CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63/177,727, filed April 21, 2021, the entirety of which is incorporated into this application by reference.

BACKGROUND

[0002] Traumatic brain injury (TBI) is a significant public health issue in the United States. In 2014, over 2.8 million TBI-related hospital visits and 50,000 deaths occurred in the U.S. Taylor, C.A., Bell, J.M., Breiding, M.J. & Xu, L. Traumatic Brain Injury -Related Emergency Department Visits, Hospitalizations, and Deaths - United States, 2007 and 2013. MMWR Surveill Summ 66, 1-16 (2017). TBI often leads to lifelong motor, cognitive, and behavioral disabilities. Potts, M.B. el al. Traumatic injury to the immature brain: inflammation, oxidative injury, and iron-mediated damage as potential therapeutic targets. NeuroRx 3, 143- 153 (2006); Elder, G.A. Update on TBI and Cognitive Impairment in Military Veterans. Curr Neurol Neurosci Rep 15, 68 (2015). It is also a risk factor for developing other neurodegenerative diseases, including Alzheimer’s Disease and Parkinson’s Disease. Elder, G.A. Update on TBI and Cognitive Impairment in Military Veterans. Curr Neurol Neurosci Rep 15, 68 (2015).

[0003] In TBI, the mechanical insult to the brain rapidly elicits an assembled series of potent immune responses in the central nervous system (CNS), collectively termed “neuroinflammation.” Potts, M.B. el al. Traumatic injury to the immature brain: inflammation, oxidative injury, and iron-mediated damage as potential therapeutic targets. NeuroRx 3, 143-153 (2006); Corps, K.N., Roth, T.L. & McGavem, D.B. Inflammation and Neuroprotection in Traumatic Brain Injury. JAMA neurology (2015); Hinson, H.E., Rowell,

S. & Schreiber, M. Clinical evidence of inflammation driving secondary brain injury: a systematic review. J Trauma Acute Care Surg 78, 184-191 (2015).

[0004] The responding circulating immune cells include monocyte-derived macrophages, which infiltrate the brain and differentiate into activated macrophages within and about the area of injury. In addition, resident innate immune cells, including microglia, become activated at the area of injury. Jassam, Y.N., Izzy, S., Whalen, M, McGavem, D.B. & El Khoury, J. Neuroimmunology of Traumatic Brain Injury: Time for a Paradigm Shift. Neuron 95, 1246-1265 (2017).

[0005] Inflammation has evolved to serve the purposes of sterilizing wounds and facilitating repair (REFS on repair). It can, however, be neurotoxic and expand brain damage following TBI as it remains active over years in humans and mice, resulting in secondary injury. Corps, K.N., Roth, T.L. & McGavem, D.B. Inflammation and Neuroprotection in Traumatic Brain Injury. JAMA neurology (2015); Martin, P. & Leibovich, S.J. Inflammatory cells during wound repair: the good, the bad and the ugly. Trends Cell Biol 15, 599-607 (2005); Loane, D.J., Kumar, A., Stoica, B.A., Cabatbat, R. & Faden, A.I. Progressive neurodegeneration after experimental brain trauma: association with chronic microglial activation. J Neuropathol Exp Neurol 73, 14-29 (2014); Johnson, V.E. et al. Inflammation and white matter degeneration persist for years after a single traumatic brain injury. Brain 136, 28-42 (2013); Erturk, A. et al. Interfering with the Chronic Immune Response Rescues Chronic Degeneration After Traumatic Brain Injury. J Neurosci 36, 9962-9975 (2016).

[0006] Because this secondary injurious response is prolonged, it offers opportunities to intervene. TBI, however lacks effective pharmacological treatment. Investigators have sought to understand innate immune cells and pathways that are harmful or protective in TBI. Hsieh, C.L. et al. Traumatic brain injury induces macrophage subsets in the brain. Eur J Immunol 43, 2010-2022 (2013); Kim, C.C., Nakamura, M.C. & Hsieh, C.L. Brain trauma elicits non- canonical macrophage activation states. J Neuroinflammation 13, 117 (2016). Precisely identifying components of neuroinflammation, including immune cell subsets and activated pathways that impact TBI, is important for informed designs of therapeutic approaches to treat TBI and to minimize brain damage. These needs and others are met by the following disclosure.

SUMMARY

[0007] In one aspect, this disclosure relates to a method of treating a traumatic brain injury, a spinal cord injury, or stroke in a subject, comprising administering to the subject an effective amount of a compound represented by Formula I, II, or III: or a pharmaceutically acceptable salt thereof, thereby treating the traumatic brain injury, the spinal cord injury, or stroke in the subject.

[0008] In a further aspect, disclosed are kits and therapeutic uses of the compound of Formula I, II, or III.

[0009] Still other objects and advantages of the present disclosure will become readily apparent by those skilled in the art from the following detailed description, which is shown and described by reference to preferred aspects, simply by way of illustration of the best mode. As will be realized, the disclosure is capable of other and different aspects, and its several details are capable of modifications in various respects, without departing from the disclosure. Accordingly, the description is to be regarded as illustrative in nature and not as restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The accompanying drawings, which are incorporated in and constitute a part of this specification and together with the description, serve to explain the principles of the disclosure.

[0011] FIGs. 1A-1D collectively show defining cell lineages of clusters from single cell RNA sequencing of white cells from ipsilateral acute TBI and normal brain tissues from Ccr2 ^_/ and WT mice. FIG 1A shows representative flow cytometry gates of LIVE CD45 ⁺ Ly6G cells sorted from ipsilateral brain hemispheres from wildtype (WT) TBI mice (4 days post-injury, n=3 animals, 1 animal/sample), Ccr2 ^/_ TBI mice (n=3 animals, 1 animal/sample) and normal control mice (n=2-3 animals/group, 1 animal/sample). FIG IB shows UMAP visualization of scRNA-seq data from 111,717 single cells, separated into clusters of microglia, macrophages and dendritic cells, a separate Ccr7 ^hl dendritic cell subset, neutrophils, T and NK cells, B cells, and a subset of cells with unclear cell lineage labelled “other”. FIG 1C shows Dot plot analysis of cell lineage marker expression used to define clusters for microglia ( Salll ), monocyte/macrophages ( Ccr2 ), dendritic cells ( Flt3 ), B cells (' Cdl9 ), T cells ( CD3e ), NK cells ( Ncrl ), neutrophils ( Ly6g ), neurons ( Slcl2a5 ), astrocytes (Aldhlll), and oligodendrocytes ( Mog ). FIG ID shows gross analysis of cell numbers of microglia and circulating leukocytes per brain hemisphere by genotype and by injury showed that TBI induced significant increases in cell numbers of the macrophage/dendritic cell cluster (ANOVA p val = 0.01, WT TBI = 793 + 315 (mean ± SD), WT normal control = 190 ± 73, adj p val=0.01), as well as increases in the T and NK cell cluster (ANOVA p=0.007, WT TBI = 345 ± 145 [mean ± SD], WT normal control = 92 ± 46, adj p val=0.04). ANOVA analysis (p val=0.01) found a significant difference by genotype in microglia and in the macrophage/dendritic cell cluster. Tukey’s post-hoc analysis showed a significant difference in macrophage/dendritic cell numbers in pairwise comparisons between WT TBI and Ccr2 ^/ TBI mice (WT TBI=793 + 315 [mean ± SD], Ccr '- TBI=220 ± 51, adj p val=0.02).

