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Title:  Methods of preventing or treating brain ischemia or brain injury
United States Patent: 
7,202,211
Issued: 
April 10, 2007

Inventors: 
Faerman; Alexander (Bnei Aish, IL), Kachalsky; Sylvia G. (Gan Yavne, IL), Idelson; Gregory Hirsch (Maale Adumim, IL)
Assignee: 
Astellas Pharma Inc. (Tokyo, JP), Quark Biotech, Inc. (Cleveland, OH)
Appl. No.: 
10/371,725
Filed: 
February 20, 2003


 

Woodbury College's Master of Science in Law


Abstract

The present invention relates to use of Narp inhibitor in order to promote or enhance recovery from ischemic events, particularly focal ischemia of the central nervous system, as well as for preventing or diminishing chronic degenerative changes.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for alleviation or reduction of the symptoms and signs associated with damaged neuronal tissues whether resulting from tissue trauma, or from chronic degenerative changes. It is an object of the present invention to provide pharmaceutical compositions to reduce or even to completely diminish tissue damage or degeneration due to acute injury to the CNS as described or due to other insults. It is a further object of the present invention to provide methods leading to functional improvement after traumatic ischemic events, including but not limited to traumatic brain injury (TBI) or cerebral stroke. These effects will be achieved by administering an agent that interacts with Narp molecules, and consequently prevents the effect of Narp on AMPA type glutamate receptors.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides polypeptides, compositions and methods for alleviation or reduction of the symptoms and signs associated with damaged neuronal tissues whether resulting from tissue trauma, or from chronic degenerative changes. It is an object of the present invention to provide pharmaceutical compositions to reduce or even to completely diminish tissue damage or degeneration. It is a further object of the present invention to provide methods leading to functional improvement after traumatic ischemic events. These effects will be achieved by administering an agent that interacts with Narp molecules, and consequently prevents the effect of Narp.

Narp (also termed neuronal activity regulated pentraxin or neuronal pentraxin II) is a secreted protein the messenger RNA (mRNA) of which is transcribed from an immediate-early gene (IEG) that is rapidly induced in neurons of the hippocampus and cortex by physiological synaptic activity. It has homology to members of the pentraxin family of secreted lectins that include C-reactive protein (CRP) and serum amyloid P (SAP) component. Narp is characterized by a cyclic pentameric structure and radial symmetry. The five identical 24-kDa protomers consist of 206 amino acids, and are noncovalently linked. Given that Narp binds to GluR1 AMPA receptor subunit in a calcium dependent manner, and that its suggested functions are neurite-outgrowth promoting activity (role in excitatory synaptogenesis) and extracellular aggregating factor for AMPA receptors, targeting against Narp may decrease the "excitotoxic" damage mediated by AMPA receptors at the early stages of the ischemic event.

The present invention is based, inlet alia, on the finding by the inventors that in animals which were subjected to middle cerebral artery occlusion (MCAO), an ischemia (stroke) model, the Narp RNA level was significantly upregulated (as compared to controls) following the onset of the ischemic event.

The present invention utilizes a polypeptide, antibody, or a small chemical compound that binds Narp, thus preventing Narp biological activity.

According to a preferred embodiment of the invention, an antibody directed to a neural activity-regulated pentraxin peptide or its immunoreactive fragments is provided.

According to another preferred embodiment of the invention, a polypeptide which binds to a neural activity-regulated pentraxin peptide or fragments thereof is provided.

According to another preferred embodiment of the invention, a small chemical compound which binds to a neural activity-regulated pentraxin polypeptide or fragments thereof is provided.

The term "Narp", as used herein, refers to the Narp (neural activity-regulated pentraxin) polypeptide and is understood to include "neuronal activated pentraxin" (or pentaxin), "pentraxin II" "pentaxin II", and "NP2", derived from any organism, preferably man or mice, and homologs thereof having similar biological activity, preferably having 70%, 80%, 90% or even 95% homology to the Narp polypeptide. Polypeptides encoded by nucleic acid sequences which bind to the Narp gene under conditions of highly stringent hybridization, which are well-known in the art (for example Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1988), updated in 1995 and 1998), are also encompassed by this term.

By "biological effect of Narp" or "Narp biological activity" is meant the effect of Narp on AMPA type glutamate receptors, which may be direct or indirect, and includes, without being bound by theory, Neurite-outgrowth promoting activity, and a function as an extracellular aggregating factor for AMPA receptors, which includes an inhibitory effect wherein Narp causes clustering of AMPA receptors on the surface of a cell. The glutamate receptors are preferably on the surface of neuronal cells; the indirect effect includes, but is not limited to, Narp binding to or having an effect on one of several molecules which are involved in a signal transduction cascade resulting in an effect on AMPA type glutamate receptors.

