COMPOSITIONS AND METHODS FOR TREATMENT DURING NON-ACUTE PERIODS FOLLOWING CNS NEUROLOGICAL INJURY

Abstract

This invention relates to treatment of neuroinjury in a post-acute window or in a chronic period following neuroinjury.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation of U.S. Ser. No. 15/357,586, filed 21 Nov. 2016, which is a continuation of U.S. Ser. No. 13/059,206, filed 29 Apr. 2011, now U.S. Pat. No. 9,498,515, issued 22 Nov. 2016, which is a national phase application under 35 U.S.C. .sctn. 371 of International Patent Application PCT Application No. PCT/US2009/004692, filed 17 Aug. 2009, which claims the benefit of U.S. Provisional Application No. 61/189,191, filed 15 Aug. 2008, the entire contents of each of which are incorporated herein by reference in their entireties.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

[0002] The contents of the text file named "43509-515N01US_v3_ST25.txt", which was created on Apr. 30, 2015, and is 41 KB in size, are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

[0003] The field of the invention relates to treatment of central nervous system neuroinjury. More specifically, the invention is directed to the administration of a neuregulin [e.g., glial growth factor 2 (GGF2)] to a subject during the non-acute or chronic period following traumatic neuroinjury.

BACKGROUND OF THE INVENTION

[0004] Central nervous system (CNS) injuries to are a serious health problem. This category of injury includes events such as ischemic injury, hemorrhagic injury, penetrating trauma and non-penetrating trauma. CNS injuries generally heal incompletely leaving the subject with some degree of permanent dysfunction ranging from extremely mild to death. The residual dysfunction may include motor, sensory, cognitive, emotional and autonomic abnormalities.

[0005] A key category of CNS neuroinjury comprises brain injury. Brain injury is a devastating condition that results in some degree of permanent disability including motor, sensory and cognitive deficits and emotional instability such as post traumatic stress disorder, attention deficit disorder, depression and emotional lability. Common causes of brain injury include ischemic stroke, hemorrhagic stroke, subdural hematoma, epidural hematoma, closed head injury (acceleration/deceleration, concussion and rotational), penetrating brain injury (gunshot wounds and other projectiles).

[0006] Stroke is the third-leading cause of death and the main cause of disability in the western world. Stroke, therefore, presents a large socioeconomic burden. The etiology of a stroke can be either ischemic, which is the case in the majority of strokes, or hemorrhagic. An ischemic stroke can be caused by a clot that forms elsewhere in the body and travels via the bloodstream to the brain (embolic stroke) or by a blood clot that forms inside the artery of the brain (thrombotic stroke). After massive cell death in the immediate infarct core due to lack of glucose and oxygen, the infarct area expands for days, owing to secondary mechanisms such as glutamate excitotoxicity, apoptotic mechanisms, and generation of free radicals. Following neuroinjuries (e.g. an ischemic event) animals and people may recover function over several days, weeks and months without any therapeutic. All too often, this recovery however is only partial and animals and people suffer from permanent disability which may include motor, sensory and cognitive deficits.

[0007] Risk factors that increase the likelihood of an individual having a stroke are well known. These include, and are not limited to, risk factors that cannot be changed: advanced age, heredity, ace, gender, prior history of stroke or heart attack; and risk factors that can be changed, treated or controlled: high blood pressure, cigarette smoking, diabetes mellitus, carotid or other artery disease, atrial fibrillation, other heart disease, sickle cell disease, high blood cholesterol, poor diet, and physical inactivity and obesity.

[0008] To date, the non-palliative treatment of ischemic stroke is confined to therapeutics administered in the acute phase following a stroke. The acute phase ranges from the time of onset of the neuroinjury (e.g., stroke) to approximately six hours post-neuroinjury. The acute phase is followed by the semi-acute phase, which ranges from approximately six hours to two days post-neuroinjury. Accordingly, current non-palliative therapeutics are used in an attempt to reverse the occlusion of blood flow, restore oxygenation of the brain and limit the extent of lost brain structure. Other than tPA for acute use, there are no drugs are approved for the treatment of stroke. Patients remain with some level of dysfunction that at best may improve somewhat endogenously for approximately 60 days. This recovery may only be augmented by physical therapy. Unfortunately, many patients are left with permanent disability with little hope for improvement.

[0009] At present, the only drug approved by the Food and Drug Administration (FDA) for the treatment of ischemic stroke is tissue plasminogen activator (tPA). tPA is a serine protease that converts plasminogen to plasmin. Plasmin then breaks fibrin which is a component of the clots that occlude the vessels in the brain and cause strokes. It is ideally administered within three hours from the onset of symptoms. Generally, only 3% to 5% of individuals who suffer a stroke reach the hospital in time to be considered for this treatment. Ideally, tPA is administered within the first three hours post-occlusion, but may be administered by some clinicians as late as six hours post-occlusion. Unfortunately, the vast majority of patients who experience a stroke fail to reach the hospital in time to be considered for this treatment. For those patients who arrive at the hospital within the efficacious temporal window, tPA is administered in an attempt to reverse the occlusion of blood flow, restore oxygenation of the brain and limit the extent of lost brain structure. However, there are some significant contraindications that limit the ongoing use of tPA. After an initial period of about 3 to 6 hours, at most, tPA can cause intracerbral bleeding and hemorrhagic stroke. For such reasons, tPA is limited to administration during the acute phase in order to achieve any therapeutic efficacy.

[0010] To date no other therapy has been approved for the treatment of stroke. Other experimental therapies such as arterially delivered pro-urokinase are under investigation as potential means for disrupting clots and restoring blood flow. The scientific literature has, however, described many agents that have proven beneficial for protecting brain matter and restoring function in experimental animal models of stroke. All these agents focus on reducing acute cell death, inflammation, and apoptosis and must, therefore, be delivered within hours (some up to 24 hours) after the ischemic event. Heretofore, it is generally accepted that treatment for CNS injury such as stroke is required acutely. (Abe et al., 2008, J Cereb Blood Flow Metab. July 23, Epub ahead of print, Sun et al., 2008, Stroke July 10, Epub ahead of print, pages not available yet); Dohare et al., 2008, Behav Brain Res. 193(2):289-97; Belayev et al., 2001, Stroke 32(2):553-60).

[0011] Such agents have not, however, been shown to limit damage to the brain, restore function or enhance recovery following stroke when administered after a lag time of several hours, at most in some experimental animal models about one day following stroke. The only therapy known to show efficacy days and weeks after a stroke is palliative or rehabilitative, such as occupational or physical therapy. Indeed, the present inventors are not aware of any agents or drugs that have been shown to enhance recovery days or weeks following stroke.

[0012] After an acute occlusion, there is often a localized area of destroyed brain matter that is surrounded by a penumbral zone that will die within hours if circulation is not restored. The time to death of this penumbral zone can be extended by a few hours in experimental models with neuroprotectants, such as NMDA antagonists, calcium channel blockers, radical scavengers and trapping agents, anti-apoptotics, caspase inhibitors, parp inhibitors, etc. For this purpose a "neuroprotectant" is something that can save neurons before they die from the variety of insults presented to them in the acute phase. After 24 to 48 hours, however, there is little hope for protecting cells from necrotic death and, while apoptotic death continues for several days (See FIG. 1 the therapeutic window for anti-apoptotic therapies has not proven to be much wider than acute protective therapies [Schulz et al., 1998, Cell Death Differ. 5(10):847-57; Komjati et al., 2004, Int J Mol Med. 13(3):373-82].

[0013] Neuregulin exhibits neuroprotective properties which, like other agents described above, have shown benefit in reducing the disability seen if delivered to animals within hours after stroke. See United States Application Serial Number 09/530,884, the entire contents of which are incorporated herein by reference.

[0014] In view of the prevalence of neuroinjury, particularly with regard to stroke, there is a need for therapeutic agents that can be administered efficaciously to subjects to limit damage to the brain, restore function and/or enhance recovery following neuroinjury.

[0015] Neuregulins (NRGs) and NRG receptors comprise a growth factor-receptor tyrosine kinase system for cell-cell signaling that is involved in organogenesis in nerve, muscle, epithelia, and other tissues (Lemke, Mol. Cell. Neurosci. 7:247-262, 1996 and Burden et al., Neuron 18:847-855, 1997). The NRG family consists of four genes that encode numerous ligands containing epidermal growth factor (EGF)-like, immunoglobulin (Ig), and other recognizable domains. Numerous secreted and membrane-attached isoforms function as ligands in this signaling system. The receptors for NRG ligands are all members of the EGF receptor (EGFR) family, and include EGFR (or ErbB1), ErbB2, ErbB3, and ErbB4, also known as HER1 through HER4, respectively, in humans (Meyer et al., Development 124:3575-3586, 1997; Orr-Urtreger et al., Proc. Natl. Acad. Sci. USA 90: 1867-71, 1993; Marchionni et al., Nature 362:312-8, 1993; Chen et al., J. Comp. Neurol. 349:389-400, 1994; Corfas et al., Neuron 14:103115, 1995; Meyer et al., Proc. Natl. Acad. Sci. USA 91:1064-1068, 1994; and Pinkas-Kramarski et al., Oncogene 15:2803-2815, 1997).

[0016] The four NRG genes, NRG-1, NRG-2, NRG-3, and NRG-4, map to distinct chromosomal loci (Pinkas-Kramarski et al., Proc. Natl. Acad. Sci. USA 91:9387-91, 1994; Carraway et al., Nature 387:512-516, 1997; Chang et al., Nature 387:509-511, 1997; and Zhang et al., Proc. Natl. Acad. Sci. USA 94:9562-9567, 1997), and collectively encode a diverse array of NRG proteins. The gene products of NRG-1, for example, comprise a group of approximately 15 distinct structurally-related isoforms (Lemke, Mol. Cell. Neurosci. 7:247-262, 1996 and Peles and Yarden, BioEssays 15:815-824, 1993). The first-identified isoforms of NRG-1 included Neu Differentiation Factor (NDF; Peles et al., Cell 69, 205-216, 1992 and Wen et al., Cell 69, 559572, 1992), heregulin (HRG; Holmes et al., Science 256:1205-1210, 1992), Acetylcholine Receptor Inducing Activity (ARIA; Falls et al., Cell 72:801-815, 1993), and the glial growth factors GGF1, GGF2, and GGF3 (Marchionni et al. Nature 362:312-8, 1993).

[0017] The NRG-2 gene was identified by homology cloning (Chang et al., Nature 387:509-512, 1997; Carraway et al., Nature 387:512-516, 1997; and Higashiyama et al., J. Biochem. 122:675-680, 1997) and through genomic approaches (Busfield et al., Mol. Cell. Biol. 17:4007-4014, 1997). NRG-2 cDNAs are also known as Neural- and Thymus-Derived Activator of ErbB Kinases (NTAK; Genbank Accession No. AB005060), Divergent of Neuregulin (Don-1), and Cerebellum-Derived Growth Factor (CDGF; PCT application WO 97/09425). Experimental evidence shows that cells expressing ErbB4 or the ErbB2/ErbB4 combination are likely to show a particularly robust response to NRG-2 (Pinkas-Kramarski et al., Mol. Cell. Biol. 18:6090-6101, 1998). The NRG-3 gene product (Zhang et al., supra) is also known to bind and activate ErbB4 receptors (Hijazi et al., Int. J. Oncol. 13:1061-1067, 1998).

[0018] An EGF-like domain is present at the core of all forms of NRGs, and is required for binding and activating ErbB receptors. Deduced amino acid sequences of the EGF-like domains encoded in the three genes are approximately 30-40% identical (pairwise comparisons). Further, there appear to be at least two sub-forms of EGF-like domains in NRG-1 and NRG-2, which may confer different bioactivities and tissue-specific potencies.

[0019] Cellular responses to NRGs are mediated through the NRG receptor tyrosine kinases EGFR, ErbB2, ErbB3, and ErbB4 of the epidermal growth factor receptor family. High-affinity binding of all NRGs is mediated principally via either ErbB3 or ErbB4. Binding of NRG ligands leads to dimerization with other ErbB subunits and transactivation by phosphorylation on specific tyrosine residues. In certain experimental settings, nearly all combinations of ErbB receptors appear to be capable of forming dimers in response to the binding of NRG-1 isoforms. However, it appears that ErbB2 is a preferred dimerization partner that may play an important role in stabilizing the ligand-receptor complex. ErbB2 does not bind ligand on its own, but must be heterologously paired with one of the other receptor subtypes. ErbB3 does not possess tyrosine kinase activity, but is a target for phosphorylation by the other receptors. Expression of NRG-1, ErbB2, and ErbB4 is known to be necessary for trabeculation of the ventricular myocardium during mouse development.

SUMMARY OF THE INVENTION

[0020] The present invention presents a novel method for treating following neuroinjury in a mammal. The method is based on the observation that therapeutic benefits of a polypeptide that contains an epidermal growth factor-like (EGF-like) domain can be achieved by administering a therapeutically effective amount of the polypeptide to a mammal; in certain embodiments the treatment is performed at or after hour 1, hour 2, hour 8, hour 12, hour 24, hour 30, hour 36 hour 42, day 2 or later following neuroinjury. In one embodiment of the invention treatment is initiated after the acute window post-injury. In one embodiment of the invention treatment is initiated after the semi-acute window post-injury. In one embodiment of the invention treatment is initiated during and yet continues after the acute window post-injury. In one embodiment of the invention treatment is initiated during and yet continues after the semi-acute window post-injury.

[0021] Accordingly, the present invention comprises administration of a polypeptide (or nucleic acid encoding same) that contains an EGF-like domain to the mammal starting at day 1, 2 or 3 even up to and including days 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days; a week or more than one week, two weeks or more than two weeks post-neuroinjury; three weeks or more than three weeks post-neuroinjury; four weeks or more than four weeks post-neuroinjury; one month or more than one month post-neural injury; two months or more than two months post-neural injury; three months or more than three months post-neural injury; four months or more than four months post-neural injury; five months or more than five months post-neural injury; six months or more than six months post-neural injury. In accordance with the present invention the EGF-like domain is encoded by a neuregulin gene. Administration in accordance with the invention comprises administration of a peptide comprising an EGF-like domain, or a nucleic acid molecule encoding same, in an amount effective to treat the chronic phase following neuroinjury in the mammal.

[0022] In accordance with another aspect of the invention, a method for promoting neurorecovery during a period outside the acute or semi-acute phase following an ischemic event in a mammal is presented. Treatment in accordance with the invention can begin within an acute or semi-acute period but includes at least one, two three, four, five, six or more than six treatments beyond the acute or semi-acute period, respectively.

[0023] A method of the invention comprises administering a polypeptide comprising an epidermal growth factor-like (EGF-like) domain to said mammal, wherein said EGF-like domain is encoded by the neuregulin (NRG)-1 gene, and said administering is performed at least two or three or four days after the ischemic event, although treatment can begin within the acute or semi acute time frame, and in a therapeutically effective amount sufficient to promote neurorecovery during the chronic phase following an ischemic event in said mammal.

[0024] In particular embodiments of the invention, the neuregulin gene may be the NRG-1 gene, the NRG-2 gene, the NRG-3 gene or the NRG-4 gene. A neuregulin polypeptide of the invention may, in turn, be encoded by any one of these four neuregulin genes; a neuregulin polypeptide of the invention may, in turn, be encoded by a variant or homologue of any one of these four neuregulin genes. See FIGS. 6A-6D for the amino and nucleic acid sequences of full length human GGF2 an isoform of NRG-1.

