WO2014183110A1 - Methods for the detection of brain injury - Google Patents

Abstract

The invention provides methods for the detection of brain injury, such as traumatic brain injury (TBE) or chronic traumatic encephalopathy (CTE), in a patient (e.g., a human) that has suffered or is at risk of suffering one or more traumatic events. The methods of the invention measure, through non-invasive or invasive means, the expression of System x_c ^- cystine/glutamate transporter expression in patient tissue (e.g., neuronal tissue). The loss of System x_c ^- cystine/glutamate transporter expression is correlated with brain injury, severity, and disease progression.

Description

METHODS FOR THE DETECTION OF BRAIN INJURY

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing date of U.S. Provisional Patent Application Ser. No. 61/822,034, filed May 10, 2013, the disclosure of which is incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Traumatic brain injury (TBI) in the United States accounts for 1.7 million injuries per year. TBI, in many cases, results in long-term cognitive impairment and neuropsychiatric disturbances. Military and civilian personnel exposed to a single severe or repeated mild-to- moderate TBI experience a loss of short and long-term memory, a decreased ability to learn new skills, difficulty in concentrating on specific tasks insomnia, headaches, and anxiety and increased susceptibility to post traumatic stress disorder (PTSD). These cognitive changes may appear immediately after the injury as found in post-concussive syndrome (PCS) or they may take years to manifest as observed in patients suffering from chronic traumatic encephalopathy (CTE). In either case, the patient experiences profound neuro-cognitive alterations that, at the present time, cannot be treated. Previous studies of PCS and CTE have demonstrated that the cognitive impairment is due to pathological alterations in the biochemistry and architecture of the brain. In the acute phase, this occurs over time as a result of an immune mediated inflammation, excitotoxicity, astrogliosis, and apoptosis that results in a loss of neurons, axons, and dendrites in and around the injury zone.

There is also a sub-acute (72 hours-4 weeks) and chronic phase (1 month-years) of TBI in which the brain damage continues. In the case of PCS, cognitive impairment and neurological dysfunction from the sub-acute and chronic phase will eventually resolve over time. However, in the case of CTE, the disease will manifest years after the TBI. CTE differs from PCS in scope and magnitude; CTE is a fatal neurodegenerative disorder that produces neuromotor abnormalities that mimic Parkinson's disease including ataxia, antalgic gate, and dysarthric speech. These changes in cognition and neuromotor function are mediated by pathological alterations in the brain structure. CTE is histologically identified by the presence of hyperphosphorylated tau leading to tau-immunoreactive neurofibrillary tangles (NFTs) with many cases displaying a TDP-43 proteinopathy and/or beta-amyloid plaques. The cerebral hemispheres, the thalamus, and the medial temporal lobe show profound atrophy. The neurofibrillary degeneration of CTE is distinguished from other tauopathies by involvement of the superficial cortical layers and marked accumulation of tau-immunoreactive astrocytes.

Behaviorally, CTE patients do not manifest neuropsychological symptoms until the disease has significantly progressed. Initially, patients suffer short memory loss, executive dysfunction, irritability, restlessness, and insomnia. Over time many patients suffer from emotional instability, volatility and depression. Many CTE patients develop drug and alcohol dependencies that further exacerbate their behavioral issues⁸.

Unfortunately, the diagnosis of CTE is made postmortem; there are currently no noninvasive diagnostic tests or biomarkers available to diagnose the disease. Clinically, cognitive and behavioral changes become the identifying symptom of the disease. However, many individuals remain undiagnosed until their death from suicide, drug overdose, or violence. Of further concern is the relative lack of knowledge regarding exactly what triggers the development of CTE in certain patients, how the disease progresses, and what molecular targets are available as treatment options.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides a non-invasive method for detecting a brain injury in a patient by identifying a patient (e.g., a human, non-human primate, ungulate, or rodent) suffering from or at risk of developing a brain injury, contacting the patient with an imaging agent, such as an antibody, that binds specifically to the System x_c ^~ cystine/glutamate transporter, detecting the imaging agent in the patient, and then comparing the expression profile of System x_c ^~ cystine/glutamate transporter expression in the patient with a control profile. A reduced level of System xc- cystine/glutamate transporter expression in the patient compared to the control profile is diagnostic of a brain injury or disease progression. The methods of the invention can be used to detect a traumatic brain injury or chronic traumatic encephalopathy. In one embodiment, the imaging agent is a radiolabeled positron emission tomography ligand and the imaging agent is detected by positron emission tomography. In another embodiment, the System xc- cystine/glutamate transporter control expression profile is derived from the patient prior to the occurrence of the brain injury. In a further embodiment, the System xc- cystine/glutamate transporter control expression profile is derived from one or more subjects that have not suffered a prior brain injury.

In a second aspect, the invention provides a method for detecting a brain injury in a patient by identifying a patient (e.g., a human, non-human primate, ungulate, or rodent) suffering from or at risk of developing a brain injury, isolating a tissue sample from the patient, contacting the tissue sample with an quantifying agent, such as an antibody or nucleic acid, that binds specifically to the System x_c ^~ cystine/glutamate transporter protein or nucleic acid, detecting the quantifying agent in the tissue sample, and then comparing the expression profile of System x_c ^~ cystine/glutamate transporter expression in the patient with a control profile. A reduced level of System xc- cystine/glutamate transporter expression in the patient compared to the control profile is diagnostic of a brain injury or disease progression. The methods of the invention can be used to detect a traumatic brain injury or chronic traumatic encephalopathy. The tissue sample can be derived from neuronal tissue, cerebral spinal fluid, or peripheral blood. In one embodiment, the quantifying agent is a nucleic acid and the quantifying agent is detected by polymerase chain reaction. In another embodiment, the System xc- cystine/glutamate transporter control expression profile is derived from the patient prior to the occurrence of the brain injury. In a further embodiment, the System xc- cystine/glutamate transporter control expression profile is derived from one or more subjects that have not suffered a prior brain injury.

