Biomarker sets for diagnosis, prognosis, and treatment of traumatic brain injury

Abstract

Here disclosed is a set of biomarkers useful for diagnosis, prognosis, treatment, determining the short- and long-term effects of traumatic brain injury (TBI) in a subject. Methods for using the biomarkers also are described.

Description

Attorney Docket No. 15969-052PC0 BIOMARKER SETS FOR DIAGNOSIS, PROGNOSIS, AND TREATMENT OF TRAUMATIC BRAIN INJURY CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is an international PCT application which claims the benefit of United States provisional application serial no. 63 / 686372, filed 23 August 2024 and United States provisional application serial no. 63 / 686630, filed 23 August 2024. The entire contents of the aforementioned applications are hereby incorporated by reference as if fully set forth herein. GOVERNMENT FUNDING SUPPORT

[0002] This invention was made with government support under grant no. HR0011262137, awarded by the Defense Advanced Research Projects Agency. The government has certain rights in the invention BACKGROUND

[0003] 1. Field of the Invention

[0004] This invention relates to the general field of medicine, in particular methods for diagnosis, prognosis, and treatment of traumatic brain injury (TBI). The application here discloses the development of biomarker sets, identified using phosphoproteomics and metaboloproteomics, which allows medical personnel to diagnose TBI, and to determine a prognosis and suitable treatment methods for TBI.

[0005] 2. Background of the invention

[0006] Traumatic brain injury (TBI) is a heterogenous disease state that can result from several sources such as ballistic, blunt, or blast exposure. TBI has recently been understood to be a serious consequence of injuries common in sport, vehicular accidents, military service, and the like.Attorney Docket No. 15969-052PC0

[0007] TBIs induced by blast exposure are a common concern and major cause of morbidity for military and civilian personnel and are not well understood. Various long-term consequences of blast exposure, including cognitive impairment and other neuropsychiatric conditions, have been well documented. However, molecular events mediating the transition from the initial injury to the subsequent psychiatric sequalae are less understood, hampering efforts of developing effective and targeted preventative therapeutics. In particular, identification of molecular changes in the immediate aftermath of blast-induced trauma that initiate the escalating cascade of physiological changes can inform opportune intervention targets in this critical time window. The lack of understanding of the mechanisms of the sequelae of traumatic brain injury has hampered progress in providing accurate diagnosis and prognosis to patients having or suspected of having traumatic brain injury, and in proving targets for the treatment of the metabolic, functional, and other effects of traumatic brain injury.

[0008] Phosphoproteomics, the large-scale analysis of protein phosphorylation sites, is a tool that allows one to define signaling network regulation and dysregulation in normal and pathological conditions. Metabolomics is a study of the set of metabolites present within a cell, tissue, or organism that allows one to obtain a detailed characterization of metabolic phenotypes.

[0009] Phosphorylation is a reversible post-translational modification that is critical in the body for regulating inter- and intra-cellular signaling networks. In mammalian cells, phosphorylation typically occurs on the side chains of three amino acids; serine, threonine and tyrosine. The addition of a phosphate group to a protein or peptide can have a variety of effects, including changes in conformation, stability, activity, subcellular localization, or its potential interactions.

[0010] Alterations in the phosphorylation pattern(s) of proteins and / or the metabolites present in a cell or tissue, including number, location, timing, duration, and intensity, play a critical role in governing signaling networks. Most major cellular processes, including proliferation, migration, apoptosis, and others, are regulated by protein phosphorylation-mediated signaling cascades.Attorney Docket No. 15969-052PC0 SUMMARY

[0011] There is a need in the art for methods to diagnose and treat TBI and to increase our understanding of traumatic brain injury and their pathological sequelae. While a growing body of literature links blast exposures to persistent neurocognitive decline, to this date, there are no FDA approved drugs or diagnostics methods to treat or mitigate the risk of blast-related or other traumatic brain injury. To address this gap, we used our established mouse and ferret models of blast exposure and both phosphoproteomics and metaboloproteomics approaches to identify phosphorylated proteins from tissue samples obtained within first few seconds through four hours following blast exposure in two different species (mice and ferrets) employing a high- fidelity advanced blast simulator, which closely mimics blast exposure experienced by the military and civilians. The invention described herein thus provides embodiments related to a set of biomarkers for this purpose. In particular, the invention relates to a set of biomarkers useful in diagnosing TBI, that includes at least 3 proteins selected from the group consisting of: (a) Serpin Family F Member 2 (SERPINF2); (b) Serpin Family D Member 2 (SERPIND1); (c) Septin 2 (SEPTIN2); (d) Protein Kinase C Beta (PRKCB); (e) Microtubule Associated Protein Tau (MAPT); (f) Microtubule Associated Protein 2 (MAP2); (g) Microtubule Associated Protein 1A (MAP1A); (h) Inter-Alpha-Trypsin Inhibitor Heavy Chain 2 (ITIH2); (i) Heat Shock Protein 90 Alpha Family Class A Member 1 (HSP90AA1); (j) DnaJ Heat Shock Protein Family Member C5 (DNAJC5); and (k) Alpha 2-HS Glycoprotein (AHSG).

[0012] In a specific embodiment, the set of biomarkers includes at least 3 proteins selected from the group consisting of: (a) SERPINF2; (b) SEPTIN2; (c) PRKCB; (d) MAPT;Attorney Docket No. 15969-052PC0 (e) MAP2; and (f) MAP1A.

[0013] In another specific embodiment, the set of biomarkers includes at least 3 proteins selected from the group consisting of: (a) SERPIND1; (b) SEPTIN2; (c) PRKCB; (d) MAPT; (e) MAP2; and (e) MAP1A.

[0014] In another specific embodiment, the set of biomarkers includes at least 3 proteins selected from the group consisting of: (a) SERPIND1; (b) ITIH2; (c) HSP90AA1; (d) DNAJC5; and (e) AHSG.

[0015] In further embodiments, the invention relates to a set of metabolite biomarkers comprising at least 2 compounds selected from the group consisting of: L-glutamic acid; and / or (a) metabolites listed herein; and / or (b) metabolites listed herein.

[0016] According to a further embodiment, a method of diagnosing traumatic brain injury (TBI) in a subject in need thereof is provided. The method involves (a) obtaining a sample of injured brain tissue from the subject; (b) testing the injured brain tissue for phosphorylation of a set of biomarkers such as described above in the injured brain tissue; and (c) diagnosing the subject with TBI when the test results in (b) exceed normal values in the assay when compared to internal standard. In a specific example, the subject is human.

[0017] A further embodiment pertains to a method of treating TBI in a subject, such as a human subject, in need thereof. The method involves (a) obtaining a sample of injured brain tissueAttorney Docket No. 15969-052PC0 from the subject; (b) testing the injured brain tissue for phosphorylation of the set of biomarkers of claim 1 in the injured brain tissue; and (c) treating the subject by administering an effective amount of a therapeutic agent to the subject, wherein the amount is based on fold changes of levels of the proteins tested compared to the internal standard levels. The methods of treating and or the method of diagnosing TBI may further involve administering to the subject an agent selected from the group consisting of an N-methyl-D-aspartate (NMDA) receptor blocker, a glutamate influx transporter upregulator, a protein kinase C beta (PKCβ) activator, a glial group II metabotropic glutamate receptor (glial GpII mGluR) antagonist, a glial group III metabotropic glutamate receptor (glial GpIII mGluR) agonist, and a glutamate remover. The glutamate remover may be selected from the group consisting of pyruvate and oxaloacetate.

[0018] Another embodiment, pertains to a method of diagnosing traumatic brain injury (TBI) in a subject in need thereof. The method involves (a) obtaining a sample of injured brain tissue, or blood plasma or serum, from the subject; (b) testing the injured brain tissue, or the blood plasma or serum, for the level of the set of metabolomic biomarkers of claim 1 in the injured brain tissue; and (c) diagnosing the subject with TBI when the test results in (b) exceed normal levels when compared to an internal standard. The method may further involve (d) treating the diagnosed subject for TBI. In a specific example, the subject is human.

[0019] According to another embodiment, provided is a method of treating TBI in a subject (e.g. human) in need thereof. The method involves (a) obtaining a sample of injured brain tissue, or blood plasma or serum, from the subject; (b) testing the injured brain tissue, or the blood plasma or serum, for elevated levels of L-glutamic acid in the injured brain tissue; and (c) treating the subject by administering an agent which decreases L-glutamic acid to the subject.

[0020] In a specific example, the agent is selected from the group consisting of an N-methyl-D- aspartate (NMDA) receptor blocker, a glutamate influx transporter upregulator, a protein kinase C beta (PKCβ) activator, a glial group II metabotropic glutamate receptor (glial GpII mGluR) antagonist, a glial group III metabotropic glutamate receptor (glial GpIII mGluR) agonist, and a glutamate remover. In a more specific example, glutamate remover is selected from the group consisting of pyruvate and oxaloacetate.

[0021] A further embodiment pertains to a method of predicting short- and long-term effects of TBI in a subject in need thereof. The method may involve (a) obtaining a sample of injured brain tissue, or blood plasma or blood serum, from the subject; (b) testing the sample of injuredAttorney Docket No. 15969-052PC0 brain tissue, or the blood plasma or blood serum for levels of L-glutamic acid in the injured brain tissue; and (c) when testing shows a trend of L-glutamic acid toward normal levels, predicting that the subject will suffer from short-term effects of TBI; or when testing shows no trend of L- glutamic acid toward normal levels, predicting that the subject will suffer from long-term effects of TBI.

[0022] An additional embodiment pertains to a method of prognosing the outcome of TBI in a subject in need thereof. The method involves (a) obtaining a sample of injured brain tissue, or blood plasma or serum, from the subject; (b) testing the injured brain tissue, or the blood plasma or serum, for levels of L-glutamic acid; and (c) when testing shows a trend of L-glutamic acid toward normal levels, predicting that the subject will recover from the TBI.

[0023] Also provided is a method of diagnosing TBI in a subject in need thereof, comprising (a) obtaining a sample of injured brain tissue, or blood plasma or serum, from the subject; (b) testing the injured brain tissue, or the blood plasma or serum, for levels of phosphorylation of proteins in the injured brain tissue, or the blood plasma or serum; (c) testing the injured brain tissue, or the blood plasma or serum, for L-glutamic acid metabolites; and (d) diagnosing the subject with TBI when the testing reveals levels of phosphorylation and L-glutamic acid outside of normal ranges.

[0024] In a further embodiment, provided is a method of treating TBI in a subject in need thereof, comprising (a) obtaining a sample of injured brain tissue, or blood plasma or serum, from the subject; (b) testing the injured brain tissue, or the blood plasma or serum, for levels of phosphorylation of proteins in the injured brain tissue, or the blood plasma or serum; (c) testing the injured brain tissue, or the blood plasma or serum, for L-glutamic acid metabolites; and (d) treating the subject for TBI when the testing reveals levels of phosphorylation and L-glutamic acid outside of normal ranges; wherein the proteins tested for phosphorylation are selected from the group consisting of Serpin Family F Member 2; Serpin Family D Member 1; Septin 2; Protein Kinase C Beta; Microtubule Associated Protein Tau; Microtubule Associated Protein 2; Microtubule Associated Protein 1A; Inter-Alpha-Trypsin Inhibitor Heavy Chain 2; Heat Shock Protein 90 Alpha Family Class A Member 1; DnaJ Heat Shock Protein Family Member C5; and Alpha 2-HS Glycoprotein.

[0025] In an additional embodiment, provided is a method of prognosing the outcome of TBI inAttorney Docket No. 15969-052PC0 a subject in need thereof, comprising (a) obtaining a sample of injured brain tissue, or blood plasma or serum, from the subject; (b) testing the injured brain tissue, or the blood plasma or serum, for phosphorylation of the set of biomarkers of claim 1 in the injured brain tissue; (c) testing the injured brain tissue, or the blood plasma or serum, for L-glutamic acid metabolites; and (d) when testing shows a trend of L-glutamic acid toward normal levels, predicting that the subject is recovering from TBI.