[0012] FIGs. 2A-2D collectively show quantification and gene expression analysis of microglia subsets in TBI reveals pathways upregulated in TBI and pathways associated with Ccr2 -deficiency. FIG 2A shows joint graph-based clustering with canonical correlation vectors separated the combined microglia from all samples into 13 distinct subclusters or subsets visualized using the UMAP dimensionality reduction technique. Each subset is named after a top differentially expressed gene (DEG) that distinguished it from all other microglia subclusters. FIG 2B shows gene expression visualization by UMAP of a few top DEGs for a few microglia subclusters and their expression in normal vs TBI brain microglia: Irf7, a DEG of Cxcll0 ^hl and Ifi27l2a ^hl microglia, Ccl4 and Clec7a (Dectin 1) are DEGS of Ccl4 ^hl microglia, and Tnf was the top DEG for Nfkbia ^hl microglia. FIG 2C shows five microglia subsets increased in cell number during acute TBI. Three out of the five subsets were decreased in Ccr2 ^y TBI mice, including a type I IFN responsive subset. Cell counts (top) and pathway enrichment analysis (middle) of DEGs for each microglia subset were assessed. Significance of cell counts analyses were determined via ANOVA followed by Tukey’s multiple comparison tests (mean +SD are shown). Differential gene expression analysis between WT TBI and Ccr2 ^y TBI (bottom). FIG 2D shows analysis of a few microglia subsets that did not increase in cell number during TBI. Cell counts and gene ontology analysis of DEGs of each subset showed that these shared a response to IL-1 (left top and bottom). Differential gene expression analysis between WT TBI and Ccr2 ^y TBI (right). [0013] FIGs. 3A-3E collectively show histological validation and quantification of CXCL10 ⁺ IBA1 ⁺ cells and KI67 ⁺ proliferating microglia at the injury site. FIG. 3A. Four days after TBI, immunohistochemistry was performed on brain samples, constraining for CXCL10, IBA1, NEUN, and DAPI. FIG. 3B. Co-expression of CXCL10 and IBA1 in cells localized in the ipsilateral hippocampus and thalamus post-TBI. FIG. 3C. The mean number of IB Al ⁺ CXCL10 ⁺ microglia/macrophages per square mm increased in ipsilateral TBI tissue compared to sham brain tissue (mean+SD; 141+73 vs 18+15, unpaired t test with Holm-Sidak correction p val=0.017, n=4/group). There was no increase in CXCL10 ⁺ cells observed between contralateral TBI and sham tissue (mean+SD; 50+36 vs 11+8, p val=0.08). FIG. 3D. Immunohistochemistry on brain tissue post-TBI for the cell proliferation marker, Ki67 (brown) showed more Ki67 ⁺ IBA1 ⁺ proliferative cells in the ipsilateral thalamus compared to the contralateral thalamus. E. There was a significant increase of Ki67 ⁺ IBA1 ⁺ cells in the ipsilateral thalamus, but not the contralateral or sham control thalami (mean+SD; 49+18 vs 2+1, unpaired t test with Benjamini, Krieger, Yekutieli correction p val p=0.003).

[0014] FIGs. 4A-4D collectively show monocyte/macrophage and dendritic cell subclusters analysis. FIG. 4A. UMAP representation of a reclustering analysis of the monocyte and dendritic cell population, which identified 9 monocyte/macrophage subclusters and two dendritic cell subclusters in addition to the Ccr7 ^hl dendritic cell cluster. FIG. 4B. Gene ontology analysis of five selected monocyte/macrophage and dendritic cell subclusters are shown. Common enriched pathways include response to type I IFN or wound healing. FIG. 4C. Quantification of cell numbers of monocyte/macrophage subsets, and dendritic cell subsets in FIG. 4D. Significance of cell counts between WT normal and WT TBI mice or between WT TBI and Ccr2 ^y TBI mice were determined by two way ANOVA analyses followed by Tukey’s multiple comparison tests (mean +SD are shown).

[0015] FIGs. 5A-5C collectively show validation of LybC ¹¹¹ protein co-expression with Chil3 ^hl expression in TBI macrophage subsets by flow cytometry. FIG. 5A. Flow cytometry gating strategy for TBI day 4 ipsilateral brain white cells that are LIVE, CD45 ¹¹¹ CD 1 lb ⁺ and Ly6G. Cells are further gated for their binding to RNA probes for expression of Chil3, Argl, and Gpnmb conjugated to Alexa Fluor 647. A control RNA probe for Dapb , which detects a bacterial gene, did not bind TBI macrophages (data are representative of three independent experiments). FIG. 5B. Histogram analysis for Ly6C surface expression on gated TBI macrophages showed that Ly6C was preferentially highly expressed in 60% of Chil3 ⁺ TBI macrophages by flow cytometry. Argl expression was distinct from Chil3 expression as Argl was predominantly found in Ly6C ^b monocyte/macrophages. Gpnmb served as a robust coexpression marker Cor . I rg I . Dapb did not bind TBI macrophages. FIG. 5C. UMAP visualization of all cells and their expression of Argl , Chil3 _, and, Mrcl which are signature M(IL-4) genes, demonstrated that each gene was preferentially expressed by distinct macrophage subsets.

[0016] FIGs. 6A-6D collectively show targeting human CCR2 pharmacologically after TBI in human CCR2 knock-in mice led to reduced macrophage infiltration into the brain, improved cognitive memory, and reduced expression of a key ISG, IRF7. FIG. 6A. HCCR2 knock-in mice were administered a small molecule inhibitor with specific blockade of hCCR2 at 30 mg/kg (mpk) or 100 mpk, or vehicle beginning 2h post-surgery. Flow cytometry analysis of proportions of microglia and macrophages in the brain one day after surgery are shown. Ly6G viable cells are shown. Data represent at least three independent experiments each with biological replicates. FIG. 6B. Absolute macrophage numbers in ipsilateral hemispheres four days post-TBI or sham surgery were quantified by flow cytometry (n=3-10) per group). A one way ANOVA was used to determine statistical significance amongst groups, followed by a Kmskal -Wallis test and Dunn’s multiple comparisons test (*p<0.05, **/><0.01, ***/><0.001, ****/><0.0001). FIG. 6C. Cued platform version of the Morris water maze was performed beginning 4 weeks post-surgery (n=17 per TBI group, n=4-9 per sham group). Cued platform trial data are shown with statistics reflecting rank summary score analysis for each measurement (*/?<() 05. ***p<0.001, ****/?<() 0001 ) Swim velocity across all groups were equivalent. FIG. 6D. Relative quantitative PCR of ipsilateral brain hemispheres four days post-TBI or sham surgery were performed in triplicate for each gene, lrf7 or Tnf with biological replicates (n=4-10 per group). Two independent experiments were performed. One-way ANOVA was used to determine statistical significance followed by Tukey’s multiple comparisons test (*/?«) 05.

**p<0.01).

[0017] FIG. 7 shows gene expression of cell lineage markers shown in uMAPs of TBI and normal brain white cells combined. Top row: Microglia markers include Sail l and Tmemll9. Dendritic cell markers include Flt3. Second row: Monocyte/Macrophage DEGs include Ccr2 and F13al. B cell markers include Cdl9. Third row: Lymphocyte genes used to define T cells include Cd3e, and to define NK cells we used Ncrl (NKp46). Ly6g was used as a neutrophil marker. Fourth row: Neuronal cell markers and macroglia genes include and used include potassium-chloride transporter member 5 ( Slcl2a5 ), aldehyde dehydrogenase- 1 ( Aldhlll ) for astrocytes, and myelin oligodendrocyte glycoprotein (Mog).

[0018] FIG. 8 shows relative expression of significant DEGs defining microglia subclusters in the TBI and normal mouse brain. A heatmap showing relative expression of the top 10 differentially expressed genes between 13 microglia subsets identified by scRNA seq.

[0019] FIG. 9 shows gene ontology analysis of the DEGs of each microglia subset. Top immune-related gene enrichment pathways, their q values, and the number of genes in each set are shown. The top DEGs of each microglia subset are also shown with the fold change value of expression of each gene shown in parentheses.

[0020] FIG. 10 shows differential expression analysis between ('xclIO ¹ ' ¹ microglia and Ifi27l2a ^hl microglia subsets. Top DEGs that are differentially expressed between the two microglia subsets and their ratio of expression in ('xclIO ¹ " /fi27l2a'" microglia (data are log2 transformed).

[0021] FIG. 11 shows microglia subset analysis by injury and by genotype of subsets that were not significantly increased in an analysis of WT TBI vs WT normal samples. Cell counts of each microglia subset as quantified in brain hemispheres by scRNA seq (n=2- 3/group). Significance was quantified by two way ANOVAs and Tukey’s multiple comparisons tests. Below: Top DEGs in a DE analysis by genotype comparing gene expression in WT TBI/Ccr2 ^_/ TBI for each microglia subset shown.

