U.S. patent application number 09/865285 was filed with the patent office on 2002-08-08 for method for reducing or preventing the establishment, growth or metastasis of cancer by administering benzimidazolone peptidomimetics par-1 antagonist and optionally par-2 antagonists.
Invention is credited to D'Andrea, Michael, Derian, Claudia, Woodrow, Hal Brent.
Application Number | 20020107204 09/865285 |
Document ID | / |
Family ID | 26839224 |
Filed Date | 2002-08-08 |
United States Patent
Application |
20020107204 |
Kind Code |
A1 |
D'Andrea, Michael ; et
al. |
August 8, 2002 |
Method for reducing or preventing the establishment, growth or
metastasis of cancer by administering benzimidazolone
peptidomimetics PAR-1 antagonist and optionally PAR-2
antagonists
Abstract
We have discovered a method of modifying the tumor cell
microenvironment to reduce or prevent the establishment, growth or
metastasis of malignant cells comprising administering to a patient
having malignant cells a pharmaceutically effective amount of a
PAR-1 inhibitor and optionally a PAR-2 inhibitor to prevent or
reduce activation of normal cells within the tumor
microenvironment. This method also has the effect in some patients
of modulating the immune system to facilitate a more efficient
immune response to malignant cells and maybe coupled with cytokine
therapy and T-cell therapy to enhance the patient's immune response
to the malignant cells.
Inventors: |
D'Andrea, Michael; (Cherry
Hill, NJ) ; Derian, Claudia; (Hatboro, PA) ;
Woodrow, Hal Brent; (Princeton, NJ) |
Correspondence
Address: |
AUDLEY A. CIAMPORCERO JR.
JOHNSON & JOHNSON
ONE JOHNSON & JOHNSON PLAZA
NEW BRUNSWICK
NJ
08933-7003
US
|
Family ID: |
26839224 |
Appl. No.: |
09/865285 |
Filed: |
May 25, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09865285 |
May 25, 2001 |
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09599826 |
Jun 22, 2000 |
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60141552 |
Jun 29, 1999 |
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Current U.S.
Class: |
424/85.2 ;
514/13.3; 514/19.8; 514/21.9; 548/304.7; 548/306.4 |
Current CPC
Class: |
H01L 2924/01078
20130101; H01L 2924/01079 20130101; H01L 2924/01044 20130101; H05K
3/4092 20130101; H01L 2924/01027 20130101; C07D 235/26 20130101;
A61K 38/00 20130101; H01L 2924/01047 20130101; H01L 2924/01046
20130101; H01L 2924/01074 20130101; H01L 2924/01013 20130101; H01L
2924/01045 20130101; H01L 2924/01023 20130101; H01L 2924/01042
20130101; H01L 2924/01029 20130101; H01L 2924/0104 20130101; H01L
2924/01076 20130101; C07K 5/06191 20130101; H01L 2924/01077
20130101; H01L 2924/01024 20130101 |
Class at
Publication: |
514/19 ;
548/304.7; 548/306.4 |
International
Class: |
A61K 038/05; C07D
235/24; C07D 43/02 |
Claims
We Claim:
1. A method of modifying the tumor cell microenvironrment to reduce
or prevent the establishment, growth or metastasis of malignant
cells that directly or indirectly activate the PAR-1 receptor of
normal cells comprising providing a pharmaceutically effective
amount of a PAR-1 inhibitor having the formula (I): 12wherein:
A.sub.1 and A.sub.2 are each independently a D- or L-amino acid
selected from the group consisting of alanine, .beta.-alanine,
arginine, homoarginine, cyclohexylalanine, citrulline, cysteine
(optionally substituted with C.sub.1-C.sub.4 alkyl, aryl, or
arC.sub.1-C.sub.4 alkyl), 2,4-diaminobutyric acid (optionally
substituted with acyl, C.sub.1-C.sub.4 alkyl, aroyl, amidino, or
MeC(NH)--), 2,3 diaminopropionic acid (optionally substituted with
acyl, C.sub.1-C.sub.4 alkyl, aroyl, amidino, or MeC(NH)--),
glutamine, glycine, indanylglycine, lysine (optionally substituted
with acyl, C.sub.1-C.sub.4 alkyl, aroyl, MeC(NH)--), valine,
methionine, proline, serine (optionally substituted with
C.sub.1-C.sub.4 alkyl, aryl, or arC.sub.1-C.sub.4 alkyl),
homoserine (optionally substituted with C.sub.1-C.sub.4 alkyl,
aryl, or arC.sub.1-C.sub.4 alkyl), tetrahydroisoquinoline-3--COOH,
threonine (optionally substituted with C.sub.1-C.sub.4 alkyl, aryl,
or arC.sub.1-C.sub.4 alkyl), ornithine (optionally substituted with
acyl, C.sub.1-C.sub.4 alkyl, aroyl, MeC(NH)--), and an
unsubstituted or substituted aromatic amino acid selected from the
group consisting of phenylalanine, heteroarylalanine,
naphthylalanine, homophenylalanine, histidine, tryptophan,
tyrosine, arylglycine, heteroarylglycine, aryl-.beta.-alanine, and
heteroaryl-.beta.-alanine wherein the substituents on the aromatic
amino acid are independently selected from one or more of halogen,
C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, hydroxy,
C.sub.1-C.sub.4 alkoxycarbonyl, amino, amidino, guanidino,
fluorinated C.sub.1-C.sub.4 alkyl, fluorinated C.sub.1-C.sub.4
alkoxy, C.sub.1-C.sub.4 alkylsulfonyl, C.sub.1-C.sub.4
alkylcarbonyl, cyano, aryl, heteroaryl, arC.sub.1-C.sub.4 alkyl,
C.sub.2-C.sub.4 alkenyl, alkynyl, or nitro; R.sub.1 is selected
from amino, C.sub.1-C.sub.8 alkylamino, C.sub.1-C.sub.8
dialkylamino, arylamino, arC.sub.1-C.sub.8 alkylamino,
C.sub.3-C.sub.8 cycloalkylamino, heteroalkylC.sub.1-C.sub.8
alkylamino, heteroalkylC.sub.1-C.sub.8 alkyl-N-methylamino,
C.sub.1-C.sub.8 dialkylaminoC.sub.1-C.sub.8 alkylamino,
--N(C.sub.1-C.sub.8alkyl)-C.sub.1-C.sup.8
alkyl-N(C.sub.1-C.sub.8alkyl).s- ub.2, N(C.sub.1-C.sub.8
alkyl)(C.sub.1-C.sub.8 alkenyl),
--N(C.sub.1-C.sub.8alkyl)(C.sub.3-C.sub.8cycloalkyl), heteroalkyl
or substituted heteroalkyl wherein the substituent on the
heteroalkyl is selected from oxo, amino, C.sub.1-C.sub.8
alkoxyC.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 alkylamino or
C.sub.1-C.sub.8 dialkylamino; R.sub.2 and R.sub.3 are each
independently selected from hydrogen, C.sub.1-C.sub.8 alkyl,
C.sub.3-C.sub.8 cycloalkyl, C.sub.3-C.sub.8
cycloalkylC.sub.1-C.sub.8 alkyl, aryl, heteroalkyl, substituted
heteroalkyl (wherein the substituent on the heteroalkyl is one or
more substituents independently selected from C.sub.1-C.sub.8
alkoxycarbonyl, C.sub.1-C.sub.8 alkyl, or C.sub.1-C.sub.4
alkylcarbonyl), heteroalkylC.sub.1-C.sub.8 alkyl, indanyl,
acetamidinoC.sub.1-C.sub.8 alkyl, aminoC.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 alkylaminoC.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8
dialkylaminoC.sub.1-C.su- b.8 alkyl, unsubstituted or substituted
heteroarylC.sub.1-C.sub.8 alkyl or unsubstituted or substituted
arC.sub.1-C.sub.8 alkyl, wherein the substituent on the aralkyl or
heteroarylalkyl group is one or more substituents independently
selected from halogen, nitro, amino, C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 alkoxy, hydroxy, cyano, C.sub.1-C.sub.4
alkylcarbonyl, C.sub.1-C.sub.8 alkoxycarbonyl,
hydroxyC.sub.1-C.sub.8 alkyl or aminosulfonyl; or R.sub.2 and
R.sub.3, together with the nitrogen to which they are attached,
alternatively form an unsubstituted or substituted heteroalkyl
group selected from piperidinyl, piperazinyl, morpholinyl or
pyrrolidinyl, wherein the substituent is one or more substituents
independently selected from C.sub.1-C.sub.8 alkyl C.sub.1-C.sub.8
alkoxycarbonyl or C.sub.1-C.sub.4 alkylcarbonyl; R.sub.4 is
selected from unsubstituted or substituted aryl, arC.sub.1-C.sub.8
alkyl, C.sub.3-C.sub.8 5 cycloalkyl, or heteroaryl, where the
substituents on the aryl, arC.sub.1-C.sub.8 alkyl, cycloalkyl or
heteroaryl group are independently selected from one or more of
halogen, nitro, amino, cyano, hydroxyalkyl, C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 alkoxy, hydroxy, C.sub.1-C.sub.4 alkylcarbonyl,
C.sub.1-C.sub.8 alkoxycarbonyl, fluorinated C.sub.1-C.sub.4 alkyl,
fluorinated C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 alkylsulfonyl;
R.sub.5 is selected from hydrogen or C.sub.1-C.sub.8 alkyl; X is
oxygen or sulfur; m is an integer selected from 0, 1, 2 or 3; n is
an integer selected from 2 or 3; and p is an integer selected from
0 or 1; and pharmaceutically acceptable salts thereof and
optionally a PAR-2 inhibitor to a patient with malignant cells that
directly or indirectly activate PAR-1 and/or PAR-2.
2. The method of claim 1 wherein the PAR-1 inhibitor is
administered with a therapeutically effective amount of at least
one PAR-2 inhibitor.
3. The method of claim 1 wherein the PAR-1 inhibitor is
administered with a therapeutically effective amount of a cytokine
selected from the group consisting of IL-2, IL-12, IL-18, G-CSF,
M-CSF, GM-CSF, INF-.alpha., INF-.beta., INF-.gamma., TNF and
combinations thereof.
4. The method of claim 3 wherein additionally administered in a
pharmaceutical effective amount is at least one conventional
chemotherapy agent.
5. The method of claim 4 wherein the chemotherapy agent is selected
from the group consisting of antiangiogenic compounds, alkylating
compounds, antimetabolites, hormonal agonist/antagonists,
monoclonal antibodies for cancer treatment, antiproliferative
compounds and combinations thereof.
6. The method of claim 1 wherein additional administered are T
cells selected from the group consisting of activated T cells,
activated NK cells and combinations thereof.
7. The method of claim 1 wherein the PAR-1 inhibitor is
administered before surgery.
8. The method of claim 1 wherein the PAR-1 inhibitor is
administered after surgery.
9. A method for the modulation of the immune system to enhance a
patient's immune response to malignant cells that directly or
indirectly activate the PAR-1 receptor of normal cells comprising
administer a pharmaceutically effective dose of a PAR-1 inhibitor
having the formula (I): 13wherein: A.sub.1 and A.sub.2 are each
independently a D- or L-amino acid selected from the group
consisting of alanine, .beta.-alanine, arginine, homoarginine,
cyclohexylalanine, citrulline, cysteine (optionally substituted
with C.sub.1-C.sub.4 alkyl, aryl, or arC.sub.1-C.sub.4 alkyl),
2,4-diaminobutyric acid (optionally substituted with acyl,
C.sub.1-C.sub.4 alkyl, aroyl, amidino, or MeC(NH)--), 2,3
diaminopropionic acid (optionally substituted with acyl,
C.sub.1-C.sub.4 alkyl, aroyl, amidino, or MeC(NH)--), glutamine,
glycine, indanylglycine, lysine (optionally substituted with acyl,
C.sub.1-C.sub.4 alkyl, aroyl, MeC(NH)--), valine, methionine,
proline, serine (optionally substituted with C.sub.1-C.sub.4 alkyl,
aryl, or arC.sub.1-C.sub.4 alkyl), homoserine (optionally
substituted with C.sub.1-C.sub.4 alkyl, aryl, or arC.sub.1-C.sub.4
alkyl), tetrahydroisoquinoline-3-COOH, threonine (optionally
substituted with C.sub.1-C.sub.4 alkyl, aryl, or arC.sub.1-C.sub.4
alkyl), omithine (optionally substituted with acyl, C.sub.1-C.sub.4
alkyl, aroyl, MeC(NH)--), and an unsubstituted or substituted
aromatic amino acid selected from the group consisting of
phenylalanine, heteroarylalanine, naphthylalanine,
homophenylalanine, histidine, tryptophan, tyrosine, arylglycine,
heteroarylglycine, aryl-.beta.-alanine, and
heteroaryl-.beta.-alanine wherein the substituents on the aromatic
amino acid are independently selected from one or more of halogen,
C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, hydroxy,
C.sub.1-C.sub.4 alkoxycarbonyl, amino, amidino, guanidino,
fluorinated C.sub.1-C.sub.4 alkyl, fluorinated C.sub.1-C.sub.4
alkoxy, C.sub.1-C.sub.4 alkylsulfonyl, C.sub.1-C.sub.4
alkylcarbonyl, cyano, aryl, heteroaryl, arC.sub.1-C.sub.4 alkyl,
C.sub.2-C.sub.4 alkenyl, alkynyl, or nitro; R.sub.1 is selected
from amino, C.sub.1-C.sub.8 alkylamino, C.sub.1-C.sub.8
dialkylamino, arylamino, arC.sub.1-C.sub.8 alkylamino,
C.sub.3-C.sub.8 cycloalkylamino, heteroalkylC.sub.1-C.sub.8
alkylamino, heteroalkylC.sub.1-C.sub.8 alkyl-N-methylamino,
C.sub.1-C.sub.8 dialkylaminoC.sub.1-C.sub.8 alkylamino,
--N(C.sub.1-C.sub.8alkyl)-C.sub.1-C.sub.8
alkyl-N(C.sub.1-C.sub.8alkyl).s- ub.2, N(C.sub.1-C.sub.8
alkyl)(C.sub.1-C.sub.8 alkenyl),
--N(C.sub.1-C.sub.8alkyl)(C.sub.3-C.sub.8cycloalkyl), heteroalkyl
or substituted heteroalkyl wherein the substituent on the
heteroalkyl is selected from oxo, amino, C.sub.1-C.sub.8
alkoxyC.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 alkylamino or
C.sub.1-C.sub.8 dialkylamino; R.sub.2 and R.sub.3 are each
independently selected from hydrogen, C.sub.1-C.sub.8 alkyl,
C.sub.3-C.sub.8 cycloalkyl, C.sub.3-C.sub.8
cycloalkylC.sub.1-C.sub.8 alkyl, aryl, heteroalkyl, substituted
heteroalkyl (wherein the substituent on the heteroalkyl is one or
more substituents independently selected from C.sub.1-C.sub.8
alkoxycarbonyl, C.sub.1-C.sub.8 alkyl, or C.sub.1-C.sub.4
alkylcarbonyl), heteroalkylC.sub.1-C.sub.8 alkyl, indanyl,
acetamidinoC.sub.1-C.sub.8 alkyl, aminoC.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 alkylaminoC.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8
dialkylaminoC.sub.1-C.su- b.8 alkyl, unsubstituted or substituted
heteroarylC.sub.1-C.sub.8 alkyl or unsubstituted or substituted
arC.sub.1-C.sub.8 alkyl, wherein the substituent on the aralkyl or
heteroarylalkyl group is one or more substituents independently
selected from halogen, nitro, amino, C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 alkoxy, hydroxy, cyano, C.sub.1-C.sub.4
alkylcarbonyl, C.sub.1-C.sub.8 alkoxycarbonyl,
hydroxyC.sub.1-C.sub.8 alkyl or aminosulfonyl; or R.sub.2 and
R.sub.3, together with the nitrogen to which they are attached,
alternatively form an unsubstituted or substituted heteroalkyl
group selected from piperidinyl, piperazinyl, morpholinyl or
pyrrolidinyl, wherein the substituent is one or more substituents
independently selected from C.sub.1-C.sub.8 alkyl C.sub.1-C.sub.8
alkoxycarbonyl or C.sub.1-C.sub.4 alkylcarbonyl; R.sub.4 is
selected from unsubstituted or substituted aryl, arC.sub.1-C.sub.8
alkyl, C.sub.3-C.sub.8 cycloalkyl, or heteroaryl, where the
substituents on the aryl, arC.sub.1-C.sub.8 alkyl, cycloalkyl or
heteroaryl group are independently selected from one or more of
halogen, nitro, amino, cyano, hydroxyalkyl, C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 alkoxy, hydroxy, C.sub.1-C.sub.4 alkylcarbonyl,
C.sub.1-C.sub.8 alkoxycarbonyl, fluorinated C.sub.1-C.sub.4 alkyl,
fluorinated C.sub.1C.sub.4 alkoxy, C.sub.1-C.sub.4 alkylsulfonyl;
R.sub.5 is selected from hydrogen or C.sub.1-C.sub.8 alkyl; X is
oxygen or sulfur; m is an integer selected from 0, 1, 2 or 3; n is
an integer selected from 2 or 3; and p is an integer selected from
0 or 1; and pharmaceutically acceptable salts thereof and
optionally a PAR-2 inhibitor to the patient to enhance the
patient's immune response to the malignant cells.
