Treatment of cerebral ischemia-reperfusion injury

Abstract

To provide a method for treating cerebral ischemia-reperfusion injury.SOLUTION: The method includes a step of administering to a subject an antibody that specifically binds to interleukin-1α.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 843,182, titled "Treatment of Brain Ischemia - Reperfusion Injury," filed on May 3, 2019.

[0002] Statement Regarding Federally Sponsored Research Not applicable

[0003] Field of the Invention The present invention generally relates to the fields of medicine, neurology, and immunology. More particularly, the present invention relates to the use of an antibody (Ab) that specifically binds to interleukin - 1α (IL - 1α) to reduce various sequelae of ischemia - reperfusion injury to the central nervous system (e.g., the brain).

Background Art

[0004] Ischemic strokes are a major cause of death and disability. It is treated by removing the clot from the occluded blood vessel using tissue plasminogen activator or mechanical thrombectomy so that blood flow to the affected tissue is restored. However, the restoration of blood flow induces the release of excitotoxic neurotransmitters, the accumulation of intracellular Ca2+, free radical damage, neuronal apoptosis, neuroinflammation, and lipolysis leading to ischemia - reperfusion injury.

Summary of the Invention

[0005] It has been discovered that a monoclonal antibody (mAb) that specifically binds to IL - 1α is useful for alleviating the pathological sequelae that occur after brain ischemia - reperfusion injury.

[0006] Accordingly, methods are described herein for reducing one or more of the pathological events that can occur after cerebral ischemia-reperfusion injury in a subject. These methods can include administering to the subject a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an agent that selectively binds to an effective amount of IL-1α to reduce edema, hemorrhagic changes, intracranial pressure, disruption of the blood-brain barrier, resulting infarct volume, and resulting neurological deficits in the subject. The agent can be an anti-IL-1α antibody, such as a monoclonal antibody (e.g., of the IgG1 isotype). The pharmaceutical composition can be administered to the subject by injection, subcutaneously, intravenously, intramuscularly, or intrathecally. In the method, the dosage can be at least 50 mg (e.g., at least 50, 75, 100, 150, 200, 300, 400, 500, 600, 700, or 800 mg). Preferably, the first dose is administered within 1, 2, 3, 4, 5, or 6 hours after observation of the first symptoms of the ischemic attack, or within 10, 20, 30, or 60 minutes after the subject seeks treatment by a medical professional. Thereafter, additional doses (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) can be administered to the subject, for example, at about 20 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, or 4 weeks after the preceding administration.

[0007] Methods for treating cerebral ischemia-reperfusion injury in a subject by administering an antibody that specifically binds to interleukin-1α (IL-1α) to the subject; methods for reducing the volume of cerebral infarction resulting from an occlusive episode in a subject by administering an antibody that specifically binds to IL-1α to the subject; methods for reducing neurological deficits resulting from an occlusive episode in a subject by administering an antibody that specifically binds to IL-1α to the subject; and methods for reducing the number of activated macrophages in the ischemic penumbra of brain injury resulting from an occlusive episode in a subject by administering an antibody that specifically binds to IL-1α to the subject are further described herein. In the above and the methods herein, the antibody that specifically binds to IL-1α can be administered to the subject after the subject develops cerebral ischemia. In the method for reducing the volume of cerebral infarction resulting from an occlusive episode in a subject, the volume of cerebral infarction resulting from an occlusive episode in the subject can be at least 20% (e.g., at least 20, 30, 40, or 50%) less than the volume of cerebral infarction that would have resulted from the occlusive episode if the subject had not been administered the antibody that specifically binds to IL-1α.

[0008] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Commonly understood definitions of biological terms can be found in Rieger et al., Glossary of Genetics: Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991; and Lewin, Genes V, Oxford University Press: New York, 1994. Commonly understood definitions of medical terms can be found in Stedman’s Medical Dictionary, 27 th Edition, Lippincott, Williams & Wilkins, 2000.