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

As used herein, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof. The term "a nucleic acid molecule" includes a plurality of nucleic acid molecules.

As used herein, the terms below have the meanings indicated.

The term "bond" refers to a covalent linkage between two atoms, or two moieties when the atoms joined by the bond are considered to be part of larger substructure. A bond may be single, double, or triple unless otherwise specified. A dashed line between two atoms in a drawing of a molecule indicates that an additional bond may be present or absent at that position.

The term "combination therapy" means the administration of two or more therapeutic agents to treat a therapeutic condition or disorder described in the present disclosure. Such administration encompasses co -administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients or in multiple, separate capsules for each active ingredient. In addition, such administration also encompasses use of each type of therapeutic agent in a sequential manner. In either case, the treatment regimen will provide beneficial effects of the drug combination in treating the conditions or disorders described herein.

An "expression profile" or "hybridization profile" of a particular sample is essentially a fingerprint of the state of the sample; while two states may have any particular gene similarly expressed, the evaluation of a number of genes simultaneously allows the generation of a gene expression profile that is unique to the state of the cell. That is, normal tissue may be distinguished from abnormal (e.g., diseased or injured) tissue, and within abnormal tissue, different prognosis states (for example, good or poor long term survival prospects) may be determined. By comparing expression profiles of tissue in different states, information regarding which genes are important (including both up- and down-regulation of genes) in each of these states is obtained. The identification of sequences that are differentially expressed in tissue, as well as differential expression resulting in different prognostic outcomes, allows the use of this information in a number of ways. For example, a particular treatment regime may be evaluated (e.g., to determine whether a therapeutic drug acts to improve the long-term prognosis in a particular patient). Similarly, diagnosis may be done or confirmed by comparing patient samples with known expression profiles. Furthermore, these gene expression profiles (or individual genes) allow screening of drug candidates that alter or normalize tissue expression profiles to impart a clinical benefit.

The term "imaging agent" as used herein refers to any moiety useful for the detection, tracing, or visualization of a compound when coupled thereto. Imaging agents include, e.g., an enzyme, a fluorescent label (e.g., fluorescein), a luminescent label, a bioluminescent label, a magnetic label, a metallic particle (e.g., a gold particle), a nanoparticle, an antibody or fragment thereof (e.g., a Fab, Fab', or F(ab')₂ molecule), and biotin. An imaging agent can be coupled to a compound by, for example, a covalent bond, ionic bond, van der Waals interaction or a hydrophobic bond. An imaging agent can be a radiolabel coupled to or a radioisotope incorporated into the chemical structure of a compound used according to the invention. Methods of detecting such imaging agents include, but are not limited to, positron emission tomography (PET), X-ray computed tomography (CT) and magnetic resonance imaging (MRI).

As used herein interchangeably, a "miR gene product," "microRNA," "miR," or "miRNA" refers to the unprocessed (e.g., precursor) or processed (e.g., mature) RNA transcript from a miR gene. As the miR gene products are not translated into protein, the term "miR gene products" does not include proteins. The unprocessed miR gene transcript is also called a "miR precursor" or "miR prec" and typically comprises an RNA transcript of about 70-100 nucleotides in length. The miR precursor can be processed by digestion with an RNAse (for example, Dicer, Argonaut, or RNAse III (e.g., E. coli RNAse III)) into an active 19-25 nucleotide RNA molecule. This active 19-25 nucleotide RNA molecule is also called the "processed" miR gene transcript or "mature" miRNA.

The term "neurodegenerative disorder" as used herein, refers to any disease, disorder, condition, or symptom characterized by the structural or functional loss of neurons. Neurodegenerative disorders include, e.g., Alzheimer's disease, Parkinson's disease, Huntington's Disease, Lewy Body dementia, and amyotrophic lateral sclerosis (ALS).

As used herein, "probe oligonucleotide" or "probe oligodeoxynucleotide" refers to an oligonucleotide that is capable of hybridizing to a target oligonucleotide. By "miR-specific probe oligonucleotide" or "probe oligonucleotide specific for a miR" is meant a probe oligonucleotide that has a sequence selected to hybridize to a specific miR gene product, or to a reverse transcript of the specific miR gene product.

"Target oligonucleotide" or "target oligodeoxynucleotide" refers to a molecule to be detected (e.g., via hybridization).

As used herein, "sample" refers to any biological matter derived from a subject (e.g., a human). Samples include, but are not limited to, blood, PBMC, plasma, platelets, serum, cerebral spinal fluid (CSF), saliva, cells, tissues, and organs. In certain embodiments of the invention, preferred samples include blood plasma, CSF, and brain tissue.

The phrase "therapeutically effective" is intended to qualify the amount of active ingredients used in the treatment of a disease or disorder. This amount will achieve the goal of reducing or eliminating the disease or disorder.

The term "therapeutically acceptable" refers to those compounds (or salts, esters, prodrugs, tautomers, zwitterionic forms, etc. thereof) which are suitable for use in contact with the tissues of patients without undue toxicity, irritation, and allergic response, are commensurate with a reasonable benefit/risk ratio, and are effective for their intended use.

As used herein, reference to "treatment" of a patient is intended to include prophylaxis. The term "patient" means mammals and non-mammals. Mammals means any member of the mammalian class including, but not limited to, humans; non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, and swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice, and guinea pigs; and the like. Examples of non-mammals include, but are not limited to, birds, and the like. The term "patient" does not denote a particular age or sex.

