HK1026640A - Treatment of infarcts through inhibition of nf-kappab - Google Patents

Description

Treatment of infarcts by inhibition of NF-KAPPAB

The present invention relates to the treatment of ischemia and reperfusion injury, which comprises preventing embolism or reducing the size of embolism caused by vascular occlusion.

All tissues are sensitive to hypo-perfusion and hence oxygen deficiency, ischemia. Prolonged ischemia can lead to cellular damage. The extent of injury and the likelihood of tissue rescue depends on the extent and length of ischemia. Prolonged ischemia can result in cell death (infarction), and in this case, the damage is irreversible. On the other hand, if the drug is applied at the right time, dead or dying cells can be rescued by drug treatment.

Major ischemic events associated with treatment include, but are not limited to, heart disease and stroke. In humans, stroke accounts for 10% of all early deaths. For those who survive the injury, 50% of them remain severely disabled and only a small fraction (10%) of patients fully recover function.

Approximately more than 1,500,000 americans suffer from myocardial infarction each year. Half of them failed to survive to the hospital. However, with the gradual acceptance of thrombolytic therapies (e.g., streptokinase or Tissue Plasminogen Activation (TPA), one month survival rates for patients arriving to the hospital can reach as high as 93.6% (Werns, S.W.textbook of International medicine, ED.Topol, E.J.WB Saunders: 1994, PP 142-153.) ischemic muscle and tissue can be saved by lysing blood clots during infarction.

Reperfusion injury results from one or more of the following causes: cellular acidosis leads to calcium overload; catabolism causes an increase in intracellular penetration, resulting in cell swelling; free radicals from neutrophils and other inflammatory cells.

Neutrophils are immediately visible in the reperfused myocardium after reperfusion. Single cell/large phage appeared within 24-48 hours. Neutrophils in the reperfused myocardium are 3-5 times as large as neutrophils in the ischemic myocardium. It is initiated by the adhesion of endothelial cells, occurring within 10 minutes of reperfusion. The neutrophils therein or themselves may be captured by the capillaries and interfere with reperfusion. Neutrophils in the blood vessel block up to 27% of the capillaries and have been shown to be associated with a reduction in local blood flow (Forman et al, inlet myodialinfringement, eds. Gersh et al. Elsevier: 1991, pp 347-) 370). This condition can lead to a "no reflow" phenomenon, in which blood flow continues to decrease after reperfusion.

It is known that neutrophils must first attach themselves to the endothelial cell wall by interaction with adsorbed molecules, and then they drive themselves between adjacent endothelial cells into the brain tissue where cytotoxic cytokinins are released. Expression of such adhesion molecules is increased following cellular injury (including ischemia). In addition, the endothelial cell wall becomes more permeable to infiltrating cells due to the release of Nitric Oxide (NO). Therefore, inhibition of this movement of neutrophils from peripheral blood vessels into damaged tissue (erythrocyte extravasation) is of value in allowing the timely recovery of dead cells from ischemic injury.

Thus, there is a need in the art for an effective treatment that can be used to prevent or reduce the consequences of ischemia.

In a first aspect, the present invention relates to a method of treating ischemic injury in a mammal comprising administering to said mammal an effective amount of an inhibitor of NF-kB activation. Preferred inhibitors of NF-kB activation are selected from the group consisting of proteasome inhibitors, ubiquitin inhibitors, inhibitors of IkB-alpha serine phosphorylation and mixtures thereof. Preferably, these agents are administered after the occurrence of temporary vascular occlusion and before the onset of permanent ischemic injury.

In a second aspect, the present invention relates to a method of preventing or reducing the severity of reperfusion injury in a mammal in need thereof, which comprises administering to said mammal an effective amount of an inhibitor of NF-kB activation. Preferred inhibitors of NF-kB activation are selected from the group consisting of proteasome inhibitors, ubiquitin inhibitors, inhibitors of IkB-alpha serine phosphorylation and mixtures thereof.