[0025] In an aspect of the invention, suitable mammals include, but are not limited to, mice, rats, rabbits, dogs, monkeys or pigs. In a more particular embodiment of the invention, the mammal is a human.

[0026] Definitions:

[0027] As used herein, the term `about" comprises the specified value plus or minus 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15% of the specified value. In one embodiment "about signifies 98-102% of the specified value. In one embodiment "about" signifies 95-105% of the specified value.

[0028] Post-injury, the "acute phase" ranges from the time of onset of the neuroinjury (e.g., stroke) to approximately six hours post-neuroinjury. The acute phase is followed by the "semi-acute phase", which ranges from approximately six hours to two days post-neuroinjury. In one embodiment the "semi-acute phase" ranges from approximately six hours to three days post-neuroinjury. The period following the semi-acute phase is referred to as the "chronic phase" post-neuroinjury. The post-acute phase includes both the semi-acute and chronic post-injury periods.

[0029] By "epidermal growth factor-like domain" or "EGF-like domain" is meant a polypeptide motif encoded by the NRG-1, NRG-2, or NRG-3 gene that binds to and activates ErbB2, ErbB3, ErbB4, or combinations thereof, and bears a structural similarity to the EGF receptor-binding domain as disclosed in Holmes et al., Science 256:1205-1210, 1992; U.S. Pat. Nos. 5,530,109; 5,716,930; 7,037,888; Hijazi et al., Int. J. Oncol. 13:1061-1067, 1998; Chang et al., Nature 387:509-512, 1997; Carraway et al., Nature 387:512516, 1997; Higashiyama et al., J Biochem. 122:675-680, 1997; and WO 97/09425). See FIGS. 7-12 for nucleic and amino acid sequences corresponding to EGFL domains 1-6 encoded by the NRG-1 gene.

[0030] By "expression vector" is meant a genetically engineered plasmid or virus, derived from, for example, a bacteriophage, adenovirus, retrovirus, poxvirus, herpesvirus, or artificial chromosome, that is used to transfer a polypeptide (e.g., a neuregulin) coding sequence, operably linked to a promoter, into a host cell, such that the encoded peptide or polypeptide is expressed within the host cell.

[0031] The terms "neuroinjury" and "injury" are often used interchangeably herein. "neurotrauma" is one embodiment of a "neuroinjury" and may generally be considered a synonym. A "neuroinjury" is an injury that causes some destruction or death of neurological tissue. A neuroinjury generally has as sequelae some loss, e.g., a diminution of mental, sensory or muscle function.

[0032] A "neuroprotectant" is something that can save neurons before they die from the variety of insults presented to them in the acute or semi-acute post-injury phase. After an acute occlusion, there is often a localized area of destroyed brain matter that is surrounded by a penumbral zone that will die within hours if circulation is not restored. The time to death of this penumbral zone can be extended by a few hours in experimental models with "neuroprotectants", such as NMDA antagonists, calcium channel blockers, radical scavengers and trapping agents, anti-apoptotics, caspase inhibitors, parp inhibitors, etc. For this purpose a "neuroprotectant" is something that can save neurons before they die from the variety of insults presented to them in the acute phase.

[0033] By "neuregulin" or "NRG" is meant a polypeptide that is encoded by an NRG-1, NRG-2, NRG-3 or NRG-4 gene or nucleic acid (e.g., a cDNA), and binds to and activates EGFR, ErbBI, ErbB2, ErbB3, or ErbB4 receptors, or combinations thereof.

[0034] By "neuregulin-1," "NRG-1," "heregulin," "GGF2," or "p185erbB2 ligand" is meant a polypeptide that binds to the ErbB2 receptor and is encoded by the p185erbB2 ligand gene described in U.S. Pat. Nos. 5,530,109; 5,716,930; and 7,037,888, each of which is incorporated herein by reference in its entirety. Binding to the erbB2 receptor may be indirect through heterologous pairing of the erbB2 receptor to erbB 1, erbB3 or erbB4.

[0035] By "neuregulin-like polypeptide" is meant a polypeptide that possesses an EGF-like domain encoded by a neuregulin gene, and binds to and activates EGFR, ErbBl, ErbB-2, ErbB-3, ErbB-4, or a combination thereof. Binding to the erbB2 receptor may be indirect through heterologous pairing of the erbB2 receptor to erbB 1, erbB3 or erbB4.

[0036] As used herein, the term "neurorecovery" is used to refer to the process by which the nervous system restores its functioning towards a normal state following injury, disease, infection or other disruption of the nervous system, the brain, spinal cord or peripheral nerves.

[0037] By "operably linked" is meant that a nucleic acid encoding a polypeptide (e.g., a cDNA) and one or more regulatory sequences are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequences.

[0038] As used herein "peptide" comprises, consists essentially of, or consists of a peptide of about: 625, 600, 575, 550, 525, 500, 475, 450, 425, 400, 375, 350, 325, 300, 275, 250, 225, 200, 175, 150, 125, 100, 75, 50 or fewer than 50 amino acids.

[0039] By "promoter" is meant a minimal sequence sufficient to direct transcription. Also included in the invention are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell type or physiological status (e.g., hypoxic versus normoxic conditions), or inducible by external signals or agents; such elements may be located in the 5' or 3' or internal regions of the native gene.

[0040] The term "therapeutically effective amount" is intended to mean that amount of a drug or pharmaceutical agent that elicits the biological or medical response of a tissue, a system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician. A therapeutic change is a change in a measured biochemical characteristic in a direction expected to alleviate the disease or condition being addressed. More particularly, a "therapeutically effective amount" is an amount sufficient to decrease the symptoms associated with a medical condition or infirmity, to normalize body functions in disease or disorders that result in impairment of specific bodily functions, or to provide improvement in one or more of the clinically measured parameters of a disease.

[0041] As used herein, the term "treating means obtaining recovery following a neuroinjury where there would otherwise be none; accelerating the rate of natural recovery following a neuroinjury; or promoting recovery to a higher functioning level. Without being bound by theory, in the chronic post-injury window it is not contemplated that there will be an ability to lessen any further neurological death, i.e., it is understood in that the nerves have died by the time the chronic window occurs. However, treatment in accordance with the invention comprises during the chronic period, e.g., lessening a decrease function or viability of tissue (e.g., muscle or bone) that would otherwise occur in the tissue served by the comprised nerve. In addition, treatment in accordance with the invention comprises during the chronic period, e.g., lessening of atrophy (e.g., of muscle or bone) that would otherwise occur in the tissue served by the comprised nerve.

[0042] By "transformed cell" is meant a cell (or a descendent of a cell) into which a DNA molecule encoding a neuregulin or polypeptide having a neuregulin EGF-like domain has been introduced, by means of recombinant DNA techniques or known gene therapy techniques.

[0043] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

BRIEF DESCRIPTION OF THE DRAWINGS

[0044] FIG. 1 shows a schematic of the progressive phases following stroke/ischemia. In the FIG. "OT" indicates occupational therapy and "PT" indicates physical therapy.

[0045] FIG. 2 depicts forelimb behavioral scores following permanent ligation of the middle cerebral artery. Rats were treated with GGF2, NRG1, FGF, or vehicle as indicated. FGF and GGF2 at 100 .mu.g/kg demonstrated significant improvements at the Day 21 behavioral test. (11, +, V, * indicate significantly different from vehicle for bFGF, NRG I, GGF2 at 6.5 uglkg and GGF2 at 100 ug/kg by ANOVA and post-Tukey).

[0046] FIG. 3 depicts hindlimb behavioral scores following permanent ligation of the middle cerebral artery. Rats were treated with GGF2, NRG1, FGF, or vehicle as indicated. GGF2 at 6.5 .mu.g/kg and NRG at 1.0 .mu.g/kg were significantly better than vehicle at the Day 7 and 14 behavioral testing, but not at the study endpoint on Day 21. GGF2 at 100 .mu.g/kg and FGF were significantly better than vehicle at all behavioral time points after treatment. (1, +, * indicate significantly different from vehicle for bFGF, NRG1, GGF2 at 6.5 ug/kg and GGF2 at 100 ug/kg by ANOVA and post-Tukey).

[0047] FIG. 4 depicts body swing behavioral scores following permanent ligation of the middle cerebral artery. Rats were treated with GGF2, NRG1, FGF, or vehicle as indicated. GGF2 at 100 .mu.g/kg and FGF were significantly improved compared to vehicle at Day 21. (1f, +, , * indicate significantly different from vehicle for bFGF, NRG1, GGF2 at 6.5 ug/kg and GGF2 at 100 ug/kg by repeated measures ANOVA and post-hoc Tukey).

[0048] FIG. 5A shows forelimb behavioral scores following permanent ligation of the middle cerebral artery. Rats were treated with GGF2 starting 1, 3 or 7 days after the ligation. GGF2 was delivered at 0.1 mg/kg, IV daily for 10 days. Forelimb behavioral scores were significantly better than vehicle with all treatment paradigms at the day 21 time point. (*, V, + indicate significantly different from vehicle for the day 1, 3 and 7 treatment groups, respectively by repeated measures ANOVA and post-hoc Tukey).

[0049] FIG. 5B shows hindlimb behavioral scores following permanent ligation of the middle cerebral artery. Rats were treated with GGF2 starting 1, 3 or 7 days after the ligation. GGF2 was delivered at 0.1 mg/kg, IV daily for 10 days. Hindlimb behavioral scores were significantly better than vehicle when treatment was initiated at 1 or 7 days after ligation, and were improved compared to vehicle with treatment initiated 3 days post ligation at the day 21 time point. (*, , + indicate significantly different from vehicle for the day 1, 3 and 7 treatment groups, respectively by repeated measures ANOVA and post-hoc Tukey).

[0050] FIG. 5C shows body swing behavioral scores following permanent ligation of the middle cerebral artery. Rats were treated with GGF2 starting 1, 3 or 7 days after the ligation. GGF2 was delivered at 0.1 mg/kg, IV daily for 10 days. Body swing scores were significantly better than vehicle when treatment was initiated 1 day after ligation, and were improved compared to vehicle with treatment initiated 3 or 7 days post ligation. (*, +indicate significantly different from vehicle for the day 1, 3 and 7 treatment groups, respectively by repeated measures ANOVA and post-hoc Tukey).

[0051] FIGS. 6A-D show the nucleic and amino acid sequences of full length GGF2. The nucleic acid sequence is designated SEQ ID NO: 6 and the amino acid sequence is designated SEQ ID NO: 7. GGF2 variant sequences are identified as follows: GGF-II 09 (SEQ ID NO: 8), GGF-II 08 (SEQ ID NO: 9), GGF-II 04 (SEQ ID NO: 10), GGF-II 01 and 11 (SEQ ID NO: 11), GGF-II 10 (SEQ ID NO: 12), GGF-II 03 (SEQ ID NO: 13), GGF-II 02 (SEQ ID NO: 14), GGF-II 06 (SEQ ID NO: 15), and GGF-II 12 (SEQ ID NO: 16).

[0052] FIG. 7 shows the nucleic and amino acid sequences of epidermal growth factor like (EGFL) domainl. The nucleic acid sequence is designated SEQ ID NO: 17 and the amino acid sequence is designated SEQ ID NO: 18.

[0053] FIG. 8 shows the nucleic and amino acid sequences of epidermal growth factor like (EGFL) domain 2. The nucleic acid sequence is designated SEQ ID NO: 19 and the amino acid sequence is designated SEQ ID NO: 20.

[0054] FIG. 9 shows the nucleic and amino acid sequences of epidermal growth factor like (EGFL) domain 3. The nucleic acid sequence is designated SEQ ID NO: 21 and the amino acid sequence is designated SEQ ID NO: 22.

[0055] FIG. 10 shows the nucleic and amino acid sequences of epidermal growth factor like

[0056] (EGFL) domain 4. The nucleic acid sequence is designated SEQ ID NO: 23 and the amino acid sequence is designated SEQ ID NO: 24.

[0057] FIG. 11 shows the nucleic and amino acid sequences of epidermal growth factor like (EGFL) domain 5. The nucleic acid sequence is designated SEQ ID NO: 25 and the amino acid sequence is designated SEQ ID NO: 26.

[0058] FIG. 12 shows the nucleic and amino acid sequences of epidermal growth factor like (EGFL) domain 6. The nucleic acid sequence is designated SEQ ID NO: 27 and the amino acid sequence is designated SEQ ID NO: 28.

[0059] FIG. 13 shows the nucleic and amino acid sequences of an epidermal growth factor like peptide from the NRG-I gene. The nucleic acid sequence is designated SEQ ID NO: 29 and the amino acid sequence is designated SEQ ID NO: 30.

[0060] FIG. 14 shows the amino acid sequence of an epidermal growth factor like (EGFL) beta fragment from NRG-1 (SEQ ID NO: 31).

[0061] FIG. 15 shows the amino acid sequence of an epidermal growth factor like (EGFL) alpha fragment from NRG-1 (SEQ ID NO: 32).

[0062] FIG. 16 shows the amino acid sequence of an epidermal growth factor like (EGFL) alpha fragment from NRG-2 alpha (SEQ ID NO: 33).

[0063] FIG. 17 shows the amino acid sequence of an epidermal growth factor like (EGFL) alpha fragment from NRG-2 beta (SEQ ID NO: 34).

[0064] FIG. 18 shows amino acid sequence alignments of various EGF-like peptides. EGF-like domain can be defined as sub-domains of NRGs for which sequence alignment reveals at least 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45% homology in amino acid sequence as compared to the human EGF molecule (sequence P011331971-1023, at the bottom of the alignment in the figure). Homologous amino acids have identical, conserved or semi-conserved physicochemical and structural properties, as denoted by the symbols `*`, `:` and `.`, respectively. EGF-like domain sequences are identified as follows: Beta2 region (SEQ ID NO: 35), Zensun (SEQ ID NO: 36), 156/416 (SEQ ID NO: 37), RnD (SEQ ID NO: 38), 151 (SEQ ID NO: 39), EGFO-1d_NRGbeta3 (SEQ ID NO: 40), 157/417seq (SEQ ID NO: 41), EGF-1d_GGF (SEQ ID NO: 42), RnD_catno296HR (SEQ ID NO: 43), 155/415_seqid_200_seqid155 (SEQ ID NO: 44), EGF-1d_NDF43 (SEQ ID NO: 45), 152 (SEQ ID NO: 46), 159/419 (SEQ ID NO: 47), 158/418 (SEQ ID NO: 48), EGF-1d_NRGlgamma (SEQ ID NO: 49), EGF-1d_GGF2 (SEQ ID NO: 50), EGF-1d_SMDF (SEQ ID NO: 51), 154/414 (SEQ ID NO: 52), P01133/971-1023 (SEQ ID NO: 53).

[0065] FIG. 19A shows the expression vector (pSV-AHSG) that CHO (dhfr-) cells were transfected with.

[0066] FIG. 19B shows the pCMGGF2 vector containing the coding sequence for human GGF2 that the dhfr.sup.-.alpha.2HSGP cells were transfected with using the cationic lipid DMRIE-C reagent (Life Technologies #10459-014).

