AU7370100A - Method of inhibiting nf-kappab with heparin - Google Patents

Description

WO 01/19376 PCTUSOO/24910 METHOD OF INHIBITING NF-KB WITH HEPARIN BACKGROUND OF THE INVENTION Nuclear factor-KB (NF-KB) is an important transcription factor involved in the regulation of a variety of genes in animal cells. Normally in a quiescent state, NF-KB resides in the cytoplasm in the form of a "Rel complex" formed by Rel proteins and NF-KB. When activated by an extracellular or intracellular signal, NF-KB translocates to the nucleus, where it attaches to cis-acting KB sites in promoters and enhancers of a variety of genes. The NF-KB binding DNA consensus sequence is 5' GGGPuNNPyPyCC-3'. The translocation of NF-KB upregulates the transcription of mRNA for a host of proteins. An important group of such proteins are cytokines including tumor necrosis factor (TNF), IL-1, IL-2, IL-6, IL-8, interferon-3, interferon-y, tissue factor-1, complement, and inducible nitric oxide synthase, and the like. See e.g., Siebenlist et al., Annu. Rev. Cell Biol. 10:405-455 (1994); see also U.S. Patent No. 5,804,374. As a result, NF-KB plays an important role in mediating signal transduction in a variety of inducible systems. For example, NF-KB is involved in the activation of various cytokines and thus plays a central role in the mediation of immune response and inflammation. NF-KB is also involved in ischemia-reperfusion, e.g., in myocardial infarction and stroke. See Meldrum et al., J. Mol. Cell. Cardiol. 29:2849-2854 (1997). NF-KB has also been shown to increase the propagation of human immunodeficiency virus (HIV) in cells infected with other viruses. See e.g., Gimble et al. J. Virol., 62:4104-4112 (1998). -1 - WO 01/19376 PCT/USOO/24910 Morigi et al. J. Clin. Invest. 101:1905-1915 (1998) discloses that leukocyte-endothelial interaction is augmented by hyperglycemia in a NF KB-dependent manner, indicating that activation of NF-KB is involved in the formation of microvascular lesions associated with diabetes, causing diabetic vascular disease. It is also known that cytokines induced by the NF-KB activity are the causes of a number of diseases. For example, it has been discovered that the NF-iB-controlled cytokine tumor necrosis factor (TNF) mediates heart failure. See e.g., Cain et al. Cell Cardiol. 31:931-947 (1999). Clinically, steroids such as dexamethasone and solumedrol, which inhibit NF-cB activities, decrease TNF-ca levels after cardiac bypass. See e.g., Hill et al. J. Thorac. Cardiovasc. Surg. 110:1658-1662 (1995). Therefore, inhibition of NF-KB can have significant clinical implications in treating many diseases. In cytoplasm, the inhibitory molecules I-KBs are associated with NF-KB in the Rel complex. See, e.g, Grimm, et al., Biochem. J. 290:297-308 (1993). Genes encoding both NF KB and a number of I-KBs have been isolated. See, e.g., U.S. Patent Nos. 5,804,374; 5,597,898; 5,849,580. Activation of NF-KB is initiated when I KB is phosphorylated by I-KB kinase. The phosphorylation leads to the recognition of I-KB by ubiquitin and subsequent proteosomal degradation. See, e.g., Thanos et al. Cell 80:529-532 (1995); Stancovski, et al., Cell, 91:299-302 (1997). The removal of I-KB from the NF-KB protein exposes a positively charged group of amino acids on NF-KB protein known as the nuclear localization site (NLF). It has been found that the nuclear translocation of NF-KB can be competitively inhibited in a dose-dependent manner by synthetic peptides containing a cell membrane permeable motif and the nuclear localization sequence. See Lin et al., J Biol. Chem. 270:14255-14258 (1995). A number of glucocorticoids have been shown to be able to block the translocation of NF-KB from the cytoplasm to nucleus. See Adcock, et al., Am. J. Physiol. 37:C331-C338 (1995); see also Ray et al., Proc. Natl. Acad Sci. USA 91:752-756 (1994). -2- WO 01/19376 PCT/USOO/24910 However, glucocorticoids when used in patients have a number of adverse effects, including induction of hypertension, glucose intolerance and bone demineralization. Thus, it would be of major advantage to develop an alternative, non-glucocorticoid based strategy for inhibiting activation of NF-KB, and treating diseases in animals. SUMMARY OF THE INVENTION This invention provides a method for inhibiting NF-cB in animal cells using heparin. According to the present invention, it has been discovered that heparin is capable of blocking the translocation of NF-KB from cytoplasm to nucleus, thus inhibiting the NF-KB-dependent gene expression. In one aspect of this invention, a method is provided for inhibiting the translocation of NF-KB from the cytoplasm to the nucleus of cells. The method can be applied either in vitro directly to cells, or in vivo to a patient. The method includes administering heparin to the cells so that the heparin is internalized into the cytoplasm of the cells to inhibit the translocation of NF-KB. In another aspect of this invention, a method for treating diabetic microvascular disease in a patient suffering the disease is provided. The method comprises administering to the patient a therapeutically effective amount of heparin to inhibit the translocation of NF-KB to cell nucleus. In addition, heparin can also be administered to prevent diabetic microvascular disease in a patient having the tendency to develop the disease, e.g., a diabetic patient. In yet another aspect of this invention, a method for treating heart failure in a patient suffering the disease is provided. The method comprises administering to the patient a therapeutically effective amount of heparin to inhibit the NF-KB activity. In addition, a therapeutically effective amount of heparin can also be administered in a patient having the tendency to suffer heart failure, to prevent the occurrence of heart failure. The heparin used in this invention can be heparin sulfate, or heparin derivatives including O-desulfated or N-desulfated, or N- and O-desulfated -3- WO 01/19376 PCT/USOO/24910 heparin, or acylated heparin. Alternatively, heparin can be modified with a lipophilic moiety or administered in a liposomal preparation to facilitate the internalization of heparin into cell cytoplasm. Preferably, a nonanticoagulant heparin or heparin having reduced anticoagulant activity is used to avoid the adverse effects from bleeding. When heparin is administered to a patient, it is readily absorbed by cells such as endothelium, smooth muscle and cardiac myocytes. In addition, it has been used clinically for over fifty years in treating and preventing thrombosis, and is proved to be relatively non-toxic and safe. Thus, the discovery of the new use of heparin in this invention offers a readily available and easily used treatment for diseases such as heart failure and diabetic vascular disease in man and other mammals. The foregoing and other advantages and features of the invention, and the manner in which the same are accomplished, will become more readily apparent upon consideration of the following detailed description of the invention taken in conjunction with the accompanying examples, which illustrate preferred and exemplary embodiments. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows that heparin and O-desulfated (ODS) heparin reduce infarct size (AN/AAR). The area at risk (AAR) is expressed as a percentage of the left ventricle (LV). The infarct size (area of necrosis, AN) is expressed as a percentage of the area at risk (AAR). Columns represent group means ± standard error of the mean. *p < 0.05 versus Control. Figure 2 shows heparin and ODS heparin reduce plasma creatine kinase activity after infarction. Plasma creatine kinase activity is shown during the time course of the experiment. Values are means ± standard error of the mean. *p <0.05 HEP and ODS-HEP versus Control and ^ p < 0.05 versus the previous time point in the same group. Figure 3 shows heparin and ODS heparin do not alter regional myocardial collateral blood flow. A. Regional collateral blood flow: AAR blood flow. Regional myocardial blood flow in the area at risk (AAR) which is in the distribution of the ischemic-reperfused left anterior -4- WO 01/19376 PCTIUSOO/24910 descending (LAD) coronary artery. B. Regional collateral blood flow: Nonischemic blood flow. Regional myocardial blood flow in the nonischemic myocardial area which is in the distribution of the non ischemic-reperfused left circumflex (LCx) coronary artery. Figure 4 demonstrates that heparin and ODS heparin reduce influx of PMNs after myocardial infarction. Myeloperoxidase activity, an index of PMN accumulation, is shown in normal, ischemic, and necrotic myocardial tissue samples from each group. *p < 0.05 HEP and ODS-HEP versus Control. Figure 5 shows that ODS heparin does not produce anticoagulation in vivo. Systemic whole blood anticoagulation was studied using the activated clotting time, measured in seconds. *p < 0.05 HEP versus other groups. Figure 6 shows that heparin and partially 0-desulfated nonanticoagulant heparin block PMN4 adherence to normal coronary artery endothelium in vitro. A. Neutrophil adherence to normal coronary endothelium was stimulated by 100 nM platelet activating factor (PAF) added to medium and was inhibited in a dose-dependent manner by heparin (HEP) or ODS-HEP. *p <0.05 HEP group versus HEP control, @p <0.05 HEP group versus 0 tg HEP group, *p < 0.05 ODS-HEP versus ODS control and 4p < 0.05 ODS-HEP versus 0 pig ODS group. B. Inhibition of neutrophil adherence PAF-stimulated normal coronary endothelium by the polyanions heparin (HEP) and ODS-HEP is antagonized in a dose dependent manner by charge neutralization with the polycation protamine (Prot). *p < 0.05 HEP-Prot group versus HEP alone, @p < 0.05 HEP-Prot group versus 0 ptg Hep-Prot group, *p < 0.05 ODS-Prot versus ODS alone and "p < 0.05 ODS-Prot versus 0 ptg ODS-Prot group. Figure 7 demonstrates that heparin and ODS heparin reduce PMN adherence to post-experimental coronary artery endothelium. Neutrophil (PMN) adherence to coronary endothelium was quantitated as the number of adherent PMNIs/mm2 of coronary endothelium. LCx = the non-ischemic reperfused left circumflex coronary artery, LAD = the ischemic-reperfused -5- WO 01/19376 PCT/USOO/24910 left anterior descending coronary artery. *p < 0.05 HEP and ODS-HEP versus LAD Control. Figure 8 shows that heparin and ODS heparin preserve the vasodilator function of ischemic-reperfused coronary arteries. A. Agonist stimulated macrovascular relaxation to acetylcholine: LAD. Response curves to incremental concentrations of acetylcholine (ACh) to the ischemic-reperfused left anterior descending (LAD) coronary artery precontracted with U46619. *p < 0.05 HEP and ODS-HEP versus Control and *p < 0.05 HEP versus Control. B. Agonist-stimulated macrovascular relaxation to Acetylcholine: LCx. Response curves to incremental concentrations of acetylcholine (ACh) to the non-ischemic-reperfused left circumflex (LCx) coronary artery precontracted with U46619. Figure 9 shows that heparin and ODS heparin prevent translocation of NF-xB from cytoplasm to the nucleus. A. Immunohistochemical staining for NF-xB in unstimulated control cells. Brown anti-p65 staining is present in the cytoplasm of HUVEC, but not in nuclei. B. HUVEC stimulated with TNFa (10 ng/ml) without addition heparin. Some but not all nuclei now stain positive for anti-p65, corresponding to translocation of NF-KI3 from cytoplasm to the nucleus. In HUVEC pre-treated with 200 Rg/ml HEP (C) or ODS-HEP (D), TNF stimulation fails to produce anti p65 nuclear staining, suggesting that heparin or nonanticoagulant heparin prevents NF-idB nuclear translocation. Figure 10 shows that ODS heparin decreases DNA binding of NF xI3 in TNF-stimulated HUVECs. HUVECs were stimulated with 10 ng/ml TNFa for one hr and nuclear protein was harvested for electrophoretic mobility shift assays (EMSAs) to detect binding of NF-KB, using the oligonucleotide consensus AGTTGAGGGGACTTTCCCAGGC, end labeled with [y 32 P]ATP. Binding reactions were performed with 10 pg nuclear protein, electrophoresed on a 6% nondenaturing polyacrylamide gel in 0.5 x TBE (45 mM Tris borate, 25 mM boric acid, 1 mM EDTA) at 40 C and autoradiographed at -80' C. A typical EMSA experiment from HUVEC is shown. Treatment of monolayers with TNF stimulates DNA binding of NF-KB (lane 2) compared to untreated controls (lane 1). Pretreatment of cells with 200 ptg/ml ODS-HEP virtually eliminates NF-KB -6- WO 01/19376 PCTIUSOO/24910 binding activity in nuclear protein extracts (lane 3), confirming that heparin prevents translocation of NF-icB from the cytoplasm to the nucleus. Figure 11 shows that ODS heparin decreases DNA binding of NF KB in ischemic-reperfused myocardium. A. Electrophoretic mobility shift assays of nuclear protein from ischemic-reperfused rat myocardium. Langendorf perfused rat hearts were subjected to 15 min warm global ischemia followed by 15 min reperfusion. Nuclear protein was then harvested for EMSAs to measure DNA binding of NF-icB. Binding reactions were performed with 15 pg of nuclear protein, electrophoresed at room temperature on a 5% nondenaturing polyacrylamide gel in 0.5 x TGE (120 mM glycine and 1 mM EDTA in 25 mM Tris, pH 8.5) and autoradiographed at -80 0 C. Compared to sham perfused control hearts (lane 1), ischemia and reperfusion typically increased DNA binding of myocardial nuclear protein to oligonucleotide sequences for NF-KB (lanes 2 and 4). Three distinct complexes were identified. Supershift