In a third aspect, the present invention relates to a method for preventing or reducing the size or reducing the severity of an infarct comprising administering to said mammal an effective amount of an inhibitor of NF-kB activation. Preferred inhibitors of NF-kB activation are selected from the group consisting of proteasome inhibitors, ubiquitin inhibitors, inhibitors of IkB-alpha serine phosphorylation and mixtures thereof. In a preferred embodiment, according to this aspect of the invention, the method can prevent or reduce the severity of infarction following closure of a cerebrovascular or cardiovascular event. In some preferred embodiments, the method can prevent stroke due to occlusion, or reduce the severity of stroke due to cerebrovascular occlusion.

In a fourth aspect, the invention relates to a method of treating ischemia or reperfusion injury, comprising preventing or reducing the severity of infarction in a mammal by administering an additional therapeutic agent to the mammal in addition to an inhibitor of NF-kB activation. Preferred inhibitors of NF-kB activation are selected from the group consisting of proteasome inhibitors, ubiquitin inhibitors, inhibitors of IkB-alpha serine phosphorylation and mixtures thereof. Some preferred additional therapeutic agents include, but are not limited to, steroids that can further inhibit NF-kB activation or inhibit the expression or action of pro-inflammatory cytokines or cell adhesion molecules; drugs that can act on the reperfused or oxidized tissue, such as anti-edema drugs, thrombolytic agents, such as TPA, streptokinase and urokinase, polyanions, such as heparin, anticoagulants; and agents that assist in temperature normalization.

Preferred inhibitors of NF-kB activation inhibit NF-kB activation through the ubiquitin proteasome pathway. In some preferred embodiments, the NF-kB activation inhibitor inhibits phosphorylation of IkB-alpha. In some preferred embodiments, the inhibitor of NF-kB activation is a proteasome inhibitor. Preferably, the proteasome inhibitor is selected from peptidyl aldehydes, boronic acids or boronic esters, lactocystine (lactacystin) or lactocystine congeners. In some preferred embodiments, the inhibitor of NF-kB activation is a ubiquitin inhibitor.

Brief description of the drawings

FIG. 1 shows the series of processes leading to NF-kB activation of reperfusion injury (including infarction). The intervention points of the method of the invention are given on the figure.

FIG. 2 shows the ubiquitin proteasome pathway.

Figure 3 shows the reduction in infarct volume following Moderate Cerebral Artery (MCA) occlusion caused by the administration of the proteasome inhibitor N- (2-pyrazine) carbonyl-L-phenylalanine-L-leucine boronic acid.

FIG. 4 shows the decrease in infarct volume following administration of the proteasome inhibitor N- (2-pyrazine) carbonyl-L-phenylalanine-L-leucine boronic acid, expressed as a percentage of the contralateral hemisphere.

Figure 5 shows the reduction in infarct volume following Moderate Cerebral Artery (MCA) occlusion caused by administration of the proteasome inhibitor 7-n-propyl-fragmentation (claspto) -lactacystin-beta-lactam.

Figure 6 shows the decrease in neurological score following Moderate Cerebral Artery (MCA) occlusion caused by administration of the proteasome inhibitor 7-n-propyl-fragmentation-lactacystin-beta-lactam.

Figure 7 shows the reduction in infarct volume resulting from administration of the proteasome inhibitor 7-n-propyl-cleavages-lactacystin-beta-lactam.

Figure 8 shows the decrease in neurological score resulting from administration of the proteasome inhibitor 7-n-propyl-cleavages-lactacystin-beta-lactam.

The present invention relates to the treatment of ischemia and reperfusion injury, which comprises preventing or reducing the severity of infarction following vascular occlusion. The patent applications, patents, and references cited herein are indicative of the knowledge to be included within this scope and are incorporated by reference in their entirety. In case of conflict, the present disclosure controls.

It has now been unexpectedly discovered that the ubiquitin proteasome pathway is a target for treating ischemia and reperfusion injury following vascular occlusion (e.g., occurring during heart disease or stroke), including preventing infarction resulting after vascular occlusion, reducing infarct size, or reducing the severity of infarction, and that these disease conditions can be effectively treated by inhibitors of NF-kB activation of the ubiquitin proteasome pathway. The present invention surprisingly provides an effective method of treating ischemia and reperfusion injury.

