Method for reducing or preventing the establishment, growth or metastasis of cancer by administering indole 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.

Description

BACKGROUND OF THE INVENTION

[0003] 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).

[0004] 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). 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 Bemdt, 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;

[0006] 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.

[0007] 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 CD3ξ 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-β) 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 Th1cytokines 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-β 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-2Rα 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; Sheu et al., 2001). The presence of these enzymes in the TME should facilitate the colonization, growth, and potentially the metastasis of malignant cells.

[0008] 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

[0009] 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 of the following formula (I): 1

[0010] wherein:

[0011] A1 and A2 are each independently a D- or L-amino acid selected from the group consisting of alanine, β-alanine, arginine, homoarginine, cyclohexylalanine, citrulline, cysteine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), 2,4-diaminobutyric acid (optionally substituted with acyl, C1-C4 alkyl, aroyl, amidino, or MeC(NH)—), 2,3-diaminopropionic acid (optionally substituted with acyl, C1-C4 alkyl, aroyl, amidino, or MeC(NH)—), glutamine, glycine, indanylglycine, lysine (optionally substituted with acyl, C1-C4 alkyl, aroyl, MeC(NH)—), valine, methionine, proline, serine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), homoserine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), tetrahydroisoquinoline-3-COOH, threonine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), ornithine (optionally substituted with acyl, C1-C4 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-β-alanine, and heteroaryl-β-alanine wherein the substituents on the aromatic amino acid are independently selected from one or more of halogen, C1-C4 alkyl, C1-C4 alkoxy, hydroxy, C1-C4 alkoxycarbonyl, amino, amidino, guanidino, fluorinated C1-C4 alkyl, fluorinated C1-C4 alkoxy, C1-C4 alkylsulfonyl, C1-C4 alkylcarbonyl, cyano, aryl, heteroaryl, arC1-C4 alkyl, C2-C4 alkenyl, alkynyl, or nitro;

[0012] R1 is selected from amino, C1-C8 alkylamino, C1-C8 dialkylamino, arylamino, arC1-C8 alkylamino, C3-C8 cycloalkylamino, heteroalkylC1-C8 alkylamino, heteroalkylC1-C8 alkyl-N-methylamino, C1-C8 dialkylaminoC1-C8 alkylamino, -N(C1-C8alkyl)-C1-C8 alkyl-N(C-C8alkyl)2, N(C1-C8 alkyl)(C1-C8 alkenyl), -N(C1- C8alkyl)(C3-C8cycloalkyl), heteroalkyl or substituted heteroalkyl wherein the substituent on the heteroalkyl is selected from oxo, amino, C1-C8 alkoxyC1-C8 alkyl, C1-C8 alkylamino or C1-C8 dialkylamino;

[0013] R2 is selected from hydrogen, halogen, C1-C8 alkyl, C3-C8 cycloalkyl, C1-C8 alkenyl, C1-C8 alkynyl, arC1-C8 alkyl, aryl or heteroaryl;

[0014] R3 and R4 are each independently selected from hydrogen, C1-C8 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C8 alkyl, aryl, heteroalkyl, substituted heteroalkyl (wherein the substituent on the heteroalkyl is one or more substituents independently selected from C1-C8 alkoxycarbonyl, C1-C8 alkyl, or C1-C4 alkylcarbonyl), heteroalkylC1-C8 alkyl, indanyl, acetamidinoC1-C8 alkyl, aminoC1-C8 alkyl, C1-C8 alkylaminoC1-C8 alkyl, C1-C8 dialkylaminoC1-C8 alkyl, unsubstituted or substituted heteroarylC1-C8 alkyl, or unsubstituted or substituted arC1-C8 alkyl, wherein the substituent on the aralkyl or heteroarylalkyl group is one or more substituents independently selected from halogen, nitro, amino, C1-C8 alkyl, C1-C8 alkoxy, hydroxy, cyano, C1-C4 alkylcarbonyl, C1-C8 alkoxycarbonyl, hydroxyC1-C8 alkyl or aminosulfonyl; or

[0015] R3 and R4, 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 C1-C8 alkyl C1-C8 alkoxycarbonyl or C1-C4 alkylcarbonyl;

[0016] R5 is selected from unsubstituted or substituted aryl, arC1-C8 alkyl, C3-C8 cycloalkyl, or heteroaryl, where the substituents on the aryl, arC1-C8 alkyl, cycloalkyl or heteroaryl group are independently selected from one or more of halogen, nitro, amino, cyano, hydroxyalkyl, C1-C8 alkyl, C1-C8 alkoxy, hydroxy, C1-C 4 alkylcarbonyl, C1-C8 alkoxycarbonyl, fluorinated C1-C4 alkyl, fluorinated C1-C4 alkoxy or C1-C4 alkylsulfonyl;

[0017] R6 is selected from hydrogen or C1-C8 alkyl,

[0018] X is oxygen or sulfur;

[0019] m is an integer selected from 0, 1, 2 or 3;

[0020] n is an integer selected from 1 or 2; and

[0021] p is an integer selected from 0 or 1;

[0022] and pharmaceutically acceptable salts thereof and optionally a PAR-2 inhibitor to a patient with malignant cells.

[0023] 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 of the following formula (I): 2

[0024] wherein:

[0025] A1 and A2 are each independently a D- or L-amino acid selected from the group consisting of alanine, β-alanine, arginine, homoarginine, cyclohexylalanine, citrulline, cysteine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), 2,4-diaminobutyric acid (optionally substituted with acyl, C1-C4 alkyl, aroyl, amidino, or MeC(NH)—), 2,3-diaminopropionic acid (optionally substituted with acyl, C1-C4 alkyl, aroyl, amidino, or MeC(NH)—), glutamine, glycine, indanylglycine, lysine (optionally substituted with acyl, C1-C4 alkyl, aroyl, MeC(NH)—), valine, methionine, proline, serine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), homoserine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), tetrahydroisoquinoline-3-COOH, threonine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), ornithine (optionally substituted with acyl, C1-C4 alkyl, aroyl, MeC(N)—), and an unsubstituted or substituted aromatic amino acid selected from the group consisting of phenylalanine, heteroarylalanine, naphthylalanine, homophenylalanine, histidine, tryptophan, tyrosine, arylglycine, heteroarylglycine, aryl-β-alanine, and heteroaryl-β-alanine wherein the substituents on the aromatic amino acid are independently selected from one or more of halogen, C1-C4 alkyl, C1-C4 alkoxy, hydroxy, C1-C4 alkoxycarbonyl, amino, amidino, guanidino, fluorinated C1-C4 alkyl, fluorinated C1-C4 alkoxy, C1-C4 alkylsulfonyl, C1-C4 alkylcarbonyl, cyano, aryl, heteroaryl, arC1-C4 alkyl, C2-C4 alkenyl, alkynyl, or nitro;

[0026] R1 is selected from amino, C1-C8 alkylamino, C1-C8 dialkylamino, arylamino, arC1-C8 alkylamino, C3-C8 cycloalkylamino, heteroalkylC1-C8 alkylamino, heteroalkylC1-C8 alkyl-N-methylamino, C1-C8 dialkylaminoC1-C8 alkylamino, -N(C1-C8alkyl)-C1-C8 alkyl-N(C1-C8 alkyl)2, N(C1-C8 alkyl)(C1-C8 alkenyl), —N(C1-C8alkyl(C3-C8cycloalkyl), heteroalkyl or substituted heteroalkyl wherein the substituent on the heteroalkyl is selected from oxo, amino, C1-C8 alkoxyC1-C8 alkyl, C1-C8 alkylamino or C1-C8 dialkylamino;

