Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Hydrolases
Reexamination Certificate
2000-07-20
2002-10-08
Tate, Christopher R. (Department: 1651)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Hydrolases
C530S327000, C530S328000, C530S329000, C530S330000
Reexamination Certificate
active
06461611
ABSTRACT:
FIELD OF THE INVENTION
The present invention concerns agents for prevention or decrease of damages caused by stress conditions, particularly ischemica and/or hypoxia and to pharmaceutical compositions comprising such agents.
BACKGROUND OF THE INVENTION
Hypoxia, defined as lack or decrease of oxygen levels and ischemia, defined as lack or decrease of a blood supply, are common medical disorders of the vascular network, which may be caused by various reasons including: lack of oxygen such as in cases of drowning or suffocation, narrowing of blood vessels due to depositions on their walls, blockage of blood vessels for example by blood clots, and damage to blood vessels which can interpret their integrity. Decline or cessation of the normal blood supply to a specific tissue causes damage, which if prolonged can be irreversible leading to massive cell death. Where ischemia is caused due to the narrowing of coronary blood vessels and decrease of the blood supply to the heart, it may lead ultimately to myocardial infarction, one of the most common causes of mortality in the western world (Sabia, et al.,
N. Engl. J. Med.,
327:1825-1831, (1992); Sasayama, S., Fujita, M.,
Circulation,
85:1197-1204, (1992)).
The myocardial vascular network is regulated to a great extent by microenvironmental induced hypoxia caused by decrease of blood flow, i.e. ischemia. The molecular mechanism of hypoxia-regulated gene expression of cell surface receptors, involved in the regulation and maintenance of the cardiovascular system, is poorly understood. Insufficient blood supply following restenosis is the leading cause of heart failure as a result of ischemic stress.
Hypoxia has been suggested as one of the microenvironmental factors that induce angiogenesis (Schaper, et al.,
Cir. Res.,
28:671-679 (1971)) and modulate the phenotype of smooth muscle cells (Rugh, K. et al.,
Cardiovac. Res.,
21:730-736 (1987); Shweiki, et al.,
Nature,
359:843-845 (1992)) and myocytes (Stenmark, et al.,
Chest.,
93:127-133 (1988)). Hypoxic and ischemic stress causes a series of well documented changes in myocardial cells and tissues, including increased anaerobic glycolysis, loss of contractility and eventually cell death. In the heart, the induction of the proto-oncogenes fos and jun in cardiac myocytes exposed to severe hypoxia has been described (Konziolka, et al.,
J. Neuroser.,
69:494-499 (1988)). This induction occurred after exposure of between 1 and 4 hours to hypoxia, and then declined, coinciding with loss of myocytes contractility but prior to irreversible cell damage.
Current attempts to treat or prevent myocardial damage due to hypoxia or ischemia involve the development of collateral circulation, an alternative source of blood supply to the myocardium, to provide adequate flow to the major epicardial branches of the coronary artery, which blood flow is prevented by failure of the original vessels. This may be achieved by the secretion of the angiogenic factor bFGF or the upregulation of VEGF under hypoxia. Thus, while cardiac myocytes undergo ischemia, collaterals may develop actively by growth, DNA replication and mitosis of endothelial and smooth muscle cell (SMC). Collateral development by heparin binding growth factors has the disadvantage that it may take a rather long period before collateral develops and functionality take over pre-existing blood vessels.
