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  Pharmaceutical Patents  

 

Title:  Compositions and methods for modulating muscle cell and tissue contractility
United States Patent: 
7,425,534
Issued: 
September 16, 2008

Inventors:
 Cines; Douglas B. (Wynnewood, PA), Higazi; Abd Al-Roof (Jerusalem, IL)
Assignee:
  The Trustees of the University of Pennsylvania (Philadelphia, PA)
Appl. No.:
 11/019,448
Filed:
 December 21, 2004


 

Executive MBA in Pharmaceutical Management, U. Colorado


Abstract

The present invention relates to compositions and methods comprising one or more domains of urokinase-type plasminogen activator (uPA) in an amount effective to modulate one or more of the contractility and angiogenic activity of a mammalian muscle or endothelial cell or tissue for use in the treatment of a disease or condition having as a symptom thereof one or more of abnormal muscle cell or tissue contractility and abnormal angiogenic activity. The one or more domains of uPA can be present in the inventive compositions and methods either as part of the full uPA molecule in either single chain or two chain form (scuPA or tcuPA), or as an isolated polypeptide, or a fragment of the uPA molecule (e.g., the amino terminal fragment "ATF"), or a deletion mutant of the uPA molecule. The inventive methods comprise administering to a mammal afflicted with such a disease or condition the inventive composition, and modulating one or more of the contractility and the angiogenic activity of the muscle or endothelial cell or tissue, thereby treating the disease or condition. Kits for treating such diseases are also included.

Description of the Invention

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.sup..DELTA.136-143 in an amount effective to enhance or disinhibit the contractility of a mammalian muscle cell or tissue, wherein the scuPA.sup..DELTA.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 polypeptide selected from the group consisting of .DELTA.kringle-scuPA and .DELTA.kringle-tcuPA in an amount effective to proteolytically activate plasminogen and to inhibit the contractility of a mammalian muscle cell or tissue, wherein the .DELTA.kringle-scuPA and the .DELTA.kingle-tcuPA each share at least about 75% homology with a polypeptide having the amino acid sequence corresponding to SEQ ID NO:7.

In a further embodiment, the composition comprises a polypeptide, the polypeptide comprising the amino terminal fragment (ATF) and the connecting peptide of uPA, wherein the polypeptide shares at least about 75% homology with a polypeptide having the amino acid sequence corresponding to SEQ ID NO:8.

In yet a further embodiment, the composition comprises a polypeptide, the polypeptide comprising the uPA kringle and the connecting peptide, wherein the polypeptide shares at least about 75% homology with a polypeptide having the amino acid sequence corresponding to SEQ ID NO:9.

In one aspect, the composition is in the form of a pharmaceutical composition.

The invention also includes a composition comprising one or more polypeptides, each of the polypeptides having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9. The one or more polypeptides are present 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 invention also includes a composition comprising an isolated nucleic acid. The isolated nucleic acid has a nucleotide sequence which shares at least about 75% homology with a nucleotide sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18. The isolated nucleic acid is present in the composition in an amount effective to transform a mammalian muscle or endothelial cell to provide transgene expression of a polypeptide at a level of expression effective to modulate one or more of the contractility and angiogenic activity of the muscle or endothelial cell after transfection with the isolated nucleic acid.

The invention includes a method of treating a mammal afflicted with a disease or condition having as a symptom thereof one or more of abnormal muscle cell or tissue contractility and abnormal muscle or endothelial cell or tissue angiogenic activity. The method comprises a) administering to the mammal a composition comprising the 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, wherein the uPA kringle shares at least about 75% homology with a polypeptide having the amino acid sequence corresponding to SEQ ID NO:1; and b) modulating one or more of the contractility and the angiogenic activity of the muscle or endothelial cell or tissue having one or more of abnormal contractility and abnormal angiogenic activity, whereby the disease or condition in the mammal is treated.

In one embodiment, the uPA kringle is a part of a polypeptide which shares at least about 75% homology with a polypeptide selected from the group consisting of SEQ ID NO:3 (tcuPA), SEQ ID NO:4 (ATF), SEQ ID NO:6 (scuPA.sup..DELTA.136-143), SEQ ID NO:8 and SEQ ID NO:9.

In one aspect, the composition further comprises one or more of an agonist of the uPA kringle, an agonist of a binding protein of the uPA kringle, an antagonist of the uPA growth factor domain, an antagonist of the connecting peptide, an antagonist of a binding protein of the uPA growth factor domain, and an antagonist of a binding protein of the connecting peptide.

In another aspect, the disease or condition is selected from the group consisting of hypotension, hypertension, atherosclerosis, stroke, heart attack, microvascular occlusions, thrombotic microangiopathies, surgically induced thrombotic disorders, angiogenic disorders, pulmonary fibrosis, asthma, tumor cell invasion, tumor cell angiogenesis, tumor cell metastasis, glaucoma diabetic retinopathy, a wound healing or clotting disorder, a uterine contraction disorder and male impotence.

The invention also includes a method for treating a mammal afflicted with a disease or condition having as a symptom thereof one or more of abnormal muscle cell or tissue contractility and abnormal muscle or endothelial cell or tissue angiogenic activity. The method comprises a) administering to the mammal a composition comprising the uPA growth factor domain 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, wherein the uPA growth factor domain shares at least about 75% homology with a polypeptide having the amino acid sequence corresponding to SEQ ID NO:2; and b) modulating one or more of the contractility and the angiogenic activity of the muscle or endothelial cell or tissue having one or more of abnormal contractility and abnormal angiogenic activity, whereby the disease or condition in the mammal is treated.

In one aspect, the composition comprises the uPA growth factor domain as part of a polypeptide which shares at least about 75% homology with a polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO:3 (scuPA), SEQ ID NO:4 (ATF), SEQ ID NO:6 (scuPA.sup..DELTA.136-143), SEQ ID NO:7 (.DELTA.kringle-scuPA or .DELTA.kringle-tcuPA) and SEQ ID NO:8.

In one embodiment, the composition further comprises one or more of an agonist of the uPA growth factor domain, an agonist of the connecting peptide, an agonist of a binding protein of the growth factor domain, an agonist of a binding protein of the connecting peptide, an antagonist of the uPA kringle, and an antagonist of a binding protein of the uPA kringle.

In one aspect, the composition is administered to the mammal in an amount effective to inhibit the contractility of a mammalian smooth muscle cell or tissue.

In another aspect, the smooth muscle cell or tissue is a vascular smooth muscle cell or tissue, and the disease or condition treated is hypertension.

In another embodiment, the disease or condition is a respiratory disease or condition selected from the group consisting of asthma, adult respiratory distress syndrome, primary pulmonary hypertension, microvascular thrombotic occlusion and a disorder associated with chronic intrapulmonary fibrin formation.

In one aspect, the uPA kringle is present in an amount effective to inhibit the contractility of a bronchial smooth muscle cell or tissue and is a part of a polypeptide selected from the group consisting of an isolated kringle, ATF, tcuPA, scuPA.sup..DELTA.136-143 SEQ ID NO:8 and SEQ ID NO:9.

In one embodiment, the disease or condition in the mammal sought to be treated has as a symptom thereof abnormally low vascular smooth muscle cell or tissue contractility.

In another aspect, the uPA kringle is present in an amount effective to enhance or disinhibit the contractility of a vascular smooth muscle cell or tissue and is a part of a polypeptide selected from the group consisting of an isolated kringle, ATF, tcuPA, scuPA.sup..DELTA.136-143, SEQ ID NO:8 and SEQ ID NO:9.

In one embodiment, the disease or condition has as a symptom thereof abnormally high vascular smooth muscle cell or tissue contractility.

In one aspect, the uPA growth factor domain is present in the composition in an amount effective to inhibit the contractility of a vascular smooth muscle cell or tissue, and is present in the composition as a part of a polypeptide selected from the group consisting of an isolated growth factor domain, scuPA, .DELTA.kringle-scuPA and .DELTA.kringle-tcuPA.

The invention also includes a method of identifying a compound which is an agonist or antagonist of one or more of the uPA kringle or a binding protein thereof, the uPA growth factor domain or a binding protein thereof, and the connecting peptide or a binding protein thereof, upon the contractility or angiogenic activity of a mammalian muscle or endothelial cell or tissue. The method comprises a) providing to a first cell and an otherwise identical second cell a composition comprising a polypeptide, the polypeptide comprising one or more of the uPA kringle, the uPA growth factor domain and the connecting peptide, wherein the polypeptide is present in the composition in an amount effective to modulate the contractility or angiogenic activity of a mammalian muscle or endothelial cell or tissue; b) providing to the first cell a test compound; c) assessing the contractility or the angiogenic activity of the first cell and the second cell prior to and after administering the composition and the test compound to the first cell, and prior to and after administering the composition to the second cell; and d) comparing the contractility or angiogenic activity of the first cell with the contractility or angiogenic activity of the second cell prior to and after administration of the composition and the test compound. When the effect of the composition upon contractility or angiogenic activity in the first cell is either increased or decreased relative to the effect of the composition upon contractility or angiogenic activity in the second cell, a compound is identified which is an agonist or antagonist of one or more of the uPA kringle or a binding protein thereof, the uPA growth factor domain or a binding protein thereof, and the connecting peptide or a binding protein thereof, upon the contractility or angiogenic activity of a mammalian muscle or endothelial cell or tissue.

The invention also includes a method of treating a disease or condition in a mammal having as a symptom thereof one or more of abnormal muscle cell or tissue contractility and abnormal angiogenic activity. The method comprises a) administering to the mammal an amount suspected to be effective for modulating the contractility or angiogenic activity of a mammalian muscle or endothelial cell or tissue of an agonist or antagonist of one or more of the uPA kringle or a binding protein thereof, the uPA growth factor domain or a binding protein thereof, and the connecting peptide or a binding protein thereof; b) providing the agonist or antagonist to a muscle or endothelial cell or tissue in the mammal having abnormal contractility or abnormal angiogenic activity, or to a tissue or fluid which is contiguous therewith; and c) modulating the effect of one or more of the uPA kringle or a binding protein thereof, the uPA growth factor domain or a binding protein thereof, and the connecting peptide or a binding protein thereof, upon the muscle or endothelial cell or tissue having abnormal contractility or abnormal angiogenic activity, whereby a disease or condition in the mammal having abnormal muscle cell or tissue contractility or abnormal angiogenic activity as a symptom thereof is treated.

