Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
2000-05-02
2002-12-03
Riley, Jezia (Department: 1656)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C424S450000
Reexamination Certificate
active
06489311
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a method for inhibiting apoptosis in ischemic-reperfused myocardium. More specifically this invention relates to a method for using heparin or noncoagulant heparin in the prevention of apoptosis.
BACKGROUND OF THE INVENTION
Cells die by one of either of two processes: necrosis or apoptosis. Whereas necrosis occurs through external injury producing cellular membrane destruction, swelling and lysis, apoptosis is endogenously mediated cellular suicide effected by activation of a series of aspartate-specific proteases called caspases and endonucleases, resulting in proteolytic destruction of cellular proteins and chromosomal elements. Apoptotic events include DNA fragmentation, chromatin condensation, membrane blebbing, cell shrinkage, and disassembly into membrane-enclosed vesicles (apoptotic bodies). In vivo, this process culminates with the engulfment of apoptotic bodies by other cells, preventing complications that would result from a release of intracellular contents. In myocardial infarction, both processes contribute to myocardial muscle injury and destruction. Overt necrosis predominates in the central zone of infarcted myocardium, and apoptosis occurs in the border zones of histologically infarcted myocardium. See, G. Olivetti, et al., “Acute myocardial infarction in humans is associated with activation of programmed myocyte cell death in the surviving portion of the heart,”
J. Mol. Cell Cardiol,
28:2005-2016, 1994; and A. Saraste, et al., “Apoptosis in human myocardial infarction,”
Circulation,
95:320-323, 1997. Also, apoptosis occurs in hypoperfused hibernating myocardium. See, C. Chen, et al., “Myocardial cell death and apoptosis in hibernating myocardium,”
J.A.C.C,
30:1407-1412, 1997. Apoptosis also contributes substantially to myocyte death in patients suffering heart failure from dilated cardiomyopathy. See, A. Haunstetter, et al., “Basic mechanisms and implications for cardiovascular diseases,”
Circ. Res.,
82:1111-1129, 1998.
Apoptosis is controlled at two distinct levels. First, cells have unique sensors, termed death receptors, on their membrane surface. Death receptors detect the presence of extracellular death signals and, in reponse, ignite the cell's intrinsic apoptosis machinery. See, A. Ashkenazi, et al., “Death receptors: signaling and modulation,”
Science,
281:1305-1308, 1998. One of the more important receptors is the member of the tumor necrosis receptor family TNFR1 (also called p55). When tumor necrosis factor (TNF) attaches to TNFR1, the receptor trimerizes, and binds a series of other proteins: TRADD ((TNFR-associated death domain); TRAF-2 (TNFR-associated factor-2; RIP (receptor-interacting protein): and FADD (Fas-associated death domain). FADD couples the TNFR1-TRADD complex to activate caspase-8, thereby initiating activation of the entire cascade of other caspases that effect apoptosis. TNF plays an important role in ischemia-reperfusion injury and in the contractile depression of myocardium following ischemia and reperfusion during myocardial infarction. See, B. S. Cain, et al., “Therapeutic strategies to reduce TNF-&agr; mediated cardiac contractile depression following ischemia and reperfusion,”
J. Mol. Cell. Cardiol.,
31:931-947. TNF plays an important role in hemorhagic shock. See, D. R. Meldrum, et al., “Hemorrhage activates myocardial NF&kgr;B and increases TNF-&agr; in the heart,”
J. Mol. Cell. Cardiol.,
29:2849-2854, 1997. Apoptosis from TNF produced endogenously by overloaded myocardium also plays a significant role in mediating cardiac apoptosis leading to initiation and progression of congestive heart failure. See, for example, J. Narula, et al., Apoptosis in myocytes in end-stage heart failure,”
New England J. Med,
335:1182-1189, 1996; and T. Kubota, et al., et al., “Dilated cardiomyopathy in transgenic mice with cardiac-specific overexpression of tumor necrosis factor-&agr;,”
Circ. Res.,
81:627-635, 1997.
