Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-04-05
2003-02-04
Pak, Michael (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C514S002600, C514S011400
Reexamination Certificate
active
06514710
ABSTRACT:
TECHNICAL FIELD
The present invention relates to certain cyclic peptides that bind to the cell surface receptor for urokinase-type plasminogen activator and, thus, are capable of inhibiting the binding of urokinase-type plasminogen activator to this cell surface receptor. The invention also relates to pharmaceutical compositions containing these peptides and to the use of these peptides to inhibit the binding of urokinase-type plasminogen activator to its cell surface receptor. Effects derived from the inhibition of binding of urokinase-type plasminogen activator to its cell surface receptor include the inhibition of proteolysis; the inhibition of programmed gene expression; the inhibition of cell motility, migration, and morphogenesis; the inhibition of the activation of certain pro-growth factors to the active form of the growth factor; the inhibition of angiogenesis; the inhibition of tumor metastasis; the inhibition of retinal neovascularization in the treatment of certain forms of blindness; and the inhibition of tissue remodeling as a treatment for inflammatory diseases, such as arthritis. The peptides of the invention that are capable of carrying a suitable radioactive, fluorogenic, chromogenic, or chemical label can also be used to quantitate urokinase-type plasminogen activator receptor levels in tissue samples and can be used, therefore, as diagnostic and prognostic tools in all diseases where the receptor plays a pathological role, including those mentioned above.
BACKGROUND OF THE INVENTION
Urokinase-type plasminogen activator (uPA) has been identified as the initiator of a major amplified cascade of extracellular proteolysis. 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. Danø et al.
Adv. Cancer Res
., 44:139-266 (1985). Cells express uPA as an inactive form, pro-uPA or single-chain uPA, which then binds to its receptor, uPAR. This binding event is necessary for activation to two-chain uPA. Ellis et al.,
J. Biol. Chem
., 264:2185-88 (1989). The amino acid sequence for human pro-uPA is as follows:
Ser Asn Glu Leu His Gln Val Pro Ser Asn Cys Asp Cys Leu Asn Gly
1 10
Gly Thr Cys Val Ser Asn Lys Tyr Phe Ser Asn Ile His Trp Cys Asn Cys
20 30
Pro Lys Lys Phe Gly Gly Gln His Cys Glu Ile
40
The sequence of amino acids of pro-uPA are represented above by their standard three-letter abbreviations as follows:
Amino Acid
Three-letter Symbol
Alanine
Ala
Arginine
Arg
Asparagine
Asn
Aspartic acid
Asp
Cysteine
Cys
Glutamine
Gln
Glutamic acid
Glu
Glycine
Gly
Histidine
His
Isoleucine
Ile
Leucine
Leu
Lysine
Lys
Methionine
Met
Phenylalanine
Phe
Proline
Pro
Serine
Ser
Threonine
Thr
Tryptophan
Trp
Tyrosine
Tyr
Valine
Val
The structure of pro-uPA is shown in FIG.
1
.
uPA is a three-domain protein comprising (1) an N-terminal epidermal growth factor-like domain, (2) a kringle domain, and (3) a C-terminal serine protease domain. The receptor for pro-uPA (uPAR) is a multi-domain protein anchored by a glycolipid to the cell membrane, thus ensuring that activation of uPA is a pericellular event. Behrendt . et al.,
Biol. Chem. Hoppe
-
Seyler
, 376.269-79 (1995). uPA activity is confined to the cell surface by plasminogen activator inhibitors (PAI-1 and PAI-2), which bind to and inactivate the 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. Enzymol
., 19:184-99 (1970). The product of uPA's action upon plasminogen, plasmin, 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 metaloproteases. 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 Matrisian,
Bioessays
, 14:455-63 (1992).
In tumor biology, the link between extracellular 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 pre-eminent angiogenic growth factors, basic fibroblast growth factor and vascular endothelial growth factor markedly up-regulate the production of uPA. (Montesano et al.,
Proc. Natl. Acad Sci. USA
, 83:7297-7301 (1986); Pepper et al.,
Biochem. Biophys. Res. Comm
., 181:902-906 (1991)) 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)). Thus uPA/uPAR has emerged as a new target for developing an anti-metastatic/anti-angiogenic therapy for cancer, where most studies have been conducted (Fazioli et al.,
Trends Pharmacological Sci
., 15:25-29 (1994).
However, the uPA/uPAR interaction goes far beyond localizing proteolysis at the cell surface. Independent of all proteolytic effects, 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-C1066 (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.
165:107-118 (1995)); and endothelial cell deformability and motility (Lu et al.,
FEBS Lett
. 380:21-24 (1996)). All of these phenomena are blocked by blocking the access of uPA to uPAR. An antagonist of uPAR that prevented the binding of uPA would thus interfere with proteolytic activity by preempting uPA activation and, further, would greatly diminish uPAR's capacity for signal transduction.
The anti-angiogenic effects accompanying uPAR antagonism (Min et al.,
Cancer Res
., 56:2428-33 (1996)) should allow a uPAR antagonist to play a role in other diseases characterized by inappropriate angiogenesis, e.g. ocular angiogenesis leading to blindness. Furthermore, it is likely that a uPAR antagonist would also play a therapeutic role in inflammatory diseases, for example, rheumatoid arthritis. (Ronday et al., Br. J. Rheum., 35:416-423 (1996).
One approach to drug therapy is to target uPA itself at its catalytic serine protease domain. Yang et al.,
Fibrinolysis
, 6 (Suppl. 1):31-34, (1992). Amiloride (Vassalli et al.,
FEBS Lett
., 214:187-191 (1987); and Kellen et al.,
Anticancer Res
. 8:1373-76 (1988)) and p-aminobenzamidine (Geratz et al.,
Thrombosis Res
. 24:73-83 (1981); and Billström et al.,
Int. J. Cancer
, 61:542-47 (1995)) are competitive inhibitors of this site and have anti-metastatic activity in vivo. Selective inhibition of uPA as compared with other serine proteases, was evident in phenylguanidines (Yang et al.,
J. Med Chem
., 33:2956-61 (1990)) and, even more so, in benzo[b]thiophene-2-carboxamidines (Bridges, Bioorganic & Medicinal Chemi
Haney David N.
Jones Terence R.
Varga Janos
Angstrom Pharmaceuticals, Inc.
Livnat Shmuel
Pak Michael
Venable
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