[0009] As used herein, "antibody" or "Ab" is an immunoglobulin (Ig), a solution of identical or heterogeneous Ig, or a mixture of Ig. "Ab" can also refer to fragments and engineered versions of Ig, such as Fab, Fab', and F(ab')2 fragments; as well as scFv, heteroconjugate Abs, and similar artificial molecules that use Ig-derived CDRs to confer antigen specificity. "Monoclonal antibody" or "mAb" is a population of Ab molecules expressed by a single clonal B cell line or containing only one antigen-binding site capable of immunoreacting with a specific epitope of a particular antigen. "Polyclonal Ab" is a mixture of heterogeneous Abs. Typically, polyclonal Abs contain numerous different Ab molecules that bind to a particular antigen, and at least some of the different Abs immunoreact with different epitopes of the antigen. As used herein, polyclonal Abs can be a mixture of two or more mAbs.

[0010] The "antigen-binding portion" of an Ab is the portion of the Ab contained within the variable region of the Fab portion of the Ab that confers antigen specificity to the Ab (i.e., typically the three-dimensional pocket formed by the CDRs of the heavy and light chains of the Ab). The "Fab portion" or "Fab region" is the proteolytic fragment of a papain-digested Ig that contains the antigen-binding portion of that Ig. The "non-Fab portion" is the portion of the Ab that is not within the Fab portion, e.g., the "Fc portion" or "Fc region". The "constant region" of an Ab is the portion of the Ab outside the variable region. The "effector portion" of an Ab, which is the portion of the Ab that causes binding to other components of the immune system that promote an immune response, is generally included within the constant region. Thus, for example, the site on an Ab that binds to a complement component or an Fc receptor (without passing through its antigen-binding portion) is the effector portion of that Ab.

[0011] When referring to a protein molecule, e.g., an Ab, "purified" means separated from components that naturally accompany such a molecule. Typically, an Ab or protein is purified when it is at least about 10% (e.g., 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.9%, and 100%) free by weight from non-Ab proteins or other naturally occurring organic molecules with which it is naturally associated. Purity can be measured by any suitable method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. A chemically synthesized protein or other recombinant protein produced in a cell type other than the cell type in which it naturally occurs is "purified".

[0012] "Bind," "binds," or "reacts with" means that one molecule recognizes and attaches to a specific second molecule in a sample, but does not substantially recognize or attach to other molecules in the sample. In general, an Ab that "specifically binds" to another molecule has a specific binding affinity of about 10 to that other molecule. 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , or 10 12 Higher K than liter / mole d An Ab that "selectively binds" a first molecule specifically binds the first molecule at a first epitope, but does not specifically bind to other molecules that do not have the first epitope. For example, an Ab that selectively binds IL-1 alpha specifically binds to an epitope on IL-1 alpha, but does not specifically bind to IL-1 beta (which does not have that epitope).

[0013] A "therapeutically effective amount" is an amount capable of producing a medically desirable effect (eg, amelioration or prevention of a disease or symptoms of a disease) in the treated animal or human.

[0014] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All applications and publications mentioned herein are incorporated by reference in their entirety. In case of conflict, this specification, including definitions, will control. Additionally, the specific embodiments discussed below are exemplary only and are not intended to be limiting. BRIEF DESCRIPTION OF THE DRAWINGS

[0015]

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[0016] Compositions and methods for reducing one or more sequelae of cerebral ischemia-reperfusion injury in a subject are described herein. The preferred embodiments described below illustrate adaptations of these compositions and methods. Nevertheless, from the description of these embodiments, other aspects of the invention can be manufactured and / or practiced based on the description provided below.

[0017] General Methodology Methods involving conventional immunological and molecular biological techniques are described herein. Immunological methods (e.g., assays for detection and localization of antigen-Ab complexes, immunoprecipitation, and immunoblotting, etc.) are generally known in the art and are described in treatises on methodology, such as Current Protocols in Immunology, edited by Coligan et al., John Wiley & Sons, New York. Techniques of molecular biology are described in detail in treatises, such as Molecular Cloning: A Laboratory Manual, 2nd ed., vols. 1-3, edited by Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; and Current Protocols in Molecular Biology, edited by Ausubel et al., Greene Publishing and Wiley-Interscience, New York. Methods of Abs are described in Handbook of Therapeutic Abs, edited by Dubel, S., Wiley-VCH, 2007. General methods of medical treatment are described in McPhee and Papadakis, Current Medical Diagnosis and Treatment 2010, 49 th Edition, McGraw-Hill Medical, 2010; and Fauci et al., Harrison’s Principles of Internal Medicine, 17 th Edition, McGraw-Hill Professional, 2008. Methods in neurology are described in Daroff R., Bradley’s Neurology in Clinical Practice, 2-Volume Set 7th Edition, Elsevier, 2015.