The present inventors have found that blocking proteasome function can reduce the effects of ischemia, such as reducing the size of infarcts after vascular occlusion. This can be achieved either by direct proteasome inhibition (shown by N- (2-pyrazine) carbonyl-L-phenylalanine-L-leucine boronic acid and 7-N-propyl-cleavage-lactacystin-beta-lactam) or by blocking ubiquitination of proteasome target proteins (e.g., IkB-alpha). Any inhibitor that affects NF-kB activation through the ubiquitin proteasome pathway in eukaryotic cells would be expected to be effective in preventing or treating infarction, including infarction caused after vascular occlusion, and therefore, are also included within the scope of the present invention.

According to the present invention, treatment of ischemia including reperfusion injury, prevention of infarction following vascular occlusion, reduction in infarct size or lessening of infarct severity can be achieved by administering to a mammal an effective amount of an inhibitor of NF-kB activation. Preferred inhibitors of NF-kB activation are selected from the group consisting of proteasome inhibitors, ubiquitin inhibitors, inhibitors of IkB-alpha serine phosphorylation and mixtures thereof.

In a first aspect, the present invention relates to a method of treating ischemia in a mammal comprising administering to said mammal an effective amount of an inhibitor of NF-kB activation. All tissues are sensitive to oxygen deficiency (ischemia) due to hypoperfusion. Major ischemic events associated with treatment include, but are not limited to, heart disease and stroke. Ischemia may also affect other tissues including retina, liver, kidney, bone, placenta, and spinal tissues. Prolonged ischemia can lead to cellular damage and, apparently, neurological dysfunction in the case of cerebral ischemia. Currently used agents for treating stroke are aimed at 1) reversing the excessive toxic phenomena associated with ischemic events; or 2) increase blood flow to the ischemic tissue.

Ischemia is usually caused by vascular closure, such as that caused by emboli or thrombi, hemorrhage, drowning, and asphyxia. Without being bound by theory, it is believed that ischemia triggers the release of a number of pro-toxic amino acids, glutamate, in the presynaptic nerve terminal of the brain, which may act on the N-methyl-D-aspartate (NMDA) receptor in neighboring cells. Once activated, NMDA receptors allow excess calcium to enter the cell, which in turn activates a series of secondary pathways that ultimately lead to degradation of cellular proteins and cell death. In the search for effective treatments, various efforts have been made to either allow presynaptic glutamate release (via stimulation of k-opiate receptors) or block NMNA receptor activation (either directly with NMDA antagonists or indirectly with glycine antagonists). Calcium channel blockers and inhibitors of calpain (calpin) have also been studied. While various drugs have shown activity in preclinical and clinical settings, their benefits are limited by the speed with which the series of reactions occurs, the time of administration, and the effectiveness of the treatment. The only drug that clinically increases blood flow is the Tissue Plasminogen Activator (TPA), which helps to block the rapid dissolution of blood clotting in blood vessels. Despite the limited degree of efficacy in stroke patients (which in fact promotes bleeding), TPA, a drug that can be lethal to those patients with cerebral hemorrhage. Thus, TPA cannot be administered until the patient is diagnosed with a stroke, rather than a hemorrhage. For stroke patients, the time required for this analysis significantly increases the time to ischemia, thus allowing a reduction in the amount of salvageable tissue. Agents that act on the ischemic cascade itself, such as inhibitors of NF-kB activation, are not affected by such extended diagnostic periods, since they are not harmful to patients with cerebral hemorrhage.

Preferred inhibitors of NF-kB activation inhibit NF-kB activation through the ubiquitin proteasome pathway. In some preferred embodiments, the NF-kB activation inhibitor inhibits phosphorylation of IkB-alpha. In some preferred embodiments, the inhibitor of NF-kB activation is a proteasome inhibitor. In such embodiments, inhibition of the proteasome is preferably less than complete inhibition. Preferred proteasome inhibitors are selected from peptidyl aldehydes, boronic acids or boronic esters, lactocystine proteins or lactocystine protein analogues. In some preferred embodiments, the inhibitor of NF-kB activation is a ubiquitin pathway inhibitor.