10. The method of claim 9 wherein additionally administered are
cytokines to facilitate the development of a Th1 response.
11. The method of claim 10 wherein the cytokines are selected from
the group consisting of IL-2, IL12, IL-18, INF-.alpha., INF-.beta.,
INF-.gamma., TNF and combinations thereof.
12. The method of claim 9 wherein additionally administered are T
cells selected from the group consisting of activated CTL cells,
activated NK cells and combinations thereof.
13. The method of claim 9 wherein additionally administered are
activated NK cells.
14. The method of claim 9 wherein additionally administered are
activated CTL cells.
15. The method of claim 9 wherein the PAR-1 inhibitor is
administered before surgery.
16. The method of claim 9 wherein the PAR-1 inhibitor is
administered after surgery.
Description
FIELD OF THE INVENTION
[0001] This invention is a continuation-in-part of serial number
09/599,826 filed Jun. 22, 2000 and claims benefit of Ser. No.
60/141,552 filed on Jun. 29, 1999 (both hereby incorporated by
reference). This invention relates to the use of PAR-1 antagonist
and optionally PAR-2 antagonist to reduce or prevent the
establishment, growth and/or metastasis of malignant cells, as well
as, immune modulation to aid in the treatment of malignant
cells.
BACKGROUND OF THE INVENTION
[0002] Malignant cells solicit the help of other cell types such as
stromal fibroblasts, mast cells, monocytes and vascular cells, to
facilitate their invasion into the surrounding tissue (Gregoire and
Lieubeau, 1995) because unrestrained growth of the tumor, by
itself, does not result in invasion and metastasis (Liotta et al.,
1991). The interface between the invading malignant cells and the
hosting stromal cells, referred to as the tumor microenvironment
(TME) (O'Meara, 1958), possesses a vast array of well-orchestrated
cell signaling molecules which function to facilitate the
proliferating tumor front to invade the stroma, degrade and remodel
the extracellular matrix and so forth (Gregoire and Lieubeau,
1995). Of the many factors secreted by the tumor cells, the two
proteolytic enzymes, thrombin and trypsin, have been correlated to
the stage and type of carcinoma and are associated with cell
invasion and extracellular matrix degradation (Koivunen et al.,
1991). Furthermore, the ratio of proteases to their inhibitors in
the TME can favor capillary sprout elongation and lumen formation
during angiogenesis (Liotta et al., 1991).
[0003] Thrombin is known for example to facilitate metastasis,
stimulate the adherence of platelets, increase vascular
permeability, attract monocytes, stimulate mitogenic activity of
endothelial cells and fibroblasts, degranulate mast cells (Fenton
et al., 1995; Vouret-Craviari et al., 1992 ; Carney, 1992;
Nierodzik et al., 1992 & 1996; Wojtukiewicz et al., 1993 &
1995; Cirino et al., 1996; Razin and Marx, 1984).
[0004] Thrombin also influences the rate of deposition of
connective tissue proteins and the development of tissue fibrosis
during normal wound healing; a process similar to cellular
metastasis (Chambers et al., 1998). Many of thrombin's effects are
mediated through a seven transmembrane G-protein coupled receptor
known as protease-activated receptor-1 (PAR-1) via proteolytic
cleavage of the amino-terminal extension unveiling a new amino
terminus that folds back on the receptor thereby activating the
receptor as a tethered peptide ligand (Vu et al., 1991). Thrombin
and PAR-1 agonist peptides promote tumor cell adhesion to
endothelium, extracellular matrix and platelets, enhance the
metastatic capacity of tumor cells, activate cell growth and
stimulate angiogenesis (Nierodzik et al., 1992 & 1995;
Dennington and Berndt, 1994; Klementsen and Jorgensen, 1997;
Wojtukiewicz et al., 1993 & 1995; Tsopanglou et al., 1997;
Mirza et al., 1996). PAR-1 has been localized in pancreas tumor
cells (Rudroff et al., 1998), carcinoma and melanoma cell lines
(Wojtukiewicz et al., 1995). In breast carcinoma cells, the level
of PAR-1 expression has been correlated to the degree of
invasiveness (Even-Ram et al., 1998). Furthermore, B16F10 melanoma
cells, transfected with PAR-1, enhanced thrombin-treated tumor cell
adhesion to fibronectin 2.5-fold in vitro and pulmonary metastasis
as high as 39-fold in vivo compared to the control thrombin-treated
tumor cells (Chen et al., 1998). However, the expression of PAR-1
in malignant and benign human tumor tissues has not been
extensively described in their histological context among the
surrounding cell types forming the TME.
[0005] Trypsin can stimulate fibroblasts to secrete procollagen,
stimulate mast cells to degranulate and is secreted by numerous
tumor cell lines that are correlated with the stage and
histological type of carcinoma (Koivunen et al., 1991; Koshikawa et
al., 1992 & 1994;Hirahara et al., 1995). Some of the actions of
trypsin are mediated by a second protease-activated receptor known
as PAR-2 (Nystedt et al., 1994; Bohm et al., 1996; Mizra et al.,
1996;Hollenberg et al., 1996) which has been described in human
tissues and tumor cell lines (Nystedt et al., 1994; Bohm et al.,
1996; D'Andrea et al., 1998). Trypsin's ability to degrade matrix
proteins suggests it may participate in the processes of invasion,
adhesion and metastasis; however, the presence of trypsin in tumors
also suggests that PAR-2 may mediate these processes (Miyata et
al., 2000). Although it is clear that tumor-derived trypsin-like
enzymes could directly regulate growth in an autocrine and/or
paracrine manner via PAR-2 activation (Bohm et al., 1996), the
function of PAR-2 activation in metastasis has not been
described.
[0006] Since both thrombin and trypsin appear to activate several
immune cells including monocytes/macrophages and mast cells.
Monocytes/macrophages and mast cells may play a significant role in
facilitating the survival, growth and metastasis of malignant
cells. The role of monocyte infiltration in tumors is somewhat
controversial. In some circumstances monocyte infiltration has been
associated both with inhibiting tumor growth and in other
circumstances with stimulating tumor growth. However, if the
macrophages/monocytes have been activated they will produce
reactive oxygen species that will lead to the decreased expression
of CD3.zeta. and results in reduced T cell response to tumor cells
(Otsuji et al., 1996). The activation and degranulation of mast
cells is associated with the expression of Th2 cytokines (e.g.
IL-4, IL-10, IL-13) and growth factors (e.g. TFG-.beta.) that would
lead be expected to lead to the recruitment of other immune cells
(i.e. CD4 T cells etc.) and the development of a localized Th2 type
immune response (Bradding et al., 1995; Moller et al. 1998). One
consequence of a localized Th2 immune response being established is
the localized suppression of Th1 cytokine expression.
Unfortunately, the Th1 cytokines are associated with the activation
of cytolytic T cells (CTL) and natural killer cells (NK cells),
which are believed to be the principle cells that the immune system
uses in attempting to respond to the presence of malignant cell and
in limiting their growth and metastasis. Studies in animals have
demonstrated that IL-4 will suppress T lymphocytes from tumor
draining lymph nodes in vivo (Fu et al., 1997). Similarly, the
expression of IL-10 also appears to block the generation of a tumor
specific Th1 immune response (Halak et al., 1999). In human
patients with various cancers the mean level of IL-4 seems to be
elevated and the levels of cytokines associated with Th1 response
are significantly reduced as compared to healthy subjects (Goto et
al., 1999). The growth factors secreted by mast cells also may play
a role in immune suppression. TGF-.beta. appears to suppress T cell
response to tumors (Jarnicki et al. 1996). Additionally, mast cells
degranulation will release among other things neutral proteases
such as tryptase (which cleaves fibrinogen and activates
collagenase) and chymase (which converts angiotensin I into
angotensin II and degrades basement membranes). Chymase also
appears to activate MMP-9/gelatinase B, which also cleaving the
IL-2R.alpha. receptor of T cells down regulating the capability of
T cells to proliferate in the tumor microenvironment in response to
IL-2 (Coussen et al., 1999; 20 Sheu et al., 2001). The presence of
these enzymes in the TME should facilitate the colonization,
growth, and potentially the metastasis of malignant cells.
[0007] We have discovered the presence of PAR-1 and PAR-2 in the
stromal fibroblast and mast cells are important in establishing a
TME that facilitates the metastasis of cancer cells. Therefore, it
is an object of the present invention to provide (1) a method of
modifying the tumor cell microenvironment to reduce or prevent the
establishment, growth or metastasis of certain types of malignant
cells; and (2) a means of modulating the immune system to more
effectively respond to malignant cells.
SUMMARY OF THE INVENTION
[0008] We have discovered a method of modifying the tumor
microenvironment to reduce or prevent the establishment, growth or
metastasis of malignant cells that activate the PAR-1 receptor of
normal cells comprising providing a pharmaceutically effective
amount of a PAR-1 inhibitor having the formula (I): 1
[0009] wherein:
[0010] A.sub.1 and A.sub.2 are each independently a D- or L-amino
acid selected from the group consisting of alanine, .beta.-alanine,
arginine, homoarginine, cyclohexylalanine, citrulline, cysteine
(optionally substituted with C.sub.1-C.sub.4 alkyl, aryl, or
arC.sub.1-C.sub.4 alkyl), 2,4-diaminobutyric acid (optionally
substituted with acyl, C.sub.1-C.sub.4 alkyl, aroyl, amidino, or
MeC(NH)--), 2,3 diaminopropionic acid (optionally substituted with
acyl, C.sub.1-C.sub.4 alkyl, aroyl, amidino, or MeC(NH)--),
glutamine, glycine, indanylglycine, lysine (optionally substituted
with acyl, C.sub.1-C.sub.4 alkyl, aroyl, MeC(NH)--), valine,
methionine, proline, serine (optionally substituted with
C.sub.1-C.sub.4 alkyl, aryl, or arC.sub.1-C.sub.4 alkyl),
homoserine (optionally substituted with C.sub.1-C.sub.4 alkyl,
aryl, or arC.sub.1-C.sub.4 alkyl), tetrahydroisoquinoline-3-COOH,
threonine (optionally substituted with C.sub.1-C.sub.4 alkyl, aryl,
or arC.sub.1-C.sub.4 alkyl), ornithine (optionally substituted with
acyl, C.sub.1-C.sub.4 alkyl, aroyl, MeC(NH)--), and an
unsubstituted or substituted aromatic amino acid selected from the
group consisting of phenylalanine, heteroarylalanine,
naphthylalanine, homophenylalanine, histidine, tryptophan,
tyrosine, arylglycine, heteroarylglycine, aryl-.beta.-alanine, and
heteroaryl-.beta.-alanine wherein the substituents on the aromatic
amino acid are independently selected from one or more of halogen,
C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, hydroxy,
C.sub.1-C.sub.4 alkoxycarbonyl, amino, amidino, guanidino,
fluorinated C.sub.1-C.sub.4 alkyl, fluorinated C.sub.1-C.sub.4
alkoxy, C.sub.1-C.sub.4 alkylsulfonyl, C.sub.1-C.sub.4
alkylcarbonyl, cyano, aryl, heteroaryl, arC.sub.1-C.sub.4 alkyl,
C.sub.2-C.sub.4 alkenyl, alkynyl, or nitro;
[0011] R.sub.1 is selected from amino, C.sub.1-C.sub.8 alkylamino,
C.sub.1-C.sub.8 dialkylamino, arylamino, arC.sub.1-C.sub.8
alkylamino, C.sub.3-C.sub.8 cycloalkylamino,
heteroalkylC.sub.1-C.sub.8 alkylamino, heteroalkylC.sub.1-C.sub.8
alkyl-N-methylamino, C.sub.1-C.sub.8 dialkylaminoC.sub.1-C.sub.8
alkylamino, --N(C.sub.1-C.sub.8alkyl)-C.sub.1- -C.sub.8
alkyl-N(C.sub.1-C.sub.8alkyl).sub.2, N(C.sub.1-C.sub.8alkyl)(C.su-
b.1-C.sub.8 alkenyl),
--N(C.sub.1-C.sub.8alkyl)(C.sub.3-C.sub.8cycloalkyl)- , heteroalkyl
or substituted heteroalkyl wherein the substituent on the
heteroalkyl is selected from oxo, amino, C.sub.1-C.sub.8
alkoxyC.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 alkylamino or
C.sub.1-C.sub.8 dialkylamino;
[0012] R.sub.2 and R.sub.3 are each independently selected from
hydrogen, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl,
C.sub.3-C.sub.8 cycloalkylC.sub.1-C.sub.8 alkyl, aryl, heteroalkyl,
substituted heteroalkyl (wherein the substituent on the heteroalkyl
is one or more substituents independently selected from
C.sub.1-C.sub.8 alkoxycarbonyl, C.sub.1-C.sub.8 alkyl, or
C.sub.1-C.sub.4 alkylcarbonyl), heteroalkylC.sub.1-C.sub.8 alkyl,
indanyl, acetamidinoC.sub.1-C.sub.8 alkyl, aminoC.sub.1-C.sub.8
alkyl, C.sub.1-C.sub.8 alkylaminoC.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 dialkylaminoC.sub.1-C.su- b.8 alkyl, unsubstituted
or substituted heteroarylC.sub.1-C.sub.8 alkyl or unsubstituted or
substituted arC.sub.1-C.sub.8 alkyl, wherein the substituent on the
aralkyl or heteroarylalkyl group is one or more substituents
independently selected from halogen, nitro, amino, C.sub.1-C.sub.8
alkyl, C.sub.1-C.sub.8 alkoxy, hydroxy, cyano, C.sub.1-C.sub.4
alkylcarbonyl, C.sub.1-C.sub.8 alkoxycarbonyl,
hydroxyC.sub.1-C.sub.8 alkyl or aminosulfonyl; or
[0013] R.sub.2 and R.sub.3, together with the nitrogen to which
they are attached, alternatively form an unsubstituted or
substituted heteroalkyl group selected from piperidinyl,
piperazinyl, morpholinyl or pyrrolidinyl, wherein the substituent
is one or more substituents independently selected from
C.sub.1-C.sub.8 alkyl C.sub.1-C.sub.8 alkoxycarbonyl or
C.sub.1-C.sub.4 alkylcarbonyl;
[0014] R.sub.4 is selected from unsubstituted or substituted aryl,
arC.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, or heteroaryl,
where the substituents on the aryl, arC.sub.1-C.sub.8 alkyl,
cycloalkyl or heteroaryl group are independently selected from one
or more of halogen, nitro, amino, cyano, hydroxyalkyl,
C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 alkoxy, hydroxy,
C.sub.1-C.sub.4 alkylcarbonyl, C.sub.1-C.sub.8 alkoxycarbonyl,
fluorinated C.sub.1-C.sub.4 alkyl, fluorinated C.sub.1-C.sub.4
alkoxy, C.sub.1-C.sub.4 alkylsulfonyl;
[0015] R.sub.5 is selected from hydrogen or C.sub.1-C.sub.8
alkyl;
[0016] X is oxygen or sulfur;
[0017] m is an integer selected from 0, 1, 2 or 3;
[0018] n is an integer selected from 2 or 3; and
[0019] p is an integer selected from 0 or 1;
[0020] and pharmaceutically acceptable salts thereof and optionally
a PAR-2 inhibitor to a patient with malignant cells.