[0022] FIG. 12 shows relative expression of significant DEGs defining monocyte/macrophage and dendritic cell subclusters found in the TBI and normal mouse brain. A heatmap showing relative expression of the top 10 differentially expressed genes between 9 microglia subsets and 2 dendritic cell subsets identified by scRNA seq.

[0023] FIG. 13 shows gene ontology analysis of the DEGs of each monocyte/macrophage and dendritic cell subset. Top immune-related gene enrichment pathways, their q values, and the number of genes in each set are shown. The top DEGs of each subset are shown with the fold change value of expression of each gene shown in parentheses. [0024] FIG. 14 shows HCCR2 knock-in mice treated with a small molecule inhibitor for hCCR2 beginning 2h post-surgery showed no differences in absolute cell numbers of microglia, neutrophils, or CD3 ⁺ T cells in the brain one day post-TBI. Flow cytometry analysis and quantification using counting beads determined cell numbers and were plotted as a fold change to the TBI vehicle group.

[0025] FIG. 15 shows open field and rotarod testing of hCCR2 knock-in mice treated with a hCCR2 small molecule inhibitor or vehicle. Tests were performed at three weeks postsurgery. Top row: Animals were placed in a novel environment and monitored by a grid of infrared beams for 10 min/day for two days. TBI animals were hyperactive relative to controls as measured by their basic movement score, less exploratory in the vertical direction as assessed by reduced rearing, and more anxious as quantified by their reduced time in the center zone. Bottom row: Motor balance and coordination across all TBI and sham groups were equivalent as measured by similar latencies to stay on the accelerating rotating rod.

DETAILED DESCRIPTION

[0026] The present invention can be understood more readily by reference to the following detailed description of the invention and the Examples included therein.

[0027] Disclosed are components that can be used to perform the disclosed methods and systems. These and other components are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these components are disclosed that while specific reference of each various individual and collective combinations and permutation of these may not be explicitly disclosed, each is specifically contemplated and described herein, for all methods and systems. This applies to all aspects of this application including, but not limited to, steps in disclosed methods. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.

[0028] The present compositions, methods, and kits may be understood more readily by reference to the following detailed description of preferred embodiments and the examples included therein.

[0029] While aspects of this disclosure can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of this disclosure can be described and claimed in any statutory class. Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be constmed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or description that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0030] Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon. Nothing herein is to be constmed as an admission that the present application is not entitled to antedate such publication by virtue of prior invention. Further, stated publication dates may be different from actual publication dates, which can require independent confirmation.

A. DEFINITIONS

[0031] Listed below are definitions of various terms used to describe this invention. These definitions apply to the terms as they are used throughout this specification, unless otherwise limited in specific instances, either individually or as part of a larger group.

[0032] As used in the specification and in the claims, the term “comprising” can include the aspects “consisting of’ and “consisting essentially of.”

[0033] As used in the specification and claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0034] Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0035] As used herein, the terms “about” and “at or about” mean that the amount or value in question can be the value designated some other value approximately or about the same. It is generally understood, as used herein, that it is the nominal value indicated ±10% variation unless otherwise indicated or inferred. The term is intended to convey that similar values promote equivalent results or effects recited in the claims. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but can be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about” or “approximate” whether or not expressly stated to be such. It is understood that where “about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0036] “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0037] The term “pharmaceutically acceptable salt,” as used herein, refers to an inorganic or organic salt of the compound of Formula I that is suitable for administration to a subject.

[0038] The term “pharmaceutically acceptable” describes a material that is not biologically or otherwise undesirable, i.e., without causing an unacceptable level of undesirable biological effects or interacting in a deleterious manner.

[0039] As used herein, the term “pharmaceutically acceptable carrier” refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. [0040] As used herein, the term “subject” can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. Thus, the subject can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. In one aspect, the subject is a mammal. A patient refers to a subject afflicted with an ailment, disease, or disorder. The term “patient” includes human and veterinary subjects.

[0041] As used herein, the terms “treatment” and “treating” refer to the medical management of a subject with the intent to cure, ameliorate, stabilize, or prevent an ailment, disease, pathological condition, disorder, or injury. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, disorder, or injury, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, disorder, or injury. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, disorder, or injury; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, disorder, or injury; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, disorder, or injury. In various aspects, the term covers any treatment of a subject, including a mammal (e.g, a human), and includes: (i) preventing the disorder or condition from occurring in a subject that can be predisposed to the disorder or condition but has not yet been diagnosed as having it; (ii) inhibiting the disorder or condition, i.e., arresting its development or exacerbation thereof; or (iii) relieving the disorder or condition, i.e., promoting healing of the disorder or condition. In one aspect, the subject is a mammal such as a primate, and, in a further aspect, the subject is a human.

[0042] As used herein, the term “prevent” or “preventing” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit, or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed. [0043] As used herein, the term “diagnosed” means having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition that can be treated by the compound of Formula I.

[0044] As used herein, the terms “administering” and “administration” refer to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In various aspects, a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. In further various aspects, a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition.

[0045] As used herein, the terms “effective amount” and “amount effective” refer to an amount that is sufficient to achieve the desired result or to have an effect on an undesired condition. For example, a “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drags used in combination or coincidental with the specific compound employed and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose.

The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. In further various aspects, a preparation can be administered in a “prophylactically effective amount”; that is, an amount effective for prevention of a disease or condition.

[0046] “C-C chemokine receptor type 2” or “CCR2” refers to a G protein-coupled receptor protein that in humans is encoded by the CCR2 gene. CCR2 is a key functional receptor for CCL2 (monocyte chemoattractant protein-1, MCP-1), which is a potent chemokine for monocytes and a variety of other immune cells

[0047] As used herein, “traumatic brain injury” or “TBG refers to an acquired brain injury or head injury in which trauma damages the brain. The damage can be localized, i.e., limited to one area of the brain, or diffuse, affecting one or more areas of the brain.

[0048] The term “spinal cord injury,” as used herein, means any injury to the spinal cord that is caused by trauma instead of disease. Depending on where the spinal cord and nerve roots are damaged, the symptoms can vary widely, for example from pain to paralysis to incontinence. Spinal cord injuries are described at various levels of “incomplete,” which can vary from having no effect on the subject to a “complete” injury which means a total loss of function. Spinal cord injuries have many causes, but are typically associated with major trauma from motor vehicle accidents, falls, sports injuries, and violence. The abbreviation “SCI” means spinal cord injury.

[0049] The term “stroke,” as used herein, refers to a condition in which blood supply to part of the brain is interrupted or reduced, preventing brain tissue from receiving oxygen and nutrients, resulting in the death of brain cells within minutes.

[0050] As used herein, “dosage form” means a pharmacologically active material in a medium, carrier, vehicle, or device suitable for administration to a subject.

[0051] As used herein, “kit” means a collection of at least two components constituting the kit. Together, the components constitute a functional unit for a given purpose. Individual member components may be physically packaged together or separately. For example, a kit comprising an instmction for using the kit may or may not physically include the instruction with other individual member components. Instead, the instmction can be supplied as a separate member component, either in a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as a recorded presentation.

[0052] As used herein, “instruction(s)” means documents describing relevant materials or methodologies pertaining to a kit. These materials may include any combination of the following: background information, list of components and their availability information (purchase information, etc.), brief or detailed protocols for using the kit, trouble-shooting, references, technical support, and any other related documents. Instructions can be supplied with the kit or as a separate member component, either as a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation. Instructions can comprise one or multiple documents, and are meant to include future updates.

[0053] Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including: matters of logic with respect to arrangement of steps or operational flow; plain meaning derived from grammatical organization or punctuation; the number or type of embodiments described in the specification.