[0067] FIG. 19C shows the GGF2 coding sequence placed after the EBV BMLF-1 intervening sequence (MIS).

DETAILED DESCRIPTION OF THE INVENTION

[0068] As indicated herein, the non-palliative treatment of ischemic stroke has heretofore been confined to therapeutics administered in the acute phase following a stroke.

[0069] Immediate cell death due, at least in part, to oxygen deprivation is observed during the acute phase. Furthermore, as shown in FIG. 1, the occlusion of blood flow results in release of intracellular stores of free radicals, glutamate, and calcium and sodium that are understood to destroy brain tissue and expand the area of the lesion.

[0070] The semi-acute phase, from approximately six hours to two days (or three days by some definitions) post-neuroinjury, is characterized by continued free radical release, glutamate dumping, calcium and sodium release, oxygen deprivation of the occluded region, and immediate localized cell death. To date, there are no known clinically approved agents for use in humans during the semi-acute phase following a stroke.

[0071] As depicted in FIG. 1, the limited window for pharmaceutical post-neuroinjury therapy is at least partially explained by the pathophysiology and temporal progression of the injury. Within minutes, e.g., of an occlusion, neurons at the core of the infarct are destroyed. In the hours after the occlusion, free radicals, excitotoxic and inflammatory agents are released/produced and these molecules continue to destroy brain tissue and expand the area of the lesion. The extent of the injury can be limited, as described above, by restoring blood flow (using a clinically approved clot-buster, i.e., tPA) and thus achieving re-oxygenation of the affected area.

[0072] As indicated by the scientific literature, various compounds appear to exhibit efficacy in the acute and semi-acute periods. However, after the 24, 36 or 48 hour marks post-CNS neuroinjury, potential therapies progressively lose the ability to treat the injury. In fact, some of the therapeutics modalities, such as tPA, that have an effect in the acute period begin to have serious, life-threatening contraindications as time occurs post-injury.

[0073] Days, weeks or months after an ischemic event, during the chronic phase post-stroke, therapies must be aimed at promoting neurorecovery. The promotion of neurorecovery following a traumatic event such as stroke in the central nervous system involves distinctly different physiologic phenomena and therapeutic strategy than employed in the pre-chronic window. The pre-chronic treatments generally involve agents that aim to restore blood flow and reduce acute cell death. In contrast, a therapeutic agent that can be efficaciously administered at 48 hours or more, or 72 hours or more post-injury is differentiated from acute phase therapeutics by its ability to restore function without altering the size of the ischemic lesion. In one embodiment, treatment in accordance with the invention begins after essentially complete post-injury death of an ischemic CNS lesion; by "essentially complete post-injury death of an ischemic CNS lesion is intended that the CNS cell death that is directly consequent to the ischemic event will have transpired, other cell death due to age or therapy (whether or not the therapy is designed to address the ischemia) is not within the scope of this definition.

[0074] As shown herein, the present inventors demonstrated that neuregulins are effective in restoring neurological function during the chronic phase of a neurotraumatic injury. In one embodiment, the neurotraumatic injury is an ischemic stroke. As described herein, the present inventors have made the surprising discovery that neuregulin is effective when dosing is initiated during the chronic phase following an ischemic event. Even more surprising is the discovery that neuregulin is effective even when dosing is initiated as late as 7 days after the ischemic event.

[0075] The present data shows that the favorable outcomes achieved have not come about by the same mechanisms found to be effective in immediate post-ischemic treatment. Neuregulin treatment during chronic phase following stroke does not alter the size of the ischemic lesion (see Table 1). This clearly demonstrates that the acute and semi-acute phases of the pathophysiology are complete at these phases, and neuregulin administered during the chronic phase is promoting neurorecovery, rather than establishing reperfusion and protecting neurons.

TABLE-US-00001 TABLE 1 Infarct Volume (%) bFGF 21.3 .+-. 3.3 NRG 1.0 .mu.g/kg 26.8 .+-. 3.0 GGF2 6.5 .mu.g/kg 27.1 .+-. 3.7 GGF2 100 .mu.g/kg 26.3 .+-. 3.5 Vehicle 25.0 .+-. 3.5

Compositions of the Invention:

[0076] As indicated above, neuregulins are polypeptides encoded by the NRG-1, NRG-2, NRG-3 or NRG-4 genes and possess EGF-like domains that allow them to bind to and activate ErbB receptors. Holmes et al. (Science 256:1205-1210, 1992) have shown that the EGF-like domain alone is sufficient to bind and activate the p 1 85erbB2 receptor. Accordingly, any polypeptide product encoded by the NRG-1, NRG-2, NRG-3 or NRG-4 gene, or any neuregulin-like polypeptide, e.g., a polypeptide having an EGF-like domain encoded by a neuregulin gene or cDNA (e.g., an EGF-like domain containing the NRG-1 peptide subdomains C-CID or C-C/D', as described in U.S. Pat. Nos. 5,530,109, 5,716,930, and 7,037,888; or an EGF-like domain as disclosed in WO 97/09425) may be used in the methods of the invention. A composition of the invention may be in unit dosage form. Kits comprising compositions of the invention and/or instructions in accordance with the invention are within the scope of the present invention as well.

[0077] Compositions of the invention may be administered to patients with a pharmaceutically-acceptable diluent, carrier, or excipient. Conventional pharmaceutical practice is employed to provide formulations or compositions to administer such compositions to patients or experimental animals. Although intravenous administration is preferred, any appropriate route of administration may be employed, for example, parenteral, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracistemal, intraperitoneal, intranasal, aerosol, oral, or transdermal (e.g., by applying an adhesive patch carrying a formulation capable of crossing the dermis and entering the bloodstream) or topical administration.

[0078] Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols. Methods well known in the art for making formulations are found in, for example, "Remington's Pharmaceutical Sciences." Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Other potentially useful parenteral delivery systems for administering molecules of the invention include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.

[0079] As a further aspect of the invention there is provided the present compounds for use as a pharmaceutical especially in the treatment or prevention of the aforementioned conditions and diseases. Also provided herein is the use of the present compounds in the manufacture of a medicament for the treatment or prevention of one of the aforementioned conditions and diseases.

[0080] With respect to intravenous injections, dose levels are generally in a range from one value in the following list to a higher value in the list: about 0.001 mg/kg, about 0.01 mg/kg, about 0.1 mg/kg, about 1 mg/kg, and about 10 mg/kg. The dosing periodicity is generally in regular time intervals of from about every 24, 48, 72, or 96 hours. In an alternative embodiment, the dosing periodicity is generally in regular time intervals of from about every 1, 2, 3, 4, 5, 6, 7, days. In an alternative embodiment, the dosing periodicity is generally in regular time intervals of from about every 1, 2, 3, 4, or 5 weeks. After a period of such dosing, less frequent dosing such as monthly, every three months, every four months, or yearly can be employed.

[0081] Transdermal doses are selected to provide essentially identical, similar or lower physiologic levels (plasma, tissue, CSF) than are achieved using injection doses.

[0082] The compounds of the invention can be administered as the sole active agent or they can be administered in combination with other agents, including other compounds that may be found to demonstrate the same or a similar therapeutic activity and that are determined to be safe and efficacious for such combined administration. Other such compounds contemplated for use in the treatment of acute or semi-acute neuroinjury include hematopoietic factors (e.g., G-CSF and/or GM-CSF); substances that have thrombolytic activities, e.g., tPA, streptokinase, urokinase, and/or Ancrod; antiplatelet agents such as acetylsalicylic acid (aspirin), clopidogrel (Plavix), aspirin combined with extended release dipyridamole (Aggrenox); anticoagulants such as warfarin (Coumadin) or Heparin; and/or substances that interfere with apoptotic signaling (e.g. inhibitors of caspases) or progesterone. It is understood, however, that the above compounds are administered for preventive purposes in advance of stroke (e.g., for antiplatelet agents or anticoagulants), or during the acute phase following stroke (tPA).

[0083] In order to evaluate the motor, sensory or cognitive deficits that are effectively treated in accordance with the present invention, several tools are available in the art. Well known indicia for monitoring treatment efficacy include Mini-Mental State Examination (MMSE), Modified Mini-Mental State Examination (3MS), Functional Impairment Measure (FIM, Barthel Test, Fugl-Meyer Motor Score, Wolf Motor Function Test, Jebsen-Taylor hand function test, Nottingham Health Profile Part 1 and Motor Assessment Scale (MAS), Sodring Motor Evaluation Scale (SMES), the Berg Balance Scale (BBS) and Barthel Activities of Daily Living (ADL), and other clinical tests such as measures of affect, speech, swallowing, cognition, motor coordination, strength, sensation and autonomic function, as well as survival rates and hospitalization rates may be used to assess disease progression.

[0084] Following injury, disease, infection or other disruption of the nervous system, the brain, spinal cord or peripheral nerves do not function properly due to any combination of gross destruction, apoptosis, disrupted pathways and synapses, inflammation, altered chemical environment, changes in cell metabolism and changes in cellular transcription and translation. As used herein, the term "neurorecovery" is used to refer to the process by which the nervous system restores its functioning towards a normal state. This process may occur by correction, circumvention, reversal or elimination of any of the causes noted above.

[0085] Furthermore, it is now believed that significant neurorecovery can occur through a process known as `plasticity` whereby the nervous system forms new connections to compensate or adapt to other changes. Injuries of the CNS cause disruption of local and long-range connections resulting in disrupted function and disability. Plasticity is an event where new connections are formed between existing neurons. Plasticity has been shown to be a mechanism of neurorecovery in CNS systems including the visual system (Pizzorusso et al.,) and the spinal cord (Fawcett JW (2009) Brain 132:1417-1418.). In plasticity, new synapses are formed or existing synapses are strengthened, weakened or removed to allow existing structures and systems to compensate for those that have been destroyed or disrupted in the injury. Other forms of plasticity may include altering the neurochemistry of existing cells to changes the direct synaptic signaling and paracrine signaling. Plasticity may also take the form of altering receptor levels to make neurons and other cells more or less sensitive to direct synaptic or paracrine signaling. These processes are well accepted as mechanism of learning and memory.

[0086] The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention before the priority date of each claim of this application.

[0087] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the appended claims. The following Examples will assist those skilled in the art to better understand the invention and its principles and advantages. It is intended that these Examples be illustrative of the invention and not limit the scope thereof.

EXAMPLES

Example 1

Materials and Methods

[0088] Animal Preparation:

[0089] Fifty (50), adult, male Sprague-Dawley were used for the study (10 extra animals were ordered). All rats were housed and handled for behavioral assessment for seven (7) days prior to surgery for acclimation purposes. At the end of the handling period, rats were randomized and assigned to different groups. Rats were given a unique identification number by tail marking. Ten extra rats were also handled.

[0090] Surgical Preparation:

[0091] Middle Cerebral Artery Occlusion (MCAO), Tamura Model:

[0092] This surgical injury model in the rat is a well accepted model of stroke in the field (Tamura et al, 1981, J Cereb Blood Flow Metab. 1(1):53-60; Tamura et al, 1981, J Cereb Blood Flow Metab. 1(1):61-9). Focal cerebral infarcts were made by permanent occlusion of the proximal right middle cerebral artery (MCA) using a modification of the method of Tamura et al. Male Sprague-Dawley rats (300-400 g at the time of surgery) were anesthetized with 2-3% halothane in the mixture of N.sub.2O:O.sub.2 (2:1), and were maintained with 1-1.5% halothane in the mixture of N.sub.2O:O.sub.2 (2:1). The temporalis muscle was bisected and reflected through an incision made midway between the eye and the eardrum canal. The proximal MCA was exposed through a subtemporal craniectomy without removing the zygomatic arch and without transecting the facial nerve. The artery was then occluded by microbipolar coagulation from just proximal to the olfactory tract to the inferior cerebral vein, and was transected. Body temperature was maintained at 37.5.degree. C..+-.0.5.degree. C. throughout the entire procedure. Cefazolin (40 mg/kg; Baxter, Lot 06014.1, Exp. January 2009) was given intraperitoneally(i.p.) one day before MCAO and just after MCAO to prevent infections. Burprenorphine (NDC 12496-0757-1, Lot #700Y02, exp: Jan. 1, 2010) s.c. (0.05-0.1 mg/kg) was given before the MCAO surgery as analgesia.

[0093] Compound Preparation and Dosing:

[0094] GGF2 and NRG-1 (NRG-EGF):

[0095] Stock solutions were prepared at Acorda Therapeutics and stored at 0-5.degree. C. Doses were made up as described below:

[0096] Cloning, Expression and Purification of NRG-1 [NRG1b2 EGF Domain (156Q)]

[0097] DNA: NRG1b2 egf domain was cloned from human brain cDNA and cloned into pet 15b vector (Novagen cat #69661-3) using Ndel and BamH1 restriction sites. The resulting protein is a 6.92 kda+.about.3kDa His tag (=9.35 kDa).

[0098] DNA sequence of NRG1b2 egf pet 15 clone

[0099] The underlined sequences are the cloning sites (Nde1 and BamH1)

TABLE-US-00002 (SEQ ID NO: 1) CATATGAGCCA TCTTGTAAAA TGTGCGGAGA AGGAGAAAAC TTTCTGTGTG AATGGAGGGG AGTGCTTCAT GGTGAAAGAC CTTTCAAACC CCTCGAGATA CTTGTGCAAG TGCCCAAATG AGTTTACTGG TGATCGCTGC CAAAACTACG TAATGGCCAG CTTCTACAAG GCGGAGGAGC TGTACCAGTA AGGATCC.

[0100] The final translated protein from pet15b vector is shown below. The egf domain is underlined.

TABLE-US-00003 (SEQ ID NO: 2) 10 20 30 MGSSHHHHHH SSGLVPRGSH MSHLVKCAEK 40 50 EKTFCVNGGE CFMVKDLSNP SRYLCKCPNE 60 70 80 FTGDRCQNYV MASFYKAEEL YQ

Theoretical pI/Mw: 7.69/9349.58

[0101] Protein Expression

[0102] The clone was transformed into B121 cells for protein expression using the Overnight Express Autoinduction System (Novagen) in LB media at 25.degree. C. for 24 hours. Expression is primarily in insoluble inclusion bodies.

[0103] Protein Refolding: Adapted from Novagen Protein Refolding Kit, 70123-3.

[0104] Protein Purification: Protein is loaded onto an anion exchange column DEAE at 2.5 ml/min. The NRG-1 fragment remains in the flow through, whereas the contaminants bind and elute at a higher salt. The loading and washing buffer is 50 mM Tris pH7.9 and elution buffer is 50 mM Tris pH7.9 with 1M NaCl. The flow through is pooled and concentrated with Centriprep YM-3 from Millipore.

[0105] Western blotting: Protein expression is assessed by western blotting. Resulting band runs at around 10 kD.

[0106] A 4-20% criterion gel (Biorad) was used for protein resolution followed by transfer onto Protran nitrocellulose paper (0.1 um pore size from Schliecher and Schull). The blot is blocked in 5% milk in TBS-T (0.1%). Primary antibody (Anti EGF Human NRG1-alpha/HRG1-alpha Affinity Purified Polyclonal Ab Cat #AF-296-NA from R&D systems) 1:1000 dilution in 5% milk in TBS-T-1 hour at RT (also works at 4.degree. C. overnight). Rabbit anti goat HRP secondary antibody was used at 1:10,000 dilution in 5% milk in TBS-T for 1 hour at RT. All washes were performed in TBS-T.