In one embodiment, the disease or condition treated is the vascular disease hypertension.

In another embodiment, the agonist or antagonist is one or more of an antagonist to the uPA kringle, an antagonist to a binding protein of the uPA kringle, an agonist of the uPA growth factor domain, an agonist of a binding protein of the UPA growth factor domain, an agonist of the connecting peptide, and an agonist of a binding protein of the connecting peptide.

In one aspect, the disease or condition treated is selected from the group consisting of asthma, adult respiratory distress syndrome, primary pulmonary hypertension, microvascular thrombotic occlusion and a disorder associated with chronic intrapulmonary fibrin formation.

In another aspect, the agonist or antagonist is one or more of an agonist to the uPA kringle and an agonist to a binding protein of the uPA kringle.

The invention also includes a method of identifying whether a test protein is a binding protein of one or more of the uPA kringle, the uPA growth factor domain and the connecting peptide. The method comprises a) assessing the contractility modulating effect or the angiogenic activity modulating effect of one or more of the uPA kringle, the uPA growth factor receptor and the connecting peptide upon a first cell or tissue, wherein the first cell or tissue comprises the test protein or is contiguous with a tissue or fluid of a mammal which comprises the test protein; b) assessing the contractility modulating effect or the angiogenic activity modulating effect of one or more of the uPA kringle, the uPA growth factor receptor and the connecting peptide upon a second, otherwise identical cell or tissue which does not comprise the test protein and which is not contiguous with a tissue or fluid which comprises the test protein; and c) comparing the contractility modulating effect or the angiogenic activity modulating effect in the first cell or tissue with the contractility modulating effect or the angiogenic activity modulating effect in the second cell or tissue. If the contractility modulating effect or the angiogenic activity modulating effect of one or more of the uPA kringle, the uPA growth factor receptor and the connecting peptide is greater in the first cell or tissue relative to the second cell or tissue, then the test protein is a binding protein of one or more of the uPA kringle, the uPA growth factor receptor and the connecting peptide.

Also included in the invention is a method of identifying a functional element of one or more of the uPA kringle, the uPA growth factor domain and the connecting peptide, the functional element participating in the modulation of contractility or angiogenic activity of a mammalian muscle or endothelial cell or tissue. The method comprises a) preparing one or more mutant polypeptides which lack a portion of the amino acid sequence of one or more of the uPA kringle, the uPA growth factor domain and the connecting peptide; b) assessing the ability of each of the mutant polypeptides to modulate the contractility or angiogenic activity of a mammalian muscle or endothelial cell or tissue once provided to the cell or tissue, or to a tissue or fluid which is contiguous with the cell or tissue; c) identifying, from b) a mutant polypeptide which is not able to modulate the contractility or angiogenic activity of a mammalian muscle or endothelial cell or tissue; and d) determining from c) and a) the corresponding deleted portion of the amino acid sequence of one or more of the uPA kringle, the uPA growth factor domain and the connecting peptide which participates in the modulation of muscle or endothelial cell or tissue contractility or angiogenic activity, whereby a functional element of one or more of the uPA kringle, the uPA growth factor domain and the connecting peptide is identified.

The invention also includes a method of treating a vascular disease or condition in a mammal having as a symptom thereof abnormally high fibrin clot formation. The method comprises a) administering to the mammal a composition comprising one or more of .DELTA.kringle-scuPA, .DELTA.kringle-tcuPA, an antagonist of the uPA kringle and an antagonist of a binding protein of the uPA kringle in an amount effective to inhibit the contractility of a mammalian vascular smooth muscle cell or tissue, wherein the .DELTA.kringle-scuPA and .DELTA.kringle-tcuPA share at least about 75% homology with the polypeptide corresponding to SEQ ID NO:7; b) providing the composition to an affected vascular smooth muscle cell or tissue of the cardiovascular system of the mammal which has or is prone to excessive fibrin clot formation, or to a tissue or fluid which is contiguous therewith; and c) vasodilating the affected vascular smooth muscle cell or tissue by inhibiting the contractility of the affected vascular smooth muscle cell or tissue, thereby promoting both fibrin clot lysis and vasodilation in the affected area of the vasculature of the mammal, thereby treating the vascular disease or condition.

The invention also includes a kit for treating a disease or condition in a mammal, the disease or condition having as a symptom thereof one or more of abnormal muscle cell or tissue contractility and abnormal angiogenic activity. The kit comprises a) a composition comprising a polypeptide, the polypeptide comprising one or more of the uPA kringle, the uPA growth factor domain, and the connecting peptide 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; and b) an instructional material.

In one aspect, the kit further comprises a sterile solvent suitable for dissolving or suspending the composition prior to administering the composition to the mammal.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the finding that several domains of the urokinase-type plasminogen activator (uPA) are effective for modulating the contractility or modulating the angiogenic activity of a mammalian muscle or endothelial cell or tissue. The invention includes compositions and methods for using these uPA domains advantageously to modulate the contractility and/or the angiogenic activity of a mammalian muscle or endothelial cell or tissue in the treatment of a disease or condition having abnormal muscle cell or tissue contractility or abnormal muscle or endothelial cell or tissue angiogenic activity as a symptom thereof, or wherein inducing relaxation of a muscle cell or tissue or modulating angiogenesis would be efficacious to the mammal.

The invention also includes methods for identifying and using binding proteins as well as agonists and antagonists of these domains of uPA in the treatment of a disease or condition having abnormal muscle cell or tissue contractility or abnormal angiogenic activity as a symptom thereof. In addition, agonists or antagonists to the binding proteins are used in the methods of the invention for the treatment of a disease or condition having abnormal muscle cell or tissue contractility or abnormal angiogenic activity as a symptom thereof.

Description

The invention includes compositions comprising the uPA kringle or the growth factor domain alone, or in combination with one or more other domains of the uPA molecule, in an amount effective to modulate one or more of the contractility of a mammalian muscle cell or tissue and the angiogenic activity of a mammalian muscle or endothelial cell or tissue. The compositions of the invention are useful for the treatment of a disease or condition in a mammal having as a symptom thereof one or more of abnormal muscle cell or tissue contractility and abnormal muscle or endothelial cell or tissue angiogenic activity. The domain or domains of uPA can be present in the inventive compositions and methods either as an isolated polypeptide, as part of the whole uPA molecule in either single chain or two chain form (scuPA or tcuPA, respectively), or as part of a deletion mutant thereof, which lacks either the uPA kringle or the connecting peptide.

The inventive method comprises administering to a mammal afflicted with such a disease or condition an effective amount of an inventive composition, and modulating the contractility and/or the angiogenic activity of the muscle or endothelial cell or tissue, thereby treating the disease or condition. The invention also includes methods for identifying and using binding proteins, agonists and antagonists for one or more domains of uPA to modulate muscle cell or tissue contractility and/or angiogenic activity, as well as using agonists and antagonists to the binding proteins themselves to modulate muscle or endothelial cell or tissue contractility and/or angiogenic activity, thereby treating the disease or condition.

In the methods and compositions of the invention, the disease or condition can be any disease or condition in a mammal in which one or more of abnormal muscle cell or tissue contractility and abnormal angiogenic activity is a symptom. Such diseases and conditions include, by way of example and not by limitation, the following: cardiovascular diseases and conditions such as hypotension, hypertension and atherosclerosis; conditions which promote thrombotic disorders such as stroke, heart attack, microvascular occlusions, thrombotic microangiopathies, and surgically induced thrombotic disorders (i.e. post angioplasty stenting); angiogenic disorders; respiratory diseases and conditions such as pulmonary fibrosis and asthma; invasion, angiogenesis and metastasis of cancer or tumor cells; ocular disorders such as glaucoma and diabetic retinopathy; wound healing disorders, disorders of fibrinolysis and clotting; and reproductive disorders such as uterine contraction disorders and male impotence.

In the methods and compositions of the invention, the cell can be any type of mammalian muscle cell or vascular endothelial cell. Types of muscle cells include a smooth muscle cell, a striated muscle cell and a cardiac muscle cell. Preferably, the cell is a smooth muscle cell. Examples of preferred smooth muscle cells include vascular and bronchial smooth muscle cells.

In preferred embodiments of the invention, the mammalian muscle or endothelial cell is a part of a mammalian muscle tissue, and is in a mammal. Preferably, the inventive composition comprises the one or more domains of uPA in an amount effective to modulate the contractility and/or angiogenic activity of a mammalian muscle or endothelial tissue.

The invention includes a composition comprising the 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 is a polypeptide portion of the urokinase-type plasminogen activator protein which shares at least about 75% homology with the polypeptide corresponding to SEQ ID NO:1 (FIG. 1A, see Original Patent). Preferably, the uPA kringle is about 80% homologous, more preferably about 85% homologous, even more preferably 90% homologous, yet more preferably 95% homologous, and most preferably about 99% homologous to the polypeptide corresponding to SEQ ID NO:1. Even more preferably, the uPA kringle is the polypeptide corresponding to SEQ ID NO:1.

The determination of percent homology (i.e. percent identity) described herein between two amino acid or nucleotide sequences can be accomplished using a mathematical algorithm. For example, a mathematical algorithm useful for comparing two sequences is the algorithm of Karlin and Altschul (1990, Proc. Natl. Acad. Sci. USA 87:2264-2268), modified as in Karlin and Altschul (1993, Proc. Natl. Acad. Sci. USA 90:5873-5877).