[0018] Treatment The compositions described herein are useful for treating cerebral ischemia-reperfusion injury in a mammalian subject by administering to the subject a pharmaceutical composition comprising an effective amount of anti-IL-1α Ab to improve at least one characteristic of the condition in the subject (e.g., edema, hemorrhagic changes, intracranial pressure, disruption of the blood-brain barrier, infarct volume, and resulting neurological deficits in the subject). The success of treating cerebral ischemia-reperfusion injury can be evaluated according to established methods. These include neurological examinations, computed tomography, magnetic resonance imaging, and cerebral angiography. The improvement can be evaluated as at least 10% (e.g., at least 10, 20, 30, 40, 50, 60, or 70%) better score in a test used to assess sequelae of cerebral ischemia-reperfusion injury at a given time point after the onset of injury (e.g., 1, 2, 3, 4, 5, 7, 10, or 30 days after the onset of injury; or 1, 2, 3, 4, 5, 6, 12, or 24 months) compared to when the subject was not administered an effective amount of anti-IL-1α Ab (e.g., compared to that estimated from historical data of genetically matched subjects or subjects with similar injuries).

[0019] The cerebral infarction volume after cerebral ischemia reperfusion injury can be determined in a living subject by magnetic resonance imaging (MRI) as described, for example, by Lovblad et al., Ann Neurol. 42:164-170, 1997. Preferably this is done when the final infarction volume has been reached (e.g., at least 30 days after injury). The amount and / or quality of neurological deficits resulting from cerebral ischemia reperfusion injury in a subject can be determined by known methods, such as by the National Institutes of Health Stroke Scale (NIHSS), which measures neurological impairment using a 15-item scale (Table 1) or the Canadian Neurological Scale. The number of activated macrophages / microglia in the ischemic penumbra of brain injury resulting from cerebral ischemia reperfusion injury can be evaluated in a living subject by MRI in which ultrasmall superparamagnetic iron oxide (USPIO) is used as a macrophage / microglia-specific contrast agent or by other known methods.

[0020] The subject can be a mammal, such as a human, rodent, cat, dog, horse, sheep, or pig, including a human who has suffered from, is suffering from, or is at risk of developing cerebral ischemia (e.g., ischemic attack, transient ischemic attack, or subarachnoid hemorrhage). The human subject can be male, female, adult, child, elderly (65 years and older), and those with other diseases or risk factors for cerebral ischemia (e.g., hypertension, diabetes, heart disease, race / ethnicity, personal or family history of cerebral ischemia, cerebral aneurysm, and / or cerebral arteriovenous malformation). As a non-limiting example, the subject can be a human diagnosed with cerebrovascular occlusion or a human with a transient ischemic attack. The subject can also be a human who has been administered tissue plasminogen activator (e.g., after being diagnosed with acute cerebrovascular occlusion). The initial dose of the agent that binds to IL-1α can be administered within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 36, or 48 hours from the onset of symptoms of the ischemic attack. For subjects at high risk of developing an ischemic attack (e.g., subjects experiencing a transient ischemic attack or having thrombosis), the agent that binds to IL-1α can be prophylactically administered once daily or once every 2, 3, 4, 5, 6, 7, or 14 days until the risk is reduced (e.g., the transient ischemic attack stops or the thrombosis resolves). It is preferred to treat a subject who has developed a human anti-human antibody response due to a previous administration of a therapeutic antibody accompanied by an anti-IL-1α Ab that is a truly human Ab (e.g., one that is naturally expressed in a human subject).

