Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2000-02-07
2004-11-16
Wax, Robert A. (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S013800, C514S014800, C514S015800, C514S016700, C514S017400, C514S018700, C530S324000, C530S325000, C530S326000, C530S327000, C530S328000, C530S329000, C530S330000, C530S331000, C530S344000, C530S412000, C530S415000, C530S856000
Reexamination Certificate
active
06818617
ABSTRACT:
FIELD OF THE INVENTION
This invention generally relates to methods and compositions for modulating cell adhesion and for inhibiting the interaction between integrins and their ligands. In particular, the invention relates to peptides that selectively inhibit &agr;4 integrins.
BACKGROUND OF THE INVENTION
A. Integrins
Integrins are a family of cell surface proteins that mediate adhesion between cells (cell-cell adhesion) and between cells and extracellular matrix proteins (cell-ECM adhesion). Integrins are heterodimeric structures composed of noncovalently bound &agr; and &bgr; subunits. In humans there are at least 15 different &agr; and eight different &bgr; subunits, and these can combine to form proteins with diverse biological activities and ligand specificities.
The integrins play important roles in many diverse biological processes including platelet aggregation, tissue repair, angiogenesis, bone destruction, tumor invasion, inflammation, and immune reactions. The integrins are, therefore, important targets for therapeutic intervention in human disease.
Integrin &agr;IIb&bgr;3 (glycoprotein IIb/IIIa complex) binds fibrinogen on the platelet surface and mediates platelet aggregation. Integrin &agr;v&bgr;3 is predominantly expressed on endothelial cells and plays an important role in angiogenesis. Integrin &agr;v&bgr;3 is also expressed on osteoclasts and participates in bone destruction. Integrin &agr;5&bgr;1 is widely distributed on a variety of cells; it plays a critical role in cell adhesion to extracellular matrix as well as in the formation of tissues and organs during embryonic development. All three integrins &agr;IIb&bgr;3, &agr;v&bgr;3 and &agr;5&bgr;1 recognize RGD sequences in the adhesive ligands.
One example of the therapeutic targeting of integrins is the use of integrin inhibitors as antithrombotic agents. Peptides and peptidomimetics that block the adhesion of GPIIb/IIIa to fibrinogen can prolong bleeding times and prevent thrombotic occlusion in vivo. One group of naturally occurring peptides that inhibit platelet aggregation by interfering with fibrinogen binding to GPIIb/IIIa has been called the “disintegrins.”
B. Disintegrins
The disintegrins are a family of low molecular weight cysteine-rich peptides that have been isolated from the venom of various snakes (reviewed in Niewiarowski et al.,
Seminars in Hematology
31(4):289-300 (1994)). Most disintegrins described to date contain an RGD motif. RGD is a recognition site for many integrins, and disintegrins inhibit fibrinogen binding to GPIIb/IIIa, as well as the binding of other ligands to RGD-dependant integrins on the surface of cells. Peptides modeled on the structure of disintegrins have potential clinical applications in the prevention and treatment of coronary thrombosis, stroke, and other vascular diseases.
The first disintegrin described in the literature, trigramin, was identified and characterized on the basis of its ability to block platelet aggregation and inhibit fibrinogen binding to &agr;IIb&bgr;3. Trigramin contains 72 amino acids including 12 cysteines, is linked by S-S bonds, and contains an RGD sequence. Subsequently several other RGD containing viper venom disintegrins of similar size were isolated.
A 49 amino acid disintegrin, called echistatin, has been isolated from the venom of
Echis carinatus
(Gan et al.,
J. Biol. Chem
. 263:19827-32 (1988)). Like other disintegrins, echistatin contains an RGD sequence and inhibits GPIIb/IIIa binding of fibrinogen. Echistatin has been called a “promiscuous disintegrin” because it blocks &agr;IIb&bgr;3, &agr;v&bgr;3 and &agr;5&bgr;1 with similar potency.
NMR studies on RGD-containing disintegrins shows that the RGD sequence is located in a mobile loop joining two strands of &bgr; sheet protruding from the protein core (reviewed in Niewiarowski et al.,
Semin. Hematol
. 31:289-300 (1994)). The disulfide bonds around the RGD sequence maintain the hair-pin loop conformation in each peptide, and this conformation seems to be important for potency and selectivity.
The disintegrin eristostatin, originally described as a potent inhibitor of &agr;IIb&bgr;3, also inhibits human melanoma cell (MV3) metastases in immune deficient mice. It has been suggested that this effect is mediated by altering the function of an &agr;4 integrin expressed on MV3 cells (Danen et al,
Exp. Cell Res
. 238:188-196 (1998)).
