliferation, and the duration of exposure, may be determined
readily by the skilled artisan.
The formulation of the instant compounds in appropriate vehicles or carriers
or drug delivery systems is
also determinable by the skilled artisan, and all such methods of delivery are
embraced herein. Examples
are provided herein by way of illustration only, and are not intended to be
limiting whatsoever.
Such pharmaceutical compositions may be for administration to a particular
site by injection,
catheterization or implantation, but may also be delivered for certain uses by
other routes including oral,
pulmonary, nasal or other forms of administration. In general, comprehended by
the invention are
pharmaceutical compositions comprising effective amounts of an agent or
agents, or derivative products,
of the invention together with pharmaceutically acceptable diluents,
preservatives, solubilizers,
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WO 02/02593 PCT/US01/20849
emulsifiers, adjuvants and/or carriers. Such compositions may include diluents
of various buffer content
(e.g., Tris-HC1, acetate, phosphate), pH and ionic strength; additives such as
detergents and solubilizing
agents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid,
sodium metabisulfite),
preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g.,
lactose, malmitol). The
foregoing examples are merely illustrative and non-limiting, as the skilled
artisan will be amply aware of
suitable excipients and other component of a pharmaceutical composition
comprising one or more agents
of the invention.
For controlled delivery, incorporation of the material into particulate
preparations of polymeric
compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes
maybe used, or the use of a
controlled release device, such as an implantable osmotic or other type of
pump. Another form of a
controlled release of this therapeutic is by a method based on the Oros
therapeutic system (Alza Corp.),
i.e. the drug is enclosed in a semipermeable membrane which allows water to
enter and push drug out
through a single small opening due to osmotic effects. Likewise, the skilled
artisan will be amply aware
of suitable delivery methods that maybe extended to the agents of the
invention to achieve the intended
therapeutic goals of the invention. Such local release may be desirably, for
example, with
antiproliferative agents for treatment of a tumor or abnormal vascularization
in the eye; and proliferative
agents at the site of grafts or transplants.
In another embodiment of the invention, cells of the body may be transfected
with a polynucleotide
encoding the various peptides of the invention, or a polynucleotide encoding a
protein which comprise a
peptide agent of the invention, including degenerate polynucleotides which
encode the same peptides or
proteins comprising the aforementioned peptides. Furthermore, such degenerate
polynucleotides may be
optimized for expression in the target mammalian cells. The transfection
carried out by any number of
means, for example, using a viral vector, wherein the transfected cells
express and secrete the peptide with
attendant local effects. The DNA, virus, or other conveyance or vector for the
polynucleotide can be
administered to the target site by catheter or other means. Such vectors
include an attenuated or defective
DNA virus, such as but not limited to herpes simplex virus (HSV),
papillomavirus, Epstein Barr virus
(EBV), adenovirus, adeno-associated virus (AAV), lentivirus and the like.
Defective viruses, which
entirely or almost entirely lack viral genes, are preferred. Defective virus
is not infective after
introduction into a cell. Use of defective viral vectors allows for
administration to cells in a specific,
localized area, without concern that the vector can infect other cells. Thus,
particular tissues can be
specifically targeted. Examples of particular vectors include, but are not
limited to, a defective herpes
virus 1 (HSV1) vector [Kaplitt et al., Molec. Cell. Neurosci. 2:320-330
(1991)], an attenuated adenovirus
vector, such as the vector described by Stratford-Perricaudet et al. [J. Clin.
Invest. 90:626-630 (1992)],
and a defective adeno-associated virus vector [Samulski et al., J. Virol.
61:3096-3101 (1987); Samulski et
al., J. Virol. 63:3822-3828 (1989)]. Alternatively, the vector can be
introduced in vivo by lipofection.
For the past decade, there has been increasing use of liposomes for
encapsulation and transfection of
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WO 02/02593 PCT/US01/20849
nucleic acids in vitro. Synthetic cationic lipids designed to limit the
difficulties and dangers encountered
with liposome mediated transfection can be used to prepare liposomes for in-
vivo transfection of a gene
encoding a marker [Feigner, et. al., Proc. Natl. Acad. Sci. U.S.A. 84:7413-
7417 (1987); see Mackey, et
al., Proc. Natl. Acad. Sci. U.S.A. 85:8027-8031 (1988)]. The use of cationic
lipids may promote
encapsulation of negatively charged nucleic acids, and also promote fusion
with negatively charged cell
membranes [Feigner and Ringold, Science 337:387-388 (1989)]. The use of
lipofection to introduce
exogenous genes into the specific organs in vivo has certain practical
advantages. Molecular targeting of
liposomes to specific cells represents one area of benefit. It is clear that
directing transfection to particular
cell types would be particularly advantageous in a tissue with cellular
heterogeneity, such as pancreas,
liver, kidney, and the brain. Lipids may be chemically coupled to other
molecules for the purpose of
targeting [see Mackey, et. al., supra]. Targeted peptides, e.g., hormones or
neurotransmitters, and proteins
such as antibodies, or non-peptide molecules could be coupled to liposomes
chemically.
It is also possible to introduce the vector in vivo as a naked DNA plasmid.
Naked DNA vectors for gene
therapy can be introduced into the desired host cells by methods known in the
art, e.g., transfection,
electroporation, microinjection, transduction, cell fusion, DEAF dextran,
calcium phosphate precipitation,
use of a gene gun, or use of a DNA vector transporter [see, e.g., Wu et al.,
J. Biol. Chem. 267:963-967
(1992); Wu and Wu, J. Biol. Chem. 263:14621-14624 (1988); Hartmut et al.,
Canadian Patent Application
No. 2,012,311, filed March 15, 1990].
In a preferred embodiment of the present invention, a gene therapy vector as
described above employs a
transcription control sequence operably associated with the sequence for the
angiogenic or angiostatic
peptide inserted in the vector. That is, a specific expression vector of the
present invention can be used in
gene therapy.
In addition, cells or tissues may be transfected to express a protein of the
invention or a protein
comprising a peptide of the invention, and then implanted at the desired site.
Such cells may be, for
example, derived from a patient's own body such that acceptance of the cells
will occur. Alternatively,
exogenous cells may be used. Such patient-derived or exogenous cells may be
prepared such that they
maybe selectively activated or destroyed, for example, by sensitivity to a
particular drug, or expression
activated by a particular drug, such that the secretion of the desired agent
may be initiated, maintained, or
terminated as appropriate for the duration of appropriate therapy. The ability
to engineer such cells with
the appropriate promoters and sensitivity markers is known in the art.
As noted above, the peptides of the invention may be conservatively
substituted, wherein functionally
equivalent amino acid residues are substituted for residues within the
sequence resulting in a conservative
amino acid substitution. Such alterations define the term "a conservatively-
substituted variant" as used
herein. For example, one or more amino acid residues within the sequence can
be substituted by another
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WO 02/02593 PCT/US01/20849
amino acid of a similar polarity, which acts as a functional equivalent.
Substitutes for an amino acid
within the sequence may be selected from other members of the class to which
the amino acid belongs.
For example, the nonpolar (hydrophobic) amino acids include alanine, leucine,
isoleucine, valine, proline,
phenylalanine, tryptophan and methionine. Amino acids containing aromatic ring
structures are
phenylalanine, tryptophan, and tyrosine. The polar neutral amino acids include
glycine, serine, threonine,
cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic)
amino acids include arginine,
lysine and histidine. The negatively charged (acidic) amino acids include
aspartic acid and glutamic acid.
Such alterations will not be expected to affect apparent molecular weight as
determined by
polyacrylamide gel electrophoresis, or isoelectric point.
Particularly preferred conservative substitutions are:
- Lys for Arg and vice versa such that a positive charge may be maintained;
- Glu for Asp and vice versa such that a negative charge may be maintained;
- Ser for Thr such that a free -OH can be maintained; and
- Gln for Asn such that a free NH2 can be maintained.
It is noted that all amino acid sequences are written starting with the amino-
terminal residue and ending
with the carboxy-terminal residue. Moreover, sequences are provided using one-
letter or three-letter
abbreviations.
The appropriate effective dosage of an agent of the invention, whether a
peptide, small-molecule
compound, DNA vector, or other active agent, may be readily determinable by
following standard
methods. Several animal models are described herein which model conditions and
diseases encountered
in the clinical setting, and as part of a drug development process,
efficacious doses in animal studies, in
particular, dose-response studies, are translated into appropriate doses for
testing in humans, by following
guidelines well known to those skilled in the art. Thus, an effective dose in
a human may be determined
following such industry-standard guidelines.
In another embodiment, a conjugate between the proliferation promoting or
antiproliferative agent of the
invention, particularly a peptide agent, and another moiety may be provided to
enhance particular
characteristics of the agent, such as but not limited to targeting, delivery,
persistence in the body or at the
site of administration, etc. As shown in the examples below, a heparan sulfate-
binding polypeptide was
added to the carboxy-terminus of an angiogenic peptide (carried out by solid-
phase peptide synthesis), and
the resultant polypeptide exhibited superior angiogenic activity. Thus, the
present invention embraces
fusion peptides or other conjugates between the peptides of the invention and
another peptide sequence,
which may be administered as described hereinabove, or cells in the body may
be transfected with a
polynucleotide sequence comprising the fusion peptide resulting in expression
of the agent within the
body. Thus, the agents of the invention need only comprise as a portion
thereof an active agent of the
invention to be embraced herein.
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Identification of Proliferation Promoting Agents
In accordance with the invention, proliferation promoting agents and in
particular peptide agents may be
identified by having the properties of 1) binding to an antibody to HGF/SF;
and 2) having proliferative
activity such as but not limited to angiogenic activity in one or more in-
vitro or in-vivo assays. Binding to
an antibody to HGF/SF refers to agents, including but not limited to peptides
and proteins, which
comprise an epitope recognized by an monoclonal or polyclonal antibody to
HGF/SF. A monoclonal
antibody is preferred. Non-limiting examples of such antibodies include a
polyclonal rabbit anti-HGF/SF
antibody (designated 813), and a monoclonal anti-HGF/SF antibodies (e.g.,
clone 23C2). Means for
assessing the binding includes various immunochemical methods known to one of
skill in the art. Binding
may be measured by binding of the antibody to the agent, wherein the agent is
conjugated to another
entity, such as the phage display method described in the examples below.
Alternatively, the binding to
the anti-HGF/SF antibody may be assessed by measuring competition by the agent
in binding of the
antibody to HGF/SF, any peptide mimetic of HGF/SF, or any agent identified by
the methods herein as an
proliferation promoting agent. Thus, small-molecule drugs on the order of the
size of a hapten may be
identified as interfering with the binding of the antibody to its binding
partner.
Moreover, an activity that may be but not necessarily exhibited by the
proliferation promoting agents of
the invention is the agonism of c-met, the HGF/SF receptor. Thus, an assay for
such agonism may be
included in the methods for identifying such active agents.
In-vivo and in-vitro assays for proliferation promoting including angiogenic
agents are known to the
skilled artisan; several non-limiting examples are described in the Examples,
below. Appropriate cells
for such assays express c-inet.
In one non-limiting example of the practice of the invention, randomly-
generated 12-amino acid peptides
expressed in a phage display system were screened for binding to either a
monoclonal antibody or
polyclonal antibody to HGF/SF, and the phages expressing a peptide binding to
the antibody were
amplified, and rescreened, and after three rounds, twenty positive clones were
selected. Other methods
for generating peptides, as well as small-molecule drugs, are useful as well.
The assay for binding to an
antibody to HGF/SF maybe carried out as above, or in a screen in which direct
binding of the candidate
agent to the antibody is assessed, or in the case of small molecules or
haptens, the assay for binding to an
anti-HGF/SF antibody may be carried out by measuring the inhibition by the
candidate molecule of the
binding of the antibody to either intact HGF/SF, a fragment of the molecule
comprising the epitope
recognized by the antibody, or inhibition of binding of the antibody to one of
the peptides or other small
molecules previously identified as an proliferation promoting or angiogenic
compound by the methods
described herein. As known to one of skill in the art, standard immunoassay
procedures, including ELISA
techniques, can be used, for example, with a bound antibody and labeled
binding partner to the antibody.
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After screening of compounds to identify those capable of binding to the anti-
HGF/SF antibody or
interfering with the binding of the antibody with an angiogenic molecule, the
candidate compound is
evaluated in one or more functional proliferation and/or angiogenesis assays.
Such assays include but are
not limited to an endothelial cell proliferation assay, which measured the
effect of the compound on the
proliferation of endothelial cells in vitro. Other assays include
proliferation of cells which express c-met,
such as but not limited to endothelial cells, epithelial cells, neuronal
cells, and smooth muscle cells. Such
methods are known in the art, examples of which are described in the examples
below. Another assay is
the vascular sprouting or aortic ring assay, in which the outgrowth of
endothelial cells from aortic rings
embedded in collagen gels is evaluated. In an in-vivo assay, a solid gel
comprising basement membrane
and the test sample is implanted subcutaneously in mice, and after a period of
time, ingrowth of blood
vessels into the matrix is determined histologically. As noted in the Examples
below, two peptides T M G
F T A P R F P H Y (SEQ ID No:l) and K V W Y H T T S I P S H (SEQ ID No:2) were
identified as
having activity in the angiogenesis assays.
Moreover, once agents have found, three-dimensional structural analysis may be
used to model other
compounds, including small-molecule drugs, as described herein.
In another embodiment, the proliferation promoting peptides may be modified to
enhance activity. One
non-limiting means is by conjugation of active peptides to a heparan sulfate
binding polypeptide. Such
modification includes chemical conjugation, co-expression on the same peptide
chain; i.e., linkage
through a peptide bond, etc. By way of non-limiting example, the lysine-rich
sequence
K V W Y H T T S I P S H C R P K A K A K A K A K D Q T K (SEQ ID No:7) was
added to the
carboxy-terminus of SEQ ID No:2 (forming SEQ ID No: 10). As shown in the
Examples, the angiogenic
activity of the conjugate was about three-fold higher than a control conjugate
in which the same
angiogenic peptide was conjugated to a non-specific sequence with the same
amino acid composition but
without heparan sulfate-binding activity (Y H T T S I P S H C Q K A K T R A K
A A K P D K K [SEQ
ID No:8]), and had comparable or greater than the mitogenic activity of SF and
bFGF. These data show
that the growth promoting and possibly the angiogenic activity of SEQ ID No:2
can be enhanced by
increasing its affinity to heparin.
Use of Proliferation promoting Agents
As described above, the proliferation promoting agents including angiogenic
agents of the invention may
be used to promote endothelial cell and microvessel growth, with the goal of
increasing vascularization
and perfusion of tissues and organs in the body. They may also be used to
promote growth of other cells
types, such as those expressing c-met. Also as noted above, the agents may be
locally applied or
delivered to the desired site or sites. While the invention embraces any and
all uses of angiogenic an
generally proliferation promoting agents for humans and other mammals, some
examples include
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treatment of ischemic tissues and organs, such as after injury, including
myocardial damage after a heart
attack, promoting vascularization of transplanted, reattached or translocated
tissues or organs, such as
following organ transplants, traumatic injury, promotion of wound healing,
skin and other organ grafting,
to name some examples. They are particularly useful for promoting the growth
of endothelial cells in
vascular grafts and transplants.
The agents of the invention may be formulated into suitable phannaceutical
compositions for
administration. As noted above, such compositions and methods of
administration include the
transfection of cells in the target tissue or organ of the body with a vector,
such as a viral vector, or naked
DNA, to induce expression of the agents of the invention in situ, including
the implantation of cells
expressing an agent or agents of the invention to act locally upon a desired
target to continuously produce
the agent. Such cells may have engineered susceptibility to, for example, a
drug such that at the
conclusion of desired therapy, the cells expressing the agent may be readily
destroyed.
Identification of Antiproliferative Agents
Agents with antiproliferative activity, including angiostatic activity, may be
identified by their ability to
bind to the HGF/SF receptor c-met. As will be shown in the Examples below, a
phage display peptide
library as described herein which expresses a combinatorial library of 12-mers
fused to the pIII coat
protein were screened for binding to the extracellular domain of HGF/SF
receptor C-met. Following this
procedure, four peptides were identified and synthesized by solid-phase
synthesis: A T W S H H L S S A
GL(SEQIDNo:3); WPQLPPRPYSTL(SEQIDNo:4);SNTSAGTPFTSL(SEQID
No:5); and D S T P K S T P W Y Y I (SEQ ID No:6). The ability of these
peptides to stimulate
endothelial proliferation or inhibit HGF/SF-mediated increase in endothelial
proliferation was
subsequently determined. The peptides did not significantly affect endothelial
growth, but completely
inhibited the SF-mediated increase in endothelial growth. These data indicate
that met peptides can bind
HGF/SF receptor C-Met thereby inhibit HGF/SF binding. Thus, these peptides may
have potential
angiostatic activity. Moreover, inhibition of growth of several tumor cells in
vitro in the absence of a
tumor vasculature demonstrates that the antiproliferative agents of the
invention have general
antiproliferative activity, and not necessarily only antiangiogenic activity.
