Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2001-06-13
2004-12-21
Carlson, Karen Cochrane (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C530S300000, C530S350000, C424S278100
Reexamination Certificate
active
06833357
ABSTRACT:
BACKGROUND OF THE INVENTION
Urokinase-type plasminogen activator (uPA) is a serine protease which has been implicated in various biological processes, including fibrinolysis, (Carmeliet et al., 1994, Nature 369:419-424; Carmeliet et al., 1996, Haemostasis 26:132-153; Pinsky et al., 1998, J. Clin. Invest. 102:919-928; Bugge et al., 1996, Proc. Natl. Acad. Sci. USA 93:5899-5904) angiogenesis, (Odekon et al., 1992, J. Cell, Physiol. 150:258-263; Bacharach et al., 1992, Proc. Natl. Acad. Sci. USA 89:10686; Pepper et al., 1990, J. Cell Biol. 111:743-755; Goldberg et al., eds., 1997, In:
Regulation of Angiogenesis
Birkhauser Verlag, Basel, pp. 391-411) neointima and aneurysm formation, (Clowes et al., 1990, Circ. Res. 67:61-67; Carmeliet et al., 1997, Circ. Res. 81:829-839; Shireman et al., 1997, J. Vasc. Surg. 25:157-164; Noda-Heiny et al., 1995, Arterioscler. Thromb. Vasc. Biol. 15:37-43; Lijnen et al., 1998, Arterioscler. Thromb. Vasc. Biol. 18:1035-1045) chemotaxis, (Pedersen et al., 1996, Br. J. Haematol. 95:45-51) and wound healing (Carmeliet et al., 1998, J. Cell Biol. 140:233-245).
Certain of these activities may require the proteolytic activity of uPA, (Kirschheimer et al., 1987, Fed. Am. Soc. of Exper. Biol. and Med. Journal 1:125-128; DePetro et al., 1994, Exp. Cell Res. 213:186-194) whereas others involve intracellular signaling through the urokinase receptor (uPAR) (Pedersen et al., 1996, Br. J. Haematol. 95:45-51; Degryse et al., 1999, Blood 94:649-662) or additional, as yet undefined receptors (Carmeliet et al., 1998, J. Cell Biol. 140:233-245; Kanse et al., 1997, Arteriosclerosis, Thromb., and Vas. Biol. 17:2848-2854; Koopman et al., 1998, J. Biol. Chem. 273:33267-33272; Rabbani et al., 1992, J. Biol. Chem. 267:14151-14156).
Urokinase is synthesized as a single chain molecule (scuPA) which exhibits little or no intrinsic enzymatic activity (Urano et al., 1988, Arch. Biochem. Biophys. 264:222-230; Gurewich et al., 1987, Semin. Thromb. Hemost. 13:146-151; Husain, 1991, Biochemistry 30:5707-5805). scuPA is a multi-domain protein composed of a C-terminal protease domain and an amino-terminal fragment (ATF). The ATF is composed of two domains: a growth factor domain (GFD) which binds to uPAR, and a kringle domain (uPA kringle), the function of which has heretofore been unknown. scuPA can be cleaved by plasmin at the Lys
158
-Ile
159
position to generate an enzymatically active, disulfide-linked two-chain urokinase molecule (tcuPA). Between the ATF and the protease domain is a region designated the connecting peptide (corresponding to amino acids 136-158).
It is hypothesized that the natural enzyme uPA is normally used in the human body to dissolve clots and to facilitate cell migration. Although uPA is known in the art as a useful therapeutic molecule for the treatment of diseases and disorders having as a symptom thereof abnormal clotting in critical blood vessels, there remains a need in the art for compositions and methods which are useful for the treatment of such diseases as well as for the treatment of diseases and disorders having as symptoms abnormally high or abnormally low muscle cell contractility or undesirable angiogenic activity. Such diseases and conditions include the following: cardiovascular diseases and conditions such as hypotension, hypertension and atherosclerosis; thrombotic conditions such as stroke, heart attack and post angioplasty stenting; angiogenic disorders; respiratory diseases and conditions such as pulmonary fibrosis and asthma; diseases and disorders related to tumor cell invasion, angiogenesis and metastasis; wound healing and clotting disorders and reproductive disorders such as premature uterine contraction and impotence. The present invention meets these needs.