[0027] R2 is selected from hydrogen, halogen, C1-C8 alkyl, C3-C8 cycloalkyl, C1-C8 alkenyl, C1-C8 alkynyl, arC1-C8 alkyl, aryl or heteroaryl;

[0028] R3 and R4 are each independently selected from hydrogen, C1-C8 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C8 alkyl, aryl, heteroalkyl, substituted heteroalkyl (wherein the substituent on the heteroalkyl is one or more substituents independently selected from C1-C8 alkoxycarbonyl, C1-C8 alkyl, or C1-C4 alkylcarbonyl), heteroalkylC1-C8 alkyl, indanyl, acetamidinoC1-C8 alkyl, aminoC1-C8 alkyl, C1-C8 alkylaminoC1-C8 alkyl, C1-C8 dialkylaminoC 1-C8 alkyl, unsubstituted or substituted heteroarylC1-C8 alkyl, or unsubstituted or substituted arC1-C8 alkyl, wherein the substituent on the aralkyl or heteroarylalkyl group is one or more substituents independently selected from halogen, nitro, amino, C1-C8 alkyl, C1-C8 alkoxy, hydroxy, cyano, C1-C4 alkylcarbonyl, C1-C8 alkoxycarbonyl, hydroxyC1-C8 alkyl or aminosulfonyl; or

[0029] R3 and R4, 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 C1-C8 alkyl C1-C8 alkoxycarbonyl or C1-C4 alkylcarbonyl;

[0030] R5 is selected from unsubstituted or substituted aryl, arC1-C8 alkyl, C3-C8 cycloalkyl, or heteroaryl, where the substituents on the aryl, arC1-C8 alkyl, cycloalkyl or heteroaryl group are independently selected from one or more of halogen, nitro, amino, cyano, hydroxyalkyl, C1-C8 alkyl, C1-C8 alkoxy, hydroxy, C1-C4 alkylcarbonyl, C1-C8 alkoxycarbonyl, fluorinated C1-C4 alkyl, fluorinated C1-C4 alkoxy or C1-C4 alkylsulfonyl;

[0031] R6 is selected from hydrogen or C1-C8 alkyl,

[0032] X is oxygen or sulfur;

[0033] m is an integer selected from 0, 1, 2 or 3;

[0034] n is an integer selected from 1 or 2; and

[0035] p is an integer selected from 0 or 1;

[0036] 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

[0037] 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 20x 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.

[0038] 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 IIα 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 900x.

[0039] 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.

[0040] Arrowheads indicate positive immunolabeling in tumor cells and arrows indicate immunolabeling in the stromal fibroblasts. The magnification was approximately 900x.

[0041] 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 900x.

[0042] 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 IIα (red) and PAR-2 (brown) and Topo IIα (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 900x.

[0043] 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 IIα (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 IIα-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 900x.

DETAILED DESCRIPTION OF THE INVENTION

[0044] 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.

[0045] 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

[0046] 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

[0047] 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.

[0048] Suitable PAR-1 antagonist include antibodies that block activation of PAR-1 receptor and compounds of the following general formula (I): 3

[0049] wherein:

[0050] A1 and A2 are each independently a D- or L-amino acid selected from the group consisting of alanine, β-alanine, arginine, homoarginine, cyclohexylalanine, citrulline, cysteine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), 2,4-diaminobutyric acid (optionally substituted with acyl, C1-C4 alkyl, aroyl, amidino, or MeC(NH)—), 2,3 diaminopropionic acid (optionally substituted with acyl, C1-C4 alkyl, aroyl, amidino, or MeC(NH)—), glutamine, glycine, indanylglycine, lysine (optionally substituted with acyl, C1-C4 alkyl, aroyl, MeC(NH)—), valine, methionine, proline, serine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), homoserine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), tetrahydroisoquinoline-3-COOH, threonine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), ornithine (optionally substituted with acyl, C1-C4 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-β-alanine, and heteroaryl-β-alanine wherein the substituents on the aromatic amino acid are independently selected from one or more of halogen, C1-C4 alkyl, C1-C4 alkoxy, hydroxy, C1-C4 alkoxycarbonyl, amino, amidino, guanidino, fluorinated C1-C4 alkyl, fluorinated C1-C4 alkoxy, C1-C4 alkylsulfonyl, C1-C4 alkylcarbonyl, cyano, aryl, heteroaryl, arC1-C4 alkyl, C2-C4 alkenyl, alkynyl, or nitro;

[0051] Preferably, A1 and A2 are each independently an L-amino acid selected from the group consisting of alanine, β-alanine, arginine, homoarginine, cyclohexylalanine, citrulline, cysteine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), 2,4-diaminobutyric acid (optionally substituted with acyl, C1-C4 alkyl, aroyl, amidino, or MeC(NH)—), 2,3 diaminopropionic acid (optionally substituted with acyl, C1-C4 alkyl, aroyl, amidino, or MeC(NH)—), glutamine, glycine, indanylglycine, lysine (optionally substituted with acyl, C1-C4 alkyl, aroyl, MeC(NH)—), valine, methionine, proline, serine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), homoserine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), tetrahydroisoquinoline-3-COOH, threonine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), ornithine (optionally substituted with acyl, C1-C4 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-β-alanine, and heteroaryl-β-alanine wherein the substituents on the aromatic amino acid are independently selected from one or more of halogen, C1-C4 alkyl, C1-C4 alkoxy, hydroxy, C1-C4 alkoxycarbonyl, amino, amidino, guanidino, fluorinated C1-C4 alkyl, fluorinated C1-C4 alkoxy, C1-C4 alkylsulfonyl, C1-C4 alkylcarbonyl, cyano, aryl, heteroaryl, arC1-C4 alkyl, C2-C4 alkenyl, alkynyl, or nitro;

[0052] R1 is selected from amino, C1-C8 alkylamino, C1-C8 dialkylamino, arylamino, arC1-C8 alkylamino, C3-C8 cycloalkylamino, heteroalkylC1-C8 alkylamino, heteroalkylC1-C8 alkyl-N-methylamino, C1-C8 dialkylaminoC1-C8 alkylamino, -N(C1-C8alkyl)-C1-C8 alkyl-N(C1-C8alkyl), N(C1-C8 alkenyl), -N(C1-C 8alkyl)(C3-C8cycloalkyl), heteroalkyl or substituted heteroalkyl wherein the substituent on the heteroalkyl is selected from oxo, amino, C1-C8 alkoxyC1-C8 alkyl, C1-C8 alkylamino or C1-C8 dialkylamino;

[0053] Preferably, R1 is selected from amino, C1-C6 alkylamino, C1-C6 dialkylamino, arylamino, arC1-C6 alkylamino, heteroalkylC1-C6 alkylamino, —N(C1-C6 alkyl)-C1-C6 alkyl-N(C1-C6 alkyl)2, heteroalkyl or substituted heteroalkyl wherein the substituent on the heteroalkyl is selected from oxo, amino, C1-C6 alkoxy C1-C6 alkyl, C1-C6 alkylamino or C1-C6 dialkylamino;

[0054] R2 is selected from hydrogen, halogen, C1-C8 alkyl, C3-C8 cycloalkyl, C1-C8 alkenyl, C1-C8 alkynyl, arC1-C8 alkyl, aryl or heteroaryl;

[0055] Preferably, R2 is selected from hydrogen, halogen or phenyl;