Both activities are useful for
the treatment of certain conditions, such as a solid tumor with both tumor
cellular and vascular targets.
These peptides and conservatively-substituted variants thereof maybe provided
in pharmaceutical
compositions for various uses to inhibit growth and proliferation of cells,
tissues and organs, as well as
inhibit vascularization of tissues and organs, as described above. Also noted
above, the foregoing
methods may be used to identify agents other than peptides with
antiproliferative including angiostatic
activity, by identifying compounds with the property of binding to c-met, and
exhibiting antiproliferative,
such as but not limited to angiostatic, activity. Moreover, once agents have
found, three-dimensional
structural analysis may be used to model other compounds, including small-
molecule drugs.
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The antiproliferative peptides of the invention were evaluated in two tumor
models:
human glioma and glioblastoma cell lines. These data show that C-met peptides
each alone or in
combination can block endogenous HGF/SF activity and associated tumor growth.
Use of Antiproliferative Agents
Expression of scatter factor (HGF/SF), and its receptor, c-met, is often
associated with malignant
progression of human tumors, including gliomas. Overexpression of HGF/SF in
experimental gliomas
enhances tumorigenicity and tumor-associated angiogenesis (i.e., growth of new
blood vessels). More
recent studies showed that human glioblastomas are HGF/SF-c-met dependent and
that a reduction in
endogenous HGF/SF or c-met expression can lead to inhibition of tumor growth
and tumorigenicity.
Thus, targeting the HGF/SF-c-met signaling pathway may be an important
approach in controlling tumor
progression.
Examples of cancers, tumors, malignancies, neoplasms, and other
dysproliferative diseases that can be
treated according to the invention include leukemias such as myeloid and
lymphocytic leukemias,
lymphomas, myeloproliferative diseases, and solid tumors, such as but not
limited to sarcomas and
carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,
osteogenic sarcoma,
chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma,
synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
colon carcinoma,
pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous
cell carcinoma, basal cell
carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma,
papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,
bronchogenic carcinoma, renal
cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma,
embryonal carcinoma,
Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell
lung carcinoma, bladder
carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma,
craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma,
meningioma,
melanoma, neuroblastoma, and retinoblastoma.
The present invention is also directed to treatment of non-malignant tumors
and other disorders involving
inappropriate cell or tissue growth by administering a therapeutically
effective amount of an agent of the
invention. For example, it is contemplated that the invention is useful for
the treatment of arteriovenous
(AV) malformations, particularly in intracranial sites. The invention may also
be used to treat psoriasis, a
dermatologic condition that is characterized by inflammation and vascular
proliferation; benign prostatic
hypertrophy, a condition associated with inflammation and possibly vascular
proliferation; and cutaneous
fungal infections. Treatment of other hyperproliferative disorders is also
contemplated. The agents may
also be used topically to remove warts, birthmarks, moles, nevi, skin tags,
lipomas, angiomas including
hemangiomas, and other cutaneous lesions for cosmetic or other purposes.
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As noted above, other uses of the agents herein include intentional ablation
or destruction of tissues or
organs in a human or animal, for example, in the area of animal husbandry, and
in the field of
reproductive biology, to reduce the number of'developing embryos; as an
abortifacient, and as a means to
achieve a biochemical castration, particularly for livestock and domesticated
animals such as pets.
The agents of the invention may be formulated into suitable phannaceutical
compositions for
administration. As noted above, such compositions and methods of
administration include vectors and
microorganisms including cells expressing the peptide agents of the invention
and larger polypeptides
comprising the peptides, and extend to the transfection of cells in the target
tissue or organ of the body
with a vector, such as a viral vector, or naked DNA, to induce expression of
the agents of the invention in
situ, including the implantation of cells expressing an agent or agents of the
invention to act locally upon a
desired target to continuously produce the agent. Such cells may have
engineered susceptibility to, for
example, a drug such that at the conclusion of desired therapy, the cells
expressing the angiogenic agent
may be readily destroyed.
In addition to the peptide agents of the invention described above, the
inventors herein have identified
various small organic molecules of molecular weight below about 1,000 Daltons
with the ability to either
mimic or antagonize the biological activities of various growth factors that
bind the tyrosine kinase
receptor, such as hepatocyte growth factor / scatter factor (HGF/SF), vascular
endothelial growth factor
(VEGF), and fibroblast growth factor (FGF). The present invention is directed
to methods for the
modulation of the various activities exhibited by such growth factors, for
example, hepatocyte growth
factor / scatter factor (HGF/SF), using small non-protein or non-peptide
molecules. The inventors herein
have identified for the first time small organic molecules that either mimic
or have HGF/SF, VEGF or
FGF activities, as well as those that are capable of inhibiting or
antagonizing the activities of HGF/SF,
VEGF and FGF. Thus, small molecule compounds having the HGF/SF-like or HGF/SF-
inhibitory
activities are described and their uses for the treatment of various
conditions and diseases embraced
herein. With the attendant difficulties in administering protein therapeutic
agents at a desired level and
for a duration effective to achieve acute and particularly chronic therapeutic
goals, notwithstanding the
cost of manufacture, the discovery by the inventors herein of small molecules
with the desirable growth
factor modulating activities offers pharmaceutically-desirable means to
address a large number of
conditions and diseases heretofore poorly or minimally treatable with
available therapies.
Small-molecule, non-protein or non-peptide compounds with HGFISF-like activity
are characterized by
one or more of the following activities: promoting proliferation of
endothelial cells in vitro or in vivo,
promoting angiogenesis in vitro or in vivo, promoting angiogenesis in wounds
in vivo, promoting the
growth of tumor cells in vitro or in vivo, promoting scatter, promoting anti-
apoptotic activity, or inducing
gene expression of angiogenic-cascade-related genes such as but not limited to
IL-8 and angiopoietin-2.
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The modulator compounds of the invention, whether exhibiting HGF/SF-like
activity, herein referred to
interchangeably as HGF/SF agonist activity, or exhibiting HGF/SF inhibitory
activity, herein referred to
interchangeably as HGF/SF antagonist activity, are by theory acting through
the HGF/SF receptor c-met.
While Applicants have no duty to disclose the theory by which the compounds of
the invention are
operating and are not bound thereto, the small-molecule compounds of the
invention modulate c-met
activity, and bind to c-met. Preferred are compounds in which the
aforementioned activity is inhibited or
competed in the presence of exogenously-added c-met receptor. The skilled
artisan can readily identify
such compounds by carrying out the foregoing assays as described in the
examples below, and the present
invention embraces the use of any and all such compounds for the purposes
described herein.
While the discussions herein describe the activities of HGF/SF agonists and
antagonists, the skilled artisan
will recognize, based on the studies described herein using other growth
factors, that similar uses are
afforded the agonists and antagonist compounds of the invention for these
other growth factors.
Small-molecule, non-protein or non-peptide compounds with HGF/SF-antagonist
activity are
characterized by one or more of the following activities: inhibiting
proliferation of endothelial cells in
vitro or in vivo, inhibiting the growth, scatter or metastasis of tumor cells
in vitro or in vivo, inhibiting
scatter, or inhibiting anti-apoptotic activity. Preferred are compounds in
which such activities are
exhibitable in the presence of exogenously-added HGF/SF. The skilled artisan
can readily identify such
compounds by carrying out the foregoing assays as described in the examples
below, and the present
invention embraces the use of any and all such compounds for the purposes
described herein.
The small organic molecules of the invention preferably have a molecular
weight below 1,000 Daltons
and more preferably of about 200 Daltons to about 1,000 Daltons; most
preferably between about 300
Daltons and about 750 Daltons, and even most preferably between about 300
Daltons and about 500
Daltons. Moreover, the compounds preferably are not proteins or peptides, but
may fall into any other
class of organic molecule.
Compounds with HGF/SF activity are therapeutically useful for the treatment of
numerous conditions and
diseases in many but not all cases related to enhancement of cellular
proliferation or vascular proliferation
(angiogenesis). These have been described in detail above. One aspect of the
invention embraces the
uses of the small molecule compounds described herein for the treatment of
these conditions and diseases.
These conditions and diseases are related to organ dysfunction and
regeneration, reducing duration of
wound healing, normalization of myocardial perfusion as a consequence of
chronic cardiac ischemia or
myocardial infarction, development or augmentation of collateral vessel
development after vascular
occlusion of or to ischemic tissues or organs, and vascularization of grafted
or transplanted tissues,
organs, or wound healing. These desired activities may be accelerated by
administration of a compound
of the invention. For example, promoting cellular proliferation, particularly
of vascular cells, may be
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applied to the treatment of an ischemic, damaged or transplanted organ.
Prophylaxis or treatment may be
provided by contacting the tissue with an effective angiogenic amount of a
compound of the invention.
Contact may be provided by any appropriate means to deliver an effective
amount of the agent for a
duration to achieve the desired results. By way of non-limiting example,
topical application may be
applied to the desired target, or by infusion, bathing, or implantation of a
sustained delivery device. For
systemic administration, oral or parenteral routes may be employed. The target
cells or tissue may be, for
example, the liver or kidney, a transplanted or grafted tissue or organ such
as skin, heart, vascular tissue or
kidney, an ischemic organ, such as a heart following myocardial infarction or
angina, a tissue or organ
damaged by wounding, surgical intervention, vascular tissue, neural tissue, a
wound, ulcer, etc. The cells
may be, by way of non-limiting example, epithelial cells, endothelial cells,
and smooth muscle cells, and
tissues and organs comprising such cells. Promotion of growth and/or
regeneration of neural tissue, teeth,
and other tissues are embraced herein. Preferred cells, organs and tissues
comprise the c-met receptor.
The aforementioned compounds with HGF/SF activity are also desirably useful
for the treatment of
various hepatic diseases including cirrhosis and liver failure; various renal
diseases including renal failure.
The compounds are also useful for inducing bone regeneration.
In one preferred embodiment, treatment of the endothelial cell dysfunction,
vasculopathy and wound
healing dysfunction that typifies diabetes mellitus is among the uses of the
methods and compounds
herein.
Thus, one aspect of the invention is directed to methods for the prophylaxis
or treatment of a condition or
disease in a mammal in which HGF/SF activity is desired or increased activity
is desired comprising
administering to the mammal an effective amount of a small-molecule compound
with HGF/SF activity.
The HGF/SF activity of a small-molecule compound of the invention is inhibited
or blocked in the
presence of or by preincubation with c-met receptor.
In another broad aspect of the invention, compounds which antagonize HGF/SF
activity have
therapeutically-desirable properties for the treatment of conditions and
diseases in which HGF/SF activity
is undesirable. The use of any and all such small-molecule compounds is
embraced herein. For example,
abnormal cellular proliferation such as occurs in dysproliferative diseases
such as various cancers and
psoriasis, are such amenable conditions. Other conditions include inflammatory
diseases, which exhibit a
proliferative component, such as the intimal thickening and smooth muscle cell
proliferation in
atherosclerosis, among other inflammatory conditions.
Thus, another embodiment of the invention is directed to methods for the
prophylaxis or treatment of a
condition or disease in a mammal in which HGF/SF activity is not desired or
decreased activity is desired
comprising administering to the mammal an effective amount of a small-molecule
compound with
HGF/SF antagonist activity. The HGF/SF antagonist activity of a small-molecule
HGF/SF antagonist
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compound of the invention may occur alone or only in the presence of
exogenously-added HGF/SF or in
cells or tissues in which HGF/SF is expressed.
Expression of scatter factor (HGF/SF), and its receptor, c-met, is often
associated with malignant
progression (metastasis) of human tumors, including gliomas. Overexpression of
HGF/SF in experimental
gliomas enhances turnorigenicity and tumor-associated angiogenesis (i.e.,
growth of new blood vessels).
More recent studies showed that human glioblastomas are HGF/SF-c-met dependent
and that a reduction
in endogenous HGF/SF or c-niet expression can lead to inhibition of tumor
growth and tumorigenicity.
Thus, targeting the HGF/SF-c-met signaling pathway using a compound as
characterized above is an
important approach in controlling tumor progression.
The present invention is also directed to treatment of non-malignant tumors
and other disorders involving
inappropriate cell or tissue growth by administering a therapeutically
effective amount of an agent of the
invention. For example, it is contemplated that the invention is useful for
the treatment of arteriovenous
(AV) malformations, particularly in intracranial sites. The invention may also
be used to treat psoriasis, a
dermatologic condition that is characterized by inflammation and vascular
proliferation; benign prostatic
hypertrophy, a condition associated with inflammation and possibly vascular
proliferation; and cutaneous
fungal infections. Treatment of other hyperproliferative disorders is also
contemplated. The agents may
also be used topically to remove warts, birthmarks, moles, nevi, skin tags,
lipomas, angiomas including
hemangiomas, and other cutaneous lesions for cosmetic or other purposes.
The small-molecule HGF/SF activity modulator compounds of the invention fall
generally into four
groups, as described below. As used herein, the term "alkyl" means straight-
chain, branched-chain or
cyclo saturated aliphatic hydrocarbon groups preferably containing from one to
about 6 carbon atoms.
Representative of such straight-chain groups are methyl, ethyl, butyl, pentyl,
hexyl and the like.
Examples of branched-chain groups include isopropyl, isobutyl and t-butyl.
Cycloalkyl includes groups
such as but not limited to cyclopropyl, cyclobutyl, cyclopentyl and
cyclohexyl. The term "aryl" refers to,
for example, phenyl, biphenyl and naphthyl groups, which are optionally
substituted by one or more
halogen (F, Cl, Br and I), Cl to C4 alkyl, or Cl to C4 alkyloxy, where
alkyloxy refers to an alkyl group as
defined above attached to the remainder of the molecule by oxygen. Examples of
alkyloxy include
methoxy, ethoxy, propoxy, isopropoxy and the like. The term "heteroaryl"
refers to heterocyclic groups
containing 4-10 ring members and 1-3 heteroatoms selected from the group
consisting of oxygen, nitrogen
and sulfur. Examples include but are not limited to isoxazolyl,
phenylisoxazolyl, furyl, pyrimidinyl,
quinolyl, tetrahydroquinolyl, pyridyl, imidazolyl, pyrrolidinyl, 1,2,4-
triazoylyl, thiazolyl, thienyl, and the
like. The aryl or heteroaryl group may be optionally substituted by one or
more halogen (F, Cl, Br and I),
Cl to C4 alkyl, Cl to C4 alkyloxy as described above, trifluoromethyl,
difluoromethyl, nitro, hydroxy,
amine (optionally alkyl substituted), or another aryl or another heteroaryl
group as described above.
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The organic compounds described herein with HGF/SF-like agonist or antagonist
activity are merely
illustrative of compounds which modulate one or more of the activities of
HGF/SF and the uses of which
are embraced herein.
Among the compounds and the formulae described below, certain of such
compounds are known, and
others are heretofore undescribed. The present invention embraces all such
novel compounds with one or
more of the activities hereindescribed, as well as pharmaceutical compositions
comprising such
compounds.
In one embodiment, the invention is directed to the use for any one or more of
the aforementioned
purposes of compounds that modulate HGF/SF activity with the general formula
I:
R5
R1_N R4
N
R3
Formula I
wherein
R3 and R5 are independently or together a straight-chain or branched CI-C6
alkyl optionally
substituted with a cyano or halogen, halogen, trifluoromethyl or
difluoromethyl groups;
RI is hydrogen, methyl, CO-Aryl, S02-Aryl, CO-heteroaryl, or CO-alkyl; and
R4 is CH2-Aryl, halogen, arylcarbonylvinyl or S-heteroaryl.
The definitions of the substituents are as described hereinabove. R3 and R5
preferably may be methyl, t-
butyl or chloro groups. The aryl group of substituent RI is preferably an
aromatic group such as phenyl,
naphthyl, or biphenyl, substituted with one or more halogen, Cl to C4 alkyl or
Cl to C4 alkyloxy groups.