[0021] Antibodies and other agents that target IL-1α Any suitable type of Ab that specifically reduces one or more sequelae of cerebral ischemia-reperfusion injury in a subject can be used in the methods described herein. For example, the anti-IL-1α Ab used can be an mAb, a polyclonal Ab, a mixture of mAbs, or an Ab fragment or engineered Ab-like molecule, such as an scFv. The Ka of the Ab is preferably at least 1×10 9 M -1 or higher (e.g., 9×1010 M -1 、 8 × 10 10 M -1 、 7 × 10 10 M -1 、 6 × 10 10 M -1 、 5 × 10 10 M -1 、 4 × 10 10 M -1 、 3 × 10 10 M -1 、 2 × 10 10 M -1 、 or 1 × 10 10 M -1 or higher). In a preferred embodiment, Ab is a fully human mAb comprising (i) an antigen-binding variable region exhibiting very high binding affinity (e.g., at least nanomolar or picomolar concentration) for human IL-1α and (ii) a constant region. The human Ab is preferably IgG1, but may be a different isotype, such as IgM, IgA, or IgE, or a subclass, such as IgG2, IgG3, or IgG4. Useful mAbs include those that neutralize IL-1α (e.g., those that prevent IL-1α from binding to the IL-1α receptor).

[0022] Since B lymphocytes expressing Ig specific for human IL-1α naturally exist in humans, the presently preferred method for producing mAbs is to first isolate such B lymphocytes from a subject and then immortalize them to enable continuous replication in culture. A subject lacking a number of naturally occurring B lymphocytes expressing Ig specific for human IL-1α may be immunized with one or more human IL-1α antigens to increase the number of such B lymphocytes. Human mAbs are prepared by immortalizing human Ab-secreting cells (e.g., human plasma cells). See, for example, U.S. Patent No. 4,634,664.

[0023] In an exemplary method, one or more (e.g., 5, 10, 25, 50, 100, 1000, or more) human subjects are screened for the presence of such human IL-1α-specific Abs in the blood. Subjects expressing the desired Ab can then be used as B lymphocyte donors. In one possible method, peripheral blood is obtained from a human donor having B lymphocytes that express a human IL-1α-specific Ab. Such B lymphocytes are then isolated from the blood sample by cell sorting (e.g., fluorescence-activated cell sorting, "FACS"; or magnetic bead cell sorting) to select, for example, B lymphocytes that express a human IL-1α-specific Ig. These cells can then be immortalized by viral transformation (e.g., using EBV) or by fusion to another immortalized cell, such as a human myeloma, according to known techniques. B lymphocytes within this population that express an Ig having at least nanomolar or picomolar binding affinity for human IL-1α can then be isolated by limiting dilution (e.g., cells in wells of a microtiter plate positive for a human IL-1α-specific Ig are selected, passaged, and the process is repeated until the desired clone line can be isolated). See, for example, Goding, MAbs: Principles and Practice, pages 59-103, Academic Press, 1986. Clone cell lines that express an Ig having at least nanomolar or picomolar binding affinity for human IL-1α are preferred. The MAbs secreted by these clone cell lines can be purified from the culture medium or body fluid (e.g., ascites) by conventional Ig purification procedures, such as salt cuts, size exclusion, ion exchange separation, and affinity chromatography.

[0024] Immortalized B lymphocytes may be used in in vitro cultures to directly produce mAbs, but in certain cases, it may be desirable to use a heterologous expression system to produce mAbs. See, for example, the method described in U.S. Patent Application No. 11 / 754,899. For example, a gene encoding an mAb specific for human IL-1α may be cloned and introduced into an expression vector (e.g., a plasmid-based expression vector) for expression in heterologous host cells (e.g., CHO cells, COS cells, myeloma cells, and E. coli cells). Since Ig contains heavy (H) and light (L) chains in an H2L2 configuration, the genes encoding each may be isolated separately and expressed in different vectors.