The term "prodrug" refers to a compound that is made more active in vivo. Certain compounds may also exist as prodrugs, as described in Hydrolysis in Drug and Prodrug Metabolism: Chemistry, Biochemistry, and Enzymology, Testa, Bernard and Wiley- VHCA, Zurich, Switzerland 2003. Prodrugs of the compounds are structurally modified forms of the compound that readily undergo chemical changes under physiological conditions to provide the compound. Additionally, prodrugs can be converted to the compound by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to a compound when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent. Prodrugs are often useful because, in some situations, they may be easier to administer than the compound, or parent drug. They may, for instance, be bio-available by oral administration whereas the parent drug is not. The prodrug may also have improved solubility in pharmaceutical compositions over the parent drug. A wide variety of prodrug derivatives are known in the art, such as those that rely on hydrolytic cleavage or oxidative activation of the prodrug. An example, without limitation, of a prodrug is a compound that is administered as an ester (the "prodrug"), but then is metabolically hydrolyzed to the carboxylic acid, the active entity. Additional examples include peptidyl derivatives of a compound.

Compounds can exist as therapeutically acceptable salts. Suitable salts include those formed with both organic and inorganic acids. Such acid addition salts will normally be pharmaceutically acceptable. However, salts of non-pharmaceutieally acceptable salts may be of utility in the preparation and purification of the compound in question. Basic addition salts may also be formed and be pharmaceutically acceptable. For a more complete discussion of the preparation and selection of salts, refer to Stahl, P. Heinrich, Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCHA, Zurich, Switzerland (2002).

The term "therapeutically acceptable salt" as used herein, represents salts or zwitterionic forms of a compound which are water or oil-soluble or dispersible and therapeutically acceptable as defined herein. The salts can be prepared during the final isolation and purification of the compounds or separately by reacting the appropriate compound in the form of the free base with a suitable acid. Representative acid addition salts include acetate, adipate, alginate, L-ascorbate, aspartate, benzoate, benzenesulfonate (besylate), bisulfate, butyrate, camphorate, camphorsulfonate, citrate, digluconate, formate, fumarate, gentisate, glutarate, glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate, hippurate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethansulfonate (isethionate), lactate, maleate, malonate, DL-mandelate, mesitylenesulfonate, methanesulfonate, naphthylenesulfonate, nicotinate, 2- naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylproprionate, phosphonate, picrate, pivalate, propionate, pyroglutamate, succinate, sulfonate, tartrate, L- tartrate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, para- toluenesulfonate (p-tosylate), and undecanoate. Also, basic groups in the compounds can be quaternized with methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and steryl chlorides, bromides, and iodides; and benzyl and phenethyl bromides. Examples of acids which can be employed to form therapeutically acceptable addition salts include inorganic acids such as hydrochloric, hydrobromic, sulfuric, and phosphoric, and organic acids such as oxalic, maleic, succinic, and citric. Salts can also be formed by coordination of the compounds with an alkali metal or alkaline earth ion.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG.l are fluorescent micrographs of xCT expression in sham and TBI-injured rat brains: Panel A is a surgical sham animal. Panel B is a animal 48 hours after TBI. Panel C is an animal 7 days post-TBI. Panel D is an animal 46 days post-TBI. Panel E is a graphical representation of the loss of and modest recovery of xCT staining that occurs over a 46 day period after TBI. Data collected by laser scanning cytometry.

FIG. 2 are fluorescent micrographs of xCT expression in sham and TBI-injured rat cortex: Panel A shows xCT expression levels in an uninjured sham animal. Panel B shows the loss of xCT expression 48 hours after a severe TBI. Panel C shows the statistical analysis. ***=p<0.001 Two tailed T-test. n=4 animals with 4 replicates each.

FIG. 3 are fluorescent micrographs of xCT staining in the CA1 region of the rat hippocampus: Panel A shows xCT expression in the hippocampus of an uninjured sham animal. Panel B shows the loss of xCT expression in the hippocampus 48 hours after a severe TBI. Panel C shows the statistical analysis of measurements taken from the pyramidal layer. Panel D shows the statistical analysis of measurements taken from the axonal layer. ***=p<0.001 Two tailed T-test. n=4 animals with 4 replicates each.

FIG. 4 are fluorescent micrographs that show glial fibrillary acidic protein (GFAP) expression in sham and TBI-injured rat brains. Panel A is a surgical sham animal. Panel B is an animal 48 hours after TBI. Panel C is an animal 7 days post-TBI. Panel D is an animal 46 days post-TBI. Panel E is a graphical representation of the astrogliosis that occurs over time. Data collected by laser scanning cytometry.

FIG. 5 are fluorescent micrographs that show rat cortical tissue, derived from the side contralateral to the injury, stained for the xc- subunit of the xCT transporter. Panel A is a control (sham) rat 48 hours post-surgery. Panel B is an animal 48 hours post-TBI. Panel C is a graphical representation of xCT expression in rat contralateral cortex in sham and TBI animals 48 hours after injury. Two-tailed unpaired T-test. ***=p<0.001; n=6.

FIG. 6 are fluorescent micrographs that show rat hippocampal (CA1 region) tissue stained for the xc- subunit of the Xct transporter. Panel A is a control (sham) rat 48 hours post- surgery. Panel B is a TBI rat 48 hours post-TBI. Two-tailed unpaired T-test. ***=p<0.001; n=6.