The heteroaryl group of substituent RI preferably is a 3-aryl-substituted
isoxazole or 3-aryl-substituted
thienyl group. The alkyl group of substituent RI preferably is t-butyl, or a
CI-C6 straight, branched or
cycloalkyl group. In a most preferred embodiment, R3 is methyl, R5 is chloro,
Rl is methyl, and R4 is 4-
cl-Aorophenylcarbonylvinyl group.
Certain of the compounds of Formula I are novel, and the present invention is
directed to all such novel
compounds. The invention is also directed to a pharmaceutical composition
comprising at least one
compound of Formula I, in a pharmaceutically-acceptable carrier, for any of
the uses described herein.
The invention is also directed to methods for the prophylaxis or treatment of
a condition or disease in a
mammal wherein the effects of HGF/SF would be beneficial by administering to
the mammal an effective
amount of a pharmaceutical composition comprising an active HGF/SF agonist of
Formula I above. As
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mentioned above, such activities include but are not limited to promoting the
proliferation of cells,
including endothelial cells, vascular cells, hepatic cells, renal cells, among
others; promoting
angiogenesis; promoting vascularization; promoting wound healing and
angiogenesis in wound healing,
improving blood flow to ischemic tissues; and other desirable activities
attendant to the desirable
biological activities of endogenously-present or exogenously-administered
HGF/SF. Those compounds of
Formula I exhibiting HGF/SF antagonist activity, determinable readily by one
of skill in the art, would be
useful for the prophylaxis or treatment of a condition or disease in a mammal
wherein the effects of
HGF/SF would be undesirable, by administering to the mammal an effective
amount of a pharmaceutical
composition comprising an active HGF/SF antagonist of Formula I above. Such
utilities include but are
not limited to inhibition of angiogenesis or neovascularization, prevention of
tumor growth or metastasis,
inhibiting scatter, and inhibiting anti-apoptotic activities.
In a further embodiment, the invention is directed to methods for the
prophylaxis or treatment of the
aforementioned conditions and diseases using a therapeutically effective
amount of a compound of
Formula I whose HGF/SF-like activities are inhibited in the presence of, or by
preincubation with, the
HGF/SF receptor c-met.
Non-limiting example of modulators of HGF/SF activity of Formula I include the
following compounds,
most of which, as will be seen in the examples below, exhibit HGF/SF agonist
activity.
3-(5-chloro-1,3 -dimethyl- 1H-pyrazol-4-yl)- 1 -(4-chlorophenyl)prop-2-en- 1 -
one
[4-(2,6-dichlorobenzyl)-3,5-dimethyl-lH-pyrazol-l-yl] [3-(2,6-dichlorophenyl)-
5-methylisoxazol-4-
yl]methanone
(4-(2-chloro-6-fluorobenzyl)-3,5-dimethyl-lH-pyrazole-l-yl)(3-(2,6-
dichlorophenyl)-5-
methylisoxazol-4-yl)methanone
4-(2-chloro-6-fluorobenzyl)-1-((3,4-dichlorophenyl)sulfonyl)-3,5-dimethyl-lH-
pyrazole
4-(2-chloro-6-fluorobenzyl)-1,3,5-timethyl-lH-pyrazole
4-(2-chloro-6-fluorobenzyl)-3, 5-dimethyl-1 H-pyrazole
(4-bromo-3,5-dimethyl-1H-pyrazol-1-yl)(3-(2,6-dichlorophenyl)isoxazole-4-
carbohydrazide)
3-(4-(2,6-dichlorobenzyl)-3,5-dimethyl-1H-pyrazol-1-yl)propanenitrile
3,5-di(tert-butyl)-4-(2-chloro-6-fluorobenzyl)-1H-pyrazole
(4-(2-chloro-6-fluorobenzyl)-3,5-dimethyl-lH-pyrazole-1-yl)(2,6-
dichlorophenyl)methanone
1-(4-(2-chloro-6-fluorobenzyl)-3,5-dimethyl-lH-pyrazole-1-yl)2,2-
dimethylpropan-l -one
(4-(2-chloro-6-fluorobenzyl)-3,5-dimethyl-lH-pyrazole-1 yl)(4-
chlorophenyl)methanone
(4-(2-chloro-6-fluorob enzyl)-3, 5-dimethyl- l H-pyrazole-1-yl) (2-
thienyl)methanone
(4-chlorophenyl)(3,5-dimethyl-4-((1-methyl-lH-imidazol-2-yl)thio)-1 H-pyrazol-
l-yl)methanone
In yet another embodiment, the invention is directed to the use for any one or
more of the aforementioned
purposes of compounds that modulate HGF/SF activity with the general formula
II:
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R3
R1
NJN,R2
03 el
R5
Formula II
wherein
R5 is a C1 to C6 branched or straight-chained alkyl group;
R3 is a substituted or unsubstituted Aryl group;
RI is hydrogen or a Cl to C4 straight-chained, branched or cycloalkyl group;
R2 is COCH2ONCH-Aryl; heteroaryl, COCH2CH2Ary1; Aryl; COS-Aryl; CO-Heteroaryl;
C1 to
C4 straight-chained alkyl, branched alkyl, or cycloalkyl; or wherein R1 and R2
form a cyclic group of 5 or
6 carbon atoms.
The substituents are as defined hereinabove. Preferably, R5 is methyl. R3 is
preferably an alkyl-,
halogen- or alkyloxy-substituted phenyl group such as 2,6-dichlorophenyl. RI
is preferably hydrogen or
methyl. R2 is preferably a substituted pyridyl group such as 2-(6-
trifluoromethyl)pyridyl, a substituted
arylthiocarbonyl group such as 2-(nitrophenyl)thiocarbonyl, or a 4-aryl-
substituted-5-
methylisoxazonecarbonyl group.
Most of the compounds of Formula II exhibit HGF/SF antagonist or inhibitory
activity, as will be seen in
the examples below. When R2 is COCH2ONCH-Aryl, the compounds may exhibit
agonist activity.
Certain of the compounds of Formula II are novel, and the present invention is
directed to all such novel
compounds. The invention is also directed to a pharmaceutical composition
comprising at least one
compound of Formula II, in a pharmaceutically-acceptable carrier, for any of
the uses described herein.
Thus, this aspect of the invention is directed to method for the prophylaxis
or treatment of a condition or
disease in a mammal wherein the effects of HGF/SF would be undesirable, by
administering to the
mammal an effective amount of a pharmaceutical composition comprising an
active HGF/SF antagonist
of Formula II above. Such utilities include but are not limited to inhibition
of angiogenesis or
neovascularization, prevention of tumor growth or metastasis, inhibiting
scatter, and inhibiting anti-
apoptotic activities. As noted above, some compounds in Formula II exhibit
HGF/SF-like activity and are
likewise useful for the prophylaxis or treatment of a condition or disease in
a mammal wherein the effects
of HGF/SF would be beneficial by administering to the mammal an effective
amount of a pharmaceutical
composition comprising an active HGF/SF agonist of Formula II above. As
mentioned above, such
activities include but are not limited to promoting the proliferation of
cells, including endothelial cells,
vascular cells, hepatic cells, renal cells, among others; promoting
angiogenesis; promoting
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vascularization; treatment of wound healing and endothelial cell dysfunction,
improving blood flow to
ischemic tissues; and other desirable activities attendant to the desirable
biological activities of
endogenously-present or exogenously-administered HGF/SF. The activity of a
compound of Formula II
would be readily determinable by one of skill in the art.
In a further embodiment, the invention is directed to methods for the
prophylaxis or treatment of the
aforementioned conditions and diseases using a therapeutically effective
amount of a compound of
Formula II whose HGF/SF antagonist activities occur in the presence of
exogenously-added or
endogenously produced HGF/SF, the latter within the cells or tissues.
Non-limiting examples of compounds of Formula II include:
N'4,5-dimethyl-N'4-(5-nitro-2-pyridyl)-3-(2,6-dichlorophenyl)isoxazole-4-
carbohydrazide
N'4-(2-(((2,4-dichlorobenzylidene)ainino)oxy)acetyl)-3-(2,6-dichlorophenyl)-5-
methylisoxazole-4-
carbohydrazide
N'4-(3-(3,4,5-trimethoxyphenyl)propanoyl)-3-(2,6-dichlorophenyl)-5-
methylisoxazole-4-
carbohydrazide
2-nitrophenyl 2-((3-(2,6-dichlorophenyl)-5-methylisoxazol-4-
yl)carbonyl)hydrazine-l-carbothioate
N'4-((2-methyl-l,3-thiazol-4-4y1)carbonyl)-3-(2,6-dichlorophenyl)-5-
methylisoxazole-4-
4carbohydrazide
N 1-((2-((3-(2,6-dichlorophenyl)-5-methylisoxazol-4-
yl)carbonyl)hydrazino)(methylthio)methylidene)benzene-l -sulfonamide
N'4-(2,4,6-trichlorophenyl)-3-3(2,6-dichlorophenyl)-5-methylisoxazole-4-
carbohydrazide
N'4,3-di(2,6-dichlorophenyl)-5-methylisoxazole-4-carbohydrazide
N'4-(3,5-dichloro-4-pyridyl)-3-(2,6-dichlorophenyl)-5-methylisoxazole-4-
carbohydrazide
N'4-phenyl-3-(2,6-dichlorophenyl)-5-methylisoxazole-4-carbohydrazide
N'4,N'4,5-trimethyl-3-(2,6-dichlorophenyl)isoxazole-4-carbohydrazide
N4-azepan-1-yl-3-(2,6-dichlorophenyl)-5-methylisoxazole-4-carboxamide
N'4-(6-(trifluoromethyl)-2-pyridyl)-3-(2,6-dichlorophenyl)-5-methylisoxazole-4-
carbohydrazide
N'4-(3,3-diethoxypropanoyl)-3-(2,6-dichlorophenyl)-5-methylisoxazole-4-
carbohydrazide
In still a further embodiment, the invention is directed to the use for the
aforementioned purposes of
compounds that modulate HGF/SF activity with the general formula III:
R1 __ N
N
R3
Formula III
wherein
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RI is SO2AIkyl, S02-Aryl, CO-t-Butyl, COAryl, CONHAIkyl; CONHAryl; and
R3 is CHCH-heteroaryl; phenoxyphenyl; heteroaryl; or Aryl substituted
heteroaryl.
Preferably, RI may be SO2Alkyl, wherein Alkyl is Cl to C4 straight-chained,
branched or cyclo, most
preferably S02CH3i S02-Aryl, wherein Aryl is halo, C1-4 alkyl or alkyloxy
substituted phenyl; COAlkyl,
wherein alkyl is C1 to C6 straight-chained alkyl, branched alkyl or
cycloalkyl, most preferably CO-t-
Butyl ; COAryl wherein Aryl is phenyl substituted with halo, CI-C4 alkyl or
alkyloxy; CONHAlkyl
wherein alkyl is Cl to C6 straight-chained alkyl, branched alkyl or
cycloalkyl, most preferably
CONHCH3; or CONHAryl, wherein aryl is phenyl substituted with halo, Cl to C4
alkyl or Cl to C4
alkyloxy. R3 may be CHCH-heteroaryl, where in heteroaryl includes but is not
limited to both cis and
trans CHCH-3-thienyl, CHCH-2-furyl and CHCH-3-furyl, and substituted CHCH-
thienyl and CHCH-
furyl, most preferably CHCH-2-thienyl; phenoxyphenyl; heteroaryl; or aryl
substituted heteroaryl.
Certain of the compounds of Formula III are novel, and the present invention
is directed to all such novel
compounds. Moreover, the invention is also directed to a pharmaceutical
composition comprising at least
one compound of Formula III, in a pharmaceutically-acceptable carrier, for any
of the uses described
herein.
The invention is also directed to methods for the prophylaxis or treatment of
a condition or disease in a
mammal wherein the effects of HGF/SF would be beneficial by administering to
the mammal an effective
amount of a pharmaceutical composition comprising an active HGF/SF agonist of
Formula III above. As
mentioned above, such activities include but are not limited to promoting the
proliferation of cells,
including endothelial cells, vascular cells, hepatic cells, renal cells, among
others; promoting
angiogenesis; promoting vascularization; improving or enhancing wound healing;
treating endothelial cell
dysfunction; improving blood flow to ischemic tissues; and other desirable
activities attendant to the
desirable biological activities of endogenously-present or exogenously-
administered HGF/SF. Those
compounds of Formula III exhibiting HGF/SF antagonist activity, detenninable
readily by one of skill in
the art, would be useful for the prophylaxis or treatment of a condition or
disease in a mammal wherein
the effects of HGF/SF would be undesirable, by administering to the mammal an
effective amount of a
pharmaceutical composition comprising an active HGF/SF antagonist of Formula
III above. Such utilities
include but are not limited to inhibition of angiogenesis or
neovascularization, prevention of tumor growth
or metastasis, inhibiting scatter, and inhibiting anti-apoptotic activities.
In a further embodiment, the invention is directed to methods for the
prophylaxis or treatment of the
aforementioned conditions and diseases using a therapeutically effective
amount of a compound of
Formula III whose HGF/SF-like activities are inhibited in the presence of, or
by preincubation with, the
HGF/SF receptor c-met.
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These compounds generally exhibit HGF/SF stimulatory or agonist activity. Non-
limiting examples of
compounds of Formula III include
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-l-yl]methanone;
1-(methylsulfonyl)-3 -(2-(2-thienyl)vinyl)- l H-pyrazole
2,2-dimethyl- l -(3-(2-(2-thienyl)vnryl)-1 H-pyrazole- l -yl)propan- l -one
N-methyl-3-(2-(2-thienyl)vinyl)-1 H-pyrazole- l -c arboxamide
(4-chlorophenyl)(3-(3-phenylisoxazol-5-yl)-1H-pyrazol-l -yl)methanone
(4-chlorophenyl)(3-(3-(4-chlorophenyl)-5-methylisoxazol-4-yl)-1H-pyrazol-l -
yl)methanone
(4-chlorophenyl)(3-(5-(2-thienyl)-2-thienyl)-IH-pyrazol-l-yl)methanone
(2,4-dichlorophenyl) (3 -(5 -(2,4-difluorophenyl)-2-furyl)-1 H-pyrazol- l -
yl)methanone
N I -phenyl-3 -(2-(2-thienyl)vinyl)-1 H-pyrazole- l -c arb oxamide
(4-chlorophenyl)(3-(2-(5-(2-thienyl)-2-thienyl)-4-methyl-1,3-thiazol-5-yl)-1H-
pyrazol-l -
yl)methanone
(3-benzhydryl-1H-pyrazol-1-yl)(4-chlorophenyl)methanone
N 1-(4-chlorophenyl)-3 -(2-(2-thienyl)vinyl)-1 H-pyrazole-1-darb oxamnide
(4-chlorophenyl) (3 -(2-methylimidazo (1,2-a)pyridin-3 -yl)-1 H-pyrazol-1-
yl)methanone
2-chloro-6-(4-(1-(4-chlorobenzyl)-1H-pyrazol-3-yl)phenoxy)benzonitrile
1-((4-chlorophenyl)sulfonyl)-3-(2-(2-thienyl)vinyl)-1 H-pyrazole
In a further embodiment, the invention is directed to the use for any one or
more of the aforementioned
purposes of compounds that modulate HGF/SF activity with the general formula
IV:
O
R1
R2
Formula IV
Wherein
Rl is Aryl or Heteroaryl; and
R2 is one or more halogen, nitro, Cl to C4 straight-chained alkyl, branched
alkyl, or cycloalkyl,
or C 1 to C4 alkyloxy groups.
The definitions of the aforementioned substituents are described hereinabove.
Preferably, R1 is a phenyl
group substituted with one or more halogen, C 1 to C4 alkyl, or C 1 to C4
alkyloxy groups, or a heteroaryl,
most preferably 4-bromo-2-thienyl, 4-pyridyl, 2-furyl, 3-thienyl, substituted
with halogens and/or Cl to
C4 alkyl. R2 preferably is halogen (F, Cl, Br), nitro, or a C1 to C4 straight-
chained alkyl, branched alkyl,
or cycloalkyl group or a Cl to C4 alkyloxy group; most preferably, R2 is a
methyl group and a chloro
group.
