Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Transferases
Reexamination Certificate
1999-12-22
2004-02-03
Prouty, Rebecca E. (Department: 1652)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Transferases
C514S012200, C435S194000
Reexamination Certificate
active
06685938
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to the field of medicine, and relates specifically to methods and compositions for modulating vascular permeability (VP).
BACKGROUND
Angiogenesis is a process of tissue vascularization that involves the growth of new developing blood vessels into a tissue, and is also referred to as neo-vascularization. The process is mediated by the infiltration of endothelial cells and smooth muscle cells. The process is believed to proceed in any one of three ways: the vessels can sprout from pre-existing vessels, de-novo development of vessels can arise from precursor cells (vasculogenesis), or existing small vessels can enlarge in diameter. Blood et al.,
Bioch. Biophys. Acta
, 1032:89-118 (1990). For angiogenesis to occur, endothelial cells must first degrade and cross the blood vessel basement membrane in a similar manner used by tumor cells during invasion and metastasis formation. Angiogenesis is generally absent in adult or mature tissues, although it does occur in wound healing and in the corpus luteum growth cycle. See, for example, Moses et al.,
Science
, 248:1408-1410 (1990).
While angiogenesis is an important process in neonatal growth, it is also important in wound healing and is a factor in the pathogenesis of a large variety of clinical diseases including tissue inflammation, arthritis, tumor growth, diabetic retinopathy, macular degeneration by neovascularization of the retina, and like conditions. These clinical manifestations associated with angiogenesis are referred to as angiogenic diseases. Folkman et al.,
Science
, 235:442-447 (1987).
It has been proposed that inhibition of angiogenesis would be a useful therapy for restricting tumor growth. Inhibition of angiogenesis has been proposed by (1) inhibition of release of “angiogenic molecules” such as bFGF (basic fibroblast growth factor), (2) neutralization of angiogenic molecules, such as by use of anti-&bgr;bFGF antibodies, (3) use of inhibitors of vitronectin receptor (&agr;
v
&bgr;
3
, and (4) inhibition of endothelial cell response to angiogenic stimuli. This latter strategy has received attention, Folkman et al.,
Cancer Biology
, 3:89-96 (1992), have described several endothelial cell response inhibitors, including collagenase inhibitor, basement membrane turnover inhibitors, angiostatic steroids, fungal-derived angiogenesis inhibitors, platelet factor 4, thrombospondin, arthritis drugs such as D-penicillamine and gold thiomalate, vitamin D
3
analogs, alpha-interferon, and the like that might be used to inhibit angiogenesis. For additional proposed inhibitors of angiogenesis, see Blood et al.,
Bioch. Biophys. Acta
., 1032:89-118 (1990), Moses et al.,
Science
, 248:1408-1410 (1990), Ingber et al.,
Lab. Invest
., 59:44-51 (1988), and U.S. Pat. No. 5,092,885, U.S. Pat. No. 5,112,946, U.S. Pat. No. 5,192,744, U.S. Pat. No. 5,202,352, U.S. Pat. No. 5,753,230 and U.S. Pat. No. 5,766,591. None of the inhibitors of angiogenesis described in the foregoing references involve the Src proteins, however.
It has been previously reported that angiogenesis depends on the interaction between vascular integrins and extracellular matrix proteins. Brooks et al.,
Science
, 264:569-571 (1994). Furthermore, it was reported that programmed cell death (apoptosis) of angiogenic vascular cells is initiated by the interaction, which would be inhibited by certain antagonists of the vascular integrin &agr;
v
&bgr;
3
. Brooks et al.,
Cell
, 79:1157-1164 (1994). More recently, it has been reported that the binding of matrix metalloproteinase-2 (MMP-2) to vitronectin receptor (&agr;
v
&bgr;
5
) can be inhibited using &agr;
v
&bgr;
5
antagonists, and thereby inhibit the enzymatic function of the proteinase. Brooks et al.,
Cell
, 85:683-693 (1996).
The brain vasculature is characterized by a highly restrictive blood-brain barrier that prohibits small molecules from extravasating into the surrounding brain tissue. The nature of the blood-brain barrier in mammals has been of special concern with pharmacological studies, as many drugs are routinely prevented from passing from the vasculature to the brain tissues because of the highly restrictive blood-brain barrier. The present invention involves the unexpected discovery that VP, as measured by vascular leakage of blood, can be modulated by src or yes. Moreover, VP has been associated with angiogenesis and other pathologies. Inflammation induced increased vascular permeability is associated with edema and swelling.
SUMMARY OF THE INVENTION
The present invention is directed to modulation of vascular permeability (VP) by tyrosine kinase Src, also referred to generically herein as Src, or the tyrosine kinase Yes, also referred to generically herein as Yes.
Thus, one aspect of the invention encompasses pharmaceutical compositions for modulating VP in target tissue of a mammal. The compositions of the invention comprise a therapeutically effective VP modulating amount of a mixture of tyrosine kinase protein Src and Yes, in a pharmaceutically acceptable carrier.
In compositions which comprise active Src and Yes kinase proteins, the expected modulation is a potentiation or increase in vascular permeability of the blood vessels in a target tissue. Where the desired Src protein is an active kinase, a preferred Src is Src-A. Another preferred active Src protein is one in which the amino acid residue at position 527 of the Src protein is any amino acid residue except for tyrosine, serine or threonine. The preferred active Yes protein will have the kinase activity of wild-type human Yes, such as that or the Yes-1 protein. Another preferred active Yes is one in which the kinase inactivating phosphorylation site of the Yes protein is mutated to abolish or minimize inactivating phosphorylation, similar to a mutation of amino acid residue 527 of Src to any amino acid residue except for tyrosine, serine or threonine.
Where the composition comprises Src and Yes protein that are inactive kinase proteins, the expected modulation is an inhibition or decrease in vascular permeability of the blood vessels in the target tissue. When the desired Src protein is an inactive protein, a preferred Src is Src 251. A further preferred inactive Src is Src K295M. A preferred inactive Yes protein will have diminished kinase activity as compared with the wild-type protein.
A further aspect of the claimed invention is a pharmaceutical composition comprising a therapeutically effective VP modulating amount of nucleic acid capable of expressing tyrosine kinase protein Src and Yes, when transfected into a target cell, in a suitable pharmaceutical carrier. The expressible nucleic acids encoding for Src or Yes protein can comprise nucleic acid segments which describe all or part of the Yes or Src protein. When transferred into target cells, the target cell transcribes and translates the nucleic acid sequence to express the desired protein.
Where the modulation is a potentiation or increase in vascular permeability of the blood vessels in the target tissue, Src encoding nucleic acid will encode active forms of Src, and Yes encoding nucleic acids will encode active forms of Yes kinase proteins. Once transferred into the target host cell, the nucleic acids will be expressed by the host cell. A preferred Src encoding nucleic acid encodes active Src A protein. A further preferred Src encoding nucleic acid encodes a mutated active Src where the amino acid residue at position 527 of the expressed Src protein is any amino acid residue except for tyrosine, serine or threonine. A preferred Yes encoding nucleic acid will encode the wild-type protein, or a protein modified to abolish or inhibit the inactivating phosphorylation site of the Yes protein, in a similar manner as the Src position 527 mutation described.
When the desired modulation is an inhibition or decrease in vascular permeability of the blood vessels in the target tissue, a preferred inactive Src encoding nucleic acid encodes Src 251 protein. A further preferred inactive Src encoding nucleic acid encodes inactive Src K295M.
Cheresh David A.
Eliceiri Brian
Olson & Hierl Ltd.
Prouty Rebecca E.
The Scripps Research Institute
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