Other

The United States of America As Represented by the Department of Health

National Institutes of Health, a Component of the US Dept. of Health & Human Services

The United States of America As Represented by the Secretary of

William G Stetler-Stevenson

Lance A Liotta

Henry C Krutzsch

Frank Cuttitta

Alfredo Martinez

Zhengping Zhuang

Mark Roth

Edward J Unsworth

Irina A Lubensky

Juan M Saavedra

Henry C Krutsch

Michael R Buck

The present disclosure concerns the use of peptides and compositions, such as pharmaceutical compositions, to influence angiogenesis. Particular methods are useful for promoting angiogenesis, while others are particularly useful for inhibiting angiogenesis.

Methods for inhibiting angiogenesis with inhibitors of proadrenomedullin N-terminal 20 peptide (PAMP)

Compositions and methods for promoting angiogenesis

Methods and compounds are described for regulating blood pressure in a subject. Specific embodiments are methods for reversing vasodilation of blood vessels, by administering to a subject a therapeutically effective amount peptide AM(11-22). The vasoconstrictor can be used for a variety of purposes, including hemostasis or the treatment of shock, for example vasodilatory shock syndromes such as septic shock. Other specific embodiments are methods for reversing vasoconstriction of blood vessels, by administering to a subject a therapeutically effect amount of an inhibitor of AM(11-22), sufficient to reduce hypertension in the subject. Compounds and pharmaceutical compositions are also provided, as are kits.

Vasoregulating compounds and methods of their use

The present invention relates to the development of a modified substrate capture immunoassay useful in the detection of a neutral metalloproteinase enzyme, type IV collagenase. Capture and immobilization of this enzyme can be achieved by coating the microtiter plate with gelatin, an alternative substrate for this enzyme. The captured enzyme can then be detected by affinity purified rabbit anti-type IV collagenase antibodies prepared against synthetic peptides from the amino terminus of the enzyme. Soluble type IV collagenase can readily be detected in samples known to contain this enzyme. Using purified enzyme this assay method can detect less than 50 ng of latent type IV collagenase. Using EDTA, an inhibitor of this metalloproteinase, gelatin binding can be shown to be independent of catalytic activity.

Method for measuring type IV collagenase

A method of direct extraction of cellular material from a tissue sample which involves: forming an image field of cells of the tissue sample utilizing a microscope, identifying at least one zone of cells of interest from the image field of cells which at least one zone of cells of interest includes different types of cells than adjacent zones of cells, and extracting the at least one zone of cells of interest from the tissue sample. The extracted zone(s) of cells is subjected to analysis. The overall process of identifying, extracting, transporting, and analyzing the extracted zones(s) of cells can be fully automated.

Isolation of cellular material under microscopic visualization using an

Inappropriate degradation of extracellular matrix molecules by metalloproteinases plays an important role in a wide variety of pathologic conditions including neoplasia and arthritis. The present invention is an isolated protein of approximately 23,000 daltons in size which binds to metalloproteinases with high affinity, can be purified using affinity chromatography on solid phase metalloproteinases, and is potentially useful for therapy of pathologic conditions involving the inappropriate production of metalloproteinases. This protein is characterized by the presence of the following amino acid sequences:

Metalloproteinase peptides

The present invention is an isolated protein of 21,600 Da which binds to both latent and activated type IV collagenase with high affinity at 1:1 molar stoichiometry, thereby abolishing enzyme activity. The protein is purified by affinity chromatography on solid phase metalloproteinase, or solid phase metalloproteinase substrates which bind the enzyme-inhibitor complex. The complete primary structure of this protein (initially called CSC-21K), as determined by sequencing overlapping peptides spanning the entire protein, reveals homology with a protein called TIMP, Tissue Inhibitor of Metalloproteinases. In addition, a cDNA for this novel inhibitor, now designated TIMP-2, was cloned from a melanoma cell and its sequence was compared with that of human TIMP-1. Northern blots of melanoma cell mRNA showed two distinct transcripts of 0.9 kb and 3.5 kb which are down-regulated by transforming growth factor-.beta., and are unchanged by phorbol ester treatment. The inhibitor of the present invention may be used for treatment of pathologic conditions resulting from inappropriate degradation of extracellular matrix molecules by matrix metalloproteinases, such as metastatic neoplasia, myocardial infarction, and arthritis. Therapeutic treatments using this inhibitor may include formulations for inhalation and inclusion complexes adapted for buccal or sublingual administration, or administration of a recombinant DNA molecule which expresses a DNA segment that encodes the matrix metalloproteinase inhibitor of this invention.

Matrix metalloproteinase inhibitor peptides

A family of metalloproteinases exist which cleave extracellular matrix molecules. These metalloproteinases are secreted in a latent inactive form and require activation in order to specifically cleave the preferred substrate. A series of peptides have been prepared based on the complete sequence analysis of type IV procollagenase. Peptide inhibitors were synthesized which correspond to cysteine repeat regions and histidine containing regions; the mechanism of action of these peptides involves inhibition of binding of the enzyme to the substrate. Peptide inhibitors were synthesized which correspond to the peptide cleaved off during activation, and constitute a novel class of metalloproteinase inhibitors. These inhibitors are members of a series of peptides which contain the core amino acid sequence RKPRC or analogs thereof. The cysteine residue is required for activity. Affinity purified antibodies directed against specific peptides can be used to a) detect any general metalloproteinase enzyme with the sequence in part VAAHE or PRCGNPD, and distinguish it from other known members of the metalloproteinase family, b) block functional domains resulting in the inhibition of enzyme activity, and c) distinguish latent from activated forms of the enzyme.

Matrix metalloproteinase peptides: role in diagnosis and therapy

Antigenic matrix metalloproteinase peptides

A family of metalloproteinases exist which cleave extracellular matrix molecules. These metalloproteinases are secreted in a latent inactive form and require activation in order to specifically cleave the preferred substrate. A series of peptides have been prepared based on the complete sequence analysis of type IV Procollagenase. Peptide inhibitors were synthesized which correspond to cysteine repeat regions and histidine containing regions; the mechanism of action of these peptides involves inhibition of binding of the enzyme to the substrate. Peptide inhibitors were synthesized which correspond to the peptide cleaved off during activation, and constitute a novel class of metalloproteinase inhibitors. These inhibitors are members of a series of peptides which contain the core amino acid sequence PRCG. The cysteine residue is required for activity. Affinity purified antibodies directed against specific peptides can be used to a) detect any general metalloproteinase enzyme with the sequence in part VAAHE or PRCGNPD, and distinguish it from other known members of the metalloproteinase family, b) block functional domains resulting in the inhibition of enzyme activity, and c) distinguish latent from activated forms of the enzyme.

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Gaithersburg, MD, United States of America

William G Stetler-Stevenson