experiments performed with antibody to p65 (lane 5), antibody to p50 (lane 6) or both antibodies (lane 7) demonstrated complex I to be shifted (arrow), identifying it as the band containing the p65 component of NF-icB. Pretreatment and perfusion with ODS-HEP (6 mg/kg iv 2 hr prior to heart perfusion; 100 ptg/ml in perfusate) prevented the ischemia-reperfusion related stimulation of NF-KB DNA binding of the p65-containing complex I (lane 3). DNA binding of the p65-containing complex I was nearly eliminated by ODS-HEP, with a reduction of 54 V 6 % as measured by densitometry in comparison to complex I of untreated ischemic-reperfused rat hearts (p < 0.05, n = 4). B. Competition experiments were performed by incubation of nuclear proteins with 1 Ox unlabeled NF-KB (lane 2) or cyclic-AMP responsive element oligonucleotides (CRE, AGAGATTGCCTGACGTCAGAGAGCTAG, Promega) (lane 3) for 5 min prior to addition of 32 P-labeled NF-KB probe. Compared to binding reactions without excess unlabeled probe (lane 1), addition of unlabeled NF-KB blocked DNA binding in all three complexes. Figure 12 shows that ODS-heparin improves recovery of contractile function following ischemia and reperfusion. Pressure Rate Product [HR x -7- WO 01/19376 PCTIUSOO/24910 (PSP-EDP)] determined in isolated rat hearts before ischemia (Baseline) and following 30 minutes of ischemia and 15 minutes of reperfusion (Reperfusion). Values are mean V SEM, n = 5 per group. Two-way Repeated Measures ANOVA indicates the following significant effects: * Overall treatment effect, overall time effect, overall treatment-time interaction, reperfusion sham vs ischemia-reperfusion (IR), reperfusion sham vs ODS heparin-treated ischemia-reperfusion (ODS HEP IR), reperfusion IR vs ODS HEP IR. DETAILED DESCRIPTION OF THE INVENTION The present invention now will be described more fully hereinafter with reference to the accompanying drawings, 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 provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. In accordance with the present invention, it has been discovered that the translocation of NF-KB from cytoplasm to cell nucleus can be inhibited by heparin. While not wishing to be bound by any theory, it is believed that this results from the interaction of heparin, a polyanion, with the positively charged nucleus localization factor (NLF) on NF-KB. The electrostatical interaction may lead to the binding of heparin to NF-KB, thus blocking its translocation to nucleus. As used herein, unless otherwise specified, the meaning of the term "heparin" is inclusive and include heparin, heparan sulfate, and derivatives thereof including, e.g., heparin or heparan sulfate linked to a peptide or protein, heparin or heparan sulfate linked to a lipophilic moiety, and the like. Typically, the term "heparin" as used herein refers to a molecule having alternating disaccharide sequences of D-glucuronic acid-N-acetyl D-glucosamine, and/or disaccharide sequences of L-iduronic acid-N-acetyl D-glucosamine. The manufacture of various types of heparin and several commercially available heparins are disclosed in Holmer in Heparin, Lane -8- WO 01/19376 PCTIUSOO/24910 and Lindahl eds., CRC Press, Inc., Boca Raton, FL (1989), the entirety of which is incorporated herein by reference. The pharmacokinetics of heparins and their application in thrombosis are also disclosed in Holmer, Id. Methods for preparing heparin from various mammalian tissues are known in the art and are disclosed in, e.g., Coyne in Chemistry and Biology ofHeparin, Lunblad et al, eds., Elsevier Publishers, North Holland, N.Y. (1981), which is incorporated herein by reference. Any conventional molecular weights known in the art will be useful. For example, the average molecular weight of suitable heparin can range from about 1,000 to about 50,000, preferably from about 2,000 to about 20,000. However, heparins having an average molecular weight beyond the above ranges may also be useful for this invention. As used herein, the term "heparin" also include heparins modified by, e.g., sulfation, desulfation, acylation, and the like, at any positions in the heparin molecule. For example, heparin can be modified at one or more of the positions of 2-0, 3-0, 6-0, and N-, and the like, by desulfation or acylation. Typically, 0-desulfated heparins, N-desulfated heparins, and N acylated heparins are preferred as they are normally non-anticoagulant and thus do not cause serious bleeding. Methods for heparin desulfation at various positions and to various extent are known in the art. Typically such methods were developed in association with the work done in the art to remove anti-coagulant activity from heparin. For example, N-desulfation can be achieved by treating pyridinium heparin salt with dimethylsulfoxide (DMSO) in five percent methanol for 1.5 hours at 50' C or by similar treatment in MDSO in 10% methanol for 18 hours at 1000 C. Partial N-desulfation from heparin can also be done by acid hydrolysis at 55'-60' C for 72 hours. For purpose of N-acylation, after N-desulfation, the N-desulfated heparin can be easily acylated by methods known in the art. Preparation of 2-0-desulfated heparin which is not anticoagulant has been described in Jaseja et al., Can. J Chem., 67:1449-1456 (1989) and Rej et al., Thrombosis and Hemostasis, 61 (3):540 (1989), both of which are incorporated herein by reference. Briefly, the method starts with a -9- WO 01/19376 PCT/USOO/24910 solution of heparin in 0.1N sodium hydroxide which is then lyophilized, thereby effecting a selective displacement of the 2-sulfate group of a-L iduronic acid 2-sulfate and leaving a 2-0-desulfated a-L-iduronic acid residue. This compound was shown to have minimal anticoagulant activity. U.S. Patent No. 5,668,118 describes an improved method for preparing 2-0-desulfated heparin in commercially useful quantities, which is incorporated herein by reference. In addition, U.S. Patent No. 5,795,875 describes a method of preparing 6-0-desulfated heparin, which is incorporated herein by reference. Heparin can be acylated at one or more of the 0- or N- positions to form, e.g., 0- or N-acetylated, butyrylated, hexanoylated, benzoylated, octanoylated, or succinylated heparin. Acylation of heparin is described in, e.g., Barzu et al. J Med Chem. 36:3546-3555 (1993), which is incorporated herein by reference. Heparin is readily bound and internalized into the cytosolic compartment by cells such as endothelium, vascular and airway smooth muscle, mesangial cells and even cardiac myocytes. See Wright, et al., In Heparin, Lane and U. Lindahl, eds. CRC Press, Inc., Boca Raton, FL, pages 295-316 (1989); Akimoto et al., Circulation, 98:810-816 (1996). In other cell types, such as epithelium, where heparin is not readily internalized, a heparin modified with a lipophilic moiety may be used. Suitable lipophilic moieties can be a long chain fatty acid or a cholesterol derivative. With a lipophilic moiety, the heparin will have an increased ability to penetrate the cell membrane to reach cell cytoplasm. Such heparins can be prepared by covalently linking the lipophilic moiety to heparin by any suitable methods known in the art. For example, acylation reactions useful for conjugating heparin to a lipophilic moiety are described in Barzu et al. J Med Chem. 36:3546-3555 (1993), which is incorporated herein by reference. Acylation can occur at free N groups, desulfated N groups, OH groups or COOH groups on a heparin molecule. Methods of conjugation disclosed in U.S. Patent No. 5,681,811 may also be adapted for use in this invention. -10- WO 01/19376 PCT/USOO/24910 In one embodiment of this invention, a method for inhibiting NF-cB activity is provided by contacting heparin to a cell in vitro such that the heparin is internalized into the cytoplasm of the cell thereby the translocation of NF-xB to nucleus is blocked. When the method is applied to an animal in vivo, the heparin can be administered to the animal such as a human by any conventional methods of administration such as parenteral, topical, intradermal or subcutaneous administration. As discussed above, it is known in the art that NF-KB activates gene expression for a variety of genes. However, the NF-KB activity requires that the NF-B protein translocate from cytoplasm to cell nucleus. In accordance with this invention, heparin is administered to cells to interfere with and to prevent the translocation of NF-KB. As a result, the expression of the genes controlled by NF-xB is inhibited. Thus, the present invention is useful in regulating the expression of a variety of genes normally controlled by NF-xB. As used herein, the term "inhibiting NF-B activity" means that, as a result of the administration of heparin, NF-KB is prevented from translocation to cell nucleus, and thus at least one of the genes normally activable by NF-KB protein is not activated. Many genes activable by NF KB are known in the art, including cytokines such as tumor necrosis factor (TNF), IL-1, IL-2, IL-6, IL-8, interferon-, interferon-y, tissue factor-1, complement, and inducible nitric oxide synthase, and the like. See e.g., Siebenlist et al., Annu. Rev. Cell Biol. 10:405-455 (1994); see also U.S. Patent No. 5,804,374. In another embodiment of this invention, a method for treating heart failure in a patient is provided. A number of studies have reported elevated circulating levels of tumor necrosis factor in patients with end-stage congestive heart failure. These reports generally suggest that serum levels of TNF correlates the severity of heart failure. See e.g., Levine et al. N. Engl. J. Med. 323:236 241 (1990); FeFerrari et al. Circulation 92:1479-1486 (1995); Torre Amione et al. J. Am. Coll. Cardiol. 27:1201-1206 (1996); Testa et al. J Am. Coll. Cardiol. 28:964-971 (1996); MacGowan et al. Am. J Cardiol. -11- WO 01/19376 PCT/USOO/24910 79:1128-1131 (1997). Transgenic mice overexpressing TNF-a exhibits the phenotype of congestive heart failure and increased mortality. Such transgenic mice display both cardiac dilatation and left ventricular dysfunction. See Kubota et al. Circ. Res. 81:627-635 (1997). Administration of TNF-a to dog decreases myocardial contractile function in dog. See Murray et al., Circ. Res. 78:154-160 (1995). Likewise, TNF-a directly depresses myocardial contractility in human. In addition, clinically TNF-a has been shown to be an important cardiodepressant factor during sepsis and heart failure. See Cain et al., J. Am. Coll. Surg. 186:337-350 (1998); Kubota et al. Circulation 97:2499-2501 (1998). As discussed above, TNF-a gene expression has been shown to be activated by NF-xB. When activated by, e.g., oxidant stress in myocardium, NF-xB in myocytes translocates into cell nucleus and binds to the concensus sequence in the promoter or enhancer of TNF-a gene resulting the activation of the TNF-a. gene expression. The expressed TNF-a protein depresses both animal and human myocardial function in a dose dependent manner, which contributes to the development of heart failure. See e.g., Cain et al. Cell Cardiol. 31:931-947 (1999). Clinically, steroids such as dexamethasone and solumedrol, which inhibit NF-KB activities, decrease TNF-ca levels after cardiac bypass. See e.g., Hill et al. J. Thorac. Cardiovasc. Surg. 110:1658-1662 (1995). Accordingly, in accordance with one aspect of this invention, heparin is used to treat heart failure in a patient suffering heart failure or to prevent the occurrence of heart failure in a patient known to have a tendency to develop heart failure. The method includes administering to the patient a therapeutically effective amount of heparin. Once heparin is administered to the patient and is absorbed into cell cytoplasm, preferably the cytoplasm of the myocytes of the patient, the heparin inhibits the NF KB activity in the cells by preventing or interfering with the translocation of NF-KB to cell nucleus. As a result, the expression of TNF-C is inhibited, and the cardiodepressant activity of TNF-a is stopped or prevented, and heart failure is thus treated or prevented. -12- WO 01/19376 PCT/USOO/24910 In accordance with another aspect of the invention, a method for treating diabetic vascular disease in a patient is provided. NF-xB has been shown to be involved in the development of diabetic vascular disease. See Morigi et al. J. Clin. Invest. 