Thrombin is an agent having multiple effects on the cells of the vascular and circulation systems. In hemostasis thrombin has a central role as a serine protease that converts fibrinogen to fibrin which clots blood. Additional functions of thrombin are widespread and diverse and appear to involve cellular activations which are mediated through cellular thrombin receptor(s). For example, thrombin is the most potent activator of platelets; it is chemotactic for monocytes; it is mitogenic for lymphocytes and mesenchymal cells including vascular smooth muscle cells and; it promotes numerous responses within the vascular endothelium. (Coughlin, et al.,
J. Clin. Invest.
89:351-355 (1992)). Because these cell activating functions of thrombin occur within the range of concentrations normally required for the clotting of blood, thrombin has been proposed to play important physiological roles not only in hemostasis and thrmobosis but may also have principle roles in mediating responses to vascular injury such as leukocyte chemotaxis (to mediate inflammation), cellular proliferation (to mediate restenosis), glomerulonephritis, wound repair (such as occurs in bone remodelling), smooth muscle cell proliferation and ligation of &agr;
v
&bgr;
3
integrin through a cryptic yet functional RGD site (Bar-Shavit, et al.,
J. Clin. Invest.,
84:1096-1104 (1989); Bar-Shavit et al.,
Cell Reg.,
1:453-463, (1990)).
The cellular receptor of thrombin (ThR) is preferentially upregulated in advanced atherosclerotic lesions following percutaneous transluminal coronary angioplasty. This receptor is present also in rate ventricular myocytes implicated in the physiological maintenance of these cells.
Thrombin receptor is a seven transmembrane G-coupled protein that belongs to a new family of receptors termed “
Protease Activated Receptors
” (PAR). Unlike most growth factor receptors, the activation of PAR does not require the traditional ligand receptor complex formation. Instead, receptors of this family serve as substrates for protease digestion to yield an irreversible form of activated receptor that conveys further cellular effects. Activation of the ThR was found to be important in various physiological systems such as myocytes (Glenbotski et al.,
J. Biol. Chem.,
268:20646-20652 (1993)), keratinocytes (Santulli et al.,
PNAS,
92:1-6 (1995)) and astrocyte proliferation (Grabham, P. and Cunningham, D.,
J. Neurochem.,
64(2):583-591 (1995)). Other PARs have been discovered to have importance in diverse physiological systems (Coughlin et al.,
PNAS,
91; 9200-12, (1994)) an example being PAR-2 receptor which is involved in the inflammation cascade systems (Nystedt et al.,
PNAS,
91:9208-9212 (1994); Nystedt et al.,
Eur. J. Biochem.,
232:84-89 (1995); Nystedt et al.,
J. Biol. Chem.,
271:19910-19915 (1996); Schmidt,
J. Biol. Chem.,
271:9307-9212 (1996)).
SUMMARY OF THE INVENTION
The present invention is based on the surprising finding that mRNA levels of the thrombin receptor, a member of the Protease-Activated Receptor (PAR) family, decrease under conditions of stress, such as hypoxia. It has further been found that activating the ThR prior to or during the exposure to hypoxia, prevents said hypoxia-induced decreased in the level of mRNA. Administration of a ThR activating agents to cells under normal conditions, i.e. normal oxygen level, did not cause an increase in the normal levels of ThR mRNA levels.
ThR is a growth promoting receptor for cells of different origins, for example: fibroblasts, smooth muscle cells, astrocytes, etc. In myocytes activation of ThR induces hypertrophy and increases atrial natriuretic factor gene expression.
Therefore, maintaining a normal level of ThR mRNA is necessary for the well being and maintenance of cells in general and of myocytes in particular. Thus a decrease in the level of ThR mRNA under stress conditions, such as hypoxia contributes to the deterioration of cells, and particularly myocytes under these conditions. Agents capable of preventing such a stress-induced decrease in the level of ThR mRNA may help prevent or decrease some of the damages caused to the cells under stress conditions.
Thus, the present invention provides an agent for preventing the decrease in the levels of protease-activated receptor (PAR) mRNA under stress conditions, said agent being an activator of the PAR.
The term “preventing the decrease of mRNA levels” refers to a complete prevention of the decrease of these levels and reversal to normal mRNA levels featured by the same type of cells under normal conditions. Alternatively, this term refers to situations where there is partial prevention, i.e. while the mRNA level of the PAR in the cells
Hadasit Medical Research Services & Development Limited
Oliff & Berridg,e PLC
Tate Christopher R.
Winston Randall
LandOfFree
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