FIG. 7 are fluorescent micrographs that show human cortical brain tissue stained for xCT. Panel A is tissue taken from a non-CTE patient. Panel B was taken from a stage IV CTE patient Two-tailed unpaired T-test. ***=p<0.001; n=4.

DETAILED DESCRIPTION OF THE INVENTION

The invention features methods of detecting, diagnosing, and tracking brain injuries, including traumatic brain injury (TBI), chronic traumatic encephalopathy (CTE), and related conditions, by evaluating the expression of the xCT (SLC7A11) subunit of the cystine/glutamate transporter System x_c ^~ in a patient (e.g., a human) that has suffered or is at risk of suffering a brain injury. The inventors have found that a single brain injury causes loss of System x_c ^~ transporter expression in experimental animal models. System x_c ^~ is an transporter that regulates cellular glutamate and cysteine exchange. The influx of cystine is the rate limiting step in the production of the critical anti-oxidant glutathione (GSH). Dysregulation of glutamate/cysteine exchange and GSH production may cause or contribute to neuronal damage experienced by patients suffering from TBI or CTE.

The invention provides for the non-invasive measurement, e.g., by positron emission tomography (PET) and radiolabeled System x_c ^"-specific ligands, of System x_c ^~ transporter expression in a patient (e.g., a human). The invention further provides for the measurement of xCT expression in tissue samples (e.g., brain tissue, blood sample, or cerebral spinal fluid sample) derived from a patient (e.g., a human), either ex vivo or post-mortem, considered to have suffered from, or is at risk of suffering from, a TBI, CTE, or other neurological defect. The methods of the invention can be used to diagnose, predict, and monitor the progression of neuronal damage caused by single or multiple traumatic events, such as those experienced in, e.g., falling accidents, active sports (e.g., boxing, wrestling, and football), vehicular or labor accidents, or by law enforcement or military personnel (i.e., victims of improvised explosive devices).

The methods of the invention include diagnostic or prognostic imaging can be performed on a patient (e.g., a human) that has suffered a traumatic event that may have caused a brain injury. For example, a football player that has suffered a concussion may be subjected to the methods of the invention to determine the presence and degree of brain injury (e.g., TBI). Alternatively, the methods of the invention can be employed to establish "baseline" neuronal System x_c ^~ transporter expression profiles in a patient (e.g., a human) population thought to be at increased risk of experiencing a brain injury in the future, such as active sports players and military personnel. Such baseline expression profiles can be used as "personalized" expression control profiles that can be used to more accurately diagnose brain injury and measure disease (e.g., CTE) progression over a period of time.

The methods of the invention can be used at a single point in time (e.g., for diagnostic purposes) or longitudinally (e.g., to measure disease progression).

The control System x_c ^~ transporter expression profiles can be derived from a panel of one or more healthy subjects, i.e., individuals that have not suffered a brain injury. In certain cases, matching certain demographic, environmental, or genetic traits between the test subject (e.g., a human patient) and the control panel may increase the sensitivity of the diagnostic test. For example, a control expression profile derived from healthy individuals that are, e.g., the same age or sex as the patient (e.g., a human) under examination. Additional System x_c ^~ transporter expression control profiles taken from individuals known to have suffered one or more traumatic events that resulted in brain injury can be used to guide clinicians in determining the severity of individual injuries. Such profiles also allow clinicians to make more accurate prognosis of disease (e.g., CTE) progression and symptomology.

The System x_c ^~ Cystine/Glutamate Transporter and Brain Injury

The present invention embraces the discovery that severe TBI produces a global loss of the xCT (SLC7A11) subunit of a key amino acid transporter, System x_c ^~ (Figures 1-7). Furthermore, System x_c ^~ transporter immunoreactivity completely disappears 48 hours after severe TBI, but gradually begins to recovers over 46 dys. It does not, however, achieve pre-TBI levels, suggesting TBI produces a permenant loss of System x_c ^~ throughout the brain.

System x_c ^~ is a cystine/glutamate antiporter comprised of two distinct subunits xCT and 4F2hc (SLC3A2) and a member of the heteromeric amino acid transporter (HAT) family. Under physiological conditions, the System x_c ^~ transporter mediates the exchange of extracellular L-cystine and intracellular L-glutamate across the plasma membrane. In the CNS, the influx of L-cystine represents the critical rate limiting step in the biosynthesis of glutathione (GSH) while the concurrent efflux of L-glutamate serve as a non- vesicular route of excitatory neurotransmitter release to initiate excitatory amino acid (EAA) signalling. GSH serves as the key cellular antioxidant responsible for scavenging reactive oxygen species (ROS) that develop as a result of physiological cellular metabolism. Thus a global loss of System x_c ^~ activity would result in decreased intracellular glutathione levels, leaving the CNS vunerable to oxidative stress due to an increase in cellular ROS. While it is likely that other antioxidant systems such as SOD1, SOD2, and catalase would initially metabolize ROS, as an individual ages these compenstatory enzymes lose scavenging efficiency resulting in a prolonged elevation in ROS. With glutathione missing and supporting antioxidant systems operating with less efficiency, ROS accumulation could result in unsurmountable oxidative stress leading to neuropathology associated with the gradual process of neurodegenerative events leading to CTE.

Methods of Diagnostic Imaging

The present invention provides for the non-invasive diagnosis and medical evaluation of patients (e.g., a human) suffering from, or at risk of suffering from brain injury, including TBI and CTE. For example, an imaging agent (e.g., an antibody) specific for System x_c ^~ can also be used, alone or in combination with other agents and compounds, in medical imaging applications to diagnose or follow the progression of diseases, disorders, conditions or symptoms related to TBI or CTE in a patient (e.g., a human). Radiologists and other medical clinicians skilled in the use of radiographic imaging devices, such as positron emission tomography (PET) scanners, and methods of imaging tracer compounds, such as a radionuclide-labeled antibody specific for the System x_c ^~ transporter protein, in a patient are widely known (e.g., Saha, Basics of PET Imaging: Physics, Chemistry, and Regulations, Springer (2010) ISBN 978-1-4419-0804-9, hereby incorporated by reference).