[0107] Purification Protocol for NRG-1

[0108] The cultures are grown at 25.degree. C. in Overnight Express Autoinduction System 1 from Novagen (cat #71300-4). There is very little soluble NRG-1 present. The culture is spun down and the pellets are extracted, solubilized and re-folded to acquire the NRG-1 before purification can take place.

[0109] Materials for Extraction, Solubilization and Re-Folding:

[0110] 10.times. Wash Buffer: 200 mM Tris-HCl, pH 7.5, 100 mM EDTA, 10% Triton X-100

[0111] 10.times. Solubilization Buffer: 500 mM CAPS, pH 11.0

[0112] 50.times. Dialysis Buffer: 1M Tris-HCl, pH 8.5

[0113] 30% N-laurylsarcosine--add as powder (Sigma 61739-5G)

[0114] 1M DTT

[0115] Reduced glutathione (Novagen 3541)

[0116] Oxidized glutathione (Novagen 3542)

[0117] A. Cell Lysis and Preparation of Inclusion Bodies

[0118] Thaw and re-suspend cell pellet in 30 mls 1.times. wash buffer. Mix as needed for full re-suspension.

[0119] dd protease inhibitors (25 ul of 10.times. per 50 mls), DNase (200 ul of 1 mg/ml per 50 ml) and MgC12 (500 ul of 1 M per 50 mls) to suspension,

[0120] Lyse the cells by sonication.

[0121] a. Cool the cells on ice throughout this step.

[0122] b. Using the square tip, sonicate for 30 seconds on level 6, 10 times until suspension become less viscous. Let suspension cool on ice for 60 seconds between each sonication. Keep volume no higher than 40 mls in 50 ml conical tube when sonicating.

[0123] When complete, transfer each suspension to 250 ml angled neck centrifuge bottles for use with F-16/250 rotor.

[0124] Collect the inclusion bodies by centrifugation at 10000.times.g for 12 minutes.

[0125] Remove the supernatant (save a sample for analysis of soluble protein) and thoroughly re-suspend the pellet in 30 mls of 1.times. Wash Buffer.

[0126] Repeat centrifugation as in Step 4 and save the pellet.

[0127] Again, thoroughly re-suspend the pellet in 30 mls of 1.times. Wash Buffer.

[0128] Collect the inclusion bodies by centrifugation at 10000.times.g for 10 minutes. Decant the supernatant and remove the last traces of liquid by tapping the inverted tube on a paper towel.

[0129] B. Solubilization and Refolding

[0130] From the wet weight of inclusion bodies to be processed, calculate the amount of 1.times. Solubilization Buffer necessary to re-suspend the inclusion bodies at a concentration of 10-15 mg/ml. If the calculated volume is greater than 250 ml, use 250 ml.

[0131] At room temperature, prepare the calculated volume of 1.times. Solubilization Buffer supplemented with 0.3% N-laurylsarcosine (up to 2% may be used if needed in further optimization) (300 mg/100 mL buffer) and 1 mM DTT.

[0132] Add the calculated amount of 1.times. Solubilization Buffer from step 2 to the inclusion bodies and gently mix. Large debris can be broken up by repeated pipetting.

[0133] Incubate in refrigerator shaker at 25.degree. C., 50-100 rpm for 4-5 hours.

[0134] Clarify by centrifugation at 10000.times.g for 10 minutes at room temperature.

[0135] C. Dialysis Protocol for Protein Refolding

[0136] Prepare the required volume of buffer for dialysis of solubilized protein. The dialysis should be performed with at least 2 buffer changes of greater than 50 times the volume of the sample.

[0137] Dilute the 50.times. Dialysis Buffer to 1.times. at the desired volume and supplement with 0.1 mM DTT.

[0138] Dialyze for at least 4 hours at 4.degree. C. Change the buffer and continue. Dialyze for an additional 4 or more hours.

[0139] Prepare additional dialysis buffer as determined in step 1, but omit DTT.

[0140] Continue the dialysis through two additional changes (min. 4 hr each), with the dialysis buffer lacking DTT.

[0141] D. Redox Refolding Buffer to Promote Disulfide Bond Formation

[0142] Prepare a dialysis buffer containing 1 mM reduced glutathione (1.2 g/4 L) and 0.2 mM oxidized glutathione (0.48 g/4 L) in 1.times. Dialysis Buffer. The volume should be 25 times greater than the volume of the solubilized protein sample. Chill to 4.degree. C.

[0143] Dialyze the refolded protein from step 1 overnight at 4.degree. C.

[0144] Purification

[0145] All procedures are done at 4.degree. C.

[0146] Chemicals:

[0147] Trizma Hydrochloride (Sigma T5941-500G)

[0148] Sodium Chloride 5M Solution (Sigma 56546-4L)

[0149] Sodium Hydroxide 10N (JT Baker 5674-02)

[0150] E. Purification on the DEAE HiPrep 16/10 Anion Column-20 mls (GE Healthcare)

[0151] Buffer A: 50 mM Tris-HCL pH8.0

[0152] Buffer B: 50 mM Tris-HCL with 1M NaCI pH 8.0

[0153] Equilibration of column: Buffer A-5 CV, Buffer B-5 CV, Buffer A-10 CV

[0154] Load 50 ml of sample per run on 20 ml column at 2.0 mUmin (NRG-1) is in the flow through).

[0155] Wash 20 ml column with 5 CV of buffer A

[0156] 20 ml column with gradient to 100% B with 5 CV. This is to elute off contaminants.

[0157] Clean with 10 CV of 100% Buffer B.

[0158] Equilibrate with 15 CV of Buffer A

[0159] Analyze fractions with a SDS-page silver stain

[0160] Pool fractions with NRG-1 (10 kDa)

[0161] F. Concentration of NRG-1

[0162] Concentrate with Millipore Centriprep 3000 MWCO 15 ml concentrator (Ultracel YM-3, 4320)

[0163] Use Modified Lowry Protein Assay to determine concentration.

[0164] G. His-Tag Removal

[0165] Removal of the His-Tag is done with A Thrombin Cleavage Capture Kit from Novagen (Cat #69022-3). Based on previous testing the best conditions are room temp for 4 hours with Thrombin at 0.005 U of enzyme per .mu.l for every 10 .mu.g of NRG-1 protein. After four hours of incubation, add 160 of Streptavidin Agarose slurry per unit of Thrombin enzyme. Rock sample for 30 min at room temp. Recover the NRG-1 through spin-filtration or sterile filtering (depending on volume). Complete cleavage is determined with an EGF and Anti-His western.

[0166] H. Storage in Final Buffer

[0167] Stored in 1.times. PBS with 0.2% BSA at 4.degree. C.

[0168] Expression and Purification of GGF2

[0169] For the cloning and background information for GGF2, see U.S. Pat. No. 5,530,109. The cell line is described in U.S. Pat. No. 6,051,401. The entire content of each of U.S. Pat. Nos. 5,530,109 and 6,051,401 is incorporated herein by reference in its entirety.

[0170] CHO-(Alpha2HSG)-GGF cell line: This cell line was designed to produce sufficient quantities of fetuin (human alpha2HSG) to support high production rates of rhGGF2 in serum free conditions.

[0171] Cho (dhfr-) cells were transfected with the expression vector (pSV-AHSG) shown in FIG. 19A. Stable cells were grown under ampicillin pressure. The cell line was designated (dhfr.sup.-/a2HSGP). The dhfr.sup.-/a2HSGP cells were then transfected with the pCMGGF2 vector shown in FIG. 19B containing the coding sequence for human GGF2 using the cationic lipid DMRIE-C reagent (Life Technologies #I0459-014) (See FIGS. 19A-19B).

[0172] Stable and high producing cell lines were derived under standard protocols using methotrexate (100 nM, 200 nM, 400 nM, 1 .mu.M) at 4-6 weeks intervals. The cells were gradually weaned from serum containing media. Clones were isolated by standard limiting dilution methodologies. Details of the media requirements are found in the above mentioned reports.

[0173] To enhance transcription, the GGF2 coding sequence was placed after the EBV BMLF-1 intervening sequence (MIS). See FIG. 19C.

TABLE-US-00004 MIS Sequence (SEQ ID NO: 3) CGAT[AACTAGCAGCATTTCCTCCAACGAGGATCCCGCAG (GTAAGAAGCTACACCGGCCAGTGGCCGGGGCC CGATAACTAGCAGCATTTCCTCCAACGAGGATCCCGCAG(GTAAGAAGCTACACC GGCCAGTGGCCGGGGCC GTGGAGCCGGGGGCATCCGCTGCCTGAGACAGAGGTGCTCAAGGCAGTCTCCAC CTITTGTCTCCCCTCTGCAG)AGAGCCACATTCTGGAA]GTT GGF2 coding sequence (SEQ ID NO: 4) atgagatgg cqacgcgccc cgcgccgctc cgggcgtccc 301 ggcccccggg cccagcgccc cggctccgcc gcccgctcgt cgccgccgct gccgctgctg 361 ccactactgc tgctgctggg gaccgcggcc ctggcgccgg gggcggcggc cggcaacgag 421 gcggctcccg cgggggcctc ggtgtgctac tcgtccccgc ccagcgtggg atcggtgcag 481 gagctagctc agcgcgccgc ggtggtgatc gagggaaagg tgcacccgca gcggcggcag 541 cagggggcac tcgacaggaa ggcggcggcg gcggcgggcg aggcaggggc gtggggcggc 601 gatcgcgagc cgccagccgc gggcccacgg gcgctggggc cgcccgccga ggagccgctg 661 ctcgccgcca acgggaccgt gccctcttgg cccaccgccc cggtgcccag cgccggcgag 721 cccggggagg aggcgcccta tctggtgaag gtgcaccagg tgtgggcggt gaaagccggg 781 ggcttgaaga aggactcgct gctcaccgtg cgcctgggga cctggggcca ccccgccttc 841 ccctcctgcg ggaggctcaa ggaggacagc aggtacatct tcttcatgga gcccgacgcc 901 aacagcacca gccgcgcgcc ggccgccttc cgagcctctt tcccccctct ggagacgggc 961 cggaacctca agaaggaggt cagccgggtg ctgtgcaagc ggtgcgcctt gcctccccaa 1021 ttgaaagaga tgaaaagcca ggaatcggct gcaggttcca aactagtcct tcggtgtgaa 1081 accagttctg aatactcctc tctcagattc aagtggttca agaatgggaa tgaattgaat 1141 cgaaaaaaca aaccacaaaa tatcaagata caaaaaaagc cagggaagtc agaacttcgc 1201 attaacaaag catcactggc tgattctgga gagtatatgt gcaaagtgat cagcaaatta 1261 ggaaatgaca gtgcctctgc caatatcacc atcgtggaat caaacgctac atctacatcc 1321 accactggga caagccatct tgtaaaatgt gcggagaagg agaaaacttt ctgtgtgaat 1381 ggaggggagt gcttcatggt gaaagacctt tcaaacccct cgagatactt gtgcaagtgc 1441 ccaaatgagt ttactggtga tcgctgccaa aactacgtaa tggccagctt ctacagtacg 1501 tccactccct ttctgtctct gcctgaatag. GGF2 Protein Sequence (SEQ ID NO: 5) MRWRRAPRRSGRPGPRAQRPGSAARSSPPLPLLPLLLLLGTAALPGAAAGNEAA PAGASVCYSSPPSVGSVQELAQRAAVVIEGKVHPQRRQQGALDRKAAAAAGEAGAW GGDREPPAAGPRALGPPAEEPLLAANGTVPSWPTAPVPSAGEPGEEAPYLVKVHQVWA VKAGGLKKDSLLTVRLGTWGHPAFPSCGRLKEDSRYIFFMEPDANSTSRAPAAFRASFP PLETGRNLKKEVSRVLCKRCALPPQLKEMKSQESAAGSKLVLRCETSSEYSSLRFKWE KNGNELNRKNKPQNIKIQKKPGKSELRINKASLADSGEYMCKVISKLGNDSASANITIVE SNATSTSTTGTSHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQN YVMASFYSTSTPFLSLPE.

[0174] GGF2 production: One vial of GGF2 at 2.2.times.10.sup.6 cells/mL was thawed into 100 mls of Acorda Medium 1 (see Table 2) and expanded until reaching sufficient numbers to seed production vessels. Cells were inoculated into the production media Acorda Medium 2 (see Table 3) at 1.0.times.10.sup.5 cells/mL in two liter vented roller bottles. Roller bottles are maintained at 37.degree. C. for 5 days and then reduced to 27.degree. C. for 26 days. The roller bottles are monitored for cell count and general appearance but they are not fed. Once viability is below 10% the cells are spun out and conditioned media harvested and sterile filtered.

TABLE-US-00005 TABLE 2 Medium 1 Item Vendor Catalog Number Final concentration CD-CHO Invitrogen 10743-029 -remove 50 ml, then add components below FeSO.sub.4.cndot.EDTA Sigma F-0518 1x (10 ml/L) L-Glutamine Cellgro 25-005-CI 4 mM (20 ml/L) Recombinant Sigma 1-9278 290 U/L (1 ml/L) Human Insulin Non-essential Cellgro 25-025-CI 1x (10 ml/L) amino acid Peptone Type 4 Sigma P0521 Powder - Made 20X Soybean-HySoy in CD-CHO (50 mI/L) Gentamicin Invitrogen 15750-078 100 .mu.g (2 ml/L)

TABLE-US-00006 TABLE 3 Medium 2 Item Vendor Catalog Number Final concentration CD-CHO Invitrogen 10743-029 50% (-50 ml first) HyQ SFX-CHO HyClone SH30187.02 50% (-50 ml first) FeSO.sub.4.cndot.EDTA Sigma F-0518 1x (10 ml/L) L-Glutamine Cellgro 25-005-CI 4 mM (20 ml/L) Recombinant Sigma 1-9278 290 U/L (1 ml/L) Human Insulin Non-essential Cellgro 25-025-C1 1x (10 ml/L) amino acid Peptone Type 4 Sigma P0521 Powder - Made 20X in Soybean-HySoy CD-CHO (50 ml/L) Gentamicin Invitrogen 15750-078 100 pg (2 ml/L)

[0175] Purification Protocol for GGF2

[0176] All procedures are done at 4.degree. C.

[0177] Chemicals:

[0178] Sodium Acetate

[0179] Glacial Acetic Acid (for pH adjustment)

[0180] 10N NaOH (for pH adjustment)

[0181] NaCl

[0182] Sodium Sulfate

[0183] L-Arginine (JT Baker cat #: 2066-06)

[0184] Mannitol (JT Baker cat #: 2553-01)

[0185] Starting material: Conditioned media supernatant. Adjust pH to 6.5.

[0186] Step 1:

[0187] Capture-Cation Exchange Chromatography

[0188] HiPrep SP 16/10 (Amersham Biosciences)

[0189] Column equilibration: Buffer A-SCV, buffer B-SCV, buffer 15%B-5 CV

[0190] Buffer A: 20 mM NaAcetate, pH 6.0

[0191] Buffer B: 20 mM NaAcetate, pH 6.0, 1M NaCl

[0192] Load sample at 2ml/min with a continuous load overnight if possible. Binding is better with continuous loading.