[0026] These and other embodiments are described in further detail herein.

[0027] In particular, the invention as claimed relates to a set of biomarkers comprising at least 3 proteins selected from the group consisting of: (a) Serpin Family F Member 2 (SERPINF2); (b) Serpin Family D Member 2 (SERPIND1); (c) Septin 2 (SEPTIN2); (d) Protein Kinase C Beta (PRKCB); (e) Microtubule Associated Protein Tau (MAPT); (f) Microtubule Associated Protein 2 (MAP2); (g) Microtubule Associated Protein 1A (MAP1A); (h) Inter-Alpha-Trypsin Inhibitor Heavy Chain 2 (ITIH2); (i) Heat Shock Protein 90 Alpha Family Class A Member 1 (HSP90AA1); (j) DnaJ Heat Shock Protein Family Member C5 (DNAJC5); and (k) Alpha 2-HS Glycoprotein (AHSG).

[0028] In particular, the invention as claimed relates to a set of biomarkers comprising In another embodiment, the invention relates to the set of biomarkers comprising at least 3 proteins selected from the group consisting of: (a) SERPINF2; (b) SEPTIN2; (c) PRKCB; (d) MAPT; (e) MAP2; and (f) MAP1A.

[0029] In another embodiment, the invention relates to the set of biomarkers of claim 1 comprising at least 3 proteins selected from the group consisting of:Attorney Docket No. 15969-052PC0 (a) SERPIND1; (b) SEPTIN2; (c) PRKCB; (d) MAPT; (e) MAP2; and (f) MAP1A.

[0030] In another embodiment, the invention relates to the set of biomarkers of claim 1 comprising at least 3 proteins selected from the group consisting of: (a) SERPIND1; (b) ITIH2; (c) HSP90AA1; (d) DNAJC5; and (e) AHSG.

[0031] In another embodiment, the invention relates to a set of metabolite biomarkers comprising at least 2 compounds selected from the group consisting of: (a) L-glutamic acid; (b) MFCD00036904; (c) MFCD00133435; (d) ST2975000; (e) N-Acetyl-L-aspartic acid; (f) L-α-glycerylphosphoryl choline; (g) Thymine; (h) Retinyl acetate; (i) L-aspartic acid; (j) Cytosine; (k) Creatine; (l) DL-glutamic acid; and; (m) Spiroxamine.

[0032] In preferred embodiments, the biomarker is L-glutamic acid.

[0033] Additionally, the invention relates to a method of diagnosing traumatic brain injury (TBI) in a subject in need thereof, comprising: (a) obtaining a sample of injured brain tissue fromAttorney Docket No. 15969-052PC0 the subject; (b) testing the injured brain tissue for phosphorylation of the set of biomarkers of claim 1 in the injured brain tissue; and (c) diagnosing the subject with TBI when the test results in (b) exceed normal values in the assay when compared to internal standard. The invention also relates to a method of diagnosing traumatic brain injury (TBI) in a subject in need thereof, comprising: (a) obtaining a sample of injured brain tissue, or blood plasma or serum from the subject; (b) testing the injured brain tissue, or the blood plasma or serum for phosphorylation of the set of metabolomic biomarkers of claim 5 in the injured brain tissue; and (c) diagnosing the subject with TBI when the test results in (b) exceed normal values in the assay when compared to internal standard.

[0034] Preferably, the subject is human.

[0035] In additional embodiments, the invention relates to a method of treating TBI in a subject in need thereof, comprising: (a) obtaining a sample of injured brain tissue from the subject; (b) testing the injured brain tissue for phosphorylation of the set of biomarkers of claim 1 in the injured brain tissue; and (c) treating the subject by administering an effective amount of a therapeutic agent to the subject, wherein the amount is based on fold changes of levels of the proteins tested compared to the internal standard levels. Alternatively, the invention relates to a method of treating TBI in a subject in need thereof, comprising: (a) obtaining a sample of injured brain tissue or blood plasma or serum from the subject; (b) testing the injured brain tissue or blood plasma or serum for the metabolite set of biomarkers of claim 5; and (c) treating the subject by administering an effective amount of a therapeutic agent to the subject, wherein the identity of the therapeutic agent is based on the metabolites identified in step (b) and the amount of the therapeutic agent is based on fold changes of levels of the metabolites tested compared to the internal standard levels. Preferably, the subject is human.

[0036] In certain preferred embodiments, the invention relates to a method of treating TBI in a subject in need thereof of claim 11, wherein the proteins tested for phosphorylation are selected from the group consisting of Serpin Family F Member 2; Serpin Family D Member 1; Septin 2; Protein Kinase C Beta; Microtubule Associated Protein Tau; Microtubule Associated Protein 2; Microtubule Associated Protein 1A; Inter-Alpha-Trypsin Inhibitor Heavy Chain 2; Heat Shock Protein 90 Alpha Family Class A Member 1; DnaJ Heat Shock Protein Family Member C5; and Alpha 2-HS Glycoprotein, or it relates to a method of diagnosing TBI of claim 12, wherein the therapeutic agent is selected from the group consisting of an N-methyl-D-aspartate (NMDA)Attorney Docket No. 15969-052PC0 receptor blocker, a glutamate influx transporter upregulator, a protein kinase C beta (PKCβ) activator, a glial group II metabotropic glutamate receptor (glial GpII mGluR) antagonist, a glial group III metabotropic glutamate receptor (glial GpIII mGluR) agonist, and a glutamate remover. In certain embodiments, the invention relates to a method of treating TBI in a subject in need thereof of claim 12, wherein the therapeutic agent is selected from the group consisting of an N- methyl-D-aspartate (NMDA) receptor blocker, a glutamate influx transporter upregulator, a protein kinase C beta (PKCβ) activator, a glial group II metabotropic glutamate receptor (glial GpII mGluR) antagonist, a glial group III metabotropic glutamate receptor (glial GpIII mGluR) agonist, and a glutamate remover. Preferably, the glutamate remover is selected from the group consisting of pyruvate and oxaloacetate.

[0037] In further embodiments, the invention relates to a method of treating TBI in a subject in need thereof, comprising: (a) obtaining a sample of injured brain tissue, or blood plasma or serum, from the subject; (b) testing the injured brain tissue, or the blood plasma or serum, for elevated levels of L-glutamic acid in the injured brain tissue; and (c) treating the subject by administering an agent which decreases L-glutamic acid to the subject. In further embodiments, the invention relates to a method of diagnosing TBI in a subject in need thereof, comprising: (j) obtaining a sample of injured brain tissue, or blood plasma or serum, from the subject; (k) testing the injured brain tissue, or the blood plasma or serum, for levels of phosphorylation of proteins in the injured brain tissue, or the blood plasma or serum; (l) testing the injured brain tissue, or the blood plasma or serum, for L-glutamic acid metabolites; and (m) diagnosing the subject with TBI when the testing reveals levels of phosphorylation and L- glutamic acid outside of normal ranges.

[0038] An additional embodiment of the invention includes a method of treating TBI in a subject in need thereof, comprising: (a) obtaining a sample of injured brain tissue, or blood plasma or serum, from the subject; (b) testing the injured brain tissue, or the blood plasma or serum, for levels of phosphorylation of proteins in the injured brain tissue, or the blood plasma or serum; (c) testing the injured brain tissue, or the blood plasma or serum, for L-glutamic acid metabolites; and (d) treating the subject for TBI when the testing reveals levels of phosphorylation and L- glutamic acid outside of normal ranges, wherein the proteins tested for phosphorylation are selected from the group consisting of Serpin Family F Member 2; Serpin Family D Member 1; Septin 2; Protein Kinase C Beta; Microtubule Associated Protein Tau;Attorney Docket No. 15969-052PC0 Microtubule Associated Protein 2; Microtubule Associated Protein 1A; Inter-Alpha-Trypsin Inhibitor Heavy Chain 2; Heat Shock Protein 90 Alpha Family Class A Member 1; DnaJ Heat Shock Protein Family Member C5; and Alpha 2-HS Glycoprotein.

[0039] The invention also relates to a method of prognosing the outcome of TBI in a subject in need thereof, comprising: (a) obtaining a sample of injured brain tissue, or blood plasma or blood serum from the subject; (b) testing the injured brain tissue, or the blood plasma or blood serum for phosphorylation of the set of biomarkers of claim 1 and testing the injured brain tissue, or the blood plasma or blood serum for glutamic acid metabolites; (c) treating the subject for TBI when the testing reveals levels of phosphorylation and L- glutamic acid metabolites outside of normal ranges; and (d) repeating testing of step (b) after treating of step (c), and when repeated testing shows a trend of phosphorylation levels and / or L-glutamic acid toward normal levels, predicting that the subject is recovering from TBI.

[0040] The invention also relates to a method of predicting short and long-term effects of TBI in a subject in need thereof, comprising: (a) obtaining a sample of injured brain tissue, or blood plasma or blood serum, from the subject; (b) testing the injured brain tissue, or the blood plasma or blood serum, for phosphorylation of the set of biomarkers of claim 1; and (c) repeating testing of step (b) at a later time, and when testing shows a trend of phosphorylation levels and / or L-glutamic acid toward normal levels, predicting that the subject will suffer from short-term effects of TBI; or when testing shows no trend of phosphorylation levels and / or L-glutamic acid toward normal levels, predicting that the subject will suffer from long-term effects of TBI. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Certain embodiments are illustrated by way of example, and not by way of limitation in the figures of the accompanying drawings.

[0042] FIG. 1A is a schematic drawing showing the brains of human, mouse, and ferret. FIG. 1B is a set of photographs showing slices of brain tissue from mouse, ferret, and human.

[0043] FIG. 2A is a photograph of the advanced blast simulator instrument. FIG. 2B is a representative blast over pressure wave recording for this instrument.

[0044] FIG. 3 is a schematic diagram showing therapeutic targets for brain injuries, including those with long-term psychiatric symptoms.

[0045] FIG. 4A, FIG. 4B, and FIG. 4C present data on MAPT hyper-phosphorylation.Attorney Docket No. 15969-052PC0

[0046] FIG. 5A, FIG. 5B, FIG. 5C, and FIG. 5D present data on MAP2 hypo-phosphorylation.

[0047] FIG. 6A and FIG. 6B are figures showing statistically significant (P < 0.05) differences in the blast-vs-sham comparison at 10-second post-blast timepoints.

[0048] FIG. 7A and FIG. 7B are figures showing statistically significant (P < 0.05) differences in the blast-vs-sham comparison at 4-hour post-blast timepoints.

[0049] FIG. 8A through FIG. 8D are sets of volcano plots showing phosphorylation patterns.

[0050] FIG. 9 is a flow chart showing methods for diagnosis and treatment of TBI patients using metabolomic (metabolite level) testing.

[0051] FIG. 10A and FIG. 10B are volcano plots showing metabolite data generated from mouse brain at 10 seconds (FIG. 10A) and at 4 hours (FIG. 10B) post-TBI.

[0052] FIG. 11A and FIG. 11B are volcano plots showing metabolite data generated from ferret brain at 10 seconds (FIG. 11A) and at 4 hours FIG. 11B) post-TBI.

[0053] FIG. 12A through FIG. 12D are graphs showing TBI-induced upregulation of brain glutamate levels in ferret and mouse compared to sham animals at both 10 seconds and 4 hours after TBI.

[0054] FIG. 13A is a flow chart showing the miRNA sequence library preparation. FIG. 13B is a graph showing representative data.

[0055] FIG. 14 is a chart showing determination of a transcriptomics study.

[0056] FIG. 15A is a set of correspondence charts for mouse and ferret, as indicated.

[0057] FIG. 16A and FIG. 16B present phosphorylation data for ferret brain 10 seconds and 4 hours after blast, respectively.

