Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1998-11-23
2002-01-15
Davenport, Avis M. (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S016700, C514S017400, C530S300000, C530S328000, C530S329000, C530S330000, C424S185100
Reexamination Certificate
active
06339062
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to peptide fragments and synthetic analogs of thrombospondin (also known as thrombin sensitive protein or TSP) with thrombospondin-like activity. The peptides mimic or inhibit the biological activity of TSP. These peptides may be used in biological and pharmaceutical applications such as: (a) inhibiting the invasive and metastatic activity of melanoma cells, (b) promoting and inhibiting cellular attachment to tissue culture flasks, (c) promoting wound healing, angiogenesis, and implant acceptance, (d) agents for anti-platelet aggregation, (e) agents for antimalarial activity, and (f) diagnostic reagents in different therapeutic applications, as well as other related areas.
BACKGROUND
Thrombospondin (TSP) is secreted by platelets in response to physiological activators such as thrombin and collagen (Lawler,
Blood,
67:112-123 (1986)). Other cells also synthesize TSP including fibroblasts (E. A. Jaffe et al.,
Proc. Natl. Acad. Sci.,
80:999-1002 (1983)), smooth muscle cells (Raugi, G. J. et al.,
J. Cell Biol.
95:351-354 (1982)), and endothelial cells (J. McPhearson et al.,
J. Biol. Chem.,
256:11330-11336). TSP has been found in certain tumor tissues, such as melanoma cells (J. Varani et al.,
Clin. Expl. Metastais,
7:265-76 (1989)), squamous lung carcinoma (B. L. Riser et al.,
Exp. Cell Res.,
174:319-329 (1988)) and breast carcinoma (D. A. Pratt et al.,
Eur. J. Cancer Clin. Oncol.
25:343-350 (1989)). In addition, certain tumor cells in culture, such as, fibrosarcoma, rhabdomyosarcoma, glioblastoma, Wilm's tumor, neuroblastoma, teratocarcinoma, choriocarcinoma, melanoma, and lung carcinoma have been shown to synthesize TSP (D. F. Mosher,
Annu. Rev. Med.,
41:85-97 (1990)).
TSP has been shown to play a role in many diverse and clinically important processes, such as: cell migration, wound healing, nerve regeneration, and tumor cell metastasis. TSP has been purified by a number or procedures including exclusion chromatography (Lawler et al.,
J. Cell Biol.,
103:1635-48 (1986)). The complete amino acid sequence of TSP has been deduced from DNA clones prepared by various groups including Lawler et al.,
J. Cell Biol.,
103:1635-48 (1986); Kobayashi et al.,
Biochemistry,
25:8418-25 (1986); Dixit et al.,
Proc. Ntl. Acad. Sci.,
83:5449-53 (1986); and Hennessy et al.,
J. Cell Biol.,
108:729-36 (1989). The structure of TSP is conserved among various animal species as indicated by the fact that the antiobody against the human protein cross-reacts with TSP from mouse, rat, pig, cow, sheep, dog, and turkey (H. I. Switalska et al.,
J. Lab Clin. Med;
106:690-700). It is now known that TSP, originally characterized from platelet released proteins, is only one member of a family of structurally related proteins encoded by different genes which include at least four new members designated TSP-2, TSP-3, TSP-4, and TSP-5/COMP (cartilage oligomeric matrix protein). Adams and Lawler,
Current Biology,
3: 188-190 (1993).
TSP-1 is composed of three identical disulfide-linked chains each consisting of 1,152 amino acids (MW 145,000), and each polypeptide chain is composed primarily of domains consisting of repeating homologous amino acid sequences. Adams and Lawler,
Current Biology,
3: 188-190 (1993). These domains are a) NH
2
-terminal globular domain; b) a procollagen homology domain; c) the type 1 or properdin repeat domain, consisting of three repeating sequences homologous to sequences found in properdin; d) the type 2 repeat domain, consisting of three repeating sequences homologous to those in epidermal growth factor; e) the type 3 repeat domain, consisting of seven repeating Ca
2+
-binding sequences, and f) a COOH-terminal globular domain. These distinct domains interact with different cell surface receptors and mediate a variety of cellular processes including cell attachment, migration, proliferation and differentiation. For example, the Cys-Ser-Val-Thr-Cys-Gly (SEQ ID NO: 1) sequence within the type 1 repeats binds the Cys-Ser-Val-Thr-Cys-Gly (SEQ ID NO: 1) receptor, recently identified and isolated from tumor and endothelial cells (Tuszynski et al.,
J. Cell Biol.,
116:209-217 (1992) and Prater et al.,
J. Cell Biol.,
112: 1031-1040 (1991) and is part of the central stalk or protease resistant region of TSP-1.