This algorithm is incorporated in the NBLAST and XBLAST programs of Altschul, et al. (1990, J. Mol. Biol. 215:403-410), and can be accessed, for example, at the National Center for Biotechnology Information (NCBI) world wide web site "www.ncbi.nlm.nih.gov/BLAST/". BLAST nucleotide searches can be performed with the NBLAST program (designated "blastn" at the NCBI web site), using the following parameters: gap penalty=5; gap extension penalty=2; mismatch penalty=3; match reward=1; expectation value 10.0; and word size=11 to obtain nucleotide sequences homologous to a nucleic acid described herein. BLAST protein searches can be performed with the XBLAST program (designated "blastn" at the NCBI web site) or the NCBI "blastp" program, using the following parameters: expectation value 10.0, BLOSUM62 scoring matrix to obtain amino acid sequences homologous to a protein molecule described herein.

To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997, Nucleic Acids Res. 25:3389-3402). Alternatively, PSI-Blast or PHI-Blast can be used to perform an iterated search which detects distant relationships between molecules (id.) and relationships between molecules which share a common pattern.

When utilizing BLAST, Gapped BLAST, PSI-Blast, and PHI-Blast, programs, the Default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

The percent identity between two amino acid or nucleotide sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically exact matches are counted.

In a preferred embodiment, the uPA kringle is present in an amount effective to enhance or disinhibit the contractility of a mammalian smooth muscle cell or tissue. In another preferred embodiment, the uPA kringle is present in an amount effective to inhibit the angiogenic activity of a vascular smooth muscle cell or tissue or a vascular endothelial cell or tissue.

Preferably, the uPA kringle is present in the inventive composition at a concentration ranging from about ten picomolar to about one hundred micromolar.

In one embodiment, the uPA kringle is present in the inventive composition as an isolated kringle. An isolated kringle is an isolated polypeptide which comprises the uPA kringle. The isolated kringle can be prepared by any method known to the skilled artisan for preparing an isolated polypeptide.

For example, the isolated kringle can be obtained by preparing and purifying a recombinant version of any of the polypeptides described herein which comprise the uPA kringle. Molecular biology techniques for the preparation of recombinant polypeptides are well known in the art, and are described for example in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York; Ausubel et al., 1994, Current Protocols in Molecular Biology, John Wiley & Sons, New York), and Gerhardt et al., eds., 1994, Methods for General and Molecular Bacteriology, American Society for Microbiology, Washington, D.C. Protein purification methods are also well known in the art, and are described, for example in Deutscher et al. (ed., 1990, Guide to Protein Purification, Harcourt Brace Jovanovich, San Diego).

In a preferred aspect, the isolated kringle is prepared by performing a limited proteolysis of scuPA, then purifying the resulting polypeptide using reverse-phase HPLC, as described herein in the Examples.

The invention also includes a composition comprising the uPA growth factor domain in an amount effective to modulate the contractility and/or the angiogenic activity of a mammalian muscle or endothelial cell. The growth factor domain is a polypeptide portion of the urokinase-type plasminogen activator protein which shares at least about 75% homology with the polypeptide corresponding to SEQ ID NO:2 (FIG. 1B, see Original Patent). Preferably, the growth factor domain is about 80% homologous, more preferably about 85% homologous, even more preferably 90% homologous, yet more preferably 95% homologous, and most preferably about 99% homologous to the polypeptide corresponding to SEQ ID NO:2. Even more preferably, the growth factor domain is the polypeptide corresponding to SEQ ID NO:2.

In a preferred embodiment, the uPA growth factor domain is present in an amount effective to inhibit the contractility of a mammalian smooth muscle cell or tissue. Preferably, the uPA growth factor domain is present in the inventive composition at a concentration ranging from about 10 picomolar to about 100 micromolar.

In one embodiment, the growth factor domain is present in the inventive composition as an isolated growth factor domain. An isolated growth factor domain is an isolated polypeptide which comprises the uPA growth factor domain. The isolated growth factor domain can be prepared by any method known to the skilled artisan for preparing an isolated polypeptide. For example, the isolated growth factor domain can be obtained by preparing and purifying a recombinant version of any of the polypeptides described herein which comprise the growth factor domain. Molecular biology techniques for the preparation of recombinant polypeptides are well known in the art, and are discussed above.

In other embodiments, the inventive composition comprises, in addition to the uPA kringle or the growth factor domain, one or more additional domains of the uPA protein selected from the group consisting of the connecting peptide, the protease domain, the uPA kringle and the growth factor domain.

In one aspect, the composition of the invention comprises the full uPA protein. The full uPA protein comprises, from the N-terminal end to the C-terminal end, respectively, the growth factor domain, the kringle, the connecting peptide, and the protease domain. The full uPA protein is a polypeptide which shares at least about 75% homology with the polypeptide corresponding to SEQ ID NO:3 (FIG. 1C, see Original Patent). Preferably, the full uPA protein is about 80% homologous, more preferably about 85% homologous, even more preferably 90% homologous, yet more preferably 95% homologous and most preferably about 99% homologous to the polypeptide corresponding to SEQ ID NO:3. Even more preferably, the full uPA protein is the polypeptide corresponding to SEQ ID NO:3.

The inventive composition can comprise the full uPA protein in nascent form, which is termed single chain urokinase (scuPA). Single chain urokinase (scuPA) is the form of the urokinase-type plasminogen activator protein which is synthesized by mammalian cells as the parent molecule. In this embodiment, the growth factor domain of scuPA is present in an amount effective to inhibit the contractility of a mammalian muscle cell. Preferably, the growth factor domain is present in an amount effective to cause a dose-dependent inhibition of acetylcholine induced contraction of a bronchial smooth muscle cell.

The molecule scuPA is capable of being converted by either an autoactivation or by a protease to a form termed two chain urokinase (tcuPA). Proteases capable of converting scuPA to tcuPA include plasmin and kallikrein. The two chain form of urokinase is produced by proteolytic cleavage of scuPA between amino acid residues number 158 and 159 of the full uPA protein, and the resulting tcuPA molecule is held together in two chain form by disulfide bonds.

In another aspect, the inventive composition comprises tcuPA, a polypeptide which shares at least about 75% homology with the polypeptide corresponding to SEQ ID NO:3 (FIG. 1C). Preferably, tcuPA is about 80% homologous, more preferably about 85% homologous, even more preferably 90% homologous, yet more preferably 95% homologous, and most preferably about 99% homologous to the polypeptide corresponding to SEQ ID NO:3. Even more preferably, tcuPA is the polypeptide corresponding to SEQ ID NO:3. In this embodiment, tcuPA is present in an amount effective to modulate the contractility of a mammalian muscle cell.

In one preferred aspect, the uPA kringle is present in the inventive composition in an amount effective to enhance or disinhibit PE-induced contraction of a mammalian vascular smooth muscle cell or tissue.

In another preferred aspect, the uPA kringle is present in the inventive composition in an amount effective to inhibit AC-induced contraction of a mammalian bronchial smooth muscle cell or tissue.

In another embodiment, the inventive composition comprises the amino terminal fragment of uPA (ATF). The ATF is a polypeptide portion of the full uPA protein which comprises the growth factor domain and the uPA kringle. The ATF shares at least about 75% homology with the polypeptide corresponding to SEQ ID NO:4 (FIG. 1D, see Original Patent). Preferably, the ATF is about 80% homologous, more preferably about 85% homologous, even more preferably 90% homologous, yet more preferably 95% homologous, and most preferably about 99% homologous to the polypeptide corresponding to SEQ ID NO:4. Even more preferably, the ATF is the polypeptide corresponding to SEQ ID NO:4.

In embodiments of the inventive composition comprising the ATF, the ATF is present in an amount effective to modulate the contractility or angiogenic activity of a mammalian muscle or endothelial cell or tissue. Preferably, the uPA kringle of ATF is present in the inventive composition in an amount effective to enhance or disinhibit PE-induced contraction of a mammalian vascular smooth muscle cell or tissue or in an amount effective to inhibit the angiogenic activity of a vascular smooth muscle cell or tissue or a vascular endothelial cell or tissue.

In one embodiment, the ATF is present in the inventive composition as an isolated ATF. An isolated ATF is an isolated polypeptide which comprises the uPA kringle and the uPA growth factor domain. The isolated ATF can be prepared by any method known to the skilled artisan for preparing an isolated polypeptide. For example, the isolated ATF can be obtained by preparing and purifying a recombinant version of any of the polypeptides described herein which comprise the ATF. Molecular biology techniques for the preparation of recombinant polypeptides are well known in the art, and are discussed above.

Preferably, the isolated ATF is prepared by autodigestion of tcuPA. In another preferred aspect, where the polypeptide of the invention comprises both the ATF and the connecting peptide, the polypeptide is prepared by matrilysin (MMP-7) or stromelysin (MMP-3) digestion of uPA.

In any of the compositions and methods of the invention described herein, a functional element, an analog, an epitope, or a chimeric peptide can be used in place of one or more of the isolated uPA kringle, the isolated growth factor domain and the isolated ATF. This enables the use of a smaller polypeptide in the inventive compositions having similar or greater activity than the corresponding larger isolated polypeptide for modulating muscle cell contractility or angiogenic activity. Methods for identifying a functional element, an epitope, an analog or a chimeric polypeptide for any of these isolated polypeptides are described herein and known in the art. By way of example and not by limitation, such functional elements, analogs, epitopes and chimeric peptides can be prepared using recombinant technology or isolated from natural sources. Alternatively, they can be prepared synthetically by using any polypeptide synthesis method known in the art, such as a solid-phase polypeptide synthesis method.

The invention also includes a composition comprising a polypeptide which comprises the connecting peptide and the protease domains of uPA, in an amount effective to modulate the contractility of a mammalian muscle cell or tissue. This polypeptide is termed LMW-uPA, and shares at least about 75% homology with the polypeptide corresponding to SEQ ID NO:5 (FIG. 1E, see Original Patent). Preferably, the LMW-uPA is about 80% homologous, more preferably about 85% homologous, even more preferably 90% homologous, yet more preferably 95% homologous, and most preferably about 99% homologous to the polypeptide corresponding to SEQ ID NO:5. Even more preferably, the LMW-uPA is the polypeptide corresponding to SEQ ID NO:5.