[0056] R3 and R4 are each independently selected from hydrogen, C1-C8 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C8 alkyl, aryl, heteroalkyl, substituted heteroalkyl (wherein the substituent on the heteroalkyl is one or more substituents independently selected from C1-C8 alkoxycarbonyl, C1-C8 alkyl, or C1-C4 alkylcarbonyl), heteroalkylC1-C8 alkyl, indanyl, acetamidinoC1-C8 alkyl, aminoC1-C8 alkyl, C1-C8 alkylaminoC1-C8 alkyl, C1-C8 dialkylaminoC1-C8 alkyl, unsubstituted or substituted heteroarylC1-C8 alkyl, or unsubstituted or substituted arC1-C8 alkyl, wherein the substituent on the aralkyl or heteroarylalkyl group is one or more substituents independently selected from halogen, nitro, amino, C1-C8 alkyl, C1-C8 alkoxy, hydroxy, cyano, C1-C4 alkylcarbonyl, C1-C8 alkoxycarbonyl, hydroxyC1-C8 alkyl or aminosulfonyl; or

[0057] R3 and R4, 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 C1-C8 alkyl C1-C8 alkoxycarbonyl or C1-C4 alkylcarbonyl;

[0058] Preferably, R3 is selected from hydrogen or C1-C6 alkyl;

[0059] R4 is selected from C1-C8 alkyl, C3-C6 cycloalkyl, C3-C6 cycloalkylC1-C6 cycloalkylC1-C6 alkyl, aryl, heteroarylC1-C6 alkyl, substituted heteroarylC1-C6 alkyl wherein the substituent is C1-C4 alkyl, heteroalkyl, heteroalkylC1-C6 alkyl, indanyl, acetamidinoC1-C6 alkyl, aminoC1-C6 alkyl, C1-C6 alkylaminoC1-C6 alkyl, C1-C6 dialkylaminoC1-C6 alkyl, arC1-C8 alkyl, substituted arC1-C8 alkyl wherein the substituent on the aralkyl group is one to five substituents independently selected from halogen, nitro, amino, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkoxycarbonyl, hydroxyalkyl or aminosulfonyl; or

[0060] R3 and R4, 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 C1-C6 alkyl;

[0061] R5 is selected from unsubstituted or substituted aryl, arC1-C8 alkyl, C3-C8 cycloalkyl, or heteroaryl, where the substituents on the aryl, arC1-C8 alkyl, cycloalkyl or heteroaryl group are independently selected from one or more of halogen, nitro, amino, cyano, hydroxyalkyl, C1-C8 alkyl, C1-C8 alkoxy, hydroxy, C1-C4 alkylcarbonyl, C1-C8 alkoxycarbonyl, fluorinated C1-C4 alkyl, fluorinated C1-C4 alkoxy or C1-C4 alkylsulfonyl;

[0062] Preferably, R5 is selected from unsubstituted or substituted aryl, arC1-C6 alkyl, C3-C6 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, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkoxycarbonyl, fluorinated C1-C4 alkyl, fluorinated C1-C4 alkoxy or C1-C4 alkylsulfonyl;

[0063] R6 is selected from hydrogen or C1-C8 alkyl; preferably, R6 is hydrogen;

[0064] X is oxygen or sulfur; preferably, X is oxygen;

[0065] m is an integer selected from 0, 1, 2 or 3;

[0066] n is an integer selected from 1 or 2;

[0067] p is an integer selected from 0 or 1; preferably, p is 1;

[0068] and pharmaceutically acceptable salts thereof.

[0069] In a preferred embodiment of the PAR-1 antagonist are of the formula (1), wherein:

[0070] A1 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, C1-C4 alkyl, C1-C4 alkoxy, hydroxy, C1-C4 alkoxycarbonyl, amino, amidino, guanidino, fluorinated C1-C4 alkyl, fluorinated C1-C4 alkoxy, C1-C4 alkylsulfonyl, C1-C4 alkylcarbonyl, cyano, aryl, heteroaryl, arC1-C4 alkyl, C2-C4 alkenyl, alkynyl, or nitro;

[0071] A2 is an L-amino acid selected from the group consisting of alanine, β-alanine, arginine, citrulline, cysteine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), 2,4-diaminobutyric acid (optionally substituted with acyl, C1-C4 alkyl, aroyl, amidino, or MeC(NH)—), 2,3-diaminopropionic acid (optionally substituted with acyl, C1-C4 alkyl, aroyl, amidino, or MeC(NH)—), glutamine, glycine, lysine (optionally substituted with acyl, C1-C4 alkyl, aroyl, MeC(NH)—), valine, methionine, serine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), homoserine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), threonine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), ornithine (optionally substituted with acyl, C1-C4 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, C1-C4 alkyl, C1-C4 alkoxy, hydroxy, C1-C4 alkoxycarbonyl, amino, amidino, guanidino, fluorinated C1-C4 alkyl, fluorinated C1-C4 alkoxy, C1-C4 alkylsulfonyl, C1-C4 alkylcarbonyl, cyano, aryl, heteroaryl, arC1-C4 alkyl, C2-C4 alkenyl, alkynyl, or nitro;

[0072] R2 is selected from hydrogen, chlorine or phenyl;

[0073] R3 is selected from hydrogen or C1-C4 alkyl;

[0074] m and n are both 1;

[0075] and all other variables are as defined previously;

[0076] and pharmaceutically acceptable salts thereof

[0077] A subclass the PAR-1 antagonist may also be of the formula: 4

[0078] A1 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, C1-C4 alkyl, C1-C4 alkoxy, hydroxy, C1-C4 alkoxycarbonyl, amino, amidino, guanidino, fluorinated C1-C4 alkyl, fluorinated C1-C4 alkoxy, C1-C4 alkylsulfonyl, C1-C4 alkylcarbonyl, cyano, aryl, heteroaryl, arC1-C4 alkyl, C2-C4 alkenyl, alkynyl, or nitro;

[0079] A2 is an L-amino acid selected from the group consisting of alanine, β-alanine, arginine, citrulline, cysteine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), 2,4-diaminobutyric acid (optionally substituted with acyl, C1-C4 alkyl, aroyl, amidino, or MeC(NH)—), 2,3-diaminopropionic acid (optionally substituted with acyl, C1-C4 alkyl, aroyl, amidino, or MeC(NH)—), glutamine, glycine, lysine (optionally substituted with acyl, C1-C4 alkyl, aroyl, MeC(NH)—), valine, methionine, serine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), homoserine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), threonine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), ornithine (optionally substituted with acyl, C1-C4 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, C1-C4 alkyl, C1-C4 alkoxy, hydroxy, C1-C4 alkoxycarbonyl, amino, amidino, guanidino, fluorinated C1-C4 alkyl, fluorinated C1-C4 alkoxy, C1-C4 alkylsulfonyl, C1-C4 alkylcarbonyl, cyano, aryl, heteroaryl, arC1-C4 alkyl, C2-C4 alkenyl, alkynyl, or nitro;

[0080] R1 is selected from diethylamino, di-(n-propyl)amino, 5

[0081] R3 is selected from hydrogen, methyl or ethyl;

[0082] R4 is selected from 2-indanyl, phenyl, cyclohexylmethyl, cyclopentyl, pyridylmethyl, furanylmethyl, 2-(4-methyl-furanyl)methyl, thienylmethyl, diphenylmethyl, 4-imidazolylethyl, 2-(4-N-methyl)imidazolylethyl, 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

[0083] R3 and R4, together with the nitrogen to which they are attached, alternatively form a heteroalkyl group selected from piperidinyl or 4-(N-methyl)piperazinyl;

[0084] R5 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;

[0085] Preferably, R5 is 2,6-dichlorophenyl or 2-methylphenyl; and all other variables are as defined previously; and pharmaceutically acceptable salts thereof.