By "Narp inhibitor" is meant any molecule, whether a polypeptide, antibody, or small chemical compound, that prevents or reduces the biological effect of Narp, as recited above. Narp inhibitor may also be an inhibitor of the Narp promoter such as inter alia, antisense RNA molecule, dominant negative peptide (see, for example, O'Brien et al., Synaptically Targeted Narp Plays an Essential Role in the Aggregation of AMPA Receptors at Excitatory Synapses in Cultures Spinal Neurons, Journal of Neuroscience 22(11): 4487 4498, 2002, which discloses Narp dominant negative mutants that inhibit Narp activity). A preferred Narp inhibitor is gamma-taipoxin.

By the term "antibody" as used in the present invention is meant both poly- and mono-clonal complete antibodies as well as fragments thereof, such as Fab, F(ab').sub.2, and Fv, which are capable of binding the epitopic determinant. These antibody fragments retain the ability to selectively bind with its antigen or receptor and are exemplified as follows, inter alia: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule can be produced by digestion of whole antibody with the enzyme papain to yield a light chain and a portion of the heavy chain; (2) (Fab').sub.2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab'.sub.2) is a dimer of two Fab fragments held together by two disulfide bonds; (3) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (4) Single chain antibody (SCA), defined as a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain linked by a suitable polypeptide linker as a genetically fused single chain molecule.

Details on how to prepare all types of antibodies are provided in Example 8 below.

By the term "epitope" as used in this invention is meant an antigenic determinant on an antigen to which the antibody binds. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.

By "Polypeptide" is meant a molecule composed of amino acids and the term includes peptides, polypeptides, proteins and peptidomimetics.

The term "Amino acid" refers to a molecule which consists of any one of the 20 naturally occurring amino acids, amino acids which have been chemically modified (see below), or synthetic amino acids.

The terms "chemical compound", "small molecule", "chemical molecule" "small chemical molecule" and "small chemical compound" are used interchangeably herein and are understood to refer to chemical moieties of any particular type which may be synthetically produced or obtained from natural sources and typically have a molecular weight of less than 2000 daltons, more preferably less than 1000 daltons or even less than 600 daltons.

By "homolog/homology", as utilized in the present invention, is meant at least about 70%, preferably at least about 75% homology, advantageously at least about 80% homology, more advantageously at least about 90% homology, even more advantageously at least about 95%, e.g., at least about 97%, about 98%, about 99% or even about 100% homology. The invention also comprehends that these polynucleotides and polypeptides can be used in the same fashion as the herein or aforementioned polynucleotides and polypeptides.

Alternatively or additionally, "homology", with respect to sequences, can refer to the number of positions with identical nucleotides or amino acid residues, divided by the number of nucleotides or amino acid residues in the shorter of the two sequences, wherein alignment of the two sequences can be determined in accordance with the Wilbur and Lipman algorithm ((1983) Proc. Natl. Acad. Sci. USA 80:726), for instance, using a window size of 20 nucleotides, a word length of 4 nucleotides, and a gap penalty of 4, and computer-assisted analysis and interpretation of the sequence data, including alignment can be conveniently performed using commercially available programs (e.g., Intelligenetics.TM. Suite, Intelligenetics Inc., Calif.). When RNA sequences are said to be similar, or to have a degree of sequence identity or homology with DNA sequences, thymidine (T) in the DNA sequence is considered equal to uracil (U) in the RNA sequence. RNA sequences within the scope of the invention can be derived from DNA sequences or their complements, by substituting thymidine (T) in the DNA sequence with uracil (U).

Additionally or alternatively, amino acid sequence similarity or homology can be determined, for instance, using the BlastP program (Altschul el al., Nucl. Acids Res. 25:3389 3402) and available at NCBI. The following references provide algorithms for comparing the relative identity or homology of amino acid residues of two polypeptides, and additionally, or alternatively, with respect to the foregoing, the teachings in these references can be used for determining percent homology: Smith et al., (1981) Adv. Appl. Math. 2:482 489; Smith et al., (1983) Nucl. Acids Res. 11:2205 2220; Devereux et al., (1984) Nucl. Acids Res. 12:387 395; Feng et al., (1987) J. Molec. Evol. 25:351 360; Higgins et al., (1989) CABIOS 5:151 153; and Thompson et al., (1994) Nucl. Acids Res. 22:4673 4680.