[0025] Chimeric mAbs (e.g., "humanized" mAbs), which are antigen-binding molecules having different portions from different animal species (e.g., variable regions of mouse Ig fused to the constant regions of human Ig), may be used, although they are generally of lower preference due to the higher likelihood that the subject will generate an anti-Ab response. Such chimeric Abs can be prepared by methods known in the art. See, for example, Morrison et al., Proc. Nat’l. Acad. Sci. USA, 81:6851, 1984; Neuberger et al., Nature, 312:604, 1984; Takeda et al., Nature, 314:452, 1984. Similarly, Abs can be humanized by methods known in the art. For example, an mAb having a desired binding specificity can be humanized by various vendors or as described in U.S. Patent Nos. 5,693,762, 5,530,101, or 5,585,089.

[0026] The mAbs described herein may be affinity matured to enhance or otherwise alter their binding specificities by known methods such as VH and VL domain shuffling (Marks et al., Bio / Technology 10:779-783, 1992), random mutagenesis of hypervariable regions (HVRs) and / or framework residues (Barbas et al., Proc Nat. Acad. Sci. USA 91:3809-3813, 1994; Schier et al., Gene 169:147-155, 1995; Yelton et al., J. Immunol. 155:1994-2004, 1995; Jackson et al., J. Immunol. 154(7):3310-9, 1995; and Hawkins et al., J. Mol. Biol. 226:889-896, 1992). Amino acid sequence variants of the Ab may be prepared by introducing appropriate changes into the nucleotide sequence encoding the Ab. Additionally, modifications to the nucleic acid sequence encoding the mAb may be made to enhance production of the mAb in a particular expression system (e.g., without changing the amino acid sequence of the mAb) (e.g., elimination of introns and / or codon optimization for a given expression system). The mAbs described herein may also be modified by conjugation to another protein (e.g., another mAb) or non-protein molecule. For example, the mAb may be conjugated to a water-soluble polymer such as polyethylene glycol or a carbon nanotube (e.g., see Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605, 2005). See U.S. Patent Application No. 11 / 754,899.

[0027] Preferably, to ensure that a high titer human IL-1α specific mAb can be administered to a subject with minimal adverse effects, the mAb composition should be pure (excluding any excipients) at a weight percentage of at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, 99.9 or higher. The mAb composition may contain only a single type of mAb (i.e., produced from a single clone of B lymphocyte lineage), or a mixture of two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) different types of mAb.

[0028] The above IL-1α-specific Abs are preferred for use, but in some cases, other agents that specifically target IL-1α may be used as long as their administration leads to a reduction in one or more sequelae of cerebral ischemia-reperfusion injury in a subject when used in the methods described herein. Since some IL-1α-specific Abs have been shown to block the action of IL-1α by preventing its interaction with the IL-1 receptor (IL-1R1), based on this mechanism of action in the treatment of various pathological conditions, other Abs or non-Ab agents that similarly block the interaction of IL-1α with IL-1R1 can also be used (e.g., other anti-IL-1α Abs or anti-IL-1R1 Abs that block the interaction of IL-1α with IL-1R1). These Abs can be made according to the methods described above. Non-Ab agents include vaccines that cause the production of anti-IL-1α Abs that block the interaction of IL-1α with IL-1R1, proteins or peptides that bind to IL-1α and block the interaction of IL-1α with IL-1R1, and small organic molecules that specifically target IL-1α and block the interaction of IL-1α with IL-1R1. Those that do not specifically bind to IL-1β are preferred. Whether a particular agent can reduce one or more sequelae of cerebral ischemia-reperfusion injury in a subject can be determined by the methods described in the following Examples section.

[0029] Pharmaceutical Compositions and Methods Anti-IL-1α Ab compositions (and other agents that specifically target IL-1α) may be administered to animals or humans in a pharmaceutically acceptable carrier (e.g., sterile saline), which is selected based on the mode and route of administration and standard pharmaceutical practice. In addition to pharmaceutically acceptable carriers, a list of pharmaceutical formulations can be found in the standard text in this field, Remington’s Pharmaceutical Sciences, and the USP / NF. Other substances can be added to the compositions and other steps can be taken to stabilize and / or preserve the compositions and / or to facilitate their administration to a subject.