Certain of the compounds of Formula IV are novel, and the present invention is
directed to all such novel
compounds. In addition, the invention is also directed to a pharmaceutical
composition comprising at
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least one compound of Formula IV, in a pharmaceutically-acceptable carrier,
for any of the uses described
herein.
Certain compounds of Formula IV exhibit HGF/SF agonist activity and others
exhibit HGF/SF antagonist
activity. The skilled artisan may readily determine the activity of the
compounds, and the dose at which
the compound exhibits such activity. Thus, the invention is also directed to
methods for the prophylaxis
or treatment of a condition or disease in a mammal wherein the effects of
HGF/SF would be beneficial by
administering to the mammal an effective amount of a pharmaceutical
composition comprising an active
HGF/SF agonist of Formula IV above. As mentioned above, such activities
include but are not limited to
promoting the proliferation of cells, including endothelial cells, vascular
cells, hepatic cells, renal cells,
among others; promoting angiogenesis; promoting vascularization; improving
wound healing,; improving
vascularization of wounds; improving endothelial cell dysfunction; improving
blood flow to ischemic
tissues; and other desirable activities attendant to the desirable biological
activities of endogenously-
present or exogenously-administered HGF/SF. Those compounds of Formula IV
exhibiting HGF/SF
antagonist activity, determinable readily by one of skill in the art, would be
useful for the prophylaxis or
treatment of a condition or disease in a mammal wherein the effects of HGF/SF
would be undesirable, by
administering to the mammal an effective amount of a pharmaceutical
composition comprising an active
HGF/SF antagonist of Formula IV above. Such utilities include but are not
limited to inhibition of
angiogenesis or neovascularization, prevention of tumor growth or metastasis,
inhibiting scatter, and
inhibiting anti-apoptotic activities. Moreover, compounds of Formula IV show
antagonistic activity to the
growth factors VEGF and FGF, and may be used for the treatment of any
condition or disease in which
inhibition of VEGF or FGF activity is desired.
In a further embodiment, the invention is directed to methods for the
prophylaxis or treatment of the
aforementioned conditions and diseases using a therapeutically effective
amount of a compound of
Formula IV whose HGF/SF-like activities are inhibited in the presence of, or
by preincubation with, the
HGF/SF receptor c-met. The antagonist activity of an HGF/SF antagonist
compound of Formula IV may
be active alone, or may be active in the presence of either exogenously-
administered HGF/SF or in cells
or tissues in which HGF/SF is expressed or induced to be expressed.
The compounds in this group may be HGF/SF agonists or antagonists. Non-
limiting examples of
modulators of Formula IV include:
1-(4-chloro-3-meth ylphenyl)-3-(2,6-dichlorophenyl)-prop-2-en-l-one
1-(4-chloro-3-methylphenyl)-3-(2-chlorophenyl)prop-2-en-l-one
3-(2-chloro-6-fluorophenyl)-1-(4-chloro-3-inethylphenyl)prop-2-en-1-one
3 -(4-bromo-2-thienyl)-1-(3,4-dichlorophenyl)prop-2-en- l -one
3 -(4-bromo-2-thienyl)-1-(4-chloro-3 -methylphenyl)prop-2-en- l -one
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3-(4-bromo-2-thienyl)-1-(4-fluorophenyl)prop-2-en-1-one
3-(4-bromo-2-thienyl)-l-(4-chlorophenyl)prop-2-en-1-one
1-(4-cl-Aorophenyl)-3-(2,4-dichlorophenyl)prop-2-en-l-one
3-(1,3-benzodioxol-5-yl)-1-(4-bromophenyl)prop-2-en-1-one
3-(3-phenoxy-2-thienyl)-1-(2-thienyl)prop-2-en-l -one
3-(3-bromo-4-inethoxyphenyl)-1-phenylprop-2-en-one
3-(3,4-dichlorophenyl)-1-(2-nitrophenyl)prop-2-en-l-one
1-(4-chlorophenyl)-3 -(3,4-dichlorophenyl)prop-2-en-1-one
1-(4-chlorophenyl)-3-(3,5-dichloro-2-hydroxyphenyl)prop-2-en-1-one
1-(2-chlorophenyl)-3-(3,5-dichloro-2-hydroxyphenyl)prop-2-en-l-one
3-(4-chlorophenyl)-1-(2,6-dicl-Aorophenyl)prop-2-en-l-one
1 -(4-bromophenyl)-3 -(4-chlorophenyl)prop-2-en-1-one
1-(2 -chlorophenyl)-3 - (2, 6-dichlorophenyl)prop-2-en-l-one
1-(4-chlorophenyl)-3-(2,6-dichlorophenyl)prop-2-en-l-one
3-(2,6-dichlorophenyl)-1-(4-methoxyphenyl)prop-2-en-l-one
3 -(4-chloro-1-methyl-1 H-pyrazol-3 -yl)-1-[4-(trifluoromethyl)phenyl]prop-2-
en- l -one
3-(2,4-dichlorophenyl)-1-(2-methylphenyl)prop-2-en-l-one
3-(2,6-dichlorophenyl)- 1 -(2-methylphenyl)prop-2-en- 1 -one
3-(3,4-dichlorophenyl)-l -(2-methylphenyl)prop-2-en-1-one
3 -(5 -broino-2 -hydroxyphenyl)-1-(3-methylphenyl)prop-2 -en-1-one
3-(5-bromo-2-hydroxyphenyl)-1-(4-methylphenyl)prop-2-en- l -one
3-(2,4-dichlorophenyl)- 1 -(3 -methylphenyl)prop-2-en-1-one
3 -(2,4-dichlorophenyl)-1-(4-methoxyphenyl)prop-2-en-1-one
1-[4-amino-2-(methylthio)-1,3-thiazol-5-yl]-3-(4-chlorophenyl)prop-2-en-l-one
1-(4-chlorophenyl)-3 - [4-(trifluoromethyl)phenyl]prop-2-en-1-one
1 -benzo[b]thiophen-3 -yl-3-(4-chlorophenyl)prop-2-en- 1 -one
1,3 -di(5 -nitro-3 -thienyl)prop-2-en-1-one
1-(4-bromophenyl)-3-(3,5-difluorophenyl)prop-2-en-l-one
3-(3,5-difluorophenyl)-1-(3-nitrophenyl)prop-2-en-l-one
The compounds of Formulae I-IV described above may be synthesized and isolated
following standard
methods readily available to one skilled in the art of synthetic organic
chemistry. Moreover, the
compounds may be readily prepared at a purity acceptable for administration to
a mammal, preferably a
human, at a dose effective to prophylax or treat any of the conditions and
diseases related to the desired or
undesired activities of HGF/SF as mentioned above.
The various uses, formulations including pharmaceutical compositions, and dose
considerations are as
described hereinabove.
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While the foregoing discussions have been directed principally to the
compounds useful for the promotion
or inhibition of HGF/SF activity, and its receptor c-met, they are generally
applicable to agonists and
antagonists of other growth factors, particularly growth factors whose
activities involve binding to a
tyrosine kinase receptor. As will be seen in the examples below, compounds of
the invention have been
shown to inhibit VEGF and FGF activities, and certain other compounds have
VEGF-like activities. The
present invention embraces these as well as other tyrosine kinase receptor
growth factors and small-
molecule compounds including but not limited to those described herein, as
agents useful for the various
agonist and antagonist activities directed to such growth factors generally.
The present invention may be better understood by reference to the following
non-limiting Examples,
which are provided as exemplary of the invention. The following examples are
presented in order to more
fully illustrate the preferred embodiments of the invention. They should in no
way be construed,
however, as limiting the broad scope of the invention.
Example 1
Methods
Peptide library. The Ph.D.-12 phage display peptide library (New England
BioLabs) was used. The
original library contained 1.5 X 109 pfu/ l. This peptide library is based on
a combinatorial library of
random peptide 12-mers fused to a minor coat protein (piII) of M13 phage. The
displayed 12-mer
peptides are expressed at the N-terminus of pIII.
Phage Display. To identify peptide mimics of HGF/SF, peptide libraries were
screened twice each with
polyclonal HGF/SF antibodies (813, rabbit) and monoclonal HGF/SF antibodies
(clone-23C2). Antibodies
were diluted in NaHCO3, pH 8 buffer (final concentration -100 g/100 l) and
96 well plates were coated
with 100 l of antibody for 16 h at 4oC. Wells were washed with TBST (Tris
buffered saline containing +
0.1% Tween-20). Ten gl of original phage (4 X 101) was diluted to 10 ml and
100 1 of this solution was
incubated with antibody coated plate with gentle rocking for 60 min at room
temperature. Non-binding
phage was removed and wells were washed 10 times with TBST. Bound phage was
eluted with 100 1 of
elution buffer. (0.2 M Glycine-HC1, pH 2.2, 1 mg/ml albumin), neutralized with
15 ml 1M Tris-HC1 (pH
9.1) and amplified. The titer of the first round eluate is 2x1010 pfu/ L. A
second round of biopanning was
carried out with 2 X 1011 pfu of the first round amplified phage as above.
This procedure was repeated
once more.
Characterization of Binding Clones and peptide synthesis. Twenty clones from
the third round were
picked to detect the consensus binding sequence. Ten clones were from the
Monoclonal antibody plate
and 10 clones were from the polyclonal antibody plate. Following amplification
DNA was extracted and
sequenced. Out of 20 clones sequenced, 9 unique sequences were identified: S G
W H M R S P F N H M
(SEQ ID No: 12); H L K P H F W P S S P Y (SEQ ID No: 13); T M G F T A P R F P
H Y (SEQ ID No:1);
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KVWYHTTSIPSH(SEQID No:2); L L A D T T H H R P W T (SEQ ID No:14); N H P H P T
P A
RGII(SEQID No:15); V S R H Q S W H P H D L (SEQ ID No:16); A L N W S R K L P V
P P (SEQ
ID No:18); and Q T G H W N A E W H T R (SEQ ID No:19). The peptides were
synthesized based on
the DNA sequence using solid phase peptide synthesizer at the North Shore
University Research Building.
Endothelial cell proliferation. Bovine aortic endothelial cells (BAEC) and
human microvascular
endothelial cells (MMEC) were used for proliferation assays (19). BAEC were
grown in minimal
essential medium containing 10% FBS. HMEC were grown in RPMI medium containing
10% FBS and
10% NuSerum. Endothelial cell proliferation was determined as described
previously. Subconfluent (50-
60%) endothelial cells were incubated in serum free medium containing various
concentrations of
peptides or growth factor for 16 h-24 h. 3H-thymidine was then added to medium
and incubation was
continued under culture conditions for another 4-5 h. The cells were washed
and 3H-thymidine
incorporation (increase in DNA synthesis) was determined.
Vascular sprouting (aortic ring) assay. Rat aortic ring assay of angiogenesis
was performed as described
previously (20). Briefly, one mm-long aortic rings were sectioned from rat
aorta and embedded in
collagen gels. Following gelation, sections were incubated in medium
containing 4% serum in the absence
(control) or presence of SEQ ID No: 2 (100 gg/ml) or HGF (100 ng/ml).
Murine angiogenesis assay. Angiogenesis was assayed as growth of blood vessels
from subcutaneous
tissue into a solid gel of basement membrane containing the test sample.
Matrigel in liquid form (0.5 ml)
was mixed with SEQ ID No:2 or basic fibroblast growth factor and injected into
the abdominal
subcutaneous tissue of mice as previously described (21). After 10 days, mice
were sacrificed and the
Matrigel plugs were removed, fixed, sectioned, stained and examined for
ingrowth of blood vessels.
Example 2
HGF/SF Antibody-binding Peptides
Two of these aforementioned nine peptides, T M G F T A P R F P H Y and K V W Y
H T T S I P S H
(designated SEQ ID No: 1 and SEQ ID No:2, respectively), showed stimulation of
endothelial
proliferation and were characterized further.
Incubation of endothelial cells with SEQ ID No:1 and SEQ ID No:2 (100 gg/ml)
significantly increased
3H-thymidine incorporation into DNA (p<0.01). This increase was comparable
with increase showed by
HGF/SF (20 ng/ml). As shown in Figure 1, subconfluent endothelial cells were
incubated with SEQ ID
No:2 (100 g/ml) or scatter factor (HGF/SF, 20 ng/ml) for 24 h in serum-free
medium under culture
conditions. 3H-thymidine was then added and DNA synthesis was assessed after 5
h. Values represent
Mean SD of six measurements. *p<0.01.
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The peptide depicted in SEQ ID No:2 stimulated growth of microvessels in an in-
vitro angiogenesis assay.
Figure 2 shows the effects of peptide 4 (SEQ ID No:2) and HGF on angiogenesis
in rat aortic ring assay:
One mm-long aortic rings were sectioned from rat aorta and embedded in
collagen gels. Following
gelation, sections were incubated in medium containing 4% serum in the absence
(control) or presence of
SEQ ID No:2 (100 .tghnl) or HGF (100 nghnl). Values represent Mean SD (n=70-
120).
The ability of the peptides to induce blood vessel growth in vivo was
evaluated. These experiments were
performed at Paragon Bioservices, Baltimore, MD. In this assay SEQ ID No:l or
SEQ ID No:2 or an
equal amount of water (control) was mixed with Matrigel, a matrix of
reconstituted basement membrane.
Samples were injected subcutaneously into mice. After 10 days, mice were
sacrificed for histologic and
morphometric analysis of Matrigel plugs. Plugs containing SEQ ID No:2 show
significantly higher
number of endothelial cells (Figure 3, p<0.0001, n=56).
Example 3
Addition of a heparin-binding sequence enhances growth promoting activity
HGF/SF is a heparin binding protein and recent studies show that HGF/SF
binding to cell surface HSPG
greatly enhances HGF/SF-induced signal transduction by c-met (11,22). To
determine whether the
growth promoting activity of SEQ ID No:2 can be enhanced by increasing its
heparin-affinity, a lysine-
rich heparin-binding sequence (23) was added to the carboxy-terminus of SEQ ID
No:2 ("HS-P4"; K V
WYHTTSIPSHCRPKAKAKAKAKDQTK[SEQIDNo:7]). Anon-specific sequence
with the same amino acid composition was added and treated as non-specific
control ("NS-P4"; K V W Y
HTTSIPSHCQKAKTRAKAAKPDKK[SEQIDNo:8]).Theability ofHS-P4 and NS-P4
to stimulate endothelial cell proliferation was compared with SEQ ID No:2
(Figure 4A) and with growth
factors (Figure 4B). Endothelial cells were incubated with peptides for 24 h
and 3H-thymidine
incorporation was determined. HS-P4 but not NS-P4 increased endothelial growth
by about three fold
(Figure 4A) and is comparable or greater than the mitogenic activity of HGF/SF
and bFGF (Figure 4B).
These data suggest that the growth promoting and possibly the angiogenic
activity of SEQ ID No:2 can be
enhanced by increasing its affinity to heparin.
Example 4
Identification of peptides that bind scatter factor receptor c-met
The Ph.D.-12 phage display peptide library was used as described above. The
extracellular domain of
HGF/SF receptor C-met was obtained from R&D Systems. Peptide libraries were
screened twice with
plates coated with C-met as described above for HGF/SF antibody screening.
Eighteen binding clones
were sequenced. Of these, 15 clones were found to contain unique sequences.
Four peptides (SEQ ID
No:3,4,5and6)weresynthesized: ATWSHHLSSAGL(SEQIDNo:3);WPQLPPRPYST
L(SEQID No:4); S N T S A G T P F T S L (SEQ ID No:5);andD S T P K S T P
WYYI(SEQID
No:6). The ability of these peptides to stimulate endothelial proliferation or
inhibit HGF/SF-mediated
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increase in endothelial proliferation was determined (as assessed by 3H-
thymidine incorporation). SEQ ID
Nos. 3-6 did not significantly affect endothelial growth (Figure 5). As shown
in Figure 5, subconfluent
endothelial cells were incubated with each of the peptides (100 g/ml),
scatter factor alone (HGF/SF, 20
ng/ml), or HGF/SF plus each of the peptides for 24 h in serum-free medium
under culture conditions. 3H-
thymidine was then added and DNA synthesis was assessed after 5 h. Values
represent Mean SD of
four measurements. HGF/SF induced endothelial growth by about 2 fold. However,
SEQ ID Nos:3-6
completely inhibited HGF/SF-inediated increase in endothelial growth. These
data suggest that these
peptides can bind HGF/SF receptor C-Met thereby inhibit HGF/SF binding. Thus,
these peptides may
have potential angiostatic activity.