The transcription factor NF-kB is a member of the Rel protein family. The Rel family of transcription activator proteins can be divided into two groups. The first group required proteolytic manipulations, including p105 and p100, which were processed to p50 and p52, respectively. The second group did not require proteolytic manipulations, including p65(Rel A), Rel (c-Rel) and Rel B. NF-kB comprises two subunits, p50 and another member of the Rel gene family (e.g., p 65). Unprocessed p105 can also be linked to p65 and other members of the Rel family. In most cells, the p50-p65 heterozygote exists in the cytoplasm in an inactive form linked to IkB-alpha. The ternary complex is activated by the separation and disruption of IkB-alpha, whereas the p65/p105 bihybrid can be activated by the processing of p 105.

The ubiquitin proteasome pathway plays an essential role in the regulation of NF-kB activity, responsible for both the processing of p105 to p50 and the degradation of the inhibitor protein IkB-. alpha. (Palommbella et al, WO 95/25533). For degradation by the proteasome, IkB-. alpha.must first undergo selective phosphorylation at serine residues 32 and 36, followed by ubiquitination (Alkalay et al, Proc. Natl. Acad. Sci. USA 92: 10599 (1995); Chen, WO 97/35014).

Upon activation, NF-kB translocates to the nucleus where it plays a central role in the regulation Of a distinctly different set Of genes involved in immune and inflammatory responses (Grilli et al, International Review Of biology 143: 1-62 (1993)). For example, the expression of many genes involved in inflammatory responses requires NF-kB, such as the TNF- α gene, and encodes the cell adhesion molecules E-selectin, P-selectin, ICAM and VCAM (Collins, T., Lab. invest. (1993) 68: 499). NF-kB is also required for expression of a number of cytokine genes (e.g., IL-2, IL-6, granulocyte colony stimulating factor, and IFN- β). Inducible nitric oxide synthase is also under the control of NF-kB.

Proteasome inhibitors block degradation of IkB-alpha and activation of NF-kB (Palommbella et al, WO95/25533, published at 9/28/95; Traneckner et al, EMBO J. (1994) 13: 5433). Proteasome inhibitors can also block IFN- α -induced expression of the leukocyte adhesion molecules E-selectin, VCAM-1 and ICAM-1 (Read et al, Immunity (1995) 2: 493). These cell adhesion molecules play a key role in supporting leukocyte extravasation from the blood stream to extravascular lesions (e.g., ischemic tissue). Although repair is desired, the resulting influx of cells (especially neutrophils) can promote damage through cytokine release, resulting in accelerated cell death and providing a signal for other cells (e.g., phage) to attack the area.

In a second aspect, the present invention relates to a method of preventing or reducing the severity of reperfusion injury in a mammal in need thereof, which comprises administering to said mammal an effective amount of an inhibitor of NF-kB activation. Reperfusion injury results from one or more of the following causes: cellular acidosis leads to calcium overload; catabolism causes an increase in intracellular penetration, resulting in cell swelling; free radicals from neutrophils and other inflammatory cells. Preferred inhibitors of NF-kB activation are selected from the group consisting of proteasome inhibitors, ubiquitin pathway inhibitors, IkB-alpha serine phosphorylation inhibitors, and mixtures thereof.

In a third aspect, the present invention relates to a method for preventing or reducing the size or reducing the severity of an infarct comprising administering to said mammal an effective amount of an inhibitor of NF-kB activation. Preferred inhibitors of NF-kB activation are selected from the group consisting of proteasome inhibitors, ubiquitin inhibitors, inhibitors of IkB-alpha serine phosphorylation and mixtures thereof. In a preferred embodiment, according to this aspect of the invention, the method prevents the development of infarct following closure of the cerebral or cardiovascular vessels, reduces infarct size, and reduces infarct severity. In some preferred embodiments, the method can prevent stroke due to occlusion, or reduce the severity of stroke due to cerebrovascular occlusion.