[0058] FIG. 17A and FIG. 17B are a volcano plot and a chart with data on significantly up- and down-regulated proteins at 4 hours in blast exposed mouse brain.

[0059] FIG. 18A and FIG. 18B are a volcano plot and a chart with data on significantly up- and down-regulated proteins at 4 hours in blast exposed ferret brain.

[0060] FIG. 19 is a flow chart showing the processes of metabolomics analysis from biospecimen processing to metabolite detection and quantification and data analysis.

[0061] FIG. 20A and FIG. 20B are drawings showing the progress of data during examination of ferret and mouse brain and plasma, as indicated.

[0062] FIG. 21A, FIG. 21B, and FIG. 21C show data for mouse brain in an example study. The data show that blast -induced metabolic dysregulation in mouse brain across timepointsAttorney Docket No. 15969-052PC0 demonstrated differential regulation of metabolites in the mouse brain at 10 seconds and at four hours after the blast injury.

[0063] FIG. FIG. 22A and FIG. 22B are figures presenting data on ferret brain at 0 seconds and four hours.

[0064] FIG. 23A and FIG. 23B show data for significantly regulated biological pathways in joint enrichment analysis of metabolomics and mRNA (FIG. 23A) and metabolomics and protein (FIG. 23B) expression levels in mouse brain at 4 hours.

[0065] FIG. 24A and FIG. 24B show metabolomics data for _ significantly regulated biological pathways in joint enrichment analysis of metabolomics and mRNA (FIG. 23A) and metabolomics and protein (FIG. 23B) expression levels in ferret brain at 4 hours.

[0066] FIG. 25A is a bar graph presenting phosphoprotein data for the indicated tissues in mouse. FIG. 25B, FIG. 25C, and FIG. 25D are diagrams representing the results in FIG. 25A.

[0067] FIG. 26A and FIG. 26B present data on metabolites in mouse and ferret, as indicated. FIG. 26C and FIG. 26D are tables showing the metabolites significant at two time points in mouse and ferret. DETAILED DESCRIPTION

[0068] 1. Overview

[0069] Blast-related traumatic brain injury (bTBI) is a major cause of neurological disorders in the U.S. military that can adversely impact some civilian populations as well and can lead to lifelong deficits and diminished quality of life. The use of heavy weapons systems in modern conflicts has raised the concern over the effect of blast waves on the brain and peripheral tissues. The focus of this study is to employ a multi-omic approach to profile rapidly occurring biochemical changes in the plasma, and brain, lung, spleen and liver tissues following blast exposure. An objective of these studies is to identify multi-omics changes in the hyper-acute phase following blast exposure using well established animal models to identify early diagnostic biomarkers and therapeutic targets for prophylaxis and therapy. The work focusses on cross- species signals that are consistent across omics layers that are likely to be conserved and translated to humans.

[0070] The present invention relates to a set of biomarkers useful in determining the presence and severity of injury to the brain after trauma, using blast-induced trauma in two animal models,Attorney Docket No. 15969-052PC0 mouse and ferret. Blast-induced changes in proteins’ reversible post-translational modifications (phosphorylation) or in brain metabolism were examined in the immediate aftermath of blast exposure. Thus, the studies are focused on understanding early phosphoproteomic and metabolomic alterations in response to blast exposure and the information they provide on the traumatic brain injury of particular patients. The invention provides molecular biomarkers that can be used to diagnose / treat / predict TBI outcomes, including blast-related TBI outcomes.

[0071] The studies described here provide data from mouse and ferret animal models which show that, following blast exposure, certain metabolites are differentially expressed in tissues. These metabolites are molecular biomarkers useful to diagnose / treat / predict the outcome of blast- related TBI. The ferret animal model is considered more translatable for humans, and it is now thought to be as acceptable as nonhuman primate (NHP) testing for brain related data considered by the FDA. See FIG. 1.

[0072] In both the mouse and ferret models, the data was obtained from tissue samples obtained at 10 seconds and four hours following TBI (the animal’s blast exposure). Biomarkers discovered here to be relevant to diagnosis, prognosis, prediction of short- and long-term effects, and treatment of TBI include up-regulation of L-glutamic acid and D,L-glutamic acid at both 10 seconds and 4 hours (blast exposure significantly increases the levels of brain glutamate, an excitatory neurotransmitter). Therefore, the main molecular mechanisms involved in the pathophysiology of blast-TBI include both glutamate excitotoxicity and glutamate-induced neuroinflammation in the brain tissue.

[0073] In addition, hyper-phosphorylation of MAPT sites at 10 seconds (hyperphosphorylation of MAPT – pTau oligomers – formation of neurofibrillary tangles) and hypo-phosphorylation of MAP2 sites at 4 hours can be used as biomarkers, separately or in conjunction with L-glutamate.

[0074] 2. Definitions

[0075] Unless defined otherwise, all technical and scientific terms use herein have the same meaning as commonly understood by one of ordinary skill in the art. Although various methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials used are described below. However, the skilled artisan understands that the methods and materials used and described are examples and may not be the only ones suitable for use in the invention. Moreover, as measurements areAttorney Docket No. 15969-052PC0 subject to inherent variability, any temperature, weight, volume, time interval, pH, salinity, molarity or molality, range, concentration, and any other measurements, quantities, or numerical expressions given herein are intended to be approximate and not exact or critical figures unless expressly stated to the contrary.

[0076] In the foregoing specification, the invention has been described with reference to specific embodiments thereof. It will, however, be evident that various modifications and changes may be made thereto without departing from the broader spirit and scope of the invention. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense. Throughout this specification and the claims, unless the context requires otherwise, the word “comprise” and its variations, such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated item, element or step or group of items, elements or steps but not the inclusion of any other item, element, step, or group of items, elements, or steps. Furthermore, the indefinite article “a” or “an” is meant to indicate one or more of the item, element, or step modified by the article.

[0077] As used herein, the term “about” means plus or minus 20 percent of the recited value so that, for example, “about 0.125” means 0.125 ± 0.025, and “about 1.0” means 1.0 ± 0.2. Notwithstanding that the numerical ranges and parameters setting for the broad scope of the invention are approximations, the numerical values set forth in specific non-limiting examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements at the time of this writing.

[0078] As used herein, the term “phosphoproteomics” refers to study of the post-translational modification of protein(s) by phosphorylation.

[0079] As used herein, the term “metabolomics” refers to studying the set of metabolites present within an organism, cell, or tissue. Here, this approach of determining the levels of certain metabolites in tissue, with and without blast-induced TBI, was used to investigate the metabolic changes resulting from TBI.

[0080] As used herein, the term “subject” can also be referred to as a "host" or a "patient," and can refer to any animal, including mammals such as humans, laboratory animals (e.g. rodents, ferrets, rabbits, and the like), companion animals (e.g. felines, canines, and the like), farm animals (e.g., equines, bovines, porcines, ovines, caprines, and the like, and sport animals (e.g.,Attorney Docket No. 15969-052PC0 equines, canines, and the like). A suitable subject for the invention preferably is a human that is suspected of having, has been diagnosed as having, or is at risk of receiving a traumatic brain injury (TBI). A “subject in need” is any subject that has, may have or likely to receive a TBI.

[0081] As used herein, the term "administer" and all its cognates refers to introducing an agent to a subject, and can be performed using any of the various methods or delivery systems for administering agents, pharmaceutical compositions, and the like which are known to those skilled in the art.

[0082] As used herein, the term “treatment” and all its cognates refer to obtaining a desired pharmacologic and / or physiologic effect in a subject. "Treatment," includes: (a) preventing the condition or disease or symptom thereof from occurring in a subject which may be predisposed to the condition or disease but has not yet been diagnosed as having it; (b) inhibiting the condition or disease or symptom thereof, such as, arresting its development; and (c) relieving, alleviating or ameliorating the condition or disease or symptom thereof, such as, for example, causing regression or improvement of the condition or disease or symptom thereof.

[0083] As used herein, the term “diagnosis” and all its cognates refers to determining whether the subject has received a TBI.

[0084] As used herein, the term “prognosis” and all its cognates refers to determining the likely outcome of a subject that has received a TBI. Specifically, this invention allows the practitioner to determine the pathophysiological state of a subject using objective biomarkers.

[0085] As used herein, the term “predicting the short- and long-term effects” of TBI refers to identification of pathophysiological state. Short-term effects of TBI include, but are not limited to those which occur less than 24 hour after the TBI. Long-term effects of TBI include, but are not limited to, those which occur more than 24 hours following the TBI.

[0086] As used herein, the tem “metabolomics” refers to the study of the metabolites present in a cell, tissue, or organism. Untargeted metabolomics is an intended comprehensive analysis of all the measurable analytes in a sample including chemical unknowns. Targeted metabolomics is the measurement of defined groups of chemically characterized and biochemically annotated metabolites.

[0087] 3. Summary of ResultsAttorney Docket No. 15969-052PC0

[0088] The findings show proteins involved in axonogenesis and microtubule cytoskeleton organization are differentially phosphorylated early in the hyperacute phase after blast injury. Converging evidence from analysis in two animal models from different species of animals indicates the role of protein phosphorylation in the hyperacute phase of blast-TBI induced excitotoxicity. The proteins showing changes in phosphorylation in both species are suitable for use as biomarkers which can diagnose and determine the prognosis of traumatic brain injury patients and to identify targets for treatment as well.

[0089] The findings presented here also have identified proteins involved in, for example, axonogenesis and microtubule cytoskeleton organization (MAPT, MAP2, MAP1A, and MAP1B) which are differentially phosphorylated early in the hyperacute phase after blast injury.

[0090] These targets identified here are useful for developing targeted therapeutic countermeasures to blast injuries that within a matter of seconds, shape biological responses to blast exposure by mitigating immediately, short-term and long term deleterious effects. In addition prophylactic measures can be developed for for first responders, military personnel, and persons in dangerous occupations. The biomarker targets can serve as a diagnostic panel for prognostic and diagnostics of blast injuries.

[0091] Significantly altered molecular changes that are shared between the two animal models tested here include hyper-phosphorylation of MAPT sites at 10 seconds (hyperphosphorylation of MAPT – pTau oligomers – formation of neurofibrillary tangles) and hypo-phosphorylation of MAP2 sites at 4 hours.

[0092] Some of the other key outcomes of the studies presented here are the identification of differentially expressed metabolites in ferrets and mouse after TBI. These metabolites can be involved in multiple metabolic pathways in an organism. In these studies the focus was mostly on metabolites that differentially changed after TBI in plasma and brain tissue of mouse and ferret. See prior provisional applications serial nos. 63 / 686372, filed 23 August 2024 and 63 / 686630, filed 23 August 2024 for raw data in ferret and mouse, containing lists of metabolites selected based on having a magnitude of log-fold-change of at least 1. For the mouse data the criterion for inclusion was a more stringent log-fold-change of 1.2.

[0093] Significantly altered molecular changes in metabolites that are shared between the two animal models particularly include up-regulation of L-glutamic acid at both 10 seconds and 4 hours. In certain embodiments, tissues can be tested also for hyper-phosphorylation of MAPTAttorney Docket No. 15969-052PC0 sites at 10 seconds and / or hypo-phosphorylation of MAP2 sites at 4 hours. These changes are biomarkers for TBI and can be used to diagnose the condition, determine the short- and long- term effects of TBI, determine the prognosis of the subject, and to design appropriate treatment for TBI.

[0094] The targets identified here are useful for developing targeted therapeutic countermeasures to blast injuries that within a matter of seconds, shape biological responses to blast exposure. By using a targeted countermeasure as soon as possible after injury, both short-term and long term deleterious effects of TBI can be ameliorated. In addition prophylactic measures can be developed for first responders, military personnel, and persons in dangerous occupations. The biomarker targets can serve as a diagnostic panel for prognostic and diagnostics of blast injuries.

[0095] In additional studies, blast exposure led to significant changes in the levels of numerous miRNA in the hyper-acute time points in multiple tissue and two species, many of these miRNAs consistently changing in more than one tissue across both time points.