In a preferred embodiment, the LMW-uPA is present in the inventive composition in an amount effective to inhibit the contractility of a mammalian smooth muscle cell or tissue.

The invention also includes a composition comprising a polypeptide deletion mutant of either scuPA or tcuPA in which the connecting peptide has been deleted. Methods for preparing deletion mutants of wild type polypeptides are known in the art, and a preferred method is described herein in the Examples.

In one embodiment, a uPA deletion mutant lacking the connecting peptide (scuPA.sup..DELTA.136-143) is present in the inventive composition in an amount effective to modulate the contractility and/or the angiogenic activity of a mammalian muscle or endothelial cell. The scuPA.sup..DELTA.136-143 shares at least about 75% homology with the polypeptide corresponding to SEQ ID NO:6 (FIG. 1F, see Original Patent). Preferably, the uPA deletion mutant is about 80% homologous, more preferably about 85% homologous, even more preferably 90% homologous, yet more preferably 95% homologous and most preferably about 99% homologous to the polypeptide corresponding to SEQ ID NO:6. Even more preferably, the uPA deletion mutant is the polypeptide corresponding to SEQ ID NO:6.

In a preferred embodiment, scuPA.sup..DELTA.136-143 is present in the inventive composition in an amount effective to enhance or disinhibit the contractility of a mammalian smooth muscle cell.

The invention also includes a composition comprising a polypeptide deletion mutant of either scuPA or tcuPA in which the uPA kringle has been deleted. Methods for preparing deletion mutants of wild type polypeptides are known in the art, and a preferred method is described herein in the Examples.

In one embodiment, a uPA deletion mutant of scuPA or tcuPA lacking the uPA kringle (.DELTA.kringle-scuPA or .DELTA.kringle-tcuPA) is present in the inventive composition in an amount effective to modulate the contractility and/or the angiogenic activity of a mammalian muscle or endothelial cell. The .DELTA.kringle-scuPA shares at least about 75% homology with the polypeptide corresponding to SEQ ID NO:7 (FIG. 1G, see Original Patent). Preferably, the uPA deletion mutant is about 80% homologous, more preferably about 85% homologous, even more preferably 90% homologous, yet more preferably 95% homologous and most preferably about 99% homologous to the polypeptide corresponding to SEQ ID NO:7. Even more preferably, the uPA deletion mutant is the polypeptide corresponding to SEQ ID NO:7.

In a preferred embodiment, one or more of .DELTA.kringle-scuPA and .DELTA.kringle-tcuPA is present in the inventive composition in an amount effective to inhibit the contractility of a mammalian smooth muscle cell and to proteolytically activate plasminogen.

In another embodiment, the inventive composition comprises a polypeptide comprising the ATF and the connecting peptide in an amount effective to modulate one or more of the contractility and angiogenic activity of a mammalian muscle or endothelial cell or tissue. In this aspect, the polypeptide comprising the ATF and the connecting peptide shares at least about 75% homology with the polypeptide corresponding to SEQ ID NO:8 (FIG. 1H, see Original Patent). Preferably, the polypeptide is about 80% homologous, more preferably about 85% homologous, even more preferably 90% homologous, yet more preferably 95% homologous, and most preferably about 99% homologous to the polypeptide corresponding to SEQ ID NO:8. Even more preferably, the polypeptide is the polypeptide corresponding to SEQ ID NO:8. Without wishing to be bound by any one theory, it is suspected that the connecting peptide domain, when present in the same polypeptide with the ATF, results in a polypeptide which is more effective at modulating angiogenic activity than the ATF alone, possibly by affecting cell adhesion.

The invention also includes a composition comprising a polypeptide which comprises the uPA kringle and the connecting peptide, 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. This polypeptide shares at least about 75% homology with the polypeptide corresponding to SEQ ID NO:9 (FIG. 1I, see Original Patent). Preferably, the polypeptide is about 80% homologous, more preferably about 85% homologous, even more preferably 90% homologous, yet more preferably 95% homologous, and most preferably about 99% homologous to the polypeptide corresponding to SEQ ID NO:9. Even more preferably, the polypeptide is the polypeptide corresponding to SEQ ID NO:9.

In another embodiment, any of the compositions of the invention described above can further comprise an inducing compound present in an amount effective to mediate contraction of a mammalian muscle cell. The inducing compound can be any compound capable of mediating the contraction of a mammalian muscle cell, and includes, by way of example and not by limitation, neurotransmitter compounds such as phenylepherine (PE), acetylcholine (AC), epinepherine and endothelin. The amount of such compounds to be effective for mediating contraction of a mammalian muscle cell will be apparent to the skilled artisan.

In another embodiment, the inventive composition comprises any of the compositions of the invention described above, and further comprises one or more compounds suitable for one or more of promoting, enhancing, prolonging and amplifying the effectiveness of the composition of the invention for modulating the contractility and/or the angiogenic activity of a mammalian muscle or endothelial cell or tissue. In one aspect, the one or more compounds are selected from the group consisting of agonists, antagonists and binding proteins of any one or more domains of uPA. Methods of identifying agonists, antagonists and binding proteins to one or more domains of uPA are described herein. Also, the one or more compounds can be agonists or antagonists to the binding proteins themselves.

The invention also includes an isolated nucleic acid encoding a polypeptide comprising one or more of the domains of uPA described herein. The isolated nucleic acid shares at least about 75% homology, preferably about 80% homology, more preferably about 85% homology, even more preferably about 90% homology, yet more preferably 95% homology, and most preferably about 99% homology with a nucleic acid selected from the group consisting of SEQ ID NO:10 (FIG. 1J), SEQ ID NO:11 (FIG. 1K), SEQ ID NO:12 (FIG. 1L), SEQ ID NO:13 (FIG. 1M), SEQ ID NO:14 (FIG. 1N), SEQ ID NO:15 (FIG. 1O), SEQ ID NO:16 (FIG. 1P), SEQ ID NO:17 (FIG. 1Q) and SEQ ID NO:18 (FIG. 1R). Even more preferably, the nucleic acid is selected from the group consisting of SEQ ID NO:10-SEQ ID NO:18. In this embodiment of the invention, the isolated nucleic acid is present in an amount effective to transform a mammalian muscle or endothelial cell to provide transgene expression of the one or more polypeptide domains of uPA at a level of expression effective to modulate the contractility and/or angiogenic activity of the mammalian muscle or endothelial cell so transfected.

The isolated nucleic acid can be either alone as a "naked" nucleic acid, such as a linearized nucleic acid, or as a component of any type of vector suitable for transfecting a mammalian muscle or endothelial cell described herein or known in the art. Preferably, the isolated nucleic acid is a recombinant polynucleotide component of a viral or plasmid expression vector suitable for transfecting a mammalian muscle or endothelial cell, and is operably linked to the appropriate regulatory elements to provide a high level of expression of the transgene once a targeted mammalian muscle or endothelial cell is transformed with the isolated nucleic acid. Examples of preferred vectors include adenovirus, retrovirus, lentivirus and adeno-associated virus vectors. Techniques for using such vectors to transfect a mammalian muscle or endothelial cell are known in the art.

When any one of the polypeptides described herein comprising one or more domains of uPA are to be administered to a mammal or to a tissue of a mammal for the purpose of exerting a beneficial effect in the mammal, the invention should be construed to include delivery of the polypeptide via delivery of an isolated nucleotide sequence encoding the peptide. Expression of the peptide from the nucleotide sequence so delivered to the desired tissue is effective administration of the peptide to the tissue.

In another embodiment, the inventive composition is in the form of a pharmaceutical composition comprising a pharmaceutically acceptable carrier. Such a pharmaceutical composition may consist of the inventive composition alone as the active ingredient, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise the inventive composition as the active ingredient and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The active ingredient may be present in the pharmaceutical composition in the form of a pharmaceutically acceptable salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.

As used herein, the term "pharmaceutically acceptable carrier" means a chemical composition with which the active ingredient may be combined and which, following the combination, can be used to administer the active ingredient to a subject.

As used herein, the term "pharmaceutically acceptable salt" means a salt form of the active ingredient which is compatible with any other ingredients of the pharmaceutical composition, which is not deleterious to the subject to which the composition is to be administered.

The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any mammal. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various mammals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation.

Pharmaceutical compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for any route of administration known to the skilled artisan, including, by way of example and not by limitation, oral, parenteral, topical, ocular, inhalation, intrauterine, intravesicular, intraurethral and buccal routes of administration. The pharmaceutical composition can be administered to a mammal by any route of administration known to the skilled artisan, such as those described above, and by any method of administering a pharmaceutical composition to a mammal known in the art. A pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses.

As used herein, a "unit dose" is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.

Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.

A formulation of a pharmaceutical composition of the invention suitable for oral administration may be prepared, packaged, or sold in the form of a discrete solid dose unit including, but not limited to, a tablet, a hard or soft capsule, a cachet, a troche, or a lozenge, each containing a predetermined amount of the active ingredient. Other formulations suitable for oral administration include, but are not limited to, a powdered or granular formulation, an aqueous or oily suspension, an aqueous or oily solution, or an emulsion.

As used herein, an "oily" liquid is one which comprises a carbon-containing liquid molecule and which exhibits a less polar character than water.