[0021] In another embodiment of the present invention we have
discovered a method for the modulation of the immune system to
facilitate a more efficient immune response to malignant cells that
activate the PAR-1 receptor comprising administer a
pharmaceutically effective dose of a PAR-1 inhibitor having the
formula (I): 2
[0022] (I)
[0023] wherein:
[0024] A.sub.1 and A.sub.2 are each independently a D- or L-amino
acid selected from the group consisting of alanine, .beta.-alanine,
arginine, homoarginine, cyclohexylalanine, citrulline, cysteine
(optionally substituted with C.sub.1-C.sub.4 alkyl, aryl, or
arC.sub.1-C.sub.4 alkyl), 2,4-diaminobutyric acid (optionally
substituted with acyl, C.sub.1-C.sub.4 alkyl, aroyl, amidino, or
MeC(NH)--), 2,3 diaminopropionic acid (optionally substituted with
acyl, C.sub.1-C.sub.4 alkyl, aroyl, amidino, or MeC(NH)--),
glutamine, glycine, indanylglycine, lysine (optionally substituted
with acyl, C.sub.1-C.sub.4 alkyl, aroyl, MeC(NH)--), valine,
methionine, proline, serine (optionally substituted with
C.sub.1-C.sub.4 alkyl, aryl, or arC.sub.1-C.sub.4 alkyl),
homoserine (optionally substituted with C.sub.1-C.sub.4 alkyl,
aryl, or arC.sub.1-C.sub.4 alkyl), tetrahydroisoquinoline-3-COOH,
threonine (optionally substituted with C.sub.1-C.sub.4 alkyl, aryl,
or arC.sub.1-C.sub.4 alkyl), ornithine (optionally substituted with
acyl, C.sub.1-C.sub.4 alkyl, aroyl, MeC(NH)--), and an
unsubstituted or substituted aromatic amino acid selected from the
group consisting of phenylalanine, heteroarylalanine,
naphthylalanine, homophenylalanine, histidine, tryptophan,
tyrosine, arylglycine, heteroarylglycine, aryl-p-alanine, and
heteroaryl-p-alanine wherein the substituents on the aromatic amino
acid are independently selected from one or more of halogen,
C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, hydroxy,
C.sub.1-C.sub.4 alkoxycarbonyl, amino, amidino, guanidino,
fluorinated C.sub.1-C.sub.4 alkyl, fluorinated C.sub.1-C.sub.4
alkoxy, C.sub.1-C.sub.4 alkylsulfonyl, C.sub.1-C.sub.4
alkylcarbonyl, cyano, aryl, heteroaryl, arC.sub.1-C.sub.4 alkyl,
C.sub.2-C.sub.4 alkenyl, alkynyl, or nitro;
[0025] R.sub.1 is selected from amino, C.sub.1-C.sub.8 alkylamino,
C.sub.1-C.sub.8 dialkylamino, arylamino, arC.sub.1-C.sub.8
alkylamino, C.sub.3-C.sub.8 cycloalkylamino,
heteroalkylC.sub.1-C.sub.8 5 alkylamino, heteroalkylC.sub.1-C.sub.8
alkyl-N-methylamino, C.sub.1-C.sub.8 dialkylaminoC.sub.1-C.sub.8
alkylamino, --N(C.sub.1-C.sub.8alkyl)-C.sub.1- -C.sub.8
alkyl-N(C.sub.1-C.sub.8alkyl).sub.2, N(C.sub.1-C.sub.8
alkyl)(C.sub.1-C.sub.8 alkenyl),
--N(C.sub.1-C.sub.8alkyl)(C.sub.3-C.sub.- 8cycloalkyl), heteroalkyl
or substituted heteroalkyl wherein the substituent on the
heteroalkyl is selected from oxo, amino, C.sub.1-C.sub.8
alkoxyC.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 alkylamino or
C.sub.1-C.sub.8 dialkylamino;
[0026] R.sub.2 and R.sub.3 are each independently selected from
hydrogen, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl,
C.sub.3-C.sub.8 cycloalkylC.sub.1-C.sub.8 alkyl, aryl, heteroalkyl,
substituted heteroalkyl (wherein the substituent on the heteroalkyl
is one or more substituents independently selected from
C.sub.1-C.sub.8 alkoxycarbonyl, C.sub.1-C.sub.8 alkyl, or
C.sub.1-C.sub.4 alkylcarbonyl), heteroalkylC.sub.1-C.sub.8 alkyl,
indanyl, acetamidinoC.sub.1-C.sub.8 alkyl, aminoC.sub.1-C.sub.8
alkyl, C.sub.1-C.sub.8 alkylaminoC.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 dialkylaminoC.sub.1-C.su- b.8 alkyl, unsubstituted
or substituted heteroarylC.sub.1-C.sub.8 alkyl or unsubstituted or
substituted arC.sub.1-C.sub.8 alkyl, wherein the substituent on the
aralkyl or heteroarylalkyl group is one or more substituents
independently selected from halogen, nitro, amino, C.sub.1-C.sub.8
alkyl, C.sub.1-C.sub.8 alkoxy, hydroxy, cyano, C.sub.1-C.sub.4
alkylcarbonyl, C.sub.1-C.sub.8 alkoxycarbonyl,
hydroxyC.sub.1-C.sub.8 alkyl or aminosulfonyl; or
[0027] R.sub.2 and R.sub.3, together with the nitrogen to which
they are attached, alternatively form an unsubstituted or
substituted heteroalkyl group selected from piperidinyl,
piperazinyl, morpholinyl or pyrrolidinyl, wherein the substituent
is one or more substituents independently selected from
C.sub.1-C.sub.8 alkyl C.sub.1-C.sub.8 alkoxycarbonyl or
C.sub.1-C.sub.4 alkylcarbonyl;
[0028] R.sub.4 is selected from unsubstituted or substituted aryl,
arC.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, or heteroaryl,
where the substituents on the aryl, arC.sub.1-C.sub.8 alkyl,
cycloalkyl or heteroaryl group are independently selected from one
or more of halogen, nitro, amino, cyano, hydroxyalkyl,
C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 alkoxy, hydroxy,
C.sub.1-C.sub.4 alkylcarbonyl, C.sub.1-C.sub.8 alkoxycarbonyl,
fluorinated C.sub.1-C.sub.4 alkyl, fluorinated C.sub.1-C.sub.4
alkoxy, C.sub.1-C.sub.4 alkylsulfonyl;
[0029] R.sub.5 is selected from hydrogen or C.sub.1-C.sub.8
alkyl;
[0030] X is oxygen or sulfur;
[0031] m is an integer selected from 0, 1, 2 or 3;
[0032] n is an integer selected from 2 or 3; and
[0033] p is an integer selected from 0 or 1;
[0034] and pharmaceutically acceptable salts thereof and optionally
a PAR-2 inhibitor to a patient to enhance the patient's immune
response to the malignant cells.
BRIEF DESCRIPTION OF THE FIGURES
[0035] FIG. 1. Is a graphic illustration of PAR-1 and PAR-2
immunolabeling in normal (n=20), benign (n=10) and malignant (n=46)
human breast tissues. Immunolabeling data was expressed as the mean
.+-.S.E. Intesity of labeled cells observed in a 20.times. viewing
field the following values were assigned to cells; (0.0) was
assigned to unlabeled cells; (1.0) was assigned to weak or light
brown labeling; (2.0) was assigned to moderate brown labeling; and
(3.0) was assigned to intense or dark brown labeling.
[0036] FIG. 2. Presents representative immunohistochemical
micrographs for normal (left panels: A, D, G, J), benign (middle
panels: B, E, H, K) and malignant (right panels: C, F, I, L) breast
tissues are presented in FIG. 2. Breast tissues were assayed for
immunohistochemistry using negative control antibodies A-C, smooth
muscle actin antibodies (D-F), DNA topoisomerase II.alpha.
antibodies (G-I), PAR-1 (J-L) antibodies, and PAR-2 (M-O)
antibodies. Arrowheads indicate normal, benign and malignant
epithelial cells in the breast. Arrows indicate areas in labeling
in the stromal fibroblasts. Mast cells (MC) and macrophages (M) are
indicated in the tissue. The magnification was approximately
900.times..
[0037] FIG. 3. Presents representative immunohistochemical
micrographs of PAR-1 (A, C, E) and PAR-2 (B, D, F) expression
observed in gastric carcinoma (A, B), undifferentiated carcinoma
(C, D) and lung adenocarcinoma (E, F) tissues. Arrowheads indicate
positive immunolabeling in tumor cells and arrows indicate
immunolabeling in the stromal fibroblasts. The magnification was
approximately 900.times..
[0038] FIG. 4. Human malignant breast carcinoma tissues were
assayed for the expression of PAR-1 (A) and PAR-2 (B) mRNA through
in situ hybridization. The positive control probe, GAP-DH (C) and
the negative control probe lac Z (D) are also presented. Arrowheads
indicate tumor cells and arrows indicate stromal fibroblasts. The
magnification was approximately 900.times..
[0039] FIG. 5. Presents representative immunohistochemical
micrographs of malignant breast tissues processed using double
immunohistochemical procedures for detecting the presence of PAR-1
(brown) and Topo II.alpha. (red) and PAR-2 (brown) and Topo
II.alpha. (red). Arrowheads indicate the presence of proliferating,
red-labeled nuclei cells with the presence of brown intracellular
and membrane PAR-1 and PAR-2 positive cells. The magnification was
approximately 900.times..
[0040] FIG. 6. Presents representative immunocytochemical
micrographs for quiescent (A, D, G, J, M), proliferating (B, E, H,
K, N) and wounded (C, F, I, L, O) human dermal fibroblasts using
negative control antibodies (A-C), antibodies to detect smooth
muscle actin (SMA) (D-F), Topo II.alpha. (G-I), PAR-1 (J-L) and
PAR-2 (M-O). No observable labeling was observed of cells using
negative control antibodies. No SMA immunolabeling was not observed
in the quiescent cultured cells (D). SMA-positive cells
(arrowheads) were observed in cultured cells in the proliferating
and wounding conditions (E-F). Proliferating cells were detected by
the presence of brown, Topo II.alpha.a-positive nuclei in the
proliferating cells (H-I), but not in the quiescent cells (G).
Positive intracellular and membrane PAR-1 (K-L) and PAR-2 (N-O)
immunoreactive cells (arrowheads) were observed in the cells in the
proliferating and wounding conditions, but were absent in the cells
in the quiescent cells for PAR-1 (J) and PAR-2 (M). The
magnification was approximately 900.times..
DETAILED DESCRIPTION OF THE INVENTION
[0041] We have discovered that the activation of PAR1 and/or PAR2
on normal host cells appears to participate in creating an
environment that allows malignant cells to become established, grow
or metastasize. This activation is associated with a variety of
cells that form the TME such as fibroblasts, mast cells and
macrophages/monocytes. PAR-1 and PAR-2 activation of the stromal
fibroblasts is believed to contribute to the elaboration of
mitogens for angiogenesis and tumor cell growth. This activation is
also believed to result in the deposition of extracellular matrix
proteins and the release of proteolytic enzymes to facilitate tumor
growth and metastasis. The activation of PAR-1 and/or PAR-2 on
monocytes/macrophages is also appears to potentially suppress CTL
and NK cells that would other wise mount an immune response to the
tumor cells. The activation of PAR1 and/or PAR2 receptors of mast
cells is also believed to result in the establishment of an
inappropriate immune response to malignant cells. This activation
appears to lead to an autocrine activation cycle of PAR1 and/or
PAR2.
[0042] Administering a PAR1 antagonist will block the activation
and degranulation of mast cells in response to thrombin and other
activator of PAR1. Co-administration of PAR1 and PAR2 antagonists
is anticipated to further reduce the potential for the activation
and degranulation of mast cells in response to thrombin and other
direct or indirect activators of PAR1 and PAR2 (e.g. trypsin).
Additionally, cytokines and other therapeutic agents may be
simultaneously or sequentially administered to facilitate a desired
immune response to the malignant cells such as the activation of
CTL and NK cell, activation of cytolytic T lymphocytes or
stimulation of antigen presenting cells. Generally the cytokines
will be those that are associated with establishing a Th1 response
such as IL-2, IL-12 and IL-18.
Diagnosis
[0043] Suspected malignant cells and surrounding tissue may be
isolated by well-known surgical techniques (such as needle biopsy).
The suspected malignant cells maybe tested for the secretion of
substances that activate PAR1 and/or PAR2. Some of the proteins
that are known to activate PAR1 are thrombin and trypsin. Suitable
means for testing for these proteins include but are not limited to
bioassay or pathologic analysis of tissue specimens. The presence,
type and relative concentration of these proteins will allow for
the qualification of these malignant cells for mast cell activation
and degranulation consequently the potential for metastasis.
Additionally the surrounding fibroblast tissue may be tested for
the relative amounts of PAR-1 and PAR-2 to determine the tumor
grade and determine its degree of malignancy.
Method for Reducing or Preventing Metastasis
[0044] Since, PAR-1 and PAR-2 are directly implicated in the
cascade of events leading to metastasis of malignant cells
expressing PAR-1 and PAR-2 activator proteins blocking this process
would disrupt the process of malignant cell metastasis.
Consequently, the use of PAR-1 and PAR-2 antagonist and the like
(e.g. antisense sequences), receptor blocking ligands or
antibodies, or the use of anti-thrombin and anti-tryptase agents
would provide a means of reducing or preventing metastasis.