At a second site, activation of caspases and subsequent apoptosis are initiated by events that disturb mitochondria. Either disruption of electron transport and aerobic oxidative phosphorylation or opening of pores in the outer mitochondrial membrane by pro-apoptotic cytoplasmic proteins of the BAX or BH3 families will allow leakage out of the mitochrondria of the respiratory chain component cytochrome c. Upon entering the cytoplasm, cytochrome c binds to a cytosolic protein called apoptotic protease activating factor-1 (Apaf-1). In the presence of ATP, the complex of cytochrome c and Apaf-1 activate procaspase 9, which initiates subsequent activation of the remainder of the caspase cascade and initiation of cellular apoptosis. See, D. R. Green, et. al., “Mitochrondria and apoptosis,”
Science,
81:1309-1312, 1998.
The death domain and mitochrondrial pathways of caspase and apoptosis activation are interrelated in that TNF can stimulate neutral membrane sphingomyelinase, resulting in production of ceramide, which disrupts mitochrondrial electron transport, also eventually effecting release into the cytoplasm of mitochondrial cytochrome c. Cytochrome c plays a prominent early role in the signal transduction of caspase activation and cardiomyocyte apoptosis induced by reactive oxygen species. See, R. von Harsdord, et al., “Signaling pathways in reactive oxygen species-induced cardiomyocyte apoptosis,”
Circ.,
99:2934-2941, 1999. Production of reactive oxygen species is greatly enhanced as a consequence of ischemia-reperfusion of myocardium and oxidant stress produced during ischemia-reperfusion induces myocardial apoptosis. See, N. Maulik, et al., “Oxidative stress developed during the reperfusion of ischemic myocardium induces apoptosis,”
Free Rad. Biol. Med,
24:869-875, 1998. Thus, the activity of cytochrome c when it is transported to the cytoplasm appears to play an important and pivotal role in activating pro-apoptotic cascades, whether the initial induction of apoptosis is effected through membrane death receptor or mitochrondrial pathways.
In view of the foregoing it is readily apparent that there is a need for treatment of myocardial reperfusion injury that inhibits or prevents apoptosis.
SUMMARY OF THE INVENTION
It is therefore the general object of this invention to provides a method of inhibiting or preventing apoptosis in ischemic-reperfused myocardium using heparin or nonanticoagulant heparin.
The present invention provides a method for inhibiting apoptosis in ischemic-reperfused myocardium by administering to a mammal an effective amount of heparin to reduce myocardial cell death in myocardial infarction. It has been found that at doses greatly exceeding those needed for anticoagulation heparin substantially reduces reperfusion injury both in the isolated perfused heart and intact whole animal models of myocardial infarction. This protective effect is independent of heparin's activity as an anticoagulant.
In accordance with another aspect of this invention, there is provided a method for inhibiting apoptosis in ischemic-reperfused myocardium by administering to a mammal an effective amount of nonanticoagulant heparin, such as O-desulfated heparin, to reduce or prevent myocardial cell death in myocardial infarction.
In yet another aspect of this invention it was found that heparin or nonanticoagulant heparin when conjugated to a lipophilic moiety such as a fatty acid or cholesterol by reaction across a carboxylic acid or free amine group can be used to enhance cellular uptake by cell types not normally concentrating heparin, such as neurons, thereby enhancing the anti-apoptotic effect of heparin or nonanticoagulant heparin. Furthermore, heparin or nonanticoagulant heparin, either alone or conjugated to a lipophilic group, can be used to block apoptosis in situations of acute trauma, such as generalized trauma, global ischemia-reperfusion injury occurring as a consequence of hemorrhagic shock, or spinal cord injury, thereby preventing cell death in organs such as the spinal cord.
REFERENCES:
patent: 4239754 (1980-12-01), Sache et al.
paten
Alston & Bird LLP
Charlotte-Mecklenburg Hospital Authoirty
Riley Jezia
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