[0086] In this subclass of PAR-1 antagonist it is preferable, that:

[0087] A1 is selected from 3,4-Difluorophenylalanine or 4-Chlorophenylalanine;

[0088] A2 is selected from 2,4-Diaminobutyric acid or 4-Pyridylalanine;

[0089] R3 is hydrogen;

[0090] R4 is selected from benzyl or 2-aminoethyl;

[0091] and all other variables are as defined previously; and pharmaceutically acceptable salts thereof.

[0092] In another aspect of the invention the PAR-1 antagonist maybe of the formula (II): 6

[0093] wherein:

[0094] A1 and A2 are each independently a D- or L-amino acid selected from the group consisting of alanine, β-alanine, arginine, homoarginine, cyclohexylalanine, citrulline, cysteine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), 2,4-diaminobutyric acid (optionally substituted with acyl, C1-C4 alkyl, aroyl, amidino, or MeC(NH)—), 2,3 diaminopropionic acid (optionally substituted with acyl, C1-C4 alkyl, aroyl, amidino, or MeC(NH)—), glutamine, glycine, indanylglycine, lysine (optionally substituted with acyl, C1-C4 alkyl, aroyl, MeC(NH)—), valine, methionine, proline, serine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), homoserine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), tetrahydroisoquinoline-3-COOH, threonine (optionally substituted with C1-C4 alkyl, aryl, or arC1-C4 alkyl), ornithine (optionally substituted with acyl, C1-C4 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-β-alanine, and heteroaryl-β-alanine, wherein the substituents on the aromatic amino acid are independently selected from one or more of halogen, C1-C4 alkyl, C1-C4 alkoxy, hydroxy, C1-C4 alkoxycarbonyl, amino, amidino, guanidino, fluorinated C1-C4 alkyl, fluorinated C1-C4 alkoxy, C1-C4 alkylsulfonyl, C1-C4 alkylcarbonyl, cyano, aryl, heteroaryl, arC1-C4 alkyl, C2-C4 alkenyl, alkynyl, or nitro;

[0095] R2 is selected from hydrogen, halogen, C1-C8 alkyl, C3-C8 cycloalkyl, C1-C8 alkenyl, C1-C8 alkynyl, arC1-C8 alkyl, aryl or heteroaryl;

[0096] R3 and R4 are each independently selected from hydrogen, C1-C8 alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkylC1-C8 alkyl, aryl, heteroalkyl, substituted heteroalkyl (wherein the substituent on the heteroalkyl is one or more substituents independently selected from C1-C8 alkoxycarbonyl, C1-C8 alkyl, or C1-C4 alkylcarbonyl), heteroalkylC1-C8 alkyl, indanyl, acetamidinoC1-C8 alkyl, aminoC1-C8 alkyl, C1-C8 alkylaminoC1-C8 alkyl, C1-C8 dialkylaminoC1-C8 alkyl, unsubstituted or substituted heteroarylC1-C8 alkyl, or unsubstituted or substituted arC1-C8 alkyl, wherein the substituent on the aralkyl or heteroarylalkyl group is one or more substituents independently selected from halogen, nitro, amino, C1-C8 alkyl, C1-C8 alkoxy, hydroxy, cyano, C1-C4 alkylcarbonyl, C1-C8 alkoxycarbonyl, hydroxy C1-C8 alkyl or aminosulfonyl; or

[0097] R3 and R4, 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 selected from C1-C8 alkyl C1-C8 alkoxycarbonyl or C1-C4 alkylcarbonyl;

[0098] R5 is selected from unsubstituted or substituted aryl, arC1-C8 alkyl, C3-C8 cycloalkyl or heteroaryl, where the substituents on the aryl, arC1-C8 alkyl, cycloalkyl or heteroaryl group are independently selected from one or more of halogen, nitro, amino, cyano, hydroxyalkyl, C1-C8 alkyl, C1-C8 alkoxy, hydroxy, C1-C4 alkylcarbonyl, C1-C8 alkoxycarbonyl, fluorinated C1-C4 alkyl, fluorinated C1-C4 alkoxy or C1-C4 alkylsulfonyl; and,

[0099] X is oxygen or sulfur; and salts thereof.

[0100] In the compounds of formula (I), the amino acid residues comprising the A1 and A2 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 46, where A1 is 3,4-difluorophenylalanine and A2 is Arg, the N-terminus of the 3,4-difluorophenylalanine (A1) is attached to the carbonylgroup and the carboxy-terminus of the 3,4-difluorophenylalanine (A1) is attached to the N-terminus of the A2 substituent (Arg), similarly, the N-terminus of the Arg (A2) is attached to the carboxy-terminus of the A1 substituent and the carboxy-terminus of the Arg (A2) is attached to the N-R3R4 group.

[0101] 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.

[0102] 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 “phenylC1-C6 alkylamidoC1-C6alkyl” substituent refers to a group of the formula: 7

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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-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.

[0107] 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.

[0108] 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 quatemized. 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.

[0109] Preferred heteroalkyl groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, azetidinyl and tetrahydrothiazolyl.

[0110] 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 which 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.

[0111] Preferred heteroaryl groups include pyridyl, pyrrolyl, pyrazinyl, thiadiazolyl, pyrazolyl, thienyl, triazolyl and quinolinyl.

[0112] 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-NH2). 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]2).

[0113] 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.

[0114] The term “oxo” refers to the group =O.

[0115] The term “carbonyl” refers to the group C(O).

[0116] The term “halogen” shall include iodine, bromine, chlorine and fluorine.

[0117] The term “N(CH2)4” as used herein (e.g., in the Tables), refers to a pyrrolidinyl group having the structure: 8

[0118] Similarly, “N(CH2)5”, “N(CH2)3”, “NCH2S(CH2)2”, and “N(CH2)2O(CH2)2” refer to: 9

[0119] respectively.

[0120] Similarly “C6H11” and “C5H9” (or “c-C6H11” and “c-C5H9”) refer to cyclohexyl and cyclopentyl groups, respectively, and the term “D-Pro-ψ-CH2OMe” refers to a group of the formula: 10

[0121] 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., C1-C6) 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.

[0122] 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 know in the art as well as those methods set forth herein.

[0123] 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.

[0124] Amino acid abbreviations are defined below:

Ala     Alanine
β-Ala   beta-Alanine
Arg     Arginine
hArg    Homoarginine
Cha     Cyclohexylalanine
Cit     Citrulline
Cys     Cysteine
Dbu     2,4-Diaminobutyric acid
Dpr     Diaminopropionic acid
Gln     Glutamine
Gly     Glycine
His     Histidine
Lys     Lysine
Met     Methionine
Nal     Naphthylalanine
Orn     Ornithine
Phe     Phenylalanine
hPhe    Homophenylalanine
Pro     Proline
Pyr-Ala Pyridylalanine
Ser     Serine
hSer    Homoserine
Tic     Tetrahydroisoquinoline-3-COOH
Tyr     Tyrosine
Val     Valine

[0125] Particularly preferred compounds of the present invention are shown in Table 1 through Table 6, following; the amino acids bear the “L” absolute configuration unless denoted otherwise.