The most common stroke is thrombotic stroke, in which closure of the cerebral blood vessels is thought to be due to a plug of aggregated platelets. Typically, these platelet plugs are released as emboli from platelet thrombi on atherosclerotic plaques in the main carotid or cerebrovascular arteries. Thrombotic stroke is usually characterized by "stuttering", the initial onset of which is mild, usually reversible, and after a more severe irreversible stroke, neurological damage occurs. The initial event usually reflects a temporary cerebrovascular blockage by platelet thrombosis, which is reversible in nature. In fact, from a clinical point of view, a mild stroke can be seen as an extreme termination of the Transient Ischemic Attack (TIA) series of reactions-a reversible neurological deficit in which the cerebral blood vessels are temporarily blocked by a thrombotic platelet thrombus, with consequent aggregation, so that flow can only be reestablished. Accordingly, the present invention provides methods of administering the agents disclosed herein after a temporary vascular closure has occurred. As previously discussed, another important form of stroke is a stroke following a cerebral hemorrhage. It is believed that the agents disclosed herein have broad therapeutic utility in preventing, reducing the size of, or reducing the severity of infarctions caused by a variety of causes, including thrombotic stroke and stroke following cerebral hemorrhage. For practical purposes, a reduction in infarct size or a reduction in infarct severity can be inferred from a reduction in infarct-related symptoms, including unrestrained neurological symptoms as well as cardiac functional symptoms.

In a fourth aspect, the invention relates to methods of treating ischemia or reperfusion injury, including but not limited to methods of reducing infarct size or reducing infarct severity in a mammal comprising administering to said mammal an additional therapeutic agent in addition to an inhibitor of NF-kB activation. Preferred inhibitors of NF-kB activation are selected from the group consisting of proteasome inhibitors, ubiquitin pathway inhibitors, IkB-alpha serine phosphorylation inhibitors, and mixtures thereof. Some preferred additional therapeutic agents include, but are not limited to, steroids that further inhibit NF-kB activation or inhibit the expression or action of pro-inflammatory cytokines or cell adhesion molecules; agents that can reperfuse or oxidize tissue, anti-edema drugs, thrombolytic agents such as TPA, streptokinase and urokinase, polyanions such as heparin, anticoagulants; and agents that assist in temperature normalization. Agents that inhibit the action of proinflammatory cytokines or cell adhesion molecules include, but are not limited to, antibodies or antibody derivatives, more preferably monoclonal antibodies, human antibodies, humanized antibodies, single chain antibodies, chimeric antibodies or antibody fragments linked to an antigen. The use of any of the agents discussed or disclosed herein in combination with any other agent used in the treatment of stroke or myocardial infarction is further included within the scope of the present invention.

In this description, the following definitions are used.

"treating" or "treatment" refers to the prevention or reduction of ischemic or reperfusion injury, which includes the prevention of infarction after vascular closure or the reduction of infarct size or the reduction of infarct severity, including unlimited infarctions. Any improvement in infarction symptoms observed with any proteasome inhibitor, ubiquitin inhibitor, or agent that interferes with the activation of NF-kB by an inhibitor, according to the method of treatment, is included within the scope of the present invention.

The term "mammal" includes humans.

"inhibitor of NF-kB activation" or "inhibitor of NF-kB activation" refers to any substance that inhibits NF-kB activation via the ubiquitin proteasome pathway, including any of 1) inhibiting proteasomes or their activity; 2) inhibit the ubiquity of IkB-alpha or p 105; or 3) inhibits phosphorylation of IkB-alpha or p 105.

"ubiquitin pathway inhibitor" refers to any substance that directly or indirectly inhibits ubiquitin ation or ubiquitin transfer to a protein. Non-limiting ubiquitin inhibitors include those described in Berleth et al, biochem.35 (5): 1664-1671 (1996). Inhibitors of IkB-alpha phosphorylation are also known (Chen, Cell 84: 853 (1996)).

"proteasome inhibitor" refers to any substance that directly or indirectly inhibits the proteasome or its activity. Non-limiting proteasome inhibitors useful in the present invention include peptidyl aldehydes (WO 95/24914 published by Stein et al at 21.9.1995; WO 91/13904 published by Siman et al at 19.9.1991; Iqbal et al, J.Med.chem.38: 2276-2277(1995)), peptidyl boronic acids (WO 96/13266 published by Adams et al at 9.5.1996; Siman et al, WO 91/13904), milk cystatin and milk cystatin congeners (Fenteany et al, Proc.Natl.Acad.Sci.USA (1994) 91: 3358; WO96/32105 published by Fenteany et al 10/17/96).

Preferably, the peptidyl aldehyde proteasome inhibitors used in the present invention are those compounds disclosed in WO 95/24914 to Stein et al, published on 9/21 of 1995 or WO 91/13904 to Siman et al, published on 9/19 of 1991, both of which are incorporated by reference in their entirety.