101:1905-1915 (1998). Activation of NF-B contributes to the formation of microvascular lesions associated with diabetes, causing diabetic vascular disease. Therefore, in one embodiment of this invention, heparin is used to inhibit the NF-B activity in a patient thus treating or preventing diabetic vascular disease. Accordingly, a therapeutically effective amount of heparin can be administered to a patient suffering from diabetic vascular disease. For prevention, a patient known to have the tendency to develop diabetic vascular disease, typically a diabetic patient, may be administered a therapeutically effective amount of heparin. In accordance with this invention, heparin is typically administered in a pharmaceutically acceptable carrier through any appropriate routes such as parenteral, intravenous, oral, intranasal, intradermal, subcutaneous, or topical administration. The heparin is administered at a therapeutically effective amount to achieve the desired therapeutic effect without causing any serious adverse effects in the patient treated. Heparin has been used commercially as anticoagulant and its pharmacokinetics is discussed in detail in, e.g., Holmer in Heparin, Lane and Lindahl eds., CRC Press, Inc., Boca Raton, FL (1989). Heparin can be effective when administered at an amount within the conventional clinical ranges determined in the art. Typically, it can be effective at an amount of from about 0.1 mg/kg to about 100 mg/kg per day, preferably from about 0.5 to about 50 mg/kg per day based on the total body weight of the patient administered. Advantageously, heparin is administered at an amount of about 2.5 mg/kg to about 10 mg/kg per day. Heparin may be administered at once, or may be divided into a number of smaller doses to be administered at predetermined intervals of time. The suitable dosage unit for each administration of heparin can be, e.g., from about 5 mg to about 2000 mg, preferably from about 50 to about 500 mg. The dosages for various heparin derivatives such as lipophilic moiety conjugates can be estimated or calculated based on the above -13- WO 01/19376 PCT/USOO/24910 dosage ranges of heparin and the molecular weights of the derivatives, or by other methods known in the art. It should be understood that the dosage ranges set forth above are exemplary only and are not intended to limit the scope of this invention. The therapeutically effective amount can vary with factors including but not limited to the activity of the compound used, stability of the active compound in the patient's body, the severity of the conditions to be alleviated, the total weight of the patient treated, the route of administration, the ease of absorption, distribution, and excretion of the active compound by the body, the age and sensitivity of the patient to be treated, and the like, as will be apparent to a skilled artisan. The amount of administration can also be adjusted as the various factors change over time. Heparin can be administered to a patient to be treated through any suitable routes of administration. Advantageously, heparin is delivered to the patient parenterally, i.e., intravenously or subcutaneously. For parenteral administration, heparin can be formulated into solutions or suspensions, or in lyophilized forms for conversion into solutions or suspensions before use. Sterile water, physiological saline, e.g., phosphate buffered saline (PBS) can be used conveniently as the pharmaceutically acceptable carriers or diluents. Conventional solvents, surfactants, stabilizers, pH balancing buffers, anti-bacteria agents, and antioxidants can all be used in the parenteral formulations, including but not limited to acetates, citrates or phosphates buffers, sodium chloride, dextrose, fixed oils, glycerine, polyethylene glycol, propylene glycol, benzyl alcohol, methyl parabens, ascorbic acid, sodium bisulfite, and the like. The parenteral formulation can be stored in any conventional containers such as vials, ampoules, and syringes. Heparin can also be delivered orally in enclosed gelatin capsules or compressed tablets. Capsules and tablets can be prepared in any conventional techniques. For example, heparin can be incorporated into a formulation which includes pharmaceutically acceptable carriers such as excipients (e.g., starch, lactose), binders (e.g., gelatin, cellulose, gum tragacanth), disintegrating agents (e.g., alginate, Primogel, and corn starch), lubricants (e.g., magnesium stearate, silicon dioxide), and sweetening or -14- WO 01/19376 PCTIUSOO/24910 flavoring agents (e.g., glucose, sucrose, saccharin, methyl salicylate, and peppermint). Various coatings can also be prepared for the capsules and tablets to modify the flavors, tastes, colors, and shapes of the capsules and tablets. In addition, liquid carriers such as fatty oil can also be included in capsules. Other forms of oral formulations such as chewing gum, suspension, syrup, wafer, elixir, and the like can also be prepared containing heparin used in this invention. Various modifying agents for flavors, tastes, colors, and shapes of the special forms can also be included. In addition, for convenient administration by enteral feeding tube in patients unable to swallow, heparin can be dissolved in an acceptable lipophilic vegetable oil vehicle such as olive oil, corn oil and safflower oil. Heparin can also be administered topically through rectal, vaginal, nasal or mucosal applications. Topical formulations are generally known in the art including creams, gels, ointments, lotions, powders, pastes, suspensions, sprays, and aerosols. Typically, topical formulations include one or more thickening agents, humectants, and/or emollients including but not limited to xanthan gum, petrolatum, beeswax, or polyethylene glycol, sorbitol, mineral oil, lanolin, squalene, and the like. A special form of topical administration is delivery by a transdermal patch. Methods for preparing transdermal patches are disclosed, e.g., in Brown, et al., Annual Review ofMedicine, 39:221-229 (1988), which is incorporated herein by reference. Exemplary formulations for nasal delivery of heparin are disclosed in U.S. Patent 5,668,118, which is incorporated herein by reference. Typically, as known in the art, heparin can be delivered in the form of micronized dry powder for aresolization into the lung. Alternatively, heparin can be dissolved in a saline solution and delivered into a patient to be treated by a conventional nebulizer. Heparin can also be delivered by subcutaneous implantation for sustained release. This may be accomplished by using aseptic techniques to surgically implant heparin in any suitable formulation into the subcutaneous space of the anterior abdominal wall. Sustained release can be achieved by incorporating the active ingredients into a special carrier -15- WO 01/19376 PCT/USOO/24910 such as a hydrogel. Typically, a hydrogel is a network of high molecular weight biocompatible polymers, which can swell in water to form a gel like material. Hydrogels are generally known in the art. For example, hydrogels made of polyethylene glycols, or collagen, or poly(glycolic-co L-lactic acid) are suitable for this invention. See, e.g., Phillips et al., J Pharmaceut. Sci. 73:1718-1720 (1984). Heparin can also be conjugated, i.e., covalently linked, to a water soluble non-immunogenic high molecular weight polymer to form a polymer conjugate. Advantageously, such polymers, e.g., polyethylene glycol, can impart solubility, stability, and reduced immunogenicity to heparin. As a result, the active compound in the conjugate when administered to a patient, can have a longer half-life in the body, and exhibit better efficacy. PEGylated proteins are currently being used in protein replacement therapies and for other therapeutic uses. For example, PEGylated adenosine deaminase (ADAGEN7) is being used to treat severe combined immunodeficiency disease (SCIDS). PEGylated L-asparaginase (ONCAPSPAR7) is being used to treat acute lymphoblastic leukemia (ALL). PEGylated taxol has also been shown to be effective and have less toxicity. For a general review of PEG-protein conjugates with clinical efficacy. See, e.g., Burnham, Am. J. Hosp. Pharm., 15:210-218 (1994). Preferably, the covalent linkage between the polymer and heparin is hydrolytically degradable and is susceptible to hydrolysis under physiological conditions. Such conjugates are known as "prodrugs" and the polymer in the conjugate can be readily cleaved off inside the body, releasing the free heparin. Alternatively, other forms of controlled release or protection including microcapsules and nanocapsules generally known in the art, and hydrogels described above can all be utilized in oral, parenteral, topical, and subcutaneous administration of the heparin. Another preferable delivery form is using liposomes as carrier. Liposomes are micelles formed from various lipids such as cholesterol, phospholipids, fatty acids, and derivatives thereof. Heparin can be enclosed within such micelles. Methods for preparing liposomal -16- WO 01/19376 PCT/USOO/24910 suspensions containing active ingredients therein are generally known in the art and are disclosed in, e.g., U.S. Pat. No. 4,522,811, which is incorporated herein by reference. Several anticancer drugs delivered in the form of liposomes are known in the art and are commercially available from Liposome Inc. of Princeton, New Jersey, U.S.A. It has been shown that liposomes can reduce the toxicity of the active ingredient contained therein, and increase their stability. Heparin can also be administered in combination with other active agents that treat or prevent another disease or symptom in the patient treated. However, it is to be understood that such other active agents should not interfere with or adversely affect the effects of heparin on the symptoms being treated. Such other active agents include but are not limited to antiviral agents, antibiotics, antifungal agents, anti-inflammation agents, antithrombotic agents, cardiovascular drugs, cholesterol lowering agents, hypertension drugs, and the like. EXPERIMENTAL Porcine intestinal mucosal heparin was used in human umbilical vein endothelial cells (HUVEC) stimulated with tumor necrosis factor (TNFa) to activate cytosolic to nucleus translocation of NF-KB. Activation of NF-mB was studied by two methods: immunohistochemical staining of actual protein location; and electrophoretic mobility shift assays of DNA binding. To probe whether effects on NF-mB activation were related to an anticoagulant function, we also studied a nonanticoagulant heparin produced by partial 0-desulfation, as described below. In addition, heparin action was also studied in an in vivo canine model. Heparin and a partially O-desulfated nonanticoagulant (ODS) heparin with greatly reduced anticomplement activity were also compared. Given at the time of coronary artery reperfusion in a canine model of myocardial infarction, both heparin and ODS 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 ODS -17- WO 01/19376 PCTIUSOO/24910 heparin also prevented dysfunction of endothelial-dependent relaxation in the coronary arterial circulation following ischemic injury. These studies showed that heparin inhibited translocation of the transcription factor NF-KB from cytoplasm to the nucleus in human endothelial cells and in isolated perfused rat hearts. Heparin decreased NF KB DNA binding in human endothelium and ischemic-reperfused rat myocardium, providing a novel explanation for the reduction in ischemia reperfusion injury by ODS heparin. The results obtained here also suggest that heparin can be effective in treating diseases including heart failure, diabetic vascular disease, asthma, and sepsis, ischemic-reperfusion injury, among others, by disrupting multiple levels of the inflammatory cascade through inhibiting activation of the transcription factor NF-xB. MATERIALS AND METHODS Acetylcholine chloride, the calcium ionophore A23187, sodium nitroprusside, and indomethacin were obtained from Sigma Chemical Company (St. Louis, MO). The final concentration of acetylcholine, A23187, sodium nitroprusside, indomethacin, and U-46619 were determined from a previous study (11). The thromboxane A 2 mimetic, U 46619, was donated by Upjohn Pharmaceuticals, Inc (Kalamazoo, MI). Ca - and Mg 2 +-free Hanks= balanced salt solution IX was purchased from Cellgro (Mediatech, Inc. Herndon, VA). Grade I-A heparin sodium salt from porcine intestinal mucosa (Sigma Chemical Company, St. Louis, MO) was resuspended with Krebs-Henseliet (K-H) buffer and administered as an intravenous bolus (3 mg/kg to dogs). Synthesis and Analysis of Alkaline Lyophilized Heparin Heparin was partially 0-desulfated (ODS heparin) by lyophilization under alkaline conditions using a modification of previously reported methods. See Rej et al., Thromb. Haemostat. 61:540 (1989); Jaseja et al., Can. J. Chem. 67:1449-1456 (1989). When heparin is lyophilized at about pH 13.0 or greater, a-L-iduronic acid(2-sulfate) residues are desulfated to 2,3-oxirane intermediates that are further hydrolyzed to nonsulfated a-L iduronic acid, forming a 2-0 desulfated heparin. The much less common 3-0 sulfate of D-glucosamine-N-sulfate (3,6-disulfate) is also removed, but -18- WO 01/19376 PCT/USOO/24910 other sulfates, including those of hexosamine residues, remain intact. Aqueous solutions (0.4 to 5.0%) of porcine intestinal mucosal heparin were alkalinized to pH 13.0 by addition of NaOH up to 0.5 M final concentration, frozen, and lyophilized to dryness. In some syntheses NaBH 4 (1% final concentration) was added prior to alkalinization. After NaOH and NaBH 4 were removed by ultrafiltration, pH of the analog in solution was adjusted to approximately 7.0 and the solution was lyophilized again to dryness. Molecular weights of unmodified and alkaline lyophilized heparins were determined by high performance size exclusion chromatography in conjunction with multiangle laser light scattering, using a miniDAWN7 detector (Wyatt Technology Corporation, Santa Barbara, CA) operating at 690 nm. Disaccharide analysis was performed by the method of Guo and Conrad. See Guo et al., Anal. Biochem. 178:54-62 (1988). In this process N-acetyl-D-glucosamine residues are deacylated with hydrazine. The heparin is then deaminated and depolymerized by exposure to nitrous acid at pH 4 to break bonds between D-glucosamine and uronic acids, and then at pH 1.5 to break bonds between D glucosamine N-sulfate and uronic acids. Both reactions leave O-sulfates intact, and convert glucosamine or glucosamine N-sulfate to anhydromannose, which is radiolabeled with NaB[ 3

