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Title:  Method for the prevention of apoptosis

United States Patent:  6,489,311

Issued:  December 3, 2002

Inventors:  Kennedy; Thomas P. (Charlotte, NC)

Assignee:  Charlotte-Mecklenburg Hospital Authoirty (Charlotte, NC)

Appl. No.:  561663

Filed:  May 2, 2000


Heparin reduces ischemia-reperfusion injury to myocardium. This effect has been attributed complement inhibition, but heparin also has other activities that might diminish ischemia-reperfusion. To further probe these mechanisms, we compared heparin and an O-desulfated nonanticoagulant heparin with greatly reduced anti-complement activity. Given at the time of coronary artery reperfusion in a canine model of myocardial infarction, both heparin and O-desulfated heparin equally reduced neutrophil adherence to ischemic-reperfused coronary artery endothelium, influx of neutrophils into ischemic-reperfused myocardium, myocardial necrosis and release of creatine kinase into plasma. Heparin and O-desulfated heparin also prevented dysfunction of endothelial-dependent coronary relaxation following ischemic injury. In addition, heparin and O-desulfated heparin inhibited translocation of the transcription factor NF-.kappa.B from cytoplasm to the nucleus in human endothelial cells and decreased NF-.kappa.B DNA binding in human endothelium and ischemic-reperfused rat myocardium. Thus, heparin and nonanticoagulant heparin decrease ischemia-reperfusion injury by disrupting multiple levels of the inflammatory cascade, including the novel observation that heparins inhibit activation of the pro-inflammatory transcription factor NF-.kappa.B.


The present invention now will be described more fully hereinafter with reference to the accompanying examples, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather these embodiments are provides so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

It has been fund that heparin in larger than usual anticoagulant doses of heparin and a variety of nonanticoagulant heparins (N-desulfated; 2-O, 3-O or 6-O desulfated; N-desulfated and reacetylated; and O-decarboxylated heparin) can attenuate ischemia-reperfusion injury in the heart and reduce myocardial infarct size as measured by the area of cellular necrosis and thus attenuate development of myocardial apoptosis. Examples of the preparation of O-desulfated nonanticoagulant heparin may be found in, for example, U.S. Pat. Nos. 5,668,118 and 5,912,237 incorporated herein by reference. "O-desulfated heparin" can include O-desulfated heparin having modifications, such as reduced molecular weight or acetylation, deacetylation, oxidation, and decarboxylation. The heparin or nonant: coagulant heparin may be given in amounts of 3 mg/kg to 100 mg/kg, but preferably in amounts from about 3.5 mg/kg to about 10 mg/kg.

The mechanisms by which heparin reduces reperfusion injury, were studied by in vivo ischemia-reperfusion in a canine infarct model using partially O-desulfated nonanticoagulant heparin (ODS-HEP). Despite greatly reduced anti-complement activity, ODS-HEP decreases PMN adherence to coronary epithelium. This was found both in vitro when stimulated by PAF (FIG. 6) and in vivo when stimulated by coronary ischemia and reperfusion (FIG. 7). Given at the time of reperfusion, ODS-HEP decreases PMN influx into ischemic-reperfused myocardium (FIG. 4) and reduces infarct size (FIGS. 1 and 2). Depressed contractile function remained initially unchanged, but function might be expected to recover over time as stunned but not irreversibly injured myocardium recovers to a normal energy state following ischemia. ODS-HEP also preserves normal vasodilator function in ischemic-reperfused coronary endothelium (FIG. 8). These benefits were produced without anticoagulation (FIG. 5).