[0045] Suitable PAR-1 antagonist include antibodies that block
activation of PAR-1 receptor and compounds of the general formula
(I): 3
[0046] wherein
[0047] A.sub.1 and A.sub.2 are each independently a D- or L-amino
acid selected from the group consisting of alanine, .beta.-alanine,
arginine, homoarginine, cyclohexylalanine, citrulline, cysteine
(optionally substituted with C.sub.1-C.sub.4 alkyl, aryl, or
arC.sub.1-C.sub.4 alkyl), 2,4-diaminobutyric acid (optionally
substituted with acyl, C.sub.1-C.sub.4 alkyl, aroyl, amidino, or
MeC(NH)--), 2,3 diaminopropionic acid (optionally substituted with
acyl, C.sub.1-C.sub.4 alkyl, aroyl, amidino, or MeC(NH)--),
glutamine, glycine, indanylglycine, lysine (optionally substituted
with acyl, C.sub.1-C.sub.4 alkyl, aroyl, MeC(NH)--), valine,
methionine, proline, serine (optionally substituted with
C.sub.1-C.sub.4 alkyl, aryl, or arC.sub.1-C.sub.4 alkyl),
homoserine (optionally substituted with C.sub.1-C.sub.4 alkyl,
aryl, or arC.sub.1-C.sub.4 alkyl), tetrahydroisoquinoline-3-COOH,
threonine (optionally substituted with C.sub.1-C.sub.4 alkyl, aryl,
or arC.sub.1-C.sub.4 alkyl), omithine (optionally substituted with
acyl, C.sub.1-C.sub.4 alkyl, aroyl, MeC(NH)--), and an
unsubstituted or substituted aromatic amino acid selected from the
group consisting of phenylalanine, heteroarylalanine,
naphthylalanine, homophenylalanine, histidine, tryptophan,
tyrosine, arylglycine, heteroarylglycine, aryl-.beta.-alanine, and
heteroaryl-.beta.-alanine wherein the substituents on the aromatic
amino acid are independently selected from one or more of halogen,
C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, hydroxy,
C.sub.1-C.sub.4 alkoxycarbonyl, amino, amidino, guanidino,
fluorinated C.sub.1-C.sub.4 alkyl, fluorinated C.sub.1-C.sub.4
alkoxy, C.sub.1-C.sub.4 alkylsulfonyl, C.sub.1-C.sub.4
alkylcarbonyl, cyano, aryl, heteroaryl, arC.sub.1-C.sub.4 alkyl,
C.sub.2-C.sub.4 alkenyl, alkynyl, or nitro;
[0048] Preferably, A.sub.1 and A.sub.2 are each independently an
L-amino acid selected from the group consisting of alanine,
.beta.-alanine, arginine, homoarginine, cyclohexylalanine,
citrulline, cysteine (optionally substituted with C.sub.1-C.sub.4
alkyl, aryl, or arC.sub.1-C.sub.4 alkyl), 2,4-diaminobutyric acid
(optionally substituted with acyl, C.sub.1-C.sub.4 alkyl, aroyl,
amidino, or MeC(NH)--), 2,3 diaminopropionic acid (optionally
substituted with acyl, C.sub.1-C.sub.4 alkyl, aroyl, amidino, or
MeC(NH)--), glutamine, glycine, indanylglycine, lysine (optionally
substituted with acyl, C.sub.1-C.sub.4 alkyl, aroyl, MeC(NH)--),
valine, methionine, proline, serine (optionally substituted with
C.sub.1-C.sub.4 alkyl, aryl, or arC.sub.1-C.sub.4 alkyl),
homoserine (optionally substituted with C.sub.1-C.sub.4 alkyl,
aryl, or arC.sub.1-C.sub.4 alkyl), tetrahydroisoquinoline-3-COOH,
threonine (optionally substituted with C.sub.1-C.sub.4 alkyl, aryl,
or arC.sub.1-C.sub.4 alkyl), omithine (optionally substituted with
acyl, C.sub.1-C.sub.4 alkyl, aroyl, MeC(NH)--), and an
unsubstituted or substituted aromatic amino acid selected from the
group consisting of phenylalanine, heteroarylalanine,
naphthylalanine, homophenylalanine, histidine, tryptophan,
tyrosine, arylglycine, heteroarylglycine, aryl-.beta.-alanine, and
heteroaryl-.beta.-alanine wherein the substituents on the aromatic
amino acid are independently selected from one or more of halogen,
C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, hydroxy,
C.sub.1-C.sub.4 alkoxycarbonyl, amino, amidino, guanidino,
fluorinated C.sub.1-C.sub.4 alkyl, fluorinated C.sub.1-C.sub.4
alkoxy, C.sub.1-C.sub.4 alkylsulfonyl, C.sub.1-C.sub.4
alkylcarbonyl, cyano, aryl, heteroaryl, arC.sub.1-C.sub.4 alkyl,
C.sub.2-C.sub.4 alkenyl, alkynyl, or nitro;
[0049] R.sub.1 is selected from amino, C.sub.1-C.sub.8 alkylamino,
C.sub.1-C.sub.8 dialkylamino, arylamino, arC.sub.1-C.sub.8
alkylamino, C.sub.3-C.sub.8 cycloalkylamino,
heteroalkylC.sub.1-C.sub.8 alkylamino, heteroalkylC.sub.1-C.sub.8
alkyl-N-methylamino, C.sub.1-C.sub.8 dialkylaminoC.sub.1-C.sub.8
alkylamino, --N(C.sub.1-C.sub.8alkyl)-C.sub.1- -C.sub.8
alkyl-N(C.sub.1-C.sub.8alkyl).sub.2, N(C.sub.1-C.sub.8
alkyl)(C.sub.1-C.sub.8 alkenyl),
--N(C.sub.1-C.sub.8alkyl)(C.sub.3-C.sub.- 8cycloalkyl), heteroalkyl
or substituted heteroalkyl wherein the substituent on the
heteroalkyl is selected from oxo, amino, C.sub.1-C.sub.8
alkoxyC.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 alkylamino or
C.sub.1-C.sub.8 dialkylamino;
[0050] Preferably, R.sub.1 is selected from amino, C.sub.1-C.sub.6
alkylamino, C.sub.1-C.sub.6 dialkylamino, arylamino,
arC.sub.1-C.sub.6 alkylamino, heteroalkylC.sub.1-C.sub.6
alkylamino,
--N(C.sub.1-C.sub.6alkyl)-C.sub.1-C.sub.6alkyl-N(C.sub.1-C.sub.6alkyl).su-
b.2, heteroalkyl or substituted heteroalkyl wherein the substituent
on the heteroalkyl is selected from oxo, amino,
C.sub.1-C.sub.6alkoxyC.sub.1-C.s- ub.6 alkyl, C.sub.1-C.sub.6
alkylamino or C.sub.1-C.sub.6 dialkylamino;
[0051] R.sub.2 and R.sub.3 are each independently selected from
hydrogen, C.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl,
C.sub.3-C.sub.8 cycloalkylC.sub.1-C.sub.8 alkyl, aryl, heteroalkyl,
substituted heteroalkyl (wherein the substituent on the heteroalkyl
is one or more substituents independently selected from
C.sub.1-C.sub.8 alkoxycarbonyl, C.sub.1-C.sub.8 alkyl, or
C.sub.1-C.sub.4 alkylcarbonyl), heteroalkylC.sub.1-C.sub.8 alkyl,
indanyl, acetamidinoC.sub.1-C.sub.8 alkyl, aminoC.sub.1-C.sub.8
alkyl, C.sub.1-C.sub.8 alkylaminoC.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 dialkylaminoC.sub.1-C.su- b.8 alkyl, unsubstituted
or substituted heteroarylC.sub.1-C.sub.8 alkyl or unsubstituted or
substituted arC.sub.1-C.sub.8 alkyl, wherein the substituent on the
aralkyl or heteroarylalkyl group is one or more substituents
independently selected from halogen, nitro, amino, C.sub.1-C.sub.8
alkyl, C.sub.1-C.sub.8 alkoxy, hydroxy, cyano, C.sub.1-C.sub.4
alkylcarbonyl, C.sub.1-C.sub.8 alkoxycarbonyl,
hydroxyC.sub.1-C.sub.8 alkyl or aminosulfonyl; or
[0052] R.sub.2 and R.sub.3 together with the nitrogen to which they
are attached, alternatively form an unsubstituted or substituted
heteroalkyl group selected from piperidinyl, piperazinyl,
morpholinyl or pyrrolidinyl, wherein the substituent is one or more
substituents independently selected from C.sub.1-C.sub.8 alkyl
C.sub.1-C.sub.8 alkoxycarbonyl or C.sub.1-C.sub.4
alkylcarbonyl;
[0053] Preferably, R.sub.2 is selected from hydrogen or
C.sub.1-C.sub.6 alkyl;
[0054] R.sub.3 is selected from C.sub.1-C.sub.8 alkyl,
C.sub.3-C.sub.6 cycloalkyl, C.sub.3-C.sub.6
cycloalkylC.sub.1-C.sub.6 alkyl, aryl, heteroarylC.sub.1-C.sub.6
alkyl, substituted heteroarylC.sub.1-C.sub.6 alkyl wherein the
substituent is C.sub.1-C.sub.4 alkyl, heteroalkyl,
heteroalkylC.sub.1-C.sub.6 alkyl, indanyl,
acetamidinoC.sub.1-C.sub.6 alkyl, aminoC.sub.1-C.sub.6 alkyl,
C.sub.1-C.sub.6 alkylaminoC.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6
dialkylaminoC.sub.1-C.su- b.6 alkyl, arC.sub.1-C.sub.8 alkyl,
substituted arC.sub.1-C.sub.8 alkyl wherein the substituent on the
aralkyl group is one to five substituents independently selected
from halogen, nitro, amino, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6
alkoxy, C.sub.1-C.sub.6 alkoxycarbonyl, hydroxyalkyl or
aminosulfonyl; or R.sub.2 and R.sub.3, together with the nitrogen
to which they are attached, alternatively form an unsubstituted or
substituted heteroalkyl group selected from piperidinyl,
piperazinyl or pyrrolidinyl, wherein the substituent is
independently one or two substituents selected from C.sub.1-C.sub.6
alkyl;
[0055] R.sub.4 is selected from unsubstituted or substituted aryl,
arC.sub.1-C.sub.8 alkyl, C.sub.3-C.sub.8 cycloalkyl, or heteroaryl,
where the substituents on the aryl, arC.sub.1-C.sub.8 alkyl,
cycloalkyl or heteroaryl group are independently selected from one
or more of halogen, nitro, amino, cyano, hydroxyalkyl,
C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 alkoxy, hydroxy,
C.sub.1-C.sub.4 alkylcarbonyl, C.sub.1-C.sub.8 alkoxycarbonyl,
fluorinated C.sub.1-C.sub.4 alkyl, fluorinated C.sub.1-C.sub.4
alkoxy or C.sub.1-C.sub.4 alkylsulfonyl; Preferably, R.sub.4 is
selected from unsubstituted or substituted aryl, arC.sub.1-C.sub.6
alkyl, C.sub.3-C.sub.6 cycloalkyl or heteroaryl, where the
substituents on the aryl, aralkyl, cycloalkyl or heteroaryl group
are independently selected from one to three substituents selected
from halogen, cyano, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy,
C.sub.1-C.sub.4 alkoxycarbonyl, fluorinated C.sub.1-C.sub.4 alkyl,
fluorinated C.sub.1-C.sub.4 alkoxy or C.sub.1-C.sub.4
alkylsulfonyl;
[0056] R.sub.5 is selected from hydrogen or C.sub.1-C.sub.8 alkyl;
preferably, R.sub.5 is hydrogen;
[0057] X is oxygen or sulfur; preferably, X is oxygen;
[0058] m is an integer selected from 0, 1, 2 or 3;
[0059] n is an integer selected from 2 or 3; and
[0060] p is an integer selected from 0 or 1; preferably, p is
1;
[0061] and pharmaceutically acceptable salts thereof.
[0062] In a preferred embodiment of the present invention:
[0063] A.sub.1 is an L-amino acid selected from the group
consisting of alanine, arginine, cyclohexylalanine, glycine,
proline, tetrahydroisoquinoline-3-COOH, and an unsubstituted or
substituted aromatic amino acid selected from the group consisting
of phenylalanine, naphthylalanine, homophenylalanine, and O-methyl
tyrosine, wherein the substituents on the aromatic amino acid are
independently selected from one to five of (preferably, one to
three of) halogen, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy,
hydroxy, C.sub.1-C.sub.4 alkoxycarbonyl, amino, amidino, guanidino,
fluorinated C.sub.1-C.sub.4 alkyl, fluorinated C.sub.1-C.sub.4
alkoxy, C.sub.1-C.sub.4 alkylsulfonyl, C.sub.1-C.sub.4
alkylcarbonyl, cyano, aryl, heteroaryl, arC.sub.1-C.sub.4 alkyl,
C.sub.2-C.sub.4 alkenyl, alkynyl, or nitro;
[0064] A.sub.2 is an L-amino acid selected from the group
consisting of alanine, .beta.-alanine, arginine, citrulline,
cysteine (optionally substituted with C.sub.1-C.sub.4 alkyl, aryl,
or arC.sub.1-C.sub.4 alkyl), 2,4-diaminobutyric acid (optionally
substituted with acyl, C.sub.1-C.sub.4 alkyl, aroyl, amidino, or
MeC(NH)--), 2,3-diaminopropionic acid (optionally substituted with
acyl, C.sub.1-C.sub.4 alkyl, aroyl, amidino, or MeC(NH)--),
glutamine, glycine, lysine (optionally substituted with acyl,
C.sub.1-C.sub.4 alkyl, aroyl, MeC(NH)--), valine, methionine,
serine (optionally substituted with C.sub.1-C.sub.4 alkyl, aryl, or
arC.sub.1-C.sub.4 alkyl), homoserine (optionally substituted with
C.sub.1-C.sub.4 alkyl, aryl, or arC.sub.1-C.sub.4 alkyl), threonine
(optionally substituted with C.sub.1-C.sub.4 alkyl, aryl, or
arC.sub.1-C.sub.4 alkyl), ornithine (optionally substituted with
acyl, C.sub.1-C.sub.4 alkyl, aroyl, MeC(NH)--), and an
unsubstituted or substituted aromatic amino acid selected from the
group consisting of phenylalanine, heteroarylalanine, and
histidine, wherein the substituents on the aromatic amino acid are
independently selected from one to five of (preferably, one to
three of) halogen, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy,
hydroxy, C.sub.1-C.sub.4 alkoxycarbonyl, amino, amidino, guanidino,
fluorinated C.sub.1-C.sub.4 alkyl, fluorinated C.sub.1-C.sub.4
alkoxy, C.sub.1-C.sub.4 alkylsulfonyl, C.sub.1-C.sub.4
alkylcarbonyl, cyano, aryl, heteroaryl, arC.sub.1-C.sub.4 alkyl,
C.sub.2-C.sub.4 alkenyl, alkynyl, or nitro;
[0065] R.sub.2 is selected from hydrogen or C.sub.1-C.sub.4
alkyl;
[0066] m is 1 and n is 2;
[0067] and all other variables are as defined previously; and
pharmaceutically acceptable salts thereof
[0068] In a class of the invention:
[0069] A.sub.1 is an L-amino acid selected from the group
consisting of alanine, arginine, cyclohexylalanine, glycine,
proline, and an unsubstituted or substituted aromatic amino acid
selected from the group consisting of phenylalanine,
naphthylalanine, homophenylalanine, and O-methyl tyrosine, wherein
the substituents on the aromatic amino acid are independently one
to two substituents selected from halogen, C.sub.1-C.sub.4 alkyl,
C.sub.1-C.sub.4 alkoxy, hydroxy, C.sub.1-C.sub.4 alkoxycarbonyl,
amino, amidino, guanidino, fluorinated C.sub.1-C.sub.4 alkyl,
fluorinated C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 alkylsulfonyl,
C.sub.1-C.sub.4 alkylcarbonyl, cyano, aryl, heteroaryl,
arC.sub.1-C.sub.4 alkyl, C.sub.2-C.sub.4 alkenyl, alkynyl, or
nitro;
[0070] A.sub.2 is an L-amino acid selected from the group
consisting of alanine, .beta.-alanine, arginine, citrulline,
cysteine (optionally substituted with C.sub.1-C.sub.4 alkyl, aryl,
or arC.sub.1-C.sub.4 alkyl), 2,4-diaminobutyric acid (optionally
substituted with acyl, C.sub.1-C.sub.4 alkyl, aroyl, amidino, or
MeC(NH)--), 2,3-diaminopropionic acid (optionally substituted with
acyl, C.sub.1-C.sub.4 alkyl, aroyl, amidino, or MeC(NH)--),
glutamine, glycine, lysine (optionally substituted with acyl,
C.sub.1-C.sub.4 alkyl, aroyl, MeC(NH)--), valine, methionine,
serine (optionally substituted with C.sub.1-C.sub.4 alkyl, aryl, or
arC.sub.1-C.sub.4 alkyl), homoserine (optionally substituted with
C.sub.1-C.sub.4 alkyl, aryl, or arC.sub.1-C.sub.4 alkyl), threonine
(optionally substituted with C.sub.1-C.sub.4 alkyl, aryl, or
arC.sub.1-C.sub.4 alkyl), ornithine (optionally substituted with
acyl, C.sub.1-C.sub.4 alkyl, aroyl, MeC(NH)--), and an
unsubstituted or substituted aromatic amino acid selected from the
group consisting of phenylalanine, heteroarylalanine, and
histidine, wherein the substituents on the aromatic amino acid are
independently one to two substituents selected from halogen,
C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, hydroxy,
C.sub.1-C.sub.4 alkoxycarbonyl, amino, amidino, guanidino,
fluorinated C.sub.1-C.sub.4 alkyl, fluorinated C.sub.1-C.sub.4
alkoxy, C.sub.1-C.sub.4 alkylsulfonyl, C.sub.1-C.sub.4
alkylcarbonyl, cyano, aryl, heteroaryl, arC.sub.1-C.sub.4 alkyl,
C.sub.2-C.sub.4 alkenyl, alkynyl, or nitro;
[0071] R.sub.1 is selected from dimethylamino, diethylamino,
di-(n-propyl)amino, 4
[0072] R.sub.2 is selected from hydrogen, methyl or ethyl;
[0073] R.sub.3 is selected from 2-indanyl, phenyl,
cyclohexylmethyl, cyclopentyl, pyridylmethyl, furanylmethyl,
2-(4-methyl-furanyl)methyl, thienylmethyl, diphenylmethyl,
4-imidazolylethyl, 2-(4-N-methyl)imidazoly- lethyl, n-octyl,
phenyl-n-propyl, aminoethyl, aminopropyl, amino-n-pentyl,
dimethylaminoethyl, 4-aminophenylsulfonylaminomethyl,
acetamidineylethyl, 2-N-pyrrolidinylethyl,
N-ethoxycarbonylpiperidinyl, unsubstituted or substituted
phenylethyl or unsubstituted or substituted benzyl wherein the
substituents on the phenylethyl or benzyl are independently one or
two substituents selected from methyl, fluorine, chlorine, nitro,
methoxy, methoxycarbonyl or hydroxymethyl; or
[0074] R.sub.2 and R.sub.3, together with the nitrogen to which
they are attached, alternatively form a heteroalkyl group selected
from piperidinyl, or 4-(N-methyl)piperazinyl;
[0075] R.sub.4 is selected from cyclohexyl, 2-naphthyl,
phenylethyl, 4-fluorophenylethyl, or unsubstituted or substituted
phenyl, where the substituents on the phenyl are independently
selected from one to two substituents selected from fluorine,
chlorine, iodine, methyl, cyano, or trifluoromethyl;
[0076] Preferably, R.sub.4 is 2,6-dichlorophenyl or 2-methylphenyl;
and all other variables are as defined previously; and
pharmaceutically acceptable salts thereof.
[0077] In a subclass of the invention,
[0078] A.sub.1 is selected from 3,4-Difluorophenylalanine or
4-Chlorophenylalanine;
[0079] A.sub.2 is selected from 2,4-Diaminobutyric acid or
4-Pyridylalanine;
[0080] R.sub.2 is hydrogen;
[0081] R.sub.3 is selected from benzyl or 2-aminoethyl;
[0082] and all other variables are as defined previously; and
pharmaceutically acceptable salts thereof.
[0083] In the compounds of formula (I), the amino acid residues
comprising the A.sub.1 and A.sub.2 substituents are attached to the
adjacent moiety according to standard nomenclature so that the
amino-terminus (N-terminus) of the amino acid is drawn on the left
and the carboxy-terminus of the amino acid is drawn on the right.
So, for example, in Compound 1 in Table 1, where A.sub.1 is
3,4-difluorophenylalanine and A.sub.2 is Dbu (2,4-Diaminobutyric
acid), the N-terminus of the 3,4-difluorophenylalanine (Al) is
attached to the carbonylgroup and the carboxy-terminus of the
3,4-difluorophenylalanine (A.sub.1) is attached to the N-terminus
of the A.sub.2 substituent (Dbu), similarly, the N-terminus of the
Dbu (A.sub.2) is attached to the carboxy-terminus of the Al
substituent and the carboxy-terminus of the Dbu (A.sub.2) is
attached to the N--R.sub.2R.sub.3 group.