A tablet comprising the active ingredient may, for example, be made by compressing or molding the active ingredient, optionally with one or more additional ingredients. Compressed tablets may be prepared by compressing, in a suitable device, the active ingredient in a free-flowing form such as a powder or granular preparation, optionally mixed with one or more of a binder, a lubricant, an excipient, a surface active agent, and a dispersing agent. Molded tablets may be made by molding, in a suitable device, a mixture of the active ingredient, a pharmaceutically acceptable carrier, and at least sufficient liquid to moisten the mixture. Pharmaceutically acceptable excipients used in the manufacture of tablets include, but are not limited to, inert diluents, granulating and disintegrating agents, binding agents, and lubricating agents. Known dispersing agents include, but are not limited to, potato starch and sodium starch glycollate. Known surface active agents include, but are not limited to, sodium lauryl sulphate. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate. Known granulating and disintegrating agents include, but are not limited to, corn starch and alginic acid. Known binding agents include, but are not limited to, gelatin, acacia, pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Known lubricating agents include, but are not limited to, magnesium stearate, stearic acid, silica, and talc.

Tablets may be non-coated or they may be coated using known methods to achieve delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the active ingredient. By way of example, a material such as glyceryl monostearate or glyceryl distearate may be used to coat tablets. Further by way of example, tablets may be coated using methods described in U.S. Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to form osmotically-controlled release tablets. Tablets may further comprise a sweetening agent, a flavoring agent, a coloring agent, a preservative, or some combination of these in order to provide pharmaceutically elegant and palatable preparation.

Hard capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. Such hard capsules comprise the active ingredient, and may further comprise additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin.

Soft gelatin capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. Such soft capsules comprise the active ingredient, which may be mixed with water or an oil medium such as peanut oil, liquid paraffin, or olive oil.

Liquid formulations of a pharmaceutical composition of the invention which are suitable for oral administration may be prepared, packaged, and sold either in liquid form or in the form of a dry product intended for reconstitution with water or another suitable vehicle prior to use.

Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents. Oily suspensions may further comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose. Known dispersing or wetting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g. polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin and acacia. Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl-para-hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol.

Liquid solutions of the active ingredient in aqueous or oily solvents may be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent. Liquid solutions of the pharmaceutical composition of the invention may comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.

Powdered and granular formulations of a pharmaceutical preparation of the invention may be prepared using known methods. Such formulations may be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each of these formulations may further comprise one or more of dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations.

A pharmaceutical composition of the invention may also be prepared, packaged, or sold in the form of oil-in-water emulsion or a water-in-oil emulsion. The oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these. Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.

As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrastemal injection, and kidney dialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e. powder or granular) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

Formulations suitable for topical administration include, but are not limited to, liquid or semi-liquid preparations such as liniments, lotions, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes, and solutions or suspensions. Topically-administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets or lozenges made using conventional methods, and may, for example, 0.1 to 20% (w/w) active ingredient, the balance comprising an orally dissolvable or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise a powder or an aerosolized or atomized solution or suspension comprising the active ingredient. Such powdered, aerosolized, or aerosolized formulations, when dispersed, preferably have an average particle or droplet size in the range from about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein.

As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" which may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Genaro, ed., 1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., which is incorporated herein by reference.

Typically dosages of the composition of the invention which may be administered to an animal, preferably a human, range in amount from 1 microgram to about 100 grams per kilogram of body weight of the animal. While the precise dosage administered will vary depending upon any number of factors, including but not limited to, the type of animal and type of disease state being treated, the age of the animal and the route of administration. Preferably, the dosage of the composition will vary from about 1 mg to about 10 g per kilogram of body weight of the animal. More preferably, the dosage will vary from about 10 mg to about 1 g per kilogram of body weight of the animal.

The composition may be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, etc.

The invention also includes a method of treating a disease or condition in a mammal which has as a symptom thereof one or more of abnormal muscle cell or tissue contractility and abnormal muscle or endothelial cell or tissue angiogenic activity. The components used in the method of the invention are the same as those described above in the inventive compositions. The mammal can be any mammal described in the inventive compositions, but is preferably a human. The muscle or endothelial cell or tissue can be any of the mammalian cell or tissue types described in the inventive compositions. A preferred muscle cell is a smooth muscle cell.

The diseases or conditions in a mammal which can be treated by the method of the invention are the same diseases and conditions described above in the inventive compositions. By way of example and not by limitation, the method of the invention can be used to treat cardiovascular diseases and conditions such as hypotension, hypertension and atherosclerosis; conditions which promote thrombotic disorders such as stroke, heart attack, microvascular occlusions, thrombotic microangiopathies, and surgically induced thrombotic disorders (i.e. post angioplasty stenting); angiogenic disorders; respiratory diseases and conditions such as pulmonary fibrosis and asthma; invasion, angiogenesis and metastasis of cancer or tumor cells; ocular disorders such as glaucoma and diabetic retinopathy; wound healing disorders, disorders of fibrinolysis and clotting; and reproductive disorders such as uterine contraction disorders and male impotence.

In the method of the invention, any of the inventive compositions can be administered to the mammal to be treated by any route of administration known in the art or described herein. In one aspect, the inventive composition is administered to the mammal in the form of a pharmaceutical composition. The pharmaceutical composition can be any of the pharmaceutical compositions described herein in the inventive compositions.

In the method of the invention, any of the compositions of the invention may comprise an inducer compound in an amount effective to mediate the contraction of a mammalian muscle cell. The inducer compound can be any of those described in the inventive compositions.

Also, in the method of the invention, any of the compositions of the invention may further comprise one or more compounds known in the art to enhance and facilitate drug administration. Other possible formulations, such as nanoparticles, liposomes, resealed erythrocytes, and immunologically based systems such as antibody targeting systems may also be used to administer the inventive composition according to the methods of the invention.

The method comprises administering to a mammal afflicted with such a disease or condition a composition comprising the 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. In one aspect, the uPA kringle shares at least about 75% homology with a polypeptide having the amino acid sequence corresponding to SEQ ID NO:1. The uPA kringle component of the inventive composition can be obtained from any of the natural or recombinant sources described herein in the inventive compositions.

In one embodiment, the composition administered to the mammal comprises the uPA kringle as an isolated 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 isolated kringle can be prepared by any method described herein or known to the skilled artisan.

In another embodiment, the composition administered to the mammal comprises the uPA kringle as part of a polypeptide which shares at least about 75% homology with a polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO:3 (tcuPA), SEQ ID NO:4 (ATF), SEQ ID NO:6 (scuPA.sup..DELTA.136-143), SEQ ID NO:8 and SEQ ID NO:9. The uPA kringle is present in the composition 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.

In yet another embodiment, the composition comprises, in place of the uPA kringle, one or more of a functional element thereof, an epitope thereof, an analog thereof and a chimeric polypeptide thereof, in an amount effective to modulate one or more of the contractility and the angiogenic activity of a mammalian muscle or endothelial cell.

In a further embodiment, the composition of the invention administered to the mammal further comprises, in addition to the uPA kringle (or the one or more functional element thereof, epitope thereof, analog thereof and chimeric polypeptide thereof), one or more of an agonist of the uPA kringle, an agonist of a binding protein of the uPA kringle, an antagonist of the uPA growth factor domain, an antagonist of the connecting peptide, an antagonist of a binding protein of the uPA growth factor domain, and an antagonist of a binding protein of the connecting peptide. These agonists, antagonists and binding proteins are described herein below.

The inventive method also includes modulating one or more of the contractility and the angiogenic activity of the muscle or endothelial cell or tissue having one or more of abnormal contractility and abnormal angiogenic activity. The contractility and/or angiogenic activity of the muscle or endothelial cell or tissue can be either enhanced, disinhibited, or inhibited, relative to an otherwise identical muscle or endothelial cell or tissue in the mammal which is not provided the inventive composition. By modulating one or more of the contractility and the angiogenic activity of the muscle or endothelial cell or tissue, the disease or condition in the mammal is treated.

By way of example and not by limitation, where the disease or condition treated is tumor angiogenesis in a mammal, the angiogenic activity in the mammal is modulated by inhibiting the endothelial cell proliferation which is required to develop the tumor neovessels which are to supply the growing tumor, lest it outgrow its blood supply. By inhibiting endothelial cell proliferation in this context, the tumor is deprived of the vascularization necessary for continued growth, thereby treating the disease or condition of tumor angiogenesis.

Also, by way of example, and not by limitation, where the disease or condition treated is an ocular disorder such as a diabetic or sickle cell or other form of ischemic retinopathy, wherein poorly constructed vascular vessels are induced to grow, but are weak and rupture, leading to bleeding and impaired vision, the angiogenic activity in the mammal is modulated by enhancing the endothelial cell proliferation which is required to develop vascular vessels of proper strength to prevent rupture, thereby treating the disease or condition of ischemic retinopathy.

By way of a further example, and not by limitation, where the disease or condition treated is any one or more of post-coronary angioplasty, carotid endarterectomy, post-cardiac transplant, and atherosclerosis, wherein the proliferation of vascular smooth muscle cells in the media and migration of these cells through the internal elastic lanina into the vascular intima and subsequent proliferation of these cells with concomitant generation of atherogenic lipids occurs, the angiogenic activity in the mammal is modulated by inhibiting the vascular smooth muscle cell proliferation and migration associated with this phenomenon, thereby treating the disease or condition.

Also by way of example, and not by limitation, where the disease or condition in the mammal sought to be prevented is congestive heart failure (CHF), the contractility of cardiac muscle tissue is modulated by inhibiting tonic contraction of cardiac muscle tissue, thereby preventing the development of CHF. In contrast, where CHF has already developed, the CHF is treated by enhancing the contractility of cardiac muscle tissue in order to increase the stroke volume upon contraction of the cardiac muscle tissue.

A further non-limiting example, where the disease or condition treated in the mammal is either an inherited or an acquired dystrophic condition of a skeletal muscle tissue, the contractility of the skeletal muscle tissue is modulated by enhancing the contractility of the skeletal muscle tissue in order to increase or preserve muscle power, thereby treating the disease. In contrast, where the disease or condition treated involves skeletal muscle spasms which are deleterious, disadvantageous, or create discomfort in a mammal, the contractility of the affected skeletal muscle tissue is modulated by inhibiting the contractility of the skeletal muscle tissue.