The methods of the invention can be used to diagnose a brain injury (e.g., TBI or CTE) in a patient (e.g., a human) that has experienced a head injury or other bodily physical shock that can damage brain cells (e.g., neurons, glia), brain tissue (e.g., cortex, medulla), or critical brain structures (e.g., synaptic junctions and pathways). Ideally, the expression of the System x_c ^~ transporter following the TBI event can be compared to a control sample taken from the patient prior to event that is suspected of causing a brain injury. Alternatively, the System x_c ^~ transporter expression profile measured in a patient can be compared to control profiles derived from one or more individuals (i.e., "population" control). In this case, the control profiles may be derived from individuals that are most similar to the patient under examination. For example, age and/or gender matched control profiles may allow greater sensitivity in making a diagnosis of brain injury. The control profiles may be derived from "healthy" individuals that have not experienced a brain injury or from "injured" individuals that have suffered one or more traumatic events resulting in neuronal damage.

The methods of the present invention are also useful for the non-invasive medical imaging and diagnosis of animals, e.g., domesticated animal, companion animals (e.g., dogs and cats), exotic animals, farm animals (e.g., ungulates, including horses, cows, sheep, goats, and pigs), and animals used in scientific research (e.g., rodents and non-human primates) using similar imaging methodologies (e.g., PET, CT) and species-relevant System x_c ^~ transporter ligands (e.g., antibodies).

Ex Vivo Diagnosis of Brain Injury

The methods of the invention also provide for the evaluation of neuronal cell damage and brain injury by determining the expression of System x_c ^~ transporter expression in brain or CNS tissue samples. While the non-invasive methods of the invention may be preferable in most clinical circumstances, the availability of tissue biopsy or necropsy samples from patients that suffered a brain injury would allow for the ex vivo quantification of System x_c ^~ transporter expression. Any method suitable to quantify System x_c ^~ transporter protein or nucleic acid expression may be used. For example, protein arrays, Western blots, immunohistochemistry, and laser scanning cytometry can be used to detect and measure the expression of System x_c ^~ transporter in a tissue sample. Likewise, nucleic acid based assays, such as quantitative PCR to measure cellular System x_c ^~ transporter mR A, can also be used to detect and measure neuronal damage in a tissue sample.

Compound Administration and Formulation

Basic addition salts can be prepared during the final isolation and purification of the compounds by reaction of a carboxy group with a suitable base such as the hydroxide, carbonate, or bicarbonate of a metal cation or with ammonia or an organic primary, secondary, or tertiary amine. The cations of therapeutically acceptable salts include lithium, sodium, potassium, calcium, magnesium, and aluminum, as well as nontoxic quaternary amine cations such as ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine, dibenzylamine, N,N-dibenzylphenethylamine, 1-ephenamine, and NN- dibenzylethylenediamine. Other representative organic amines useful for the formation of base addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine, and piperazine.

A salt of a compound can be made by reacting the appropriate compound in the form of the free base with the appropriate acid. The novel compounds described herein can be prepared in a form of pharmaceutically acceptable salts that will be prepared from nontoxic inorganic or organic bases including but not limited to aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc, and the like. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally-occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, ethylamine, 2-diethylaminoethano, 1 ,2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydroxylamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, trishydroxylmethyl amino methane, tripropyl amine, and tromethamine.

If the compounds of the invention are basic, salts could be prepared in a form of pharmaceutically acceptable salts that will be prepared from nontoxic inorganic or organic acids including but not limited to hydrochloric, hydrobromic, phosphoric, sulfuric, tartaric, citric, acetic, fumaric, alkylsulphonic, naphthalenesulphonic, /?ara-toluenesulphonic, camphoric acids, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, gluconic, glutamic, isethonic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, and succinic.

While it may be possible for the compounds of the invention to be administered as the raw chemical, it is also possible to present them as a pharmaceutical formulation. Accordingly, the present invention provides a pharmaceutical formulation comprising a compound or a pharmaceutically acceptable salt, ester, prodrug or solvate thereof, together with one or more pharmaceutically acceptable carriers thereof and optionally one or more other therapeutic ingredients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Proper formulation is dependent upon the route of administration chosen. Any of the well-known techniques, carriers, and excipients may be used as suitable and as understood in the art; e.g., in Remington's Pharmaceutical Sciences. The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compression processes.

The formulations include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous, intraarticular, and intramedullary), intraperitoneal, transmucosal, transdermal, rectal and topical (including dermal, buccal, sublingual and intraocular) administration although the most suitable route may depend upon for example the condition and disorder of the recipient. When used in the diagnostic imaging methods of the invention, the compounds of the invention are preferably administered to the patient (e.g., a human) by intravenous injection. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association a compound of the present invention or a pharmaceutically acceptable salt, ester, prodrug or solvate thereof ("active ingredient") with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.

Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.

Pharmaceutical preparations which can be used orally include tablets, push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Tablets may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with binders, inert diluents, or lubricating, surface active or dispersing agents. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein. All formulations for oral administration should be in dosages suitable for such administration. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

The compounds of the invention may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in powder form or in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or sterile pyrogen-free water, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Formulations for parenteral administration include aqueous and non-aqueous (oily) sterile injection solutions of the active compounds which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

In addition to the formulations described previously, the compounds of the invention may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

For buccal or sublingual administration, the compounds of the invention may take the form of tablets, lozenges, pastilles, or gels formulated in conventional manner. Such compositions may comprise the active ingredient in a flavored basis such as sucrose and acacia or tragacanth.