B. TREATMENT METHODS

[0054] The present invention is based in part an established model of controlled cortical impact (CCI) TBI in mice, which induces a focal brain injury and recapitulates key features of human TBI, including bleeding, blood-brain barrier (BBB) damage, edema, progressive loss of neurons, robust inflammatory leukocyte infiltration into the brain, upregulation of specific cytokines, and functional deficits in animal behavior. Jassam, Y.N., Izzy, S., Whalen, M, McGavem, D.B. & El Khoury, J. Neuroimmunology of Traumatic Brain Injury: Time for a Paradigm Shift. Neuron 95, 1246-1265 (2017); Hsieh, C.L. et al. Traumatic brain injury induces macrophage subsets in the brain. Eur J Immunol 43, 2010-2022 (2013); Xiong, Y., Mahmood, A. & Chopp, M. Animal models of traumatic brain injury. Nat Rev Neurosci 14, 128-142 (2013); Hsieh, C.L. et al. CCR2 deficiency impairs macrophage infiltration and improves cognitive function after traumatic brain injury. J Neurotrauma 31, 1677-1688 (2014).

[0055] Mice deficient in C-C chemokine receptor-2 ( Ccr2 ) exhibit reduced macrophage infiltration, improved hippocampal-dependent cognitive outcomes, and preserved viable hippocampal neurons. Hsieh, C.L. et al. CCR2 deficiency impairs macrophage infiltration and improves cognitive function after traumatic brain injury. J Neurotrauma 31, 1677-1688 (2014). Other studies similarly support an overall deleterious role for the recruitment of peripheral monocytes to the brain in TBI. Semple, B.D., Bye, N., Rancan, M., Ziebell, J.M. & Morganti-Kossmann, M.C. Role of CCL2 (MCP-1) in traumatic brain injury (TBI): evidence from severe TBI patients and CCL2-/- mice. J Cereb Blood Flow Metab 30, 769-782 (2010); Morganti, J.M. et al. CCR2 antagonism alters brain macrophage polarization and ameliorates cognitive dysfunction induced by traumatic brain injury. J Neurosci 35, 748-760 (2015).

[0056] Mice deficient in a major ligand for CCR2, Ccl2, exhibit less macrophage accumulation, less astrogliosis, and improved functional recovery after TBI. Semple, B.D., Bye, N., Rancan, M., Ziebell, J.M. & Morganti-Kossmann, M.C. Role of CCL2 (MCP-1) in traumatic brain injury (TBI): evidence from severe TBI patients and CCL2-/- mice. J Cereb Blood Flow Metab 30, 769-782 (2010).

[0057] In WT mice, treatment prior to injury with a small molecule hCCR2 inhibitor (the compound of Formula I) results in diminished macrophage infiltration, reduced inflammation, and preserved cognitive function. In humans, CCL2 protein is produced locally within hours post-TBI and remains elevated in the CSF for up to 9 days, demonstrating that this pathway is steadily active in acute human TBI. Semple, B.D., Bye, N., Rancan, M., Ziebell, J.M. & Morganti-Kossmann, M.C. Role of CCL2 (MCP-1) in traumatic brain injury (TBI): evidence from severe TBI patients and CCL2-/- mice. J Cereb Blood Flow Metab 30, 769-782 (2010); Morganti, J.M. et al. CCR2 antagonism alters brain macrophage polarization and ameliorates cognitive dysfunction induced by traumatic brain injury. J Neurosci 35, 748- 760 (2015).

[0058] The present disclosure demonstrates a role for CCR2 in the damaging roles of neuroinflammation that follows TBI, leaving open the possibility that healing monocyte/macrophages are present but are overwhelmed by inflammatory myeloid cells. Macrophage subtypes were defined in TBI with a hypothesis that beneficial macrophages and/or phenotypes would emerge in the absence of CCR2. While a number of studies have characterized macrophage and microglia responses following TBI in bulk population studies, the study of average expression obscures the actions of cellular subsets. Mahata, B. et al. Single-cell RNA sequencing reveals T helper cells synthesizing steroids de novo to contribute to immune homeostasis. Cell reports 7, 1130-1142 (2014); Patel, A.P. el al. Single-cell RNA- seq highlights intratumoral heterogeneity in primary glioblastoma. Science 344, 1396-1401 (2014); Shalek, A.K. el al. Single-cell RNA-seq reveals dynamic paracrine control of cellular variation. Nature 509, 363-369 (2014); Trapnell, C. Defining cell types and states with single-cell genomics. Genome Res 25, 1491-1498 (2015); Wu, A.R. et al. Quantitative assessment of single-cell RNA-sequencing methods. Nature methods 11, 41-46 (2014); Ricardo -Gonzalez, R.R. et al. Tissue signals imprint ILC2 identity with anticipatory function. Nat Immunol 19, 1093-1099 (2018).

[0059] Recent studies have begun to unravel the heterogeneity of brain macrophage and microglia subtypes primarily in chronic neurodegenerative diseases of Alzheimer’s Disease and experimental autoimmune encephalitis, development, and aging. Jordao, M.J.C. et al. Single-cell profiling identifies myeloid cell subsets with distinct fates during neuroinflammation. Science 363 (2019); Keren-Shaul, H. et al. A Unique Microglia Type Associated with Restricting Development of Alzheimer's Disease. Cell 169, 1276-1290 el217 (2017); Hammond, T.R. et al. Single-Cell RNA Sequencing of Microglia throughout the Mouse Lifespan and in the Injured Brain Reveals Complex Cell-State Changes. Immunity 50, 253-271 e256 (2019); Mathys, H. et al. Temporal Tracking of Microglia Activation in Neurodegeneration at Single-Cell Resolution. Cell reports 21, 366-380 (2017); Sala Frigerio, C. et al. The Major Risk Factors for Alzheimer's Disease: Age, Sex, and Genes Modulate the Microglia Response to Abeta Plaques. Cell reports 27, 1293-1306 el296 (2019).

[0060] The present disclosure identifies myeloid cell subtypes in the setting of acute neurology and traumatic brain injury. Alterations of microglia phenotypes that are associated with the functional benefit of targeting Ccr2 were identified. Because CCR2 is restricted to circulating immune cells, such as monocytes and dendritic cells, and it is not expressed by microglia, the effects of Ccr2 deficiency on microglia are likely not direct. The present disclosure indicates the presence of crosstalk between microglia and infiltrating monocytes. Mizutani, M. el al. The fractalkine receptor but not CCR2 is present on microglia from embryonic development throughout adulthood. J Immunol 188, 29-36 (2012).

[0061] Translational studies were performed in which human CCR2 was targeted therapeutically in hCCR2 knock-in mice by therapeutic administration of a small-molecule inhibitor specific for hCCR2 (the compound of Formula I). These studies showed that using a clinically relevant administration of the compound of Formula 12h after injury blocked infiltrating macrophages after TBI, rescued cognitive function, and that the effects previously seen in genetically targeted mice were not due to developmental differences. In addition, administration of a therapeutic amount of the compound of Formula I between 5 minutes and 6 hours after TBI reduced monocyte, macrophage, and dendritic cell infiltration into the central nervous system. Without wishing to be bound by theory, in vivo data corroborate ex vivo scRNA-seq data to suggest that altering microglia towards a reduction in the type I IFN response may be the mechanism of neuroprotection. Infiltration of macrophages as an inflammatory response mediated by CCR2-CCL2 binding is also known to occur in other acute conditions or injuries, including spinal cord injury and stroke.

[0062] Thus, disclosed is a method of treating a traumatic brain injury, a spinal cord injury, or stroke in a subject, comprising administering to the subject an effective amount of a compound represented by Formula I, II, or III: or a pharmaceutically acceptable salt thereof, thereby treating the traumatic brain injury, the spinal cord injury, or stroke in the subject.

[0063] In some aspects, the compound of Formula I, II, or III can be administered to the subject within 24 hours following the traumatic brain injury, the spinal cord injury, or stroke. In a further aspect, the compound of Formula I, II, or III can be administered to the subject within 12 hours following the traumatic brain injury, the spinal cord injury, or stroke. In a further aspect, the compound of Formula I, II, or III can be administered to the subject within 6 hours following the traumatic brain injury, the spinal cord injury, or stroke. In a still further aspect, the compound of Formula I, II, or III can be administered to the subject within 6 hours following the traumatic brain injury, the spinal cord injury, or stroke.