The present invention is based, inter alia, on the observation by the inventors that occlusion of a cerebral artery, which serves as a model for stroke or other ischemic and hypoxic events, induces significant elevation in Narp transcription as determined by the levels of Narp RNA compared to controls, and on the fact that Narp interacts with certain subunits of AMPA receptors, and subsequently causing their clustering.

The present invention discloses for the first time the utility of Narp inhibition and the utilization of anti-Narp antibodies, Narp inhibitors, use of gamma-taipoxin, small molecules, antisense RNAs, or ribozymes for inhibition of Narp function. Thus, without being bound by theory, we suggest that NARP antibody or other peptide Narp inhibitors or small molecule Narp inhibitors prevent the effect of Narp on Glutamate receptors, possibly the clustering of Glutamate receptors, thereby improving clinical and recovery outcome after brain ischemia or tissue trauma.

It is known in the art, that in certain neurological diseases, for example, brain ischemia or stroke, the blood brain barrier (BBB) is relatively open compared to that of a normal subject, thus enabling penetration of even large molecules such as macromolecules, including antibodies into the brain, and subsequently allowing interaction of the latter with Narp at the post synaptic region. Further information on delivery into the brain is provided in Example 6 herein below.

Without being bound by theory, we suggest that the effect of NARP in the clustering of Glutamate receptors in post-synaptic terminals in the brain as well as its up-regulation following stroke are detrimental. We further suggest that inhibition of the interaction between NARP and the glutamate receptors in the post-synaptic terminals may inhibit the clusters of AMPA receptors in the synapse and inhibit and/or decrease AMPA receptor driven excitotoxicity.

Without being bound by theory, other inhibitors of Narp, apart from gamma taipoxin or anti-Narp antibody, may inhibit (prevent) the effect of Narp on Glutamate receptors. Such inhibitors are inter alia polypeptides capable of inhibiting the effect of NARP (both dominant negative peptides and/or extracellular polypeptides that inhibit the clustering-see, for example, Mi et al., Differing Mechanisms for Glutamate Receptor Aggregation on Dendritic Spines and Shafts in Cultures Hippocampal Neurons. The Journal of Neuroscience, 22(17): 7606 7616, 2002) and antisense oligonucleotides such as peptide antagonists, synthetic small molecule antagonists, antisense RNAs, or ribozymes.

The present approach has several distinct advantages over any hitherto available or suggested therapies, including a longer therapeutic effect while preserving the favorable or beneficial effects.

One aspect of this invention provides for a polypeptide that binds to Narp. This polypeptide may be, but is not limited to, an antibody or a portion of a toxin.

Binding of said polypeptide to Narp may occur through a specific binding site or epitope. This binding site is characterized by the fact that it confers to Narp the possibility of executing any of the activities attributed to Narp, including but not limited to neurite-outgrowth promoting activity and function as an extracellular aggregating factor for AMPA receptors. This binding site is further characterized by the fact that binding of the polypeptide of the invention to Narp through this binding site prevents or reduces the biological activity of Narp, including but not limited to Neurite-outgrowth promoting activity and function as an Extracellular aggregating factor for AMPA receptors.

In one embodiment of this invention, the claimed polypeptide is an antibody that inhibits the binding of a murine antibody to Narp, preferably through the same binding site. This inhibition may be tested by methods known to those skilled in the art.

Another aspect of this invention provides for a pharmaceutical composition comprising a Narp inhibitor, preferably a polypeptide, preferably an antibody or a portion of a toxin, preferably taipoxin. In one embodiment of this invention, this pharmaceutical composition is used for alleviation or reduction of the symptoms and signs associated with damaged neuronal tissues whether resulting from tissue trauma, or from chronic degenerative changes.

The Narp inhibitor may cause inhibition of Narp biological activity through several pathways, preferably through binding. Bound Narp may cease to possess Narp activity due to inactivation of a site or an epitope which is crucial to Narp activity (as is possible, for example, in the case of an inhibitor which is a small chemical compound or a portion of a toxin), or due to a spatial interference caused by the bound inhibitor (as is possible for example in the case of an antibody or a portion of a toxin). As a result of binding of the Narp inhibitor to Narp, Narp may no longer possess the possibility of Narp biological activity, which may include, but is not limited to Neurite-outgrowth promoting activity, and a function as an extracellular aggregating factor for AMPA receptors (which includes an inhibitory effect wherein Narp causes clustering of AMPA receptors on the surface of a cell). This prevention of Narp biological activity may aid in alleviation or reduction of the symptoms and signs associated with damaged neuronal tissues whether resulting from tissue trauma, or from chronic degenerative changes.

