Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2000-04-06
2004-06-15
Lankford, Jr., Leon B. (Department: 1654)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C530S329000
Reexamination Certificate
active
06750201
ABSTRACT:
FIELD OF THE INVENTION
The field of the invention is regulation of cell adhesion and migration.
BACKGROUND OF THE INVENTION
Plasminogen activators convert the inactive zymogen plasminogen into the broad-spectrum proteolytic enzyme, plasmin (Higgins et al., 1990, Annu. Rev. Pharmacol. Toxicol. 30:91-121; Holden, 1990, Radiology 174:993-1001; Mayer, 1990, Clin. Biochem. 23:197-211). One type of plasminogen activator, designated urokinase-type plasminogen activator (uPA), is a component of the circulatory system and other fluid compartments of the mammalian body.
uPA is the principal cell-associated plasminogen activator and has been implicated in several biological processes including angiogenesis, organogenesis, ovulation, inflammation, cancer, tumor cell invasion and metastasis, atherosclerosis, and other biological and pathological processes characterized by cell migration through physiological barriers such as fibrin and basement membranes (Gyetko et al., 1994, J. Clin. Invest. 93:1380-1387; Gyetko et al., 1996, J. Clin. Invest. 97:1818-1826; Shapiro et al., 1997, Am. J. Pathol. 150:359-369; Dado et al., 1994, Fibrinolysis 8(Suppl. 1):189-203).
uPA is synthesized as a single chain zymogen, designated single chain uPA (scuPA), which exhibits little urokinase activity (Ellis et al., 1987, J. Biol. Chem. 262:14998-15003; Petersen et al., 1988, J. Biol. Chem. 263:11189-11195; Husain, 1991, Biochemistry 30:5707-5805; Colleen et al., 1986, J. Biol. Chem. 261:1259-1266). Activation of scuPA occurs by enzymatic cleavage of scuPA, yielding two-chain uPA (tcuPA). Physiological formation of tcuPA from scuPA is catalyzed primarily by plasmin (Robbins et al., 1967, J. Biol. Chem. 242:2333-2342). scuPA may also be activated by binding of scuPA to the cell-surface receptor, uPAR. In the case of scuPA binding to uPAR, scuPA remains a single chain molecule, but is active (Higazi et al., 1995, J. Biol. Chem. 270:17375-17380).
The activity of uPA is regulated, in part, by plasminogen activator inhibitor-1 (PAI-1), which is a member of the serine protease inhibitor (SERPIN) family of proteins (Kruithof, 1988, Enzyme 40:113-121; Potempa et al., 1994, J. Biol. Chem. 269:15957-15960; Lijnen et al., 1994, Eur. J. Biochem. 224:567-574). PAI-1 is thought to be the most relevant inhibitor of uPA activity in the fluid phase, due to its high second order rate constant of inhibition, 1.7×10
−8
M
−1
.s
−1
, which is higher than any other protease inhibitor(Hekman et al., 1988, Arch. Biochem. Biophys. 262:199-210).
A soluble recombinant form of uPAR, designated suPAR, is known and differs from uPAR by lacking the portion of uPAR that links the receptor to the cell surface. suPAR possesses the same properties as uPAR with respect to binding and activating scuPA and promoting the adhesivity of the uPA-uPAR complex (Higazi et al., 1995, J. Biol. Chem. 270:17375-17380; Higazi et al., 1996, Blood 87:3545-3549).
Binding of scuPA to uPAR enhances urokinase activity of scuPA (Higazi et al., 1995, J. Biol. Chem. 270:17375-17380). Formation of the scuPA-uPAR complex also dampens the capacity of PAI-1 to inhibit scuPA activity, relative to the capacity of PAI-1 to inhibit tcuPA activity (Higazi et al., 1996, Blood 87:3545-3549). Formation of a complex between scuPA and uPAR also alters the regulation of scuPA enzymatic activity by peptide substrates of plasmin and promotes binding of scuPA to vitronectin (Higazi et al., 1996, Thromb. Res. 84:243-252; Higazi et al., 1996, Blood 88:542-551; Wei et al., 1994, J. Biol. Chem. 269:32380-32388; Wei et al., 1996, Science 273:1551-1555; Deng et al., 1996, J. Cell Biol. 134:1563-1571; Stefansson et al., 1996, Nature 383:441-443; O'Reilly et al., 1996, Nature Med. 2:689-692).