H

4 ], converting anhydromannose to anhydromannitol. Radiolabeled disaccharides are then separated by reverse-phase, ion-pairing high pressure liquid chromatography. The in vitro anticoagulant activity of heparins was studied in the activated partial thromboplastin time in the U.S. P. anticoagulant assay (United States Pharmacopeial Convention). (APTT) See Miletich, J.P., Williams Hematology, 5th ed., Beutler et al. eds., McGraw-Hill, Inc., New York, 1995. The anticogulant activity was also studied in anti-Xa clotting and amidolytic assays. Jesty et al., Methods Enzymol. 45:95-107 (1976); Teien et al., Thromb. Res. 10:399-410 (1977). Disaccharide analysis showed that alkaline lyophilization of porcine mucosal heparin produces an analog that is 2-0 desulfated on a-L iduronic acid (2-sulfate) saccharides and 3-0 desulfated at D-glucosamine -19- WO 01/19376 PCT/USOO/24910 N-sulfate (3,6-disulfate). This partially O-desulfated heparin analog (ODS heparin) had an average molecular weight of 10,500 daltons, compared to 11,500 daltons for the starting material. However, ODS heparin was much more polydisperse. Whereas only 30% of the starting heparin was less than 10,000 daltons and none was less than 6,000 daltons, over 60% of ODS fragments were less than 10,000 daltons and 30% were less than 6,000 daltons. Eight separately synthesized lots (100-1,000 gm) of ODS heparin showed 7.7 V 0.9 units/mg anticoagulant activity in the USP assay and 4.9 V 0.8 units/mg anti-Xa activity in the amidolytic assay, compared to 170 USP units/mg anticoagulant activity and 150 units/mg anti-Xa activity for the unmodified procine intestinal heparin from which all lots were manufactured. 1. In vitro studies PMN degranulation Supernatant myeloperoxidase (MPO) activity was measured as the product of canine PMN degranulation using a modification of the method by Ely (17). Canine PMNs (20 x 106 cells/ml) were incubated in the presence or absence of different concentrations of nonanticoagulant partially 0-desulfated heparin (ODS-HEP) for 5 minutes at 370 C. The cells were then incubated with platelet activating factor (PAF, final concentration 10 ptM) and cytochalasin B (5 ptg/ml). After being incubated at 370 C for 10 minutes and centrifuged (500 g for 15 min), the cell-free supernatant was mixed in a 1:1 ratio with MPO solution [6 ml 0.01 M potassium phosphate, 100 pl 0-dianisidine (100 mg/ml), and 60 1d hydrogen peroxide (0.3%)]. MPO activity of the resulting supernatants were immediately measured on a SpectroMax UV-Vis Plate Reader (Molecular Devices, Palo Alto, CA) at a wavelength of 450 nm for 5 min. PMN adherence to normal coronary artery endothelium The adherence of PMNs to normal canine epicardial arteries was assessed using coronary segments and PMNs obtained from normal animals, and prepared and labeled as described above. Unstimulated, fluorescent-labeled PMNs were coincubated as described above with -20- WO 01/19376 PCT/USOO/24910 normal canine artery segments in the presence or absence of HEP or ODS HEP. Platelet activating factor (PAF, 100 nM final concentration) was then added 5 min later, and the preparation was incubated for an additional 20 min. Adherent PMNs were then counted as outlined earlier. Preparation and experiments with human umiblical vein endothelial cells (HUVEC) HUVEC were isolated according to the method of Jaffe, et al. (18) and cultured using endothelial cell growth medium (Clonetics). Cells of donors were pooled to exclude the influence of blood group antigens and donor variability. Morphology was confirmed by phase-contrast light microscopy showing typical cobblestone monolayer appearance of cells. To confirm that the cells were endothelial and to determine the purity of the cultures, they were tested with antibodies to von Willebrand's factor and found to be essentially pure endothelial cultures. Primary or secondary cultures were used in all experiments, although endothelial morphology remained through 6 passages. HUVEC were used within 1-2 days of confluence and were re-fed 24 h prior to use. As endothelial growth medium contains many stimulators (ECGF, EGF, hydrocortisone plus the hormones and growth factors in bovine brain extract and fetal calf serum) the experiment was carried out in Neuman/Tytell serum free medium (Gibco/BRL). For experiments, cells were washed twice with PBS and then incubated in Neuman/Tytell medium alone for 24 h, followed by incubation with lipopolysaccharide (1 pg/ml) plus 10-20 ng/ml TNFa for 2 h, or in heparin or ODS-HEP (200 tg/ml) for 4 h with the addition of lipopolysaccharide and TNFca after 2 h. At the end of each experimental set, translocation of the transcription factor nuclear factor-B (NF-KB) from cytoplasm to the nucleus was studied in cells fixed for immunohistochemical staining or extracted of nuclear protein for performance of electrophorectic mobility shift assays (EMSAs). Immunohistochemical localization of NF-K B p65 was performed in HUVECs grown on sterile coverslips, stimulated as above, and then fixed for 20 min on ice with 4% paraformaldehyde in CEB (10 mM Tris-HCl, pH 7.9, 60 mM KCl, 1 mM EDTA, 1 mM dithiothreitol) with protease inhibitors, PI (1 mM Pefabloc, 50 tg/ml antipain, 1 pg/ml leupeptin, 1 -21- WO 01/19376 PCT/USOO/24910 pg/ml pepstatin, 40 pg/ml bestatin, 3 tg/ml E-64, and 100 tg/ml chymostatin). Cells were permeabilized by treating for 2 min with 0.1% NP40 in CEB/PI, washed once with cold CEB and fixed as before for 10 min. Coverslips were incubated in 3% hydrogen peroxide for 30 min to suppress any remaining peroxidase, and washed three times in cold PBS. The permeabilized and fixed cells were blocked for 2 h with 2% bovine serum albumin (BSA) in PBS on ice and incubated overnight at 4' C with 1 gg/ml of anti-p65 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted in 0.1% BSA/PBS. Unbound anti-p65 was washed away with 2% BSA/PBS and bound antibody was stained by incubation with biotinylated swine anti-rabbit immunoglobulin diluted 1:1000 in 0.1% BSA/PBS for 45 min on ice. Excess secondary antibody was washed away by 3 washes with 2% BSA/PBS on ice. After washing, the tissue was incubated with a streptavidin biotin peroxidase complex at room temperature for 1 h, washed again, and incubated in 0.03% wt/vol 3-3'diaminobenzidine with 0.003% vol/vol hydrogen peroxide until a brown reaction product could be seen. Cells were then counterstained with eosin and mounted with a top coverslip before viewing under light microscopy. Electrophoretic mobility shift assays (EMSAs) were also used to study the translocation of NF-K B from the cytoplasm to the nucleus. Nuclear proteins were obtained from HUVEC as described by Digman, et al. (19) with the addition of the following proteinase inhibitors: 1 mM\4 phenylmethylsulfonyl fluoride, 1 ptg/ml pepstatin A, 0.5 ptg/ml chymostain, 1 pg/ml antipain, 1 ptg/ml leupeptin and 4 pg/ml aprotinin. Protein content was determined spectroscopically using the Bio-Rad protein reagent and 10 ptg of nuclear protein was used in all experiments. Aliquots were frozen at -80' C to prevent repeated freezing and thawing. The DNA probe of the NF-KI3 consensus sequence AGTTGAGGGGACTTTCCCAGGC (see Lenardo et al., Cell 58:227-229 (1989), was obtained from Santa Cruz Biotechnology, Inc. and supplied as a double stranded oligonucleotide designed with 5'OH blunt ends which were labeled to high specific activity with [y 32 P]ATP using polynucleotide kinase. Free radionucleotide was removed using a Sephadex G-25 column. The probe (0.5 ng) was incubated with nuclear extract (10 ptg) prepared from HUVEC after various -22- WO 01/19376 PCTIUSOO/24910 treatments in 20 pl buffer containing a final concentration of 10 mM HEPES, pH 7.5, 50 mM KCl, 5 mM MgCl 2 , 1 mM dithiothreitol, 1 mM EDTA and 5% glycerol, plus 5 ptg of poly (dI-dC) to reduce nonspecific binding. Incubations were carried out at room temperature for 20 min. Reactions were electrophoresed at 14 V/cm for 1.5-2.0 hr on a 6% nondenaturing polyacrylamide gel in 0.5 x TBE (45 mM Tris borate, 25 mM boric acid, 1 mM EDTA) at 40 C. After electrophoresis the glass plates were separated, and the plate with the gel was wrapped in plastic film and autoradiographed at -800 C. Preparation and experiments with isolated perfused rat hearts Male Sprague-Dawley rats (300-400 g) were anesthetized with sodium pentobarbital (40 mg/kg, i.p.), and the hearts were quickly excised and placed in ice-cold saline until contractions ceased. The aorta was cannulated and hearts were perfused in a Langendorff perfusion apparatus. An incision was made in the pulmonary artery, and the left ventricle was perforated to allow drainage of Thebesian venous flow. A cellophane balloon was placed into the left ventricle through the mitral valve. Compliance tests, performed on each balloon before placement in the ventricle, indicated that the balloon volume was large enough to accommodate 250 ptl of fluid without changing pressure. The balloon was connected to a pressure transducer (Gould P23) and monitored by a Grass amplifier system with an AstroMed Dash 10 recorder to allow measurement of left ventricular pressures. The following parameters of left ventricular function were monitored via this balloon: end diastolic pressure (LVEDP), peak systolic pressure (LVPSP) and autorhythmic heart rate (HR). Left ventricular developed pressure (LVDP) was then calculated as LVPSP minus LVEDP, and pressure-rate product was calculated as HR x LVDP. The hearts and buffer were maintained at a constant temperature of 370 C, using jacketed, heated heart chambers, perfusion tubing and buffer reservoirs. Hearts were perfused with modified Krebs-Henseleit bicarbonate buffer (KHB), consisting of 118 mM NaCl, 4.7 mM KCl, 1.2 mM KH 2

PO

4 , 1.2 mM MgSO 4 7 H20, 3.0 mM CaCl 2 -2H 2 0 (yielding 2.5 mM free Ca 2 in the presence of EDTA), 0.5 mM EDTA, 11 mM dextrose, and 25 mM -23- WO 01/19376 PCT/USOO/24910 NaCHO 3 . The perfusate was equilibrated with 95% 02:5% CO 2 (pH 7.4). The final composition of the KHB was monitored using a NOVA UltraStat analyzer (NOVA Biomedical Corporation, Waltham, MA) and the components were adjusted to obtain the described composition. Hearts were perfused at 60 mM Hg hydrostatic perfusion pressure for 15 min to establish preischemic control values for each parameter. Left ventricular end diastolic pressure was adjusted to zero mm Hg by varying the balloon volume during this initial period to establish these preischemic values. Balloon volume was then maintained constant throughout the remainder of the experiment. Ischemic hearts received 15 min of zero-flow global ischemia (370 C), and values obtained at the end of this ischemic period were plotted as 0 min of reperfusion. Hearts were then reperfused for 15 min at 60 mm Hg hydrostatic perfusion pressure. Nonischemic hearts were perfused for a period equal to the entire perfusion period (45 min). Three groups of animals were studied: 1) a nonischemic control group in which hearts were perfused 45 min continuously without interruption; 2) an ischemia-reperfusion group in which hearts were subjected to 15 min global ischemia and 15 min reperfusion as described above; and 3) an ODS-HEP ischemia-reperfusion group which was injected intravenously with 6 mg/mg ODS heparin in 1.0 ml saline 120 min before heart excision, and subjected to 15 min each of global ischemia and reperfusion, with 100 pg/ml ODS-HEP added to the perfusion buffer. At the end of the perfusion protocols, ventricles were frozen with Wollenberger clamps precooled in liquid nitrogen, and pulverized under liquid nitrogen temperature. Nuclear proteins were then immediately isolated from the frozen powders of the myocardial samples by a modification of the method of Li et al. (21). Briefly, 0.25 g of pulverized myocardial sample was homogenized in 2 ml of ice-cold hypotonic buffer [10 mM HEPES pH 6.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT); protease inhibitors: 0.5 mM phenylmethylsulfonyl fluoride, aprotinin, pepstatin, leupeptin (10 Ig/ml each); and phosphatase inhibitors: 50 mM NaF, 30 mM p glycerolphosphate, 1 mM Na 3

