Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Nitrogen containing other than solely as a nitrogen in an...
Reexamination Certificate
2001-06-21
2004-01-13
Reamer, James H. (Department: 1614)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Nitrogen containing other than solely as a nitrogen in an...
Reexamination Certificate
active
06677377
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to methods for diagnosing and treating conditions involving matriptase activity, particularly cancer. In particular, the invention is based on the elucidation of matriptase activity and involvement in the development of precancerous and cancerous conditions. More particularly, the invention relates to the design of bioassay testing methods for cancer diagnosis based on the detection of matriptase activity, identification of compounds capable of inhibiting matriptase activity and design of cancer therapy protocols based on the inhibition of matriptase employing small molecule inhibitors.
2. Discussion of the Related Art
Serine Proteases and Other Cancer Related Proteases. Elevated proteolytic activity has been implicated in neoplastic progression. While the exact role(s) of proteolytic enzymes in the progression of tumor remains unclear, it seems that proteases may be involved in almost every step of the development and spread of cancer. A widely proposed view is that proteases contribute to the degradation of extracellular matrix (ECM) and to tissue remodeling, and are necessary for cancer progression and metastasis. A wide array of ECM-degrading proteases has been discovered, the expression of some of which correlates with tumor progression. These include matrix metalloproteases (MMPS) family, plasmin/urokinase type plasminogen activator system and lysosomal proteases cathepsins D and B reviewed by Mignatti et al.,
Physiol. Rev.
73: 161-95 (1993). The plasmin/urokinase type plasminogen activator system is composed of plasmin, the major ECM-degrading protease; the plasminogen activator, uPA; the plasmin inhibitor &agr;2-anti-plasmin, the plasminogen activator inhibitors PAI-1 and PAI-2; and the cell membrane receptor for uPA (uPAR) (Andreasen et al.,
Int. J. Cancer
72: 1-22 (1997)).
The MMPs are a family of zinc-dependent enzymes with characteristic structures and catalytic properties. The plasmin/urokinase type plasminogen activator system and the 72-kDa gelatinase (MMP-2)/membrane-type MMP system have received the most attention for their potential roles in the process of progression of breast cancer and other carcinomas. However, both systems appear to require indirect mechanisms to recruit and activate the major ECM-degrading proteases on the surface of cancer cells. For example, uPA is produced in vivo (Nielson et al.,
Lab. Invest.
74: 168-77 (1996); Pyke et al.,
Cancer Res.
53: 1911-15 (1993); Polette et al.,
Virchows Arch.
424: 641-45 (1994); and Okada et al.,
Proc. Natl. Acad. Sci. USA
92: 2730-34 (1995)) in human breast carcinomas by myofibroblasts adjacent to cancer cells and must diffuse to the cancer cells for receptor-mediated activation and presentation on the surfaces of cancer cells. However, the uPA receptor (uPAR) is detected in macrophages that infiltrate tumor foci in ductal breast cancer. Somewhat analogously, the majority of the MMP family members, such as 72-kDa/Gelatinase A (MMP-2) (Lin et al.,
J. Biol. Chem.
272: 9147-52 (1997)), stromelysin-3 (MMP-11) (Matsudaira,
J. Biol. Chem.
262: 10035-38 (1987)), MTMMP (MMP-14), are expressed by fibroblastic cells of tumor stroma, or surrounding noncancerous tissues, or both. Indirect mechanisms of activation and recruitment of Gelatinase A in the close vicinity of the surfaces of cancer cells have been proposed, such that an unidentified cancer cell-derived membrane receptor(s) of Gelatinase A could serve as membrane anchor for Gelatinase A; cleaved MT-MMP from stroma cells could then diffuse to the surfaces of cancer cells to activate Gelatinase A. Matrilysin (MMP-7; Pump-1) appears to be the only MMP which is found predominantly in the epithelial cells.