Infiltration of PMNs plays a critical role in producing myocardial reperfusion injury. See, T. Yamszaki, et al., "Expression of intercellular adhesion molecule-1 in rat heart with ischemia/reperfusion and limitation of infarct size by treatment with antibodies against cell adhesion molecules," Am. J Pathol., 143:410-418, 1993; and P. J. Simpson, et al., "Reduction of experimental canine myocardial reperfusion injury by a monoclonal antibody (anti-Mo1, andti-CD11b) that inhibits leukocyte adhesion," J. Clin. Invest. 81:625-629, 1988. One of the earliest events mediating PMN influx from ischemia and reperfusion is the increase in surface expression of various endothelial cell adhesion molecules (ECAMs), including intercellular adhesion molecule-1 (ICAM-1), E-selectin and P-selectin, which increase rolling and adhesion of PMNs to coronary endothelium. See, T. Yamszaki, et al., supra. Enhanced expression of adhesion molecules during ischemia-reperfusion is result of the activation of nuclear factor-.kappa.B (NF-.kappa.B), See, T. Yamszaki, et al., supra, which promotes expression of many inflammatory and immune response genes. NF-.kappa.B is cytosolic when complexed with its inhibitor, I.kappa.B, but is activated by phosphorylation, ubiquitination and proteolytic degration of I.kappa.B. I. Stancovski, et al., "NF-.kappa.B activation: the I.kappa.B kinase revealed?," Cell, 91:299-302, 1997. Release from I.kappa.B exposes the NF-.kappa.B nuclear localization sequence (NLF), a highly cationic domain of eight amino acids (VQRDRQKLM, single-letter amino acid code) that targets nuclear translocation. Y.-Z. Lin, et al., "Inhibition of nuclear translocation of transcription fractor NF-.kappa.B by a synthetic peptide containing a cell membrane-permeable motif and nuclear localization sequence," J. Biol. Chem., 270:14255-14258, 1995; and S. T. Malek, et al., "I.kappa.B.alpha. functions through direct contacts with the nuclear localization signals and the DNA binding sequences of NF-.kappa.B," J. Biol. Chem., 273:25427-25435, 1998. NF-.kappa.B is activated in the heart and cultured myocytes by ischemia or ischemia and reperfusion, C. Li, et al., "Early activation of transcription factor NF-.kappa.B during ischemia in perfused rat hearts," Am. J. Physiol, 276 (Heart Circ. Physiol. 45):H543-H552, 1999; and R. Kacimi, et al., "Expression and regulation of adhesion molecules in cardiac cells by cytokines, response to acute hypoxia," Circ. Res. 82:576-586, 1998, with subsequent upregulation of adhesion molecules on the myocyte surface. See, R. Kacimi, et al., supra. Nuclear translocation of NF-.kappa.B is prevented by synthetic permeable peptides containing the NF-.kappa.B NLF, which competes for nuclear uptake. See, Y.-Z. Lin, et al., supra. Heparin is readily bound and internalized into the cytosolic compartment by endothelium, vascular and airway smooth muscle, mesangial cells and even cardiac myocytes. T. C. Wright, et al., "Regulation of cellular proliferation by heparin and heparin sulfate. In Lane DA, Lindahl U, eds. Heparin Chemical And Biological Properties, Clinical Applications. Boca Raton, Fla.:CRC Press, Inc. 1989, p.295-316; and H. Akimoto, et al., "Heparin and heparin sulfate block angiotensin-II-induced hypertrophy in cultured rat cardiomyocytes. A possible role of intrinsic heparin-like molecules in regulation of cardiomyocyte hypertrophy," Circulation, 93:810-816, 1996.

It was found that the polyanion heparin binds electrostatically to the positively charged amino acids of the NLF and prevent it from targeting NF-.kappa.B to the nuclear pore. Heparin and O-desulfated nonanticoagulant heparin prevented TNF-induced endothelial cell translocation of NF-.kappa.B from cytoplasm to the nucleus, studied immunohistochemically (FIG. 9), and reduced binding of NF-.kappa.B to DNA in electrophoretic mobility shift assays performed with HUVEC nuclear protein (FIG. 10). ODS heparin also prevented enhanced DNA binding of NF-.kappa.B in ischemic-reperfused myocardium (FIG. 11). Thus, inhibition of NF-.kappa.B activation appears specific for heparin. These results are consistent with the possibility that heparin electrostatically blocks the NLF, exposed when NF-.kappa.B dissociates from its inhibitor I.kappa.B.