In the following additional non-limiting examples, where the disease or condition treated in the mammal has as a symptom thereof abnormally high contractility of a smooth muscle cell or tissue, the contractility is modulated by inhibiting the contractility of the affected smooth muscle cell or tissue. By way of example and not by limitation, for abnormal uterine contraction such as premature labor, the contractility of uterine smooth muscle tissue is inhibited; for male impotence, the contractility of vascular smooth muscle tissue is inhibited to promote vasodilation; for angina, the contractility of vascular smooth muscle tissue is inhibited to promote vasodilation; for any type of inherited or acquired cause of thrombosis associated with premature fetal demise or spontaneous abortion, wherein the pathophysiology involves thrombosis/vascular contraction (including eclampsia), the contractility of vascular smooth muscle tissue is inhibited to promote vasodilation and to increase blood flow to the fetus; for asthma or for primary or secondary pulmonary hypertension, the contractility of vascular smooth muscle tissue is inhibited to promote vasodilation; for enhancing delivery of a thrombolytic agent to any part of the cardiovascular system of a mammal the contractility of vascular smooth muscle tissue is inhibited to promote vasodilation; and for inhibiting the spastic contraction of vascular smooth muscle tissue after vascular intervention such as after angioplasty, endarterectomy, thrombectomy br other vascular interventions, the contractility of vascular smooth muscle tissue is inhibited to reduce the frequency and intensity of the spastic contractions and to promote vasodilation.

In one preferred embodiment, the uPA kringle or functional element thereof, epitope thereof, analog thereof or chimeric polypeptide thereof is administered to the mammal in an amount effective to enhance or disinhibit the contractility of a mammalian smooth muscle cell or tissue and/or in an amount effective to inhibit the angiogenic activity of a vascular smooth muscle or vascular endothelial cell or tissue. Preferably, the smooth muscle cell or tissue is a vascular smooth muscle cell or tissue and the disease or condition treated is one or more of hypotension and cancer or tumor cell invasion, angiogenesis, growth and metastasis.

The invention also includes a method for treating a mammal afflicted with a disease or condition having as a symptom thereof one or more of abnormal contractility and abnormal angiogenic activity. The method comprises administering to the mammal a composition comprising the uPA growth factor domain 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. In one aspect, the uPA growth factor domain shares at least about 75% homology with a polypeptide having the amino acid sequence corresponding to SEQ ID NO:2. The uPA growth factor domain component of the inventive composition can be obtained from any of the natural or recombinant sources described herein in the inventive compositions.

In one embodiment, the composition administered to the mammal comprises the uPA growth factor domain as an isolated growth factor domain 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 isolated growth factor domain can be prepared by any method described herein or known to the skilled artisan.

In another embodiment, the composition administered to the mammal comprises the uPA growth factor domain as part of a polypeptide which shares at least about 75% homology with a polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO:3 (scuPA), SEQ ID NO:4 (ATF), SEQ ID NO:6 (scuPA.sup..DELTA.136-143), SEQ ID NO:7 (.DELTA.kringle-scuPA or .DELTA.kringle-tcuPA) and SEQ ID NO:8). The uPA growth factor domain is present in the polypeptide 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.

In yet another embodiment, the composition comprises, in place of the uPA growth factor domain, one or more of a functional element thereof, an epitope thereof, an analog thereof and a chimeric polypeptide thereof, 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.

In a further embodiment, the composition of the invention administered to the mammal further comprises, in addition to the uPA growth factor domain (or the one or more functional element thereof, epitope thereof, analog thereof and chimeric polypeptide thereof), one or more of an agonist of the uPA growth factor domain, an agonist of the connecting peptide, an agonist of a binding protein of the growth factor domain, an agonist of a binding protein of the connecting peptide, an antagonist of the uPA kringle, and an antagonist of a binding protein of the uPA kringle. These agonists, antagonists and binding proteins are described herein below.

The inventive method also includes one or more of modulating the contractility and modulating the angiogenic activity of the muscle or endothelial cell or tissue having one or more of abnormal contractility and abnormal angiogenic activity. The contractility of the muscle cell or tissue can be either inhibited, disinhibited or enhanced, relative to an otherwise identical muscle cell or tissue in the mammal which is not provided the inventive composition. By modulating one or more of the contractility and the angiogenic activity of the muscle or endothelial cell or tissue, the disease or condition in the mammal is treated.

In one preferred aspect, the uPA growth factor domain or functional element thereof, epitope thereof, analog thereof or chimeric polypeptide thereof is administered to the mammal in an amount effective to inhibit the contractility of a mammalian smooth muscle cell or tissue and/or to modulate the angiogenic activity of the smooth muscle or endothelial cell. In one preferred aspect, the smooth muscle cell or tissue is a vascular smooth muscle cell or tissue and the disease or condition treated is hypertension.

Optionally, the inventive method can include providing the inventive composition to the interior of a muscle or endothelial cell or tissue having abnormal contractility and/or abnormal angiogenic activity. The inventive composition can be provided to the cell or tissue either alone in "naked" form, for example as an isolated polypeptide, or formulated in a vehicle suitable for delivery, such as, by way of example and not by limitation, in a complex with a cationic molecule or a liposome forming lipid, in a vector, or as a component of a pharmaceutical composition. Such vehicles are well known to the skilled artisan.

The inventive composition can be provided to the cell either directly, by contacting the inventive composition with the cell sought to be modulated, or indirectly, such as through the action of any biological process. By way of example and not by limitation, the inventive composition can be provided to the cell by using a liposome, by contacting the composition with the cell, by transfecting the cell using a vector, by injecting the composition into a tissue or fluid surrounding the cell, by simple diffusion of the composition across the cell membrane, or by any active or passive transport mechanism across the cell membrane. In addition, any of the methods known in the art for providing targeted or in situ delivery of the inventive composition to the interior of a desired cell or to a targeted tissue can be used. By way of example and not by limitation, antibody targeting is one of such methods.

In embodiments of the inventive method where the disease or condition in the mammal sought to be treated is a respiratory disease or condition having as a symptom thereof abnormally high bronchial smooth muscle cell contractility (i.e. excessive bronchoconstriction), the inventive method comprises administering an inventive composition to the mammal which is effective at inhibiting the acetylcholine (AC) mediated contraction of a bronchial smooth muscle cell or tissue. Inventive compositions comprising the uPA kringle as an isolated kringle or as a part of the ATF, tcuPA or the deletion mutant scuPA.sup..DELTA.136-143 are capable of inhibiting the contractility of a bronchial smooth muscle cell or tissue, and thus are potent bronchodilators which can be used to treat such respiratory diseases or conditions. Examples of preferred respiratory diseases and conditions in this embodiment include asthma, adult respiratory distress syndrome, primary pulmonary hypertension, microvascular thrombotic occlusion and disorders associated with chronic intrapulmonary fibrin formation.

In contrast, in embodiments of the inventive method where the disease or condition in the mammal sought to be treated is characterized by abnormally low vascular smooth muscle cell or tissue contractility (e.g., hypotension) the uPA kringle is administered as part of an inventive composition capable of enhancing PE-induced contraction of a mammalian vascular smooth muscle cell or tissue. For example, in these embodiments, the uPA kringle is administered as an isolated kringle or as a part of a polypeptide of the invention such as ATF, tcuPA, scuPA.sup..DELTA.136-143, ATF+connecting peptide or uPA kringle+connecting peptide.

In other embodiments, where the disease or condition of the mammal sought to be treated is characterized by abnormally high vascular smooth muscle cell or tissue contractility (e.g., hypertension), the inventive method comprises administering to the mammal an inventive composition comprising the uPA growth factor domain as an isolated growth factor domain, or as a part of a polypeptide of the invention such as scuPA or .DELTA.kringle-scuPA or .DELTA.kringle-tcuPA, in an amount effective to inhibit PE-induced contraction of a vascular smooth muscle cell or tissue.

The invention also includes a method of identifying a compound which is an agonist or antagonist of one or more of the uPA kringle or a binding protein thereof, the uPA growth factor domain or a binding protein thereof, and the connecting peptide or a binding protein thereof, upon the contractility or angiogenic activity of a mammalian muscle or endothelial cell or tissue. The compound can be any type of compound, and includes by way of example and not by limitation, a drug, a peptide, a polypeptide, a protein, an oligonucleotide, a nucleic acid, a ligand, and an antibody.

The agonist can be any compound capable of enhancing, amplifying or promoting the effect of the domain of uPA or binding protein thereof upon the contractility or angiogenic activity of a mammalian muscle or endothelial cell or tissue. The agonist can also be any compound which is capable of enhancing, amplifying or promoting a transduction pathway or degradation pathway of the domain of uPA or binding protein thereof. The antagonist can be any compound which is a competitive inhibitor or other inhibitor of the effect of the domain of uPA or binding protein thereof upon the contractility or angiogenic activity of a mammalian muscle or endothelial cell or tissue. The antagonist can also be any compound which is a competitor or inhibitor of a transduction pathway or degradation pathway of the domain of uPA or binding protein thereof. The binding protein can be any protein capable of specifically binding with one or more domains of uPA.

The method comprises assessing the contractility or angiogenic activity of a first mammalian muscle or endothelial cell and an otherwise identical second mammalian muscle or endothelial cell. The contractility or angiogenic activity of the cells can be assessed by any method known in the art or described herein for evaluating the contractility or angiogenic activity of a muscle or endothelial cell or tissue. A preferred method for assessing contractility is described herein in the experimental Examples.

The angiogenic activity of the cells and the effectiveness of a test compound for modulating angiogenic activity can be assessed by examining the growth and migration of cultured endothelial cells or smooth muscle cells in progressively less luxurious media and in response to known chemotactic agents in vitro in the presence or absence of test compound. Cultures can be wounded, and the proliferation and capacity of cells to repair the wound in the presence or absence of test compound can be examined by assays known in the art. In vitro assays can be used such as placing growth factors in subcutaneous pockets of laboratory animals and measuring the ingrowth of vessels. One assay known in the art involves the use of chicken allantoic membranes (CAMs), where CAMs are implanted, and the vessel growth in response to locally infused agents for promoting angiogenesis is measured. A test compound can also be administered to an animal with a spontaneous or implanted tumor, and the angiogenic response of the tumor is evaluated by evaluating its vasculature.