[0084] When a particular group is "substituted" (e.g., Phe, aryl,
heteroalkyl, heteroaryl), that group may have one or more
substituents, preferably from one to five substituents, more
preferably from one to three substituents, most preferably from one
to two substituents, independently selected from the list of
substituents.
[0085] Under standard nomenclature used throughout this disclosure,
the terminal portion of the designated side chain is described
first, followed by the adjacent functionality toward the point of
attachment. Thus, for example, a "phenylC.sub.1-C.sub.6
alkylamidoC.sub.1-C.sub.6alky- l" substituent refers to a group of
the formula 5
[0086] The compounds of the present invention may also be present
in the form of a pharmaceutically acceptable salt. The
pharmaceutically acceptable salt generally takes a form in which
the basic nitrogen is protonated with an inorganic or organic acid.
Representative organic or inorganic acids include hydrochloric,
hydrobromic, hydriodic, perchloric, sulfuric, nitric, phosphoric,
acetic, propionic, glycolic, lactic, succinic, maleic, fumaric,
malic, tartaric, citric, benzoic, mandelic, methanesulfonic,
hydroxyethanesulfonic, benzenesulfonic, oxalic, pamoic,
2-naphthalenesulfonic, p-toluenesulfonic, cyclohexanesulfamic,
salicylic, saccharinic or trifluoroacetic.
[0087] Where the compounds according to this invention have at
least one chiral center, they may accordingly exist as enantiomers.
Where the compounds possess two or more chiral centers, they may
additionally exist as diastereomers. It is to be understood that
all such isomers and mixtures thereof are encompassed within the
scope of the present invention. Furthermore, some of the
crystalline forms for the compounds may exist as polymorphs and as
such are intended to be included in the present invention. In
addition, some of the compounds may form solvates with water (i.e.,
hydrates) or common organic solvents, and such solvates are also
intended to be encompassed within the scope of this invention.
[0088] The term "subject" as used herein, refers to an animal,
preferably a mammal, most preferably a human, who has been the
object of treatment, observation or experiment.
[0089] The term "therapeutically effective amount" as used herein,
means that amount of active compound or pharmaceutical agent that
elicits the biological or medicinal response in a tissue system,
animal or human that is being sought by a researcher, veterinarian,
medical doctor or other clinician, which includes alleviation of
the symptoms of the disease or disorder being treated.
[0090] As used herein, unless otherwise noted alkyl and alkoxy
whether used alone or as part of a substituent group, include
straight and branched chains having 1 to 8 carbon atoms, or any
number within this range. For example, alkyl radicals include
methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl,
t-butyl, n-pentyl, 3-(2-2 5 methyl)butyl, 2-pentyl, 2-methylbutyl,
neopentyl, n-hexyl, 2-hexyl and 2-methylpentyl. Alkoxy radicals are
oxygen ethers formed from the previously described straight or
branched chain alkyl groups. Cycloalkyl groups contain 3 to 8 ring
carbons and preferably 5 to 7 carbons. Similarly, alkenyl and
alkynyl groups include straight and branched chain alkenes and
alkynes having 1 to 8 carbon atoms, or any number within this
range.
[0091] The term "aryl" as used herein refers to an unsubstituted or
substituted aromatic group such as phenyl and naphthyl. The term
"aroyl" refers to the group --C(O)-aryl.
[0092] The term "heteroalkyl" as used herein represents an
unsubstituted or substituted stable three to seven membered
monocyclic saturated ring system which consists of carbon atoms and
from one to three heteroatoms selected from N, O or S, and wherein
the nitrogen or sulfur heteroatoms may optionally be oxidized, and
the nitrogen heteroatom may optionally be quaternized. The
heteroalkyl group may be attached at any heteroatom or carbon atom
which results in the creation of a stable structure. Examples of
such heteroalkyl groups include, but are not limited to azetidinyl,
piperidinyl, pyrrolidinyl, piperazinyl, oxopiperazinyl,
oxopiperidinyl, oxoazepinyl, azepinyl, tetrahydrofuranyl,
dioxolanyl, tetrahydroimidazolyl, tetrahydrothiazolyl,
tetrahydrooxazolyl, tetrahydropyranyl, morpholinyl,
thiomorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone
and oxadiazolyl. Preferred heteroalkyl groups include pyrrolidinyl,
piperidinyl, piperazinyl, morpholinyl, azetidinyl and
tetrahydrothiazolyl.
[0093] The term "heteroaryl" as used herein represents an
unsubstituted or substituted stable five or six membered monocyclic
aromatic ring system or an unsubstituted or substituted nine or ten
membered benzo-fused heteroaromatic ring system or bicyclic
heteroaromatic ring system which consists of carbon atoms and from
one to four heteroatoms selected from N, O or S, and wherein the
nitrogen or sulfur heteroatoms may optionally be oxidized, and the
nitrogen heteroatom may optionally be quaternized. The heteroaryl
group may be attached at any heteroatom or carbon atom that results
in the creation of a stable structure. Examples of heteroaryl
groups include, but are not limited to pyridyl, pyridazinyl,
thienyl, furanyl, imidazolyl, isoxazolyl, oxazolyl, pyrazolyl,
pyrrolyl, thiazolyl, thiadiazolyl, triazolyl, benzimidazolyl,
benzofuranyl, benzothienyl, benzisoxazolyl, benzoxazolyl,
benzopyrazolyl, indolyl, benzothiazolyl, benzothiadiazolyl,
benzotriazolyl adeninyl or quinolinyl. Prefered heteroaryl groups
include pyridyl, pyrrolyl, pyrazinyl, thiadiazolyl, pyrazolyl,
thienyl, triazolyl and quinolinyl.
[0094] The term "aralkyl" means an alkyl group substituted with
one, two or three aryl groups (e.g., benzyl, phenylethyl,
diphenylmethyl, triphenylmethyl). Similarly, the term "aralkoxy"
indicates an alkoxy group substituted with an aryl group (e.g.,
benzyloxy). The term aminoalkyl refers to an alkyl group
substituted with an amino group (i.e., -alkyl-NH.sub.2). The term
"alkylamino" refers to an amino group substituted with an alkyl
group (i.e., --NH-alkyl). The term "dialkylamino" refers to an
amino group which is disubstituted with alkyl groups wherein the
alkyl groups can be the same or different (i.e.,
--N-[alkyl].sub.2).
[0095] The term "acyl" as used herein means an organic radical
having 1 to 6 carbon atoms (branched or straight chain) derived
from an organic acid by removal of the hydroxyl group.
[0096] The term "oxo" refers to the group .dbd.O.
[0097] The term "carbonyl" refers to the group C(O).
[0098] The term "halogen" shall include iodine, bromine, chlorine
and fluorine.
[0099] The term "N(CH.sub.2).sub.4" as used herein (e.g., in the
Tables), refers to a pyrrolidinyl group having the structure 6
[0100] Similarly "C.sub.6H.sub.11" and "C.sub.5H.sub.9" (or
"c-C.sub.6H.sub.11" and "c-C.sub.5H.sub.9") refer to cyclohexyl and
cyclopentyl groups, respectively.
[0101] Whenever the term "alkyl" or "aryl" or either of their
prefix roots appear in a name of a substituent (e.g., aralkyl,
dialkylamino) it shall be interpreted as including those
limitations given above for "alkyl" and "aryl." Designated numbers
of carbon atoms (e.g., C.sub.1-C.sub.6) shall refer independently
to the number of carbon atoms in an alkyl or cycloalkyl moiety or
to the alkyl portion of a larger substituent in which alkyl appears
as its prefix root.
[0102] As used herein, the term "phosgene equivalent" represents
the class of carbonic acid derivatives which include 4-nitrophenyl
chloroformate, phosgene or "COCl.sub.2," phenyl chloroformate,
triphosgene or "(CCl.sub.3O).sub.2CO," carbonyldiimidazole, diethyl
carbonate or diphenyl carbonate.
[0103] It is intended that the definition of any substituent or
variable at a particular location in a molecule be independent of
its definitions elsewhere in that molecule. It is understood that
substituents and substitution patterns on the compounds of this
invention can be selected by one of ordinary skill in the art to
provide compounds that are chemically stable and that can be
readily synthesized by techniques known in the art as well as those
methods set forth herein.
[0104] As used herein, the term "composition" is intended to
encompass a product comprising the specified ingredients in the
specified amounts, as well as any product which results, directly
or indirectly, from combinations of the specified ingredients in
the specified amounts. Accordingly, pharmaceutical compositions
containing the compounds of the present invention as the active
ingredient as well as methods of preparing the instant compounds
are also part of the present invention. Particularly preferred
compounds of the present invention and their biological data are
shown in Tables 1 and 2, following; the amino acids bear the "L"
absolute configuration unless denoted otherwise. The Tables contain
IC.sub.50 values (.mu.M) of the compounds in a thrombin receptor
binding assay, and IC.sub.50 values (.mu.M) against platlet
aggregation stimulated by thrombin.
1TABLE 1 6-Substituted Benzimidazolone Peptidomimetics 7 IC.sub.50
(.mu.M) Thr GFP Thr Recptr Compd R.sub.1 R.sub.4 A.sub.1
A.sub.2NR.sub.2R.sub.3 Aggr.sup.a Bdg.sup.b 1
CH.sub.2N(CH.sub.2).sub.4 2,6-DiCl-Ph.sup.c 3,4-DiF-Phe.sup.d
Dbu.sup.e-NHBn 0.39 1.6 4 CH.sub.2NMe.sub.2 2-Me-Ph 3,4-DiF-Phe
Dbu-NHBn 0.23 0.7 5 CH.sub.2N(CH.sub.2).sub.4 2-Me-Ph 3,4-DiF-Phe
Dbu-NHBn 0.28 0.4 6 CH.sub.2N(CH.sub.2).sub.4 4-F-Ph 3,4-DiF-Phe
Dbu-NHBn 6 0.6 8 CH.sub.2N(CH.sub.2).sub.4 4-F-Ph 3,4-DiF-Phe
4-PyrAla.sup.f-NH(CH.sub.2).sub.2NH.sub.2 5.8 0.5
[0105]
2TABLE 2 5-Substituted Benzimidazolone Peptidomimetics 8 IC.sub.50
(.mu.M) Thr GFP Thr Recptr Compd R.sub.1 R.sub.4 A.sub.1
A.sub.2NR.sub.2R.sub.3 Aggr.sup.a Bdg.sup.b 2
CH.sub.2N(CH.sub.2).sub.4 2-Me-Ph 3,4-DiF-Phe Dbu-NHBn 7 3 3
CH.sub.2NMe.sub.2 2-Me-Ph 3,4-DiF-Phe Dbu-NHBn 7.7 1.9 7
CH.sub.2N(CH.sub.2).sub.4 2,6-DiCl-Ph 3,4-DiF-Phe Dbu-NHBn 5.5 1.7
.sup.aThrombin-induced gel-filtered platelet aggregation assay.
.sup.bThrombin receptor (PAR-1) binding assay.
.sup.c2,6-Dichlorophenyl. .sup.d3,4-Difluorophenylalanine.
.sup.e2,4-Diaminobutyric acid. .sup.f4-Pyridylalanine.
[0106] Suitable PAR-2 antagonists include antibodies that block
activation of the PAR-2 receptor and antisense sequences that
hybridize to uniquely conserved regions of the PAR-2 protein.
Suitable antisense sequences include the SLIGKV sequence of
nucleotides 38-43. A description of these antisense sequences may
be found in WO 00/8150 published Feb. 1, 2000 hereby incorporated
herein by reference.
[0107] Suitable antibodies for the PAR-1 and the PAR-2 receptors
that block activation of these receptors can be readily developed
using conventional monoclonal or polyclonal technology. PAR-1
(Smith-Swintosky et al., 1997; Cheung et al., 1999; Festoff et al.,
2000) and PAR-2 (Smith-Swintosky et al., 1997; D'Andrea et al.,
1998; Damiano et al., 1999) antibodies have been previously
described and characterized. These antibodies would have to be
tested for the presence of crossreative species and the cross
reactive species removed by appropriate techniques (e.g. using the
crossreactive antigen bound to columns to extract the cross
reactive species). Additionally, antibodies effectiveness in
blocking PAR-1 and PAR-2 activation would have to be monitored.
[0108] The daily dosage of the PAR-1 and/or PAR-2 antagonist may be
varied over a wide range from about 0.01 mg to about 1,000 mg per
adult human per day. For oral administration of PAR-1 antagonist,
the compositions are preferably provided in the form of tablets
containing about 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0,
25.0, 50.0, 100, 150, 200, 250 and 500 mg of the active ingredient
for the symptomatic adjustment of the dosage to the patient to be
treated. An effective amount of the drug is ordinarily supplied at
a dosage level of from about 0.03 mg/kg to about 100 mg/kg of body
weight per day. Preferably, the range is from about 0.1 mg/kg to
about 30 mg/kg of body weight per day. The compounds may be
administered on a regimen of about 1 time to about 4 times per
day.
[0109] Optimal dosages to be administered may be determined by
those skilled in the art. Since each malignant cell line will
differ it is expected that the physician treating the patient will
have to vary the dosage of the particular compound used, depending
on the mode of administration, the strength of the preparation, the
physical condition of the patient and the advancement of the
disease condition. In addition, factors associated with the
particular patient being treated, including patient age, weight,
diet and time of administration, will result in the need to adjust
dosages. Response to the treatment may be monitored by conventional
means including by CAT scan, MRI, ultrasound or other imaging
techniques.
Immune Modulation
[0110] To restore the immune modulation disrupted in the TME
pharmaceutically active compounds maybe administered prior to,
concurrently with, or subsequent to, the administration of PAR-1
and/or PAR-2 antagonists to deactivate mast cells and/or activate
CTL or NK cells. Suitable cytokines include but are not limited to
interleukin-2 (IL-2), interleukin-12 (IL-12 ) interleukin-18 (IL-1
8), granulocyte colony stimulating factor (G-CSF), macrophage
colony stimulating factor (M-CSF), granulocyte-macrophage colony
stimulating factor (GM-CSF), interferon alpha, interferon beta,
interferon-gamma, tumor necrosis factor (TNF) and combinations
thereof. Generally, a pharmaceutically effective amount of an
cytokines will be in the range of from about 1,000 to about
3000,000 U/kg/day; more preferably in an amount of from about 3,000
to about 1000,000 U/kg/day; and more preferably in an amount from
about 5,000 to about 20,000 U/kg/day.
[0111] Other methods of enhancing the immune response to malignant
cells have been described in the art such as enhancing the
activation of NK cells which was described by Hellstrand et al. in
U.S. Pat. No. 6,071,509 (hereby incorporated by reference herein).
Similarly, T cell activation can be used as a means of treating
cancer. T cell can be activated by isolating tumor infiltrating
autologous T cells, activating the T-cells in vitro with IL-2,
allowing the T cells to proliferate and injected into the patient.
Additionally, T cells activity can be increased by utilizing
blocking antibodies against CTLA-4 as described by E. D. Kwon et
al., Proc. Natl. Acad. Sci. U.S.A. 94, 8099 (1997) and Allison et
al. in U.S. Pat. No. 6,051,227 (hereby incorporated by reference
herein). Similarly antiCD3 and antiCD28 and B-7 may also be
employed to enhance the immune response.
Therapeutic Use
[0112] PAR-1 and/or PAR-2 antagonist may be used in subjects having
malignant cells that characteristically activate fibroblasts,
monocytes/macrophages, mast cells and combinations thereof.
Generally malignant cell that secrete proteases or otherwise
directly or indirectly activate the PAR-1 and/or PAR-2 receptors in
the TME maybe treated by the present therapy. Representative
malignant cell types characteristically associated with
fibroblasts, monocytes and/or mast cells include but are not
limited to lung cancer (e.g. non-small-cell lung cancer), skin
cancer (e.g. melanomas), stomach cancer, intestinal cancer,
colorectal cancer, pancreatic cancer, liver cancer, thyroid cancer,
uterine cancer, cervical cancer, ovarian cancer, testicular cancer,
prostrate cancer and breast cancer.
[0113] The use of PAR-1 and/or PAR-2 antagonist to reduce or
prevent metastasis maybe employed as soon as malignant cells are
detected with or without immune modulation techniques or
conventional therapeutic methodologies (e.g. chemotherapy agents or
radiation). Suitable chemotherapy agents include but are not
limited to anti-angiogenic compounds, alkylating compounds,
antimetabolites, hormonal agonist /antagonists, monoclonal
antibodies for cancer treatment, antiproliferatives, etc. and
combinations thereof. Any anti-angiogenic compound can be used.
Exemplary anti-angiogenic compounds include O-substituted
fumagillol and derivatives thereof, such as TNP-470, described in
U.S. Pat. Nos. 5,135,919, 5,698,586, and 5,290,807 to Kishimoto, et
al.; angiostatin and endostatin, described in U.S. Pat. Nos.