Heparin and nonanticoagulant heparin prevent myocardial apoptosis by three mechanisms:

1. Inhibition of Endogenous TNF Production by Myocardium

Decreasing endogenous production of TNF by myocardium itself would reduce the amount of hormone locally available to attach to TNFR1 receptors and stimulate death domain-dependent induction of apoptosis in an autocrine fashion. TNF expression is heavily regulated by the transcription factor nuclear factor-.kappa.B. See, F. G. Wulczyn, et al., "The NF-.kappa.B and I .kappa.B gene families: mediators of immune response and inflammation," J. Mol. Med 74:749-769, 1996.

Heparin in higher than anticoagulant doses or nonanticoagulant heparin both inhibit NF-.kappa.B activation in myocardium. See, V. H. Thournai, supra. In myocardial tissue samples from the isolated perfused rat hearts studied in the above publication, we measured myocardial TNF levels after 10 minutes ischemia and 30 minutes reperfusion. TNF levels in nonanticoagulant heparin treated ischemic-reperfused hearts were only 30% of those in untreated, ischemic reperfused hearts, reducing the potential stimulus for TNFR1 mediated apoptosis (5.74.+-.1.65 for ischemic-reperfused hearts vs 1.78.+-.0.61 pg/g dry weight for nonanticoagulant heparin treated ischemic-reperfused hearts, p<0.05).

2. Reduction of Exogenous TNF Production by Reduction of Inflammatory Cell Influx into Ischemic-reperfused Myocardium

It has been also demonstrated that larger than anticoagulant doses of heparin and nonanticoagulant heparin reduce the influx of inflammatory cells into ischemic-reperfused myocardium. V. H. Thournai, supra. Blood inflammatory elements such as neutrophils and macrophages are rich sources of TNF production, and apoptotic myocardial cell death is highly correlated with inflammatory cell influx into myocardium following ischemic and reperfusion. See, D. Velez, et al., "Inflammatory cell infiltration and apoptotic cell death after myocardial ischemia and reperfusion," Circ. 100 (Supplement):I-691, 1999. Thus, inhibition of inflammatory cell influx should reduce exogenous TNF available to induce TNFR1 death domain mediated apoptotis of following myocardial infarction.

3. Direct Inhibition of Cytochrome c Mediated Activation of Apaf-1

The activation of Apaf-1, enabling it to convert procaspase 9 to its own active form requires binding to cytochrome c. It is the lack of availability of cytochrome c within normal cytoplasm that prevents activation of this pathway of apoptosis. Events which increase the permeability of the outer mitochondrial membrane, allowing flux of cytochrome c out into the cytoplasm, are the initiating events for mitochrondrial regulated cell death. The entire anti-apoptotic Bcl-2 family and pro-apoptotic Bax and BH3 families of proteins regulate apoptosis by blocking (Bcl-2) or opening (Bax and BH3) pores in the mitochondrial membrane through which cytochrome c might flux outward. See, J. M. Adams, et al., "The Bcl-2 protein family:arbiters of cell survival," Science 281:1322-1326, 1998. Thus, cytochrome c performs a pivotal function in initiating the cellular apoptosis cascade.

Mitochrondrial cytochrome c is a basic protein with a positive charge of +9.5 at neutral pH. See, L. C. Petersen, et al., "The effect of complex formation with polyanions on the redox properties of cytochrome c," Biochem. J., 192:687-693, 1980; Bagelova, et al., "Studies on cytochrome c-heparin interactions by differential scanning calorimetry," Biochem. J., 297:99-101. 1994. Within the cell, cytochrome c forms complexes with its natural electron chain redox partners such as cytochrome bc1 complex and cytochrome c oxidase. These complexes are electrostatic in nature and involve charge-dependent binding to the positive lysine residues surrounding the exposed edge of the "haem moiety" to negatively charged amino acids on its respiratory chain partners. It is this haem-edge area on the cytochrome c molecule that is also involved in the exchange of electrons with its natural redox partners and the site of reaction with small molecules such as the reducing agent ascorbate. Thus, the cytochrome c molecule electrostatically binds in the same region as it is functionally active in redox reactions.