[0064] It is also contemplated that, if possible, the compound of Formula I, II, or III can be administered to subject within shorter time periods following the traumatic brain injury, the spinal cord injury, or stroke. For example, the compound of Formula I, II, or III can be administered to the subject within 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 20 minutes, 10 minutes, or 5 minutes following the traumatic brain injury, the spinal cord injury, or stroke.

[0065] In a further aspect, the compound of Formula I, II, or III can first be administered to the subject as soon as possible following the acute injury and subsequently administered to the subject on a daily basis for at least 5 days following the injury. For example, a daily dose of the compound of Formula I, II, or III can be administered for 5 days, 10 days, 20 days, 30 days, or longer after the injury.

[0066] In some aspects, administration of the compound of Formula I, II, or III inhibits CCR2 binding to CCL2, thereby arresting the inflammatory response associated with traumatic brain injury, spinal cord injury, or stroke. For example, administration of the compound can reduce inflammation or macrophage infiltration at the site of injury, e.g., the site of the traumatic brain injury, spinal cord injury, or stroke. In some aspects, administration of a therapeutic amount of the compound of Formula I, II, or III at a suitable time after TBI, spinal cord injury, or stroke, can reduce the infiltration of monocytes, macrophages, dendritic cells, and other T cells that express the CCR2 receptor, thereby effecting a therapeutic response after injury. Likewise, administration of the compound of Formula I, II, or III can rescue or improve cognitive function following the traumatic brain injury, spinal cord injury, or stroke, as measured by methods known in the art.

[0067] In one aspect, administering an effective amount of the compound of Formula I, II, or III after a subject’s traumatic brain injury can improve the subject’s overall global disability following TBI. Overall global disability can be assessed using the Glasgow Outcome Scale Extended (GOS-E). The GOS-E is scored from 1-8: 1 = dead, 2 = vegetative, 3-4 = severe disability, 5-6 = moderate disability, 7-8 = good recovery. Moderate disability (GOS-E 5-6) is defined as one or more of the following: (1) inability to work to previous capacity, (2) inability to resume much of regular social and leisure activities outside the home, (3) psychological problems that have frequently resulted in ongoing family disruption or disruption of friendships. Severe disability (GOS-E 3-4) is defined as one or more of the following: (1) inability to drive or travel locally without assistance, (2) inability to shop or run errands without assistance, (3) support required for activities of daily living.

Standardized, structured interviews were performed per published guidelines. Wilson JT, Pettigrew LE, Teasdale GM, “Structured interviews for the Glasgow Outcome Scale and the extended Glasgow Outcome Scale: guidelines for their use. J Neurotrauma 1998;15:573-585. In addition, in some aspects, administering an effective amount of the compound of Formula

I, II, or III after a subject’s traumatic brain injury, spinal cord injury, or stroke, can improve the subject’s performance on the mini-mental state examination, which is a known method for measuring cognitive function after certain injuries to the central nervous system.

[0068] In a further aspect, administering an effective amount of the compound of Formula I,

II, or III after a subject’s traumatic brain injury can improve the subject’s neurologic assessment. Neurological assessment can be measured according to a structured interview designed for patients with TBI (Neurobehavioral Rating Scale-Revised [NRS] . Levin HS, High WM, Goethe KE, et al. “The neurobehavioural rating scale: assessment of the behavioural sequelae of head injury by the clinician.” J Neurol Neurosurg Psychiatry 1987;50:183-193. Neurological assessment can also be measured using headache interviews capturing frequency and intensity (Migraine Disability Assessment [MIDAS], (Stewart WF, Lipton RB, Whyte J, et al. “An international study to assess reliability of the Migraine Disability Assessment (MIDAS) score.” Neurology 1999;53:988-994), the Headache Impact Test [HIT]-6 (Kosinski M, Bayliss MS, Bjomer JB, et al. “A six-item short-form survey for measuring headache impact: the HIT-6.” Qual Life Res 2003;12:963-974), the Neurologic Outcome Scale for TBI (NOS-TBI) (Wilde EA, McCauley SR, Kelly TM, et al. “The neurological outcome scale for traumatic brain injury (NOS-TBI): I: constmct validity.” J Neurotrauma 2010;27:983-989), which is designed to assess focal neurologic deficits associated with TBI, and a TBI history intake interview modified from the Brain Injury Screening Questionnaire (Cantor JB et al. “Screening for traumatic brain injury: findings and public health implications. J Head Trauma Rehabil 2014;29:479-489), to confirm life history of head injury exposure and identify new head injuries sustained since last evaluation. [0069] A variety of subjects can be treated using the method. In one aspect, the subject is a mammal. In another aspect, the subject is a human. In a further aspect, the subject has been diagnosed with a need for treatment of the traumatic brain injury, the spinal cord injury, or stroke prior to the administering step. In a still further aspect, the method further comprises the step of identifying a subject in need of treatment of the traumatic brain injury, spinal cord injury, or stroke.

[0070] The compound of Formula I, II, or III can be administered to a subject as a pharmaceutically acceptable salt. Non-limiting examples of pharmaceutically acceptable salts are mineral acid (hydrochloric acid, hydrobromic acid, phosphoric acid, and the like) salts, organic acid (acetic acid, propionic acid, glutamic acid, citric acid and the like) salts, and quaternary ammonium (methyl iodide, ethyl iodide, and the like) salts. Other non-limiting examples include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, phosphonic acid, isonicotinate, lactate, salicylate, citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., l,r-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Still other salts include, but are not limited to, salts with inorganic bases including alkali metal salts such as sodium salts, and potassium salts; alkaline earth metal salts such as calcium salts, and magnesium salts; aluminum salts; and ammonium salts. Other salts with organic bases include salts with diethylamine, diethanolamine, meglumine, and N,N'-dibenzylethylenediamine. It is understood that the pharmaceutically acceptable salts are non-toxic. Additional information on suitable pharmaceutically acceptable salts can be found in Remington’s Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, which is incorporated herein by reference.

[0071] Pharmaceutically acceptable salts of the compound of Formula I, II, or III can be salts formed with bases, namely cationic salts such as alkali and alkaline earth metal salts, such as sodium, lithium, potassium, calcium, magnesium, as well as ammonium salts, such as ammonium, trimethyl-ammonium, diethylammonium, and tris-(hydroxymethyl)-methyl- ammonium salts. Similarly, acid addition salts, such as mineral acids, organic carboxylic and organic sulfonic acids, e.g., hydrochloric acid, methanesulfonic acid, maleic acid, are also contemplated. Neutral forms of the compound can be regenerated by contacting the salt with a base or acid and isolating the parent compound in a conventional manner.

1. PHARMACEUTICALLY ACCEPTABLE CARRIERS AND DOSAGE FORMS

[0072] In various aspects, the compound of Formula I, II, or III can be administered to a subject as a composition or formulation comprising a pharmaceutically acceptable carrier. Non-limiting examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

[0073] Pharmaceutically acceptable carries can also comprise adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms can be made by forming microencapsule matrices of the compound of Formula I, II, or III in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of dmg to polymer and the nature of the particular polymer employed, the rate of dmg release can be controlled. Depot injectable formulations can also be prepared by entrapping the dmg in liposomes or microemulsions which are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial -retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers can include sugars such as lactose.

[0074] In some aspects, the pharmaceutically acceptable carrier can include an excipient. Suitable excipients include, without limitation, saccharides, for example, glucose, lactose, or sucrose, mannitol, or sorbitol, cellulose derivatives, and/or calcium phosphate, for example, tricalcium phosphate or acidic calcium phosphate.

[0075] In further aspects, the pharmaceutically acceptable carrier can include a binder. Suitable binders include, without limitation, tare compounds such as starch paste, for example, com, wheat, rice, and potato starch, gelatin, tragacanth, methylcellulose, hydro xypropyl methylcellulose, carboxymethylcellulose, and/or polyvinylpyrrolidone. In still further aspects, there can be a disintegrating agent, such as the aforementioned starches and carboxymethyl starch, crosslinked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate.