Preferably, the boronic acid or ester compounds used in the present invention are those disclosed in WO 96/13266 published by Adams et al on 9.5.1996 or those mentioned in WO 91/13904 by Siman et al, both of which are incorporated herein by reference in their entirety.

More preferably, the boronic acid or ester compound used in the present invention is selected from: n- (4-morpholine) carbonyl-beta- (1-naphthyl) -L-alanine-L-leucine boronic acid. N- (8-quinoline) sulfonyl-beta- (1-naphthyl) -L-alanine-leucine boronic acid. N- (2-pyrazine) carbonyl-L-phenylalanine-L-leucine boronic acid and N- (4-morpholine) carbonyl- [ O- (2-picolyl) ] -L-tyrosine-L-leucine boronic acid.

Preferably, the lactocystine proteins and lactocystine protein analogs used in the present invention are those compounds mentioned in WO96/32105, published by Fenteany et al at 10/17/96, which is incorporated by reference in its entirety. More preferably, the milk cystine protein congener used in the method of the invention is selected from the group consisting of milk cystine, split-milk cystine-beta-lactam, 7-ethyl-split-milk cystine-beta-lactam and 7-n-propyl-split-milk cystine-beta-lactam. The most preferred lactocystine congener is 7-n-propyl-split-lactocystine-beta-lactam.

The agents disclosed herein may be administered by any route, including intradermal, subcutaneous, oral, intra-articular, intravenous, and the like. The preferred route of administration is intravenous. The preferred mode of parenteral administration is by single injection or intravenous drip.

The concentration of the disclosed compounds in the pharmaceutical mixture may vary depending on several factors, including the dosage of the compound administered, the pharmacokinetic properties of the compound used, and the route of administration. The effective dosage of the agents for treating ischemia or reperfusion injury can vary over a wide range, from about 10 μ g to about 50mg/kg of subject mammal body weight. The agent may be administered to the subject in single or repeated doses. Treatment may be carried out daily or in a more frequent manner depending on factors including the overall health of the patient, the formulation of the compound selected and the route of administration.

The disclosed compounds can be administered at any time, such as before, during or after the onset of ischemia. In some preferred embodiments, the ischemia occurs after, but has time to reverse, some or all of the damage to the affected tissue. Preferably, the agent is administered within 12 hours, more preferably within 6 hours, and most preferably within 3 hours after the occurrence of ischemia. Treatment may be initiated before, during or after reperfusion of the ischemic tissue. In many cases, the timing of reperfusion cannot be precisely determined, but it is preferred to start treatment before, during or just after reperfusion, with the aim of preventing or mitigating additional damage caused by reperfusion.

In the event of ischemia due to coagulation, an agent that disrupts coagulation, like TPA, may be administered to reduce potential tissue damage. Once administered, the drug acts rapidly to remove intravascular obstructions, thus allowing the time to terminate ischemia to be determined. In a preferred embodiment, the inhibitor of NF-kB activation is administered at the same time as or just after the coagulation is dissolved in the drug.

In some other preferred embodiments, the inhibitor of NF-kB activation is administered prior to the onset of ischemia. In some medical procedures (e.g., surgery), the occurrence of ischemia may be predicted. In other preferred embodiments, the disclosed compounds are administered prior to or just after the onset of ischemia and reperfusion during the above-described medical procedure (e.g., angioplasty).

The following examples will further illustrate certain preferred embodiments of the invention but are not intended to limit the invention in any way. Examples example 1 method

Six male Sprague Dawley rats (300g) were anesthetized with haloethane and then their cerebral arteries (MCA) were closed for 2 hours using nylon fiber. Subsequently, the fibers were removed and the infarcted tissue was reperfused for 24 hours before the rats were decapitated.

Coronary sections (2.0 mm. times.7-8) from the whole brain were stained with triphenyltetrazolium chloride and evaluated by image analysis under double-blind procedure to determine infarct size.