Because of its positive charge, cytochrome c naturally binds to other polyanions such as heparin and dextran sulfate. See, L. C. Petersen, et al., supra. Binding of cytochrome c to heparin greatly decreases its reactivity in redox reactions. An example is the 200 fold reduction in reaction with ascorbate effected by addition of 40 .mu.g/ml heparin to 10 .mu.M cytochrome c and 0.4 M sodium ascorbate in 10 mM Tris buffer, pH 7.4 (see Table 2, Peterson, et al., cited above). The complex of heparin and cytochrome c occurs whether cytochrome c is in the reduced or oxidized state. See, M. Antalik, M., et al., "Spectrophotometric detection of the interaction between cytochrome c and heparin," Biochem. Biophys, Acta 1100:155-159, 1992). Heparin is readily taken up and internalized by endothelium, smooth muscle. See, T. C. Wright, et al, "Regulation of cellular proliferation by heparin and heparin sulfate." Heparin. Chemical and Biological properties. Clinical applications, D. A. Lane and U. Lindahl, editors, CRC Press, Inc., Boca Raton, Fla., 295-316. Heparin is readily taken up and internalized by myocardium. See, H. Akimoto, et al., "Heparin and heparin sulfate block angiotensin-II-induced hypertrophy in cultured neonatal rat cardiomyocytes. A possible role of intrinsic heparin-like molecules in regulation of cardiomyocyte hypertrophy," Circ, 93:810-816, 1996.

It has been shown that heparin and nonanticoagulant heparin inhibits activation of nuclear factor-.kappa.B in cultured human umbilical vein endothelial cells and in whole ischemic-reperfused rat hearts. In its unactivated state, nuclear factor-.kappa.B is a cytosolic protein. Therefore, at doses higher than used for anticoagulation, heparin or nonanticoagulant heparin both concentrates in myocardial endothelium and myocardium itself in levels sufficient to affect cytosolic events.

Positively charged cytochrome c binds to Apaf-1 on a negatively charged region of the Apaf-1 molecule characterized by 12 WD (tryptophan-aspartic acid) amino acid repeats. See, H. Zou, et al., "Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3," Cell, 90:405-413, 1997. Because the 6 aspartic acids are all acidic, this is a very negatively charged region of the Apaf-1 molecule that likely binds to the same positively charged lysine residues adjacent to the haem edge region, where cytochrome c binds other negatively charged partners. Binding of positively charged cytochrome c to the negatively charged 12 WD repeat region of Apaf-1 induces conformational changes that allow Apaf-1 to bind, in turn, to caspase-9 and activate it. See, J. C. Reed, "Cytochrome c: Can't live with it--Can't live without it," Cell, 91:559-562, 1997. As strong polyanions, heparin or nonanticoagulant heparin would naturally compete with cytosolic Apaf-1 for binding to positively charged cytochrome c. Electrostatic interaction with proteins is the basis for other inhibitory effects of heparin, such as the ability of heparin and other sulfated polysaccharides to inhibit the positively charged granular neutrophil proteases human leukocyte elastase and cathepsin G. See, N. V. Rao, et al., "Sulfated polysaccharides prevent human leukocyte elastase-induced lung injury and emphysema in hamsters," Am. Rev. Respir. Dis, 142:407-412, 1990. By binding any cytochrome c entering the cytoplasm, heparin or nonanticoagulant heparin would prevent the interaction of cytochrome c with Apaf-l, and thereby prevent procaspase-9 activation leading to apoptotis.