[0193] Maximum capacity for a starting sample: 5 mg GGF2/ml media

[0194] Flow rate: 3 ml/min

[0195] First wash: 15%B, 10 CV

[0196] Second wash: 35% B, 10 CV

[0197] GGF2 elution: 60%B, 8 CV

[0198] Column wash: 100%B, 8 CV

TABLE-US-00007

[0198] Buffers: Composition Conductivity Use 15% B 20 mM NaAcetate, pH 6.0, Preequilibration 150 mM NaCl First wash 35% B 20 mM NaAcetate, pH 6.0, Second wash 350 mM NaCl 60% B 20 mM NaAcetate, pH 6.0, GGF2 elution 600 mM NaCl 100% B 20 mM NaAcetate, pH 6.0, 88 mS/cm Column wash 1000 mM NaCl

[0199] Step 2:

Refinement-Gel Filtration Chromatography



[0200] Sephacryl 5200 26/60

[0201] Elution buffer: 20 mM NaAcetate, 100 mM Sodium Sulfate, 1% mannitol, 10 mM L-Arginine, pH 6.5

[0202] Buffer conductivity:

[0203] Sample: SP GGF2 elution pool concentrated up to.about.AU280 1.0

[0204] Flow rate: 1.3 ml/min

[0205] Peak elution: at .about.0.36 CV from injection start

[0206] Step 3: DNA and endotoxin removal-filtration through Intercept Q membrane.

[0207] Preequilibration buffer: 20 mM NaAcetate, 100 mM Sodium Sulfate, 1% Mannitol,

[0208] 10 mM L-Arginine, pH 6.5

[0209] Collect flow through

[0210] Step 4: Final formulation and sample preparation

[0211] Add additional 90 mM L-Arginine to the sample

[0212] Concentrate

[0213] Sterile Filter

[0214] The vehicle/control article used herein is 0.2% Bovine Serum Albumin (BSA), 0.1 M Sodium Phosphate, pH 7.6 or 0.1 M sodium phosphate, pH 7.6 as indicated.

[0215] Basic FGF:

[0216] The bFGF (PeproTech Inc., 100-18B, Lot 1206CY08 G2407) was reconstituted as directed by PeproTech to a stock solution of 0.1 mg/ml and stored at -20.degree. C. before use. On the day of the injection, 100 .mu.g/ml of BSA (Roche Diagnostics, Lot 12403328 exp. Mar. 31, 2008) solution was made as diluent, to make a final bFGF concentration of 20 .mu.g/ml (1 .mu.g/50 ul). This material was only used in study 1.

[0217] Randomization and Blinding: Five animals were operated on per day. The investigator doing the surgery and behavioral assessments was blinded to treatment assignment of each animal (except the bFGF treated group) until all of the data were collected.

[0218] Behavioral Tests:

[0219] Sensorimotor functional activities were evaluated using forelimb and hindlimb placing, and body swing behavioral tests. These tests were performed one (1) day before surgery, one (1) day after surgery and at three (3), seven (7), fourteen (14) and twenty-one (21) days after MCAO.

[0220] 1. Limb Placing

[0221] Limb placing tests were divided into both forelimb and hindlimb tests. For the forelimb placing test, the examiner held the rat close to a tabletop and scored the rat's ability to place the forelimb on the tabletop in response to whisker, visual, tactile, or proprioceptive stimulation. Similarly, for the hindlimb placing test, the examiner assessed the rat's ability to place the hindlimb on the tabletop in response to tactile and proprioceptive stimulation. Separate sub-scores were obtained for each mode of sensory input (halfpoint designations possible), and added to give total scores (for the forelimb placing test: 0=normal, 12=maximally impaired; for the hindlimb placing test: 0=normal; 6=maximally impaired).

[0222] 2. Body Swing Test

[0223] The rat was held approximately one inch from the base of its tail. It was then elevated to an inch above a surface of a table. The rat was held in the vertical axis, defined as no more than 10.degree. to either the left or the right side. A swing was recorded whenever the rat moved its head out of the vertical axis to either side. The rat must return to the verticalposition for the next swing to be counted. Thirty (30) total swings were counted. A normal rat typically has an equal number of swings to either side. Following focal ischemia, the rat tends to swing to the contralateral (left) side.

[0224] On all the days of the behavioral tests, animals were tested before drug administration. Time points are designated with the day of surgery (Day 0) as a reference.

[0225] Sacrifice and Infarct Volume:

[0226] Following behavioral evaluations at twenty one (21) days after MCAO, rats were deeply anesthetized with Ketamine (50-100 mg/kg) and Xylazine (5-10 mg/kg) mixture, intraperitoneally. Animals were perfused transcardially with normal saline (with heparin, 2 unit/ml) followed by 10% formalin. Brains were then removed and stored in 10% formalin. Fixed brains were then embedded with paraffin, and 5 micron coronal sections were cut using a microtome. Sections were then stained with hematoxylin and eosin (H&E). Seven sections (+4.7, +2.7, +0.7, -1.3, -3.3, -5.3 and -7.3, compared to bregma respectively) from each brain were photographed by a digital camera, and the infarct area on each slice was determined by NIH Image (Image J) using the "indirect method" (area of the intact contralateral [left] hemisphere--area of intact regions of the ipsilateral [right] hemisphere) to correct for brain edema. Infarct areas were then summed among slices and multiplied by slice thickness to give total infarct volume, which was expressed as a percentage of intact contralateral hemispheric volume.

Example 2

Effect Of GGF2 (an NRG-1) In Enhancing Neurological Recovery In A Model Of Permanent Middle Cerebral Artery Occlusion (MCAO) In Rats--Treatment Initiated During The Acute Phase And Continuing Thereafter

[0227] Effects of GGF2 and NRG-1: Functional recovery following MCA occlusion (MCAO) in rats

[0228] Study 1 Experimental groups (n=10):

[0229] NRG-1, 1.0 .mu./kg, 1 ml/kg intravenous; 1 hr after, & once per day (q24) for 10 days after MCAO

[0230] GGF2, 6.5 .mu.g/kg, 1 ml/kg intravenous; 1 hr after, & q24 for 10 days after MCAO

[0231] GGF2, 100 .mu.g/kg, 1 ml/kg intravenous; 1 hr after, & q24 for 10 days after MCAO

[0232] bFGF, intracistemal, 1 g/50 .mu.l; Day 1 and Day 3 (positive control) after MCAO

[0233] Vehicle, 1 ml/kg intravenous; 1 hr after, & q24 for 10 days after MCAO (Vehicle=0.2% BSA/0.1 M sodium phosphate pH 7.6)

[0234] All data are expressed as mean.+-.S.E.M. Behavioral and body weight data were analyzed by repeated measures of ANOVA (treatment X time), unless otherwise specified. Positive F-values for overall ANOVAs including all groups enabled pairwise ANOVAs between groups. Infarct volume data were analyzed by one-way ANOVA.

[0235] Results:

[0236] Forelimb Placing Test:

[0237] Recovery in the GGF2, 100 .mu.g/kg group was superior to the vehicle group (p<0.001). Recovery in the bFGF group was superior to the vehicle group (p<0.05). There was no significant difference in recovery of the GGF2, 6.5 .mu.g/kg or NRG, 1.0 .mu.g/kg groups compared to the vehicle group. See FIG. 2.

[0238] Hindlimb Placing Test:

[0239] Recovery in the bFGF and GGF2, 100 .mu.g/kg group was significantly better than the vehicle group (p<0.001) on all behavioral testing days. Recovery in the GGF2, 6.5 .mu.g/kg group and NRG, 1.0 .mu.g/kg group was significantly improved compared to vehicle on behavioral testing Days 7 and 14 but this effect was not maintained to the 21 day end point. See FIG. 3.

Body Swing Test:

[0240] Recovery in the bFGF group and GGF2, 100 .mu.g/kg group was significantly improved compared to vehicle (p<0.05) at the behavioral testing Day 21 end point. There was no significant difference in recovery of the NRG, 1.0 .mu.g/kg group, or the GGF2, 6.5 .mu.g/kg group compared to the vehicle group. See FIG. 4.

Weight Changes:

[0241] There were no significant differences among the groups. Infarct Volume: There were no significant differences among the groups. See Table 1 above.

[0242] Summary:

[0243] These results demonstrate that GGF2 is acting in a dose responsive manner and promotes functional recovery in this model of permanent stroke. The hindlimb data using the lower dose of neuregulin shows that continued treatment can lead to continued improvements.

Example 3

Effect of Timing of Administration (Post-Injury Delay) of Dosing of GGF2 in Enhancing Neurological Recovery in a Permanent Middle Cerebral Artery Occlusion (MCAO) in Rats including Chronic Period Dosing

[0244] Effects of GGF2 on functional recovery following MCAO in rats Experimental groups (n =10):

[0245] GGF2, 0.1 mg/kg, (1 ml/kg) intravenous; 10 daily injections, starting at Day 1 after MCAO

[0246] GGF2, 0.1 mg/kg, (1 ml/kg) intravenous; 10 daily injections, starting at Day 3 after MCAO

[0247] GGF2, 0.1 mg/kg, (1 ml/kg) intravenous; 10 daily injections, starting at Day 7 after MCAO

[0248] Vehicle, 1 ml/kg intravenous; 10 daily injections, starting at Day 1 after MCAO

[0249] (Vehicle=0.1 M sodium phosphate pH 7.6)

[0250] Animals start to receive GGF2 or vehicle intravenously at Day 1, Day 3 or Day 7 for 10 days after MCAO. All solutions were made fresh every day.

[0251] Results:

[0252] Forelimb Placing:

[0253] Recovery in the Day 1 GGF2 treatment group significantly improved compared to the vehicle group at all time points of testing after treatment (on Day 3 (p<0.0001), on Day 7 (p<0.005), on Day 14 (p<0.0001) and on Day 21 (p<0.0001)). The Day 3 GGF2 treatment group demonstrated significant improvements compared to vehicle on day 7 (p<0.05) and day 21 (p<0.05) The Day 7 GGF2 treatment group demonstrated a clear deflection in the slope of recovery compared to vehicle from Day 7 (start of treatment) to Day 14, this difference became significant by Day 21 (p<0.05). These data indicated that treatment as late as three and even 7 days after injury can produce significant improvements in neurological function. More prolonged treatment may be beneficial and result in greater and more sustained effects. See FIG. 5A.

Hindlimb Placing:

[0254] Recovery in the Day 1 GGF2 treatment group was significantly improved compared to the vehicle group at all time points of testing after treatment (on Day 3 (p<0.05) and on Days 7, 14 and 21 (p<0.0001)). The Day 3 GGF2 treatment group demonstrated improvements that were significantly better than vehicle on testing Day 7 (p<0.001) and Day 14 (p<0.05, day after treatment ended), and were trending toward significance on Day 21 (p<0.065). The Day 7 GGF2 treatment group was significantly improved compared to vehicle at the testing Day 21 end point of the study (p<0.05). FIG. 5B.

[0255] Body swing: Recovery in the Day 1 GGF2 treatment group was significantly better than the vehicle group (p<0.001). There was a trend toward recovery in the Day 3 GGF2 group and Day 7 GGF2 group, compared to the vehicle group FIG. 5C.

[0256] There were no significant differences in body weights between the groups.

[0257] Infarct Volume: There were no significant differences among all groups as shown in Table 4. Table 4:

TABLE-US-00008 Group % Infarct volume Day 1 Vehicle 32.89 .+-. 2.63 GGF2 Day 1 27.62 .+-. 2.48 GGF2 Day 3 33.31 .+-. 3.84 GGF2 Day 7 27.30 .+-. 2.87

Summary:

[0258] By the Day 21 study endpoint it was found that treatment initiated on Day I, Day 3 or Day 7 post MCAO resulted in significant improvements in forelimb function compared to vehicle treated animals. Treatment initiated on Day 1, Day 3 or Day 7 post MCAO resulted in significant improvements in hindlimb function compared to vehicle treatment during specific behavioral testing points that correlated with treatment time, indicating that continued treatment may prove beneficial Functional recovery with interventions given at this later time points post injury eliminates the possibility that the effect is due to acute neuroprotection. Indeed, this data coupled with the lack of a significant change in infarct volume demonstrates that the improvements are due to the promotion of neurorecovery with GGF2 treatment. This shows that a long temporal window exists (post-acute and post-semi acute) during which time GGF2 may be administered as an effective therapeutic for the chronic phase of stroke

[0259] It is, noteworthy that the significant (under ANOVA statistics) improvements observed following GGF2 administration at 3 and 7 days post-MCAO represent a dramatic expansion in the therapeutic window offered by previously available strategies for treatment of acute phase stroke. For the first time in the art, the data presented herein demonstrated that GGF2 administration is efficacious even during the chronic phase of stroke. The data presented herein further suggest that GGF2 contributes to neurorecovery following neuroinjury.

Example 4

Treatment of Ischemic Stroke

[0260] A patient presents to a medical facility with signs and symptoms of an ischemic stroke. The patient is revascularized with tPA or other therapy to restore blood flow. Although blood flow has been restored, some level of brain injury has occurred. Three days after the injury the patient is assessed neurologically and shown to have measurable sensory and/or motor deficits. Beginning on day four, after day two and after day 3, this patient would is treated with neuregulin at a dose between 0.01 and 1.0 mg/kg per dose, intravenously for 10 days to 3 months. The patient successfully regains sensory and motor function without the concomitant use of Occupational or Physical Therapy. This recovery is better than would have been clinically predicted without neuregulin therapy. This recovery is better than would have been clinically predicted without use of Occupational or Physical Therapy.

Example 5

Treatment Of Stroke And Resulting Paralysis of the Right Hand

[0261] A patient presents to the Emergency Department with paralysis of the right hand. Following evaluation and imaging it is determined that the patient has suffered an ischemic stroke. The patient receives tPA according to approved methods, and blood flow is restored through the thrombosis. However, a week after tPA treatment, the patient has residual paralysis of the right hand as measured by standard neurological measures of hand motor activity. This patient is treated with neuregulin (0.01 to 1.0 mg/kg, IV) once per week for 4 weeks. Improvement in hand function is measured periodically by a neurologist or other physician with standard neurological testing including dynamometer and other strength testing. The patient successfully regains sensory and motor function in their right hand without the concomitant use of Occupational or Physical Therapy. This recovery is better than would have been clinically predicted without neuregulin therapy. This recovery is better than would have been clinically predicted without use of Occupational or Physical Therapy.

Example 6

Treatment of Ischemic Stroke

[0262] A patient presents to a medical facility with signs and symptoms of an ischemic stroke. They are found to have paralysis of their left side. The patient does not arrive in time for revascularization therapy with. Upon clinical evaluation it is found that some brain injury has occurred. Three days after the injury the patient is assessed neurologically and shown to have measurable sensory and motor deficits. This patient is treated with neuregulin at a dose between 0.01 and 1.0 mg/kg per dose, intravenously each day for four weeks; thereafter they receive weekly doses for six months. They also receive physical therapy. Improvement is noticed as early as the second week of treatment; recovery continues throughout the period of neuregulin therapy. The patient successfully regains sensory and motor function of their left side. This recovery is seen as excellent; and is much better than would have been clinically predicted without with the use of Physical Therapy alone.