5,290,807, 5,639,725 and 5,733,876 to O'Reilly; thalidomide, as
described in U.S. Pat. Nos. 5,629,327 and 5,712,291 to D'Amato; and
other compounds, such as the anti-invasive factor, retinoic acid,
and paclitaxel, described in U.S. Pat. No. 5,716,981 to Hunter, et
al., and the metalloproteinase inhibitors described in U.S. Pat.
No. 5,713,491 to Murphy, et al. Other well known chemotherapeutic
agents may also be used such as doxorubicin, decarbazine,
irinotecan, etoposide phosphate, asparaginase, gemcitabine,
carboplatinum, cisplatinum, tomoxifen, methotrexate, ifosfamide,
cyclophosphamide, 5-fluorouracil, vinorelbine tartrate,
anastrozole, trastuzumab and combinations thereof. These and other
chemotherapeutic agents for the treatment of cancer may be found in
the Physicians Desk Reference.
[0114] The method of prevention or reduction of the establishment,
growth and/or metastasis of malignant cells may be used
preoperatively and post-operatively as an adjunct to surgery.
Dosage
[0115] It is contemplated by this invention that the administration
of the compositions described herein for reducing or preventing
metastasis or immune modulation may be accomplished by any of the
methods known to the skilled artisan. The compound may be
administered to a patient by any conventional route of
administration, including, but not limited to, intravenous, oral,
subcutaneous, intramuscular, intradermal and parenteral. The
compounds used in reducing or preventing metastasis or immune
modulation will generally be provided in association with a
pharmaceutically acceptable carrier recognized as suitable by those
skilled in the art.
[0116] The term "subject" as used herein, refers to an animal,
preferably a mammal, most preferably a human, who has been the
object of treatment, observation or experiment.
[0117] It is understood that the dosage of a pharmaceutical
compound or composition of the present invention administered in
vivo or in vitro will be dependent upon the age, sex, health, and
weight of the recipient, kind of concurrent treatment, if any,
frequency of treatment, and the nature of the pharmaceutical effect
desired. The ranges of effective doses provided herein are not
intended to be limiting and represent preferred dose ranges. The
most preferred dosage will be tailored to the individual subject,
as is understood and determinable by one skilled in the relevant
arts. See, e.g., Berkow et al., eds., The Merck Manual, 16th
edition, Merck and Co., Rahway, N. J. (1992); Goodman et al., eds.,
Goodman and Gilman's The Pharmacological Basis of Therapeutics, 8th
edition, Pergamon Press, Inc., Elmsford, N.Y. (1990); Avery's Drug
Treatment: Principles and Practice of Clinical Pharmacology and
Therapeutics, 3rd edition, ADIS Press, LTD., Williams and Wilkins,
Baltimore, Md. (1987); Ebadi, Pharmacology, Little, Brown and Co.,
Boston (1985); Osol et al., eds., Remington's Pharmaceutical
Sciences, 17th edition, Mack Publishing Co., Easton, Pa. (1990);
Katzung, Basic and Clinical Pharmacology, Appleton and Lange,
Norwalk, Conn., (1992), which references are entirely incorporated
herein by reference. The term "therapeutically effective amount" as
used herein, means that amount of active compound or pharmaceutical
agent that elicits the biological or medicinal response in a tissue
system, animal or human that is being sought by a researcher,
veterinarian, medical doctor or other clinician, which includes
alleviation of the symptoms of the disease or disorder being
treated.
[0118] The total dose required for each treatment can be
administered by multiple doses or in a single dose. The
diagnostic/pharmaceutical compound or composition can be
administered alone or in conjunction with other diagnostics and/or
pharmaceuticals directed to the pathology, or directed to other
symptoms of the pathology.
Chemistry of PAR-1 Antagonist
[0119] The antagonists of the present invention may be prepared via
a convergent solution-phase synthesis by coupling an
aminobenzimidazolone intermediate AAG4 with a dipeptide amine AAG6
via a urea linkage as described in the general Scheme AAGeneric.
The appropriately nitro substituted benzimidazolone AAG1 (Scheme
AAGeneric) was alkylated with a substituted aralkyl or heteroaryl
alkyl halide and a base such as sodium hydride in a dipolar aprotic
solvent such as DMF to give AAG2 as a mixture of two regioisomers.
Two isomers were separated by silica gel column and then alkylated,
with an aminoalkyl halide and a base such as sodium hydride in a
dipolar aprotic solvent such as DMF to give two regioisomers AAG3,
respectively. Reduction of nitro group in AAG3 in a classical
manner with for example iron and acetic acid or with a newer method
such as dimethyl hydrazine and iron to gave aminobenzimidazolone
intermediate AAG4.
[0120] Dipeptide amine AAG6 can be synthesized from the
corresponding protected amino acids using standard peptide coupling
conditions. Thus, an Fmoc protected amino-acid (A.sub.2) AAG5
(Scheme AAGeneric) was coupled to amine R.sub.2R.sub.3NH using a
coupling agent, such as dicyclohexylcarbodiimide (DCC) or
diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBT) in
a dipolar aprotic solvent such as DMF to give the amide. The amide
was then Fmoc deprotected with a dialkylamine in a dipolar aprotic
solvent, such as diethylamine in acetonitrile. The resulting amine
was coupled to the second Fmoc protected amino-acid (A.sub.1) in
the same way with a coupling agent, such as DIC and HOBT in a
dipolar aprotic solvent such as DMF to give the dipeptide. The
dipeptide was then Fmoc deprotected as above with a dialkylamine in
a dipolar aprotic solvent such as acetonitrile to afford dipeptide
amine AAG6.
[0121] Aminobenzimidazolone intermediate AAG4 was treated with a
phosgene equivalent such as 4-nitrophenyl chloroformate, phosgene
or "COCl.sub.2," phenyl chloroformate, triphosgene or
"(CCl.sub.3O).sub.2CO," carbonyldiimidazole, diethyl carbonate or
diphenyl carbonate and a base such as diisopropylethylamine in a
solvent such as dichloromethane, and to this was then added
dipeptide amine AAG6 to give a urea. Removal of the protecting
group, if necessary, such as the Boc group with an acid such as
trifluoroacetic acid from the side chain of dipeptide afforded
final targets AAG7.
Scheme AAGeneric
[0122] 9
[0123] As a typical example of this convergent solution-phase
method, Compound 1 may be synthesized as described in Scheme AA.
Thus, treatment of 5-nitrobenzimidazolone AA1 with 2,6-diCl-Bn-Br
in the presence of NaH in DMF afforded two regioisomers AA2a and
AA3a (ca. 1:1 ratio), which were then separated by flash column. A
small amount of bis-alkylated product was also obtained. The
isolated AA3a was further alkylated with 2-chloroethylpyrrolidine
by using NaH as a base in DMF to give the di-alkylated product,
which was then subjected to nitro reduction with
Me.sub.2NNH.sub.2/FeCl.sub.3 to provide aminobenzimidazolone
intermediate AA4. Coupling of
N-.alpha.-Fmoc-N-.gamma.-Boc-diaminobutyric acid (AA5) with benzyl
amine in the presence of DCC and HOBt was followed by de-protection
of the Fmoc group with diethylamine. The resulting intermediate was
coupled with Fmoc-3,4-diF-Phe-OH using DIC/HOBt and treated with
diethylamine to give dipeptide amine AA6. Urea formation between
the dipeptide amine AA6 and aminobenzimidazolone intermediate AA4
in the presence of 4-nitrophenylchloroformate was followed by
de-protection of the Boc group with TFA to afford the target
compound 1.
[0124] In an alternative approach to the targets, the preferred
intermediate AA3 could be prepared via a regioselective method. For
example, reductive alkylation of 4-nitro-1,2-phenylenediamine AB1
with one equivalent of 4-fluorobenzaldehyde in the presence of
sodium triacetoxyborohydride gave mono-alkylated product AB2 which
was then cyclized with N,N'-disuccinimidyl carbonate in
acetonitrile to give AA3c (Scheme AB). By following the same
procedure as described in Scheme AA, the target 6 was obtained from
intermediate AA3c. 10 11
[0125] The side-chain amine in antagonists such as Compound 1 and
Compound 8 may be converted to other functional groups such as
acetamidine and guanidine by using standard procedures. For
example, the acetamidine and guanidine groups can be introduced by
treating the side-chain amine with S-2-naphthylmethyl
thioacetimidate hydrobromide and 2-methyl-2-thiopseudourea,
respectively.
[0126] The thioureidoindoles [when X is S, as in general formula
(I)] may be prepared as described hereinafter. The
aminobenzimidazolone substrate is reacted with
thiocarbonyldiimidazole in a chlorinated solvent and then with the
imidazole by-product filtered from the solution. The solution can
then be concentrated to afford the
N-imidazolyl-N'-benzimidazolonyl-t- hioamide. This intermediate is
then reacted with a peptide amine in a polar, aprotic solvent with
heating (from about 80 .degree. C. to about 100.degree. C.) to
afford the N-peptido-N'-benzimidazolonyl-thiourea product.
[0127] Amidobenzimidazolone targets [when p is 0 and X is O, as in
general formula (I)] may be prepared from a dipeptide amine AAG6
(Scheme AAGeneric) and a benzimidazolone carboxylic acid
intermediate by using standard coupling conditions such as
DCC/HOBt. The required benzimidazolone carboxylic acid
intermediates can be prepared by using the method as described for
aminobenzimidazolone intermediate AAG4 in Scheme AAGeneric from the
appropriate benzimidazolone carboxylic acid esters.
[0128] Where the processes for the preparation of the compounds
according to the invention give rise to mixture of stereoisomers,
these isomers may be separated by conventional techniques such as
preparative chromatography. The compounds may be prepared in
racemic form, or individual enantiomers may be prepared either by
enantiospecific synthesis or by resolution. The compounds may, for
example, be resolved into their components enantiomers by standard
techniques, such as the formation of diastereomeric pairs by salt
formation with an optically active acid, such as
(-)-di-p-toluoyl-d-tartaric acid and/or (+)-di-p-toluoyl-1-tartaric
acid followed by fractional crystallization and regeneration of the
free base. The compounds may also be resolved by formation of
diastereomeric esters or amides, followed by chromatographic
separation and removal of the chiral auxiliary. Alternatively, the
compounds may be resolved using a chiral HPLC column.
[0129] During any of the processes for preparation of the compounds
of the present invention, it may be necessary and/or desirable to
protect sensitive or reactive groups on any of the molecules
concerned. This may be achieved by means of conventional protecting
groups, such as those described in Protective Groups in Organic
Chemistry, ed. J. F. W. McOmie, Plenum Press, 1973; and T. W.
Greene & P. G. M. Wuts, Protective Groups in Organic Synthesis,
John Wiley & Sons, 1991. The protecting groups may be removed
at a convenient subsequent stage using methods known from the
art.
[0130] To prepare the pharmaceutical compositions of this
invention, one or more compounds of formula (I) or salt thereof of
the invention as the active ingredient, is intimately admixed with
a pharmaceutical carrier according to conventional pharmaceutical
compounding techniques, which carrier may take a wide variety of
forms depending on the form of preparation desired for
administration, e.g., oral or parenteral such as intramuscular. In
preparing the compositions in oral dosage form, any of the usual
pharmaceutical media may be employed. Thus, for liquid oral
preparations, such as, for example, suspensions, elixirs and
solutions, suitable carriers and additives include water, glycols,
oils, alcohols, flavoring agents, preservatives, coloring agents
and the like; for solid oral preparations such as, for example,
powders, capsules, caplets, gelcaps and tablets, suitable carriers
and additives include starches, sugars, diluents, granulating
agents, lubricants, binders, disintegrating agents and the like.
Because of their ease in administration, tablets and capsules
represent the most advantageous oral dosage unit form, in which
case solid pharmaceutical carriers are obviously employed. If
desired, tablets may be sugar coated or enteric coated by standard
techniques. For parenterals, the carrier will usually comprise
sterile water, though other ingredients, for example, for purposes
such as aiding solubility or for preservation, may be included.
Injectable suspensions may also be prepared, in which case
appropriate liquid carriers, suspending agents and the like may be
employed. The pharmaceutical compositions herein will contain, per
dosage unit, e.g., tablet, capsule, powder, injection, teaspoonful
and the like, an amount of the active ingredient necessary to
deliver an effective dose as described above. The pharmaceutical
compositions herein will contain, per unit dosage unit, e.g.,
tablet, capsule, powder, injection, suppository, teaspoonful and
the like, from about 0.03 mg/kg to about 100 mg/kg (preferred from
about 0.1 mg/kg to about 30 mg/kg) of a compound of the present
invention and may be given at a dosage from about 0.1 mg/kg/day to
about 300 mg/kg/day (preferred from about 1 mg/kg/day to about 50
mg/kg/day). The dosages, however, may be varied depending upon the
requirement of the patients, the severity of the condition being
treated and the compound being employed. The use of either daily
administration or post-periodic dosing may be employed.
[0131] The following non-limiting examples are provided to further
illustrate the present invention.
EXAMPLE 1
[0132] Protease-activated receptors (PARs) belong to a family of
G-coupled seven transmembrane receptors that are activated by a
proteolytic cleavage of their N-termini. Recent studies suggest the
involvement of protease-activated receptors-1 and -2 (PAR-1, PAR-2)
activators in mast cell degranulation in various physiological and
pathophysiological processes in inflammatory responses. Although
PAR-1 and PAR-2 activating proteases, thrombin and tryptase, have
been associated with mast cell activation, PAR-1 and PAR-2 have not
been localized within these cells. We describe here the
localization of PAR-1 and PAR-2 in all mast cells from various
normal human tissues using immunohistochemical and double
immunofluorescence techniques. The presence of these receptors on
the membrane may explain the actions of accessible extracellular
thrombin and tryptase for mast cell activation. In addition to the
membrane labeling, these receptors are also localized on the
membrane of the intracellular tryptase-positive granules, which may
function to sustain further mast cell degranulation upon
exocytosis. The localization of these two receptors in mast cells
suggests a novel mechanism for controlling mast cell activation
through regulation of PAR-1 and PAR-2.
Materials and Methods
[0133] Human checkerboard tissue blocks (Biomeda, Foster City,
Calif.) were routinely processed for immunohistochemistry (D'Andrea
et al. 1998a) and for double immunofluorescence (D'Andrea 1998b).
Primary antibodies include monoclonal anti-human mast cell tryptase
(1:500, Dako, Carpenturia, Calif.), polyclonal anti-human PAR-1 (1
.mu.g/ml) raised against the C-terminus of the receptor
(Smith-Swintosky et al. 1997), and polyclonal anti-human PAR-2C (1
.mu.g/ml) raised against a sequence spanning the cleavage domain
(D'Andrea et al, 1998a). Briefly, slides were incubated (30 min at
room temperature) with primary antibodies, followed by incubation
with specific biotin conjugated secondary antibodies (Vector Labs,
Burlingham, Calif.) which were then detected using the
ABC-horseradish peroxidase system (Vector Labs). Slides were
treated with 3'-diaminobenzidine (Biomeda, Foster City, Calif.) as
the chromogen, stained in Mayer's hematoxylin and coverslipped with
Permount (Fisher, Pittsburgh, Pa.).
[0134] For the double immunofluorescence (D'Andrea 1998b), the
first primary antibody was incubated on the tissues and followed by
FITC-conjugated secondary antibody (1:200, Vector Labs, Burlingham,
Calif.). Subsequently, the second primary antibody was incubated on
the tissues and followed by Texas Red-conjugated secondary antibody
(1:50, Vector Labs, Burlingham, Calif.). Slides were coverslipped
with anti-fade media containing the nuclear DAPI stain (Vector
Labs, Burlingham, Calif.). Co-localization of the FITC and Texas
Red signals were visualized as yellow fluorescence. A negative
control for each antibody included the same species isotype
nonimmune serum.
Results
[0135] Single immunohistochemical techniques were used to identify
human mast cells in various normal human tissues using an antibody
to mast cell tryptase (MCT). Mast cells were localized in a lymph
node and in the submucosa of the large intestine. Extracellular
labeling of MCT in the immediate pericellular vicinity was
interpreted as an indication of mast cell degranulation (Buckley et
al. 1998, Johnson et al. 1998), which was observed using mast cell
tryptase immunolabeling. Localization of MCT was indicated by the
presence of brown precipitate within the intracellular mast cell
granules, whereas cells negative for MCT had blue nuclei only.
Immunolocalization of MCT on the cell surface of degranulated mast
cell located in the submucosa of the large intestine was observed.
Antibodies specific to PAR-1 and PAR-2 were used to localize their
respective antigens in the same normal human tissues. PAR-1 and
PAR-2 were localized to the plasma membrane and the intracellular
granule membranes of the mast cells localized in sections of the
lymph node and the submucosa of the large intestine.