C. The &agr;4 Integrins
The &agr;4 integrins, &agr;4&bgr;1 and &agr;4&bgr;7, are expressed on leukocytes and lymphoid cells, and play a major role in inflammation and auto-immune diseases.
The &agr;4&bgr;1 integrin (which has also been called VLA4, very late activation antigen 4) mediates cell adhesion to vascular cell adhesion molecule-1 (VCAM-1), an adhesive molecule belonging to the IgG superfamily which is expressed on endothelial cells at sites of inflammation. The integrin &agr;4&bgr;1 also binds to alternatively spliced variants of fibronectin containing connecting segment 1 (CS-1).
The &agr;4&bgr;7 integrin binds to the gut homing receptor mucosa addressin cell adhesion molecule-1 (MadCAM-1) and to a lesser extent to CS-1 and VCAM-1.
Cytokine activated leukocytes express &agr;4&bgr;1 and &agr;4&bgr;7 integrins. Interaction of these integrins with VCAM-1 or MadCAM-1 (which are also up-regulated by cytokines) on endothelium mediates capillary infiltration by leukocytes, which can lead to tissue and organ destruction. Selectins and &bgr;2 integrins also contribute to this process. Altevost et al. reported that the &agr;4 subunit itself is a ligand for &agr;4&bgr;1 and &agr;4&bgr;7 integrins (
J. Exp. Med
. 182:345-55 (1995)), suggesting that &agr;4 integrins may play a role in leukocyte communication during the immune response.
Activation and up-regulation of &agr;4&bgr;1 or &agr;4&bgr;7 on lymphocytes or macrophages is believed to play a significant role in the progression of many disease states, including insulin dependent diabetes mellitus, multiple sclerosis, rheumatoid arthritis, ulcerative colitis, arteriosclerosis, asthma, allergy, organ rejection, and restenosis of arteries after surgery or angioplasty. The &agr;4 integrins are therefore targets for therapeutic intervention in a variety of inflammatory, auto-immune, and other diseases.
D. The &agr;4 Integrins as Therapeutic Targets
There are several animal models of inflammatory and autoimmune diseases in which endothelial infiltration by lymphocytes and organ destruction are blocked by anti-&agr;4 monoclonal antibodies. As an example, anti-&agr;4 antibody inhibits lymphocyte infiltration of Langerhans islets in NOD mice, thus preventing development of spontaneous insulin dependent diabetes (Yang et al.,
Proc. Natl. Acad. Sci. USA
4 91:12604-08 (1994)). Anti-&agr;4 monoclonal antibodies have also shown in vivo efficacy in animal models of asthma (Abraham et at.,
J. Clin. Invest
. 93:776 (1994)), multiple sclerosis (Yednock et al.,
Nature
356:63 (1992)), inflammatory bowel disease (Podoisky et al.,
J. Clin. Invest
. 92:372 (1993)), contact hypersensitivity (Chisholm et al.,
Eur. J. Immunol
. 23:682 (1993)), and cardiac allograft rejection (Isobe et al.,
J. Immunol
. 153:5810 (1994)).
There have been several attempts to isolate naturally occurring or to develop synthetic inhibitors of &agr;4&bgr;7 and &agr;4&bgr;1. Synthetic inhibitors that have been reported include cyclic RGD peptides and short peptides based on the sequences of MadCAM-1, VCAM-1, and CS-1. These peptides are typically active in vitro at the micromolar level.
Molossi et al. (
J. Clin. Invest
. 95:2601-10 (June 1995)) reported that blockade of &agr;4&bgr;1 (VLA-4) integrin binding to fibronectin with CS-1 peptide reduces accelerated coronary arteriopathy in rabbit cardiac allografts. The sequence of the CS-1 peptide was phenylacetic acid-Leu-Asp-Phe-d-Pro-amide.
Kogan et al. (WO 96100581) reported that cyclic peptides modeled after a portion of the CS-1 peptide inhibited the binding of &agr;4&bgr;1 integrin to VCAM-1 at concentrations of peptide less than about 10 &mgr;M.
Kogan et al. (U.S. Pat. No. 5,510,332) report
Marcinkiewicz Cezary
Niewiarowski Stefan
Drinker Biddle & Reath LLP
Temple University- Of The Commonwealth System of Higher Educatio
Wax Robert A.
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