Example 7

Treatment of Ischemic Stroke

[0263] A patient presents to the Emergency Department with paralysis of the left hand. The patient reports that the problem with their hand began "over a week ago". Following evaluation and imaging it is determined that the patient has suffered an ischemic stroke. The patient does not receive tPA. Upon neurological exam it is found that The patient has residual paralysis of the left hand as measured by standard neurological measures of hand motor activity; the patient has a sensory deficit as well. The patient refuses to participate in physical or occupational therapy. This patient treated with neuregulin (0.01 to 1.0 mg/kg, IV) once per week for 12 weeks. Improvement in hand function is measured periodically by a neurologist or other physician with the standard neurological testing including dynamometer and other strength testing. Improvement is noticed as early as the second week of treatment; recovery continues throughout the period of neuregulin therapy. The patient successfully regains sensory and motor function in their left hand without the concomitant use of Occupational or Physical Therapy. This recovery is better than would have been clinically predicted without neuregulin therapy. This recovery is better than would have been clinically predicted without use of Occupational or Physical Therapy.

Example 8

Traumatic Brain Injury

[0264] A patient presents to a medical facility following a traumatic event with signs and symptoms of a head injury and resultant brain injury. Some level of brain injury has occurred as assessed by imaging and neurological testing including Glasgow Coma Scale and more detailed neurocognitive testing. Five days after the injury, the patient is assessed neurologically and shown to have measurable sensory and motor deficits. This patient is treated with neuregulin at a dose between 0.01 and 1.0 mg/kg per dose, intravenously for 3 months. Initially the patient is not willing to participate in physical therapy. Improvement in brain function is noticed as early as the first week of treatment; recovery continues throughout the period of neuregulin therapy. This recovery is better than would have been clinically predicted without neuregulin therapy. Beginning at three months the patient begins to receive neuregulin once per week, and they also begin to receive physical therapy. The concomitant therapies continue for up until one year from the day of original injury. On the anniversary of the patient's injury their recover is extraordinary. Clinically is it far better than would have been anticipated in the absence of any therapy and is also better than would have been expected from the use of physical therapy.

Example 9

Treatment of Cerebral Hemorrhage

[0265] A patient presents to a medical facility with signs and symptoms consistent with an ischemic stroke or cerebral hemorrhage. The patient is stabilized. Upon neurological assessment it is found that some level of brain injury has occurred. One week after the injury the patient is again assessed neurologically and shown to have measurable sensory and/or motor deficits. This patient is treated with neuregulin at a dose between 0.01 and 1.0 mg/kg daily, intravenously for 10 days, followed by administration of this dose weekly for 2 months, at which point all treatment is discontinued. Improvement in brain function is noticed as early as the first week of treatment; recovery continues throughout the period of neuregulin therapy. Upon cessation of neuregulin therapy the patients recover is deemed clinically excellent. The patient returns for evaluation six months and then 12 months from the date of the initial injury; at each assessment the patient's recovery is deemed clinically excellent.

Example 10

NRG Treatment of Injury including Treatment in Semi-Acute and Chronic Periods

[0266] For a comprehensive trial, inclusion criteria include: adults, male and female, with clinical evidence of neuronjury.

[0267] Indications Explored:

[0268] Ischemic stroke with thrombolytics,

[0269] Ischemic stroke without thrombolytics,

[0270] Hemorrhagic stroke,

[0271] Closed head traumatic brain injury,

[0272] Penetrating traumatic brain injury

[0273] Dose Ranges Explored:

[0274] 0.001mg/kg to 10.0 mg/kg per dose

[0275] Dose Frequencies Explored:

[0276] daily

[0277] on alternate days

[0278] every fourth day

[0279] once per week

[0280] once every other week

[0281] once per month

[0282] Mixed Periodicity Regimens:

[0283] daily for one or two weeks and then weekly, biweekly, or monthly for the remainder of the study

[0284] on alternate days for one or two weeks and then weekly, biweekly or monthly thereafter

[0285] Initiation of Treatment Explored:

[0286] As soon as possible after injury

[0287] Within 6, 12, 24 and 48 hours after injury

[0288] After 72 hours following injury

[0289] 7, 14, 30, 60, 90, 120 days following injury

[0290] Treatment Duration Explored:

[0291] treatment for 1, 2, 4, 10, 30 weeks.

[0292] treatment for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months

[0293] Function Explored:

[0294] hand motor function, face motor function, speech

[0295] cognitive function survival

[0296] time to return to work

[0297] Recovery is measured by standard neurological measures.

[0298] Adverse Events are mild and well-tolerated.

[0299] Results: Patients treated with neuregulin show statistically greater improvement in mental, sensory or muscle function as measured by methodologies known in the art, as compared to those patients treated with placebo.

[0300] In alternative embodiments combinations less than all of the above parameters are explored.

Example 11

NRG Treatment of Ischemic Stroke with Unilateral Hand Weakness and/or Paralysis (Without Thrombolysis)

[0301] Inclusion criteria include: adults, male and female, evidence of stroke based on loss of consciousness, disorientation, speech difficulty, facial or limb paralysis. Ischemic stroke confirmed with radiographic imaging.

[0302] Patients are selected for those with unilateral hand weakness and/or paralysis that are not candidates for tPA (or other thrombolytic) or who did not previously receive tPA for any reason. Consents are obtained from the patients and/or someone with authority to sign for the patients.

[0303] Patients are enrolled and randomized to receive neuregulin or placebo starting as soon as they present to a medical facility including hospital or physician's office, diagnosis, imaging is obtained.

[0304] For this trial, treatment is initiated between 1 hour and 7 days after injury. Treatment is continued for 3 months with dosing on alternate days for 1 week and then weekly for the remainder of the treatment period. Patients are dosed with 0.0001 to 1.0 mg per kg, IV, IM or SC.

[0305] Recovery is measured by standard neurological measures of hand motor activity every other week for the duration of the study.

[0306] Adverse Events are mild and well-tolerated.

[0307] Results: Patients treated with neuregulin show statistically greater improvement in hand function as measured by methodologies known in the art, as compared to those patients treated with placebo.

Example 12

NRG Treatment of Unilateral Facial Paralysis Without Thrombolytics

[0308] Patients are selected for those with unilateral facial paralysis who did not or cannot receive thrombolytics. Function is assessed by methodologies known in the art, on alternate weeks during the 3 month dosing period. Consents are obtained from the patients and/or someone with authority to sign for the patients.

[0309] Patients are enrolled and randomized to receive neuregulin or placebo. Treatment is initiated between 1 hour and 7 days after injury. Treatment is continued for 3 months with dosing on alternate days for 1 week and then weekly for the remainder of the treatment period. Patients are dosed with 0.0001 to 1.0 mg per kg, IV, IM or SC.

[0310] Adverse Events are mild and well-tolerated.

[0311] Results: Patients treated with neuregulin show statistically greater improvement in facial movement than those patients treated with placebo.

Example 13

NRG Treatment of Aphasia or Dysarthria Without Thrombolytics

[0312] Patients are selected for those with aphasia or dysarthria who did not or cannot receive thrombolytics. Function is assessed by methodologies known in the art. on alternate weeks during the 3 month dosing period. Consents are obtained from the patients and/or someone with authority to sign for the patients.

[0313] Patients are enrolled and randomized to receive neuregulin or placebo. Treatment is initiated between 1 hour and 7 days after injury. Treatment is continued for 3 months with dosing on alternate days for 1 week and then weekly for the remainder of the treatment period. Patients are dosed with 0.0001 to 1.0 mg per kg, IV, IM or SC.

[0314] Adverse Events are mild and well-tolerated.

[0315] Results: Patients treated with neuregulin show statistically greater improvement in speech capability than those patients treated with placebo.

Example 14

NRG Treatment of Ischemic Stroke (with Thrombolysis)

[0316] Inclusion criteria include: adults, male and female, evidence of stroke based on loss of consciousness, disorientation, speech difficulty, facial or limb paralysis. Ischemic stroke is confirmed with radiographic imaging.

[0317] Patients are selected for those with unilateral hand weakness and/or paralysis that and are successfully treated with tPA or other thrombolytic. Consents are obtained from the patients and/or someone with authority to sign for the patients

[0318] Patients are enrolled and randomized to receive neuregulin or placebo starting as soon as they present to a medical facility, diagnosis, and imaging is completed.

[0319] Treatment is initiated between 1 hour and 7 days after injury. Treatment is continued for 3 months with dosing on alternate days for 1 week and then weekly for the remainder of the treatment period. Patients are dosed with 0.0001 to 1.0 mg per kg, IV, IM or SC.

[0320] Recovery is measured by standard neurological measures of hand motor activity every other week for the duration of the study.

[0321] Adverse Events are generally mild and well tolerated.

[0322] Results: Patients treated with neuregulin show statistically greater improvement in hand function as measured by methodologies known in the art as compared to those patients treated with placebo.

Example 15

NRG Treatment of Patients with Unilateral Facial Paralysis (With Thrombolytics)

[0323] Inclusion criteria include: adults, male and female, evidence of stroke based on loss of consciousness, disorientation, speech difficulty, facial or limb paralysis. Ischemic stroke is confirmed with radiographic imaging. Patients are selected for those with aphasia or dysarthria and did receive thrombolytics.

[0324] Treatment is initiated between 1 hour and 7 days after injury. Treatment is continued for 3 months with dosing on alternate days for 1 week and then weekly for the remainder of the treatment period. Patients are dosed with 0.0001 to 1.0 mg per kg, IV, IM or SC.

[0325] Function is assessed by methodologies known in the art, on alternate weeks during the 3 month dosing period.

[0326] Adverse Events are mild and well-tolerated.

[0327] Results: Patients treated with neuregulin show statistically greater improvement in speech capability than those patients treated with placebo.

Example 16

NRG Treatment of Patients with Traumatic Brain Injury

[0328] Inclusion criteria include: adults, male and female, evidence of traumatic brain injury with loss of consciousness, disorientation, speech difficulty, facial or limb paralysis with evidence or history of trauma. Patients with evidence of penetrating injury are excluded. Consents are obtained from the patients and/or someone with authority to sign for the patients.

[0329] Patients are enrolled and randomized to receive neuregulin or placebo starting as soon as they present to a medical facility including hospital or physician's office, diagnosis, imaging and consent is obtained.

[0330] For this trial treatment is initiated between 1 hour and 7 days after injury. Treatment is continued for 3 months with dosing on alternate days for 1 week and then weekly for the remainder of the treatment period. Patients are dosed with 0.0001 to 1.0 mg per kg, IV, IM or SC.

[0331] Functional recovery is assessed by methodologies known in the art.

[0332] Adverse Events are mild and well-tolerated.

[0333] Results: Patients treated with neuregulin show statistically greater improvement than those patients treated with placebo.

Example 17

NRG Treatment Of Patients With Penetrating Brain Injury

[0334] Inclusion criteria include: adults, male and female, evidence of traumatic brain injury with loss of consciousness, disorientation, speech difficulty, facial or limb paralysis with evidence or history of trauma. Consents are obtained from the patients and/or someone with authority to sign for the patients.

[0335] Patients are stabilized surgically or through other measures. Diagnosis and imaging are obtained.

[0336] Patients are enrolled and randomized to receive neuregulin or placebo starting as soon as they present to a medical facility including hospital or physician's office.

[0337] Treatment is initiated between 1 hour and 7 days after injury. Treatment is continued for 3 months with dosing on alternate days for 1 week and then weekly for the remainder of the treatment period. Patients are dosed with 0.0001 to 1.0 mg per kg, IV, IM or SC.

[0338] Functional recovery is assessed by methodologies known in the art.

[0339] Adverse Events are mild and well-tolerated.

[0340] Results: Patients treated with neuregulin show statistically greater improvement in speech capability than those patients treated with placebo.

Example 18

NRG Treatment of Patients with Hemorrhagic Stroke

[0341] Inclusion criteria include: adults, male and female, evidence of stroke based on loss of consciousness, disorientation, speech difficulty, facial or limb paralysis. Hemorrhagic stroke confirmed with radiographic imaging. Consents are obtained from the patients and/or someone with authority to sign for the patients.

[0342] Patients are selected for those with unilateral hand weakness

[0343] Patients are enrolled and randomized to receive neuregulin or placebo starting as soon as they present to a medical facility including hospital or physician's office, diagnosis, imaging and consent is obtained.

[0344] Treatment is initiated between 1 hour and 7 days after injury. Treatment is continued for 3 months with dosing on alternate days for 1 week and then weekly for the remainder of the treatment period. Patients are dosed with 0.0001 to 1.0 mg per kg, IV, IM or SC.

[0345] Recovery is measured by standard neurological measures of hand motor activity every other week for the duration of the study.

[0346] Adverse Events are mild and well-tolerated.

[0347] Results: Patients treated with neuregulin show statistically greater improvement in hand function as measured by methodologies known in the art as compared to those patients treated with placebo.

Example 19

Kits

[0348] Kits comprise an exemplary embodiment of the invention. The kit can comprise an outer receptacle or container configured to receive one or more inner receptacles/containers, utensils and/or instructions. The utensil can comprise item(s) to administer the drug, such as a patch, inhalation apparatus, syringe or needle. A composition of the invention can be comprised within a receptacle of the invention. A receptacle of the invention can contain sufficient quantity of a composition of the invention to be useful for multiple doses, or may be in unit or single dose form. Kits of the invention generally comprise instructions for administration in accordance with the present invention. Any mode of administration set forth or supported herein can constitute some portion of the instructions. In one embodiment the instructions indicate that the composition of the invention is to be taken one or more than one times during the semi-acute post-injury period. In one embodiment the instructions indicate that the composition of the invention is to be taken one or more than one times during the chronic post-injury period. The instructions may be affixed to any container/receptacle of the invention. Alternatively, the instructions can be printed on or embossed in or formed as a component of a receptacle of the invention.