This activity of heparin or nonanticoagulant heparin to inhibit apoptosis would not be predicted based upon current knowledge. Ischemic preconditioning, or exposure of the heart to short noninjurious periods of ischemia, has been found to decrease apoptosis as a result of longer periods of ischemia and reperfusion. See, C. A. Piot, et al., Circ., 96:1598-1604, 1997. Nuclear factor-.kappa.B plays an essential role in myocardial ischemic preconditioning. See, Y-T Zuan, et al., "Nuclear factor-.kappa.B plays an essential role in the late phase of ischemic preconditioning in conscious rabbits," Circ. Res, 84:1095-1109, 1999. Nuclear factor-.kappa.B has also been reported to be essential in preventing apoptosis from TNF. See, A. A. Beg, et al., "An essential role for NF-.kappa.B in preventing TNF-.alpha.-induced cell death," Science, 274:782-784, 1996; and C. Y. Wang, et al., "TNF- and cancer therapy induced apoptosis: potential by inhibition of NF-.kappa.B," Science, 274:784-789, 1996. Thus, because, it has been shown that heparin or nonanticoagulant heparin inhibit NF-.kappa.B, the prior art would suggest that heparin or nonanticoagulant heparin would also reduce the anti-apoptotic effects of NF-.kappa.B and have the overall effect of enhancing apoptosis.

The strategy of using high doses of heparin or nonanticoagulant heparin to inhibit apoptosis will also have benefit in the treatment of stroke. Recent evidence points to the fact that as many as 50% of neurons that are lost as a consequence of stroke are dying by the process of apoptosis. See, M. Barinaga, "Stroke-damaged neurons may commit cellular suicide," Science, 281:1302-1303, 1998; and J-M. Lee, et al., "The changing landscape of ischemic brain injury mechanisms," Nature, 399 (Supplement): A7-A14, 1999.

Recently, activation of NF-.kappa.B has been shown to play an essential role in ischemic preconditioning. While possibly related to anti-apoptotic genes induced by NF-.kappa.B, ischemic preconditioning bears similarity to the tolerance against lethal endotoxemia conferred by prior exposure to sublethal doses of lipopolysaccharide. Tolerance to endotoxin induces several events which negatively regulate subsequent NF-.kappa.B activation. First, endotoxin related NF-.kappa.B activation induces transcriptional upregulation of I.kappa.B-.alpha. and p105, trapping NF-.kappa.B in the cytoplasmic compartment. See, C. Stratowa, et al., "Transcriptional regulation of the human intercellular adhesion molecule-1 gene: a short review," Immunobiol. 193:293-304, 1995. Second, increased production of p105 also leads to enhanced formation of p50 homodimers, which lack transcription-activation domains but compete with active Rel proteins at NF-.kappa.B binding sites. See, T. S. Blackwell, et al., "the role of nuclear factor-.kappa.B in cytokine gene regulation," Am. J Respir. Cell Miol. Biol., 17:3-9, 1997. Finally, endotoxin tolerance is associated with depletion of latent cytoplasmic p65 containing NF-.kappa.B heterodimers in tolerant cells. See, T. S. Blackwell, et al., "Induction of endotoxin tolerance depletes nuclear factor-.kappa.B and suppresses its activation in rat alveolar macrophages," J. Leukoc. Biol., 62:885-891, 1997. These events would also be expected from NF-.kappa.B activation by sublethal ischemia, providing an explanation for how NF-.kappa.B can mediate both protection from short periods of ischemic preconditioning and injury from more prolonged ischemia with reperfusion related infarction.

When given at the time of coronary reperfusion, nonanticoagulant heparin decreases myocardial infarct size, reduces neutrophilic influx into necrotic myocardium and preserves endothelial vasodilator function within the ischemic-reperfused coronary artery at risk without producing anticoagulation. Inhibition of NF-.kappa.B activation and myocardial reperfusion injury is unlikely from the previously reported anti-complement activity of heparin, since the nonanticoagulant heparin we used has low inhibitory activity against complement. These findings provide new insight into the mechanisms of anti-inflammatory activity of heparin, and disclose a true nonanticoagulant heparin with potential for interrupting both the pathophysiologic consequences of ischemia-reperfusion syndromes and NF-.kappa.B mediated inflammation.

Claim 1 of 14 Claims

That which is claimed:

1. A method for inhibiting apoptosis in ischemic-reperfused myocardium comprising administering to a human in need thereof from 3 mg/kg to 100 mg/kg of heparin to reduce apoptisis cell death in myocardial infarction.

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