SUMMARY OF THE INVENTION
The present invention provides thrombospondin fragments and analogs that mimic or inhibit the biological activity of intact thrombospondin and are, thus, useful in a variety of biological, prophylactic or therapeutic areas. These peptides are capable of modifying and inhibiting tumor cell metastasis, cell adhesion and platelet aggregation in mammals in vivo. The peptides are also useful in wound healing, for antimalarial activity, atherosclerosis, thrombotic, and thrombolytic conditions, angiogenesis, and as cell attachment promoters, complement modulators, and diagnostic reagents and in other related areas.
Analogs based on the type I repeat of thrombospondin described by Lawler et al.,
Seminars in Thrombosis
&
Hemostasis,
13:245-254 (1987), Robson et al.,
Nature,
335:79-82 (1988), and Groundis et al.,
Nature,
335:82-85 (1988) have been shown to have thrombospondin-like activity. Specifically, analogs based around and including at least a portion of the sequence motif Trp-Ser-Pro-Cys-Ser-Val-Thr-Cys-Gly (SEQ ID NO: 2) have been shown to have thrombospondin-like activity.
This invention is directed to polypeptide compounds of formula (I):
Z
1
-Xaa
2
-Xaa
3
-Xaa
4
-Xaa
5
-Xaa
6
-Xaa
7
-Xaa
8
-Xaa
9
-Xaa
10
-Z
2
(SEQ ID NO: 3)
wherein:
Xaa
2
is a neutral
on-polar/large/cyclic amino acid residue;
Xaa
3
is a neutral/polar/small or neutral/polar/large
on-cyclic or acidic amino acid residue;
Xaa
4
is a neutral
onpolar/large/cyclic or neutral
on-polar/large
on-cyclic or neutral/polar/large
on-cyclic or neutral/polar/small amino acid residue;
Xaa
5
is a neutral/polar/small amino acid residue
Xaa
6
is a neutral/polar/small or neutral/polar/large
on-cyclic amino acid residue;
Xaa
7
is a neutral
onpolar/large
on-cyclic or neutral/polar/large
on -cyclic amino acid residue;
Xaa
8
is a neutral/polar/large
on-cyclic or neutral/polar/small amino acid residue;
Xaa
9
is a neutral/polar/small amino acid residue;
Xaa
10
is a neutral/polar/small amino acid residue;
Z
1
is hydrogen, amino, acetyl or at least one amino acid residue or the desamino form thereof;
Z
2
is hydroxyl, carboxyl, non-amino acids such as agmatine, or at least one amino acid residue, including carboxyamide or alkylamide forms thereof.
Preferably, the polypeptide compounds of this invention have formula (II):
R
1
-Cys-Xaa
11
-Xaa
12
-Xaa
13
-Cys-R
2
(SEQ ID NO: 4)
wherein:
R
1
is a protected or unprotected terminal amino group, including hydrogen, amino, acetyl or at least one amino acid residue or the desamino form thereof;
Xaa
11
, Xaa
12
, and Xaa
13
are the same or different neutral
on-polar/large
on-cyclic or neutral/polar/large
on-cyclic or neutral/polar/small or basic
on-cyclic amino acid residues, preferably selected from the group consisting of valine, threonine, serine, and arginine;
R
2
is a protected or unprotected terminal carboxyl group including hydroxyl, carboxyl, or at least one amino acid residue, including carboxyamide or alkylamide forms thereof, preferably selected from the group consisting of lysine, glycine, and arginine;
wherein the structure of the polypeptide is optionally cyclized through a bond between the cysteines, such as a disulfide bond, or a bond between R
1
and R
2
.
This invention also includes polypeptides having the retroinverso form of L-amino acid polypeptides of formulae (I) and (II), i.e, polypeptides comprising D-amino acids in reverse order. In particular, retroinverso peptides of formula Cys-Ser-Val-Thr-Cys-Gly (SEQ ID NO: 1), i.e., d-Gly-Cys-Thr-Val-Ser-Cys (SEQ ID NO: 5) are preferred. Preferably, the cysteine residues are modified by a sulfhydral blocking group, such as —CH
2
—NH—COCH
3
, that is adhesive t
Actor Paul
Tuszynski George
Williams Taffy
Davenport Avis M.
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Inkine Pharmaceutical Company, Inc.
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