The compounds of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter, polyethylene glycol, or other glycerides.

Compounds of the invention may be administered topically, that is by non-systemic administration. This includes the application of a compound of the present invention externally to the epidermis or the buccal cavity and the instillation of such a compound into the ear, eye and nose, such that the compound does not significantly enter the blood stream. In contrast, systemic administration refers to oral, intravenous, intraperitoneal and intramuscular administration.

Formulations suitable for topical administration include solid, liquid or semi-liquid preparations suitable for penetration through the skin to the site of inflammation such as gels, liniments, lotions, creams, ointments or pastes, and drops suitable for administration to the eye, ear or nose. The active ingredient may comprise, for topical administration, from 0.001% to 10% w/w, for instance from 1% to 2% by weight of the formulation. It may however comprise as much as 10%> w/w but preferably will comprise less than 5% w/w, more preferably from 0.1% to 1% w/w of the formulation.

Via the topical route, the pharmaceutical composition according to the invention may be in the form of liquid or semi liquid such as ointments, or in the form of solid such as powders. It may also be in the form of suspensions such as polymeric microspheres, or polymer patches and hydrogels allowing a controlled release. This topical composition may be in anhydrous form, in aqueous form or in the form of an emulsion. The compounds are used topically at a concentration generally of between 0.001 % and 10% by weight and preferably between 0.01% and 1% by weight, relative to the total weight of the composition.

For administration by inhalation, the compounds according to the invention are conveniently delivered from an insufflator, nebulizer pressurized packs or other convenient means of delivering an aerosol spray. Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Alternatively, for administration by inhalation or insufflation, the compounds according to the invention may take the form of a dry powder composition, for example a powder mix of the compound and a suitable powder base such as lactose or starch. The powder composition may be presented in unit dosage form, in for example, capsules, cartridges, gelatin or blister packs from which the powder may be administered with the aid of an inhalator or insufflator.

Preferred unit dosage formulations are those containing an effective dose, as herein below recited, or an appropriate fraction thereof, of the active ingredient.

It should be understood that in addition to the ingredients particularly mentioned above, the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.

The compounds of the invention may be administered orally or via injection at a dose of from 0.1 to 500 mg/kg per day. The dose range for adult humans is generally from 5 mg to 2 g/day. Tablets or other forms of presentation provided in discrete units may conveniently contain an amount of compound of the invention which is effective at such dosage or as a multiple of the same, for instance, units containing 5 mg to 500 mg, usually around 10 mg to 200 mg.

Compounds according to the invention can be administered at a daily dose of about 0.001 mg/kg to 100 mg/kg of body weight, in 1 to 3 dosage intakes. Further, compounds can be used systemically, at a concentration generally of between 0.001 % and 10% by weight and preferably between 0.01 % and 1 % by weight, relative to the weight of the composition.

The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.

The compounds of the invention can be administered in various modes, e.g. orally, topically, or by injection. The precise amount of compound administered to a patient will be the responsibility of the attendant physician. The specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diets, time of administration, route of administration, rate of excretion, drug combination, the precise disorder being treated, and the severity of the indication or condition being treated. Also, the route of administration may vary depending on the condition and its severity. In certain instances, it may be appropriate to administer at least one of the compounds of the invention described herein (or a pharmaceutically acceptable salt, ester, or prodrug thereof) in combination with another therapeutic agent. By way of example only, if one of the side effects experienced by a patient upon receiving one of the compounds herein is hypertension, then it may be appropriate to administer an anti-hypertensive agent in combination with the initial therapeutic agent. Or, by way of example only, the therapeutic effectiveness of one of the compounds described herein may be enhanced by administration of an adjuvant (i.e., by itself the adjuvant may only have minimal therapeutic benefit, but in combination with another therapeutic agent, the overall therapeutic benefit to the patient is enhanced). Or, by way of example only, the benefit of experienced by a patient may be increased by administering one of the compounds described herein with another therapeutic agent (which also includes a therapeutic regimen) that also has therapeutic benefit. By way of example only, in a treatment for pain involving administration of one of the compounds described herein, increased therapeutic benefit may result by also providing the patient with another therapeutic agent for pain. In any case, regardless of the disease, disorder or condition being treated, the overall benefit experienced by the patient may simply be additive of the two therapeutic agents or the patient may experience a synergistic benefit.

Specific, non-limiting examples of possible combination therapies include use of the compounds of the invention together with inert or active compounds, or other drugs including wetting agents, flavor enhancers, preserving agents, stabilizers, humidity regulators, pH regulators, osmotic pressure modifiers, emulsifiers, UV-A and UV-B screening agents, antioxidants, depigmenting agents such as hydroquinone or kojic acid, emollients, moisturizers, for instance glycerol, PEG 400, or urea, antiseborrhoeic or antiacne agents, such as S- carboxymethylcysteine, S-benzylcysteamine, salts thereof or derivatives thereof, or benzoyl peroxide, antibiotics, for instance erythromycin and tetracyclines, chemotherapeutic agent, for example, paclitaxel, antifungal agents such as ketoconazole, agents for promoting regrowth of the hair, for example, minoxidil (2,4-diamino-6-piperidinopyrimidine 3-oxide), non-steroidal anti-inflammatory agents, carotenoids, and especially p-carotene, antipsoriatic agents such as anthralin and its derivatives, eicosa-5,8,11,14-tetraynoic acid and eicosa-5,8,11-triynoic acid, and esters and amides thereof, retinoids, e.g., RAR or RXR receptor ligands, which may be natural or synthetic, corticosteroids or oestrogens, alpha-hydroxy acids and a-keto acids or derivatives thereof, such as lactic acid, malic acid, citric acid, and also the salts, amides or esters thereof, or p-hydroxy acids or derivatives thereof, such as salicylic acid and the salts, amides or esters thereof, ion-channel blockers such as potassium-channel blockers, or alternatively, more particularly for the pharmaceutical compositions, in combination with medicaments known to interfere with the immune system, anticonvulsant agents include, and are not limited to, topiramate, analogs of topiramate, carbamazepine, valproic acid, lamotrigine, gabapentin, phenytoin and the like and mixtures or pharmaceutically acceptable salts thereof. A person skilled in the art will take care to select the other compound(s) to be added to these compositions such that the advantageous properties intrinsically associated with the compounds of the invention are not, or are not substantially, adversely affected by the envisaged addition.