The method thereafter comprises providing to the first cell and the second cell a composition of the invention comprising one or more of the uPA kringle, the uPA growth factor domain and the connecting peptide, in an amount effective to modulate the contractility or angiogenic activity of a mammalian muscle or endothelial cell or tissue. The composition is provided to the first and second cells by any method of providing a composition to a cell described herein. The composition need not be provided to the interior of the cells, but instead, where the first and second cells are in a mammal or in a tissue, the composition of the invention need only be provided to the vascular system of the tissue or mammal.

The method also includes providing to the first cell a compound suspected to be an agonist or antagonist of one or more of the uPA kringle or a binding protein thereof, the uPA growth factor domain or a binding protein thereof, and the connecting peptide or a binding protein thereof, upon the contractility or angiogenic activity of a mammalian muscle or endothelial cell or tissue. The compound is provided to the first cell by any of the methods described herein or known in the art for providing a composition or compound to a cell. The compound need not be provided to the interior of the cell, since a binding protein of the uPA kringle, the uPA growth factor domain, and the connecting peptide can be found either inside or outside of the cell (i.e., as a soluble protein). The compound is provided to the first cell in an amount suspected to be effective to modulate the effect of the composition of the invention upon the contractility or angiogenic activity of a mammalian muscle or endothelial cell or tissue. The compound can be provided to the first cell either simultaneous with, prior to, or subsequent to providing the first cell the composition of the invention.

The method includes assessing the contractility or the angiogenic activity of the first cell after providing to the first cell both the composition of the invention and the compound, and assessing the contractility or the angiogenic of the second cell, which was not provided the compound, after providing to the second cell the composition of the invention.

A preferred method for evaluating the contractility of a mammalian cell or tissue is described herein in the experimental Examples. Briefly, the contractility of a cell and the effect upon the contractility of a cell can be assessed by determining whether an increase or decrease is observed in the EC.sub.50 (50% effective concentration) for an inducing compound which mediates contraction of the cell. For example, the EC.sub.50 can be evaluated for an inducing compound such as PE or AC to determine whether the EC.sub.50 changes in the first cell after administration of the compound and the composition of the invention. In comparing any change in the EC.sub.50 observed in the first cell with the EC.sub.50 for PE in the second cell which is provided the composition of the invention but not the compound, a decrease in the EC.sub.50 of a cell for PE is an indication of enhanced or disinhibited contractility in the cell, whereas an increase in the EC.sub.50 of a cell for PE is an indication of inhibited contractility in the cell. Such methods are described herein in the experimental Examples, which describe methods for determining the EC.sub.50 for an inducing compound such as PE or AC in a muscle cell during contraction.

The method also comprises determining the effect upon the contractility or angiogenic activity of the first cell by comparing the contractility or angiogenic activity of the first cell before providing the composition of the invention with the contractility or angiogenic activity of the first cell after providing both the compound and the composition of the invention. The effect upon the contractility or angiogenic activity of the second cell is also determined by comparing the contractility or angiogenic activity of the second cell before providing the composition of the invention with the contractility or angiogenic activity of the second cell after providing the composition of the invention.

The method thereafter comprises comparing the effect upon contractility or angiogenic activity observed in the first cell with the effect upon contractility or angiogenic activity observed in the second cell. If the effect of the composition of the invention upon contractility or angiogenic activity in the first cell is either increased or decreased by at least about ten percent relative to the effect of the composition of the invention upon contractility or angiogenic activity in the second cell, then a compound is identified which is an agonist or antagonist of one or more of the uPA kringle or a binding protein thereof, the uPA growth factor domain or a binding protein thereof, and the connecting peptide or a binding protein thereof, upon the contractility or angiogenic activity of a mammalian muscle or endothelial cell or tissue.

The invention also includes a method of treating a disease or condition in a mammal having as a symptom thereof one or more of abnormal muscle cell or tissue contractility and abnormal angiogenic activity. The disease or condition can be any of the diseases or conditions described herein. Preferably, the disease or condition is one which has as a symptom thereof abnormal vascular smooth muscle cell or tissue contractility or abnormal bronchial smooth muscle cell or tissue contractility.

The method comprises administering to the mammal an amount suspected to be effective for modulating the contractility and/or angiogenic activity of a mammalian muscle or endothelial cell or tissue of an agonist or antagonist of one or more of the uPA kringle or a binding protein thereof, the uPA growth factor domain or a binding protein thereof, and the connecting peptide or a binding protein thereof. The agonist or antagonist can be administered to the mammal either alone or in the form of a pharmaceutical composition as described herein. The compound can be administered by any route of administration described herein or known to the skilled artisan for administering a compound to a mammal.

Preferably, the agonist or antagonist is administered to the mammal in an amount effective to modulate the effect of one or more of the uPA kringle or a binding protein thereof, the uPA growth factor domain or a binding protein thereof, and the connecting peptide or a binding protein thereof upon the contractility of a muscle tissue in the mammal.

The method comprises providing the agonist or antagonist to a muscle or endothelial cell or tissue in the mammal having abnormal contractility or abnormal angiogenic activity, or to a tissue or fluid of the mammal which is contiguous therewith. The abnormal contractility or angiogenic activity of the muscle or endothelial cell or tissue can be either abnormally high or abnormally low contractility. The agonist or antagonist can be provided to the cell or tissue by any method described herein for providing a compound to a cell directly, such as by contacting the compound with the cell, or indirectly, such as through the action of any biological process. The agonist or antagonist need not be provided to the interior of the cell, since a binding protein of the uPA kringle, a binding protein of the uPA growth factor domain, and a binding protein of the connecting peptide can be found either inside or outside of the cell (i.e. as a soluble protein).

The method also comprises modulating the effect of one or more of the uPA kringle or a binding protein thereof, the uPA growth factor domain or a binding protein thereof, and the connecting peptide or a binding protein thereof, upon the muscle or endothelial cell or tissue having abnormal contractility or abnormal angiogenic activity.

By modulating the effect of one or more of the uPA kringle or a binding protein thereof, the uPA growth factor domain or a binding protein thereof, and the connecting peptide or a binding protein thereof upon the contractility or angiogenic activity of the muscle or endothelial cell or tissue having abnormal contractility or abnormal angiogenic activity, a disease or condition in the mammal having abnormal muscle cell or tissue contractility or abnormal angiogenic activity as a symptom thereof is treated.

By way of example, and not by limitation, in one aspect of the method, the disease or condition treated is the vascular disease hypertension, which has as a symptom thereof abnormally high smooth muscle cell contractility in a vascular smooth muscle cell or tissue. In this embodiment, the mammal is administered an amount suspected to be effective of one or more of an antagonist to the uPA kringle, an antagonist to a binding protein of the uPA kringle, an agonist of the uPA growth factor domain, an agonist of a binding protein of the uPA growth factor domain, an agonist of the connecting peptide, and an agonist of a binding protein of the connecting peptide. In this embodiment, the vascular condition of hypertension in the mammal is treated by inhibiting the contractility enhancing effect of the uPA kringle or one of its binding proteins, or by enhancing the vasodilating effect of the growth factor domain and the connecting peptide and their binding proteins.

In another embodiment of the method, where the disease or condition treated in the mammal is a disease or condition having as a symptom thereof abnormally high bronchial smooth muscle cell or tissue contractility (e.g., asthma), the method comprises administering to the mammal an amount suspected to be effective of one or more of an agonist to the uPA kringle and an agonist to a binding protein of the uPA kringle. In this embodiment, the effect of the uPA kringle in naturally occurring tcuPA at inhibiting bronchial smooth muscle cell contractility (i.e. bronchodilation) is enhanced by the agonist in order to enhance bronchodilation in asthma. Preferably the agonist is administered in an amount effective to promote, increase or amplify the effect of the uPA kringle in naturally occurring tcuPA at inhibiting the contractility of a bronchial smooth muscle tissue, thereby treating asthma in the mammal.

The invention also includes a method of determining whether a test protein is a binding protein of one or more of the uPA kringle, the uPA growth factor domain and the connecting peptide. The method comprises assessing the contractility modulating effect or the angiogenic activity modulating effect of one or more of the uPA kringle, the uPA growth factor receptor and the connecting peptide, in an amount effective to modulate the contractility or angiogenic activity of a mammalian muscle or endothelial cell, upon a first cell or tissue, which either comprises the test protein or which is contiguous with a tissue or fluid of a mammal which comprises the test protein. The test protein need not be present in the first cell or tissue, but can instead be a soluble or circulating protein of a tissue or fluid which is contiguous with the first cell or tissue. For example, the test protein can be a known soluble protein of the bloodstream or lymphatic tissue of a mammal which is capable of direct molecular signaling or signaling indirectly through another molecule to the first cell or tissue.

As with the test protein, the one or more of the uPA kringle, the uPA growth factor receptor and the connecting peptide can either be provided to the first cell or tissue, or to a tissue or fluid which is contiguous with the first cell or tissue, such as a blood or lymph cell or tissue.

The contractility modulating effect or angiogenic activity modulating effect of one or more of the uPA kringle, the uPA growth factor receptor and the connecting peptide, in an amount effective to modulate the contractility or angiogenic activity of a mammalian muscle or endothelial cell, is also assessed in a second, otherwise identical, cell or tissue which does not comprise the test protein and which is not contiguous with a tissue or fluid which comprises the test protein. The one or more of the uPA kringle, the uPA growth factor receptor and the connecting peptide can either be provided to the second cell or tissue, or to a tissue or fluid which is contiguous with the second cell or tissue, such as a blood or lymph cell or tissue.