Example 5
Antiproliferative Activity In vivo
A human glioblastoma cell line (U87 MG, ATCC Cat. No. HTB-14) and glioma cell
line (Hs 683, ATCC
Cat. NO. HTB-138) were obtained from American Type Culture Collection (ATCC),
Va. U87 MG cells
were maintained in Minimal Essential Medium (MEM) containing 10% fetal bovine
serum, 1 mM-
pyruvate and 0.1 mM non-essential amino acids. Hs 683 cells were maintained in
DMEM containing
10% FBS. Cells were plated in a 48 well plate (10, 000 cells per well). Twenty-
four h following seeding,
medium was replaced with serum free medium. After 8 h cells were incubated
with medium with or
without peptides (100 .1g/ml final concentration) for 20 h. 3H-thymidine was
then added and DNA
synthesis was determined for 4 h.
Peptides corresponding to SEQ ID Nos:3-6 and A K T Y A G S S Y Q F G (SEQ ID
No:l l) were
evaluated.
The peptide designated as SEQ ID No:5 was most effective in inhibiting the
growth of Hs 683 cells
(p<0.001) (Figure 6A). However peptides designated as SEQ ID No:4, 5 and 6
showed significant
inhibition on U87 MG cell growth (Figure 6B). These data show that C-met
peptides each alone or in
combination could block endogenous HGF/SF activity and associated tumor
growth.
Example 6
Assay Methods for Small-molecule Compounds
The following assays were used to evaluate the activity of the various
compounds discussed herein.
Certain of the compounds and groups thereof express HGF/SF-like activity,
i.e., they induce scatter, cell
proliferation, inhibit apoptosis, among other activities, and if acting
through the c-inet receptor, agonize or
stimulate c-met receptor activity. The specificity for such compounds working
through the c-met receptor
may be identified by performing the stimulation assay in the presence of free
c-met receptor. Reduction
of a proliferative response in the presence of c-met indicates such
specificity.
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To evaluate inhibitors of activity, compounds may be evaluated directly for
anti-proliferative activities,
such as the inhibition of cellular proliferation, inhibition of tumor growth,
inhibition of scatter, and
inhibition of gene expression, and may also be evaluated on their ability to
inhibit activity when exposed
to cells together with HGF/SF. In such instances, the scatter and/or
proliferative activities induced by
added will be inhibited by the attendant presence of an inhibitory compound of
the invention. Thus,
certain inhibitors may be inhibitory in the absence of exogenously-added
HGF/SF; these and/or other
compounds may exhibit inhibitory activity only in the presence of HGF/SF.
It is noted that Applicants have no duty to disclose the theory or mechanism
by which or through the
compounds of the invention operate, nor are they in any way bound by such
disclosure.
Cell proliferation assays Endothelial cells (HUVECs) were seeded in 48 well
plates at a density of 10,000
to 20,000 cells per well in the normal growth medium (EGM-2- Clonetics)
containing 2% fetal bovine
serum, FGF, VEGF, IGF, ascorbic acid, EGF, GA, heparin and hydrocortisone. The
cells were grown
normally in the growth medium for 24 hr at 37 C and 5% CO2. The cells were
then rinsed with RPMI-l%
BSA and starved for 1-2 hrs. The stock solutions of all the compounds were
made at a concentration of 10
mg/ml in DMSO and diluted in RPMI-1% BSA at a final concentrations of 1 to 12
microgram/ml. The
cells were then washed and treated with the compounds and incubated for
another 24 hr at 37 C. Then 3H
thymidine (0.5 microgram/ml in RPMI-BSA) was added to the cells and incubated
at 37 C for 4 to 5
hours. The unincorporated thymidine was removed by washing the cells four
times with lx PBS. Then the
cells were lysed with 0.5M NaOH for 30 min and the radioactivity counted in
the beta counter.
In other experiments, human iliac artery endothelial cells were used under
similar conditions as those
described above.
Effect on growth of tumor cells. The activity of the compounds herein to
promote or inhibit the growth of
tumor cells was evaluated using human endometrial cancer cells.
Scatter Assay. A standard assay for scatter of MDCK cells was performed.
Results were evaluated by
microscopic examination.
Anti-apoptotic Activity Assay. The ability of compounds of the present
invention to protect cells from
apoptosis was performed using a MTT viability assay with MDCK cells exposed to
adriamycin (15
micromolar) to induce apoptosis. HGF/SF was evaluated as a positive control.
Effect of the compounds on gene expression patterns. Additional data on the
cellular effects of the
compounds of the invention was obtained from GeneChip studies. In particular,
effects on induction of
angiogenic-cascade-related genes including interleukin-8 and angiopoietin-2
were evaluated.
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Example 7
Compounds
The following compounds were evaluated herein.
(4-chlorophenyl)[5-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]mnethanone
(C16H11CIN2OS/315)
1-(methylsulfonyl)-5-(2-(2-thienyl)vinyl)-1H-pyrazole
2,2-dimethyl-l-(3-(2-(2-thienyl)vinyl)-1H-pyrazole-1-yl)propan-l -one
(4-chlorophenyl)(3,5-di(tert-butyl)-1H-pyrazol-1-yl)methanone
N-methy-3 -(2-(2thienyl)vinyl)-1 H-pyrazole- l -carb oxamide
1-(4-chlorobenzoyl)-5-cyclopropyl-1 H-pyrazole-4-carbonitrile
(4-chlorophenyl)(3-(3-phenylisoxazol-5-yl)-1H-pyrazol-1-yl)methanone
5-(2-(2-thienyl)vinyl)-1 H-pyrazole
ethyl 1-(4-chlorobenzoyl)-3-methyl-lH-pyrazol-5-carboxylate
(4-chlorophenyl)(3,5-dimethyl-4-(pyrimidui-2-ylthio)-lH-pyrazol-1-yl)methanone
(4-chlorophenyl)(3-(3-(4-chlorophenyl)-5-methylisoxazol-4-yl)-1 H-pyrazol-l -
yl)methanone
(4-chlorophenyl)(3-(5-(2-thienyl)-2-thienyl)-1 H-pyrazol-1-yl)methanone
(2,4-dichlorophenyl)(3-(5-(2,4-difluorophenyl)-2-furyl)-1H-pyrazol-1-
yl)methanone
(4-(2-chloro-6-fluorob enzyl)-3, 5-dimethyl- l H-pyrazol-1-yl)(4-
chlorophenyl)methanone
methyl 4-(1-(4-chlorobenzoyl)-1H-pyrazol-5-yl)-5-methylisoxazole-3-carboxylate
(4-clilorophenyl)(5-(methylthio)-3 -(4-phenoxyphenyl)-1 H-pyrazol-l -
yl)methanone
(4-chlorophenyl)(3,5-dimethyl-4-((1-methyl-lH-imidazol-2-yl)thio)-1H-pyrazol-l-
yl)methanone
N 1-phenyl-3-(2-(2-thienyl)vinyl)-1H-pyrazole-l-carboxamide
(4-chlorophenyl)(3-(2-(5-(2-thienyl)-2-thienyl)-4-methyl- l,3-thiazol-5-yl)-1H-
pyrazol-1-yl)methanone
(4-chlorophenyl)(3,5-dimethyl-4-phenoxy-1H-pyrazol-1-yl)methanone
(3-benzhydryl-1 H-pyrazol- l -yl) (4-chlorophenyl)methanone
(4-chlorophenyl)(3,5-dimethyl-4-((5-(trifluoromethyl)-2-pyridyl)thio)-1 H-
pyrazol-l -yl)methanone
N 1-(4-chlorophenyl)-3-(2-(2-thienyl)vinyl)-1H-pyrazole-l-carboxamide
methyl 1-(4-chlorobenzoyl)-5-(dimethoxymethyl)-1H-pyrazole-4-carboxylate
(4-cl-Aorophenyl)(3-(2-methylimidazo(1,2-a)pyridin-3-yl)-1H-pyrazol-1-
yl)methanone
(4-chlorophenyl)(3,5-dimethyl-4-)((l -phenyl-1 H-1,2,3,4-tetraazol-5-yl)thio)-
1H-pyrazol-l-
yl)methanone
methyl 1-(4-chlorobenzoyl)-5-isoxazol-5-yl-3-methyl-lH-pyrazole-4-carboxylate
(3-(tert-butyl)5-(methylthio)-1H-pyrazol-1-yl)(4-chlorophenyl)methanone
2-chloro-6-(4-(1-(4-chlorobenzyl)-1H-pyrazol-5-yl)phenoxy)benzonutrile
(4-chlorophenyl)(5-(5-methyl-3-phenylisoxazol-4-yl)-1H-pyrazol-1-yl)methanone
1-((4-chlorophenyl)sulfonyl)-3-(2-(2-thienyl)vinyl)-1H-pyrazole
(4-chlorophenyl)(3,5-dimethyl-4-((4-methyl-5-(trifluoromethyl)-4H-1,2,4-thazol-
3-yl)thio)-1H-pyrazol-
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1-yl)methanone
methyl 1-(4-chlorobenzoyl)-3-methyl-5-(4-methyl-1,2,3-thiadiazol-5-yl)-1H-
pyrazole-4-carboxylate
[4-(2,6-dichlorobenzyl)-3,5-dimethyl-1H-pyrazol-l-yl] [3-(2,6-dichlorophenyl)-
5-methylisoxazol-4-
yl]methanone (C23H17C14N302/509)
4-(2-chloro-6-fluorobenzyl)-3, 5 -dimethyl- l -(phenylsulfonyl)-1 H-pyrazol e
(4-(2-chloro-6-fluorobenzyl)-3,5-dimethyl-1H-pyrazole-1-yl)(3-(2,6-
dichlorophenyl)-5-methylisoxazol-
4-yl)methanone
4-(2-cllloro-6-fluorobenzyl)-1-((3,4-dichlorophenyl)sulfonyl)-3,5-dimethyl-1H-
pyrazole
4-(2-chloro-6-fluorobenzyl)-1,3,5-trimethyl-1H-pyrazole
4-(2-chloro-6-fluorobenzyl)-3, 5-dimethyl-1 H-pyrazole
(4-bromo-3,5-dimethyl-1 H-pyrazol-1-yl)(3-(2,6-dichlorophenyl)isoxazole-4-
carbohydrazide)
N'4,5-dimethyl-N'4-(5-nitro-2-pyridyl)-3-(2,6-dichlorophenyl)isoxazole-4-
carbohydrazide
N'4-(2-(((2,4-dichlorobenzylidene)amino)oxy)acetyl)-3-(2,6-dichlorophenyl)-5-
methylisoxazole-4-
carbohydrazide
3-(2,6-dichlorophenyl)-5-methylisoxazole-4-carbohydrazide
N'4-(3-(3,4,5-trimethoxyphenyl)propanoyl)-3-(2,6-dichlorophenyl)-5-
methylisoxazole-4-
carbohydrazide
2-nitrophenyl 2-((3-(2,6-dichlorophenyl)-5-methylisoxazol-4-
yl)carbonyl)hydrazine-1-carbothioate
4-(2, 6-dichlorobenzyl)-1-((3,5pdi(trifluoromethyl)phenyl)sulfonyl)-3,5-
dimethyl-1 H-pyrazole
1-((4-chlorophenyl)sulfonyl)-4-(2,6-dichlorobenzyl)-3,5-dimethyl-1H-pyrazol
(4-(2,6-dichlorobenzyl)-3,5-dimethyl-l H-pyrazol- l -yl)(2,6-
dichlorophenyl)methanone
3-(4-(2,6-dichlorobenzyl)-3,5-dimethyl-1H-pyrazol-1-yl)propanenitrile
N'4-((2-methyl-1,3-thiazol-4-4y1)carbonyl)-3-(2,6-dichlorophenyl)-5-
methylisoxazole-4-
4carbohydrazide
N 1-((2-((3-(2,6-dichlorophenyl)-5-methylisoxazol-4-
yl)carbonyl)hydrazino)(methylthio)mnethylidene)benzene-l-sulfonamide
N'4-(2,4,6-trichlorophenyl)-3-3(2,6-dichlorophenyl)-5-methylisoxazole-4-
carbohydrazide
N'4,3-di(2,6-dichlorophenyl)-5-methylisoxazole-4-carbohydrazide
3, 5-di(tert-butyl)-4-(2-chloro-6-fluorobenzyl)-1 H-pyrazole
N'4-(3,5-dichloro-4-pyridyl)-3-(2,6-dichlorophenyl)-5-methylisoxazole-4-
carbohydrazide
N'4-phenyl-3-(2,6-dichlorophenyl)-5-methylisoxazole-4-carbohydrazide
(4-(2-chloro-6-fluorobenzyl)-3,5-dunethyl-1H-pyrazole-l -yl)(2,6-
dichlorophenyl)methanone
1-(4-(2-chloro-6-fluorobenzyl)-3,5-dimethyl-1H-pyrazole-1-yl)2,2-
dimethylpropan-l-one
N'4,N'4,5-trimethyl-3-(2,6-dichlorophenyl)isoxazole-4-carbohydrazide
N4-azepan-1-yl-3-(2,6-dichlorophenyl)-5-methylisoxazole-4-carboxamide
N'4-(6-(trifluoromethyl)-2-pyridyl)-3-(2,6-dichlorophenyl)-5-methylisoxazole-4-
carbohydrazide
(4-(2-chloro-6-fluorobenzyl)-3,5-dimethyl-1H-pyrazole-1-yl)(4-
chlorophenyl)methanone
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N4Piperidino-3-(2,6-dichlorophenyl)-5methylisoxazole-4-carboxamide
N'4-(3,3-diethoxypropanoyl)-3-(2,6-dichlorophenyl)-5-methylisoxazole-4-
carbohydrazide
(4-(2-chloro-6-fluorobenzyl)-3,5-dimethyl-lH-pyrazole-l-yl)(2-
thienyl)methanone
N'4-(2,5-dichlorophenyl)-3-(2,6-dichlorophenyl)-5-methylisoxazole-4-
carbohydrazide
1-(4-cl-Aoro-3-methylphenyl)-3-(2,6-dichlorophenyl)-prop-2-en-1-one
1-(4-chloro-3-inethylphenyl)-3-(2-chloroplienyl)prop-2-en-1 -one
3 -(2-chloro-6-fluorophenyl)-1-(4-chloro-3 -(4-chloro-3-methylplienyl)prop-2-
en-1 -one
3-(4-bromo-2-thienyl)-1-(3,4-dichlorophenyl)prop-2-en- l -one
3 -(4-bromo-2-thienyl)- 1-(4-chloro-3-methylphenyl)prop-2-en- l -one
3-(4-bromo-2-thienyl)-1-(4-fluorophenyl)prop-2-en-l-one
3 -(4-bromo-2-thienyl)-1-(4-chlorophenyl)prop-2-en- l -one
1-(4-chlorophenyl)-3-(2,4-dichlorophenyl)prop-2-en-1-one
3-(1,3-benzodioxol-5-yl)-1-(4-bromophenyl)prop-2-en-1-one
3 -(3-phenoxy-2-thienyl)-1-(2-thienyl)prop-2-en-1-one
3 -(3 -bromo-4-methoxyphenyl)-1-phenylprop-2-en-one
3-(3,4-dichlorophenyl)-1-(2-nitrophenyl)prop-2-en- l-one
1-(4-chlorophenyl)-3 -(3,4-dichlorophenyl)prop-2-en- l -one
1-(4-chlorophenyl)-3-(3,5-dichloro-2-hydroxyphenyl)prop-2-en-l-one
1-(2-chlorophenyl)-3-(3,5-dichloro-2-hydroxyphenyl)prop-2-en-1-one
3 -(4-chlorophenyl)-1-(2,6-dichlorophenyl)prop-2-en-1-one
1-(4-bromophenyl)-3-(4-cl-Aorophenyl)prop-2-en-1-one
1-(2-chlorophenyl)-3-(2,6-dichlorophenyl)prop-2-en-l-one
1-(4-chlorophenyl)-3-(2,6-dichlorophenyl)prop-2-en-1-one
3 -(2, 6-dichlorophenyl)-1-(4-methoxyphenyl)prop-2-en-1-one
3-(4-chloro-1-methyl-1 H-pyrazol-3 -yl)-1-[4-(trifluoromethyl)phenyl]prop-2-en-
l -one
3 -(2,4-dichlorophenyl)-1-(2-methylphenyl)prop-2-en-l-one
3 -(2, 6-dichlorophenyl)-1-(2-methylphenyl)prop-2-en- l -one
3-(3,4-dichlorophenyl)-1-(2-iethylphenyl)prop-2-en-1-one
3-(2,6-dichlorophenyl)-1-(3-methylphenyl)prop-2-en-l-one
3-(5-bromo-2-hydroxyphenyl)-1-(3-methylphenyl)prop-2-en-l-one
3 -(5 -bromo-2-hydroxyphenyl)-1-(4-methylphenyl)prop-2-en- l -one
3-(2,4-dichlorophenyl)-1-(3-methylphenyl)prop-2-en-1-one
3-(2,4-dichlorophenyl)-1-(4-methoxyphenyl)prop-2-en-1-one
1-[4-amino-2-(methylthio)-1,3-thiazol-5-yl]-3-(4-chlorophenyl)prop-2-en-l -one
1-(4-chloroplenyl)-3-[4-(trifluoromethyl)phenyl]prop-2-en- l-one
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1-benzo[b]thiophen-3-yl-3-(4-chlorophenyl)prop-2-en-l-one
1,3-di(5-nitro-3-thienyl)prop-2-en-1-one
1-(4-methyl-2-(3-thienyl)-1,3-thiazol-5-yl]-3-(2-thienyl)prop-2-en-1-one
1-(4-bromophenyl)-3-(3,5-difluorophenyl)prop-2-en-l-one
3-(3,5-difluorophenyl)-1-(3-nitrophenyl)prop-2-en-1-one
Example 8
HGF/SF-like cellular proliferative activity of a compound of the invention
Using the endothelial cell proliferation assay described above, the compound
(4-chlorophenyl)[3-(2-(2-
thienyl)vinyl)-1H-pyrazol-l-yl]methanone was shown to increase HUVEC
proliferation by two to five
fold. The specificity of the stimulation of endothelial cell growth by the
compound as measured by 3H-
thymidine incorporation was tested by pre-incubation of cells with the HGF/SF
receptor c-met. In Figure
7, the first bar represents control cells; the second bar (4-chlorophenyl)[3-
(2-(2-thienyl)vinyl)-1H-pyrazol-
1-yl]methanone at 6 microgram/ml; and the third bar: (4-chlorophenyl)[3-(2-(2-
thienyl)vinyl)-IH-pyrazol-
1-yl]methanone at 6 microgram/ml plus c-met receptor, 100 microgram/ml. (4-
Chlorophenyl)[3-(2-(2-
thienyl)vinyl)-1H-pyrazol-l-yl]methanone by itself stimulated 3H-thymidine
incorporation by 84%. Thus,
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone is as
effective as HGF/SF in
stimulating HUVEC proliferation. In the presence of c-met, the (4-
chlorophenyl)[3-(2-(2-thienyl)vinyl)-
1H-pyrazol-1-yl]methanone stimulation of 3H-thymidine incorporation was
inhibited by 75%. Although
Applicants are not bound by theory, this study also demonstrates that (4-
chlorophenyl)[3-(2-(2-
thienyl)vinyl)-1H-pyrazol-1-yl]methanone promotes proliferation of HUVECs via
the c-met receptor. In
another related experiment, (4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-
1-yl]methanone (12
microgram/ml) was incubated with the initial target molecule C-met receptor (5
microgram/ml) for 30 min
and then added to the cells. Compound-induced EC proliferation was blocked by
40% in the presence of
C-met receptor.