[0096] Blast-induced TBI may induce dysregulation of neurotransmitters’ responses, and may affect anti-inflammatory and antioxidant mediators in the ferret brain very early after blast exposure. Stimulation of metabolism of membrane glycerophospholipids and sphingomyelins was also observed at 4 hours post-exposure.

[0097] Plasma-brain correlation in the mouse showed some common biological compounds linked to nucleotide metabolism at both the acute timepoints post-bTBI.

[0098] Joint metabolic pathway analysis showed enriched pathways includes alanine, aspartate and glutamate metabolism, and Aminoacyl-tRNA biosynthesis.

[0099] These findings are not only provide prime candidates for diagnostic and prognostic biomarkers, but also for identifying / designing novel therapeutic strategies that mitigate the long- term sequalae of blast exposure.

[0100] Significantly altered molecular changes that are shared between the two animal models include: (1) up-regulation of L-Glutamic acid at both 10 seconds and 4 hours (blast exposure significantly increases the levels of brain glutamate, an excitatory neurotransmitter); (2) hyper- phosphorylation of MAPT sites at 10 seconds (hyperphosphorylation of MAPT – pTau oligomers – formation of neurofibrillary tangles); and (3) hypo-phosphorylation of MAP2 sites at 4 hours. Given these cross-species conserved molecular changes, we hypothesize that the mainAttorney Docket No. 15969-052PC0 molecular mechanisms that are involved in pathophysiology of blast-TBI include: glutamate excitotoxicity and glutamate induced neuroinflammation. 4. Description of Embodiments of the Invention

[0101] A. Introduction, Methods, and Models

[0102] The studies described herein were performed on mice or ferrets as follows. For example, 40 male mice were divided into groups to be exposed to a model blast exposure to induce a traumatic brain injury or to serve as sham controls. Tissues were examined from animals euthanized at two early timepoints, 10 seconds and 4 hours post-blast exposure.

[0103] Mass-spectrometry (MS) based profiling of phosphoproteomic abundances was performed using homogenized whole brain samples. After standard quality-control and preprocessing steps, differentially phosphorylated proteins in the blast-exposed group were compared to sham control using a linear regression method.

[0104] Ferrets have genetically conserved proteins that function in the cellular activities similar to that of humans. More specifically, ferrets have gyrencephalic (folds of the brain) brains, which may result in higher degree of translation to humans. See FIG. 1. Observing similar findings in two different animal models creates a higher chance of successful translation to humans in accordance with the principles of Food and Drug Administration animal rules.

[0105] To produce traumatic brain injury in the experimental animals, mice and ferrets were subjected to single blast overpressure (about 20 psi) waves using an advanced blast simulator (20 psi peak pressure, impulse: 29.87 psi*msec). See FIG. 2A, which shows a photograph of the instrument and FIG. 2B, which shows blast pressure profiles generated from the advanced blast simulator that closely resemble the exposures experiences by military and civilian personnel from an explosion or detonation or weapon systems exposures. This simulated blast injury can be approximately equivalent to a mild to moderate or severe brain injury in a human patient (or moderate to severe in mice and mild to moderate in ferrets). See also Table 1, below.

[0106] Table 1. Test Groupings, FIG. 2B. Mouse FerretAttorney Docket No. 15969-052PC0 Blood Blast 11 10 10 10 Sham 11 10 10 9

[0107] B. Traumatic Brain Injury

[0108] Traumatic brain injury (TBI) is an injury to the brain caused by an external force (or a combination of both movement and sudden impact) and can range from a mild concussion to severe TBI. TBI can result in physical, cognitive, social, cognitive, and behavioral symptoms. Outcome after TBI can include permanent disability or death. TBI causes an initial injury to the brain, but also results in a cascade of events such as inflammation and swelling that can result in even further injury.

[0109] Thus, symptoms which occur after TBI include loss of consciousness, headache, nausea, vomiting, lack of motor coordination, dizziness, poor balance, blurred vision, tinnitus, dilated pupils, confusion, weakness, numbness, cognitive changes, alexithymia, poor executive function, and more. Diagnosis currently is usually based on magnetic resonance imaging (MRI), but often is not used in the acute phase of injury because MRI produces relatively poor imaging not useful for detection of bleeds and fractures, and the inability to access and stabilize or treat the injured patient during the lengthy MRI procedure.

[0110] For example, in war zones, persons exposed to blast waves can experience prolonged cognitive and metabolic impairments, which commonly start with mild changes that can progress and worsen over time. Brain health is largely dependent upon the metabolic regulation of endogenous compounds; thus, mapping acute changes associated with neurometabolic homeostasis following blast-induced TBI can be very useful in diagnosis of the condition so that medical personnel can intervene. The focus of the current work is to identify rapidly occurring molecular precursor changes in brain metabolism following blast exposures, employing measurements within a matter of seconds to hours.

[0111] The present invention provides a new way to diagnose and determine the prognosis of TBI during the acute phase after injury, from 10 second to about 4 hours or more after the injury. This study establishes a metabolomic profile response and protein phosphorylation response to blast overpressure and identifies significant changes in the ferret model at early time points post- blast exposure. Correlation analysis of metabolic and phosphorylation alterations in brain versus plasma over time has revealed biomarkers and drug targets for treating the acute phase of blast-Attorney Docket No. 15969-052PC0 TBI. Overall, these findings can lead to development of rapid treatments for use in multi-domain operations.

[0112] C. Biomarker Proteins

[0113] After testing the phosphorylation changes in a bank of proteins in injured brain tissue and plasma in two different animal models, the particular proteins which displayed differentially expressed phosphorylation in both ferret and mouse include those listed in Table 2, below. Table 2. Biomarker Proteins. SERPINF2 (Serpin Family F Member 2) SERPIND1 (Serpin Family D Member 1) SEPTIN2 (Septin 2) PRKCB (Protein Kinase C Beta) MAPT (Microtubule Associated Protein Tau) MAP2 (Microtubule Associated Protein 2) MAP1A (Microtubule Associated Protein 1A) ITIH2 (Inter-Alpha-Trypsin Inhibitor Heavy Chain 2) HSP90AA1 (Heat Shock Protein 90 Alpha Family Class A Member 1) DNAJC5 (DnaJ Heat Shock Protein Family (Hsp40) Member C5) AHSG (Alpha 2-HS Glycoprotein)

[0114] This invention also takes advantage of metabolomics to study the set of metabolites that are changed in tissues after a blast-induced traumatic brain injury. The biomarkers according to this invention include L-glutamic acid. The results of the testing here show cross-species conserved molecular changes, which indicate that the main molecular mechanisms involved in the pathophysiology of TBI include glutamate excitotoxicity and glutamate induced neuroinflammation. Therefore, therapeutic targets for treatment of TBI patients and prophylaxis for TBI include, but are not limited to, those listed in Table 3, below. Table 3. Therapeutic and Prophylactic Treatment Modalities for TBI (metabolomics). NMDA receptor blockers (such as dextromethorphan and the like) Glutamate influx transporter upregulators (such as GLT-1 transporters and similar classes of drugs) Activators of PKCβAttorney Docket No. 15969-052PC0 (such as bryostatin-1 and the like) Antagonists of Glial GpII mGluR (such as LY341495 and the like) Agonists of Glial GpIII mGluR (such as cyclobutylene AP5 and the like) Glutamate removers (such as pyruvate and oxaloacetate)

[0115] These proteins therefore are biomarkers for TBI useful in determining diagnosis, prognosis, and treatment in other animals, including humans. The raw data was collected in the initial studies on mice and ferrets at different time points and is contained in prior provisional application serial nos. 63 / 686372, filed 23 August 2024 and 63 / 686630, filed 23 August 2024, and is hereby incorporated by reference in its entirety. Diagnosis of a subject typically is carried out using at least 3 of the biomarkers, and preferably 4 or more of the biomarkers. Prognostic and Treatment methods likewise are carried out using at least 3 and preferably 4 or more of the biomarkers.

[0116] The modalities discovered by use of metabolomics methods can be used as targeted therapeutic countermeasures for subjects with blast or other traumatic brain injuries that can, within a matter of seconds of the injury, shape biological responses to blast exposure by mitigating immediately deleterious pathophysiological cascades, reduce the subsequent evolution of pathology resulting hours, days, and months after the blast exposure. The treatment modalities also can be used as prophylaxis for those likely to receive TBI such as military personnel, first responders, professional athletes, and the like. See FIG. 3 for a schematic diagram showing glutamic acid metabolism and targets for treatment of TBI.

[0117] Levels that indicate a positive result for TBI are either higher or lower levels than normal, as compared to internal standard. Blood-based tests can be processed using plasma or serum samples separated from whole blood collected in specialized tubes. The sample is used to assess the abundance of the metabolite levels against internal controls at the baseline levels. If the levels exceed / decrease in comparison to the internal standard, the test indicates a positive result for diagnosis. Once the metabolite(s) return to the baseline level, the metabolites can be assessed for prognosis potential. Depending on the treatment specifically targeted to the proteins in Tables 1 and 2, diagnostic and / or prognostic assays can be used to test the efficacy of treatment. Similar to glucose monitoring in diabetes tests, handheld devices can be developed to diagnose the patients based on the results of the test.Attorney Docket No. 15969-052PC0

[0118] The methods of the invention are contemplated for use in the immediate aftermath of the TBI or within about 4 hours of the TBI, but also can be used in the hours, days, or weeks after the TBI. Preferably a subject is diagnosed and treated as soon as feasible after injury. Also prognostic information is gathered as soon as possible as well so that a treatment program can be initiated.

[0119] D. Therapy and Therapeutic Targets

[0120] Proteins that are significantly altered in phosphorylation and that are shared between the two animal models can be used for diagnostic testing of subjects believed to have TBI. Diagnostic testing involves testing injured tissue (preferably, brain tissue for diagnosis of brain injury) for phosphorylation of one or more of these protein biomarkers. Preferably, at least 4 of these biomarkers are tested for phosphorylation levels. Levels that indicate a positive result for TBI are increased or decreased phosphorylation of proteins as compared to identified controls.

[0121] The blood-based test can be used to assess the concentration of the protein levels that are phosphorylated. Internal standards are used for comparisons and will be assessed as positive or negative for diagnosis. Once the phosphorylated proteins return to baseline levels, these can have prognostic potential. Depending on the treatment specifically targeted to the proteins, diagnostic and / or prognosis assays can be used to test the efficacy of treatment, similarly to glucose tests in diabetes using handheld devices for routine monitoring.

[0122] Metabolites that are significantly altered in both species tested (shared between the two animal models) can be used for diagnostic testing of subjects believed to have TBI. Diagnostic testing involves testing injured tissue (preferably, plasma brain tissue for diagnosis of brain injury) for changes in the metabolism or presence of one or more of these metabolite biomarkers. Preferably, at least 2 of these biomarkers are tested, but a single metabolite can be used in some embodiments of the invention. Levels of L-glutamate that indicate a positive result for TBI are those that are above the normal range or internal standard for the assay in uninjured subjects. A person of skill can readily determine this level.

[0123] In some embodiments of the invention, treatment includes administering to the subject an agent designed to reduce the effects of glutamic acid in the brain. Preferred such agents are selected from one or more of the group consisting of an N-methyl-D-aspartate (NMDA) receptor blocker, a glutamate influx transporter upregulator, a protein kinase C beta (PKCβ) activator, a glial group II metabotropic glutamate receptor (glial GpII mGluR) antagonist, a glial group IIIAttorney Docket No. 15969-052PC0 metabotropic glutamate receptor (glial GpIII mGluR) agonist, and a glutamate remover such as pyruvate or oxaloacetate.