VO

4 , and 20 mM p-nitrophyl phosphate]. After incubation on ice for 5 min to allow unbroken tissue to settle, the -24- WO 01/19376 PCT/USOO/24910 supernatants were decanted and incubated on ice for another 30 min, vortexed for 30 s after addition of 125 pl of 10% Nonidet P-40, and then centrifuged for 5 min at 2,000 rpm and 40 C to isolate nuclei. After washing with hypotonic buffer without Nonidet P-40, nuclear pellets were suspended in ice-cold hypertonic salt buffer (20 mM HEPES pH 6.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, protease inhibitor, and phosphatase inhibitors), incubated on ice for 30 min, mixed frequently, and microfuged at 14,000 rpm for 15 min at 4' C. Aliquots of supernatants were collected as nuclear extracts and stored at -80' C. The concentration of total proteins in the samples was determined by the Pierce protein assay reagent (Pierce Chemical, Rockford, IL). EMSAs were performed as described earlier, using 15 pg of nuclear protein in each binding reaction. Competition experiments were performed by incubation of nuclear proteins with 1 Ox unlabeled NF-KB or cyclic-AMP responsive element oligonucleotides (CRE, AGAGATTGCCTGACGTCAGAGAGCTAG, Promega) for 5 min prior to addition of 32 P-labeled NF-xB probe. Supershift assays were performed by adding 0.5 pg of antibodies to p65 and p50 components of NF-xB (Santa Cruz) to the binding reaction after addition of labeled probe. Reactions were electrophoresed at 100 V for 2 hr at room temperature on a 5% nondenaturing polyacrylamide gel in 0.5 x TGE (120 mM glycine and 1 mM EDTA in 25 mM Tris, pH 8.5), followed by autoradiography. 2. In vivo studies Surgical procedure All animals were handled in compliance with the AGuide for the Care and Use of Laboratory Animals~ published by the National Institutes of Health (NIH Publication No. 85-23, revised 1985). The Institutional Animal Care and Use Committees of Emory University and Carolinas Medical Center approved the study protocols. Twenty-four heartworm-free adult dogs of either sex were anesthetized with sodium pentobarbital (20 mg/kg) and endotracheally intubated. Anesthesia was supplemented with fentanyl citrate (0.3 ptg/kg/min) and diazepam (0.03 pg/kg/min) administered intravenously as -25- WO 01/19376 PCT/USOO/24910 needed to maintain deep anesthesia. Each dog was ventilated with a volume-cycled respirator using oxygen-enriched room air. A rectal temperature probe was inserted to measure core body temperature. The right femoral artery and vein were cannulated with polyethylene catheters for arterial blood sampling and for intravenous access, respectively. Serial arterial blood gases were measured to maintain the arterial oxygen tension greater than 100 mmHg. Arterial carbon dioxide tension was maintained between 30 and 40 mmHg, and arterial pH was maintained between 7.35 and 7.45 by adjustment of the ventilatory rate, and acidemia was counteracted with intravenous sodium bicarbonate. After median sternotomy, the superior and inferior vena cava were looped with umbilical tapes and the heart suspended using a pericardial cradle. Millar catheter-tipped pressure transducers (Millar Instruments, Houston, Texas) were placed in the proximal aorta and in the left ventricular cavity to measure aortic and left ventricular pressure, respectively. A polyethylene catheter was inserted into the left atrium for colored microsphere injection. A one centimeter portion of the left anterior descending (LAD) coronary artery distal to the first diagonal branch was dissected and loosely encircled with a 2-0 silk suture. A pair of opposing ultrasonic crystals were placed intramyocardially within the proposed ischemic area at risk within the left anterior descending coronary artery distribution, and were used to assess regional function within the area at risk (13). Experimental protocol The dogs were randomized to one of three groups (n=8 in each group): 1) Control (saline), 2) unmodified heparin (HEP, 3 mg/kg) and 3) modified heparin (ODS-HEP, 3 mg/kg). The LAD was occluded for 90 min producing ischemia and then released for four hours of reperfusion. Each pharmaceutical agent (saline, HEP, ODS-HEP) was infused as an intravenous bolus 10 min prior to initiation of reperfusion and at 90 and 180 min during reperfusion. Analog hemodynamic and cardiodyamic data were sampled by a personal computer using an analog-to-digital converter (Data Translation, Marlboro, MA). The data were captured, stored, and analyzed using -26- WO 01/19376 PCT/USOO/24910 SPECTRUM cardiovascular acquisition and analysis software (Wake Forest University, Winston-Salem, NC). Cardiodynamic, and hemodynamic measurements and arterial blood samples were taken prior to coronary artery occlusion (baseline), at the end of 90 min of LAD occlusion, and at 15 min and one, two, three, and four hours of reperfusion. Hemodynamic and cardiodynamic data were averaged and output was obtained from no fewer than 10 cardiac cycles. Percent systolic shortening, segmental work, and the characteristics of segmental stiffness described by exponential curve-fitting analysis were determined as previously described (13). Activated clotting time (ACT, in seconds) was measured throughout the experiment and was used as a marker for systemic anticoagulation. Briefly, four ml of arterial blood was inserted into Hemochron Celite ACT tubes (International Techidyne Corporation, Edison, NJ) and anticoagulation in seconds was assessed using the Hemochron 401 Whole Blood Coagulation System (International Techidyne Corporation, Edison, NJ). Arterial blood samples for measuring creatine kinase activity and protein were analyzed spectrophotometrically using kits from Sigma Diagnostics (St. Louis, MO). Creatine kinase activity was expressed as international units per gram of protein. The experiment was terminated with a bolus of intravenous sodium pentobarbital (100 mg/kg). The heart was immediately excised for further analysis and placed into ice-cold Krebs-Henseleit (K-H) buffer of the following composition: 118 mM NaCl, 4.7 mM KCl, 1.2 mM KH 2