Example 9
Scatter of MDCK cells
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-l-yl]methanone was further
tested for HGF/SF
activity in a standard scatter assay which is specific for HGF/SF. The ability
to scatter was demonstrated
for the first time using a non-peptide candidate compound. Scatter of MDCK
cells by (4-chlorophenyl)[3-
(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone further demonstrates that its
actions are mediated through
stimulation of the c-met receptor. As shown in Figure 8, the compound caused
scattering of MDCK cells
similar to that seen with HGF/SF. Figure 8A: Control cells; Figure 8B: (4-
chlorophenyl)[3-(2-(2-
thienyl)vinyl)-1H-pyrazol-1-yl]methanone, 6 microgram/ml.
Example 10
Anti-apoptotic activity of (4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-
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1H-pyrazol-1-yl]methanone
HGF/SF has significant anti-apoptotic activity in a number of cultured cell
lines. Using the MTT cell
viability assay the ability of (4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-
pyrazol-1-yl]methanone to
protect cells from adriamycin-induced apoptosis was evaluated. Like HGF/SF, (4-
chlorophenyl)[3-(2-(2-
thienyl)vinyl)-1H-pyrazol-l-yl]methanone was able to significantly block
adriamycin-induced apoptosis
in MDCK cells (Figure 9). Cell viability was unchanged by either HGF/SF alone
(column 2), (4-
chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone alone (column
5) or HGF/SF and (4-
chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-l-yl] methanone combined
(column 7). Adriamycin (15
mM) decreased cell viability to 56% of control (column 3). Treatment with
either HGF/SF (column 4) or
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-l-yl]methanone (column 6)
effected nearly complete
(94%) protection from adriamycin-induced apoptosis.
In another cell line, 90% protection was afforded by (4-chlorophenyl)[3-(2-(2-
thienyl)vinyl)-1H-pyrazol-
l-yl]methanone.
Example 11
Effect of (4-chlorophenyl)(3-2-(2-thienyl)vinyl)-1H-pyrazol-1-yl-methanone on
HUVEC
proliferation
Figure 10 shows a dose-response relationship between the level of level (4-
chlorophenyl)(3-2-(2-
thienyl)vinyl)-1H-pyrazol-1-yl-methanone and HUVEC proliferation.
Example 12
Gene Expression
Preliminary GeneChip studies using the compounds of the invention demonstrate
similar gene stimulation
profiles including stimulation of interleukin-8 and angiopoetin-2, both of
which have important roles in
the angiogenic cascade.
Example 13
Effect of [4-(2,6-dichlorobenzyl)-3,5-dimethyl-1H-pyrazol-1-yl][3-(2,6-
dichlorophenyl)-5-
methylisoxazol-4-yl]methanone on HGF/SF-mediated HUVEC proliferation
Figure 11 shows the results of a HUVEC growth experiment in the presence of
HGF/SF and [4-(2,6-
dichlorobenzyl)-3,5-dimethyl-lH-pyrazol-1-yl] [3-(2,6-dichlorophenyl)-5-
methylisoxazol-4-yl]methanone.
While the addition of HGF/SF increases the proliferation of HUVEC (second
bar), and the compound
alone has no effect on baseline proliferation, the combination of both HGF/SF
and the compound (fourth
bar) results in significant suppression of HGF/SF-mediated stimulation.
Example 14
In vivo blood vessel ingrowth assay
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Angiogenesis was assayed as growth of blood vessels from subcutaneous tissue
into a solid gel of
basement membrane containing the test compound. Matrigel in liquid form (0.5
ml) was mixed with a
compound of the invention, or basic fibroblast growth factor as a control, and
injected into the abdominal
subcutaneous tissue of mice as previously described (Kibbey, M. C., Grant, D.
S. Auerbach, R. and
Kleinman, H. K. [1992] Role of the SIKVAV site of laminin in promotion of
angiogenesis and tumor
growth: an in vivo Matrigel model. J. Natl. Can. Inst. 84, 1633-38). After 10
days, mice were sacrificed
and the Matrigel plugs were removed, fixed, sectioned, stained and examined
for ingrowth of blood
vessels. In Figure 12A, the effect of 1-(4-chloro-3-methylphenyl)-3-(2,6-
dichlorophenyl)-prop-2-en-1-
one was seen as a nearly complete inhibition of blood vessel ingrowth, less
than control. In contrast, in
Figure 12B, (4-(2-chloro-6-fluorobenzyl)-3,5-dimethyl-1H-pyrazole-1-yl)(3-(2,6-
dichlorophenyl)-5-
methylisoxazol-4-yl)methanone showed significant stimulation of blood vessel
ingrowth.
Example 15
Clonogenic assays
The effect of (4-(2-chloro-6-fluorobenzyl)-3,5-dimethyl-1H-pyrazole-1-yl)(3-
(2,6-dichlorophenyl)-5-
methylisoxazol-4-yl)methanone (Figure 13) and 1-(4-chloro-3-methylphenyl)-3-
(2,6-dichlorophenyl)-
prop-2-en-l-one (Figure 14) were evaluated in a clonogenic assay over 7 days.
Both Figures 13 and 14
show dose responsive inhibition of DU145 cell growth during the experiment.
Example 16
In vivo blood flow improvement assay
Figure 15 shows the results of an in-vivo experiment in which (4-
chlorophenyl)[3-(2-(2-thienyl)vinyl)-
1H-pyrazol-1-yl]methanone was administered to mice for seven days following
removal of the femoral
artery. The results show significant improvement in blood flow with the
compound.
Example 17
Other compounds with activities
D
In a similar manner as described above, the following compounds related were
evaluated for stimulation
of endothelial cell proliferation. Three different rounds of testing were
performed.
Compound Stimulation/Inhibition Stimulation/Inhibition
at 5 micrograms/ml at 10 micrograms/ml
1-(rnethylsulfonyl)-3-(2-(2-thienyl)vinyl)-1H- 74% stimulation Not significant
pyrazole
2,2-dimethyl-l-(3-(2-(2-thienyl)vinyl)-1H- No effect 55% stimulation
pyrazole-1-yl)propan-l-one
N-methy-3-(2-(2thienyl)vinyl)-1H-pyrazole-l- 70% stimulation 42% stimulation
carboxamide
(4-chlorophenyl)(3-(3-phenylisoxazol-5-yl)-1H- 54% stimulation 60% stimulation
pyrazol-1-yl)methanone
(4-chlorophenyl)(3-(5-(2-thienyl)-2-thienyl)-1H- 40% stimulation 40%
stimulation
pyrazol-1-yl)methanone
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Compound Stimulation/Inhibition Stimulation/Inhibition
at 5 micrograms/ml at 10 micrograms/nil
(4-(2-chloro-6-fluorobenzyl)-3,5-dimethyl-1H- 41% stimulation 35% stimulation
pyrazol-1-yl) (4-chlorophenyl)methanone
(4-chlorophenyl)(5-(methylthio)-3-(4- No effect 40 % inhibition
phenoxyphenyl)-1 H-pyrazol-1-yl)methanone
(4-chlorophenyl)(3,5-dimethyl-4-((1-methyl-1H- 52% stimulation 50% stimulation
imidazol-2-yl)thio)-1 H-pyrazol-1-yl)methanone
N1-phenyl-3-(2-(2-thienyl)vinyl)-1H-pyrazole-l- 40% stimulation Not
significant
carboxamide
(4-chlorophenyl)(3-(2-(5-(2-thienyl)-2-thienyl)-4- 20% stimulation 33%
stimulation
methyl-1, 3 -thiazol-5-yl)-1 H-pyrazol- l -
yl)methanorne
(3-benzhydryl-1H-pyrazol-1-yl)(4- No effect 25% stimulation
chlorophenyl)methanone
N1-(4-chlorophenyl)-3-(2-(2-thienyl)vinyl)-1H- No effect 55% stimulation
pyrazole-l-carboxamide
methyl 1-(4-chlorobenzoyl)-5-isoxazol-5-yl-3- Not significant 33% stimulation
methyl- 1 H-pyrazole-4-carb oxylate
2-chloro-6-(4-(1-(4-chlorobenzyl)-1H-pyrazol-5- 60% stimulation 90%
stimulation
yl)phenoxy)benzonitrile
The following compounds were re-evaluated.
Compound Stimulation/Inhibition Stimulation/Iffllibition
at 5 micrograms/ml at 10 micrograms/ml
4(5-chlorobenzo(b)thiophen-3-yl)-1- 1.8 fold stimulation Not significant
(2chlorophenyl) sulfonyl)-3, 5 dimethyl- l -H-
pyrazole
4-(2,6-dichlorobenzyl)-3-methyl-l-phenyl-1H- 2.3 fold stimulation two fold
stimulation
pyrazol-5-ol
3-methyl-4-(2-methylallyl)-1-(phenylsulfonyl)- two fold stimulation Not
significant
1H-pyrazol-5-ol
[3-(2,6-difluorophenyl)-4-ethyl-1H-pyrazol-l- 2.5 fold stimulation two fold
stimulation
yl] (2-thienyl)methanone
4-[(5-chloro-l-benzothiophen-3-yl)methyl] N, two fold stimulation two fold
stimulation
3,5-trimethyl-lH-pyrazole-l-carboxamide
3-(2,6-difluorophenyl)-4-ethyl-lH-pyrazole two fold stimulation 86%
stimulation
N1-(3-chlorophenyl)-4-[(5- 86% stimulation 40% stimulation
chlorobenzo [b]thiophen-3-yl)methyl]-3,5 -
dimethyl-lH-pyrazole-l-carboxamide
{4-[(5-chlorobenzo[b]thiophen-3-yl)methyl]-3,5- 74% stimulation 2.5 fold
stimulation
dimethyl-1 H-pyrazol- l -yl } (4-
nitrophenyl)methanone
N1-phenyl-4-[(5-chlorobenzo[b]thiophen-3- two fold stimulation 45% stimulation
yl)methyl]-3,5-dimethyl-lH-pyrazole-l-
carboxamide
4-[(5-chloro-l-benzothiophen-3-yl)methyl] N- 75% stimulation 35% stimulation
(2,4-dichlorophenyl)-3,5-dimethyl-lH-pyrazole-
1-carboxamide
1-[3-(2,6-difluorophenyl)-4-ethyl-1H-pyrazol-l- 58% stimulation not
significant
yl]-2,2-dimethylpropan-l-one
4-(2-chloro-6-fluorobeuzyl)-1-{[3,5- 3.1 fold stimulation 2.4 fold stimulation
di(trifluoromethyl)phenyl]sulfonyl} -3,5-
dimethyl-1H-pyrarole
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Example 18
Inhibitors of Cellular Proliferation
[4-(2,6-dichlorobenzyl)-3,5-dimethyl-1 H-pyrazol-1-yl] [3 -(2,6-
dichlorophenyl)-5-methylisoxazol-4-
yl]methanone was evaluated for the ability to block. HGF/SF induced
proliferation of HUVEC as
described above. It demonstrated 40-60% blockage at 12 micrograms/ml.
Example 19
Inhibition of Tumor Growth
[4-(2,6-Dichlorobenzyl)-3,5-dimethyk-lH-pyrazol-1-yl][3-(2,6-dichlorophenyl)-5-
methylisoxazol-4-
yl]methanone was evaluated for inhibition of human endometrial cancer tumor
growth. Growth was
inhibited by 40-50% at 40 micrograms/ml.
Example 20
Anti-proliferative activity of compounds of the invention
Using the above-described assays, the following compounds were found to block
proliferation of HUVEC
in the absence or presence of exogenously-added HGF/SF.
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Compound Activity at 2 mnicrogram/ml Activity at 6 microgram/ml.