By "portion of a toxin" is meant a complete subunit or fragment thereof, having the capacity to bind Narp, preferably derived from the toxin taipoxin, most preferably from gamma-taipoxin.

In one aspect of the claimed invention, a portion of a toxin is used in a pharmaceutical composition comprising as an active ingredient a Narp inhibitor (said portion of a toxin) further comprising a pharmaceutically acceptable diluent or carrier. Preferably, said toxin is taipoxin.

A preferred embodiment of this invention is the usage of gamma-taipoxin, or a fragment thereof, as a Narp inhibitor in a pharmaceutical composition comprising as an active ingredient a Narp inhibitor further comprising a pharmaceutically acceptable diluent or carrier, for alleviation or reduction of the symptoms and signs associated with neuronal damage. Gamma-taipoxin was found to be non-toxic by the inventors of the present invention, as detailed below in Example 3. The pharmaceutical composition described in this invention may further contain a diluent or carrier.

The term "gamma-taipoxin" as used herein refers to the gamma subunit of the taipoxin polypeptide, fragments thereof retaining binding activity, and homologs thereof, preferably having at least 70%, more preferably at least 80%, even more preferable at least 90% or 95% homology thereto. This term is understood to encompass polypeptides resulting from minor alterations in the gamma-taipoxin coding sequence, such as, inter alia, point mutations, deletions and insertions which may cause a difference in a few amino acids between the resultant polypeptide and the naturally occurring gamma-taipoxin. Polypeptides encoded by nucleic acid sequences which bind to the gamma-taipoxin coding sequence or genomic sequence under conditions of highly stringent hybridization, which are well-known in the art (for example Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1988), updated in 1995 and 1998), are also encompassed by this term. Chemically modified gamma-taipoxin or chemically modified fragments of gamma-taipoxin are also included in the term, so long as the binding activity is retained. The polypeptide sequence of gamma-taipoxin is depicted in FIG. 5 (SEQ ID No: 7). Particular fragments of the gamma-taipoxin polypeptide include amino acids 1 20, 21 40, 41 60, 61 80, 81 100, 101 120 and 121 133 of SEQ ID No: 7. Further particular fragments of the gamma-taipoxin polypeptide include amino acids 10 30, 31 50, 51 70, 71 90, 91 110 and 111 133 of SEQ ID No: 7.

The term "binding activity" as used herein in connection with gamma-taipoxin, refers to the ability of gamma-taipoxin to bind to Narp.

A preferred embodiment of the present invention concerns a method for alleviating or reducing damage to the central nervous system in a patient who has suffered an injury to the central nervous system, comprising administering to the patient a pharmaceutical composition comprising as an active ingredient a Narp inhibitor further comprising a pharmaceutically acceptable diluent or carrier, in a sufficient dosage to alleviate or reduce the damage

Another preferred embodiment of the present invention concerns a method for promoting or enhancing recovery in a patient who has suffered an injury to the central nervous system, the method comprising administering to the patient a pharmaceutical composition comprising as an active ingredient a Narp inhibitor further comprising a pharmaceutically acceptable diluent or carrier, in a sufficient dosage to promote or enhance the recovery.

In one aspect of this invention, the injury to the central nervous system which said pharmaceutical composition is aimed at alleviating or reducing, or from which said pharmaceutical composition is aiming to promote or enhance recovery, is an ischemic episode, which may be, but is not limited to, a global or focal cerebral episode.