PO

4 , 1.2 mM MgSO 4 7 H 2 0, 2.5 mM CaCl 2 2 H 2 0, 12.5 mM NaHCO 3 , and 11 mM glucose at pH 7.4. Determination of area at risk, infarct size and regional myocardial blood flow After postexperimental excision of the heart, the myocardial area at risk and infarct size were determined histologically as previously described (13) using Unisperse blue dye and 1% triphenyltetrazolium chloride (TTC, Sigma Chemical, St. Louis, MO), respectively. The area at risk (AAR) was calculated as the sum of the weights of the infarcted and necrotic tissue -27- WO 01/19376 PCT/USOO/24910 within the area at risk, divided by the weight of the left ventricle (AAR/LV) and expressed as a percentage. The infarct size (area of necrosis, AN) was calculated as the weight of necrotic tissue divided by the weight of the left ventricle (AN/LV) or the area of risk (AN/AAR) and expressed as a percentage. Regional myocardial blood flow measurements were obtained with use of dye-release colored microspheres (Triton Technology, San Diego, CA) to measure the myocardial blood flow in the AAR and non-ischemic myocardium throughout the experiment. Left atrial injections and reference blood sampling were performed at baseline, at the end of 90 min of ischemia, and at 15 min and four hours of reperfusion. Colored microspheres in arterial reference blood samples and myocardial tissue samples from the ischemic (LAD) and non-ischemic (left circumflex coronary artery; LCx) regions underwent dye release treatment and spectrophotometric analysis to determine myocardial blood flow. Measurement of myocardial neutrophil accumulation Tissue samples weighing approximately 0.4 g were taken from the non-ischemic zone and from the non-necrotic and necrotic regions of the area at risk for spectrophotometric analysis of myeloperoxidase (MPO) activity (5 absorbance/minute), as an assessment of neutrophil accumulation in myocardium as described previously (14). Myeloperoxidase activity was described as the rate of hydrogen peroxide degradation-induced color change per min/100 mg tissue, and expressed as absorbance units/gram myocardial tissue. PMN isolation and adherence to post-experimental coronary artery endothelium Neutrophil (PMN) adherence to post-experimental coronary arteries was used as a bioassay of basal endothelial function. Arterial blood was withdrawn immediately after femoral artery cannulation, and canine PMNs were isolated using the Ficoll-Paque (Sigma Chemical , St. Louis, MO) density gradient technique previously described (15). The isolated cell preparation contained greater than 95% PMNs and cell viability was greater than 90% when studied by trypan blue exclusion (15). PMNs were then -28- WO 01/19376 PCT/USOO/24910 labeled with Zynaxis PKH26 vital fluorescent dye (Zynaxis Cell Science, Malvern, PA) as previously described (15). After the experiment, ischemic-reperfused LAD and non-ischemic LCx segments were isolated after the heart was harvested, cut into 3-mm segments, and carefully opened to expose the endothelium while being submerged in ice-cold K-H buffer. The segments were then placed in dishes containing K-H buffer at 370 C. Unstimulated, fluorescent-labeled PMNs (final concentration 6 x 106 cells/dish) were incubated with postexperimental segments for 15 minutes. After incubation, the coronary segments were washed of non-adherent PMNs, mounted on glass slides, and adherent PMNs were counted under epifluorescence microscopy (490 nm excitation, 504-nm emission), as described previously (16). Agonist-stimulated macrovascular relaxation. Vasoreactivity in epicardial macrovessels was studied as described previously (11) to assess postexperimental endothelial cell function. Briefly, LAD and LCx segments were carefully transected into 2- to 3-mm rings and placed into organ chambers (Radnoti Glass, Monrovia, CA) containing oxygenated (95% oxygen and 5% carbon dioxide) K-H solution at 37'C. Indomethacin (10 ptmol/L) was used to inhibit the release of prostaglandins. The coronary rings were precontracted with an optimal concentration of thromboxane A 2 mimetic agent, U-46619 (5 nmol/L). Endothelial function was assessed by comparing the vasorelaxation responses to incremental concentrations of acetylcholine (1 to 686 tmol/L) and A23187 (I to 191 ptmol/L), whereas smooth muscle function was assessed with sodium nitroprusside (I to 381 tmol/L). Statistical analysis The data were analyzed by one-way analysis of variance or repeated measures two-way analysis of variance for analysis of group, time and group-time interactions. If significant interactions were found, Tukey=s or Student-Newman-Keuls post hoc multiple comparisons tests were applied to locate the sources of differences. Differences in the densities of the p-65 containing NF-KB gel band between treated and untreated ischemic reperfused rat hearts were compared using the t test. A p < 0.05 was -29- WO 01/19376 PCT/USOO/24910 considered significant, and mean _ standard error of the mean (SEM) are reported. RESULTS Heparin and ODS heparin reduce infarct size Heparin (HEP) and ODS heparin (ODS-HEP) significantly reduced infarct size as a consequence of ischemia-reperfusion injury (Figure 1). HEP treatment significantly decreased infarct size (area of necrosis, AN), expressed as a percentage of the area at risk (AN/AAR), by 35% compared to the Control group. Similarly, ODS-HEP reduced infarct size by 38% compared to Control hearts. There was no statistical difference in size of infarcts between the HEP and ODS-HEP groups, and the area placed at risk from LAD coronary artery occlusion, expressed as a percentage of the left ventricular mass (AAR/LV), was comparable among the groups. Plasma creatine kinase (CK) activity was used to confirm histologic measurement of infaret size. There were no significant differences in plasma CK activity at baseline among the three groups (Figure 2). There was also no significant increase in CK activity after regional ischemia. Hearts in the Control group showed the steepest rise in CK activity within the initial hour of reperfusion, while both of the heparin groups showed significantly smaller increases in CK activity (Figure 2). At four hours of reperfusion, CK activity was significantly lower in both treatment groups, consistent with the smaller infarct sizes in these groups. Despite their favorable effects on infarct size, HEP and ODS-HEP produced no significant changes in myocardial blood flow. Subendocardial blood flow in the ischemic-reperfused left anterior descending coronary artery region was statistically comparable among the three groups at baseline (Figure 3A). Transmural blood flow in the area at risk was significantly decreased during ischemia, with no group differences. All groups showed a comparable hyperemic response in the area at risk at 15 minutes of reperfusion that was significantly greater than baseline values, after which blood flow was diminished to similar levels in all groups by four hours of reperfusion. In the non-ischemic-reperfused left circumflex coronary artery region, transmural blood flow was comparable in all groups -30- WO 01/19376 PCT/USOO/2491 0 throughout the experimental protocol (Figure 3B). Thus, infarct size reduction with HEP or ODS-HEP can not be attributed to influences on local coronary blood flow during ischemia or reperfusion within the area at risk. Differences in infarct size were also not a consequence of hemodynamic or cardiodynamic differences. All hemodynamic variables at baseline were comparable among groups. Heart rate (HR, beats/min) was significantly increased after ischemia and during reperfusion as compared to baseline in all three experimental groups (Table 1). Mean arterial pressure (MAP, mmHg) in all three groups was similar throughout the study period (Table 1). Left ventricular end diastolic pressure (LVEDP, mmHg) was comparably elevated during ischemia in all three groups, and there were no statistically significant differences in heart rate, mean arterial pressure or left ventricular end diastolic pressure among groups during the reperfusion period. Following ischemia, hearts in all groups demonstrated dyskinesis in the area at risk. All hearts showed poor recovery of percent systolic shortening throughout the four hours of reperfusion, and segmental work (mmHg mm) during reperfusion was reduced (Table 2). Similarly, diastolic stiffness (as measured by the valueless p-coefficient) increased following ischemia to comparable levels in all groups (Table 2). Thus, neither HEP nor ODS-HEP had any effect on the hemodynamic or cardiodynamic consequences of in vivo ischemia-reperfusion in this model. Heparin and ODS heparin reduce neutrophil accumulation in reperfused myocardium The influx of PMNs into ischemic myocardium has previously been suggested as a major mechanism underlying lethal myocardial injury occurring during reperfusion. We therefore studied whether HEP or ODS HEP might inhibit PMIN accumulation in the area at risk. PMN accumulation within normal myocardium was low and comparable among the Control, HEP and ODS-HEP groups (16 ± 8, 18 ± 11, and 18 ± 8 6 absorbance units/minute, respectively). HEP and ODS-HEP both decreased myeloperoxidase activity in the nonnecrotic area at risk, but these changes did not achieve statistical significance (p > 0.10). However, -31- WO 01/19376 PCT/USOO/24910 treatment with HEP or ODS-HEP significantly reduced myeloperoxidase activity in necrotic myocardium by 50% compared to the Control group (Figure 4). ODS heparin does not produce anticoagulation Despite reducing infarct size, ODS-HEP did not produce anticoagulation in the whole animal. Activated clotting time (ACT, seconds) was used as a measure of anticoagulation in vivo. At baseline, ACT measurements were similar among groups (Figure 5). At four hours of reperfusion, ACT was increased greater than ten-fold after administration of HEP compared with Control (1425 ± 38 seconds versus 123 10 seconds, respectively). In contrast, ACT in the ODS-HEP group (145 10 seconds) was not different compared to Control (123 ± 10 seconds, p=0.768). Thus, ODS-HEP was able to effect the same benefits as HEP without anticoagulation. Heparin and ODS heparin reduce neutrophil adherence and endothelial dysfunction in coronary arteries To explore mechanisms of how HEP and ODS-HEP might protect against PMN migration into ischemic-reperfused myocardium, we first studied their effect on PMN degranulation, shown in Table 3. The rate of PMN degranulation without stimulation was low. As expected, the PMN degranulation was greatly enhanced by treatment of neutrophils with platelet activating factor (PAF). However, increasing concentrations of ODS-HEP did not significantly reduce PAF-stimulated PMN degranulation, suggesting that ODS-HEP has little direct effect on neutrophil activity. The attachment of PMNs to endothelium is in part based upon the function of L- and P-selectins, which are blocked by heparin. We therefore explored whether HEP and ODS-HEP might block PMN attachment to coronary endothelium in vitro. Adherence of neutrophils to