[0030] For example, the Ab compositions may be lyophilized (see Draber et al., J. Immunol. Methods. 181:37, 1995; and PCT / US90 / 01383); may be dissolved in a solution containing sodium and chloride ions; may be dissolved in a solution containing one or more stabilizers, such as albumin, glucose, maltose, sucrose, sorbitol, polyethylene glycol, and glycine; may be filtered (e.g., using 0.45 and / or 0.2 micron filters); may be contacted with beta-propiolactone; and / or may be dissolved in a solution containing a bactericide (e.g., a surfactant, an organic solvent, and a mixture of a surfactant and an organic solvent).

[0031] The Ab compositions may be administered to animals or humans by any suitable technique. Typically, such administration is parenteral (e.g., intravenous, subcutaneous, intramuscular, intrathecal, or intraperitoneal introduction). The compositions may also be administered directly to a target site (e.g., the brain or a lesion site) by application using a catheter, for example, using X-ray guidance. Other methods of delivery, such as liposome delivery or diffusion from a device impregnated with the composition, are known in the art. The compositions may be administered in a single bolus, in multiple injections, or by continuous infusion (e.g., intravenously or by peritoneal dialysis).

[0032] A therapeutically effective amount is an amount that can produce a medically desirable result in the treated animal or human. The effective amount of the anti-IL-1α Ab composition is an amount that shows clinical effectiveness in a patient as measured by reduction of one or more sequelae of cerebral ischemia reperfusion injury. As is well known in the medical field, the dosage for any one animal or human depends on many factors, including the size of the subject, body surface area, age, the specific composition administered, sex, time and route of administration, general health status, as well as other drugs being administered concurrently. Preferred dosages are in the range of about 3 to 100 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, or 100) mg / kg body weight. In some cases, a single dose may be effective. In other cases, the dosage may be repeated, for example, at 20 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, or 4 weeks after a preceding administration.

Examples

[0033] Example 1: Twelve-week-old male C57BL / 6 wild-type (WT) mice were subjected to transient middle cerebral artery occlusion (tMCAO) for 45 minutes. To induce ischemia / reperfusion (I / R) brain injury, transient middle cerebral artery occlusion (tMCAO) was performed as shown in Figure 1. Briefly, mice were anesthetized using 3% and 1.5% isoflurane for induction and maintenance, respectively. For analgesia, buprenorphine HCl was infiltrated into the incision flank (0.1 mg / Kg). Ischemia was induced by inserting a 6-0 silicone-coated filament into the common carotid artery up to the origin of the left MCA after dissection of the common, internal, and external carotid arteries. Next, mice were randomly given either mouse anti-mouse IL-1α antibody (i.e., Flo1-2a) or isotype control at different dosages (10 or 65 μg / g). IL-1α inhibition was performed after the ischemic event during reperfusion as would be the case if the patient presented to the emergency department and was eligible for thrombolytic therapy. More specifically, animals were randomized and either anti-IL-1α antibody or appropriate isotype control antibody was given via tail vein injection at the time of filament withdrawal (i.e., the start of the reperfusion period).

[0034] Forty-eight hours after tMCAO, the infarct volume was determined by 2,3,5-triphenyltetrazolium chloride (TTC) staining, and neurological deficits were determined by the rotarod test in addition to the 4-point scale neurological score (Bederson index; Bederson et al., Stroke. 1986; 17:472-476) in mice treated with control (isotype-matched) antibody exposed to tMCAO. Tissue IL-1α levels were elevated in the ipsilateral hemisphere, highlighting the pathophysiological relevance of this cytokine for infarction (Figure 1). Forty-eight hours after reperfusion, mice treated with the lower dose of anti-IL-1α antibody showed a minor reduction in infarct volume as evaluated by TTC staining, but neurological deficits after infarction were not improved (not shown). Treatment with the higher dose of anti-IL-1α antibody reduced the infarct size by 36% compared to the isotype control and improved the neurological outcome as determined by the Bederson and rotarod tests (Figures 2-4).

[0035] Post-ischemic blood-brain barrier (BBB) injury has an important impact on seizure outcome. Immunohistochemical analysis of IgG extravasation demonstrated a slight (not statistically significant) tendency towards increased BBB permeability in anti-IL-1α treated animals. Similarly, the rate of hemorrhagic changes evaluated macroscopically did not differ between groups. Similarly, the endothelial expression of occludin, claudin 5 and VE-cadherin, which are regulators of paracellular BBB permeability, did not differ between the treatment and control groups.