[0136] To determine the co-localization patterns of MCT and PAR-1
and PAR-2 in mast cells, we performed double immunofluorescence
techniques on similar human tissues. Co-localization of a MCT Texas
Red-positive signal with a PAR-1 or PAR-2 FITC-positive signal were
interpreted as distinct yellow fluorescence. The co-localization of
PAR-1 and MCT in mast cells was demonstrated in a lymph node. A
similar approach was employed to demonstrate the co-expression of
MCT and PAR-2 in a normal human uterus. We were not able to resolve
the PAR-1 or PAR-2 plasma membrane signals due to the significant
fluorescence obtained from the intracellular granules, which
appeared to mask lower antigen signals within close proximity.
Similarly, the co-expression of PAR-1 and PAR-2 was performed on
similar tissues such as a lymph node.
[0137] Mast cells with positive MCT, PAR-1 and PAR-2
immunolabeling, were localized in the following tissues (sample
size): large intestine (3), lung (5), pancreas (5), prostate (5),
skin (3), small intestine (1), spleen (5), stomach (1), testis (2),
tonsil (3), and uterus (3). A series of negative controls employed
for the double immunofluorescence techniques included 1)
replacement of the first primary antibody with similar isotype
nonimmune serum; 2) replacement of the second primary antibody with
similar isotype nonimmune serum; 3) replacement of both primary
antibodies with negative control antibodies. All of these negative
control antibodies did not yield detectable labeling.
Discussion
[0138] We describe here the presence of PAR-1 and PAR-2 on mast
cells in various normal human tissues using immunohistochemical and
double immunofluorescence techniques. More specifically, PAR-1 and
PAR-2 are distributed on the plasma membrane and on the membranes
of the intracellular tryptase-positive granules of the mast
cells.
[0139] Mast cells are involved in numerous normal and
pathophysiological conditions. Upon activation, they secrete a
range of soluble mediators including histamine, heparin,
leukotrienes, cytokines, growth factors and neutral proteases
(Fawcett 1955, Galli et al. 1989, Gordon 1990, Bradding et al.
1995, Irani 1995,He and Walls 1997, Buckley et al. 1998, Johnson et
al. 1998, Laine et al. 1999). Thus, mast cells represent an
efficient system through which powerful mediating factors and
enzymes are deposited locally upon activation. Current proposed
mechanisms of mast cell activation include most notably the binding
of an allergen to an IgE receptor on the mast cell membrane.
Subsequent binding leads to degranulation and ensuing release of
the mediators of immediate hypersensitivity from their storage
sites in mast cell granules (Dvarak 1985, Galli et al. 1989, Gordon
et al. 1990, Irani 1995, Johnson et al. 1998). In addition to the
IgE pathway, mast cells may also be activated by a "histamine
releasing factor" secreted from immunocompetent cells such as T
lymphocytes (Segwick et al. 1981) and macrophages (Liu et al.
1986). Thrombin, which activates PAR-1 (Vu et al. 1991), has been
reported to induce mast cell activation (Razin and Marx 1984,
Cirino et al. 1996). Also, tryptase, a reported PAR-2 activator
(Fox et al. 1997, Molino et al. 1997a) and a key secretory
component of all human mast cells regardless of anatomical site
(Craig et al. 1989, Irani 1995), can activate mast cells in vivo
(He and Walls 1997). Other studies suggest that mast cell tryptase
and SLIGRL, a PAR-2 activating peptide, (Kawabata et al. 1998)
elicits PAR-2 mediated inflammation. These observations suggest the
presence of mast cell receptors for thrombin and tryptase that
initiate or potentiate mast cell activation. Our discovery of PAR-1
and PAR-2 on the plasma membrane of mast cells suggest a mechanism
by which thrombin via PAR-1 (Razin and Marx 1984, Cirino et al.
1996) and tryptase via PAR-2 (Molino et al. 1997a, Fox et al. 1997,
Kawabata et al. 1998) might mediate these processes.
[0140] These receptors can then be translocated with the granules
to fuse to the mast cell surface through the process of exocytosis
(Lawson et al. 1978, Ishizaka 1984, Dvorak et al. 1985, Burgess and
Kelly 1987). Subsequently, PAR-1 and PAR-2 activation will
transduce intracellular Ca.sup.2+ mobilization (Blackhart et al.
1996, Magazine et al. 1996), which is necessary for mast cell
degranulation (Lawson et al. 1978). Therefore, it is possible that
the function of PAR-1 and PAR-2 on the membranes of intracellular
granules may be to sustain further mast cell degranulation by
replenishing internalized PAR-1 and PAR-2 from the plasma membrane
upon activation (Molino et al. 1997b ).
[0141] We have localized PAR-1 and PAR-2 on mast cell plasma
membrane and the membranes of the intracellular tryptase-positive
granules through immunohistochemistry. The presence of PAR-1 and
PAR-2 may explain the actions of PAR-1 and PAR-2 activators to
stimulate mast cell degranulation. In addition, PAR-1 and PAR-2 may
also function to sustain mast cell activation in a paracrine or
autocrine fashion. In vivo and in vitro functional studies are
necessary to elucidate the roles of PAR-1 and PAR-2 in mast cell
activation in normal and pathological conditions. Thus, the
discovery of two additional mast cell receptors suggests novel
mechanisms to control mast cell activation through the regulation
of PAR-1 and PAR-2. Ultimately, specific antagonists of PAR-1 and
PAR-2 could be used as tools to probe this hypothesis. In addition,
these antagonists might prove to be effective therapeutic agents
for inflammatory driven conditions.
EXAMPLE 2
[0142] The serine proteases, thrombin and trypsin, are among many
factors that malignant cells secrete into the extracellular space
to mediate metastatic processes such as cellular invasion,
extracellular matrix degradation, angiogenesis and tissue
remodeling. The degree protease secretion has been correlated to
their metastatic potential. Protease activated receptors (PAR)-1
and -2, which are activated by thrombin and trypsin respectively,
have not been extensively characterized in human tumors in situ. We
investigated the presence of PAR-1 and -2 in human normal, benign
and malignant tissues using immunohistochemistry and in situ
hybridization. Our results demonstrated PAR-1 and -2 expression in
the hosting stromal fibroblasts, mast cells, macrophages,
endothelial cells and vascular smooth muscle cells of the
metastatic tumor microenvironment. Interestingly, the up-regulation
of PAR-1 and -2 in reactive stromal fibroblasts surrounding the
carcinoma cells was not observed in normal or benign conditions.
Furthermore, in vitro studies using proliferating, smooth muscle
actin (SMA) positive, human dermal fibroblasts demonstrated the
presence of functional PAR-1 and -2 not detected in quiescent, SMA
negative cultures. PAR-1 and -2 in the cells forming the tumor
microenvironment suggest that these receptors mediate the signaling
of secreted thrombin and trypsin in the processes of cellular
metastasis.
[0143] Reagents.
[0144] Primary antibodies used in these experiments include the
following: desmin (Dako, CA), endothelial cell (CD3 1) (Dako, CA),
fibroblast prolyl 4-hydroxylase) (Dako, CA), macrophage (CD68)
(Dako, CA), mast cell tryptase (Dako, CA), non-immuno serum (Vector
Labs, CA), PAR-1 (RWJPRI, PA) (Smith-Swintosky et al. 1997; Cheung
et al. 1999; Festoff et al. 2000), PAR-2 (RWJPRI, PA) (D'Andrea et
al. 1998; Smith-Swintosky et al. 1997; Damiano et al. 1999), smooth
muscle actin (Dako, CA), DNA topoisomerase IIa (Pharmingen, CA)
(D'Andrea et al. 1994) and vimentin (Dako, CA).
[0145] 3 '-biotinylated molecular probes used for in situ
hybridization include the following: PAR-1 (5' TTC ATT TTT CTC CTC
CTC CTC CTC ATC C) (Research Genetics, AL) (Cheung et al. 1999;
Festoff et al. 2000), PAR-2 (5.degree. CAA TAA TGT AGA CGA CCG GAA
GAA AGA) (Research Genetics, AL) (Daminano et al. 1999),
glyceraldehyde-3-phosphate dehydrogenase (GAP-DH) (5' GAC GCC TGC
TTC TCC TCC TTC TTG) (Ransom Hill, CA), poly d(T) (5' TTT TTT TTT
TTT TTT TTT TTT TTT) (Research Genetics, CA), lac Z (5' CAC AGC GGA
TGG TTC GGA TAA TG) (Ransom Hill, CA). Immunohistochemistry.
Commercial human checkerboard tissue slides (Dako, Carpenteria,
Calif.; Biomeda, Foster City, Calif.) representing normal breast
tissues (n=26), benign breast fibroadenomas (n--14), malignant
breast carcinomas (n=46) and six other non-breast human carcinomas
(n=4-6) were deparaffinized, hydrated and processed for routine
immunohistochemistry (IHC) as previously described (D'Andrea et
al., 1998). Briefly, slides were microwaved in Target buffer
(Dako), cooled, placed in phosphate-buffered saline (pH 7.4, PBS)
and treated with 3.0% H.sub.2O.sub.2 for 10 min. Slides were
processed through an avidin-biotin blocking system according to the
manufacturer's instructions (Vector Labs, Burlingame, Calif.) and
then placed in PBS. All subsequent reagent incubations and washes
were performed at room temperature. Normal blocking serum (Vector
Labs) was placed on all slides for 10 min. After briefly rinsing in
PBS, primary antibodies were placed on slides for 30 min. PAR-1
(Smith-Swintosky et al., 1997; Cheung et al., 1999; Festoff et al.,
2000) and PAR-2 (Smith-Swintosky et al., 1997; D'Andrea et al.,
1998; Damiano et al., 1999) antibodies had been previously
characterized. The slides were washed and biotinylated secondary
antibodies, goat anti-rabbit (polyclonal antibodies) or horse
anti-mouse (monoclonal antibodies) were placed on the tissue
sections for 30 min (Vector Labs). After rinsing in PBS, the
avidin-horseradish peroxidase-biotin complex reagent (ABC, Vector
Labs) was added for 30 min. Slides were washed and treated with the
chromogen 3,3'-diaminobenzidine (DAB, Biomeda) twice for five min
each, then rinsed in dH20, and counterstained with hematoxylin. A
monoclonal antibody to vimentin, the widely conserved ubiquitious
intracellular filament protein, were utilized as a positive control
to demonstrate tissue antigenicity and control reagent quality. The
negative control included replacement of the primary antibody with
pre-immune serum or with the same species IgG isotype non-immune
serum.
[0146] Analysis of PAR-1 and PAR-2 Immunoreactivity.
[0147] The tissues were scored for the intensity of PAR-1 and PAR-2
immunoreactivity to compare the relative amounts of PAR-1 and PAR-2
in the stromal fibroblasts and epithelial cells in the normal
(n=26), benign (n=14) and malignant (n=46) breast tissues. For each
tissue, epithelial cells (n=25) and fibroblasts (n=15-25) the
presence of PAR-1 and PAR-2 immunoreactivity in the stromal
fibroblasts were scored under a 20.times. objective according to
the following criteria: 1) no immunoreactivity IR (0.0); 2) weak,
light brown immunoreactivity IR (1.0); 3) moderate brown
immunolabeling IR (2.0), and 4) intense, dark brown
immunoreactivity IR (3.0) (Table 1). The negative controls did not
produce observable labeling IR (0.0). The data from each tissue was
averaged and then grouped according to normal, benign and malignant
tissues for PAR-1 and PAR-2 expression.
[0148] Double Immunohistochemistry.
[0149] In an effort to determine if there was a correlation between
the up-regulation of PAR-1 and -2 with cell proliferation, we used
double immunohistochemical methods (IHC:IHC) to detect PAR-1 or -2
expression simultaneously with detection of a proliferation marker,
DNA topoisomerase Ia (Topo IIa) (D'Andrea et al., 1994). Protocols
for IHC:IHC have been previously described (D'Andrea et al., 1999).
Briefly, slides were first processed for single IHC labeling
protocols for detection of PAR-1 or PAR-2 as described above.
Without processing the slides for hematoxylin, Topo Ia antibodies
(Pharminigen, San Diego, Calif.) were placed on the tissues for 30
min. After brief PBS washes, the biotinylated horse anti-mouse
secondary antibodies (Vector Labs) were similarly incubated. The
presence of Topo II.alpha. positive cells were visualized using an
alkaline phosphatase detection system through incubation with
alkaline phosphatase conjugated ABC (Vector Labs) followed by
development using the Fast Red chromogen (Sigma). Slides were then
routinely counterstained and mounted.
[0150] In Situ Hybridization.
[0151] Slides were routinely dewaxed, rehydrated, placed in 3%
H.sub.2O.sub.2 for 10 min at room temperature and processed for in
situ hybridization (ISH) as previously described (Cheung et al.,
1999; Damiano et al., 1999; Festoff et al., 2000). Briefly, after a
5 min wash in water, slides were placed in Universal Buffer
(Research Genetics, Huntsville, Ala.) and the tissue sections were
digested with pre-diluted pepsin (Research Genetics) for 10 min at
42.degree. C. Sections were washed and then dehydrated in 100%
alcohol for 1 min. Each probe was diluted to 1.0 .mu.g/ml in
commercially formulated hybridization buffer (Biomeda) and heated
for 5 min at 103.degree. C. in a microcentrifuge tube on a heat
block. Anti-sense, biotinylated oligonucleotide probes to PAR-1
(Cheung et al., 1999; Festoff et al., 2000) and PAR-2 (Damiano et
al., 1999) mRNAs have been previously characterized. The ISH probes
were maintained at 42.degree. C. in a water bath until placement
onto the tissue sections. Ten microliters of probe was added to
each section and a coverslip was gently placed to cover the
solution and prevent evaporation. Slides were placed into a humid
chamber and incubated at 42.degree. C. for 2 h. After
hybridization, they were then immediately placed into a low
stringency wash (2.times. SSC) for 5 min at 42.degree. C., followed
by a high stringency wash (0.1.times. SSC) for 5 min at 42.degree.
C. Sections were washed in PBS and treated with ABC (Vector Labs)
for 1 h at room temperature. After washing, sections were placed in
DAB (Biomeda) for 2 times 5 min, washed, briefly stained with
hematoxylin, dehydrated in graded ethanols, cleared in xylene,
coverslipped in Pennount (Fisher Scientific, Pittsburgh, Pa.) and
photographed with an Olympus BX50 light microscope. Positive
controls included two biotinylated mRNA oligonucleotide probes:
GAPDH mRNA and a poly d(T) probe that hybridizes non-specifically
to all mRNA. Negative controls included 1) the absence of probe in
the probe cocktail; 2) a biotinylated probe that hybridizes to lac
Z operon mRNA (Table 2); and 3) pre-digestion of the tissues with
RNase, DNase free (10 .mu.g/.mu.l, Boehinger Mannheim, City, State)
for 2 hours at 42.degree. C. before probe hybridization.
[0152] Cell Culture.
[0153] Human neonatal dermal fibroblasts and their culture media
were obtained from Clonetics/BioWhittaker (Walkersville, Md.).
Cells were incubated for either 2 days (proliferating) or 9 days
(quiescent) prior to evaluation without serum exchange. Cell
suspensions (5.times.10.sup.4/ml) were plated in 96-well microtiter
plates for calcium mobilization studies and were seeded in 4-welled
chamber slides (NUNC, Naperville, Ill.) for immunocytochemistry. In
an effort to mimic the in vivo activation of differentiated,
quiescent fibroblasts in vitro, the 9-day, quiescent cells were
subjected to random scrape wounding induced by the end of a pipette
and cultured for 5 additional days without medium exchange. As a
control, other 9-day cultures without scrapes continued to grow
similarly.
[0154] Immunocytochemistry.
[0155] Four-chambered culture slides were routinely fixed with 10%
neutral buffered-saline for 10 min at room temperature, rinsed in
PBS and then assayed for immunocytochemistry as previously
described (Chen et al., 1998; Smith-Swintosky et al., 1998).
Hyper-confluent (quiescent) sub-confluent (proliferating) and
wounded cultured slides were processed for immunocytochemistry
using antibodies to PAR-1, PAR-2, SMA, Topo II.alpha. and
pre-immune serum. All buffered steps were performed using
Automation Buffer (Research Genetics) with Tween-20. Primary
antibodies were added to the wells for min at room temp. After
washes, the secondary antibodies were similarly incubated on the
cells. Subsequently, the presence of the primary antibodies were
detected using the ABC (Vector Labs) followed by DAB development
for 2 times 5 min each. Chambers were removed and then were
counterstained using hematoxylin then coverslipped.
Results
[0156] In situ PAR-1 and PAR-2 Protein Expression.