[0076] In some aspects, the pharmaceutically acceptable carrier can include an additive. Examples of additives include, but are not limited to, diluents, buffers, binders, surface-active agents, lubricants, humectants, pH adjusting agents, preservatives (including anti-oxidants), emulsifiers, occlusive agents, opacifiers, antioxidants, colorants, flavoring agents, gelling agents, thickening agents, stabilizers, and surfactants, among others. Thus, in various further aspects, the additive is vitamin E, gum acacia, citric acid, stevia extract powder, Luo Han Gou, Monoammonium Glycyrhizinate, Ammonium Glycyrrhizinate, honey, or combinations thereof. In a still further aspect, the additive is a flavoring agent, a binder, a disintegrant, a bulking agent, or silica. In a further aspect, the additive can include flowability -control agents and lubricants, such as silicon dioxide, talc, stearic acid and salts thereof, such as magnesium stearate or calcium stearate, and/or propylene glycol.

[0077] In various aspects, when the compound of Formula I, II, or III can be formulated for oral use, such as for example, a tablet, pill, or capsule, and the composition can include a coating layer that is resistant to gastric acid. Such a layer, in various aspects, can include a concentrated solution of saccharides that can comprise gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol, and/or titanium dioxide, and suitable organic solvents or salts thereof.

[0078] Dosage forms can comprise the compound of Formula I, II, or III, together in combination with a pharmaceutically acceptable excipient, such as a preservative, buffer, saline, or phosphate buffered saline. Dosage forms can be made using conventional pharmaceutical manufacturing and compounding techniques. Dosage forms can comprise inorganic or organic buffers (e.g., sodium or potassium salts of phosphate, carbonate, acetate, or citrate) and pH adjustment agents (e.g., hydrochloric acid, sodium or potassium hydroxide, salts of citrate or acetate, amino acids and their salts) antioxidants (e.g., ascorbic acid, alpha- tocopherol), surfactants (e.g., polysorbate 20, polysorbate 80, polyoxyethylene9-10 nonyl phenol, sodium desoxycholate), solution and/or cryo/lyo stabilizers (e.g., sucrose, lactose, mannitol, trehalose), osmotic adjustment agents (e.g., salts or sugars), antibacterial agents (e.g., benzoic acid, phenol, gentamicin), antifoaming agents (e.g., polydimethylsilozone), preservatives (e.g., thimerosal, 2-phenoxyethanol, EDTA), polymeric stabilizers and viscosity -adjustment agents (e.g., polyvinylpyrrolidone, poloxamer 488, carboxymethylcellulose) and co-solvents (e.g., glycerol, polyethylene glycol, ethanol). A dosage form formulated for injectable use can have a disclosed composition or a product of a disclosed method of making, suspended in sterile saline solution for injection together with a preservative.

2. DOSAGE AND ROUTES OF ADMINISTRATION

[0079] The effective amount or dosage of the composition or an ingredient thereof can vary within wide limits. Such a dosage can be adjusted to the individual requirements in each particular case including the specific composition(s) being administered and the injury being treated, as well as the subject being treated. In general, single dose compositions can contain such amounts or submultiples thereof of the composition to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. In some aspects, the effective amount is a therapeutically effective amount.

In a further aspect, the effective amount is a prophylactically effective amount, e.g., an amount effective for preventing or ameliorating the CCR2-CCL2 mediated inflammatory response following a traumatic brain injury, spinal cord injury, or stroke.

[0080] The compound of Formula I, II, or III can be administered through any known route of administration. In one aspect, the compound is administered intravenously at least initially following the acute injury, and can optionally be administered daily thereafter as described above either intravenously or orally. In some aspects, the compound of Formula I, II, or III can be administered intraveneously at a suitable dose in aqueous hydro xypropyl methylcellulose. [0081] A variety of dosages of the compound of Formula I, II, or III can be effective for treating the traumatic brain injury, spinal cord injury, or stroke. For example, the dosage of the compound of Formula I, II, or III can range from about 0.5 mg/kg to about 1,000 mg/kg. In some aspects, the compound is administered at a dose of 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 10 mg/kg, 30 mg/kg, 50 mg/kg, 75 mg/kg, 100 mg/kg, 500 mg/kg, 750 mg/kg, or 1,000 mg/kg, based on the body weight of the subject, or in some aspects, based on a 75 kg human as a reference subject.

C. MANUFACTURE OF A MEDICAMENT

[0082] In one aspect, disclosed is the use of the compound represented by Formula I, II, or III or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of a traumatic brain injury, spinal cord injury, or stroke.

[0083] In various aspects, the method for the manufacture of a medicament comprises combining a therapeutically effective amount of the compound of Formula I, II, or III or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or diluent.

D. KITS

[0084] In a further aspect, disclosed is a kit comprising the compound of Formula I, II, or III or a pharmaceutically acceptable salt thereof and instructions for the use thereof for the treatment of a traumatic brain injury, spinal cord injury, or stroke in a subject in need of treatment.

[0085] In various aspects, the compound of Formula I, II, or III or a pharmaceutically acceptable salt thereof, and the instructions for the use thereof for the treatment of the injury can be co-packaged. In a still further aspect, the compound or pharmaceutically acceptable salt thereof and the instmctions are not co-packaged.

[0086] The kits can also comprise compounds and/or products co-packaged, co-formulated, and/or co-delivered with other components. For example, a drug manufacturer, a dmg reseller, a physician, a compounding shop, or a pharmacist can provide a kit comprising a disclosed compound and/or product and another component for delivery to a patient.

[0087] It is understood that the disclosed kits can be prepared from the disclosed compound and pharmaceutical formulations. It is also understood that the disclosed kits can be employed in connection with the disclosed methods of using the compound and pharmaceutical formulations.

E. EXAMPLES

[0088] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the methods and products claimed herein are made and evaluated, and are intended to be purely exemplary and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric. The Examples are provided herein to illustrate the invention, and should not be construed as limiting the invention in any way.

1. EXPERIMENTAL MODEL, SUBJECT, AND METHOD DETAILS a. ANIMALS

[0089] All mice were housed in a controlled environment (12h light/ 12h dark cycle, ~20°C). WT C57BL/6 male (RRID:IMSR_JAX:000664) cage mate mice (aged 12-16 weeks) were received from Jackson Laboratories (Sacramento, CA) and served as controls. Ccr2 knockout mice (Boring, L., Gosling, J., Cleary, M. & Charo, I.F. Decreased lesion formation in CCR2- /- mice reveals a role for chemokines in the initiation of atherosclerosis. Nature 394, 894-897 (1998)) were backcrossed onto a C57BL/6 background for nine generations and were from Jackson Laboratories (Bar Harbor, ME). Human Ccr2 knock-in mice were provided by ChemoCentryx (Mountain View, CA). CCR2 transgenic mouse breeding colonies were established and maintained at the San Francisco VA Medical Center. Mouse experiments were performed according to the rules and guidelines established by the Institutional Animal Care and Use Committee at the San Francisco VA Medical Center. b. CONTROLLED CORTICAL IMPACT SURGERY AND DRUG TREATMENT

[0090] Controlled cortical impact (CCI) or sham surgery was performed as approved by the VA Animal Care Committee. Animals were anesthetized with 3% isoflurane with oxygen and were administered bupivacaine (4 mg/kg) subcutaneously. A midline incision across the scalp was made, and a craniectomy was performed over the right parietal cortex. The target for the impact of coordinates was 1.5 mm right lateral and 2.3 mm posterior from the Bregma point. No animals used in this study showed excessive bleeding or indication of breaching the dura during the craniectomy. For TBI animals, a circular, flat-tipped piston induced an injury at 3 m/s, 150 ms duration, with a depth of 1.5 mm (Amscien Instruments, Richmond VA, with extensive modifications by H&R Machine, Capay, CA). After the bleeding was stopped, the skin was stapled close together. Sham-injured mice received surgical procedures without piston impact. All mice received buprenex (0.05 mg/kg up to two times/day for 24h) or sustained release buprenorphine (1 mg/kg) post-operation and 2 mis of saline s.c. to prevent dehydration.