TABLE 1
6-Ureidoindoles
11
Cpd					ES/MS
No.	R1	R5	A1	A2—NR3R4	(MH+)
1	N(CH2)4	4-F-Ph	Tyr(Me)	Arg-NHBn	790
2	N(CH2)4	4-Me-Ph	Tyr(Me)	Arg-NHBn	786
3	NH c-C5H9	4-F-Ph	Tyr(Me)	Arg-NHBn	804
4	N(CH2)4	4-F-Ph	Tyr(Me)	Arg-NH(CH2)3Ph	818
5	MEt2	4-F-Ph	Tyr(Me)	Arg-NH(CH2)3Ph	820
6	N(CH2)4	3-F-Ph	Tyr(Me)	Arg-NHBn	790
7	N(CH2)4	2-F-Ph	Tyr(Me)	Arg-NHBn	790
8	N(CH2)5	4-F-Ph	Tyr(Me)	Arg-NHCH2C6H11	810
9	N(CH2)4	3-Me-Ph	Tyr(Me)	Arg-NHCH2C6H11	792
10	N(CH2)5	4-CO2Me-Ph	Tyr(Me)	Arg-NHCH2C6H11	850
11	N(CH2)4	4-OCF3-Ph	Tyr(Me)	Arg-NHCH2C6H11	862
12	N(CH2)4	4-SO2Me-Ph	Tyr(Me)	Arg-NHBn	850
13	N(CH2)4	4-CN-Ph	Tyr(Me)	Arg-NHBn	797
14	N(CH2)4	2,6-DiCl-Ph	Tyr(Me)	Arg-NHBn	840
15	N(CH2)4	2-Naphthalene	Tyr(Me)	Arg-NHBn	822
16	N(CH2)4	3-OMe-Ph	Tyr(Me)	Arg-NHBn	802
17	N(CH2)4	2-Me-Ph	Tyr(Me)	Arg-NHBn	786
18	N(CH2)4	4-CF3-Ph	Tyr(Me)	Arg-NHBn	840
19	N(CH2)4	4-Cl-Ph	Tyr(Me)	Arg-NHBn	806
20	N(CH2)4	4-OMe-Ph	Tyr(Me)	Arg-NHBn	802
21	N(CH2)4	3,4-diF-Ph	Tyr(Me)	Arg-NHBn	808
22	N(CH2)4	Ph	Tyr(Me)	Arg-NHBn	772
23	N(CH2)4	4-I-Ph	Tyr(Me)	Arg-NHBn	898
24	N(CH2)4	4-F-Ph	4-Pyridyl-Ala	Arg-NHBn	761
25	N(CH2)4	4-F-Ph	Cha	Arg-NHBn	766
26	N(CH2)4	4-F-Ph	4-F-Phe	Arg-NHBn	778
27	N(CH2)4	4-F-Ph	homoPhe	Arg-NHBn	774
28	N(CH2)4	4-F-Ph	2-Thienyl-Ala	Arg-NHBn	766
29	N(CH2)4	2-Me-Ph	Phenyl-Gly	Arg-NHBn	742
30	N(CH2)4	4-F-Ph	Tic	Arg-NHBn	772
31	N(CH2)4	2-Me-Ph	3-Pyridyl-Ala	Arg-NHBn	757
32	Nme(CH2)3Nme2	4-F-Ph	Tyr(Me)	Arg-NHBn	835
33	NH(CH2)3—N(CH2)4	4-F-Ph	Tyr(Me)	Arg-NHBn	847
34	N(CH2)4	3-Me-Ph	Tyr(Allyl)	Arg-NHBn	812
35	NEt2	2-Me-Ph	4-NH2-Phe	Arg-NHBn	773
36	N(CH2)4	2-Me-Ph	Tyr(Bn)	Arg-NHBn	862
37	N(CH2)4	2-Me-Ph	1-Naphthyl-Ala	Arg-NHBn	806
38	N(CH2)4	2,6-DiCl-Ph	2-Naphthyl-Ala	Arg-NHBn	860
39	N(CH2)4	2-Me-Ph	4-Ph-Phe	Arg-NHBn	832
40	N(CH2)2O(CH2)2	4-F-Ph	Tyr(Me)	Arg-NHBn	806
41	N-(n-Pr)2	2,6-DiCl-Ph	Tyr(Me)	Arg-NHBn	870
42	N(CH2)4	3-Me-Ph	Tyr(Et)	Arg-NHBn	800
43	NEt2	3-Me-Ph	Tyr(i-Pr)	Arg-NHBn	816
44	N(CH2)3	4-F-Ph	Tyr(Me)	Arg-NHBn	776
45	NH-c-C3H5	4-F-Ph	Tyr(Me)	Arg-NHBn	776
46	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Arg-NHBn	847
47	N(CH2)4	2,6-DiCl-Ph	3-F-Phe	Arg-NHBn	828
48	N(CH2)4	2-Me-Ph	4-Cl-Phe	Arg-NHBn	790
49	N(CH2)4	4-F-Ph	4-NO2-Phe	Arg-NHBn	805
50	N(CH2)4	2-Me-Ph	penta-F-Phe	Arg-NHBn	846
51	N(CH2)4	2,6-DiCl-Ph	2-F-Phe	Arg-NHBn	828
52	N(CH2)4	2,6-DiCl-Ph	4-CN-Phe	Arg-NHBn	835
53	N(CH2)4	2,6-DiCl-Ph	4-Thiazolyl-Ala	Arg-NHBn	817
54	N(CH2)4	2-Me-Ph	3-CF3-Phe	Arg-NHBn	824
55	N(CH2)4	2-Cl-Ph	3,4-DiF-Phe	Arg-NHBn	812
56	N(CH2)4	2,6-DiCl-Ph	DiPh-Ala	Arg-NHBn	886
57	N(CH2)4	2-Me-Ph	3,4-DiF-Phe	Arg-NH(CH2)2Ph	806
58	N-(n-Bu)2	2-Me-Ph	3,5-DiF-Phe	Arg-NHBn	850
59	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Arg-NH-4-F-Bn	864
60	N(CH2)4	4-F-Ph	3,4-DiF-Phe	Arg-NH—CHPh2	872
61	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Arg-NH-4-MeO-Bn	876
62	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Arg-NHCH2-4-pyridyl	847
63	N(CH2)5	2,6-DiCl-Ph	4-Br-Phe	Arg-NHBn	902
64	N(CH2)4	2-Me-Ph	3,4-DiF-Phe	Arg-NH—(CH2)2-2-Cl-Ph	840
65	N(CH2)4	2-Me-Ph	3,4-DiF-Phe	Arg-NH—(CH2)2-2-MeO-Ph	836
66	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Orn-NHBn	804
67	N(CH2)5	2,6-DiCl-Ph	3,4-DiF-Phe	Lys(iPr)-NHBn	874
68	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Lys(3-Pyridyl-CO)-NHBn	923
69	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	D-Arg-NHBn	846
70	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Lys-NHBn	818
71	N(CH2)4	2,6-DiCl-Phe	3,4-DiF-Phe	Lys(Ac)-NHBn	860
72	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	His-NHBn	827
73	N(CH2)5	4-F-Ph	3,4-DiF-Phe	Arg-NH—(CH2)7Me	832
74	N(CH2)4	c-C6H11	3,4-DiF-Phe	Arg-NHBn	784
75	NEt2	(CH2)2-Ph	3,4-DiF-Phe	Arg-NHBn	808
76	NEt2	CH2-4-F-Ph	3,4-DiF-Phe	Arg-NHBn	812
77	N(CH2)4	2,6-DiCl-Ph	3-F-Phe	Cit-NHBn	829
78	N(CH2)4	2,6-DiCl-Ph	4-Cl-Phe	Dbu-NHBn	788
79	N(CH2)4	2,6-DiCl-Ph	4-Cl-Phe	Dpr-NHBn	774
80	N(CH2)4	2,6-DiCl-Ph	Arg	3,4-DiF-Phe-NHBn	846
81	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Arg-NH—(CH2)5—NH2	841
82	N(CH2)4	4-F-Ph	3,4-DiF-Phe	Arg-NHCH2-2-furanyl	786
83	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Arg-NH—CH2-[R-CH(Me)Ph]	874
84	N(CH2)4	2,6-DiCl-Ph	D-3,4-DiF-Phe	Arg-NHBn	846
85	N(CH2)5	2,6-DiCl-Ph	4-I-Phe	Arg-NHBn	950
86	N(CH2)5	2,6-DiCl-Ph	2-Cl-Phe	Arg-NHBn	858
87	D-Pro-ψ-CH2OMe	2,6-DiCl-Ph	4-Cl-Phe	Arg-NHBn	888
88	N(CH2)2CO(CH2)2	2,6-DiCl-Ph	3-F-Phe	Arg-NHBn	856
89	N(CH2)4	4-F-Ph	3-F-Phe	Arg-NH-2-naphthyl	828
90	NCH2CH(NMe2)(CH2)2	2-Me-Ph	D-3,4-DiF-Phe	Arg-NHBn	835
91	D-Pro-ψ-CH2OMe	2,6-DiCl-4-Pyr	3-F-Phe	Arg-NHBn	873
92	N(CH2)4	2,6-DiCl-Ph	3-F-Phe	Arg-NHCH2-4-NH2SO2-Ph	907
93	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Orn-OCH2CHCH2	755
94	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Orn(Ac)-NHBn	846