By "ischemic episode" is meant any circumstance that results in a deficient supply of blood to a tissue. Cerebral ischemic episodes result from a deficiency in the blood supply to the brain. The spinal cord, which is also part of the central nervous system, is equally susceptible to ischemia resulting from diminished blood flow. An ischemic episode may be caused by hypertension, hypertensive cerebral vascular disease, rupture of aneurysm, a constriction or obstruction of a blood vessel- as occurs in the case of a thrombus or embolus, angioma, blood dyscrasias, any form of compromised cardiac function including cardiac arrest or failure, systemic hypotension, cardiac arrest, cardiogenic shock, septic shock, spinal cord trauma, head trauma, seizure, bleeding from a tumor, or other blood loss. It is expected that the invention will also be useful for treating injuries to the central nervous system that are caused by mechanical forces, such as a blow to the head or spine. Trauma can involve a tissue insult such as an abrasion, incision, contusion, puncture, compression, etc., such as can arise from traumatic contact of a foreign object with any locus of or appurtenant to the head, neck, or vertebral column. Other forms of traumatic injury can arise from constriction or compression of CNS tissue by an inappropriate accumulation of fluid (for example, a blockade or dysfunction of normal cerebrospinal fluid or vitreous humor fluid production, turnover, or volume regulation, or a subdural or intracarnial hematoma or edema). Similarly, traumatic constriction or compression can arise from the presence of a mass of abnormal tissue, such as a metastatic or primary tumor.

By "focal ischemia" as used herein in reference to the central nervous system, is meant the condition that results from the blockage of a single artery that supply blood to the brain or spinal cord, resulting in the death of all cellular elements (pan-necrosis) in the territory supplied by that artery.

By "global ischemia" as used herein in reference to the central nervous system, is meant the condition that results from general diminution of blood flow to the entire brain, forebrain, or spinal cord, which causes the death of neurons in selectively vulnerable regions throughout these tissues. The pathology in each of these cases is quite different, as are the clinical correlates. Models of focal ischemia apply to patients with focal cerebral infarction, while models of global ischemia are analogous to cardiac arrest, and other causes of systemic hypotension.

In another aspect of this invention, an additional pharmaceutically effective compound is administered in conjunction with the aforementioned pharmaceutical composition.

By "in conjunction with" is meant that the additional pharmaceutically effective compound is administered prior to, at the same time as, or subsequent to administration of Narp inhibitor.

One embodiment of the claimed invention provides for the preparation of a medicament for the treatment of a patient who has suffered an injury to the central nervous system, using a Narp inhibitor. The Narp inhibitor can be any one of those described herein, and is preferably a polypeptide.

The treatment regimen according to the invention is carried out, in terms of administration mode, timing of the administration, and dosage, so that the functional recovery of the patient from the adverse consequences of the ischemic events or central nervous system injury is improved; i.e., the patient's motor skills (e.g., posture, balance, grasp, or gait), cognitive skills, speech, and/or sensory perception (including visual ability, taste, olfaction, and proprioception) improve as a result of antibody administration according to the invention.

Administration of the antibody or polypeptide or small chemical compound according to the invention can be carried out by any known route of administration, including intravenously, intra-arteially, subcutaneously, or intracerebrally. Using specialized formulations, particularly in the case of active fragments of the anti-Narp antibodies, it may also be possible to administer these orally or via inhalation. Suitable doses and treatment regimens for administering antibodies to an individual in need thereof are discussed in detail below.

The invention can be used to treat the adverse consequences of central nervous system injuries that result from any of a variety of conditions. Thrombus, embolus, and systemic hypotension are among the most common causes of cerebral ischemic episodes. Other injuries may be caused by hypertension, hypertensive cerebral vascular disease, rupture of an aneurysm, an angioma, blood dyscrasias, cardiac failure, cardiac arrest, cardiogenic shock, septic shock, head trauma, spinal cord trauma, seizure, bleeding from tumor, or other blood loss.

Where the ischemia is associated with stroke, it can be either global or focal ischemia, as defined below. It is believed that the administration of an antibody according to the invention is effective, even though administration occurs a significant amount of time following the injury.

A preferred embodiment of the present invention concerns a pharmaceutical composition comprising gamma-taipoxin and a pharmaceutically acceptable carrier.

In an additional embodiment, a composition comprising gamma-taipoxin in an amount effective to treat an injury to the nervous system and a carrier is provided. The carrier may be a pharmaceutically acceptable carrier; further, the composition may be used to treat a patient who has suffered an injury to the central nervous system, such as an ischemic episode, which may be global or focal, or a stroke, by administering- the pharmaceutical composition in a dose sufficient to promote recovery and thereby treat the patient. The administration of the pharmaceutical composition may be periodical. By "periodical" in the context of the administration of a pharmaceutical composition and as used herein is meant administering the pharmaceutical composition in fixed intervals, preferably at fixed times. Such intervals may range from once an hour or every few hours, to once a day, or once every few days or even once a week. More information on dosage and administration regimens is provided in Example 5 below.