normal control coronary arteries was relatively low (Figure 6A). PAF significantly increased the adherence of PMNs to coronary endothelium (Figure 6A). Both HEP and ODS-HEP reduced the adherence of PAF-stimulated PMNs to endothelium in a dose-dependent manner. At concentrations of 1 mg/ml for HEP and 10 mg/ml for ODS-HEP, PMN adherence was not statistically -32- WO 01/19376 PCT/USOO/2491 0 different from unstimulated control arteries. Inhibition of PMN adherence to PAF-stimulated coronary endothelium was charge dependent. Charge neutralization by addition of increasing concentrations of the polycation protamine (Prot) to the incubation medium eliminated the activity of the polyanions HEP or ODS-HEP to prevent adherence of PMNs (Figure 6B). These results raised the possibility that HEP and ODS-HEP might also reduce PMN adherence to ischemic-reperfused coronary endothelium in vivo. To explore this potential, we studied the effect of HEP or ODS HEP treatment in vivo on PMN adherence to post-ischemic coronary artery endothelium. PMN adherence to the ischemic-reperfused LAD coronary artery was increased by 300% in the untreated Control group compared to the non-ischemic-reperfused LCx artery, indicating that ischemia reperfusion caused endothelial dysfunction (Figure 7). In contrast, neutrophil adherence in the ischemic-reperfused LAD was significantly reduced by treatment with HEP (51%) or ODS-HEP (42%), compared to the untreated Control (Figure 7). HEP and ODS-HEP treatment might also be expected to have effects on coronary vasodilator function. To quantify agonist-stimulated endothelial dysfunction in epicardial coronary arteries, we studied the vascular response to incremental concentrations of the vasodilators acetylcholine (endothelial-dependent; receptor-dependent), A23187 (endothelial-dependent; receptor-independent), and sodium nitroprusside (direct smooth muscle) in post-ischemic coronary vascular ring preparations. Figure 8 illustrates vasodilator responses to acetylcholine in isolated coronary rings, expressed as a percentage of U46619-induced precontraction, in the ischemic-reperfused LAD (Figure 8A) and non ischemic reperfused LCx (Figure 8B). In the Control group (Figure 8A), there is a general and statistically significant shift to the right in the concentration-response curve, with significantly reduced relaxation to acetylcholine at concentrations greater than 10 nmol/L. In contrast, the relaxant effect of coronary vessels to acetylcholine was preserved in HEP or ODS-HEP-treated groups (Figure 8A). Furthermore, the concentration of acetylcholine required to effect 50% relaxation (EC 5 o; -log [M]), was significantly greater for the Control group (-6.975 ± 0.06) compared to the -33- WO 01/19376 PCT/USOO/24910 HEP (-7.298 ± 0.06) and ODS-HEP (-7.201 ± 0.05) groups (p < 0.05). These differences in maximal relaxation were generally not observed in non-ischemic-reperfused ring preparations from LCx (Figure 8B). Although at specific concentrations, the ODS-HEP group did demonstrate significantly greater relaxation than Control or HEP groups, the maximal relaxation among groups was comparable, and the values for EC 50 to acetylcholine were similar. In addition, there were no significant group differences between vasodilator responses of isolated coronary rings from the LAD versus LCx vessels in response to the endothelium-dependent receptor-independent calcium ionophore A23187, or to the smooth muscle dependent vasodilator sodium nitroprusside (Table 4). Taken together, these results suggest that treatment with HEP or ODS-HEP preserves the receptor-mediated vasodilator responses of coronary endothelium following ischemia and reperfusion. ODS heparin prevents activation of nuclear factor-K3 The increase in neutrophil adherence following ischemia reperfusion has been associated with enhanced expression of endothelial cell adhesion molecules (22,23). Heparin is avidly concentrated by endothelium and once internalized can be detected within the nucleus (24). Therefore, heparin might also attenuate adhesion molecule responses at a transcriptional level. Endothelial expression of adhesion molecules and chemotactactic proteins for leukocytes such as ICAM-1, L- and P-selectins, and interleukin-8 (IL-8) is strongly influenced by activation of the nuclear transcription factor NF-B (25,26). NF-iB has recently been shown to be activated as a consequence of myocardial ischemia-reperfusion (21), and could promote many of the inflammatory events leading to PMN influx into ischemic-reperfused myocardium. Normally cytosolic in location when quiescent, NF-KIB is targeted for transport to the nucleus when dissociation from its inhibitory peptide I-xB uncovers a strongly cationic nuclear localization sequence of peptides. We reasoned that a polyanion such as heparin could bind to and charge neutralize the nuclear localization sequence, preventing translocation of NF-xB to the nucleus. Figure 9A shows results of immunohistochemical staining for NF KB in unstimulated control cells. Brown anti-p65 staining is present in the -34- WO 01/19376 PCT/USOO/24910 cytoplasm of HUVEC, but not in nuclei. Figure 9B shows results of stimulated HUVEC without heparin. Some, but not all nuclei, now stain positive for anti-p65, corresponding to translocation of NF-KB from cytoplasm to the nucleus. In HUVEC pre-treated with HEP (Figure 9C) or ODS-HEP (Figure 9D), TNF stimulation fails to produce anti-p65 nuclear staining, suggesting that heparin or nonanticoagulant heparin prevents NF KB nuclear translocation. Interruption of endothelial NF-KB activation was confirmed by electrophoretic mobility shift assays (EMSAs). Figure 10 illustrates a typical EMSA experiment from HUVEC. Treatment of monolayers with TNF stimulates DNA binding of NF-xB (lane 2) compared to untreated controls (lane 1). In contrast, pretreatment of cells with 200 pg/ml ODS HEP virtually eliminates NF-KB binding activity in nuclear protein extracts (lane 3), confirming that heparin prevents translocation of NF-KB from the cytoplasm to the nucleus. ODS-HEP also reduced DNA binding of NF-KB in ischemic reperfused myocardium. Exposure of rat hearts to 15 min warm global ischemia followed by 15 min reperfusion typically increased DNA binding of myocardial nuclear protein to oligonucleotide sequences for NF-B (Figure 11 A, lane 2). Three distinct bands of increased DNA binding were observed (Figure 11 A, lane 2), all of which were eliminated by addition of excess unlabeled NF-xB oligonucleotide probe (Figure 1 1B, lane 2). Supershift experiments identified complex I as the band containing the p65 component of NF-xB (Figure 11 A, lane 5). Pretreatment and perfusion with ODS-HEP reduced ischemia-reperfusion related stimulation of NF-KB binding to DNA in all three bands (Figure 11 A, lane 3). DNA binding of the p65-containing complex I was nearly eliminated by ODS-HEP, with a reduction of 54 V 6 % as measured by densitometry in comparison to complex I of untreated ischemic-reperfused rat hearts (p < 0.05, n = 4). Taken together, the results in Figures 10 and 11 suggest that in addition to directly attenuating vascular adherence of PMINs to coronary endothelium, decreasing PMN accumulation in the area at risk and reducing myocardial necrosis, HEP or ODS-HEP might also protect the heart by interrupting the -35- WO 01/19376 PCT/USOO/24910 activation of NF-B and preventing enhanced expression of adhesion molecules by ischemic-reperfused endothelium and myocardium. ODS-heparin reduces contractile dysfunction following ischemia and reperfusion of isolated rat hearts Two-way repeated measures ANOVA indicated no significant treatment, time or interaction effects in function of the hearts that were subjected to 15 minutes of ischemia followed by 15 minutes of reperfusion. Both groups of hearts subjected to this brief period of ischemia and reperfusion recovered high contractile function (95% of baseline, ischemia reperfusion; and 93% of baseline, ODS-HEP ischemia-reperfusion). Thus, the activation of NF-KB that was observed in these hearts occurred as an early event which preceded the development of significant contractile dysfunction. Therefore, in additional studies, the period of ischemia was increased to determine if treatment with ODS-HEP might reduce contractile dysfunction in isolated rat hearts following reperfusion. Values of contractile function were not significantly different among the three groups of rat hearts before the induction of ischemia (Figure 12), indicating that ODS-HEP did not alter normal heart autorhythmic function. Changes in function observed in hearts subjected to ischemia and reperfusion were due to the treatment injury, rather than instability in the perfusion system, since nonischemic hearts showed no significant change in pressure-rate product through the time of perfusion. Both groups of hearts subjected to 30 minutes of ischemia and 15 minutes of reperfusion had significantly reduced contractile function at the end of reperfusion compared with the sham control hearts. The model of ischemia and reperfusion produced severe reduction in contractile function, since the hearts that did not receive ODS-HEP only recovered 10% of their preischemic pressure-rate product (Figure 12). Hearts treated with ODS-HEP, however, had significantly improved recovery of contractile function, which was 2.4 times better than that observed in hearts that did not receive ODS-HEP (Figure 12). Thus, in this severe model, ODS-HEP reduces not only molecular but also the physiologic consequences of ischemia and reperfusion in myocardium. Thus, in accordance with this invention, it has been discovered that -36- WO 01/19376 PCT/USOO/24910 heparin can inhibit NF-KB activity both in cultured cells and in vivo in animal myocardium. Heparin blocks the translocation of NF-xB to cell nucleus thus inhibiting the expression of a variety of genes such as cytokines for example TNF-t. NF-KB is known to be involved in the development of diabetic vascular disease. TNF-a has been shown to be a cardiodepressant directly causing heart failure. Thus, the present invention provides methods for inhibiting NF-xB activity by administering to a patient heparin. In addition, this invention also provides methods for treating and preventing both heart failure and diabetic vascular disease by administering to the patient a therapeutically effective amount of heparin. Many modifications and other embodiments of the invention will come to mind to one skilled in the art to which this invention pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the invention is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. -37- WO 01/1 9376 PCTIUSOO/2491 0 o~~~~ to 0 ~'0 J - -H -1 -H -H - - -H v~ oo a C-~ N00 'f N%0 00 N o ) 0 -H o -H t- 00 r- No -HH I- 00 -0 -1 10 0% enA 00 0 000 00 ) O0 a ON "0 1. 0% 0% 2 i -n =0 00 r- N en en' 0 . rl 0 ooN 0 <A A < < < cz r4 N- 00 - ' C. 00 N00 00 Nt- V -H -H -H -H -H. -H -0 C -H -H 41 U) %0 ol CDID n Itc -- 00 0% 00 Ns 00I 0 '0 % CU, -P44 -a- 4 0 \O N '~o u~ *- 44 H 4 N 00 % 00 % 00 ~ No -38-.