A region of uPA, comprising the protein sequence RHRGGS (SEQ ID NO:1) at amino acid positions 179-184, is required for inhibition of uPA activity by PAI-1 (Madison et al., 1990, J. Biol. Chem. 265:21423-21426). Conservation of this sequence among mammalian uPA proteins has been demonstrated (Adams et al., 1981, J. Biol. Chem. 266:8476-8482). Working with a different plasminogen activator protein, namely tissue-type plasminogen activator (tPA), Madison et al. have identified a region of PAI-1 which is involved in inhibition of tPA by PAI-1 (1990, J. Biol. Chem. 265:21423-21426). This region of PAI-1 comprises the sequence RMAPEEIIMDR (SEQ ID NO:2) at amino acids 346-356. It has been postulated that electrostatic interactions between this region of PAI-1 and tPA play a role in stabilizing a tPA-PAI-1 complex. Similarly, it has been postulated that electrostatic interactions between a region of PAI-1 and uPA may contribute to formation of a PAI-1-uPA complex. It has been observed, however, that the scuPA-uPAR complex is less susceptible to inhibition by PAI-1 (Higazi et al., 1996, Blood 87:3545-3549) than is tcuPA or uPAR-bound tcuPA (Higazi et al., 1996, Blood 87:3545-3549; Ellis et al., 1990, J. Biol. Chem. 265:9904-9908).
In addition to inhibiting urokinase activity of uPA, PAI-1 also promotes the internalization and lysosomal degradation of uPA, which involves the &agr;
2
-macroglobulin receptor/low density lipoprotein-related receptor protein (&agr;
2
MR/LRP; Nykjaer et al., 1994, J. Biol. Chem. 269:25668-25676). The complex formed between PAI-1 and tcuPA binds to &agr;
2
MR/LRP with considerably higher affinity than does either component alone. Although it has been demonstrated that the increased affinity of the complex results from an independent contribution of epitopes present in each ligand, a possible conformation-altering effect of PAI-1 upon uPA has not been excluded (Nykjaer et al., 1994, J. Biol. Chem. 269:25668-25676).
When scuPA is bound to uPAR, scuPA is protected from inactivation by PAI-1. Furthermore, binding of scuPA to uPAR inhibits binding of scuPA to &agr;
2
MR/LRP and internalization of scuPA caused by such binding (Nykjaer et al., 1994, J. Biol. Chem. 269:25668-25676; Higazi et al., 1996, Blood 87:3545-3549). Two mechanisms have been postulated for the reduced affinity of uPAR-bound scuPA for &agr;
2
MR/LRP. Nykjaer et al. (supra) proposed that the site at which scuPA contacts &agr;
2
MR/LRP is shielded by uPAR. An alternative mechanism is that binding of scuPA to uPAR induces a conformational change that both promotes scuPA binding to integrin ligands and leads to a loss of the scuPA epitope recognized by &agr;
2
MR/LRP (Higazi et al., 1996, Blood 88:542-551). The latter proposed mechanism is consistent with the observation that soluble scuPA has a higher affinity for &agr;
2
MR/LRP than does tcuPA and with the observation that tcuPA loses affinity for &agr;
2
MR/LRP when the active site of tcuPA is occupied by diisofluoryl phosphate (Nykjaer et al., 1994, J. Biol. Chem. 269:25668-25676).
scuPA bound to uPAR is active, protected from inactivation by PAI-1, and protected from clearance from the cell surface mediated by binding of scuPA to &agr;
2
MR/LRP and subsequent degradation. Furthermore, scuPA that dissociates from uPAR reverts to an inactive conformation and becomes essentially insusceptible to inactivation by PAI-1. Thus, unbound scuPA retains the capacity to rebind to uPAR and revert once again to its active conformation.
There are abundant epidemiological data which indicate that the expression or uPA and uPAR in human tissue correlates with the conversion of cells from a benign to a neoplastic state. Furthermore, expression of uPA and uPAR is associated with a wide variety of common malignancies, and is predictive of future development of those malignancies. Interference with uPA activity by binding an antibody to uPA, by expression of an antisense oligonucleotide complementary to mRNA encoding uPA, or by overexpression of catalytically inactive forms of uPA impede tumor progression in several experimental murine models of human cancers (Ossowski, 1988, J. Cell Biol. 107:2437-2445; Ossowski et al., 1991, Canc. Res. 51:275-281; Kook et al., 1994, EMBO J. 13:3983-3991; Crowley et al., 1993, Proc. Natl. Acad. Sci. USA 90:5021-5025; Jankun et al
Cines Douglas
Higazi Abd Al-Roof
Gupta Anish
Lankford , Jr. Leon B.
Morgan & Lewis & Bockius, LLP
The Trustees of the University of Pennsylvania
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