[0349] Several publications and patent documents are referenced in this application in order to more fully describe the state of the art to which this invention pertains. All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

Sequence CWU 1

1

531198DNAHomo sapiens 1catatgagcc atcttgtaaa atgtgcggag aaggagaaaa ctttctgtgt gaatggaggg 60gagtgcttca tggtgaaaga cctttcaaac ccctcgagat acttgtgcaa gtgcccaaat 120gagtttactg gtgatcgctg ccaaaactac gtaatggcca gcttctacaa ggcggaggag 180ctgtaccagt aaggatcc 198282PRTHomo sapiens 2Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro1 5 10 15Arg Gly Ser His Met Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys 20 25 30Thr Phe Cys Val Asn Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser 35 40 45Asn Pro Ser Arg Tyr Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp 50 55 60Arg Cys Gln Asn Tyr Val Met Ala Ser Phe Tyr Lys Ala Glu Glu Leu65 70 75 80Tyr Gln3236DNAArtificial SequenceRecombinant sequence 3cgataactag cagcatttcc tccaacgagg atcccgcagg taagaagcta caccggccag 60tggccggggc ccgataacta gcagcatttc ctccaacgag gatcccgcag gtaagaagct 120acaccggcca gtggccgggg ccgtggagcc gggggcatcc ggtgcctgag acagaggtgc 180tcaaggcagt ctccaccttt tgtctcccct ctgcagagag ccacattctg gaagtt 23641269DNAHomo sapiens 4atgagatggc gacgcgcccc gcgccgctcc gggcgtcccg gcccccgggc ccagcgcccc 60ggctccgccg cccgctcgtc gccgccgctg ccgctgctgc cactactgct gctgctgggg 120accgcggccc tggcgccggg ggcggcggcc ggcaacgagg cggctcccgc gggggcctcg 180gtgtgctact cgtccccgcc cagcgtggga tcggtgcagg agctagctca gcgcgccgcg 240gtggtgatcg agggaaaggt gcacccgcag cggcggcagc agggggcact cgacaggaag 300gcggcggcgg cggcgggcga ggcaggggcg tggggcggcg atcgcgagcc gccagccgcg 360ggcccacggg cgctggggcc gcccgccgag gagccgctgc tcgccgccaa cgggaccgtg 420ccctcttggc ccaccgcccc ggtgcccagc gccggcgagc ccggggagga ggcgccctat 480ctggtgaagg tgcaccaggt gtgggcggtg aaagccgggg gcttgaagaa ggactcgctg 540ctcaccgtgc gcctggggac ctggggccac cccgccttcc cctcctgcgg gaggctcaag 600gaggacagca ggtacatctt cttcatggag cccgacgcca acagcaccag ccgcgcgccg 660gccgccttcc gagcctcttt cccccctctg gagacgggcc ggaacctcaa gaaggaggtc 720agccgggtgc tgtgcaagcg gtgcgccttg cctccccaat tgaaagagat gaaaagccag 780gaatcggctg caggttccaa actagtcctt cggtgtgaaa ccagttctga atactcctct 840ctcagattca agtggttcaa gaatgggaat gaattgaatc gaaaaaacaa accacaaaat 900atcaagatac aaaaaaagcc agggaagtca gaacttcgca ttaacaaagc atcactggct 960gattctggag agtatatgtg caaagtgatc agcaaattag gaaatgacag tgcctctgcc 1020aatatcacca tcgtggaatc aaacgctaca tctacatcca ccactgggac aagccatctt 1080gtaaaatgtg cggagaagga gaaaactttc tgtgtgaatg gaggggagtg cttcatggtg 1140aaagaccttt caaacccctc gagatacttg tgcaagtgcc caaatgagtt tactggtgat 1200cgctgccaaa actacgtaat ggccagcttc tacagtacgt ccactccctt tctgtctctg 1260cctgaatag 12695422PRTHomo sapiens 5Met Arg Trp Arg Arg Ala Pro Arg Arg Ser Gly Arg Pro Gly Pro Arg1 5 10 15Ala Gln Arg Pro Gly Ser Ala Ala Arg Ser Ser Pro Pro Leu Pro Leu 20 25 30Leu Pro Leu Leu Leu Leu Leu Gly Thr Ala Ala Leu Ala Pro Gly Ala 35 40 45Ala Ala Gly Asn Glu Ala Ala Pro Ala Gly Ala Ser Val Cys Tyr Ser 50 55 60Ser Pro Pro Ser Val Gly Ser Val Gln Glu Leu Ala Gln Arg Ala Ala65 70 75 80Val Val Ile Glu Gly Lys Val His Pro Gln Arg Arg Gln Gln Gly Ala 85 90 95Leu Asp Arg Lys Ala Ala Ala Ala Ala Gly Glu Ala Gly Ala Trp Gly 100 105 110Gly Asp Arg Glu Pro Pro Ala Ala Gly Pro Arg Ala Leu Gly Pro Pro 115 120 125Ala Glu Glu Pro Leu Leu Ala Ala Asn Gly Thr Val Pro Ser Trp Pro 130 135 140Thr Ala Pro Val Pro Ser Ala Gly Glu Pro Gly Glu Glu Ala Pro Tyr145 150 155 160Leu Val Lys Val His Gln Val Trp Ala Val Lys Ala Gly Gly Leu Lys 165 170 175Lys Asp Ser Leu Leu Thr Val Arg Leu Gly Thr Trp Gly His Pro Ala 180 185 190Phe Pro Ser Cys Gly Arg Leu Lys Glu Asp Ser Arg Tyr Ile Phe Phe 195 200 205Met Glu Pro Asp Ala Asn Ser Thr Ser Arg Ala Pro Ala Ala Phe Arg 210 215 220Ala Ser Phe Pro Pro Leu Glu Thr Gly Arg Asn Leu Lys Lys Glu Val225 230 235 240Ser Arg Val Leu Cys Lys Arg Cys Ala Leu Pro Pro Gln Leu Lys Glu 245 250 255Met Lys Ser Gln Glu Ser Ala Ala Gly Ser Lys Leu Val Leu Arg Cys 260 265 270Glu Thr Ser Ser Glu Tyr Ser Ser Leu Arg Phe Lys Trp Phe Lys Asn 275 280 285Gly Asn Glu Leu Asn Arg Lys Asn Lys Pro Gln Asn Ile Lys Ile Gln 290 295 300Lys Lys Pro Gly Lys Ser Glu Leu Arg Ile Asn Lys Ala Ser Leu Ala305 310 315 320Asp Ser Gly Glu Tyr Met Cys Lys Val Ile Ser Lys Leu Gly Asn Asp 325 330 335Ser Ala Ser Ala Asn Ile Thr Ile Val Glu Ser Asn Ala Thr Ser Thr 340 345 350Ser Thr Thr Gly Thr Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys 355 360 365Thr Phe Cys Val Asn Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser 370 375 380Asn Pro Ser Arg Tyr Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp385 390 395 400Arg Cys Gln Asn Tyr Val Met Ala Ser Phe Tyr Ser Thr Ser Thr Pro 405 410 415Phe Leu Ser Leu Pro Glu 42062003DNAHomo sapiensmisc_feature(31)..(32)n is a, c, g, or t 6ggaattcctt tttttttttt tttttttctt nntttttttt tgcccttata cctcttcgcc 60tttctgtggt tccatccact tcttccccct cctcctccca taaacaactc tcctacccct 120gcacccccaa taaataaata aaaggaggag ggcaaggggg gaggaggagg agtggtgctg 180cgaggggaag gaaaagggag gcagcgcgag aagagccggg cagagtccga accgacagcc 240agaagcccgc acgcacctcg caccatgaga tggcgacgcg ccccgcgccg ctccgggcgt 300cccggccccc gggcccagcg ccccggctcc gccgcccgct cgtcgccgcc gctgccgctg 360ctgccactac tgctgctgct ggggaccgcg gccctggcgc cgggggcggc ggccggcaac 420gaggcggctc ccgcgggggc ctcggtgtgc tactcgtccc cgcccagcgt gggatcggtg 480caggagctag ctcagcgcgc cgcggtggtg atcgagggaa aggtgcaccc gcagcggcgg 540cagcaggggg cactcgacag gaaggcggcg gcggcggcgg gcgaggcagg ggcgtggggc 600ggcgatcgcg agccgccagc cgcgggccca cgggcgctgg ggccgcccgc cgaggagccg 660ctgctcgccg ccaacgggac cgtgccctct tggcccaccg ccccggtgcc cagcgccggc 720gagcccgggg aggaggcgcc ctatctggtg aaggtgcacc aggtgtgggc ggtgaaagcc 780gggggcttga agaaggactc gctgctcacc gtgcgcctgg ggacctgggg ccaccccgcc 840ttcccctcct gcgggaggct caaggaggac agcaggtaca tcttcttcat ggagcccgac 900gccaacagca ccagccgcgc gccggccgcc ttccgagcct ctttcccccc tctggagacg 960ggccggaacc tcaagaagga ggtcagccgg gtgctgtgca agcggtgcgc cttgcctccc 1020caattgaaag agatgaaaag ccaggaatcg gctgcaggtt ccaaactagt ccttcggtgt 1080gaaaccagtt ctgaatactc ctctctcaga ttcaagtggt tcaagaatgg gaatgaattg 1140aatcgaaaaa acaaaccaca aaatatcaag atacaaaaaa agccagggaa gtcagaactt 1200cgcattaaca aagcatcact ggctgattct ggagagtata tgtgcaaagt gatcagcaaa 1260ttaggaaatg acagtgcctc tgccaatatc accatcgtgg aatcaaacgc tacatctaca 1320tccaccactg ggacaagcca tcttgtaaaa tgtgcggaga aggagaaaac tttctgtgtg 1380aatggagggg agtgcttcat ggtgaaagac ctttcaaacc cctcgagata cttgtgcaag 1440tgcccaaatg agtttactgg tgatcgctgc caaaactacg taatggccag cttctacagt 1500acgtccactc cctttctgtc tctgcctgaa taggagcatg ctcagttggt gctgctttct 1560tgttgctgca tctcccctca gattccacct agagctagat gtgtcttacc agatctaata 1620ttgactgcct ctgcctgtcg catgagaaca ttaacaaaag caattgtatt acttcctctg 1680ttcgcgacta gttggctctg agatactaat aggtgtgtga ggctccggat gtttctggaa 1740ttgatattga atgatgtgat acaaattgat agtcaatatc aagcagtgaa atatgataat 1800aaaggcattt caaagtctca cttttattga taaaataaaa atcattctac tgaacagtcc 1860atcttcttta tacaatgacc acatcctgaa aagggtgttg ctaagctgta accgatatgc 1920acttgaaatg atggtaagtt aattttgatt cagaatgtgt tatttgtcac aaataaacat 1980aataaaagga aaaaaaaaaa aaa 20037422PRTHomo sapiens 7Met Arg Trp Arg Arg Ala Pro Arg Arg Ser Gly Arg Pro Gly Pro Arg1 5 10 15Ala Gln Arg Pro Gly Ser Ala Ala Arg Ser Ser Pro Pro Leu Pro Leu 20 25 30Leu Pro Leu Leu Leu Leu Leu Gly Thr Ala Ala Leu Ala Pro Gly Ala 35 40 45Ala Ala Gly Asn Glu Ala Ala Pro Ala Gly Ala Ser Val Cys Tyr Ser 50 55 60Ser Pro Pro Ser Val Gly Ser Val Gln Glu Leu Ala Gln Arg Ala Ala65 70 75 80Val Val Ile Glu Gly Lys Val His Pro Gln Arg Arg Gln Gln Gly Ala 85 90 95Leu Asp Arg Lys Ala Ala Ala Ala Ala Gly Glu Ala Gly Ala Trp Gly 100 105 110Gly Asp Arg Glu Pro Pro Ala Ala Gly Pro Arg Ala Leu Gly Pro Pro 115 120 125Ala Glu Glu Pro Leu Leu Ala Ala Asn Gly Thr Val Pro Ser Trp Pro 130 135 140Thr Ala Pro Val Pro Ser Ala Gly Glu Pro Gly Glu Glu Ala Pro Tyr145 150 155 160Leu Val Lys Val His Gln Val Trp Ala Val Lys Ala Gly Gly Leu Lys 165 170 175Lys Asp Ser Leu Leu Thr Val Arg Leu Gly Thr Trp Gly His Pro Ala 180 185 190Phe Pro Ser Cys Gly Arg Leu Lys Glu Asp Ser Arg Tyr Ile Phe Phe 195 200 205Met Glu Pro Asp Ala Asn Ser Thr Ser Arg Ala Pro Ala Ala Phe Arg 210 215 220Ala Ser Phe Pro Pro Leu Glu Thr Gly Arg Asn Leu Lys Lys Glu Val225 230 235 240Ser Arg Val Leu Cys Lys Arg Cys Ala Leu Pro Pro Gln Leu Lys Glu 245 250 255Met Lys Ser Gln Glu Ser Ala Ala Gly Ser Lys Leu Val Leu Arg Cys 260 265 270Glu Thr Ser Ser Glu Tyr Ser Ser Leu Arg Phe Lys Trp Phe Lys Asn 275 280 285Gly Asn Glu Leu Asn Arg Lys Asn Lys Pro Gln Asn Ile Lys Ile Gln 290 295 300Lys Lys Pro Gly Lys Ser Glu Leu Arg Ile Asn Lys Ala Ser Leu Ala305 310 315 320Asp Ser Gly Glu Tyr Met Cys Lys Val Ile Ser Lys Leu Gly Asn Asp 325 330 335Ser Ala Ser Ala Asn Ile Thr Ile Val Glu Ser Asn Ala Thr Ser Thr 340 345 350Ser Thr Thr Gly Thr Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys 355 360 365Thr Phe Cys Val Asn Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser 370 375 380Asn Pro Ser Arg Tyr Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp385 390 395 400Arg Cys Gln Asn Tyr Val Met Ala Ser Phe Tyr Ser Thr Ser Thr Pro 405 410 415Phe Leu Ser Leu Pro Glu 420811PRTHomo sapiens 8Val Cys Leu Leu Thr Val Ala Ala Leu Pro Pro1 5 10915PRTHomo sapiens 9Ala Ser Pro Val Ser Val Gly Ser Val Gln Glu Leu Val Gln Arg1 5 10 15108PRTHomo sapiens 10Trp Phe Val Val Ile Glu Gly Lys1 5119PRTHomo sapiens 11Lys Val His Glu Val Trp Ala Ala Lys1 5126PRTHomo sapiensmisc_feature(5)..(5)Xaa can be any naturally occurring amino acid 12Asp Leu Leu Leu Xaa Val1 51313PRTHomo sapiensmisc_feature(12)..(12)Xaa can be any naturally occurring amino acid 13Leu Gly Ala Trp Gly Pro Pro Ala Phe Pro Val Xaa Tyr1 5 101413PRTHomo sapiensmisc_feature(10)..(10)Xaa can be any naturally occurring amino acid 14Tyr Ile Phe Phe Met Glu Pro Ala Ala Xaa Ser Ser Gly1 5 10154PRTHomo sapiens 15Leu Val Leu Arg11613PRTHomo sapiensmisc_feature(12)..(12)Xaa can be any naturally occurring amino acid 16Lys Ala Ser Leu Ala Asp Ser Gly Glu Tyr Met Xaa Lys1 5 1017198DNAHomo sapiens 17agccatcttg tcaagtgtgc agagaaggag aaaactttct gtgtgaatgg aggcgagtgc 60ttcatggtga aagagctttc aaatccctca agatacttgt gcaagtgccc aaatgagttt 120actggtgatc gctgccaaaa ctacgtaatg gccagcttct acagtacctc cactcccttt 180ctgtctctgc ctgaatag 1981865PRTHomo sapiens 18Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn1 5 10 15Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr 20 25 30Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr 35 40 45Val Met Ala Ser Phe Tyr Ser Thr Ser Thr Pro Phe Leu Ser Leu Pro 50 55 60Glu6519192DNAHomo sapiens 19agccatcttg tcaagtgtgc agagaaggag aaaactttct gtgtgaatgg aggcgagtgc 60ttcatggtga aagacctttc aaatccctca agatacttgt gcaagtgcca acctggattc 120actggagcga gatgtactga gaatgtgccc atgaaagtcc aaacccaaga aaaagcggag 180gagctctact aa 1922063PRTHomo sapiens 20Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn1 5 10 15Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr 20 25 30Leu Cys Lys Cys Gln Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn 35 40 45Val Pro Met Lys Val Gln Thr Gln Glu Lys Ala Glu Glu Leu Tyr 50 55 6021183DNAHomo sapiens 21agccatcttg tcaagtgtgc agagaaggag aaaactttct gtgtgaatgg aggcgagtgc 60ttcatggtga aagacctttc aaatccctca agatacttgt gcaagtgccc aaatgagttt 120actggtgatc gctgccaaaa ctacgtaatg gccagcttct acaaagcgga ggagctctac 180taa 1832260PRTHomo sapiens 22Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn1 5 10 15Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr 20 25 30Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr 35 40 45Val Met Ala Ser Phe Tyr Lys Ala Glu Glu Leu Tyr 50 55 6023210DNAHomo sapiens 23agccatcttg tcaagtgtgc agagaaggag aaaactttct gtgtgaatgg aggcgagtgc 60ttcatggtga aagacctttc aaatccctca agatacttgt gcaagtgccc aaatgagttt 120actggtgatc gctgccaaaa ctacgtaatg gccagcttct acaagcatct tgggattgaa 180tttatggaga aagcggagga gctctactaa 2102469PRTHomo sapiens 24Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn1 5 10 15Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr 20 25 30Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr 35 40 45Val Met Ala Ser Phe Tyr Lys His Leu Gly Ile Glu Phe Met Glu Lys 50 55 60Ala Glu Glu Leu Tyr6525267DNAHomo sapiens 25agccatcttg tcaagtgtgc agagaaggag aaaactttct gtgtgaatgg aggcgagtgc 60ttcatggtga aagacctttc aaatccctca agatacttgt gcaagtgcca acctggattc 120actggagcga gatgtactga gaatgtgccc atgaaagtcc aaacccaaga aaagtgccca 180aatgagttta ctggtgatcg ctgccaaaac tacgtaatgg ccagcttcta cagtacgtcc 240actccctttc tgtctctgcc tgaatag 2672688PRTHomo sapiens 26Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn1 5 10 15Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr 20 25 30Leu Cys Lys Cys Gln Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn 35 40 45Val Pro Met Lys Val Gln Thr Gln Glu Lys Cys Pro Asn Glu Phe Thr 50 55 60Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser Phe Tyr Ser Thr Ser65 70 75 80Thr Pro Phe Leu Ser Leu Pro Glu 8527252DNAHomo sapiens 27agccatcttg tcaagtgtgc agagaaggag aaaactttct gtgtgaatgg aggcgagtgc 60ttcatggtga aagacctttc aaatccctca agatacttgt gcaagtgcca acctggattc 120actggagcga gatgtactga gaatgtgccc atgaaagtcc aaacccaaga aaagtgccca 180aatgagttta ctggtgatcg ctgccaaaac tacgtaatgg ccagcttcta caaagcggag 240gagctctact aa 2522883PRTHomo sapiens 28Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn1 5 10 15Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr 20 25 30Leu Cys Lys Cys Gln Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn 35 40 45Val Pro Met Lys Val Gln Thr Gln Glu Lys Cys Pro Asn Glu Phe Thr 50 55 60Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser Phe Tyr Lys Ala Glu65 70 75 80Glu Leu Tyr29183DNAHomo sapiensmisc_feature(183)..(183)n is a, c, g, or t 29agccatcttg tcaagtgtgc agagaaggag aaaactttct gtgtgaatgg aggcgagtgc 60ttcatggtga aagacctttc aaatccctca agatacttgt gcaagtgccc aaatgagttt