[0091] For a subset of experiments, an antagonistic hCCR2 small molecule inhibitor (the compound of Formula I) (30 or 100 mg/kg) or vehicle (1% hypomethylcellulose) was administered subcutaneously beginning at 2h post-surgery (day 0), and then 1 time per day daily thereafter through day 5 or until sacrifice, whichever occurred earlier. Drugs and vehicle were provided by ChemoCentryx (Mountain View, CA). c. BRAIN LEUKOCYTE ISOLATION, FLOW CYTOMETRY, AND CELL SORTING

[0092] Four days after TBI, animals were euthanized and perfused through the heart with ice- cold GKN buffer (8 g/L NaCl, 0.4 g/L KC1, 1.41 g/L Na ₂ HP04, 0.6 g/L NaH ₂ P0 ⁴ , and 2 g/L D(+) glucose, pH 7.4). Hsieh, C.L. el al. Traumatic brain injury induces macrophage subsets in the brain. Eur J Immunol 43, 2010-2022 (2013); Kim, C.C., Nakamura, M.C. & Hsieh,

C.L. Brain trauma elicits non-canonical macrophage activation states. J Neuroinflammation 13, 117 (2016); Hsieh, C.L. et al. CCR2 deficiency impairs macrophage infiltration and improves cognitive function after traumatic brain injury. J Neurotrauma 31, 1677-1688 (2014). Ipsilateral brain hemispheres were harvested and the olfactory bulbs removed.

Tissues were minced and washed in cold GKN buffer. Tissue chunks were resuspended in 2.5 mL of digestion buffer (NOSE buffer (4 g/L MgCl ₂ , 2.55 g/L CaCl ₂ , 3.73 g/L KC1, 8.95 g/L NaCl, pH 6-7) with 200 U/ml DNase I (Sigma-Aldrich) and 0.2 mg/mL Collagenase I (Worthington Biochemical) and incubated at 37°C in a dry incubator for lh with shaking every 15 min. All samples were placed on ice to halt enzymatic activity. Tissues were crushed through a 100 micron nylon filter cup (BD Biosciences) and the cell suspension was washed with GKN buffer. An isotonic Percoll solution (90% Percoll (GE Biosciences), 10% 1.5M NaCl) was made and brought to room temperature. Cells were resuspended in 20 mL of a 1.03 g/mL isotonic Percoll solution in GKN buffer and underlayed with 10 mL of a 1.095 g/L isotonic Percoll solution in PBS. Cells were spun at ~850g at room temperature for 20 min. Buffy layers were isolated.

[0093] Cells were blocked with 10% rat serum for 10 min on ice and then stained with the following antibodies: anti-CD45 PE-Cy5.5 (Clone 30-F11, Invitrogen), anti-Ly6GPE- eFluor610 (Clone 1A8, Invitrogen), anti-CDllb PE (Clone Ml/70, BD Biosciences), anti- Ly6C PE-Cy7 (Clone AL-21, BD Biosciences), and anti-CD3 FITC (Clone 17 A2, BD Biosciences). DAPI (Invitrogen) was used at 1 uM to gate out dead cells. Cells were sorted on a FACS Ariallu (BD Biosciences) at the San Francisco VA Medical Center Flow Cytometry Core Facility.

[0094] Intracellular RNA flow cytometry was performed using PrimeFlow RNA reagents (Affymetrix). Kim, C.C., Nakamura, M.C. & Hsieh, C.L. Brain trauma elicits non-canonical macrophage activation states. J Neuroinflammation 13, 117 (2016). Fixable viability dye eFluor506 was used to exclude dead cells. Cell surface markers were stained using antibodies against CD45 (Clone 30-F11), CD1 lb (Clone Ml/70), Ly6G (Clone 1A8), and Ly6C (Clone AL-21). RNA probes for Argl, Chil3, and Gpnmb were used. A DapB RNA probe, a probe for RNA of a bacterial gene, served as a negative control. Cell staining was analyzed on a FACS Ariallu at the San Francisco VA Health Care System Flow Cytometry Core Facility. Data was analyzed using FlowJoX software (Treestar). d. SINGLE CELL RNA SEQUENCING

[0095] Eleven individual mice were used for scRNA-seq (3 WT TBI ipsilateral hemispheres,

3 Ccr2 ^y TBI ipsilateral hemispheres, 3 WT normal brains, and 2 Ccr2 ^~A normal brains). Dissected ipsilateral hemispheres were individually sorted for CD45 ⁺ Ly6G live singlets using a FACSAriallu at the San Francisco VA Health Care System Flow Cytometry Core Facility. Single cell RNA seq was performed at the Genomics Core Facility at the Institute for Human Genetics (University of California, San Francisco) using the 10X Genomics platform with gel emulsion bead technology. Chromium Single Cell 3 ’ Reagent Kits v3 was used according to manufacturer protocols and each sample was run on separate lanes. Libraries were sequenced on a NovaSeq6000 at the UCSF Center for Advanced Technologies with an average of 2967 average UMI per cell. e. RELATIVE QUANTITATIVE PCR

[0096] Perfused brain tissues were stored in RNA later at -20°C. RNA was isolated using TRIzol reagent (Invitrogen) and a Kinematica homogenizer (Polytron PT 10-35 GT). Reverse transcription was performed with the iScript cDNA Synthesis Kit (Bio-Rad) using a Mastercycler EP Gradient S (Eppendorf). Quantitative PCR was run using TaqMan reagents. Primer sequences used were: Irf7-Mm00516793_gl, Tnf- Mm00443258_ml, and Gapdh- Mm99999915_gl (FAM-MGB, ThermoFisher) as an endogenous control. PCR amplification was performed on QuantStudio 7 Flex (Applied Biosystems) at the San Francisco VA Health Care System Molecular Biology Core Facility. f. FLUORESCENT IMMUNOHISTOCHEMISTRY

[0097] Anesthetized mice were perfused with ice cold saline followed by 4% paraformaldehyde. Brains were post-fixed overnight in 4% paraformaldehyde and then immersed in 15% sucrose for 6h followed by 30% sucrose for 6h. Brains were embedded in Tissue-Tek optimal cutting temperature (OCT) compound (Sakura Finetech, Torrance, CA), frozen on dry ice and stored at -80°C. Brains were cut coronally into 40um thick free-floating sections into PBS. Sections were blocked for lh with 10% donkey serum in TBS containing 3% BSA and 0.4% Triton X-100 and incubated with primary antibodies overnight. Primary antibodies were against Iba-1 (rabbit polyclonal; Wako, Richmond, VA), CxcllO (goat IgG, R&D Systems, Minneapolis, MN) and NeuN (clone A60, Millipore, Burlington, MA). The following secondary antibodies were used: Donkey anti-goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488; Donkey anti-rabbit IgG (H+L) Highly Cross- Adsorbed Secondary Antibody, Alexa Fluor 568; Donkey anti-mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 (Thermo Fisher Scientific, Waltham, MA) for 2h at room temperature. DAPI (1:2000, Sigma-Aldrich, St Louis, MO) was used to identity nuclei before mounting with Fluoromount-G (Thermo Fisher Scientific, Waltham, MA). Images were acquired with a confocal laser scanning microscope (Zeiss LSM510 meta). Pictures were analyzed with Zeiss Zen microscope software. Three animals/group were analyzed. g. CHROMOGENIC IMMUNOHISTOCHEMISTRY

[0098] Formalin-fixed mouse brains were sectioned coronally through the middle of the visualized CCI lesion. The rest of the brain was sectioned into 2mm thick coronal sections as a 6-piece coronal trim. Pieces were placed rostrally face down, emanating from the mid- lesional section. These coronal sections were processed into FFPE blocks. Blocks were sectioned at 5pm on a DNS AS-400 Autosectioner. H&E and immunoperoxidase stains were performed (Ibal-Ki67) on Ventana Ultra IHC machines.