Without With Without With
HGF/SF HGF/SF HGF/SF HGF/SF
(4-(2-chloro-6-fluorobenzyl)-3,5- No effect 40-90% not significant 50-100%
dimethyl-lH-pyrazole-1-yl)(3-(2,6- inhibition inhibition
dichlorophenyl)-5-methylisoxazol-4-
yl)methanone
4-(2-chloro-6-fluorobenzyl)-l-((3,4- 73% not significant not significant not
significant
dichlorophenyl)sulfonyl)-3,5-dimethyl- stimulation
1 H-pyrazole
4-(2-chloro-6-fluorobenzyl)-1,3,5- 50% No effect No effect blocked SF by
trimethyl-lH-pyrazole stimulation 30%
4-(2-chloro-6-fluorobenzyl)-3,5- 54% No effect No effect No effect
dimethyl-lH-pyrazole stimulation
(4-bromo-3,5-dimethyl-lH-pyrazol-l- 74% No effect Not significant No effect
yl)(3-(2,6-dichlorophenyl)isoxazole-4- stimulation
carbohydrazide)
N'4,5-dimethyl-N'4-(5-nitro-2-pyridyl)- No effect No effect No effect blocked
SF by
3-(2,6-dichlorophenyl)isoxazole-4- 43%
carbohydrazide
N'4-(2-(((2,4- 32% 30% No effect No effect
dichlorobenzylidene)amino)oxy)acetyl)- stimulation stimulation
3-(2,6-dichlorophenyl)-5-
methylisoxazole-4-carbohydrazide
3-(2,6-dichlorophenyl)-5- not significant not significant not significant not
significant
methylisoxazole-4-carbohydrazide
N'4-(3-(3,4,5- 50% inhibition Blocked SF by Cytotoxic
trimnethoxyphenyl)propanoyl)-3-(2,6- 50%
dichlorophenyl)-5-methylisoxazole-4-
carbohydrazide
2-nitrophenyl 2-((3-(2,6- No effect No effect 80% inhibition 80% inhibition
dichlorophenyl)-5-methylisoxazol-4-
yl)carbonyl)hydrazine-l-carbothioate
1-((4-chlorophenyl)sulfonyl)-4-(2,6- No effect No effect No effect 50 %
dichlorobenzyl)-3,5-dimethyl-1H- inhibition
pyrazol
3-(4-(2,6-dichlorobenzyl)-3,5-dimethyl- No effect 36% inhibition 34%
inhibition 65% inhibition
1 H-pyrazol-1-yl)propanenitrile
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Compound Activity at 2 microgram/ml Activity at 6 micro am/ml
Without With Without With
HGF/SF HGF/SF HGF/SF HGF/SF
N'4-((2-inethyl-1,3-thiazol-4- No effect 50% inhibition 52% 50% inhibition
4y1)carbonyl)-3-(2,6-dichlorophenyl)-5- stimulation
methylisoxazole-4-4carbohydrazide
Nl-((2-((3-(2,6-dichlorophenyl)-5- No effect No effect No effect 25 %
inhibition
methylisoxazol-4-
yl)carbonyl)hydrazino)(methylthio)meth
ylidene)benzene-l -sulfonamide
N'4-(2,4,6-trichlorophenyl)-3-3(2,6- No effect No effect No effect 60%
inhibition
dichlorophenyl)-5-methylisoxazole-4-
carbohydrazide
N'4,3-di(2,6-dichlorophenyl)-5- No effect not significant 30% inhibition 60 %
inhibition
methylisoxazole-4-carbohydrazide
3,5-di(tert-butyl)-4-(2-chloro-6- 51% 25% inhibition No effect 50% inhibition
fluorobenzyl)-1H-pyrazole stimulation
N'4-(3,5-dichloro-4-pyridyl)-3-(2,6- No effect 25% inhibition No effect 25%
inhibition
dichlorophenyl)-5-methylisoxazole-4-
carbohydrazide
N'4-phenyl-3-(2,6-dichlorophenyl)-5- No effect 40% inhibition No effect 80%
inhibition
methylisoxazole-4-carbohydrazide
(4-(2-chloro-6-fluorobenzyl)-3,5- not significant 23% inhibition not
significant 90% inhibition
dimethyl- l H-pyrazole- l -yl) (2, 6-
dichlorophenyl)methanone
1-(4-(2-chloro-6-fluorobenzyl)-3,5- No effect 25 % inhibition No effect 25 %
inhibition
dimethyl-1H-pyrazole-1-yl)2,2-
dimethylpropan-l-one
N'4,N'4,5-trimethyl-3-(2,6- No effect No effect No effect 40% inhibition
dichlorophenyl)isoxazole-4-
carbohydrazide
N4-azepan-1-yl-3-(2,6-dichlorophenyl)- No effect No effect 50% inhibition 50%
inhibition
5-methylisoxazole-4-carboxamide
N'4-(6-(trifluoromethyl)-2-pyridyl)-3- 40% inhibition 50% inhibition 50%
inhibition 80%inhibition
(2,6-dichlorophenyl)-5-inethylisoxazole-
4-carbohydrazide
(4-(2-chloro-6-fluorobenzyl)-3,5- 40% blocked SF by 50% inhibition 90%
inhibition
dimethyl-1H-pyrazole-1-yl)(4- stimulation 30%
chlorophenyl)methanone
56
CA 02452445 2003-12-29
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Compound Activity at 2 micro graxn/ml Activity at 6 microgram/ml
Without With Without With
HGF/SF HGF/SF HGF/SF HGF/SF
N'4-(3,3-diethoxypropanoyl)-3-(2,6- No effect No effect No effect
35%inhibition
dichlorophenyl)-5-methylisoxazole-4-
carbohydrazide
(4-(2-chloro-6-fluorobenzyl)-3,5- No effect No effect No effect 61% inhibition
dimethyl-1 H-pyrazole- l -yl)(2-
thienyl)methanone
Example 21
Activities of other compounds
The following compounds, structurally unrelated to those described in the
foregoing examples, were also
evaluated for effects on endothelial cell proliferation in the absence and
presence of exogenously-added
HGF/SF. Several compounds with potential antiproliferative activity showed
activity, including those
which showed activity only in the presence of exogenous HGF/SF. Moreover,
several compounds
demonstrated stimulatory activity, and have potential growth promoting
activities useful as described
hereinabove.
Compound Stimulation/Inhibition at 6-12 micrograms/ml
Without HGF/SF With
tetraphenylthiophene 50% inhibition No effect
pentaphenylbenzene two to three fold stimulation 50% inhibition
1,3,5-triphenylbenzene 40-60% stimulation two fold stimulation
(3- Biphenyl) Trimethyl silane 40% stimulation No effect
16 methyl-16 No effect 30% stimulation
Dehydropregnenolone
9-biphenyl-4-ylmethylene-9H- 30-40% stimulation 20-30% stimulation
tri-benzo(A,C,E)-cycloheptene
1,1,3-triphenylinedene 50-100% stimulation 30-40% stimulation
9,9-Biphenanthrene No effect 20-40% inhibition
N-(furfurylidene)-2,4-xylidine 30-40% inhibition 50-60% inhibition
1-(4-Chloro-3 Methyl No effect at 1 ug/ml 40-80% inhibition
Phenyl)3-2(2,6-
dichlorophenyl)Prop-2-ene-1-
one
3-(4-Bromophenyl)-1- 40-80% stimulation at 1 ug/ml some additive effect
phenylprop-2-en- 1 -one
57
CA 02452445 2003-12-29
WO 02/02593 PCT/US01/20849
Activity at lug/ml concentration
Without HGF/SF With HGF/SF
8-Benzyledene -2,4 Diphenyl- no significant effect 40% inhibition
5,6,7,8
Tetrahydrophosphinoline
6-(3,5 -Dimethylphenyl) no significant effect 30% inhibition
Thio)-3-Phenyl (1,2,4-
Triazolo(4,3-b)pyridazine
Example 22
Inhibition of HGF/SF, VEGF and FGF Activities
In endothelial cell proliferation assays as described above, the effect of the
compound 1-(4-chloro-3-
methylphenyl)-3-(2,6-dichlorophenyl)-prop-2-en-l-one was evaluated in the
presence of HGF/SF, VEGF
or FGF for its effects on antagonizing the growth factor-induced stimulation
of endothelial cell
proliferation. As shown in Figure 16A, at 1.5 micromolar, the compound
suppressed the HGF/SF-,
VEFG- and FGF-mediated increase in endothelial cell proliferation. Similar
results were seen with 3
micromolar 1-(4-chloro-3 -methylphenyl)-3-(2,6-dichlorophenyl)-prop-2-en- 1 -
one, Figure 16B.
Example 23
Compounds with VEGF-like Activity
Compounds were screened for VEGF-like activity in a standard assay using 2
microgram/ml compound.
As shown in Figure 17, 3,3-dibromo-l-phenyl-1,2,3,4-tetrahydroquinoline-2,4-
dione (VC8) and 4-(4-
chlorophenyl)-6-(dimethylamino)-2-phenyl-5-pyrimidinecarbonitrile (VC 14)
showed positive activity,
both of which were better than VEGF (1259 104 cpm). The present invention is
also directed to
methods of use of these and structurally-related VEGF agonists or mimics for
the treatment of various
conditions and diseases for which VEGF would be useful for therapy in a
mammal, preferably a human,
such as but not limited to acceleration of wound healing, and in particular,
diabetic wound healing. The
compounds are generally useful for promoting proliferation of vascular
endothelial cells and promoting
vascularization, for such other uses as restenosis for treatment of coronary
artery disease, angina and other
ischemic diseases, including stroke.
Example 24
Further HGF/SF-like activity of
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone
One of the compounds identified with HGF/SF-like activity, (4-chlorophenyl)[3-
(2-(2-thienyl)vinyl)-1H-
pyrazol-1-yl]methanone, was able to stimulate endothelial cell proliferation
in vitro (Figure 18: first bar:
control cells; second bar (4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-l-
yl]methanone, 38 mM;
third bar: HGF/SF, 20 ng/ml; fourth bar: (4-chlorophenyl)[3-(2-(2-
thienyl)vinyl)-1H-pyrazol-l-
yl]methanone + HGF/SF). The specificity of the stimulation by this compound of
growth of HUVECs by
3H-thymid ne incorporation was tested by pre-incubation of cells with the
HGF/SF receptor c-met. (4-
Chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-l-yl]methanone by itself
stimulated 3H-thymidine
58
CA 02452445 2003-12-29
WO 02/02593 PCT/US01/20849
incorporation by more than 5 fold (Figure 18, bar 2). (4-chlorophenyl)[3-(2-(2-
thienyl)vinyl)-1H-pyrazol-
1-yl]methanone is as effective as HGF/SF in stimulating HUVEC proliferation.
In the presence of c-met
the (4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone
stimulation of 3H-thymidine
incorporation was inhibited by 75%. Scattering of MDCK cells in culture is a
known specific effect of
scatter factor and (4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-
yl]methanone also has this ability,
the first demonstration of this activity in a non-peptide compound. (4-
Chlorophenyl)[3-(2-(2-
thienyl)vinyl)-1H-pyrazol-l-yl]methanone, like HGF/SF, did not stimulate the
growth of fibroblast cell
lines and both showed similar inhibitory effects in HepG2 hepatoma cell lines.
Example 25
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone
causes phosphorylation of c-met and Erk
Using immunoprecipitation and Western blotting we were able show that like
HGF/SF, (4-
chlorophenyl)[3-(2-(2-thienyl)vinyl)-lH-pyrazol-l-yl]methanone causes
phosphorylation of the signaling
protein Erk (Figure 19). Both HGF/SF (lane 4) and (4-chlorophenyl)[3-(2-(2-
thienyl)vinyl)-1H-pyrazol-
1-yl]methanone (lane 2) showed significant amounts of phosphorylated Erk
compared to unstimulated
control cells (lane 1). A small molecule antagonist, (4-(2-chloro-6-
fluorobenzyl)-3,5-dimethyl-lH-
pyrazole-l-yl)(3-(2,6-dichlorophenyl)-5-methylisoxazol-4-yl)methanone had no
effect on phosphorylation
of Erk (Lane 3). Total Erk is shown on the bottom.
Example 26
Wound healing studies
The angiogenic properties of (4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-
pyrazol-1-yl]methanone were
further tested in a pig model of wound healing. Full thickness 8-mm skin
wounds were produced in pigs
and five days later the wounds were excised, stained with H&E and blood
vessels counted in five areas
from each section under high power. Wounds treated with (4-chlorophenyl)[3-(2-
(2-thienyl)vin yl)-1H-
pyrazol-1-yl]methanone (500 micrograms) demonstrated a 33% greater density of
blood vessels compared
to vehicle-treated (DMS) controls (Figure 20).
Example 27
Increase in capillary numbers by
(4-chlorophenyl)[3-(2-(2-thienyl)vinyl)-lH pyrazol-1-yl]methanone.
Mice were subjected to unilateral hindlimb ischemia and treated with either
the HGF/SF agonist, (4-
chlorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl]methanone (25
micrograms/day) or vehicle for
either two or three weeks prior to sacrifice. Hindlimb muscles were frozen in
liquid nitrogen and
capillaries stained by the alkaline phosphatase technique and the number of
capillaries per muscle fiber
counted in 6 to 12 random areas of the muscle by a blinded observer.
59
CA 02452445 2003-12-29
WO 02/02593 PCT/US01/20849
Recovery in mice with hindlimb ischemia by increasing the number of
capillaries in the ischemic muscle
was observer (Figure 21). At 2 weeks there was a 42% greater number of
capillaries per muscle fiber in
(4-c1-Aorophenyl)[3-(2-(2-thienyl)vinyl)-1H-pyrazol-l-yl]inethanone-treated
mice compared to vehicle
treated controls. This increased number of capillaries persisted at 3 weeks,
the last time point for which
samples were analyzed.
Example 28
(4-chlorophenyl) [3-(2-(2-thienyl)vinyl)-1H-pyrazol-1-yl] methanone
produces a dose-dependent phosphorylation of c-met
Studies of c-met phosphorylation have been extended to demonstrate that the
phosphorylation is dose-
related and occurs in both HUVECs as well as MDCK cells (Figure 22). HUVECs
(left set) or MDCK
cells (right set) were treated with either HGF/SF or (4-chlorophenyl)[3-(2-(2-
thienyl)vinyl)-1H-pyrazol-l-
yl]methanone, solubilized lysates were prepared from cells and
immunoprecipitation of phosphorylated c-
met and total c-met using specific antibodies was performed using standard
techniques.
Immunoprecipitates were separated on SDS-polyacrylamide gels and proteins were
transferred to
nitrocellulose membranes and detection of phosphorylated (top) and total c-met
(bottom) was performed
using an ECL chemiluminescence system (Amersham). Both HGF/SF and (4-
chlorophenyl)[3-(2-(2-
thienyl)vinyl)-1H-pyrazol-1-yl]methanone showed significant amounts of
phosphorylated c-met
compared to unstimmulated control cells. Total c-met is shown on the bottom.
This result further substantiates the findings that, like HGF/SF, (4-
chlorophenyl)[3-(2-(2-thienyl)vinyl)-
1H-pyrazol-l-yl]methanone produces its effects through activation of the c-met
receptor.
Example 29
Tumor and angiogenesis inhibition by c-met antagonists.
Experiments were performed to determine the ability of HGF/SF antagonists to
inhibit the growth of
tumors and improve survival in in vivo experiments. DU145 tumor cells (5 x
106) were injected
subcutaneously into the lower right flank of male nude mice. On day 14, mice
with established tumors
(tumor size, 25-30 mm2) were treated with a single intra-tumor injection of 1-
(4-chloro-3-methylphenyl)-
3-(2,6-dichlorophenyl)-prop-2-en-1-one (500 ng/50 ml in 50% EtOH/50%
cremophor). 1-(4-Chloro-3-
methylphenyl)-3-(2,6-dichlorophenyl)-prop-2-en-l-one caused a significant
inhibition of the growth of
tumors (Figure 23A). In addition, survival was significantly prolonged (Figure
23B).
Survival of mice recorded as the percentage of surviving animals on a given
day. For all animal
experiments, the tumor size was measured biweekly using a caliper and
expressed as the product of the
maximal perpendicular diameters (mm2). Animals were sacrificed when the tumor
diameter exceeded 150
mm, which is the end point for the experiment.
CA 02452445 2010-01-21
The present invention is not to be limited in scope by the specific
embodiments describe herein. Indeed,
various modifications of the invention in addition to those described herein
will become apparent to those
skilled in the art from the foregoing description and the accompanying
figures. Such modifications are
intended to fall within the scope of the appended claims.
1. Matsumoto, K, and Nakamura, T. (1997) Hepatocyte growth factor (HGF) as a
tissue organizer for
organogenesis and regeneration. Biochem. Biophys. Res. Commun. 239, 639-44.
2. Boros, P. and Miller, C.M. (1995) Hepatocyte growth factor: a
multifunctional cytokine. Lancet 345,
293-5.
3. Morishita, R, Nakamura, S, Nakamura, Y, Aoki, M, Moriguchi, A, Kida, I, Yo,
Y, Matsumoto, K,
Nakamura, T, Higaki, J, Ogihara, T (1997) Potential role of an endothelium-
specific growth factor,
hepatocyte growth factor, on endothelial damage in diabetes. Diabetes 46:138-
42.
4. Grant, D.S, Kleinman, H.K., Goldberg, I.D., Bhargava, M.M., Nickoloff,
B.J., Kinsella, J.L.,
Polverini, P., Rosen, E.M. (1993) Scatter factor induces blood vessel
formation in vivo. Proc. Natl. Acad.
Sci. U S A 90:1937-41.
5. Morishita, R., Nakamura, S., Hayashi, S., Taniyama, Y., Moriguchi, A.,
Nagano, T., Taiji, M.,
Noguchi, H., Takeshita, S., Matsumoto, K., Nakamura, T., Higalci, J., Ogihara,
T. (1999) Therapeutic
angiogenesis induced by human recombinant hepatocyte growth factor in rabbit
hind limb ischemia model
as cytokine supplement therapy. Hypertension 33:1379-84.
