Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
2000-06-22
2003-11-18
Wehbe, Anne M. (Department: 1632)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C514S002600, C435S320100, C435S069100
Reexamination Certificate
active
06649597
ABSTRACT:
BACKGROUND OF INVENTION
Urokinase plasminogen activator (uPA) is expressed in all mammalian species. It is produced by many cultured cell types of neoplastic origin and has been found more abundantly in explants of tumor tissue than in the corresponding normal tissue. uPA and its receptor, urokinase plasminogen activator receptor (uPAR), have been identified in extracts from human lung, colon, endometrial, breast, prostate and renal carcinomas, human melanomas, murine mammary tumors, the murine Lewis lung tumor, in ascites from human peritoneal carcinomatosis and human fibroblasts (Stopelli et al,
Proc. Natl Acad. Sci. USA,
82:4939-43 (1985); Vassalli et al.,
J. Cell. Biol.
100:86-92(1985), Plow et al.,
J. Cell. Biol.
103:2411-2420 (1986), Boyd et al,
Cancer Res.,
48:3112-6 (1988); Nielsen et al.,
J. Biol. Chem.,
263:2358-2363 (1988); Bajpai and Baker,
Biochem. Biophys. Res. Commun.,
133:994-1000 (1985); Needham et al.,
Br. J. Cancer,
55:13-16 (1987)).
uPA has been identified as the initiator of a major amplified cascade of extracellular proteolysis and/or cell migration, presumably through a breakdown of the extracellular matrix, caused by plasmin together with other proteolytic enzymes. This cascade, when regulated, is vital to certain normal physiological processes but, when dysregulated, is strongly linked to pathological processes, such as cell invasion and metastasis in cancer. Dano et al.,
Adv. Cancer Res.
44:139-266 (1985). There have also been reports that uPA plays a role in (1) the degradative phase of inflammation, (2) the interference of lymphocyte-mediated cytotoxicity against a variety of cells, (3) angiogenesis, (4) endothelial cell migration which is important in tumor growth, and (5) in the cytotoxic effect of natural killer cells.
uPA is a multidomain serine protease comprising (1) an N-terminal epidermal growth factor-like domain, (2) a kringle domain, and (3) a C-terminal serine protease domain. The single chain pro-uPA is activated by plasmin, which cleaves the chain into the disulfide-linked two chain active form.
The cellular receptor for uPA is uPAR, which is a multi-domain protein that is anchored by a glycolipid to the cell membrane, thus ensuring that activation of uPA is a pericellular event. Behrendt et al.,
Biol. Chem.,
376:269-79 (1995). uPAR binds the active uPA, as well as pro-uPA and uPA bound to an inhibitor molecule DFP which binds to uPA's active site. While the receptor binding domain of uPA has been localized to amino acids in the N-terminal growth factor-like domain region, there are varying studies which have yielded differing results regarding the span of the receptor binding domain.
For example, Stopelli et al. (
Proc. Natl. Acad. Sci. USA,
82:4939-43 (1985)) first reported that the N-terminal fragment of uPA (amino acids 1-135) was sufficient for high affinity, sub-nanomolar binding to uPAR. Further work confined the uPAR binding domain to amino acids 1-48 (Robbiati et al.,
Fibrinolysis,
4:53-60 (1990)). Dano et al. showed that a region spanning amino acids 12-32 could block the binding of the full-length wild-type uPA to uPAR (published PCT application WO 90/12091). Other studies have shown that residues 20-30 confer the specificity of binding, but that residues 13-19 are also needed to attain the proper binding confirmation (Appella et al.,
J. Biol. Chem.,
262:4437-40 (1987). Correspondingly, studies have shown that residues 20-30 can inhibit the binding of full-length uPA to uPAR but that a longer peptide comprising residues 17-34 is significantly more potent, requiring 10-fold less to achieve the same result. Kobayashi et al.,
J. Cancer,
57:727-33 (1994). Quax et al. (
Arterioscler. Thromb. Vasc. Biol.,
18:693-701 (1998) have reported that the receptor binding domain of uPA is localized between amino acids 20-32. In addition, Jones et al., in U.S. Pat. No. 5,942,492 have shown that cyclic peptides comprising residues 20-30 are sufficient to bind uPAR and act as antagonists of binding of uPA to uPAR and that residues N-terminal to residue 20 and C-terminal to residue 30 are not essential for high affinity binding. In view of these studies, the precise location of the uPAR high specificity binding domain in uPA is unclear.