WO 01/19376 PCT/USOO/24910 Table 2. Regional systolic and diastolic cardiodynamic variables. Baseline Ischemia r15min rlhr r2hr r3hr r4hr EDL (mm) Control 15 ±0.9 17+1 14+1 14 ±1 14 ±1 14 ±1 14 ±I HEP 14 ±1 18 1^ 15t1^ 14 ±1 14 ±1 14 ±1 15 ±1 ODS-HEP 13 ±0.9 16 1^ 14 1 14 ±1 14 ±1 14 ±1 14 ±1 ESL (mm) Control 11 ±0.8 18 2^ 14 2 15 ±1 15 ±1 15 ±1 15 1 HEP 11 ±1 19± 2^ 15 2 14 ±1 15 ±1 15 ±1 15 ±1 ODS-HEP 10 ±0.9 16 ±1 14 1 15 ±1 15 ± 1 15 ±1 15 ±1 %SS Control 26 ±2 -7± 2 -4 2 -4 ±2 -5 1 -5 ±1 -6± 2 HEP 26±3 -4 ±1 -0.4 ±2 -2 ±2 -4 ±2 -6±3 -7± 3 ODS-HEP 24 ±4 -5 ±1 -2 ±3 -2 ±3 -9 ±3 -6±4 -6± 4 Segmental Work (mmHg*mm) Control 306 ±28 -6 8^ 13 ±7 9±8 4 ±8 -4 6 -13±13 HEP 314 ±51 14 10" 73 26" 34 ±12 10 ±14 -5 10 -12 ±15 ODS-HEP 231 ±23 -5 6" 12 5" 0.8 ±7 -18 ±17 -36 8* -28 8 Diastolic Stiffness (unitless p Coefficient) Control 0.2 ±0.05 0.5+0.2 1.0 ±0.4 1.0 ±0.3 1.0 ±0.3 0.6 0.2 0.7 ±0.1 HEP 0.2 ± 0.04 0.6 ±0.1 0.8 ± 0.2 1.0 ±0.2 1.0 ±0.2 1.0 0.2 1.0 ±0.2 ODS-HEP 0.2 ±0.04 0.8 0.2" 1.0 ±0.6 1.0 ±0.2 0.9±0.5 0.7 0.2 0.5 ±0.2 EDL = end diastolic length, ESL = end systolic length, % SS = percent systolic shortening, dP/dt..= maximum developed pressure over time. Baseline = prior to left anterior descending (LAD) coronary artery occlusion; Ischemia = at the end of 90 minutes of LAD ischemia; r1 5min, rihr, r2hr, r3hr, and r4hr = minutes or hours of reperfusion following ischemia. Values are mean ± standard error of the mean. * p<0.05 ODS HEP versus all other groups in the same time period and ^ p<0.05 versus previous time point within the same treatment group. -39-- WO 01/19376 PCT/USOO/24910 Table 3. Neutrophil degranulation (supernatant myeloperoxidase activity) Groups Myeloperoxidase Activity (A abs/min) PMNs only 0.70 ± 0.10 PMNs + PAF 55.60 ± 7.37* PMNs + PAF + 1 ptg ODS 54.39 ± 5.78* PMNs + PAF + 10 pg ODS 49.83 ± 5.79* PMNs + PAF + 100 pig ODS 52.97 ± 6.97* PMNs + PAF + 1 mg ODS 53.07 ± 9.85* A abs/min = slope of spectrophotometric absorbance per minute; PMNs = neutrophils, PAF = platelet activating factor. Values are mean + standard error of the mean. *p<0.05 versue PMNs only. -40- WO 01/19376 PCTIUSOO/2491 0 -o ~~0 0 04 0 1 00 01 0. H -H -H -H -H -H 00 CD ON I"- C1 0 0 r-C--~-C ~ 0 -41 -H - -iq -H -H 0 00 0- t- C14 (4 C14 >14 "s' C1 (4 C14 C14 to 0 -l -u -H - L) 0N 00 000 "04 ca -. (' N ' C-0 V)~ < 00. 4))cq4) W. (NW 0 C*u 0. 0' 0- ca 0 . C~~ c*4 (Ni~ - - - - - xC CU -;~~~~- 1-__ _ __ _ _ _ WO 01/19376 PCT/USOO/24910 REFERENCES 1. Matsumura, K., Jeremy, R.W., Schaper, J., and Becker, L.C. 1998. Progression of myocardial necrosis during reperfusion of ischemic myocardium. Circulation. 97:795-804. 2. Farb, A., Kolodgie, F.D., Jenkins, M., and Virmani, R. 1993. Myocardial infarct extension during reperfusion after coronary artery occlusion: pathologic evidence. . Am. Coll. Cardiol. 21:1245-1253. 3. Saliba, M.J., Covell, J.W., and Bloor, C.M. 1976. Effects of heparin in large doses on the extent of myocardial ischemia after acute coronary occlusion in the dog. Am. J. Cardiol. 37:599-604. 4. Friedrichs, G.S., Kilgore, K.S., Manley, P.J., Gralinski, M.R., and Lucchesi, B.R. 1994. 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Claims (24)

1. A method for inhibiting the NF-xB activity in a patient comprising administering heparin to the cells in the patient, such that the heparin is internalized into the cytoplasm of cells in the patient and the translocation of NF-KB from cell cytoplasm to cell nucleus is inhibited.

2. The method of Claim 1, wherein said heparin is 0 desulfated or N-desulfated, or N- and 0-desulfated.

3. The method of Claim 1, wherein said heparin is desulfated in at least one positions selected from the group consisting of 2-0, 3-0 and

6-0. 4. The method of Claim 1, wherein said heparin is 2-0 desulfated. 5. The method of Claim 1, wherein said heparin is an acylated heparin. 6. The method of Claim 5, wherein said heparin is 0-acylated heparin.

7. The method of Claim 1, wherein said cells are endothelial cells, smooth muscle cells, and cardiac myocytes.

8. The method of Claim 1, wherein said cells are epithelial cells.

9. The method of Claim 8, wherein said heparin is administered in a liposomal preparation.

10. The method of Claim 8, wherein said heparin is modified with a lipophilic molecule. -49- WO 01/19376 PCTIUSOO/24910

11. A method for treating diabetic vascular disease in a patient, comprising administering to the patient a therapeutically effective amount of heparin.

12. The method of Claim 11, wherein said heparin is 0 desulfated or N-desulfated, or N- and 0-desulfated.

13. The method of Claim 11, wherein said heparin is desulfated in one or more positions selected from the group consisting of 2-0, 3-0 and 6-0.

14. The method of Claim 11, wherein said heparin is 2-0 desulfated.

15. The method of Claim 11, wherein said heparin is an acylated heparin.

16. The method of Claim 15, wherein said heparin is O-acylated heparin.

17. The method of Claim 11, wherein said heparin is administered in a liposomal preparation.

18. The method of Claim 11, wherein said heparin is modified with a lipophilic moiety.

19. A method for treating heart failure in a patient, comprising administering to the patient a therapeutically effective amount of heparin.

20. The method of Claim 19, wherein said heparin is 0 desulfated or N-desulfated, or N- and 0-desulfated. -50- WO 01/19376 PCT/USOO/24910

21. The method of Claim 19, wherein said heparin is desulfated in one or more positions selected from the group consisting of 2-0, 3-0 and 6-0.

22. The method of Claim 19, wherein said heparin is 2-0 desulfated.

23. The method of Claim 19, wherein said heparin is an acylated heparin.

24. The method of Claim 23, wherein said heparin is 0-acylated heparin.

25. The method of Claim 19, wherein said heparin is administered in a liposomal preparation.

26. The method of Claim 19, wherein said heparin is modified with a lipophilic moiety. -51-