The contractility modulating effect or the angiogenic activity modulating effect in the first cell or tissue is then compared with the contractility modulating effect or the angiogenic activity modulating effect in the second cell or tissue. If the degree of modulation of contractility or angiogenic activity by one or more of the uPA kringle, the uPA growth factor receptor and the connecting peptide is greater in the first cell or tissue relative to the second cell or tissue, then the test protein is a binding protein of one or more of the uPA kringle, the uPA growth factor receptor and the connecting peptide. The contractility or angiogenic activity of the cells and the contractility modulating effect or angiogenic activity modulating effect of one or more of the uPA kringle, the uPA growth factor receptor and the connecting peptide upon the cells can be assessed using any method known in the art or described herein.

In one embodiment, the contractility modulating effect or the angiogenic activity modulating effect of one or more of an isolated kringle, the ATF, tcuPA, and a peptide or functional element of the uPA kringle is assessed and compared in a first and second cell or tissue. In this embodiment, if a greater increase in contractility or a greater decrease in angiogenic activity is observed in the first cell or tissue relative to the second cell or tissue, then the test protein is a binding protein of one or more of an isolated kringle, the ATF, tcuPA and a peptide or functional element of the uPA kringle.

When present inside the first or second cells, the test protein and/or the one or more of the uPA kringle, the uPA growth factor receptor and the connecting peptide can be present, by way of example and not by limitation, either naturally or as an expressed recombinant polypeptide, or by providing the test protein and/or one or more of the uPA kringle, the uPA growth factor receptor and the connecting peptide to the cells as a component of any of the compositions of the invention described herein using any of the methods described herein for providing a composition or compound to a cell.

The test protein and/or one or more of the uPA kringle, the uPA growth factor receptor and the connecting peptide can also be present in the cells as a naturally occurring protein or as an expressed recombinant polypeptide resulting from the transfection of the cell with any of the vectors described herein comprising a recombinant polynucleotide which encodes the test protein and/or one or more of the uPA kringle, the uPA growth factor receptor and the connecting peptide using any method known in the art or described herein or known in the art to transfect a mammalian cell. The recombinant polynucleotide encoding such a recombinant polypeptide test protein can be prepared by any method known in the art or described herein.

In addition to the method described above, a binding protein of one or more of the uPA kringle, the uPA growth factor receptor and the connecting peptide can be identified using conventional methods known in the art. For example, immunological methods can be used to identify a binding complex of one or more of the uPA kringle, the uPA growth factor receptor and the connecting peptide with a test protein, the specificity of binding and the affinity of the binding complex being an indication that the test protein is a binding protein of one or more of the uPA kringle, the uPA growth factor receptor and the connecting peptide. In addition, the identification of an effect upon any signal transduction event (e.g. calcium release) associated with one or more of the uPA kringle, the uPA growth factor receptor and the connecting peptide can be used to identify a test protein as a binding protein thereof in assays known in the art for assessing signal transduction. Test proteins identified as having an effect upon signal transduction by one or more of the uPA kringle, the uPA growth factor receptor and the connecting peptide are thus identified as binding proteins thereof.

The invention also includes a method of identifying a functional element of one or more of the uPA kringle, the uPA growth factor receptor and the connecting peptide. The functional element participates in the modulation of contractility or angiogenic activity of a mammalian muscle or endothelial cell or tissue. The method comprises preparing one or more mutant polypeptides which lack a portion of the amino acid sequence of one or more of the uPA kringle, the uPA growth factor receptor and the connecting peptide. These mutant polypeptides can be prepared by any method known in the art for preparing deletion mutant proteins or polypeptides. Examples of such methods are described herein in the experimental Examples. The portions of the amino acid sequence which are to be deleted can be determined either randomly or in a directional manner proceeding from either the N-terminal or C-terminal end of the polypeptide and proceeding in a single direction.

The method also comprises assessing the ability of each deletion mutant so prepared to modulate the contractility or to modulate the angiogenic activity of a mammalian muscle or endothelial cell or tissue once provided to the cell or tissue, or to a tissue or fluid which is contiguous with the cell or tissue, as discussed herein. If provided to the cell or tissue; the mutant polypeptide can be provided using any method described herein for providing a composition to a cell or tissue. The modulation of the contractility or angiogenic activity can be either an enhancement, a disinhibition or an inhibition of the contractility or angiogenic activity of any of the muscle or endothelial cell or tissue types described herein. The contraction of the cell can either be mediated or not mediated by any of the inducing compounds described herein. The modulation of contractility or angiogenic activity in a muscle or endothelial cell which is provided a mutant polypeptide can be assessed by any of the methods described herein or known in the art. For example, a decrease or increase in the EC.sub.50 of an inducing compound during contraction of the muscle cell can be used to assess the ability of the mutant polypeptide to modulate the contractility of the cell or tissue, once provided to the cell or tissue, or to a tissue or fluid which is contiguous therewith.

The method also includes identifying, from the assessment described above, a mutant polypeptide which is not able to modulate the contractility or which is not able to modulate the angiogenic activity of a mammalian muscle or endothelial cell. Based on the identification of a specific mutant polypeptide, the corresponding deleted portion of the amino acid sequence of one or more of the uPA kringle, the uPA growth factor receptor and the connecting peptide which participates in the modulation of contractility or angiogenic activity of a muscle or endothelial cell is determined. Thus, a functional element of one or more of the uPA kringle, the uPA growth factor receptor and the connecting peptide is identified.

In addition to the method discussed above, other methods known in the art for identifying a functional element of a polypeptide can be used. For example, anti-peptide antibodies, deletion/substitution mutants, chimeric polypeptides and other peptide inhibition methods can be used to identify a functional element of one or more of the uPA kringle, the uPA growth factor receptor and the connecting peptide by methods known in the art.

The invention also includes a method of treating a vascular disease or condition in a mammal having as a symptom thereof abnormally high fibrin clot formation. The method comprises administering to the mammal a composition comprising one or more of kringle-scuPA, .DELTA.kringle-tcuPA, an antagonist of the uPA kringle and an antagonist of a binding protein of the uPA kringle in an amount effective to inhibit the contractility of a mammalian vascular smooth muscle cell or tissue. The polypeptides .DELTA.kringle-scuPA and .DELTA.kringle-tcuPA are deletion mutants of the urokinase-type plasminogen activator protein lacking the uPA kringle, which share at least about 75% homology with the polypeptide corresponding to SEQ ID NO:7 (FIG. 1G). Preferably, .DELTA.kringle-scuPA and .DELTA.kringle-tcuPA are about 80% homologous, more preferably about 85% homologous, even more preferably 90% homologous, yet more preferably 95% homologous, and most preferably about 99% homologous to the polypeptide corresponding to SEQ ID NO:7. Even more preferably, .DELTA.kringle-scuPA and .DELTA.kringle-tcuPA are each the polypeptide corresponding to SEQ ID NO:7. The antagonist of the uPA kringle and of the uPA kringle binding protein can be any of those described herein.

The method also comprises providing the composition to an affected cell or tissue of the cardiovascular system of the mammal which has or is prone to develop excessive fibrin clot formation, or to a tissue or fluid of the mammal which is contiguous therewith. The composition can be provided using any method described herein for providing to a cell or tissue a composition of the invention. A sufficient period of time is permitted for the uPA protease domain component of the composition, if present, to initiate the breakdown of fibrinogen and the process of fibrin clot lysis and for the composition to cause vasodilation in the affected area by inhibiting the contractility of the vascular smooth muscle cell or tissue in the affected area. Thus, the method results in the promotion of both fibrin clot lysis and vasodilation in the affected area of the vasculature of the mammal, thereby treating the vascular disease or condition.

The method can be used to promote both clot lysis and vasodilation in any vascular tissue in a mammal. The clot can be of any natural origin, such as from any of the diseases or conditions described herein, or of any artificially induced origin, for example, post-traumatic clotting, such as after vascular surgery, stenting or angioplasty. Conventional therapy involves the use of tcuPA to promote clot lysis. However, using tcuPA has the disadvantage of also causing vasoconstriction in the affected area, thus partially defeating the purpose of clot lysis (i.e. to increase blood flow through a vessel). Thus, using .DELTA.kringle-tcuPA to promote clot lysis instead of tcuPA has the advantage of overcoming the deleterious vasoconstrictive properties of tcuPA which arise from the contractility enhancing effect of the uPA kringle, while retaining the clot lysis activity of the uPA protease domain. Alternatively, .DELTA.kringle-scuPA can be administered to the mammal along with a recombinant version of soluble uPA receptor (suPAR). In this embodiment, the single chain molecule undergoes a conformational change which invests it with clot lysis activity and resistance to its plasma inhibitors. Thus, both .DELTA.kringle-scuPA and .DELTA.kringle-tcuPA are useful in the inventive method of promoting both fibrin clot lysis and vasodilation in an affected vascular tissue of a mammal.

The invention also includes a kit for treating a disease or condition in a mammal, the disease or condition having as a symptom thereof one or more of abnormal muscle cell or tissue contractility and abnormal angiogenic activity. The kit comprises a composition of the invention 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 kit also comprises an instructional material which describes administering the composition to a muscle or endothelial cell or tissue of a mammal having abnormal contractility and/or abnormal angiogenic activity.

In another embodiment, this kit comprises a (preferably sterile) solvent suitable for dissolving or suspending the composition of the invention prior to administering the composition to the mammal.
 

Claim 1 of 1 Claim

1. A method of treating a mammal afflicted with asthma having as a symptom thereof abnormal muscle cell or tissue contractility, said method comprising a) administering to the mammal a pharmaceutical composition comprising the amino acid sequence consisting of SEQ ID NO:9 in an amount effective to modulate the contractility of a mammalian muscle or endothelial cell or tissue and a pharmaceutically acceptable carrier; and b) modulating the contractility of said muscle or endothelial cell or tissue having abnormal contractility, whereby said asthma in the mammal is treated.

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