BRIEF SUMMARY OF THE INVENTION
The invention includes a composition comprising the urokinase-type plasminogen activator (uPA) kringle in an amount effective to modulate one or more of the contractility and the angiogenic activity of a mammalian muscle or endothelial cell or tissue. The uPA kringle shares at least about 75% homology with a polypeptide having the amino acid sequence corresponding to SEQ ID NO:1.
In one embodiment, the composition further comprises one or more domains of uPA selected from the group consisting of the growth factor domain, the connecting peptide and the protease domain.
The invention also includes a composition comprising the growth factor domain of uPA in an amount effective to modulate the contractility of a mammalian muscle cell or tissue. The growth factor domain shares at least about 75% homology with a polypeptide having the amino acid sequence corresponding to SEQ ID NO:2.
In one embodiment, the composition further comprises one or more domains of uPA selected from the group consisting of the uPA kringle, the connecting peptide and the protease domain.
The invention also includes a composition comprising a polypeptide, the polypeptide (LMW-uPA) comprising the connecting peptide and protease domains of uPA in an amount effective to inhibit the contractility of a mammalian muscle cell or tissue. The polypeptide shares at least about 75% homology with a polypeptide having the amino acid sequence corresponding to SEQ ID NO:5.
In one aspect, the cell is in a mammal.
In another aspect, the muscle cell is selected from the group consisting of a smooth muscle cell, a striated muscle cell and a cardiac muscle cell, and the muscle tissue is selected from the group consisting of a smooth muscle tissue, a striated muscle tissue and a cardiac muscle tissue.
In one embodiment, the composition further comprises an inducing compound in an amount effective to mediate the contraction of a mammalian muscle cell or tissue. The inducing compound is selected from the group consisting of phenylepherine, epinepherine, acetylcholine and endothelin.
In another aspect, the composition comprises two chain urokinase (tcuPA). The tcuPA shares at least about 75% homology with a polypeptide having the amino acid sequence corresponding to SEQ ID NO:3.
In one embodiment, the composition comprises single chain urokinase (scuPA). The scuPA shares at least about 75% homology with a polypeptide having the amino acid sequence corresponding to SEQ ID NO:3.
In another embodiment, the composition comprises the amino terminal fragment (ATF) of uPA. The ATF shares at least about 75% homology with a polypeptide having the amino acid sequence corresponding to SEQ ID NO:4.
In one aspect, the uPA kringle is an isolated kringle.
In another aspect, the growth factor domain is an isolated growth factor domain.
In yet another aspect, the ATF is an isolated ATF.
In one embodiment, modulating the contractility of the muscle cell or tissue comprises enhancing or disinhibiting the contractility of the muscle cell or tissue.
In another embodiment, modulating the contractility of the muscle cell or tissue comprises enhancing or disinhibiting the contractility of the muscle cell or tissue.
In one aspect, the cell or tissue is a vascular smooth muscle or endothelial cell or tissue, and the uPA kringle is present in an amount effective to modulate the angiogenic activity of the cell or tissue.
In another aspect, the cell or tissue is a vascular smooth muscle cell or tissue or a vascular endothelial cell or tissue.
In one embodiment, modulating the contractility of the muscle cell or tissue comprises inhibiting the contractility of the muscle cell or tissue.
In another embodiment, modulating the contractility of the muscle cell or tissue comprises inhibiting the contractility of the muscle cell or tissue.
In one aspect, the cell or tissue is a bronchial smooth muscle cell or tissue.
In one embodiment, the composition comprises the deletion mutant polypeptide scuPA
&Dgr;136-143
in an amount effective to enhance or disinhibit the contractility of a mammalian muscle cell or tissue, wherein the scuPA
&Dgr;136-143
shares at least about 75% homology with a polypeptide having the amino acid sequence corresponding to SEQ ID NO:6.
In another embodiment, the composition comprises a deletion mutant polypept
Cines Douglas B.
Higazi Abd Al-Roof
Carlson Karen Cochrane
Liu Samuel Wei
Morgan & Lewis & Bockius, LLP
The Trustees of the University of Pennsylvania
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