[0036] After seizures, local elevations of damage-associated molecular patterns and other inflammatory mediators mobilize circulating leukocytes to the site of injury and promote their effector functions. Leukocyte migration depends on a complex pattern of adhesion molecules expressed by both brain microvascular endothelial cells and leukocytes, including selectins, adhesion molecules of the immunoglobulin superfamily, and integrins. Confocal microscopy of the penumbra demonstrated a decrease in the endothelial expression of P-selectin, ICAM-1 and VCAM-1 in animals treated with an IL-1α inhibitory antibody compared to control littermates (Figs. 5-7). After ischemia, in addition to the activation of endogenous immune cells in the brain (i.e., microglia), infiltration of monocytes from the circulating pool contributes importantly to brain tissue damage. Neutralization of IL-1α after ischemia significantly reduced the number of activated macrophages in the seizure compartment compared to control mice as evaluated by Iba-1 immunostaining (Fig. 8). When activated, macrophages secrete several pro-inflammatory mediators such as TNF-α, ILs and MMPs, thereby exacerbating brain parenchymal damage. Among these, MMP9 can exert a direct neurotoxic effect. Consistent with the Iba-1 data, further immunohistochemical analysis of the penumbra revealed a reduction in MMP9 tissue levels in animals given an IL-1α neutralizing antibody compared to controls (Fig. 9).

[0037] Other embodiments The present invention has been described in combination with its detailed description, but the above description is intended to illustrate examples and is not intended to limit the scope of the present invention, which should be understood to be defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A method for treating cerebral ischemia-reperfusion injury, the method comprising administering to a subject an antibody that specifically binds to interleukin-1α (IL-1α).

2. The method according to claim 1, wherein the antibody that specifically binds to IL-1α is administered to the subject after the subject has developed cerebral ischemia.

3. A method for reducing the volume of a cerebral infarction resulting from an occlusive episode in a subject, the method comprising administering to the subject an antibody that specifically binds to IL-1α.

4. The method according to claim 3, wherein the antibody that specifically binds to IL-1α is administered to the subject after the subject has developed cerebral ischemia.

5. The method according to claim 3, wherein the volume of the cerebral infarction resulting from the occlusive episode in the subject is at least 20% less than the volume of the cerebral infarction that would have resulted from the occlusive episode if the subject had not been administered the antibody that specifically binds to IL-1α.

6. A method for reducing neurological deficits resulting from an occlusive episode in a subject, the method comprising administering to the subject an antibody that specifically binds to IL-1α.

7. The method according to claim 6, wherein the antibody that specifically binds to IL-1α is administered to the subject after the subject has developed cerebral ischemia.

8. A method for reducing the number of activated macrophages in the ischemic penumbra of brain injury resulting from an occlusive episode in a subject, the method comprising administering to the subject an antibody that specifically binds to IL-1α.

9. The method according to claim 8, wherein the antibody that specifically binds to IL-1α is administered to the subject after the subject has developed cerebral ischemia.

10. Use of an antibody that specifically binds to IL-1α for treating cerebral ischemia-reperfusion injury.

11. Use of an antibody that specifically binds to IL-1α for reducing the volume of a cerebral infarction resulting from an occlusive episode in a subject.

12. The use according to claim 11, wherein the volume of the cerebral infarction resulting from the occlusive episode in the subject is at least 20% less than the volume of the cerebral infarction that would have resulted from the occlusive episode if the subject had not been administered the antibody that specifically binds to IL-1α.

13. Use of an antibody that specifically binds to IL-1α for reducing neurological deficits resulting from an occlusive episode in a subject. **Claim 14** Use of an antibody that specifically binds to IL-1α for reducing the number of activated macrophages in the ischemic penumbra of brain injury resulting from an occlusive episode in a subject. **Claim 15** The use according to claim 11, 12, 13, or 14, wherein the antibody that specifically binds to IL-1α is administered to the subject after the subject has developed cerebral ischemia.

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