[0124] Determining the prognosis of a subject with TBI is performed, for example, using a commercial assay kit using a method such as ELISA or a handheld reader with a cartridge calibrated to the phosphoprotein biomarkers and / or determining the presence of the metabolite biomarkers. Determining the short- and long-term effects of blast-related TBI is performed, for example, using a commercial assay kit such as ELISA or a handheld reader with a cartridge calibrated to the phosphoprotein biomarkers.

[0125] The invention is contemplated for use in the military, where TBI is an increasing concern, but also can be used in other scenarios including any dangerous occupation or sport where TBI occurs, or in a civilian hospital setting after accidental injuries such as in falls or vehicular accidents and the like.

[0126] E. Pharmaceutical Compositions

[0127] In a preferred embodiment, the therapeutic agents of some embodiments are administered as a pharmaceutical composition that includes a pharmaceutically acceptable carrier or vehicle. A suitable carrier depends on the route of administration contemplated for the pharmaceutical composition. Routes of administration are determined by the person of skill according to convenience, the health and condition of the subject to be treated, and the location and stage of the condition to be treated.

[0128] Such routes can be any route which the practitioner deems to be most effective or convenient using considerations such as the patient, the patient’s general condition, and the specific condition to be treated. For example, routes of administration can include, but are not limited to: local or parenteral, including: oral, intravenous, intraarterial, intrathecal, injection into the cerebrospinal fluid, intraperitoneal, nasal, local injection, and the like. The administration can be given by transfusion or infusion, and can be administered by an implant, an implanted pump, or an external pump, or any device known in the art. Other potential methods of administration include transdermal patches and the like, or any method suitable for enteral or parenteral administration.

[0129] Therefore, the forms which the pharmaceutical composition can take will include, but are not limited to: tablets, capsules, granules, oral solutions, powders for dilution, sterile solutionsAttorney Docket No. 15969-052PC0 or other liquids for injection or infusion, inserts and implants, suspensions, emulsions, lipid vesicles, and the like.

[0130] Treatment regimens include a single administration or a course of administrations lasting two or more days, including a week, two weeks, several weeks, a month, two months, several months, a year, or more, including administration for the remainder of the subject’s life. The regimen can include multiple doses per day, one dose per day or per week, for example, or a long infusion administration lasting for an hour, multiple hours, a full day, or longer.

[0131] Dosage amounts per administration include any amount determined by the practitioner, and will depend on the size of the subject to be treated, the state of the health of the subject, the route of administration, the severity of the condition to be treated or prevented, and the like. A therapeutically effective dose can be administered in a single dose or in a series of doses over a period of time. In general, it is contemplated that for the majority of subjects, a dose in the range of about 0.01 mg / kg to about 100 mg / kg is suitable, preferably about 0.1 mg / kg to about 50 mg / kg, more preferably about 0.1 mg / kg to about 10 mg / kg, and most preferably about 0.2 mg / kg to about 5 mg / kg are useful. This dose can be administered weekly, daily, or multiple times per day. A dose of about 0.1 mg, 0.2 mg, 0.25 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 20 mg, 40 mg, 80 mg, 100 mg, 250 mg, 500 mg, or 1000 mg can be administered.

[0132] 5. Examples

[0133] This invention is not limited to the particular processes, compounds, compositions, or methods described, as these may vary. The terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred methods, devices, systems, compounds, compositions and materials are now described.

[0134] Example 1: General Materials and Methods.

[0135] Reagents were purchased from commercial suppliers. The PierceTM660 nm Protein Assay Kit and the Thermo Scientific EasyPrep MiniTMMS Sample Prep Kit were obtained from Thermo ScientificTM(this kit includes the lysis solution for protein extraction, and a universalAttorney Docket No. 15969-052PC0 nuclease supplemented in the extraction solution). Data acquisition was performed on a LumosTMMS / MS Instrument. Software such as Proteome DiscoverTMwas used for protein identification. Data acquisition was done using a LumosTMMS / MS instrument. Metabolite extraction from the plasma and various tissues was slightly adapted for each sample type based on methods known in the art and described in the published literature.

[0136] RNA extraction from tissue was performed in TRIzol using an miRNeasyTMMini Kit, following manufacturer’s directions. The PreAnalytixTMPAXgene Blood miRNA Kit was used for blood samples. Small RNA libraries were prepared using the TruSeqTMSmall RNA Sample Preparation kit.

[0137] Sequencing was done on the Illumina HiScan SQTMinstrument using 50 + 7 (index) sequencing cycles, resulting in 3 to 9 million reads per sample. Demultiplexing was done with BCL2FASTQTM1.8.4 software. Quality filtering and trimming of adaptor reads was done using CutAdaptTMv1.2.1. Mapping was performed on CLCTMsoftware using miRbase release 22.1. The mouse and ferret data were mapped with mouse and human databases, respectively.

[0138] Sample preparation was done using commercial kits and involved making a tissue homogenate or using whole plasma, and then a total protein lysate, which was subjected to reduction, alkylation, protease digestion, and cleanup to yield peptides. Further preprocessing and analysis of samples was conducted using the edgeR R package. MiRNA with low median expression count across samples are removed.

[0139] LC / MS-MS-based untargeted proteomics was performed using liquid chromatography separation of peptides on a C18 reverse phase nano-spray column followed by detection on a tribrid (orbi-ion trap fusion) tandem mass spectrometer. MS1 and MS2 analysis of peptides (data dependent acquisition) was performed by higher-energy collision dissociation (HCD) fragmentation and monitored in positive ion and centroid modes.

[0140] Proteome DiscovererTMv2.3 software was used to annotate the raw MS data to known protein ids / names and to determine their relative abundances, using workflows with spectral matching to species-specific protein databases (Uniprot KB) and integration of signal intensities. The general workflow was as follows: raw data → phosphoproteomics analysis to produce mass spectroscopy (MS) features → Proteome DiscovererTManalysis for protein identification and quantitation → further analysis to produce a text file. Peptide mass losses representing phosphate groups was assessed.Attorney Docket No. 15969-052PC0

[0141] For metabolomic methods, after the primary blast injury at 20 psi peak pressure (impulse: 29.87 psi*msec) in the mouse or ferret animal model, animals were sacrificed at 10 seconds and 4 hours after injury and samples were collected, typically brain and plasma. Tissues were homogenized and the metabolites extracted with 80% methanol containing a QressTMinternal standard. Metabolite profiles were obtained by assay using an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) technique. Identification of the metabolites was assisted using Compound DiscovererTMv3.3 SP2 software. Post QC filtering, further preprocessing and analysis was conducted using the limma RTMpackage. Using a criterion of p < 0.05 and log fold change > 1, differential analysis then was performed.

[0142] LC-MS / MS based untargeted metabolomics was performed using an OrbitrapTMFusion LumosTM Mass Spectrometer. Metabolite separations were performed by RPLC on Hypersil GOLD™ C18 Selectivity reversed phase HPLC columns. Data-dependent acquisition (DDA) for MS2 analysis used higher-energy collisional dissociation (HCD) and collision-induced dissociation (CID) approaches, and the data were obtained in the centroid mode.

[0143] Compound DiscovererTM3.3 SP2 software was used to annotate the untargeted LC-MS raw data to identify metabolites using a processing workflow ‘Untargeted Metabolomics with Statistics Detect Unknowns with ID using Online Databases and mzLogic.’ Identification of compounds was done using the mzCloudTM(ddMS2) and ChemSpiderTM(formula or exact mass) databases, and the mzLogic algorithm was applied to rank order ChemSpiderTMresults. The general workflow was as follows: raw data → metabolomics analysis to produce mass spectra features (MS) → Compound DiscovererTM analysis to identify and measure metabolites → further analysis to produce results for the desired metabolites in an xlsx file.

[0144] Example 2: Animal Models.

[0145] In the studies disclosed herein, two animal models, mouse and ferret, were used. In particular, the ferret model is a good animal model to predict human application and can be translated for use in humans. To produce a 20 psi blast-induced TBI in the experimental animal, an advanced blast simulator that consists of a compression chamber that is separated from a transition / expansion with a 1 foot by 1 foot test section was used for this study. See FIG. 2A and FIG. 2B.Attorney Docket No. 15969-052PC0

[0146] The anesthetized mice or ferrets were secured in the test section in a prone position and in an on-axis (head-on) orientation relative to the oncoming shock wave. The compression chamber was pressurized with room air, causing membranes to rupture at a pressure that is dependent upon the thickness of the specific membrane sheet separating the two chambers, yielding a supersonic blast wave that impacts the experimental subject in the test section at 20 psi incidence or static pressure. The exposure for incident or static pressure was measured for every animal exposed using a custom-made pitot pressure gauge at 800,000 samples per second.

[0147] Example 3: Protein Phosphorylation after Blast-TBI.

[0148] Significantly altered molecular changes that are shared between the two animal models tested here include: hyper-phosphorylation of MAPT sites at 10 seconds (hyperphosphorylation of MAPT – pTau oligomers – formation of neurofibrillary tangles) and hypo-phosphorylation of MAP2 sites at 4 hours. See results in FIG. 4 (MAPT) and FIG. 5 (MAP2).

[0149] Example 4: Blast Exposure Triggers Significant Protein Phosphorylation Changes in Mouse Brain.

[0150] In this study, 40 male mice (n = 10 animals / group / timepoint) were used to model blast exposure and to serve as sham controls. Two early timepoints were investigated: 10 seconds and 4 hours post-blast. Mass-spectrometry (MS)-based profiling of phosphoproteomic abundances were made from homogenized whole brain samples. After standard quality-control and preprocessing steps, differentially phosphorylated proteins in the blast-exposed group were compared to the sham control using a linear regression method.

[0151] The data were generated using 11 animals / group for the 10-second time point and 10 animals / group at the 4-hour time point. LC / MS-MS based untargeted proteomics profiling was performed using a data-dependent acquisition approach. Proteome DiscovererTMv2.3 software was used to annotate the raw MS data to known protein ids / names and quantify their relative abundances. This involves a pipeline that matches a given spectral data with UniprotTMKB database and calculating mass losses to assess phosphate groups.

[0152] For data preprocessing and analysis, features that were not quantitated in at least 20% of samples in at least one of the groups were filtered out. That is, features measured only in a single sample per group were removed from further analysis. After the data was log transformed, tail-Attorney Docket No. 15969-052PC0 based imputation was performed (for those features with data available for at least 40% of samples in a groups). Normalization with median scaling was used before further downstream analysis. Blast versus sham differential analysis at the two timepoints was performed using the limma package in R statistical software. Multiple testing correction was done using the Benjamini-Hochbery method.

[0153] See FIG. 6 and FIG. 7 for results. Multiple phosphoproteins showed statistically significant (P < 0.05) differences in the blast-vs-sham comparison at both the ten-second and 4- hour timepoints. The significant phosphoproteomic alterations included ones that were timepoint-specific as well as others that were shared at both timepoints. The shared alterations included proteins that are persistently hyperphosphorylated over time. Proteins with significant phosphorylation changes in the mouse brain at 10 seconds included SYN1, SEPTIN5, MAPT, MAP1A, GM49601, and DMXL2, while the topmost significant phosphorylation changes in the mouse brain at the 4-hour timepoint included ANK2, MAP1A, MYO5A, ANK2, MAP2, DMXL2, MAPT, and PRKCB.

[0154] Overall, the findings show that certain proteins involved in axonogenesis and microtubule cytoskeleton organization are differentially phosphorylated early in the hyperacute phase after blast injury (MAPT, MAP2, MAP1A, and MAP1B). In particular, STX1A [S14] is significantly hyperphosphorylated in the blast group at both timepoints. Other proteins that show differential phosphorylation at both timepoints, albeit not at the same site, include DMXL2, MAP1A, MAP2 and MAPT.

[0155] In conclusion, blast exposure triggers significant protein phosphorylation changes in mouse brain that occur within a few seconds or hours following blast injury. These molecular changes initiate a cascade of pathophysiological processes that can lead to long-term neuropsychiatric disorders. Enhanced understanding of molecular changes that occur in response to blast exposure enables development of secondary preventative therapeutic interventions and prognostic biomarkers.