95	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Orn[(MeC(NH)]-NHBn	845
96	N(CH2)4	2,6-DiCl-Ph	3-F-Phe	Orn-NH-2-indanyl	812
97	N(Me)CH2CH═CH2	2,6-DiCl-Ph	3-F-Phe	Orn-NH-2-indanyl	812
98	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Orn-NHCH2-4-pyridyl	805
99	N(CH2)4	2,6-DiCl-Ph	4-Cl-Phe	Orn-NHCH2-2-Cl-Ph	836
100	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Orn(Me2)-NHBn	832
101	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Dpr-NHBn	776
102	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Dbu-NHBn	790
103	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Gln-NHBn	818
104	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Met-NHBn	821
105	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	4-Pyridyl-Ala-NHBn	838
106	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	4-NH2-Phe-NHBn	852
107	N(CH2)4	2,6-DiCl-Ph	Pro	Arg-NHBn	760
108	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	β-Ala-Arg-NHBn	917
109	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Orn-NH(CH2)2-4-pyridyl	819
110	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Orn-N(Et)-CH2-4-pyridyl	833
111	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Orn-N(Me)-CH2Ph	818
112	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Orn-NH-3,4-DiCl-Bn	872
113	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Orn-NH—CH2-2-thienyl	810
114	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Orn-4-N-Me-piperazinyl	797
115	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Orn-piperidinyl	782
116	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Orn-NHCH2-3-pyridyl	805
117	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Orn-NHCH2-2-pyridyl	805
118	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Dbu[(MeN(NH)]-NHBn	831
119	N(Me)-c-C5H9	2,6-DiCl-Ph	4-Cl-Phe	Orn-NH—(CH2)2-2-Cl-Ph	878
120	NH(CH2)2—NMe2	2,6-DiCl-Ph	3,4-DiF-Phe	Orn-NH-2-indanyl	861
121	NH(CH2)3—NMe2	2,6-DiCl-Ph	3,4-DiF-Phe	Orn-NH-2-indane	875
122	N(Me)-CH2-2-	2,6-DiCl-Ph	4-Cl-Phe	Orn-NH-2-indane	874
	dioxolanyl
123	N(CH2)4	2,6-DiCl-Ph	4-Cl-Phe	Orn-NH(CH2)2-N-morpholinyl	839
124	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Orn-NH(CH2)2-4-(N-Me)imidazolyl	822
125	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	OrnNH-c-C5H9	782
126	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Orn-NH(CH2)2-NMe2	785
127	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Orn-NH-Ph	790
128	N(CH2)4	2,6-DiCl-Ph	hPhe	Dbu-NHBn	768
129	N(CH2)4	2,6-DiCl-Ph	2-NaI	Dbu-NHBn	804
130	N(CH2)4	2,6-DiCl-Ph	3-Pyridyl-Ala	Dbu-NHBn	755
131	N(CH2)4	2,6-DiCl-Ph	2-Thienyl-Ala	Dbu-NHBn	760
132	N(CH2)4	2,6-DiCl-Ph	4-Thiazolyl-Ala	Dbu-NHBn	761
133	N(CH2)4	2,6-DiCl-Ph	4-Cl-Phe	Phe-NH(CH2)2—NH2	788
134	N(CH2)4	2,6-DiCl-Ph	4-Cl-Phe	His-NH(CH2)2NH2	778
135	N(CH2)4	2,6-DiCl-Ph	4-Cl-Phe	4-Pyr-Ala-NH(CH2)2NH2	789
136	N(CH2)4	2,6-DiCl-Ph	4-Cl-Phe	2-Thienyl-Ala-NH(CH2)2NH2	794
137	N(CH2)4	2,6-DiCl-Ph	4-Cl-Phe	4-Thiazolyl-AlaNH(CH2)2NH2	795
138	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	3-F-Phe-NH(CH2)2NH2	808
139	N(CH2)4	2,6-DiCl-Ph	4-Cl-Phe	4-Pyr-AlaNH(CH2)2NHC(NH)Me	830
140	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Dbu[C(NH)Me]-	825
				NH(CH2)2NHC(NH)Me
141	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Dbu-NH(CH2)2-N-pyrrolidinyl	797
142	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Dbu-NH(CH2)2-4-imidazolyl	794
143	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Met-NH(CH2)2NH2	774
144	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Dbu-4-NH—N—CO2Et-piperidinyl	855
145	N(CH2)4	2,6-DiCl-Ph	4-Cl-Phe	4-Pyr-Ala-NH(CH2)3NH2	803
146	N(CH2)4	2,6-DiCl-Ph	4-Cl-Phe	β-Ala-NH(CH2)2NH2	712
147	N(CH2)4	2,6-DiCl-Ph	3-F-Phe	Gly-NH(CH2)2NH2	682
148	N(CH2)4	2,6-DiCl-Ph	4-Cl-Phe	Val-NH(CH2)2NH2	740
149	N(CH2)4	2,6-DiCl-Ph	4-Cl-Phe	Cys(Et)-NH(CH2)2NH2	772
150	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Ser-NH(CH2)2NH2	730
151	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Cys(Tr)-NH(CH2)2NH2	989
152	N(CH2)4	2,6-DiCl-Ph	D-4-Cl-Phe	D-2Thienyl-Ala-NH(CH2)2NH2	779
153	N(CH2)4	3-Me-Ph	3,4-DiF-Phe	2-Thienyl-Ala-NH(CH2)3NH2	756
154	NCH2S(CH2)2	2,6-DiCl-Ph	3,4-DiF-Phe	Dbu-NHBn	808
155	N(CH2)4	2,6-DiCl-Ph	Cha	Dbu-NHBn	760
156	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	hSer(Me)-NH(CH2)2NH2	758
157	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Dbu-NH-[R-CH(Me)Ph]	803
158	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Dbu-NH-4-NO2-Bn	834
159	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Dbu-NHCH2-2-(4-Me-furanyl)	793
160	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Dbu-NH-[S-CH(CO2Me)Ph]	847
161	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Dbu-NH-[S-CH(CH2OH)Ph]	819