In any case, the multiple therapeutic agents (at least one of which is a compound of the present invention) may be administered in any order or even simultaneously. If simultaneously, the multiple therapeutic agents may be provided in a single, unified form, or in multiple forms (by way of example only, either as a single pill or as two separate pills). One of the therapeutic agents may be given in multiple doses, or both may be given as multiple doses. If not simultaneous, the timing between the multiple doses may be any duration of time ranging from a few minutes to four weeks.

Thus, in another aspect, methods for treating diseases, disorders, conditions, or symptoms in a patient (e.g., a human or animal) in need of such treatment are presented herein, the methods comprising the step of administering to the patient an amount of a compound of the invention effective to reduce or prevent the disease, disorder, condition, or symptom, in combination with at least one additional agent for the treatment of said disorder that is known in the art.

Examples

Example 1. Gene Array Analysis Before and After TBI in Rodent Model.

System x_c ^~ is a cystine/glutamate antiporter comprised of two distinct subunits xCT and 4F2hc (SLC3A2) and a member of the heteromeric amino acid transporter (HAT) family. Under physiological conditions, System x_c ^~ mediates the exchange of extracellular L-cystine and intracellular L-glutamate across the plasma membrane. In the CNS, the influx of L-cystine represents the critical rate limiting step in the biosynthesis of glutathione (GSH) while the concurrent efflux of L-glutamate serve as a non-vesicular route of excitatory neurotransmitter release to initiate excitatory amino acid (EAA) signalling. GSH serves as the key cellular antioxidant responsible for scavenging reactive oxygen species (ROS) that develop as a result of physiological cellular metabolism. Thus a global loss of System x_c ^~ activity would result in decreased intracellular glutathione levels, leaving the CNS vunerable to oxidative stress due to an increase in cellular ROS. While it is likely that other antioxidant systems such as SOD1, SOD2, and catalase would initially metabolize ROS, as an individual ages these compenstatory enzymes lose scavenging efficiency resulting in a prolonged elevation in ROS. With glutathione missing and supporting antioxidant systems operating with less efficiency, ROS accumulation could result in unsurmountable oxidative stress leading to neuropathology associated with the gradual process of neurodegenerative events leading to CTE.

In animal studies we have found that a single TBI produced a rapid, global, long-term loss of a key transporter protein subunit, xCT (SLC7A11; FIG. 1 and 2). Over time (46 days post-TBI), the levels of xCT gradually returned but never reached pre -TBI levels suggesting the injury induced a long-term loss of xCT. xCT is the catalytic subunit of System xc-, a ubiquitous antiporter responsible for the biosynthesis of gluthathione (GSH) in the brain. GSH is the primary cellular anti-oxidant that scavenges damaging reactive oxygen species (ROS) that develop as a result of normal metabolism or neuronal injury.

Example 2. xCT Protein Expression Studies in Animal TBI Model.

Twenty-seven male Wistar rats were given a severe lateral fluid percussion TBI. Severity was determined by neurological severity scoring 24 hours post-TBI. Animals were neurologically assessed weekly and at the end of the study were cognitively assessed in the Morris Water Maze to determine and compare the level of impairment to uninjured shams. Animals were harvested at 48h (primary damage) and 45d (secondary damage) post-TBI to assess both acute and long- term effects.

Coronal whole brain sections were antibody stained for the xCT subunit. At 48h post- TBI we observed an almost complete global loss (both hemispheres and all regions) of the xCT subunit (-4% of the un-injured control). At 46 days post-TBI, xCT gradually returned to approximately 25% of the pre-TBI levels (as determined by comparison of surgical shams) but never achieved complete recovery in any of the animals studied. In an effort to understand how the brain may compensate for the loss of functional System xc- we performed an array analysis of brain tissue to assess the activity of other reactive oxygen species (ROS) defense mechanisms. We fully expected the other ROS defense genes to upregulate to compensate; the exact opposite occurred. SOD1, SOD2, ApoE, UCP3 and GCLC (the first rate limiting step in the production of GSH) are all antioxidant defense genes that were significantly downregulated 48 hours after TBI. In an effort to understand how they were downregulated, we analyzed miRNAs. miRNAs are small, potent inhibitory molecules that cross the blood brain barrier and can be found in the plasma. From the TBI rat brains, we found a signature panel of 10 miRNAs that are directly linked to System xc- supression that were significantly altered as a result of TBI.

Example 3. xCT Expression Profiles in Human Brain Tissue.