The extent of infarction can also be expressed as a percentage of the contralateral (non-infarcted) hemisphere to provide an index: the procedure described above is exactly how much damage is done to the ipsilateral (infarcted) hemisphere. The percentage of injured ipsilateral hemispheres cannot generally be determined directly as a result of edema in the infarcted hemisphere. Dosage regimen

Rats were given a single injection (1.0mL/kg) of vehicle (10% PEG 200/saline; N ═ 3) or N- (2-pyrazine) carbonyl-L-phenylalanine-L-leucine boronic acid (0.03 mg/kg; N ═ 3) intravenously 30 minutes, 2 hours, and 6 hours after the onset of closure. Results

The infarct volume in the ipsilateral hemisphere was reduced by 62% in the treated animals (see figure 1). This result reflects a 19% -2% reduction in total damage to the hemisphere (fig. 2). EXAMPLE 2 method

Male Sprague Dawley rats (250-400g) were anesthetized with haloethane and then their cerebral arteries (MCA) were occluded for 2 hours using nylon fiber. Subsequently, the fibers were removed and the infarcted tissue was reperfused for 24 hours before the rats were decapitated.

Immediately after fiber removal, animals were evaluated using a neurological scoring system. Neurological scores are expressed as numbers from 0 to 10, with 0 representing no neurological damage and 10 representing severe neurological damage. After 24 hours and before decapitation, animals were evaluated a second time using the same neurological scoring system.

Coronary sections (2.0 mm. times.7-8) from the whole brain were stained with triphenyltetrazolium chloride and evaluated by image analysis under double-blind procedure to determine infarct size. Dosage regimen

Rats were given a one-time injection (1.0mL/kg) of vehicle (50% propylene glycol/saline; n ═ 8) or 7-n-propyl-split-lactacystin- β -lactam (0.1 mg/kg; n ═ 6) intravenously 2 hours after the onset of closure. Results

Animals treated with 7-n-propyl-lactocystine-beta-lactam had 70% reduction in infarct volume (see figure 5).

The neurological score of all animals was 10 ± 0 at 2 hours immediately after the ischemic event. At 24 hours, the neurological score of vehicle-treated rats was 8.7 ± 0.6, whereas the mean score of animals treated with a single dose (0.1mg/kg) of 7-n-propyl-fractalkine-lactacystin- β -lactam was 5.5 ± 1 (see fig. 6). These data indicate that it produces a 40% neurological improvement for the drug treated animals. Conclusion

The 7-n-propyl-fragmentation-lactocystine-beta-lactam has obvious protective effect on the damage of nerve defects and infarcted brains. Example 3 method

Male Sprague Dawley rats (250-400g) were anesthetized with haloethane and then their cerebral arteries (MCA) were occluded for 2 hours using nylon fiber. Subsequently, the fibers were removed and the infarcted tissue was reperfused for 24 hours before the rats were decapitated.

Immediately after fiber removal, animals were evaluated using a neurological scoring system. Neurological scores are expressed as numbers from 0 to 10, with 0 representing no neurological damage and 10 representing severe neurological damage. After 24 hours and before decapitation, animals were evaluated a second time using the same neurological scoring system.

Coronary parts (2.0 mm. times.7-8) from the whole brain were stained with triphenyltetrazolium chloride, and evaluated by image analysis under blind operation to determine the infarct size. Dosage regimen

Rats were given a one-time injection (1.0mL/kg) of vehicle (50% propylene glycol/saline; n ═ 8) or 7-n-propyl-split-lactacystin- β -lactam (0.3 mg/kg; n ═ 7) intravenously 2 hours after the onset of closure. The other two groups of rats were given 7-n-propyl-split-lactocystine- β -lactam by intravenous injection (1.0mL/kg) at 0 min, 2 hours and 6 hours after the initiation of closure. One group (0.1mg/kg x 3; n ═ 6) received 0.1mg/kg at each of the above times, and the other group (0.3mg/kg x 3; n ═ 7) received 0.3mg/kg at each of the above times. Results

Animals treated with a single dose of 7-n-propyl-split-lactacystin-beta-lactam had a 50% reduction in infarct volume (see figure 3). There was no significant change in infarct volume in the 0.1mg/kg X3 dose group or the 0.3mg/kg X3 dose group (see FIG. 7).