The activity of uPA, when bound to uPAR, is confined to the cell surface by plasminogen activator inhibitors (PAI-1 and PAI-2), which bind to and inactivate the uPAR bound uPA. This tight control of uPA activity is necessary because uPA acts upon a substrate, plasminogen, that is present at a high concentration in plasma. Robbins,
Meth. Ensemble.,
19:184-99 (1970). uPA's action on plasminogen produces plasmin which is a powerful broad spectrum protease that not only degrades extracellular matrix proteins directly, but also activates the latent forms of other proteases, including several metalloproteases. Werb et al.,
N. Eng. J. Med.,
296:1017-1023 (1977); Mignatti et al.,
Cell,
47:487-98 (1986); He et al.,
Proc. Natl. Acad. Sci. USA,
86:2632-36 (1989); and Martrisian,
Bioessays,
14:455-63 (1992).
In tumor biology, the link between extracelluar proteolysis and angiogenesis is clearly evident. Break-up and dissolution of existing extracellular matrix is necessary in order to create new space for blood vessels to grow into. The processes of proteolysis and angiogenesis are highly coordinated. For example, two angiogenic growth factors, basic fibroblast growth factor and vascular endothelial growth factor markedly up-regulate the production of uPA and the expression of uPAR by endothelial cells. Mignatti et al.
J. Cell. Biol.,
113:1193-1201 (1991); Mandriota et al,
J. Biol. Chem.,
270:9709-9716 (1995). Therefore, uPA and uPAR have emerged as a target for developing an anti-metastatic/anti-angiogenic therapy for cancer. Fazioli et al,
Trends Pharmacological Sci.,
15:25-29 (1994).
The uPA/uPAR interaction goes far beyond localizing proteolysis at the cell surface however. The mere occupation of uPAR by uPA induces, by indirect means, signal transduction events leading to one or more of the following effects: mitogenesis (Rabbani et al.,
J. Biol. Chem.,
267:14151-56 (1992)); expression of the c-fos gene (Dumler et al.,
FEBS Lett.,
322:37-40 (1994)); cysteine- and metalloprotease expression by macrophages (Rao et al,
J. Clin. Invest.,
96:465-74 (1995)); transfer of mechanical force leading to increased cytoskeletal stiffness (Wang et al.,
Am. J. Physiol.,
268:C1062-66 (1995)); endothelial cell migration (Odekon et al.,
J. Cellul. Physiol.,
150:258-63 (1992)); endothelial cell morphogenesis into tubular structures (Schnaper et al.,
J. Cellul. Physiol.,
165:101-118 (1995)); and endothelial cell deformability and motility (Lu et al.,
FEBS Lett.
380:21-24 (1996). All of these phenomenon are blocked by blocking the access of uPA to uPAR.
In addition to binding uPA, uPAR serves as a cellular adhesion receptor for vitronectin and as a signaling receptor. Wei et al.,
J. Biol. Chem.,
269:32380-88 (1994); Robinson,
Signal transduction via GPI
-
anchored membrane proteins. ADP ribosylation in animal tissue.
Plenum Press, NY (1997); Wei et al.,
J. Cell. Biol.,
144:1285-1294 (1999). uPAR also interacts with several cell surface proteins including integrins, low-density lipoprotein receptor-related peptide, very-low-density lipoprotein receptor, megalin and the mannose-6-phosphate/insulin-like growth factor-II receptor. Moestrup et al.,
J. Biol. Chem.,
268:16564-70 (1993); Heegaard et al,
J. Biol. Chem.,
270:20855-61 (1995); Czekay et al.,
Mol. Biol. Cell,
8:517-32 (1997).
uPAR is further involved in both clathrin-dependent and clathrin-independent endocytosis. Vilhardt et al.,
Mol. Biol. Cell,
10:179-195 (1999). Clatherin-dependent endocytosis of uPAR is believed to depend on binding of uPA:PAI-1 to uPAR and subsequent interaction with internalization receptors for the low-density lipoprotein receptor family, which are internalized through clathrin-coated pits. This interaction is inhibited by receptor-associated protein (RAP). In contrast, clathrin ind
Drapkin Paola T.
Welsh Michael J.
Baker & Botts L.L.P.
Li Janice
The University of Iowa Research Foundation
Wehbe Anne M.
LandOfFree
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