120actggtgatc gctgccaaaa ctacgtaatg gccagcttct acaaagcgga ggagctctac 180can 1833061PRTHomo sapiens 30Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn1 5 10 15Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr 20 25 30Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr 35 40 45Val Met Ala Ser Phe Tyr Lys Ala Glu Glu Leu Tyr Gln 50 55 603150PRTHomo sapiens 31Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys1 5 10 15Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys Cys 20 25 30Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser 35 40 45Phe Tyr 503250PRTHomo sapiens 32Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys1 5 10 15Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys Cys 20 25 30Gln Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn Val Pro Met Lys 35 40 45Val Gln 5033330PRTHomo sapiens 33Met Arg Arg Asp Pro Ala Pro Gly Phe Ser Met Leu Leu Phe Gly Val1 5 10 15Ser Leu Ala Cys Tyr Ser Pro Ser Leu Lys Ser Val Gln Asp Gln Ala 20 25 30Tyr Lys Ala Pro Val Val Val Glu Gly Lys Val Gln Gly Leu Val Pro 35 40 45Ala Gly Gly Ser Ser Ser Asn Ser Thr Arg Glu Pro Pro Ala Ser Gly 50 55 60Arg Val Ala Leu Val Lys Val Leu Asp Lys Trp Pro Leu Arg Ser Gly65 70 75 80Gly Leu Gln Arg Glu Gln Val Ile Ser Val Gly Ser Cys Val Pro Leu 85 90 95Glu Arg Asn Gln Arg Tyr Ile Phe Phe Leu Glu Pro Thr Glu Gln Pro 100 105 110Leu Val Phe Lys Thr Ala Phe Ala Pro Leu Asp Thr Asn Gly Lys Asn 115 120 125Leu Lys Lys Glu Val Gly Lys Ile Leu Cys Thr Asp Cys Ala Thr Arg 130 135 140Pro Lys Leu Lys Lys Met Lys Ser Gln Thr Gly Gln Val Gly Glu Lys145 150 155 160Gln Ser Leu Lys Cys Glu Ala Ala Ala Gly Asn Pro Gln Pro Ser Tyr 165 170 175Arg Trp Phe Lys Asp Gly Lys Glu Leu Asn Arg Ser Arg Asp Ile Arg 180 185 190Ile Lys Tyr Gly Asn Gly Arg Lys Asn Ser Arg Leu Gln Phe Asn Lys 195 200 205Val Lys Val Glu Asp Ala Gly Glu Tyr Val Cys Glu Ala Glu Asn Ile 210 215 220Leu Gly Lys Asp Thr Val Arg Gly Arg Leu Tyr Val Asn Ser Val Ser225 230 235 240Thr Thr Leu Ser Ser Trp Ser Gly His Ala Arg Lys Cys Asn Glu Thr 245 250 255Ala Lys Ser Tyr Cys Val Asn Gly Gly Val Cys Tyr Tyr Ile Glu Gly 260 265 270Ile Asn Gln Leu Ser Cys Lys Cys Pro Asn Gly Phe Phe Gly Gln Arg 275 280 285Cys Leu Glu Lys Leu Pro Leu Arg Leu Tyr Met Pro Asp Pro Lys Gln 290 295 300Ser Val Leu Trp Asp Thr Pro Gly Thr Gly Val Ser Ser Ser Gln Trp305 310 315 320Ser Thr Ser Pro Ser Thr Leu Asp Leu Asn 325 33034298PRTHomo sapiens 34Met Arg Arg Asp Pro Ala Pro Gly Phe Ser Met Leu Leu Phe Gly Val1 5 10 15Ser Leu Ala Cys Tyr Ser Pro Ser Leu Lys Ser Val Gln Asp Gln Ala 20 25 30Tyr Lys Ala Pro Val Val Val Glu Gly Lys Val Gln Gly Leu Val Pro 35 40 45Ala Gly Gly Ser Ser Ser Asn Ser Thr Arg Glu Pro Pro Ala Ser Gly 50 55 60Arg Val Ala Leu Val Lys Val Leu Asp Lys Trp Pro Leu Arg Ser Gly65 70 75 80Gly Leu Gln Arg Glu Gln Val Ile Ser Val Gly Ser Cys Val Pro Leu 85 90 95Glu Arg Asn Gln Arg Tyr Ile Phe Phe Leu Glu Pro Thr Glu Gln Pro 100 105 110Leu Val Phe Lys Thr Ala Phe Ala Pro Leu Asp Thr Asn Gly Lys Asn 115 120 125Leu Lys Lys Glu Val Gly Lys Ile Leu Cys Thr Asp Cys Ala Thr Arg 130 135 140Pro Lys Leu Lys Lys Met Lys Ser Gln Thr Gly Gln Val Gly Glu Lys145 150 155 160Gln Ser Leu Lys Cys Glu Ala Ala Ala Gly Asn Pro Gln Pro Ser Tyr 165 170 175Arg Trp Phe Lys Asp Gly Lys Glu Leu Asn Arg Ser Arg Asp Ile Arg 180 185 190Ile Lys Tyr Gly Asn Gly Arg Lys Asn Ser Arg Leu Gln Phe Asn Lys 195 200 205Val Lys Val Glu Asp Ala Gly Glu Tyr Val Cys Glu Ala Glu Asn Ile 210 215 220Leu Gly Lys Asp Thr Val Arg Gly Arg Leu Tyr Val Asn Ser Val Ser225 230 235 240Thr Thr Leu Ser Ser Trp Ser Gly His Ala Arg Lys Cys Asn Glu Thr 245 250 255Ala Lys Ser Tyr Cys Val Asn Gly Gly Val Cys Tyr Tyr Ile Glu Gly 260 265 270Ile Asn Gln Leu Ser Cys Lys Cys Pro Val Gly Tyr Thr Gly Asp Arg 275 280 285Cys Gln Gln Phe Ala Met Val Asn Phe Ser 290 2953563PRTHomo sapiens 35Thr Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val1 5 10 15Asn Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg 20 25 30Tyr Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn 35 40 45Tyr Val Met Ala Ser Phe Tyr Lys Ala Glu Glu Leu Tyr Gln Lys 50 55 603661PRTHomo sapiens 36Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn1 5 10 15Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr 20 25 30Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr 35 40 45Val Met Ala Ser Phe Tyr Lys Ala Glu Glu Leu Tyr Gln 50 55 603760PRTHomo sapiens 37Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn1 5 10 15Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr 20 25 30Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr 35 40 45Val Met Ala Ser Phe Tyr Lys Ala Glu Glu Leu Tyr 50 55 603871PRTHomo sapiens 38Thr Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val1 5 10 15Asn Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg 20 25 30Tyr Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn 35 40 45Tyr Val Met Ala Ser Phe Tyr Lys His Leu Gly Ile Glu Phe Met Glu 50 55 60Ala Glu Glu Leu Tyr Gln Lys65 703950PRTHomo sapiens 39Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys1 5 10 15Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys Cys 20 25 30Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser 35 40 45Phe Tyr 504050PRTHomo sapiens 40Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys1 5 10 15Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys Cys 20 25 30Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser 35 40 45Phe Tyr 504169PRTHomo sapiens 41Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn1 5 10 15Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr 20 25 30Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr 35 40 45Val Met Ala Ser Phe Tyr Lys His Leu Gly Ile Glu Phe Met Glu Lys 50 55 60Ala Glu Glu Leu Tyr654250PRTHomo sapiens 42Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys1 5 10 15Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys Cys 20 25 30Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser 35 40 45Phe Tyr 504365PRTHomo sapiens 43Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn1 5 10 15Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr 20 25 30Leu Cys Lys Cys Gln Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn 35 40 45Val Pro Met Lys Val Gln Asn Gln Glu Lys Ala Glu Glu Leu Tyr Gln 50 55 60Lys654463PRTHomo sapiens 44Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn1 5 10 15Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr 20 25 30Leu Cys Lys Cys Gln Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn 35 40 45Val Pro Met Lys Val Gln Thr Gln Glu Lys Ala Glu Glu Leu Tyr 50 55 604550PRTHomo sapiens 45Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys1 5 10 15Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys Cys 20 25 30Gln Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn Val Pro Met Lys 35 40 45Val Gln 504650PRTHomo sapiens 46Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys1 5 10 15Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys Cys 20 25 30Gln Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn Val Pro Met Lys 35 40 45Val Gln 504783PRTHomo sapiens 47Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn1 5 10 15Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr 20 25 30Leu Cys Lys Cys Gln Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn 35 40 45Val Pro Met Lys Val Gln Thr Gln Glu Lys Cys Pro Asn Glu Phe Thr 50 55 60Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser Phe Tyr Lys Ala Glu65 70 75 80Glu Leu Tyr4888PRTHomo sapiens 48Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn1 5 10 15Gly Gly Glu Cys Phe Met Val Glu Asp Leu Ser Asn Pro Ser Arg Tyr 20 25 30Leu Cys Lys Cys Gln Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn 35 40 45Val Pro Met Lys Val Gln Thr Gln Glu Lys Cys Pro Asn Glu Phe Thr 50 55 60Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser Phe Tyr Ser Thr Ser65 70 75 80Thr Pro Phe Leu Ser Leu Pro Glu 854931PRTHomo sapiens 49Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys1 5 10 15Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys 20 25 305061PRTHomo sapiens 50Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys1 5 10 15Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys Cys 20 25 30Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser 35 40 45Phe Tyr Ser Thr Ser Thr Pro Phe Leu Ser Leu Pro Glu 50 55 605161PRTHomo sapiens 51Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys1 5 10 15Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys Cys 20 25 30Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser 35 40 45Phe Tyr Ser Thr Ser Thr Pro Phe Leu Ser Leu Pro Glu 50 55 605265PRTHomo sapiens 52Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn1 5 10 15Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr 20 25 30Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr 35 40 45Val Met Ala Ser Phe Tyr Ser Thr Ser Thr Pro Phe Leu Ser Leu Pro 50 55 60Glu655353PRTHomo sapiens 53Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His1 5 10 15Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45Trp Trp Glu Leu Arg 50

Claims

1. A method for administering a polypeptide comprising an epidermal growth factor-like (EGF-like) domain to a mammal, said method comprising: administering a polypeptide comprising an epidermal growth factor-like (EGF-like) domain following neuroinjury in said mammal; and, initiating the administering at least two days after the neuroinjury.

2. A method of claim 1, wherein said initiating step begins at a three days after the neuroinjury.

3. The method of claim 1, wherein said initiating step begins at least seven days after the neuroinjury.

4. A method of claim 1 wherein the EGF-like domain is encoded by the neuregulin (NRC)-1 gene, (NRG)-2 gene, (NRG)-3 gene, (NRC)-4 gene.

5. The method of claim 1, wherein said polypeptide is OGF2.

6. The method of claim 1, wherein said mammal is a human

7. A method for administering a polypeptide comprising an epidermal growth factor-like (EGF-like) domain to a mammal, said method comprising: administering a polypeptide comprising an epidermal growth factor-like (EGF-like) domain following neuroinjury in said mammal; initiating the administering at within six hours after the neuroinjury; and, continuing the administering step into a time period more than six hours post injury.

8. The method of claim 7, wherein the continuing step comprises continuing the administering step into a time period more than 48 hours post injury.

9. The method of claim 7, wherein the continuing step comprises continuing the administering step into a time period more than 72 hours post injury.

10. A method of claim 7 wherein the EGF-like domain is encoded by the neuregulin (NRG)-1 gene, (NRG)-2 gene, (NRG)-3 gene, (NRG)-4 gene.

11. The method of claim 7, wherein said polypeptide is GGF2.

12. The method of claim 7, wherein said mammal is a human.

13. A method for administering a polypeptide comprising an epidermal growth factor-like (EGF-like) domain following an ischemic central nervous system neuroinjury in a mammal, said method comprising: administering the peptide following neuroinjury in said mammal; and, initiating the administering after the mammal has achieved a complete post-injury ischemic lesion cell death volume.

14. A method of claim 13, wherein the EGF-like domain is encoded by the neuregulin (NRG)-1 gene, (NRG)-2 gene, (NRG)-3 gene, (NRG)-4 gene.

15. The method of claim 13, wherein said polypeptide is GGF2.