6. Jeffers, M., Rong, S., Woude, G.F. (1996) Hepatocyte growth factor/scatter
factor-Met signaling in
tumorigenicity and invasion/metastasis. J. Mol. Med. 74:505-13.
7. Moriyama, T., Kataoka, H., Koono, M., Wakisaka, S. (1999) Expression of
hepatocyte growth
factor/scatter factor and its receptor c-met in brain tumors: evidence for a
role in progression of astrocytic
tumors Int. J. Mol. Med. 3:531-6.
8. Gherardi, E., Hartmann, G., Hepple, J., Chirgadze, D., Srinivasan, N.,
Blundell, T. (1997) Domain
structure of hepatocyte growth factor/scatter factor (HGF/SF). Ciba Found Symp
212:84-93.
61
CA 02452445 2003-12-29
WO 02/02593 PCT/US01/20849
9. Naldini, L., Tamagnone, L., Vigna, E., Sachs, M.., Hartmann, G.,
Birchmeier, W., Daikuhara, Y.,
Tsubouchi, H., Blasi, F., Comoglio, P.M. (1992) Extracellular proteolytic
cleavage by urokinase is
required for activation of hepatocyte growth factor/scatter factor. EMBO J.
11:4825-33.
10. Bardelli, A., Ponzetto, C., Comoglio, P.M. (1994) Identification of f
nctional domains in the
hepatocyte growth factor and its receptor by molecular engineering. J.
Biotechnol . 37:109-22.
11. Sakata, H, Stahl, S. J, Taylor, W. G, Rosenberg, J. M, Sakaguchi, K,
Wingfield, P. T, and Rubin, J.
S. (1997) Heparin binding and oligomerization of hepatocyte growth
factor/scatter factor isoforms.
Heparan sulfate glycosaminoglycan requirement for Met binding and signaling. J
Biol Chem 272, 9457-
9463.
12. Lokker, N.A., Mark, M.R., Luis, E.A., Bennett, G.L., Robbins, K.A., Baker,
J.B., Godowski, P.J.
(1992) Structure-function analysis of hepatocyte growth factor: identification
of variants that lack
mitogenic activity yet retain high affinity receptor binding. EMBO J. 11:2503-
10.
13. O'Neil, K. T. and Hoess, R. H. (1995) Phage Display: Protein engineering
by directed evolution.
Curr. Opin. Struc. Biol. 5, 443-449.
14. Wrighton, N. C., Farrell, F. X., Chang, R., Kashyap, A. K., Barbone, F.
P., Mulcahy, L. S., Johnson,
D. L., Barrett, R. W., Jolliffe, L. K. and Dower, W. J. (1996) Small peptides
as potent mimetics of the
protein hormone erythropoietin. Science 273, 458-461.
15. Widersten, M. and Mannervik, B. (1995) Glutathione S transferase with
novel active sites isolated by
phage display from a library of random mutants. J. Mol. Biol. 250, 115-122.
16. Saggio, I. And Laufer, R. (1993) Biotin binders selected from a random
peptide library expressed on
phage. Biochem. J. 293, 613-616.
17. Pasqualini R, Koivunen, E. and Ruoslahti, E. (1995) A peptide isolated
from phage display libraries
is a structural and functional mimic of an RGD-binding site on integrins. J
Cell Biol. 130, 1189-1196.
18. Koivunen E, Wang, B. and Ruoslahti, E. (1994) Isolation of a highly
specific ligand for the alpha 5
beta 1 integrin from a phage display library. J Cell Biol. 124, 373-380.
19. S. Paka, Goldberg, I. J., Choi, S. Y. Obunike, J., Saxena, U. Goldberg, I.
D. and Pillarisetti, S. (1999)
Perlecan mediates the anti-proliferative effect of apolipoprotein E on smooth
muscle cells J. Biol. Chem.
274, 36403.
62
CA 02452445 2003-12-29
WO 02/02593 PCT/US01/20849
20. Nicosa, R. F. and Ottinetti, A. (1990) Growth of microvessels in serum-
free matrix culture of rat
aorta. Lab. Invest. 63: 115-122.
21. Kibbey, M. C., Grant, D. S. Auerbach, R. and Kleinunan, H. K. (1992) Role
of the SIKVAV site of
laminin in promotion of angiogenesis and tumor growth: an in vivo Matrigel
model. J. Natl. Can. Inst. 84,
1633-38.
22. van der Voort, R., Taher, T.E., Wielenga, V.J., Spaargaren, M., Prevo, R.,
Smit, L., David, G.,
Hartmann, G., Gherardi, E., Pals, S.T. (1999) Heparan sulfate-modified CD44
promotes hepatocyte
growth factor/scatter factor-induced signal transduction through the receptor
tyrosine kinase c-met. J.
Biol. Chem. 274, 6499-506.
23. Liu, S., Julian, J., Carson, D.D. (1998) A peptide sequence of
heparin/heparan sulfate (HP/HS)-
interacting protein supports selective, high affinity binding of HP/HS and
cell attachment. J. Biol. Chem.
273, 9718-26.
Matsumoto, K, and Nakamura, T. (1997) Hepatocyte growth factor (HGF) as a
tissue organizer for
organogenesis and regeneration. Biochem. Biophys. Res. Commun. 239, 639-44
Boros, P. and Miller, C.M. (1995) Hepatocyte growth factor: a multifunctional
cytokine. Lancet 345,
293-5.
Morishita, R, Nakamura, S, Nakamura, Y, Aoki, M, Moriguchi, A, Kida, I, Yo, Y,
Matsumoto, K,
Nakamura, T, Higaki, J, Ogihara, T (1997) Potential role of an endothelium-
specific growth factor,
hepatocyte growth factor, on endothelial damage in diabetes. Diabetes 46:138-
42.
Grant, D.S, Kleinman, H.K., Goldberg, I.D., Bhargava, M.M., Nickoloff, B.J.,
Kinsella, J.L., Polverini, P.,
Rosen, E.M. (1993) Scatter factor induces blood vessel formation in vivo.
Proc. Natl. Acad. Sci. U S A
90:1937-41.
Morishita, R., Nakamura, S., Hayashi, S., Taniyama, Y., Moriguchi, A., Nagano,
T., Taiji, M., Noguchi,
H., Takeshita, S., Matsumoto, K., Nakamura, T., Higaki, J., Ogihara, T. (1999)
Therapeutic angiogenesis
induced by human recombinant hepatocyte growth factor in rabbit hind limb
ischemia model as cytokine
supplement therapy. Hypertension 33:1379-84.
Jeffers, M., Rong, S., Woude, G.F. (1996) Hepatocyte growth factor/scatter
factor-Met signaling in
tumorigenicity and invasion/metastasis. J. Mol. Med. 74:505-13.
63
CA 02452445 2003-12-29
WO 02/02593 PCT/US01/20849
Moriyama, T., Kataoka, H., Koono, M., Wakisaka, S. (1999) Expression of
hepatocyte growth
factor/scatter factor and its receptor c-met in brain tumors: evidence for a
role in progression of astrocytic
tumors Int. J. Mol. Med. 3:531-6.
Gherardi, E., Hartmann, G., Hepple, J., Chirgadze, D., Srinivasan, N.,
Blundell, T. (1997) Domain
structure of hepatocyte growth factor/scatter factor (HGF/SF). Ciba Found Symp
212:84-93.
Naldini, L., Tamagnone, L., Vigna, E., Sachs, M.., Hartmann, G., Birchmeier,
W., Daikuhara, Y.,
Tsubouchi, H., Blasi, F., Comoglio, P.M. (1992) Extracellular proteolytic
cleavage by urokinase is
required for activation of hepatocyte growth factor/scatter factor. EMBO J.
11:4825-33.
Bardelli, A., Ponzetto, C., Comoglio, P.M. (1994) Identification of functional
domains in the hepatocyte
growth factor and its receptor by molecular engineering. J Biotechnol. 37:109-
22.
Sakata, H, Stahl, S. J, Taylor, W. G, Rosenberg, J. M, Sakaguchi, K,
Wingfield, P. T, and Rubin, J. S.
(1997) Heparin binding and oligomerization of hepatocyte growth factor/scatter
factor isoforms. Heparan
sulfate glycosaminoglycan requirement for Met binding and signaling. J Biol
Chem 272, 9457-9463.
Lokker, N.A., Mark, M.R., Luis, E.A., Bennett, G.L., Robbins, K.A., Baker,
J.B., Godowski, P.T. (1992)
Structure-function analysis of hepatocyte growth factor: identification of
variants that lack mitogenic
activity yet retain high affinity receptor binding. EMBO J 11:2503-10.
O'Neil, K. T. and Hoess, R. H. (1995) Phage Display: Protein engineering by
directed evolution. Curr.
Opin. Struc. Biol. 5, 443-449.
Wrighton, N. C., Farrell, F. X., Chang, R., Kashyap, A. K., Barbone, F. P.,
Mulcahy, L. S., Johnson, D.
L., Barrett, R. W., Jolliffe, L. K. and Dower, W. J. (1996) Small peptides as
potent mimetics of the protein
hormone erythropoietin. Science 273, 458-461.
Widersten, M. and Mannervik, B. (1995) Glutathione S transferase with novel
active sites isolated by
phage display from a library of random mutants. J. Mol. Biol. 250, 115-122.
Saggio, I. And Laufer, R. (1993) Biotin binders selected from a random peptide
library expressed on
phage. Biochem. J. 293, 613-616.
Pasqualini R, Koivunen, E. and Ruoslahti, E. (1995) A peptide isolated from
phage display libraries is a
structural and functional mimic of an RGD-binding site on integrins. J Cell
Biol. 130, 1189-1196.
64
CA 02452445 2003-12-29
WO 02/02593 PCT/US01/20849
Koivunen E, Wang, B. and Ruoslahti, E. (1994) Isolation of a highly specific
ligand for the alpha 5 beta 1
integrin from a phage display library. J Cell Biol. 124, 373-380.
S. Paka, Goldberg, I. J., Choi, S. Y. Obunike, J., Saxena, U. Goldberg, I. D.
and Pillarisetti, S. (1999)
Perlecan mediates the anti-proliferative effect of apolipoprotein E on smooth
muscle cells J. Biol. Chem.
274, 36403.
Nicosa, R. F. and Ottinetti, A. (1990) Growth of microvessels in serum-free
matrix culture of rat aorta.
Lab. Invest. 63: 115-122.
Kibbey, M. C., Grant, D. S. Auerbach, R. and Kleinman, H. K. (1992) Role of
the SIKVAV site of
laminin in promotion of angiogenesis and tumor growth: an in vivo Matrigel
model. J. Natl. Can. Inst. 84,
1633-38.
van der Voort, R., Taher, T.E., Wielenga, V.J., Spaargaren, M., Prevo, R.,
Smit, L., David, G., Hartmann,
G., Gherardi, E., Pals, S.T. (1999) Heparan sulfate-modified CD44 promotes
hepatocyte growth
factor/scatter factor-induced signal transduction through the receptor
tyrosine kinase c-met. J. Biol. Chem.
274, 6499-506.
Liu, S., Julian, J., Carson, D.D. (1998) A peptide sequence of heparin/heparan
sulfate (HP/HS)-interacting
protein supports selective, high affinity binding of HP/HS and cell
attachment. J. Biol. Chem. 273, 9718-
26.
CA 02452445 2004-05-14
SEQUENCE LISTING
<110> North Shore - Long Island Jewish Health System
<120> MODULATORS OF CELLULAR PROLIFERATION AND ANGIOGENESIS,
METHODS FOR USE AND IDENTIFICATION THEREOF
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<150> US 09/606,628
<151> 2000-06-29
<150> UNKNOWN
<151> 2001-03-15
<160> 19
<170> Patentln version 3.1
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<213> synthetic
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Thr Met Gly Phe Thr Ala Pro Arg Phe Pro His Tyr
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<220>
<223>
<400> 2
Lys Val Trp Tyr His Thr Thr Ser Ile Pro Ser His
1 5 10
65a
CA 02452445 2004-05-14
<210> 3
<211> 12
<212> PRT
<213> synthetic
<220>
<223>
<400> 3
Ala Thr Trp Ser His His Leu Ser Ser Ala Gly Leu
1 5 10
<210> 4
<211> 12
<212> PRT
<213> synthetic
<220>
<223>
<400> 4
Trp Pro Gln Leu Pro Pro Arg Pro Tyr Ser Thr Leu
1 5 10
<210> 5
<211> 12
<212> PRT
<213> synthetic
<220>
<223>
<400> 5
Ser Asn Thr Ser Ala Gly Thr Pro Phe Thr Ser Leu
1 5 10
<210> 6
<211> 12
<212> PRT
<213> synthetic
<220>
<223>
65b
CA 02452445 2004-05-14
<400> 6
Asp Ser Thr Pro Lys Ser Thr Pro Trp Tyr Tyr Ile
1 5 10
<210> 7
<211> 28
<212> PRT
<213> synthetic
<220>
<223>
<400> 7
Lys Val Trp Tyr His Thr Thr Ser Ile Pro Ser His Cys Arg Pro Lys
1 5 10 15
Ala Lys Ala Lys Ala Lys Ala Lys Asp Gln Thr Lys
20 25
<210> 8
<211> 25
<212> PRT
<213> synthetic
<220>
<223>
<400> 8
Tyr His Thr Thr Ser Ile Pro Ser His Cys Gln Lys Ala Lys Thr Arg
1 5 10 15
Ala Lys Ala Ala Lys Pro Asp Lys Lys
20 25
<210> 9
<211> 40
<212> PRT
<213> synthetic
<220>
<223>
<400> 9
Thr Met Gly Phe Thr Ala Pro Arg Phe Pro His Tyr Lys Val Trp Tyr
1 5 10 15
His Thr Thr Ser Ile Pro Ser His Cys Arg Pro Lys Ala Lys Ala Lys
20 25 30
65c
CA 02452445 2004-05-14
Ala Lys Ala Lys Asp Gin Thr Lys
35 40
<210> 10
<211> 40
<212> PRT
<213> synthetic
<220>
<223>
<400> 10
Lys Val Trp Tyr His Thr Thr Ser Ile Pro Ser His Lys Val Trp Tyr
1 5 10 15
His Thr Thr Ser Ile Pro Ser His Cys Arg Pro Lys Ala Lys Ala Lys
20 25 30
Ala Lys Ala Lys Asp Gin Thr Lys
35 40
<210> 11
<211> 11
<212> PRT
<213> synthetic
<220>
<223>
<400> 11
Lys Thr Tyr Ala Gly Ser Ser Tyr Gin Phe Gly
1 5 10
<210> 12
<211> 12
<212> PRT
<213> synthetic
<220>
<223>
<400> 12
Ser Gly Trp His Met Arg Ser Pro Phe Asn His Met
1 5 10
65d
CA 02452445 2004-05-14
<210> 13
<211> 12
<212> PRT
<213> synthetic
<220>
<223>
<400> 13
His Leu Lys Pro His Phe Trp Pro Ser Ser Pro Tyr
1 5 10
<210> 14
<211> 12
<212> PRT
<213> synthetic
<220>
<223>
<400> 14
Leu Leu Ala Asp Thr Thr His His Arg Pro Trp Thr
1 5 10
<210> 15
<211> 12
<212> PRT
<213> synthetic
<220>
<223>
<400> 15
Asn His Pro His Pro Thr Pro Ala Arg Gly Ile Ile
1 5 10
<210> 16
<211> 12
<212> PRT
<213> synthetic
<220>
<223>
65e
CA 02452445 2004-05-14
<400> 16
Val Ser Arg His Gln Ser Trp His Pro His Asp Leu
1 5 10
<210> 17
<211> 12
<212> PRT
<213> synthetic
<220>
<223>
<400> 17
Val Ser Arg His Gln Ser Trp His Pro His Asp Leu
1 5 10
<210> 18
<211> 12
<212> PRT
<213> synthetic
<220>
<223>
<400> 18
Ala Leu Asn Trp Ser Arg Lys Leu Pro Val Pro Pro
1 5 10
<210> 19
<211> 12
<212> PRT
<213> synthetic
<220>
<223>
<400> 19
Gln Thr Gly His Trp Asn Ala Glu Trp His Thr Arg
1 5 10
65f