[0156] Example 5: Tissue-Specific Phospho-Proteome Alterations Post-Blast Exposure Using a Ferret Animal Model.

[0157] For this study, ferrets were randomized into sham or blast-exposed groups. Following exposure to blast, ferrets were euthanized at 10 seconds after exposure or 4 hours after exposure,Attorney Docket No. 15969-052PC0 and tissues were harvested immediately. The tissues were homogenized, and protein was extracted using EasyPepTMLysis Buffer with nuclease and protease / phosphatase inhibitors. Peptides were prepared for Lumos LC-MS / MS analysis. Raw data was processed through proteome profiler.

[0158] See example results in FIG. 8. These volcano plots demonstrate significantly altered phosphorylation patterns in blast-exposed brain tissue at 4 hours (FIG. 8A), and 10 seconds (FIG. 8B) and in blast-exposed plasma samples at 4 hours (FIG. 8C) and 10 seconds (FIG. 8D). Detailed results are provided in Table 4 and Table 5, below, showing raw data and statistical information. Table 34 provides data on significantly altered protein phosphorylation in the brain tissue of ferrets at the indicated time points after blast exposure, compared to sham-treated animals. Table 5 provides data on significantly altered protein phosphorylation in plasma. Table 4. Significantly Altered Protein phosphorylation Results in Ferret Brain. Ferret Brain – Significant Proteins Phosphorylation Sites tAttorney Docket No. 15969-052PC0 Table 5. Significantly Altered Protein Phosphorylation Results in Ferret Plasma. Ferret Plasma– Significant Proteins Phosphorylation Sites Protein Phosphorylation Site logFC p-value Time Point

[0159] This study established a phosphoproteomic profile for brain and plasma and identified significant differences in the ferret animal model at early time points post-blast exposure. A higher number of statistically significant alterations in phosphoproteins were identified immediately post-trauma (10 seconds) when compared to the 4-hour time point in plasma samples. Brain tissue was highly perturbed when compared to the plasma samples at the 4-hour time point. The test for immediate effect (10 seconds) showed highly evident alterations in plasma samples as compared to the brain tissue. The data from the post-blast exposure mouse model discussed in the above examples can be used in combination with this study for a cross- species analysis to provide a list of potential biomarkers and novel targets for therapeutics.

[0160] Example 6: Glutamic Acid Metabolism

[0161] In certain embodiments of the invention, the phosphoproteomics methods described herein can be combined with metabolomic methods. For example, the injured brain tissue can be tested for glutamic acid content / metabolism as part of the diagnostic or prognostic methods, andAttorney Docket No. 15969-052PC0 treatment can consist of adding to the treatment protocol, or as part of a prophylactic protocol, an agent that reduces glutamate in the brain. Such agents can include, but are not limited to, an N- methyl-D-aspartate (NMDA) receptor blocker, a glutamate influx transporter upregulator, a protein kinase C Beta (PKCβ) activator, a glial group II metabolomic glutamate receptor (glial GpII mGluR) antagonist, a glial group III metabolomic glutamate receptor (glial GpIII mGluR) agonist, and a glutamate remover such as pyruvate or oxaloacetate. See FIG. 9 for a flow chart showing an example of such methods and FIG. 3 for a schematic diagram showing glutamic acid related targets for treatment.

[0162] Example 7: Traumatic Brain Injury Related Metabolites.

[0163] Mice and ferrets (n=10) were subjected to single blast overpressure (20 psi) waves using an advanced blast simulator. The animals were then euthanized, and their brain tissue collected at two different timepoints (10 seconds and 4 hours) after TBI and were flash frozen and stored at -80°C. Brain tissue was homogenized in 80% methanol containing an internal standard followed by metabolite extraction. The extract was then filtered through 0.22 µm nylon membrane spin tube filters and assayed for metabolite profiles using an ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) technique. Identification of metabolites was done using Compound DiscovererTM3.3 SP2 software.

[0164] Several metabolites related to TBI and their effects on biological pathways were identified. Post QC filtering, 4623 and 2852 features survived in ferret and mouse brain respectively at 10 seconds. Using a criterion of P < 0.05 and log fold change > 1, differential analysis identified 64 up- and 14 down-regulated features in ferret brain and 74 up- and 10 down-regulated features in mouse brain. In particular, in ferret brain there were 64 up- and 69 down-regulated features at 10 seconds and there were 81 up- and 9 down-regulated features at 4 hours.

[0165] These observed metabolic patterns reflect pathophysiological mechanisms triggered by brain trauma that are conserved in the two animal models tested here. Thus, these metabolites or metabolic pathways can be used to establish therapeutic targets at acute time points to suppress secondary molecular cascades after blast exposure. Additionally, these findings promote the development of optimal methods for metabolite detection within the field of the military or other relevant physical and psychological injuries.Attorney Docket No. 15969-052PC0

[0166] Example 8: Exposure to Blast Overpressure Affects Mouse Brain Metabolite Levels.

[0167] After subjecting mice to blast-induced TBI and testing of brain tissue as described above, metabolic data were collected and are shown in the volcano plots of FIG. 10A and FIG. 10B. The volcano plots show the metabolite data generated from mouse brain at 10 seconds (FIG. 10A) and at 4 hours post-exposure (FIG. 10B). Using a criterion of p < 0.05 and |logFC| > 1, there are 64 up- and 69 down-regulated features at 10 seconds and there are 81 up- and 9 down-regulated features at the 4-hour timepoint.

[0168] Example 9: Blast-Induced Alterations in Ferret Brain Metabolism at Two Acute Timepoints.

[0169] The study described above in Example 8 was repeated in ferrets here. Results are shown in FIG. 11A and FIG. 11B. These volcano plots show the metabolic changes generated after TBI in ferret brain. FIG. 11A shows the blast-induced dysregulation of brain metabolism in the ferret at 10 seconds and FIG. 11B at 4 hours post-exposure. Using a criterion of p < 0.05 and |logFC| > 1, there are 64 up- and 14 down-regulated metabolites at 10 seconds and 74 up- and 10 down- regulated metabolites at the 4-hour timepoint.

[0170] Example 10: Common Metabolites Differently Expressed in Mouse and Ferret Brain at Two Acute Timepoints Post-Blast.

[0171] The data collected in Examples 8 and 9, above, were analyzed to determine which metabolic changes occurred in both species at the two timepoints. See Table 6 and Table 7, below. Table 6 presents the number of differentially expressed metabolites in the mouse brain tissue that upregulated or downregulated across two timepoints post-blast exposure. Table 7 presents the number of differentially expressed metabolites in the ferret brain tissue that upregulated across two timepoints post-blast exposure. Table 6. Differently Expressed Metabolites in Mouse Brain Post-Blast Exposure. MetabolitelogFC (10logFC P Value P Value )Attorney Docket No. 15969-052PC0 MFCD00133435 1.666 1.711 0.002 0.026 ST2975000 1.417 1.715 0.007 0.019Table 7. Differently Expressed Metabolites in Ferret Brain Post-Blast Exposure. Metabolite logFC logFC P Value P Value (10 seconds) (4 hours) (10 seconds) (4 hours) [, g observed at both 10 seconds and at 4 hours after TBI. Metabolites that are consistently altered across the mouse and ferret models include creatine and L-Aspartic acid at 10 seconds, as well as cytosine at 4 hours. Glutamic acid, the major excitatory neurotransmitter, is up-regulated at both timepoints in both animal models. These findings indicate that these metabolites, or their metabolic pathways, are diagnostic and prognostic biomarkers, and also therapeutic targets for strategies that mitigate the long-term consequences of blast exposure.

[0173] Example 11: Differential Analysis Cross-Species and Cross-Timepoints.

[0174] The data also were analyzed to determine the number of differentially expressed metabolite levels in brain tissue across mouse and ferret that are upregulated or downregulated at acute timepoints at 10 seconds and 4 hours post TBI. See Table 8 and Table 9, below. Table 8. Cross-Species and Cross-Timepoint Differential Analysis (Ten Seconds). MetabolitelogFC (10 sec,logFC (10 sec, P Value (10 sec, P value (10 sec,Attorney Docket No. 15969-052PC0 MFCD00036904 2.19776 1.92247 0.00226 0.0005 Cyanopyrazine -1.61252 -1.32424 0.00537 0.03409Table 9. Cross-Species and Cross-Timepoint Differential Analysis (Four Hours). Metabolite logFC (4 hour, logFC (4 hour, P Value (4 hour, P value (4 hour, ferret) mouse) ferret) mouse)

[0175] Example 12: Dysregulation of brain glutamate levels at early timepoints post-blast TBI.

[0176] This example provides data showing TBI-induced up-regulation of brain glutamate in the ferret (FIG. 12A and FIG. 12B) and the mouse (FIG. 12C and FIG. 12D) compared to sham- treated animals at the indicated time points. The bars on the left of each figure are sham-treated animals and the bars on the right are TBI animals. FIG. 12A and FIG. 12C show the blast- induced dysregulation of brain metabolism in the mouse at 10 seconds and FIG. 12B and FIG. 12D at 4 hours post-exposure.

[0177] Example 13: Phosphoproteomic Data.

[0178] In certain embodiments of the invention, a subject’s tissues or other sample can be subjected to both metabolomic and phosphoproteomic analysis to provide diagnostic, prognostic, or treatment information. Thus, the samples can be tested for any or all of the metabolomic biomarkers discussed above (i.e., up-regulation of L-glutamic acid at both 10 seconds and 4 hours) and also tested for hyper-phosphorylation of MAPT sites (preferably at about 10 seconds after injury) and / or hypo-phosphorylation of MAP2 sites (preferably at about 4 hours after injury).

[0179] Example 14: Untargeted Metabolomics.Attorney Docket No. 15969-052PC0

[0180] In this study, anesthetized adult male ferrets were exposed to single blast wave at 20 psi using an advanced blast simulator to mimic the type of blast exposure. The animals were then euthanized, and their tissues and plasma collected at two different timepoints (10 seconds and 4 hours) after TBI and were immediately flash frozen. Untargeted metabolomics was performed by ultra-high performance liquid chromatography / tandem mass spectrometry (UHPLC / MS- MS). Identification of metabolites was done using Compound DiscovererTM3.3 SP2 software.

[0181] Using a criterion of P < 0.05 and log fold change > 1, differential analysis identified 64 up- and 14 down-regulated metabolites in ferret brain at 10 seconds and 74 up- and 10 down- regulated metabolites at 4 hours post-blast. Metabolic analysis of the plasma samples at 4 hours following blast exposure identified 2486 features consisting of 36 dysregulated species belonging to a variety of lipid and amino acid classes that play a vital role in various biological functions. Furthermore, the results revealed significant brain metabolic changes, including pathways associated with synaptic plasticity, fatty acid oxidation, and anti-inflammatory function, as early as 10 seconds post-blast TBI. Interestingly, some of these metabolic changes were consistent across both time points and tissues.Example 15: Acute Proteome Changes in the Brain and Plasma of Ferrets after Blast Exposure.

[0182] Adult male ferrets were subjected, while sedated, to overpressure waves in an advanced blast simulator. Age matched shams were also included. Animals were euthanized by anesthesia followed by decapitation at 10 seconds and 4 hours post-exposure (n = 10 each), and their brains and plasma collected and flash frozen. Whole brain lysates were prepared, and protein content assayed. Samples of brain protein (100 µg) and equivalent plasma (1.5 µl) were converted to total peptides by controlled protease digestion. Peptides were directly analyzed using ultra-high performance liquid chromatography / orbitrap-tribrid tandem mass spectrometry. Identities of constituent proteins were reconstructed from the peptide mass spectral data, using proteomics workflow software, as matched to a ferret-specific amino acid sequence library.