[0126]

TABLE 2
5-Ureidoindoles
12
Cpd					ES/MS
No.	R1	R5	A1	A2—NR3R4	(MH+)
162	N(CH3)2	4-F-Ph	Tyr(Me)	Arg-NHBn	804
163	NCH2SCH2CH2	4-F-Ph	Tyr(Me)	Arg-NHBn	808
164	N(CH2)4	4-F-Ph	3,4-DiF-Phe	Dbu-NHBn	740

[0127]

TABLE 3
4-Ureidoindoles
13
Cpd					ES/MS
No.	R1	R5	A1	A2—NR3R4	(MH+)
165	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Dbu-NHBn	790
166	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Dbu-NH(CH2)2—N(CH2)4	797

[0128]

TABLE 4
7-Ureidoindoles
14
Cpd					ES/MS
No	R1	R5	A1	A2—NR3R4	(MH+)
167	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Dbu-NHBn	790
168	N(CH2)4	2-Me-Ph	3,4-DiF-Phe	Dbu-NHBn	736
169	N(CH2)4	2-Me-Ph	3,4-DiF-Phe	Dbu-NH(CH2)4	743
170	N(CH2)4	2,6-DiCl-Ph	3,4-DiF-Phe	Dbu-NH(CH2)4	797

[0129]

TABLE 5
2-Substituted 6-Ureidoindoles
15
					ES/MS
Cpd No	R2	R5	A1	A2—NR3R4	(MH+)
171	Ph	4-F-Ph	3,4-DiF-Phe	Arg-NH-4-F-Bn	890
172	Cl	2-Me-Ph	3,4-DiF-Phe	Arg-NHBn	826
173	Cl	2-Me-Ph	3-F-Phe	Arg-NH-4-F-Bn	826

[0130]

TABLE 6
6-Amidoindoles
16
				ES/MS
Cpd No	R5	A1	A2—NR3R4	(MH+)
174	4-Me-Ph	Tyr(Me)	Arg-NHBn	771
175	2,6-DiCl-Ph	3,4-DiF-Phe	4-Pyr-Ala-NH(CH2)2NH2	776
176	2,6-DiCl-Ph	D-3,4-DiF-Phe	D-4-Pyr-Ala-NH(CH2)2NH2	776
177	2,6-DiCl-Ph	4-Cl-Phe	2-Thienyl-Ala-NH(CH2)2NH2	779
178	2,6-DiCl-Ph	D-4-Cl-Phe	D-2-Thienyl-Ala-NH(CH2)2NH2	779

[0131] 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.

[0132] 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. This 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.

[0133] 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.

[0134] 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

[0135] 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-18), 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 3,000,000 U/kg/day; more preferably in an amount of from about 3,000 to about 1,000,000 U/kg/day; and more preferably in an amount from about 5,000 to about 20,000 U/kg/day.

[0136] 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

[0137] 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.

[0138] 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 0-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. (incorporated by reference herein); angiostatin and endostatin, described in U.S. Pat. Nos. 5,290,807, 5,639,725 and 5,733,876 to O'Reilly (incorporated by reference herein); thalidomide, as described in U.S. Pat. Nos. 5,629,327 and 5,712,291 to D'Amato (incorporated by reference herein); 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. (incorporated by reference herein), and the metalloproteinase inhibitors described in U.S. Pat. No. 5,713,491 to Murphy, et al. (incorporated by reference herein). 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 (incorporated by reference herein).

[0139] 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

[0140] 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.

[0141] 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.

[0142] 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.

[0143] 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

[0144] The antagonists of the present invention may be prepared via either solution-phase or solid-phase methods. All compounds presented in Table 1 through Table 4 may be prepared by a convergent solution-phase synthesis as described in the general Scheme AAGeneric. A typical example was represented by the synthesis of Compound 46 (Scheme AA). Alternatively, compounds in Tables 1 through Table 4 can also be prepared via solid-phase approaches as represented by the synthesis of Compound 102 and Compound 135 (Schemes AB and AC). The antagonists in

[0145] Table 5 and Table 6 can be prepared by using the methods as described in Schemes AD and AE.

[0146] The appropriately nitro substituted indole AAG1 (Scheme AAGeneric) was alkylated with a substituted aralkyl or heteroaryl alkyl halide and a base such as cesium or potassium carbonate in a dipolar aprotic solvent such as DMF to give an intermediate, which was reduced in a classical manner with for example iron and acetic acid or with a newer method such as dimethyl hydrazine and iron to give AAG2.

[0147] An Fmoc protected amino-acid (A2), AAG3 (Scheme AAGeneric), was coupled to amine R3R4NH 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, which was Fmoc deprotected with a dialkylamine in a dipolar aprotic solvent—such as diethylamine in acetonitrile. This amine was coupled to the second Fmoc protected amino-acid (A1) 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, which was deprotected as above with a dialkylamine in a dipolar aprotic solvent such as acetonitrile to afford the dipeptide amine AAG4. Amine AAG2 was treated with a phosgene equivalent such as 4-nitrophenyl chloroformate, phosgene or “COCl2,” phenyl chloroformate, triphosgene or “(CCl3O)2CO,” carbonyldiimidazole, diethyl carbonate or diphenyl carbonate and a base such as diisopropylethylamine in a solvent such as dichloromethane to which was then added the dipeptide amine AAG4 to give the urea AAG5. The indole intermediate AAG5 was combined with formaldehyde, an amine such as pyrrolidine and an acid such as acetic acid, either neat or diluted with another solvent such as 1,4-dioxane or tetrahydrofuran. Upon work-up and deprotection with an acid, if necessary such as trifluoroacetic acid, the target product AAG6 was obtained. 17

[0148] Scheme AA presents a typical example of the convergent solution-phase synthesis of Compound 46. 18

[0149] Scheme AB presents the solid-phase approach to producing the antagonist Compound 102. N-α-Fmoc-N-γ-Boc-2,4-diaminobutyric acid was coupled with benzyl amine in the presence of DCC and HOBt. The resulting benzylamide was treated with TFA in DCM to give AB2, which was then loaded onto 2-Cl-trityl-Cl resin in the presence of DIEA to afford AB3. Deprotection of the Fmoc group in AB3 with piperidine was followed by coupling with Fmoc-3,4-diF-Phe-OH in the presence of HBTU and HOBt. The resulting coupled product was deprotected again with piperidine to afford the resin-bound dipeptide AB4. Urea formation between AB4 and amino-indole AA2b was accomplished by using 4-nitrophenylchloroformate to provide AB5. Mannich reaction of AB5 with pyrrolidine and formaldehyde followed by resin cleavage with TFA afforded the crude product Compound 102 with >95% purity, which was further purified by reverse-phase HPLC. 19

[0150] Similarly, Scheme AC represents another efficient solid-phase approach to the antagonists having an amine group at carboxy-terminus of the A2.

[0151] Antagonists having an acetamidine group (e.g., Compounds 95, 118 and 139, as in Table 1) may be prepared by treating the corresponding amine with S-2-naphthylmethyl thioacetimidate hydrobromide. 20

[0152] Scheme AD presents the preparation of 2-substituted 6-ureidoindoles (as in Table 5) by the synthesis of antagonist Compound 171. Palladium-mediated heteroannulation of terminal alkynes can be used as a key step for the preparation of 2-substituted indole intermediates. For example, to prepare the 2-phenyl-6-ureidoindole Compound 171, commercially available AD1 was converted to methanesulfonamide AD2 and subjected to palladium-mediated heteroannulation with phenylacetylene to give 2-phenylindole AD3. Deprotection of methylsulfonyl group in AD3 with KOH/MeOH afforded AD4. N-Alkylation of AD4 with 4-F-Bn-Br was followed by nitro reduction using Me2NNH2/FeCl3 to afford intermediate AD5. Amino-indole AD5 was coupled with dipeptide AD6 (prepared as described in Scheme AAGeneric) in the presence of 4-nitrophenylchloroformate to give an urea, which was subjected to Mannich reaction followed by TFA cleavage to provide target 171.