The neurological score of all animals was 10 ± 0 at 2 hours immediately after the ischemic event. At 24 hours, the neurological score of vehicle-treated rats was 8.7 ± 0.6, whereas the mean score of animals treated with a single dose (0.3mg/kg) of 7-n-propyl-fractalkine-lactacystin- β -lactam was 4 ± 1 (see fig. 8). These data indicate that it produces a 60% neurological improvement for the drug treated animals. No significant neurological improvement was observed for either the 0.1mg/kg X3 dose group or the 0.3mg/kg X3 dose group (see FIG. 8). Conclusion

The 7-n-propyl-fragmentation-lactocystine-beta-lactam has obvious protective effect on the damage of nerve defects and infarcted brains.

While certain specific preferred embodiments have been described above, it will be understood that they are not intended to limit the invention. Various modifications to the disclosed embodiments will be apparent to those skilled in the art having the benefit of this disclosure, and such modifications are intended to be included within the scope of the present invention as defined by the following claims.

Claims (24)

1. A method of treating ischemic injury in a mammal comprising administering to said mammal an effective amount of an inhibitor of NF-kB activation, an agent that interferes with NF-kB activation through the ubiquitin proteasome pathway, and mixtures thereof.

2. A method of treating reperfusion injury resulting from ischemia in a mammal comprising administering to said mammal an effective amount of an inhibitor of NF-kB activation selected from the group consisting of proteasome inhibitors, ubiquitin inhibitors, agents that interfere with NF-kB activation via the ubiquitin proteasome pathway, and mixtures thereof.

3. A method of preventing embolism, reducing the size of embolism, or reducing the severity of infarction resulting from ischemia or reperfusion injury in a mammal comprising administering to said mammal an effective amount of an inhibitor of NF-kB activation selected from the group consisting of proteasome inhibitors, ubiquitin inhibitors, agents that interfere with the activation of NF-kB through the ubiquitin proteasome pathway, and mixtures thereof.

4. The method of claims 1-3, wherein the ischemia is due to vascular occlusion.

5. The method of claim 4 wherein the occlusion is a cerebral vessel.

6. The method of claim 4 wherein the occlusion is cardiovascular.

7. The method of claims 1-3, wherein the method prevents or reduces the severity of stroke.

8. The method of claims 1-3, wherein the method prevents or reduces the severity of stroke caused by cerebrovascular occlusion.

9. The method of claim 6, wherein the method prevents or reduces the severity of myocardial infarction resulting from cardiovascular closure.

10. A method as claimed in claims 1 to 3 wherein the inhibitor of NF-kB activation inhibits phosphorylation of IkB- α.

11. The method of claims 1-3, wherein the inhibitor of NF-kB activation comprises a proteasome inhibitor.

12. The method of claim 11, wherein the proteasome inhibitor is a peptidyl aldehyde.

13. The method of claim 11, wherein the proteasome inhibitor is a peptidyl boronic acid or a peptidyl boronic ester.

14. The method of claim 11, wherein the proteasome inhibitor is a cystatin analog.

15. The method of claim 13, wherein the proteasome inhibitor is N- (2-pyrazine) carbonyl-L-phenylalanine-L-leucine boronic acid.

16. The method of claim 14, wherein the proteasome inhibitor is 7-n-propyl-cleavant-lactacystin- β -lactam.

17. The method of claims 1-3 wherein the inhibitor of NF-kB activation is administered to the mammal within 12 hours after onset of ischemia.

18. The method of claims 1-3 wherein the inhibitor of NF-kB activation is administered to the mammal within 6 hours after onset of ischemia.

19. The method of claims 1-3 wherein the inhibitor of NF-kB activation is administered to the mammal prior to the onset of ischemia.

20. The method of claims 1-4, further comprising the step of administering a second agent.

21. The method of claim 21, wherein the second agent is selected from the group consisting of an inhibitor of NF-kB activation, an agent that inhibits the expression or action of a pro-inflammatory cytokine or cell adhesion molecule, an agent that reperfused or oxidizes tissue, and an agent that contributes to temperature normalization.

22. The method of claim 21, wherein the second agent is selected from the group consisting of steroids, antiedemic drugs, thrombolytic agents, clot solubilizing agents, polyanions, and anticoagulants.

23. The method of claim 22, wherein the second agent comprises a thrombolytic agent and a clot solubilizing drug.

24. The method of claim 23, wherein the clot solubilizing agent comprises a tissue plasminogen activator.