[0183] Preliminary findings for the ferret brain and plasma at 4 hours post-exposure identified 9,445 and 881 proteins, respectively, having high confidence peptide match scores. Using a cutoff criterion of and a ≥ 2-fold change and P ≤ 0.05, differential analysis for blast versus shams identified 142 up- and 172 down-regulated proteins in brain and 20 up- and 60 down-regulated inAttorney Docket No. 15969-052PC0 plasma. A large portion (~33%) showed very robust changes, i.e., ≥1000-fold. Many are key components of metabolic pathways related to neuronal injury responses.

[0184] The observed protein changes so far likely reflect pathophysiological mechanisms triggered by systemic polytrauma resulting from blast exposure. Correlation analysis of proteome alterations in brain versus plasma over time can help reveal optimal biomarkers and drug targets for treating the acute phase of blast-TBI. Overall, these findings can lead to development of rapid treatments for use in multi-domain operations.

[0185] Example 16: Analysis of miRNA Data.

[0186] Tissue homogenation and RNA extraction was performed. Sequencing then was performed on IlluminaTMHiScan SQ. Demultiplexing was performed using BCL2FASTQ 1.8.4 software. Quality filtering and trimming of adaptor reads was done using CutAdapt v1.2.1. Mapping was done using miRbase release 22.1. The mouse and ferret data was mapped with mouse and human databases, respectively. Further preprocessing and analysis were conducted using edgeR R package. miRNA with low median expression count across samples was removed. See FIG. 13.

[0187] Example 17: Differentially Expressed miRNAs in Multiple Tissues of Mouse and Ferret Immediately After Blast Exposure.

[0188] Changes in miRNA abundances of miRNAs are analyzed in five different tissues (brain, lung, liver, spleen and blood) of mouse and ferret at 10 seconds and 4 hours after blast exposure. Significant and various levels of miRNAs were altered in the five tissues in both species. Larger magnitude change is observed at 4 hours, particularly ferret blood showing its potential for biomarker identification. Significant up-regulation of several miRNAs is also found in the mouse brain indicating potential therapeutic targets for early post-blast interventions.

[0189] See results in FIG. 14.

[0190] Example 18: Consistent Direction of Change across Species and Timepoints in Blood and Brain Tissue.Attorney Docket No. 15969-052PC0

[0191] We sought to identify miRNAs that are consistently altered across species in blood and brain tissues of ferret and mouse. The Venn diagrams show the number of overlapping miRNAs identified in blood and brain at 10 seconds and 4 hours after blast exposure (FIG. 15A). A heatmap is shown for select miRNA showing direction and magnitude of fold-change between blast exposed and unexposed animals (FIG. 15B), the colors corresponding to the direction of change (up-regulation shown in red and down-regulation shown in blue) and the darkness of the colors being proportional to the fold-change value. A few miRNAs show consistent direction of change in the two species at the two acute timepoints in blood and brain. Notable ones include miR-215-5p, miR-18a-5p and miR-200b-3p. These molecular changes that are shared between the mouse and ferret datasets are likely to be evolutionally conserved and are more likely to be translatable to humans.

[0192] See results in FIG. 15.

[0193] Example 19: Blast-Induced Phosphoproteomic Changes in Ferret Brain.

[0194] Differentially phosphorylated proteins were identified in blast-exposed ferret brain at 10 seconds and 4 hours after blast. Strict quality-control and preprocessing steps were applied to phosphoproteimics data obtained using mass-spectrometry (MS) based profiling. Few phosphoproteins show statistically significant (P < 0.05) differences in the blast exposed ferret brain compared to unexposed sham ferret group at both timepoints. Both time specific as well as alterations that are shared between the two timepoints are observed. Overall, blast overpressure dysregulated phosphorylation of microtubule-associated protein tau, neurofilament-M and protein kinase-Cb at 10 seconds; and microtubule stability, axonal outgrowth / synapse formation and maintaining structure of neuronal cytoskeleton.

[0195] See results in FIG. 16.

[0196] Example 20: Proteomics Changes in Mouse Brain at Four Hours Post-Blast.

[0197] Total protein levels were detected and quantified in mouse brain using mass-spectrometry based workflow and are matched to a mouse-specific amino acid sequence library. Differential expression analysis was performed on proteins identified with a high confidence peptide match score. Using a cutoff criterion of ≥ 2-fold change and P ≤ 0.05, differential expression analysis for blast exposed versus unexposed shams identified several up- and down-regulated proteins inAttorney Docket No. 15969-052PC0 brain, with a large proportion showing very robust changes. Many are key components of metabolic pathways related to neuronal injury responses. These includes ATP12A, SV2A and MYH4 protein abundances. These early molecular responses may reflect pathophysiological mechanisms triggered by systemic trauma induced by blast exposure.

[0198] See results in FIG. 17.

[0199] Example 21: Proteomics Changes in Ferret Brain at Four Hours Post-Blast.

[0200] Total protein levels were detected and quantified in ferret brain using mass-spectrometry based workflow and are matched to a ferret-specific amino acid sequence library. Differential expression analysis was performed on proteins identified with a high confidence peptide match score. Using a cutoff criterion of ≥ 2-fold change and P ≤ 0.05, differential expression analysis for blast exposed versus unexposed shams identified several up- and down-regulated proteins in brain, with a large proportion showing very robust changes. Many are key components of metabolic pathways related to neuronal injury responses. These includes PLP1, KRT74 and ABLIM1 protein abundances. These early molecular responses may reflect pathophysiological mechanisms triggered by systemic trauma induced by blast exposure.

[0201] See results in FIG. 18.

[0202] Example 22: LC / MS Based Untargeted Metabolomics.

[0203] Workflow for profiling of untargeted global metabolomics includes the following steps. Metabolite extraction from the plasma, brain and various tissues should be slightly adapted for each sample type based on best practices, standard / recommended protocols and published literature. In house LC-MS / MS based untargeted metabolomics was done using Orbitrap Fusion Lumos Mass Spectrometer. Metabolite separations were performed by RPLC on a Hypersil GOLD™ C18 Selectivity Reversed Phase HPLC columns. Data-dependent acquisition (DDA) for MS2 analysis with higher-energy collisional dissociation (HCD) and collision-induced dissociation (CID) approaches, and the data were obtained in the centroid mode. Compound identification and annotation is performed using online databases including mzCloud (ddMS2) and ChemSpider (formula or exact mass) databases. The raw quantitative metabolomics data is preprocesed, quality-checked and analyzed using inhouse code and software.

[0204] See results in FIG. 19.Attorney Docket No. 15969-052PC0

[0205] Example 23: Blast Exposure Induces Widespread Metabolite Abundancy Changes in both Mouse and Ferret Brain and Plasma in the Immediate Aftermath of Blast Exposure.

[0206] Global untargeted metabolomics activity was analyzed and compared in blast exposed ferret (FIG. 20A) and mouse (FIG. 20B) brain and plasma. Number of metabolites altered at a statistically significant levels is shown in the bar plots. Two example volcano plots show criterion used for significance threshold (p-value and fold-change). Larger changes were observed in in the brain compared to the changes in plasma metabolites. Overall, blast exposure induced widespread metabolite abundances changes in both mouse and ferret brain and plasma in the immediate aftermath (10 seconds and 4 hours) of the blast exposure.

[0207] See results in FIG. 20.

[0208] Example 24: Plasma-Brain Correlation of Metabolite Changes in the Mouse.

[0209] Differentially blast induced changes in brain and plasma of mouse at 10 second- and 4- hours post-blast are compared to identify overlapping sets of metabolites that are consistently altered in the two tissue and / or the two timepoints. Common metabolites include thymine, cytosine and dezaguanine. The changes in the thymine levels may be associated with reduced oxidative stress in the brain after TBI.

[0210] See results in FIG. 21.

[0211] Example 25: Correlation of Metabolic Changes in the Ferret Plasma and Brain.

[0212] A similar analysis as above for ferret metabolomics data was performed. Blast induced changes in brain and plasma of ferret at 10 second- and 4-hours post-blast are compared to identify overlapping sets of metabolites that are consistently altered in the two tissue and / or the two timepoints. Common metabolites include ones involved in energy metabolism, neurodevelopment and synaptic plasticity. Another common metabolite include a precursor for biosynthesis of histamine, a neuromodulator influencing sleep, wakefulness and appetite, and regulating pain perception and immune response.

[0213] See results in FIG. 22.

[0214] Example 26: Mouse Brain, Four Hours Post-Blast.Attorney Docket No. 15969-052PC0

[0215] Integrative omics analysis of joint metabolomics and genes expression at mRNA (FIG. 23A) and protein (FIG. 23B) levels at 4 hours in mouse brain. A number of biological pathways that comprise both significantly altered small molecules (metabolites) and gene activity (at mRNA and protein levels) were significantly enriched. The enrichment statistics is shown in the bubble plots. Each circle represents a pathway, and the position, size and color of the circle correspond to the strength of the enrichment. Enriched pathways include arginine biosynthesis, taurine and hypotaurine metabolism.

[0216] See results in FIG. 23.

[0217] Example 27: Ferret Brain, Four Hours Post-Blast.

[0218] A similar analysis as above was performed for the ferret data. Integrative omics analysis of joint metabolomics and genes expression at mRNA (FIG. 24A) and protein (FIG. 24B) levels at 4 hours in ferret brain. A number of biological pathways that comprise both significantly altered small molecules (metabolites) and gene activity (at mRNA and protein levels) were significantly enriched. The enrichment statistics is shown in the bubble plots. Each circle represents a pathway, and the position, size and color of the circle correspond to the strength of the enrichment. Enriched pathways include Glutathione metabolism, alanine, aspartate and glutamate metabolism.

[0219] See results in FIG. 24.

[0220] Example 28: Common Phosphorylation Patterns in Ferret and Mouse.

[0221] In order to identify cross-species and / or cross-tissue phosphoproteomics changes, the number / magnitude of changes in each species and tissue are compared at two timepoints immediately post blast. Comparing the magnitude of change (FIG. 25A) show the largest change was observed in mouse brain tissue at 4 hours after blast exposure while a smaller magnitude change was observed in the liver tissue of both species and timepoints. A larger proportions of phosphoprotein changes tend to be time-specific (FIG. 25B and FIG. 25C) as well as species- specific (FIG. 25B).

[0222] See results in FIG. 25.

[0223] Example 29: Common Metabolomics Patterns in Ferret and Mouse.Attorney Docket No. 15969-052PC0

[0224] A similar analysis as above for metabolomics data was performed to identify cross- species and / or cross-tissue metabolomics changes. Comparison of number / magnitude of changes was performed for the two species and five tissue and two timepoints. The largest magnitude of change was observed in the liver tissue, perhaps due to the overall abundance of small molecules. Overall, larger number of metabolites was detected and reached significance threshold in the mouse tissues compared to the ferret tissues (FIG. 26A). About half of the significantly altered metabolites in the ferret tissue were also found significant in the mouse tissues (FIG. 26B), showing a largely consistent cross-species metabolomics response to blast exposure.

[0225] Metabolites significantly altered at two timepoints in mouse brain includes N-Acetyl-L- aspartic acid, L-alpha-Glycerylphosphorylcholine, Thymine, Retinyl acetate, L-Glutamic acid, L- Aspartic acid, Cytosine and Creatine (FIG. 26C). Metabolites significantly altered at two timepoints in ferret brain includes spiroxamine, L-Glutamic acid, DL-Glutamic acid (FIG. 26D).

[0226] See results in FIG. 26.Attorney Docket No. 15969-052PC0

[0227] 6. References.