Brain sections taken from the cortex of four deceased CTE patients (provided by Boston University) clearly showed a significant decrease in xCT staining when compared to non-CTE controls. To further confirm the animal data with human testing, we performed a miRNA analysis of the human CTE brain tissue. We found that showed that six of the inhibitory miRNAs we discovered in the rat TBI brain tissue were also significantly up or down regulated in the CTE brain.

Example 4. Mechanism of Action.

xCT is a subunit of a key glutamate transporter, System xc-. System xc- is an exchange transporter that moves glutamate out of the cell and cystine in. The cystine the rate limiting step in the production of the anti-oxidant glutathione (GSH). Previous research has shown that a loss of System xc- function results in a profound loss of GSH. This in turn leads to a significant increase in ROS mediated lipid damage. Our finding that TBI results in a profound, global loss of of functional System xc- suggests that TBI is inducing a prolonged loss of GSH and thus an increase in ROS mediated damage throughout the brain. This finding is further substantiated by our array data showing that TBI results in the profound downregulation of SOD1, SOD2, GCLC, ApoE, and UCP3 at 48 hours after TBI. All of these genes play a key role in the reduction of ROS. The fact that these genes are being downregulated in conjunction with the loss of System xc- suggests that the TBI brain has very little defense against ROS mediated damage.

We hypothesize that the loss of functional System xc- occurs after every TBI. We further hypothesize that, over time, System xc- function gradually recovers. If, however, the individual suffers another TBI prior to full recovery, System xc- is further reduced. Once an individual experiences a sufficient number of TBIs, System xc- never fully recovers leaving the brain without sufficient GSH protection. The brain compensates over time by increasing the activity of other antioxidant defense mechanisms such as SOD1, SOD2, and catalase. Thus, the brain adequately neutralizes harmful ROS as they are produced from cellular metabolism. However, as the individual ages the enzyme -based ROS defense mechanisms begin to lag. This allows the balance of ROS to shift upwards leading to gradual lipid peroxidation and neuronal death that takes years to occur. We propose it is this mechanism of System xc- loss and GSH reduction that occurs leading to the ROS-mediated development of CTE. We further propose that decrease in System xc- is mediated by miRNAs the suppress the production of System xc- in the brain. Further supporting our hypothesis is the work of Shea et al that found that deficiencies in apolipoprotein E (ApoE) (as found in the epsilon 4 allele) resulted in a significant increase in the production of ROS. Furthermore, there was a compensatory increase in glutathione to, presumably, neutralize increased ROS. This finding is crucial because many individuals that are post-mortem diagnosed with CTE have an ApoE anomaly. The loss of System xc- may explain why ApoE individuals are more susceptible to the development of CTE; TBI induces a global loss of the xCT subunit that results in reduced GSH levels in a brain already undergoing ROS damage.

All Embodiments

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Other embodiments are within the claims.

Claims

CLAIMS What is claimed is:

1. A method for detecting a brain injury in a patient comprising the steps of

identifying a patient suffering from or at risk of developing a brain injury;

contacting said patient with an imaging agent that binds specifically to System x_c ^~ cystine/glutamate transporter;

detecting said imaging agent in said patient; and

comparing the expression profile of System x_c ^~ cystine/glutamate transporter expression in said patient with a control profile;

wherein a reduced level of System x_c ^~ cystine/glutamate transporter expression in said patient compared to said control profile is diagnostic of a brain injury or disease progression.

2. The method of claim 1, wherein said brain injury is traumatic brain injury or chronic traumatic encephalopathy.

3. The method of claim 1, wherein said patient is a human.

4. The method of claim 1, wherein said patient is a non-human primate, ungulate, or rodent.

5. The method of claim 1, wherein said imaging agent is an antibody.

6. The method of claim 1, wherein said imaging agent is a radiolabeled positron emission tomography ligand.

7. The method of claim 1, wherein said detecting step comprises position emission tomography.

8. The method of claim 1, wherein said control profile comprises a System x_c ^~ cystine/glutamate transporter expression profile derived from said patient prior to the occurrence of said brain injury.

9. The method of claim 1, wherein said control profile comprises a System x_c ^~ cystine/glutamate transporter expression profile derived from one or more subjects that have not suffered prior brain injury.

10. A method for detecting a brain injury in a patient comprising the steps of

identifying a patient suffering from or at risk of developing a brain injury;

isolating a tissue sample from said patient;

contacting said tissue sample with a quantifying agent that binds specifically to System x_c ^~ cystine/glutamate transporter protein or nucleic acid;

detecting said quantifying agent in said tissue sample; and

comparing the expression profile of System x_c ^~ cystine/glutamate transporter expression in said patient with a control profile;

wherein a reduced level of System x_c ^~ cystine/glutamate transporter expression in said patient compared to said control profile is diagnostic of a brain injury or disease progression.

11. The method of claim 10, wherein said brain injury is traumatic brain injury or chronic traumatic encephalopathy.

12. The method of claim 10, wherein said patient is a human.

13. The method of claim 10, wherein said patient is a non-human primate, ungulate, or rodent.

14. The method of claim 10, wherein said tissue sample is neuronal tissue or cerebral spinal fluid.

15. The method of claim 10, wherein said tissue sample is peripheral blood.

16. The method of claim 10, wherein said quantifying agent is a nucleic acid.

17. The method of claim 10, wherein said detecting step comprises polymerase chain reaction.

18. The method of claim 10, wherein said quantifying agent is an antibody.

19. The method of claim 10, wherein said control profile comprises a System x_c ^~ cystine/glutamate transporter expression profile derived from said patient prior to the occurrence of said brain injury.

20. The method of claim 10, wherein said control profile comprises a System x_c ^~ cystine/glutamate transporter expression profile derived from one or more subjects that have not suffered prior brain injury.