Synthetic analogs of thrombospondin and therapeutic use thereof

Details Synthetic analogs of thrombospondin and therapeutic use thereof Synthetic analogs of thrombospondin and therapeutic use thereof

1995-06-07

2001-05-29

Davenport, Avis M. (Department: 1653)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C514S015800, C530S327000, C530S329000, C530S330000, C424S185100

Reexamination Certificate

active

06239110

ABSTRACT:

TECHNICAL FIELD
The present invention relates generally to peptide fragments and synthetic analogs of thrombospondin (TSP) which retain thrombospondin-like activity. The peptides retain and mimic the bioactivity of TSP as a potent promoter or inhibitor of cell adhesion and attachment to different cell lines. The peptides find use as agents in inhibiting metastasis since TSP has previously been shown to mediate tumor cell metastasis presumably by mechanisms involving the cell adhesive domain of TSP. These peptides also find use in different biological and pharmaceutical applications such as: (a) promoting and inhibiting cellular attachment to tissue culture flasks, (b) promoting wound healing, angiogenesis, and implant acceptance, (c) use as agents for anti-platelet aggregation, (d) use as agents for antimalarial activity, and (e) use as diagnostic reagents in different therapeutic applications.
BACKGROUND
Thrombospondin (also known as thrombin sensitive protein or TSP) is a 450,000 molecular weight protein composed of three identical disulfide-linked polypeptide chains (Lawler et al.
J. Cell Biol
(1986) 101:1059-71). TSP is secreted by platelets in response to physiological activators such as thrombin and collagen (Lawler,
J. Blood
(1986) 67:112-123). TSP comprises 3% of the total platelet protein and 25% of the total platelet alpha granular protein (Tuszynski, G. P. et al. (1985)
J. Biol. Chem.
260:12240-12245). Other cells also synthesize TSP including fibroblasts (Jaffe, E. A. et al., (1983)
Natl. Acad. Sci. USA
80:999-1002), smooth muscle cells (Raugi, G. J. et al., (1982)
J. Cell Biol.
95:351-354), and endothelial cells (McPhearson, J. et al.
J. Biol. Chem.
256:11330-11336). TSP has been found in certain tumor tissues, such as melanoma cells (Varani, J. et al., (1989)
Clin. Expl. Metastais
7:319-329), squamous lung carcinoma (Riser, B. L. et al., (1988)
Exp. Cell Res.
174:319-329) and breast carcinoma (Pratt, D. A. et al., (1989)
Eur. J. Cancer Clin. Oncol.
25:343-350). In addition, the following tumor cells in culture have been shown to synthesize TSP: fibrosarcoma, rhabdomyosarcoma, glioblastoma, Wilm's tumor, neuroblastoma, teratocarcinoma, choriocarcinoma, melanoma, and lung carcinoma (Mosher, D. F., (1990)
Annu. Rev. Med.
41:85-97). A number of recent studies have shown that TSP plays a major role in cell-cell and cell substatum adhesion (Tuszynski, G. P. et al., (1987)
Seminars in Thrombosis Hemostasis
(13:361-368, Mosher, D. F., (1990) Annu. Rev. Med. 41:85-97). TSP promotes cell attachment, platelet aggregation, and lung tumor colony formation in a murine model of experimental metastasis (Tuszynski, G. P. et al., (1987)
Science
236:1570-1573, Tuszynski, G. P. et al., (1988)
Blood
72:109-115). The role of TSP in adhesion is further supported by the observation that the extracellular matrix of most tissues contains TSP.
TSP is composed of linear polypeptide domains that specifically interact with various macromolecules such as plasma and matrix components. For example, TSP forms a complex with heparin (Yabkowitz, R. et al. (1989)
J. Biol. Chem.
264:10888-10896), fibrinogen (Tuszynski, G. P. et al. (1985)
J. Biol. Chem.
260:12240-12245), collagen (Mumby, S. M. et al. (1984)
J. Cell Biol.
98:10888-10896, and plasminogen (Depoli. P. et al. (1989)
Blood
73:976-902). 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 (Switalska, H. I. et al.,
J. Lab Clin. Med.
106:690-700).
Thrombospondin has been purified by a number of procedures including exclusion chromatography (Lawler et al.,
J. Biol. Chem
. (1978) 253:8609-16), heparin affinity chromatography (Lawler et al.,
Thromb. Res
. (1981) 22:267-269), fibrinogen affinity chromatography (Tuszynski et al.,
J. Biol. Chem