[0228] All publications listed below and throughout the specification are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. 1. Arun, et al. Metabolic acetate therapy for the treatment of traumatic brain injury. J Neurotrauma. 2010 Jan;27(1):293-8. 2. Bykowski, et al. Urinary metabolomic signatures as indicators of injury severity following traumatic brain injury: A pilot study. IBRO Neurosci Rep. 2021 Oct 27;11:200-206. 3. Casado et al., Integrative phosphoproteomics defines two biologically distinct groups of KMT2A rearranged acute myeloid leukaemia with different drug response phenotypes. Springer Nature Signal Transduction and Targeted Therapy. 2023;8(80). 4. Elkjaer, et al. CSF proteome in multiple sclerosis subtypes related to brain lesion transcriptomes. Sci Rep. 2021 Feb 18;11(1):4132. 5. Emdal et al., Phosphoproteomics of primary AML patient samples reveals rationale for AKT combination therapy and p53 context to overcome selinexor resistance. Cell Reports. 2022;40. 6. Fazakerley et al., Phosphoproteomics reveals rewiring of the insulin signaling network and multi-nodal defects in insulin resistance. Nature Communications. 2023;14(923). 7. Flavin, et al. Traumatic brain injury and the pathways to cerebral tau accumulation. Front Neurol. 2023 Aug 11;14:1239653. 8. Gerritsen and White, Phosphoproteomics: a valuable tool for uncovering molecular signaling in cancer cells. Expert Rev Proteomics. 2021;18(8):661-74. 9. Goulding, et al. Inter-α-inhibitor deficiency in the mouse is associated with alterations in anxiety-like behavior, exploration and social approach. Genes Brain Behav. 2019 Jan;18(1):e12505. 10. Javaid, et al. Dynamics of Choline-Containing Phospholipids in Traumatic Brain Injury and Associated Comorbidities. Int J Mol Sci. 2021 Oct 20;22(21):11313.Attorney Docket No. 15969-052PC0 rin, et al. Subacute and chronic proteomic and phosphoproteomic analyses of a mouse model of traumatic brain injury at two timepoints and comparison with chronic traumatic encephalopathy in human samples. Mol Brain. 2022 Jul 18;15(1):62. mer, et al. Traumatic brain injury-induced excitotoxicity assessed in a controlled cortical impact model. J Neurochem. 1993 Dec;61(6):2015-24. ng, et al. The regulatory role of Pin1 in neuronal death. Neural Regen Res. 2023 Jan;18(1):74-80. aswathy, Phosphoproteomics. Concepts and Techniques in Genomics and Proteomics. Science Direct: Woodhead Publishing; 2011. p. 203-11. mas, et al. CENTER-TBI Participants and Investigators. Serum metabolome associated with severity of acute traumatic brain injury. Nat Commun. 2022 May 10;13(1):2545. ng and White. Quantitative Proteomic Analysis of Phosphotyrosine-Mediated Cellular Signaling Networks. Methods in Molecular Biology. 2007;359. opean Patent No. EP3660172. opean Patent No. EP3663764 rnational Publication NO. WO2021022109 ited States Patent Publication No. US2020-0254068. ited States Patent Publication No. US2022-0252618 ited States Patent Publication No. US2023-0338305. ited States Patent Publication No. US2024-0280559.

Claims

Attorney Docket No. 15969-052PC0 CLAIMS What is claimed is:

1. A set of biomarkers comprising at least 3 proteins selected from the group consisting of: (a) Serpin Family F Member 2 (SERPINF2); (b) Serpin Family D Member 2 (SERPIND1); (c) Septin 2 (SEPTIN2); (d) Protein Kinase C Beta (PRKCB); (e) Microtubule Associated Protein Tau (MAPT); (f) Microtubule Associated Protein 2 (MAP2); (g) Microtubule Associated Protein 1A (MAP1A); (h) Inter-Alpha-Trypsin Inhibitor Heavy Chain 2 (ITIH2); (i) Heat Shock Protein 90 Alpha Family Class A Member 1 (HSP90AA1); (j) DnaJ Heat Shock Protein Family Member C5 (DNAJC5); and (k) Alpha 2-HS Glycoprotein (AHSG).

2. The set of biomarkers of claim 1 comprising at least 3 proteins selected from the group consisting of: (a) SERPINF2; (b) SEPTIN2; (c) PRKCB; (d) MAPT; (e) MAP2; and (f) MAP1A.

3. The set of biomarkers of claim 1 comprising at least 3 proteins selected from the group consisting of: (a) SERPIND1; (b) SEPTIN2; (c) PRKCB; (d) MAPT; (e) MAP2; andAttorney Docket No. 15969-052PC0 (e) MAP1A.

4. The set of biomarkers of claim 1 comprising at least 3 proteins selected from the group consisting of: (a) SERPIND1; (b) ITIH2; (c) HSP90AA1; (d) DNAJC5; and (e) AHSG.

5. A set of metabolite biomarkers comprising at least 2 compounds selected from the group consisting of: (a) L-glutamic acid; (b) MFCD00036904; (c) MFCD00133435; (d) ST2975000; (e) N-Acetyl-L-aspartic acid; (f) L-α-glycerylphosphoryl choline; (g) Thymine; (h) Retinyl acetate; (i) L-aspartic acid; (j) Cytosine; (k) Creatine; (l) DL-glutamic acid; and; (m) Spiroxamine.

6. The biomarker of claim 5 comprising L-glutamic acid.

7. A method of diagnosing traumatic brain injury (TBI) in a subject in need thereof, comprising: (a) obtaining a sample of injured brain tissue from the subject; (b) testing the injured brain tissue for phosphorylation of the set of biomarkers of claim 1 in the injured brain tissue; andAttorney Docket No. 15969-052PC0 (c) diagnosing the subject with TBI when the test results in (b) exceed normal values in the assay when compared to internal standard.

8. A method of diagnosing traumatic brain injury (TBI) in a subject in need thereof, comprising: (a) obtaining a sample of injured brain tissue, or blood plasma or serum from the subject; (b) testing the injured brain tissue, or the blood plasma or serum for phosphorylation of the set of metabolomic biomarkers of claim 5 in the injured brain tissue; and (c) diagnosing the subject with TBI when the test results in (b) exceed normal values in the assay when compared to internal standard.

9. The method of diagnosing TBI in a subject in need thereof of claim 8, wherein the subject is human.

10. The method of diagnosing TBI in a subject in need thereof of claim 9, wherein the subject is human.

11. A method of treating TBI in a subject in need thereof, comprising: (a) obtaining a sample of injured brain tissue from the subject; (b) testing the injured brain tissue for phosphorylation of the set of biomarkers of claim 1 in the injured brain tissue; and (c) treating the subject by administering an effective amount of a therapeutic agent to the subject, wherein the amount is based on fold changes of levels of the proteins tested compared to the internal standard levels.

12. A method of treating TBI in a subject in need thereof, comprising: (a) obtaining a sample of injured brain tissue or blood plasma or serum from the subject; (b) testing the injured brain tissue or blood plasma or serum for the metabolite set of biomarkers of claim 5; and (c) treating the subject by administering an effective amount of a therapeutic agent to the subject, wherein the identity of the therapeutic agent is based on the metabolites identified in stepAttorney Docket No. 15969-052PC0 (b) and the amount of the therapeutic agent is based on fold changes of levels of the metabolites tested compared to the internal standard levels.

13. The method of treating TBI in a subject in need thereof of claim 11, wherein the subject is human.

14. The method of treating TBI in a subject in need thereof of claim 12, wherein the subject is human.

15. The method of treating TBI in a subject in need thereof of claim 11, wherein the proteins tested for phosphorylation are selected from the group consisting of Serpin Family F Member 2; Serpin Family D Member 1; Septin 2; Protein Kinase C Beta; Microtubule Associated Protein Tau; Microtubule Associated Protein 2; Microtubule Associated Protein 1A; Inter-Alpha- Trypsin Inhibitor Heavy Chain 2; Heat Shock Protein 90 Alpha Family Class A Member 1; DnaJ Heat Shock Protein Family Member C5; and Alpha 2-HS Glycoprotein.

16. The method of diagnosing TBI of claim 12, wherein the therapeutic agent is selected from the group consisting of an N-methyl-D-aspartate (NMDA) receptor blocker, a glutamate influx transporter upregulator, a protein kinase C beta (PKCβ) activator, a glial group II metabotropic glutamate receptor (glial GpII mGluR) antagonist, a glial group III metabotropic glutamate receptor (glial GpIII mGluR) agonist, and a glutamate remover.

17. The method of treating TBI in a subject in need thereof of claim 12, wherein the therapeutic agent is selected from the group consisting of an N-methyl-D-aspartate (NMDA) receptor blocker, a glutamate influx transporter upregulator, a protein kinase C beta (PKCβ) activator, a glial group II metabotropic glutamate receptor (glial GpII mGluR) antagonist, a glial group III metabotropic glutamate receptor (glial GpIII mGluR) agonist, and a glutamate remover.

18. The method of treating TBI in a subject in need thereof of claim 17 wherein the glutamate remover is selected from the group consisting of pyruvate and oxaloacetate.Attorney Docket No. 15969-052PC0 19. A method of treating TBI in a subject in need thereof, comprising: (a) obtaining a sample of injured brain tissue, or blood plasma or serum, from the subject; (b) testing the injured brain tissue, or the blood plasma or serum, for elevated levels of L-glutamic acid in the injured brain tissue; and (c) treating the subject by administering an agent which decreases L-glutamic acid to the subject.

20. A method of diagnosing TBI in a subject in need thereof, comprising: (j) obtaining a sample of injured brain tissue, or blood plasma or serum, from the subject; (k) testing the injured brain tissue, or the blood plasma or serum, for levels of phosphorylation of proteins in the injured brain tissue, or the blood plasma or serum; (l) testing the injured brain tissue, or the blood plasma or serum, for L-glutamic acid metabolites; and (m) diagnosing the subject with TBI when the testing reveals levels of phosphorylation and L-glutamic acid outside of normal ranges.

21. A method of treating TBI in a subject in need thereof, comprising: (a) obtaining a sample of injured brain tissue, or blood plasma or serum, from the subject; (b) testing the injured brain tissue, or the blood plasma or serum, for levels of phosphorylation of proteins in the injured brain tissue, or the blood plasma or serum; (c) testing the injured brain tissue, or the blood plasma or serum, for L-glutamic acid metabolites; and (d) treating the subject for TBI when the testing reveals levels of phosphorylation and L- glutamic acid outside of normal ranges, wherein the proteins tested for phosphorylation are selected from the group consisting of Serpin Family F Member 2; Serpin Family D Member 1; Septin 2; Protein Kinase C Beta; Microtubule Associated Protein Tau; Microtubule Associated Protein 2; Microtubule Associated Protein 1A; Inter-Alpha-Trypsin Inhibitor Heavy Chain 2; Heat Shock Protein 90 Alpha Family Class A Member 1; DnaJ Heat Shock Protein Family Member C5; and Alpha 2-HS Glycoprotein.Attorney Docket No. 15969-052PC0 22. A method of prognosing the outcome of TBI in a subject in need thereof, comprising (a) obtaining a sample of injured brain tissue, or blood plasma or blood serum from the subject; (b) testing the injured brain tissue, or the blood plasma or blood serum for phosphorylation of the set of biomarkers of claim 1 and testing the injured brain tissue, or the blood plasma or blood serum for glutamic acid metabolites; (c) treating the subject for TBI when the testing reveals levels of phosphorylation and L- glutamic acid metabolites outside of normal ranges; and (d) repeating testing of step (b) after treating of step (c), and when repeated testing shows a trend of phosphorylation levels and / or L-glutamic acid toward normal levels, predicting that the subject is recovering from TBI.

23. A method of predicting short and long-term effects of TBI in a subject in need thereof, comprising: (a) obtaining a sample of injured brain tissue, or blood plasma or blood serum, from the subject; (b) testing the injured brain tissue, or the blood plasma or blood serum, for phosphorylation of the set of biomarkers of claim 1; and (c) repeating testing of step (b) at a later time, and when testing shows a trend of phosphorylation levels and / or L-glutamic acid toward normal levels, predicting that the subject will suffer from short-term effects of TBI; or when testing shows no trend of phosphorylation levels and / or L-glutamic acid toward normal levels, predicting that the subject will suffer from long-term effects of TBI.

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