[0157] PAR-1 and PAR-2 proteins were localized in formalin-fixed,
paraffin embedded tissues. Normal (n=26), benign (n=14) and
malignant (n=46) human breast tissues and six non-breast carcinomas
(n=4-6 of each) were assayed simultaneously in a multi-tissue
format to eliminate potential staining artifacts such as
slide-to-slide and run-to-run variability. The relative amounts of
PAR-1 and PAR-2 immunoreactivity in the epithelial cells and the
surrounding sromal fibroblasts in the normal benign and malignant
breast tissues are presented in FIG. 1. Marginal increases of PAR-1
and PAR-2 expression were observed in the malignant cells as
compared to the normal and benign epithelial cells. Striking
changes in PAR-1 and PAR-2 expression were noted in the stromal
fibroblasts surrounding the malignant cells as compared to the
fibroblasts surrounding the normal and benign epithelial cells. No
PAR-1 or PAR-2 immunolabeling was observed in the stromal
fibroblasts of the benign (n=14) or normal (n=26) breast tissues.
In contrast, most malignant tissues had prominent moderate to
strong PAR-1 (n=39/46) and PAR-2 (n=37/46) labeling in the stromal
fibroblasts.
[0158] We applied additional immunohistochemical markers to further
characterize these tissues (FIG. 1). No immunolabeling was detected
using negative control antibodies in normal FIG. 2A), benign (FIG.
2B) and malignant (FIG. 2C) breast tissue. Smooth muscle actin
(SMA)-positive immunolabeling was localized in the myoepithelial
cells (large arrowheads) around the epithelial ducts and in the
vascular smooth muscle cells in the normal (FIG. 2D) and benign
fibroadenoma (FIG. 2E) breast tissues. SMA immunolabeling was
absent from stromal fibroblasts in the normal (FIG. 2D) and benign
(FIG. 2E) tissues, which were immunoreactive to the fibroblast
marker (data not presented). In the malignant breast carcinoma
tissues, SMA immunolabeling (small arrowheads) was prominent in the
stromal fibroblasts surrounding the tumor cells, in addition to the
vascular smooth muscle cells (FIG. 2F). Carcinoma cells (large
arrowheads) did not express SMA. Positive, nuclear Topo II.alpha.
immunolabeling (large arrowhead), a marker for proliferating cells
(D'Andrea and Cheung, 1994), was sparsely observed in normal breast
epithelial cells (FIG. 2G) and absent in stromal fibroblasts (small
arrowheads). Topo II.alpha. nuclear immunolabeling was observed in
the benign, fibroadenoma cells (large arrowheads), but was
similarly absent in the surrounding stromal fibroblasts (small
arrowheads) in FIG. 2H. In contrast, Topo II.alpha. nuclear
immunolabeling was observed in stromal fibroblasts (small
arrowheads) and tumor cells (large arrowheads) of the malignant
tissues (FIG. 21). Furthermore, the stromal fibroblasts surrounding
the malignant cells also expressed vimentin but did not express
desmin (data not presented).
[0159] Immunolocalization studies indicated that PAR-1 and PAR-2
were co-expressed in the different cell types in normal, benign and
malignant tissues. In normal breast tissues, immunolabeling (large
arrowheads) was confined to the normal breast ductal epithelial
cells and myoepethelial cells (FIGS. 2J and 2M). Immunolabeling
(large arrowheads) was also observed in the fibroadenoma cells
(FIGS. 2K and 2N). In both cases, normal and benign tissues,
surrounding stromal fibroblasts (small arrowheads) did not express
detectable PAR-1 or PAR-2. In the breast carcinoma tissues, PAR-1
and PAR-2 positive immunoreactivity was observed in many cell types
forming the TME such as in the malignant cells (large arrowheads)
and the stromal fibroblasts (small arrowheads) (FIGS. 2L and 2O).
Although not present in these photomicrographs, PAR-1 and PAR-2
immunolabeling was also observed in endothelial cells, vascular
smooth muscle cells as well as in the mast cells and macrophages.
The PAR-1 and PAR-2 mast cell labeling pattern was consistent with
our previous report (D'Andrea et al., 2000) and was similarly
localized to the plasma membrane and to the membranes of the
secretory vesicles. PAR-1 and PAR-2 positive macrophages were also
observed around these cancerous tissues. Labeling in the
macrophages was observed in or on the plasma membrane as well as
intracellularly. The identity of the mast cells and macrophages
were confirmed in these tissue sections using antibodies to mast
cell tryptase (MCT) and macrophages (CD68) (data not
presented).
[0160] PAR-1 and PAR-2 Expression in other Tumors.
[0161] Other human non-breast malignant tumors demonstrated similar
PAR-1 and PAR-2 expression in the tumor cells (large arrowheads),
stromal fibroblasts (small arrowheads), mast cells and macrophages
(arrows), as well as in the endothelial and vascular smooth muscle
cells (FIG. 3). FIG. 3 shows PAR-1 (FIGS. 3A, 3C and 3E) and PAR-2
(FIGS. 3B, 3D and 3F) immunolabeling in tissues representing a
gastric carcinoma (n=4, FIGS. 3A and 3B), an undifferentiated
carcinoma (n=4, FIGS. 3C and 3D) and a lung adenocarcinoma (n==4,
FIGS. 3E and 3F). PAR-1 and PAR-2 immunoreactivity was similarly
present in heptacarcinomas (n=6), thyroid carcinomas (n=4) and
ovarian carcinomas (n=6) (data not shown). Positive PAR-1 and PAR-2
immunoreactivity was also observed on surrounding endothelial and
vascular smooth muscle cells, as well as in the stromal fibroblasts
in contrast to the absence of PAR-1 and PAR-2 immunoreactivity in
the stromal fibroblasts on the normal tissue counterparts (data not
presented).
[0162] In situ PAR-1 and PAR-2 mRNA Expression.
[0163] The PAR-1 and PAR-2 protein expression correlated well with
their respective mRNA levels in the same tissues as determined by
in situ hybridization. The localization patterns of PAR-1 (FIG. 4A)
and PAR-2 (FIG. 4B) mRNA were observed in human breast carcinoma
tissues (n=48). FIG. 4A shows the intracellular localization of
PAR-1 mRNA in the malignant tumor cells (large arrowheads) and in
the surrounding stromal fibroblasts (small arrowheads). PAR-1 mRNA
was not present in the stromal cells of the normal (n=26) and
benign (n=10) breast tissues (data not presented). Similar
localization patterns were observed for PAR-2 in the same breast
carcinoma tissues as shown in FIG. 4B, and PAR-2 mRNA was also not
present in the stromal cells of the normal and benign breast
tissues (data not presented). When the same tissues were probed
with the lac Z biotinylated mRNA probe (negative control), no
observable labeling was observed in tumor cells (large arrowheads)
or stromal fibroblasts (small arrowheads) (FIG. 4C). As a positive
control probe, cells also expressed GAPDH mRNA (FIG. 4D).
[0164] In addition, PAR-1 and PAR-2 mRNA was similarly observed in
the following tissues: gastric carcinomas (n=4), undifferentiated
carcinomas (n=4), lung adenocarcinomas (n=4), heptacarcinomas
(n=6), thyroid carcinomas (n=4) and ovarian carcinomas (n=6) (data
not shown).
[0165] PAR-1 and PAR-2 Expression is Associated with Proliferating
Cells.
[0166] The results of double immunohistochemical labeling using
antibodies to PAR-1 or PAR-2 with antibodies to Topo II.alpha.,
demonstated that all proliferating cells expressed PAR-1 and PAR-2
immunolabeling. FIGS. 5A and 5B show representative examples of
these results. Co-localization (arrowheads of PAR-1 or PAR-2 with
Topo II.alpha. positive immunolabeling was observed in both
malignant cells and stromal fibroblasts.
[0167] In Vitro PAR-1 and PAR-2 Expression.
[0168] Our IHC and ISH results indicated that PAR-1 and PAR-2
expression was induced in stromal fibroblasts during the transition
to a myofibroblast phenotype. We utilized ICC to determine if this
transition could be mimicked in vitro. Hyper-confluent, fibroblast
cultures (quiescent conditions) were compared to 1) sub-confluent
cultures with visible mitotics (proliferative conditions), and 2)
confluent cultures subjected to a mechanical scrape and allowed to
recover for 5 days without changing the media (wound conditions).
FIG. 6 (FIGS. 6A-6C) shows the lack of observable immunolabeling
using negative control antibodies in all three tissue culture
conditions. SMA immunolabeling was present in the proliferating
cells (FIG. 6E, arrowheads) and in the cells migrating over the
scraped area (FIG. 6F, arrowheads), but was absent in the confluent
cultured cells (FIG. 6D) suggesting that the confluent conditions
produced quiescent, differentiated cells, which were not
myofibroblasts. Immunoreactivity to the proliferation marker, Topo
II.alpha., was present in the nuclei of the proliferating cells in
the sub-confluent cultures (FIG. 6H, arrowheads) and in the cells
migrating over the scraped area in the wounded cultures (FIG. 6I,
arrowheads) but was absent in the cell nuclei of the quiescent
cells (FIG. 6G) further confirming the quiescent, non-proliferating
status of these differentiated fibroblasts when grown to
confluency.
[0169] Positive intracellular and membrane PAR-1 and PAR-2
immunoreactivity (arrowheads) was not observed in the quiescent,
non-proliferating cells (FIGS. 6J and 6M, respectively). However,
positive PAR-1 and PAR-2 immunolabeling (arrowheads) was observed
in the proliferating cells in the sub-confluent (FIGS. 6K and 6N,
respectively) and wounded (FIGS. 6L and 6O, respectively)
conditions.
Discussion
[0170] One of the most important features in cell metastasis is the
ability of tumor cells to produce extracellular conditions
conducive to their growth through degradation and subsequent
remodeling of the extracellular matrix. This study provides
evidence for the presence of PAR-1 and PAR-2 not only on the
malignant carcinoma cells, but also on the cell types forming the
tumor microenvironment (TME), including mast cells, vascular
endothelial cells, smooth muscle cells, macrophages and most
interestingly, on reactive stromal fibroblasts. By expressing PAR-1
and PAR-2, these cell types may act as proteolytic sensors to
extracellular thrombin and trypsin, initiating a cellular response
to tissue damage incurred through the processes of cell metastasis.
The remodeling of the tumor stroma provides a permissive
environment for tumor metastasis, relying on the interplay of all
the cells within the TME.
[0171] PAR-1 and PAR-2 expression has previously been shown on
endothelial cells, vascular smooth muscle cells and mast cells. It
is therefore not surprising to find similar results for these cell
types within the TME. Activation of either PAR-1 or PAR-2 on these
cells results in characteristic events associated with inflammatory
responses such as generation of cytokines, expression of adhesion
molecules and increased vascular permeability. However, little is
known about the presence of PAR-1 and PAR-2 on macrophages. It has
been reported that macrophages can secrete thrombin (Lindahl et
al., 1989) and that thrombin has been localized in pulmonary
alveolar macrophages (Zacharski et al., 1995), suggesting an
association between macrophages and thrombin. The presence of PAR-1
and PAR-2 on human macrophages in malignant tumors in situ has not
been reported previously, although PAR-2 immunoreactivity has been
reported on macrophage-like cells in the adventitia of the mouse
isolated ureter (Webber et al., 1999). Here, we show that
macrophages express both PAR-1 and PAR-2. PAR-1 and PAR-2
activation may provide a stimulus for macrophages to proliferate,
migrate and/or phagocytize degraded stromal proteins, in addition
to synthesizing and secreting thrombin and growth factors into the
TME.
[0172] The most striking observation from our study is the presence
of PAR-1 and PAR-2 on the stromal fibroblasts surrounding the
metastatic tumor cells but not on the stromal fibroblasts
surrounding the benign, non-metastatic or normal epithelial cells.
The exact origin of the PAR-1 and PAR-2 expressing stromal
fibroblasts is unclear, i.e. local dedifferentiated stromal
fibroblasts, vascular smooth muscle cells or migrating
undifferentiated stem cells such as pericytes (Ronnov-Jessen et
al., 1995). In breast cancer, it has been shown that primarily
fibroblasts convert to the myofibroblast phenotype when exposed to
tumor cells; vascular smooth muscle cells and pericytes can also
differentiate to myofibroblasts, but to a lesser extent
(Ronnov-Jessen et al., 1995). The stromal fibroblasts associated
with metastatic tumors in our study were characterized by the
positive expression of smooth muscle actin (SMA), prolyl
4-hydroxylase (a fibroblast marker), Topo II.alpha. (a
proliferation marker) and vimentin, as well as the absence of the
vascular markers desmin and CD3 1, confirming the fibroblastic
nature of these cells (Ronnov-Jessen et al., 1995), (Webber et al.,
1999; Chiavegato et al., 1993; Sappino et al., 1990; Babij et al.,
1993).
[0173] Reactive stromal myofibroblasts are frequently associated
with cancers of epithelial origin, a process known as desmoplasia
(Schmitt-Graff et al., 1994). The induction of this phenotype has
not been well characterized, however in vitro studies have
indicated that diffusible signals, such as TGF-.beta., generated
from primed or initiated carcinoma cells are involved
(Ronnov-Jessen et al., 1995; Olumi et al., 1999; Noel, 1998;
Lieubeau et al., 1994). The stromal myofibroblasts, in turn,
influence the invasive and metastatic potential of carcinoma cells
by an unidentified mechanism (Gregoire et al., 1995) once the
carcinoma cells invade the basement membrane surrounding the
epithelial cells. Elaboration of matrix degrading proteases,
deposition of new extracellular matrix proteins to facilitate tumor
cell adhesion, cell motility and cell proliferation (Gregoire and
Lieubeau, 1995; Chambers et al., 1998), and release of cytokines
and growth factors by these myofibroblasts, emphasizes the
importance of this phenotypic change to the invasiveness of the
tumor. PAR-1 and PAR-2 activation results in many of these
biochemical events indicating that they are likely participants in
the balance of tumor containment and/or metastasis (Hung et al.,
1992; Vouret-Craviari et al, 1992; Dawes et al., 1993; Grubber et
al., 1997; Akers et al., 2000; Vrana et al., 1996). Moreover, the
expression of tissue factor, an essential co-factor for plasma
coagulation factor VII/VIIa, was reported to be consistently
observed in stromal cells of invasive breast carcinomas but not in
the benign breast tumors (Vrana et al., 1996). The increased
presence of tissue factor/factor VIIa within the TME, which in turn
can generate thrombin via the extrinsic coagulation pathway on
fibroblasts, parallels our observation of increased PAR-1
expression.
[0174] Benign proliferative disorders are characterized by a
continuous basement membrane separating the epithelium from the
stroma, similar to the normal tissue organization (Liotta et al.,
1991) It is possible that the presence of a continuous basement
membrane may actually quarantine any tumor-derived thrombin or
trypsin from the stromal fibroblasts. Thus, the actions of thrombin
and trypsin within the TME may be accentuated through up-regulation
of PAR-1 and PAR-2 in the stromal fibroblasts as they
de-differentiate (ie. SMA-negative to SMA-positive). The activation
of PAR-1 and PAR-2 on tumor cells contributes to migration by
increasing their adhesive properties as well releasing urokinase,
both of which are early changes during the initiation of metastasis
(Nierodzik et al., 1996; Nguyen et al., 1998; Evans et al.,
1997).
[0175] We were able to mimic our in situ observations in vitro
using cultured human dermal fibroblasts. Quiescent, SMA-negative,
non-proliferating (Topo II.alpha.-negative) cell cultures did not
express detectable PAR-1 or PAR-2, similar to those of the stromal
fibroblasts in normal and benign human tissues in situ. Most
notably, we were able to mimic the transformation of PAR-1 and
PAR-2-negative to PAR-1 and PAR-2-positive fibroblasts in vitro,
after the quiescent cells were subjected to scrape wounding
indicating that tissue damage relays a signal for PAR
induction.
[0176] In summary, this is the first in situ histological
comparative report describing the presence of PAR-1 and PAR-2
protein and mRNA in human malignant tumor cells as well as in local
mast cells, macrophages, endothelium and vascular smooth muscle
cells of the TME. More importantly, we observed PAR-1 and PAR-2
immunolabeling in the stromal fibroblasts immediately surrounding
the malignant cells which was absent in the surrounding stromal
fibroblasts of the normal and benign breast epithelial cells. The
presence of both PARs and their activating proteases within the TME
suggests an autocrine and/or paracrine cascade in the processes of
cellular metastasis, perhaps as natural mechanisms of tissue
injury. It will be important to investigate if there is a
correlation between the relative amounts of PAR-1 or PAR-2 in the
tumors cells and in the stromal fibroblasts with tumor grade, and
to expand our investigations into the expression of all of the
members of the PARs into other pathological tissues. Since the
degree of tumor cell malignancy has been classified by the amounts
of secreted thrombin or trypsin, theoretically, the amounts of
PAR-1 and PAR-2 in the TME cells may also be a valid predictor of
metastatic activity, thereby acquiring diagnostic and prognostic
value. More importantly, these data suggest attractive targets for
therapeutic approaches, whereby PAR-1 and PAR-2 antagonists and
anti-thrombin and anti-tryptase agents may be directed to disrupt
some of the processes of cell metastasis.
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