An additional embodiment of the present invention concerns the use of gamma-taipoxin in the preparation of a medicament, which may be used for treating a patient who has suffered an injury to the central nervous system, such as, inter alia, a stroke. Another embodiment of the present invention involves a process of identifying a species that modulates binding between Narp and gamma-taipoxin, comprising the steps of: a) contacting Narp with gamma-taipoxin under binding conditions; b) contacting Narp, gamma-taipoxin and a species to be tested under the conditions of step a); and c) comparing the level of binding between Narp and gamma-taipoxin in step a to the level of binding between Narp and gamma-taipoxin in step b), wherein a change in the level of binding is indicative of the ability of the species to modulate the binding between Narp and gamma-taipoxin.

The so identified species may enhance the binding between Narp and gamma-taipoxin.

In general, the term "species" encompasses, inter alia, small chemical molecules, antibodies, antisense oligonucleotides, antisense DNA or RNA molecules, proteins, polypeptides and peptides including peptido-mimetics, expression vectors, lipids, carbohydrates and any other molecule capable of interacting with a naturally occurring molecule.

In an additional embodiment, the present invention provides for a process of identifying a species that possesses the binding activity of gamma-taipoxin comprising the steps of: a) contacting Narp with gamma-taipoxin under binding conditions; b) contacting Narp, gamma-taipoxin and a species to be tested under the conditions of step a); and c) comparing the level of binding between Narp and gamma-taipoxin in step a to the level of binding between Narp and gamma-taipoxin in step b), wherein a lower level of binding between Narp and gamma-taipoxin in step b) (i.e., a decrease in the binding in the presence of the species as compared to the binding in the absence of the species) is indicative of the species possessing gamma-taipoxin-like binding activity.

An additional aspect of the present invention comprises a process of identifying a species that possesses the binding activity of gamma-taipoxin comprising the steps of: a) contacting cells expressing Narp with a species to be tested; b) contacting cells lacking normal Narp expression with the species of step a); and c) assaying for the presence of the species within the cells of step a) and step b), wherein a higher level of the species in the cells of step a) as compared to the level of the species in the cells of step b) is indicative of the species possessing gamma-taipoxin-like binding activity.

In an additional aspect, the present invention provides for a process of identifying a species that possesses the binding activity of gamma-taipoxin comprising the steps of: a) contacting cells expressing Narp with a species to be tested under binding conditions; b) contacting cells expressing Narp with gamma-taipoxin and a species to be tested under the conditions of step a) and c) assaying for the presence of the species within the cells of step a) and step b), wherein a lower level of the species in the cells of step b) as compared to the level of the species in the cells of step a) is indicative of the species possessing gamma-taipoxin-like binding activity.

An additional embodiment of the present invention concerns a process of identifying a species that possesses the binding activity of gamma-taipoxin comprising the steps of: a) contacting cells expressing Narp with gamma-taipoxin under binding conditions; b) contacting cells expressing Narp with gamma-taipoxin and a species to be tested under the conditions of step a); and c) assaying for the presence of gamma-taipoxin within the cells of step a) and step b), wherein a lower level of gamma-taipoxin in the cells of step b) is indicative of the species possessing gamma-taipoxin-like binding activity.

The species identified according to any one of the preceding methods may be a chemical compound.

The detection of binding between Narp and gamma-taipoxin may be performed according to methods known in the art; one preferable method is to perform the screening processes of the present invention with an immuno-fluoresent detection system. For further details on screening assays see Example 7 below.

An additional embodiment of the present invention concerns a process of producing an essentially pure non-toxic preparation of gamma-taipoxin comprising the steps of: a) obtaining crude taipoxin, containing the .alpha., .beta., and .gamma. subunits; b) separating the gamma subunit by gel chromatography; and c) purifying the gamma subunit by performing ion exchange on the preparation resulting from step b.

The methods of the invention have several advantages. First, an antibody, a polypeptide or a small chemical compound can be administered hours, days, or even weeks, following an injury to the central nervous system. This is advantageous because there is no way to anticipate when such an injury will occur. All the events that cause ischemia or trauma, as discussed above, are unpredictable. Second, this therapeutic regimen improves functional performance without adverse side effects.
 


Claim 1 of 18 Claims

1. A method of treating a patient who has suffered an injury to the central nervous system, comprising administering to the patient a dose of a pharmaceutical composition consisting essentially of gamma-taipoxin as an active ingredient effective to alleviate the symptoms associated with damaged neuronal tissues, thereby treating the patient, wherein the gamma-taipoxin consists of the amino acid sequence of SEQ ID NO:7.

 

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If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

     
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