The stromal origins of these well-characterized extracellular matrix-degrading proteases may suggest that cancer progression is an event which either depends entirely upon stromal-epithelial cooperation or which is controlled by some other unknown epithelial-derived proteases. Search for these epithelial-derived proteolytic systems that may interact with plasmin/urokinase type plasminogen activator system and/or with MMP family could provide a missing link in the understanding of malignant progression.
Matriptase was initially identified from T-47D human breast cancer cells as a major gelatinase with a migration rate between those of Gelatinase A (72-kDa, MMP-2) and Gelatinase B (92-kDa, MMP-9). It has been proposed to play a role in the metastatic invasiveness of breast cancer. (See U.S. Pat. No. 5,482,848, the contents of which are incorporated herein by reference in their entirety.) The primary cleavage specificity of matriptase was identified to be arginine and lysine residues, similar to the majority of serine proteases, including trypsin and plasmin. In addition, matriptase, like trypsin, exhibits broad spectrum cleavage activity, and such activity is likely to contribute to its gelatinolytic activity. The trypsin-like activity of matriptase distinguishes it from Gelatinases A and B, which may cleave gelatin at glycine residues, the most abundant (almost one third) of amino acid residues in gelatin.
Kunitz-Type Serine Protease Inhibitors. Hepatocyte growth factor (HGF) activator inhibitor-1 (HAI-1) is a Kunitz-type serine protease inhibitor which is able to inhibit HGF activator, a blood coagulation factor XII-like serine protease. The mature form of this protease inhibitor has 478 amino acid residues, with a calculated molecular mass of 53,319. A putative transmembrane domain is located at its carboxyl terminus. HAI-1 contains two Kunitz domains (domain I spans residues 246-306; domain II spans residues 371 to 431) separated by a LDL receptor domain (residues 315 to 360). The presumed P1 residue of active-site cleft is likely to be arginine-260 in Kunitz domain I and lysine 385 in domain II by alignment with bovine pancreatic trypsin inhibitor (BPTI, aprotinin) and with other Kunitz-type inhibitors. Thus, HAI-1 has specificity against trypsin-type proteases. Although HGF activator is exclusively expressed by liver cells, HAI-1 was originally purified from the conditioned media of carcinoma cells as a 40-kDa fragment doublet, rather than the proposed, mature, membrane-bound, 53-kDa form (Shimomura et al.,
J. Biol. Chem.
272: 6370-76 (1997)).
The protein inhibitors of serine proteases can be classified into at least 10 families, according to various schemes. Among them, serpins, such as maspin (Sheng et al.,
Proc. Natl. Acad. Sci. USA
93: 11669-74 (1996)) and Kunitz-type inhibitors, such as urinary trypsin inhibitor (Kobayashi et al.,
Cancer Res.
54: 844-49 (1994)) have been previously implicated in suppression of cancer progression. The Kunitz-type inhibitors form very tight, but reversible complexes with their target serine proteases. The reactive sites of these inhibitors are rigid and can simulate optimal protease substrates. The interaction between a serine protease and a Kunitz-type inhibitor depends on complementary, large surface areas of contact between the protease and inhibitor. The inhibitory activity of the recovered Kunitz-type inhibitor from protease complexes can always be reconstituted. The Kunitz-type inhibitors may be cleaved by cognate proteases, but such cleavage is not essential for their inhibitory activity. In contrast, serpin-type inhibitors also form tight, stable complexes with proteases; in most of cases these complexes are even more stable than those containing Kunitz-type inhibitors. Cleavage of serpins by proteases is necessary for their inhibition, and serpins are always recovered in a cleaved, inactive form from protease reactions.
Thus, serpins are considered to be suicide substrate inhibitors, and their inhibitory activity will be lost after encounters with proteases. The suicide nature of serpin inhibitors may result in regulation of proteolytic activity in vivo by direct removal of unwanted proteases via other membrane-bound endocytic receptors (in the case of uPA inhibitors). However, the Kunitz type inhibitors ma
Dickson Robert B.
Enyedy Istvan
Lee Sheau-Ling
Lin Chen-Yong
Wang Shaomeng
Georgetown University School of Medicine
Pillsbury & Winthrop LLP
Reamer James H.
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