. (1985) 260:12240-5), barium chloride precipitation (Alexander et al.,
Biochem. J
. (1984) 217:67-71) and anion exchange chromatography with HPLC (Clezarolin et al.,
J. Chromatog
. (1984) 296:249-56).
The complete amino acid sequence of TSP has been deduced from DNA clones prepared by various groups including Lawler et al.,
J. Cell Biol
. (1986) 103:1635-48; Kobayashi et al.,
Biochemistry
(1986) 25:8418-25; Dixit et al.,
Proc. Ntl. Acad. Sci
. (1986) 83:5449-53; and Hennessy et al.,
J. Cell Biol
. (1989) 108:729-36.
Cell adhesion is critical to the development and survival of multicellular organisms. The process of cell adhesion is complex requiring numerous extracellular proteins such as fibronectin, vitronectin, collagen, laminin, and TSP and numerous families of cellular receptors such as the integrins and cellular adhesion molecules (CAMS). These molecules are involved in the adhesion of both normal and tumor cells and have been studied quite intensively in recent years.
The amino acid sequence, Arg-Gly-Asp (RGD), was established as a cell attachment domain in fibronectin (Pierschbacher, M. D. and Ruoslahti, E., (1984)
Nature
(London) 309:30-32). The same or related sequences have been discovered in many proteins and serve as cell binding sites for such macromolecules as fibrinogen (Ginsberg, M. D. et al., (1985)
J. Biol. Chem.
260:11891-11896). However, it appears that the adhesive function of laminin may not be based on the RGD sequence, but on a peptide segment of the Bi chain containing the amino acid sequence tyrosine-isoleucine-glycine-serine-arginine (YIGSR) (SEQ ID NO: 33) (Sasaki, M. 1987,
Proc. Natl. Acad. Sci.
84:935-938). Synthetic peptides containing the RGD and YIGSR (SEQ ID NO: 33) sequence promote cell adhesion.
The therapeutic use of synthetic peptides based on the adhesive domains of fibronectin and laminin have recently been reported. Humphries et al. (2986)
Science
233:467-470) were the first to demonstrate that co-injection of the pentapeptide GRGDS with B16-F1O murine melanoma cells dramatically inhibited the formation of lung colonies in C57BL/6 mice. Another synthetic peptide which was derived from laminin (YIGSR) (SEQ ID NO: 33) also dramatically inhibited B16-F1O melanoma cell metastasis in C57B1/6 mice (Kleinman, H. K. et al., (1987)
Science
238:1132-1133; Kleinman, H. K. et al., (1990)
Proc. Natl. Acad. Sci. USA
87:2279-2283). The inhibitory activity of these peptides may be due to competition with endogenous laminin and fibronectin-dependent adhesion of tumor cells to the vascular bed of the target organ during the metastatic dissemination of the tumor cells.
Because metastasis is a step-by-step process involving the transfer of tumor cells from one site to another through the lymphatic and blood circulation and platelet. reduction in animals effectively blocked metastasis in animals (Gasic et al, (1968)
Proc. Natl. Acad. Sci. USA
48:46-52), platelets have been thought to play a special role in the development of metastasis. Since TSP comprises 25% of the total alpha granular platelet secreted-protein, TSP would be expected to have a major role in the hemotagenous transfer of tumor cells to distant organs. Indeed, TSP has been shown to promote tumor cell metastasis in a murine model (Tuszynski et al, (1987) 47:4130-4133). In addition, events which accompany platelet activation, such as: secretion of adhesive proteins, platelet aggregation, activation of proteolytic enzymes, and activation of the clotting cascade have all been shown to play a significant role in tumor cell metastasis (Gasic, G. J., (1984)
Cancer Metastasis Rev.
3:99-116).
Adhesive proteins which are part of the extracellular matrix control the movement, growth, and morphology of many cell types. Extracellular matrix proteins interact with tumor cell receptors and affect tumor cell adhesion to basement membrane collagen in different ways. For example, exposure of melanoma cells in vitro to laminin resulted in increased capacity of tumor cells to adhere to the basement membrane and to produce lung tumor colonies (Barsky, S. H. et al., (1984)
J. Clin. Inv.
74:843-848; Terranova, V. P. et al., (

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