[0153] By using the same method, 2-aryl, heteroaryl, alkyl, cycloalkyl, alkenyl, aralkyl-6-ureidoindoles can be prepared from the appropriate terminal alkynes.

[0154] To prepare the 2-halo-6-ureidoindoles (e.g., Compounds 172 and 173), AD2 was subjected to palladium-mediated heteroannulation with (trimethylsilyl)acetylene. The resulting 2-(trimethylsilyl)indole was converted to 2-haloindoles by treating with N-chlorosuccinimide, N-bromosuccinimide, or N-iodosuccinimide. The 2-haloindole intermediates can then be converted to the targets using the method described in Scheme AD. 21

[0155] Scheme AE presents the synthesis of 6-amidoindoles (as in Table 6). Commercially available AE1 was converted to intermediate AE2 via alkylation followed by Mannich reaction and hydrolysis of methyl ester. The acid AE2 was then converted to the targets by coupling with a resin-bound dipeptide amine (e.g., AE3, prepared using the method as described for AC4 in Scheme AC) followed by resin cleavage with TFA. The coupling reaction may also be done in solution phase. 22

[0156] The thioureidoindoles [when X is S, as in general formula (I)] may be prepared as described hereinafter. Aminoindole substrate is reacted with thiocarbonyldiimidazole in a chlorinated solvent and then the imidazole by-product filtered from the solution. The solution then can be concentrated to afford the N-imidazolyl-N′-aminoindolyl-thiourea. This intermediate is then reacted with resin-bound peptide amine in a polar, aprotic solvent with heating (from about 80 to about 100° C.) to afford the resin-bound N-peptido-N′-aminoindolyl-thiourea product. The product is then liberated from the resin and purified as previously mentioned.

[0157] Extending the carbon chain from n being 1 to n being 2 at the 3-position of the indole [see general formula (I)] may be achieved by treating the dimethylamino Mannich base (when n is 1, R1 is NMe2) with KCN followed by reducing the cyano group to an amine.

[0158] Where the processes for the preparation of the compounds described above 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.

[0159] 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.

[0160] The protecting groups may be removed at a convenient subsequent stage using methods known from the art.

[0161] To prepare the PAR-1 inhibitor (antagonist) 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 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 of 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.

[0162] The following non-limiting examples are provided to further illustrate the present invention.

EXAMPLE 1

[0163] 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

[0164] 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 μg/ml) raised against the C-terminus of the receptor (Smith-Swintosky et al. 1997), and polyclonal anti-human PAR-2C (1 μg/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.).

[0165] 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

[0166] Single immunohistochemical techniques were used to identify human mast cells in various normal human tissues using an antibody to mast cell tryptase (MCT).

[0167] 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.

[0168] 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.

[0169] 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), tonsi (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

[0170] 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.

[0171] 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.

[0172] 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 Ca2+ 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).

[0173] 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

[0174] 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.

[0175] Reagents

[0176] Primary antibodies used in these experiments include the following:

[0177] desmin (Dako, CA), endothelial cell (CD31) (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 IIα (Pharmingen, CA) (D'Andrea et al. 1994) and vimentin (Dako, CA).

[0178] 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′ 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).

[0179] 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% H2O2 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 dH2O, 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.

[0180] Analysis of PAR-1 and PAR-2 Immunoreactivity

[0181] 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 20x 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.

[0182] Double Immunohistochemistry

[0183] 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 IIα (Topo IIα) (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 IIα 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 IIα 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.

[0184] In Situ Hybridization

[0185] Slides were routinely dewaxed, rehydrated, placed in 3% H2O2 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° C. Sections were washed and then dehydrated in 100% alcohol for 1 min. Each probe was diluted to 1.0 μg/ml in commercially formulated hybridization buffer (Biomeda) and heated for 5 min at 103° 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° 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° C. for 2 h. After hybridization, they were then immediately placed into a low stringency wash (2×SSC) for 5 min at 42° C., followed by a high stringency wash (0.1×SSC) for 5 min at 42° 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 Permount (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 μg/μl, Boehinger Mannheim, City, State) for 2 hours at 42° C. before probe hybridization.

[0186] Cell Culture

[0187] 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 (5×104/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.

[0188] Immunocytochemistry

[0189] 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 IIα 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 30 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

[0190] In situ PAR-1 and PAR-2 Protein Expression

[0191] 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.

[0192] 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 IIα 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 IIα 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 IIα nuclear immunolabeling was observed in stromal fibroblasts (small arrowheads) and tumor cells (large arrowheads) of the malignant tissues (FIG. 2I). Furthermore, the stromal fibroblasts surrounding the malignant cells also expressed vimentin but did not express desmin (data not presented).

[0193] 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).

[0194] PAR-1 and PAR-2 Expression in other Tumors

[0195] 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).

[0196] In situ PAR-1 and PAR-2 mRNA Expression

[0197] 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).

[0198] 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).

[0199] PAR-1 and PAR-2 Expression is Associated with Proliferating Cells

[0200] The results of double immunohistochemical labeling using antibodies to PAR-1 or PAR-2 with antibodies to Topo IIα, demonstated that all proliferating cells expressed PAR-1 and PAR-2 immunolabeling. FIG. 5A and 5B show representative examples of these results. Co-localization (arrowheads of PAR-1 or PAR-2 with Topo IIα positive immunolabeling was observed in both malignant cells and stromal fibroblasts.

[0201] In Vitro PAR-1 and PAR-2 Expression

[0202] 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 IIα, 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.

[0203] 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

[0204] 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.

[0205] 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.

[0206] 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 IIα (a proliferation marker) and vimentin, as well as the absence of the vascular markers desmin and CD31, 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).

[0207] 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-β, 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.

[0208] 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).

[0209] We were able to mimic our in situ observations in vitro using cultured human dermal fibroblasts. Quiescent, SMA-negative, non-proliferating (Topo IIα-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.

[0210] 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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Claims

1. A method of modifying the tumor cell microenvironment 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 of the following formula (I):

2. The method of claim 1 wherein the PAR-1 inhibitor is a compounds of the formula:

3. The method of claim 1 wherein the PAR-1 inhibitor is administered with a therapeutically effective amount of at least one PAR-2 inhibitor.

4. The method of claim 2 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-α, INF-β, INF-γ, TNF and combinations thereof.

5. The method of claim 4 wherein additionally administered in a pharmaceutical effective amount is at least one conventional chemotherapy agent.

6. The method of claim 5 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.

7. The method of claim 2 wherein additional administered are T cells selected from the group consisting of activated T cells, activated NK cells and combinations thereof.

8. The method of claim 1 wherein the PAR-1 inhibitor is administered before surgery.

9. The method of claim 1 wherein the PAR-1 inhibitor is administered after surgery.

10. 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 of the following formula (I):

11. The method of claim 10 wherein additionally administered are cytokines to facilitate the development of a Th1 response.

12. The method of claim 11 wherein the cytokines are selected from the group consisting of IL-2, IL12, IL-18, INF-α, INF-β, INF-γ, TNF and combinations thereof.

13. The method of claim 10 wherein additionally administered are T cells selected from the group consisting of activated CTL cells, activated NK cells and combinations thereof.

14. The method of claim 10 wherein additionally administered are activated NK cells.

15. The method of claim 10 wherein additionally administered are activated CTL cells.

16. The method of claim 10 wherein the PAR-1 inhibitor is administered before surgery.

17. The method of claim 10 wherein the PAR-1 inhibitor is administered after surgery.