[0099] Stained slides were scanned at 20x using a Panoramic P250 slide scanner. Images were analyzed using Visiopharm software and custom image analysis algorithms. Slides were analyzed for stain Ki67 and Ibal. Ki67 and Ibal were analyzed with a morphology algorithm to detect percent area of total immunoreactivity and morphology parameters based on detected positivity. Data was compared among CCI and sham groups using 2-factor ANOVAs. h. BEHAVIOR STUDIES

[00100] The cued platform version of the Morris Water Maze was performed starting at 4 weeks post-TBI at the San Francisco VA Animal Behavior Core Facility. A swimming pool (Maze Engineers) filled with opaque water was monitored using video tracking software, Ethovision XT13 (Noldus), to analyze animals’ swim paths. A platform with a cue on top was placed in opaque water. Animals were trained to locate the platform with three trials per session for two sessions in one day. The maximum time per trial was 60s. If an animal had not located the platform after 60s, a handler blinded to the animal group would guide the animal to the platform. Hsieh, C.L. et al. CCR2 deficiency impairs macrophage infiltration and improves cognitive function after traumatic brain injury. J Neurotrauma 31, 1677-1688 (2014); Liu, Z. et al. Chronic treatment with minocycline preserves adult new neurons and reduces functional impairment after focal cerebral ischemia. Stroke; a journal of cerebral circulation 38, 146-152 (2007); Raber, J. et al. Irradiation attenuates neurogenesis and exacerbates ischemia-induced deficits. Annals of neurology 55, 381-389 (2004); Suh, S.W., Aoyama, K., Matsumori, Y., Liu, J. & Swanson, R.A. Pyruvate administered after severe hypoglycemia reduces neuronal death and cognitive impairment. Diabetes 54, 1452- 1458 (2005); Hong, S.M. et al. Reduced hippocampal neurogenesis and skill reaching performance in adult Emxl mutant mice. Experimental neurology 206, 24-32 (2007).

[00101] The open field test assessed animals’ spontaneous locomotor activity and baseline anxiety starting at 3 weeks post-TBI. Animals were placed in a novel environment inside a plexiglass enclosure (40 ^c 40 inches) surrounded by automated infrared photocells connected to a computer with KinderScientific software (Hamilton & Kinder) to record data. Beam breaks generated by movement were observed, allowing measurements of spontaneous locomotor activity. The amount of time spent in the center of the open field arena was used as an indicator of baseline anxiety. Decreased time spent in the center zone was used as an indicator of anxiety -like behavior. Animals were tested for 10 min/day for two days. Hsieh, C.L. el al. CCR2 deficiency impairs macrophage infiltration and improves cognitive function after traumatic brain injury. J Neurotrauma 31, 1677-1688 (2014); Raber, J. el al. Irradiation attenuates neurogenesis and exacerbates ischemia-induced deficits. Annals of neurology 55, 381-389 (2004); Suh, S.W., Aoyama, K., Matsumori, Y., Liu, J. & Swanson, R.A. Pyruvate administered after severe hypoglycemia reduces neuronal death and cognitive impairment. Diabetes 54, 1452-1458 (2005); Hong, S.M. et al. Reduced hippocampal neurogenesis and skill reaching performance in adult Emxl mutant mice. Experimental neurology 206, 24-32 (2007).

[00102] Rotarod (TSE Systems) was used to assess motor balance and coordination in mice three weeks after TBI. Mice were placed on a rotating rod that accelerated to 40 rpm over 300 seconds. The length of time the mouse could stay on the rod before falling off was recorded. Animals were assessed five trials/day for two days. Hsieh, C.L. et al. CCR2 deficiency impairs macrophage infiltration and improves cognitive function after traumatic brain injury. J Neurotrauma 31, 1677-1688 (2014); Liu, Z. et al. Chronic treatment with minocycline preserves adult new neurons and reduces functional impairment after focal cerebral ischemia. Stroke; a journal of cerebral circulation 38, 146-152 (2007); Raber, J. et al. Irradiation attenuates neurogenesis and exacerbates ischemia-induced deficits. Annals of neurology 55, 381-389 (2004); Suh, S.W., Aoyama, K., Matsumori, Y., Liu, J. & Swanson, R.A. Pyruvate administered after severe hypoglycemia reduces neuronal death and cognitive impairment. Diabetes 54, 1452-1458 (2005); Hong, S.M. et al. Reduced hippocampal neurogenesis and skill reaching performance in adult Emxl mutant mice. Experimental neurology 206, 24-32 (2007).

2. QUANTIFICATION AND STATISTICAL ANALYSIS a. STATISTICAL ANALYSIS

[00103] Statistical analysis of flow cytometry data was performed using Prism 7.0 & 8.0 (Graphpad). A one way ANOVA was used to determine statistical significance amongst groups, followed by a Kruskal-Wallis test and Dunn’s multiple comparisons test (*p< 0.05,

**p<0.01, *** ?<0.001, **** ><0.0001).

[00104] Relative quantification PCR data was analyzed using Prism 7.0 software (Graphpad). PCR datasets per gene of interest were analyzed using ordinary one-way ANOVA and Tukey’s multiple comparisons test, with a single pooled variance. b. SINGLE-CELL RNA SEQUENCING ANALYSIS

[00105] STAR Solo v2.7.2b was used to align reads to the mouse genome (mmlO) and aggregate UMI counts per gene per cell. STAR Solo count matrix outputs were then imported into Seurat and samples were combined following v3 integration methodology. Samples were aligned using 30 dimensions during integration and used for downstream analysis, including clustering and visualization with UMAP. After using the join graph-based clustering at a variety of resolutions, we settled on 20 total groups. Marker genes for each cluster were determined using Seurat’s “FindMarkers” function with the parameters logfc.threshold =

0.25, mimpct = 0.25, only.pos = T. Celltype identities were determined from cell lineage genes (increasing CC vectors and higher resolutions for clustering lead to cumbersome results, due to an extremely high number of clusters emerging before this group of cells would cluster independently). Each cluster was assigned to a cell type and subtype, and differential expression testing between wild type and Ccr2 ^A was done with FindMarkers function utilizing the Wilcoxon rank sum test (parameters logfc.threshold =

0.1, min.pct = 0.1, only.pos = F) inside each cell type and subtype. Wilcoxon rank sum test p- values were then corrected for multiple testing using the Bonferroni correction method based on the number genes tested. Celltype, cell subtype, and WT vs Ccr2 ^~A heatmaps were generated from top differentially expressed gene lists using Complex Heatmap.

[00106] Statistical analysis found significant differences in cell numbers in microglia and in the macrophage/dendritic cell clusters by using two-way ANOVAs followed by Tukey’s analysis. c. FLUORESCENT IMMUNOHISTOCHEMISTRY ANALYSIS

[00107] Fluorescent images were captured using a Leica SP5 laser scanning confocal microscope at the UCSF Biological Imaging Development CoLab. Quantification of IBA1 ⁺ CXCL10 ⁺ microglia/macrophages was performed using the Zeiss LSM510META confocal microscope and Zen microscope software. Three animals/group were analyzed. Unpaired t tests with Holm-Sidak correction were performed post hoc. d. CHROMOGENIC IMMUNOHISTOCHEMISTRY

[00108] Slides were analyzed for staining with anti-KI67 and anti-IB A1 antibodies using Visiopharm software. Ki67 and IB A1 staining were analyzed with a morphology algorithm to detect percent area of total immunoreactivity and morphology parameters based on detected positivity. Data was compared among TBI and sham groups utilizing two-way ANOVAs to assess for statistical significance, followed by an unpaired t test with Benjamini, Krieger, Yekutieli correction. e. BEHAVIOR S TUDIES S TATISTICAL ANALYSIS

[00109] For Morris Water Maze, TBI groups (n=17/group) and sham groups (n=4-9) were analyzed. Rank summary scores followed by a one way analysis of variance (ANOVA) (Prism 7.0, Graphpad) were used for evaluation of statistical significance. Possin, K.L. et al. Cross-species translation of the Morris maze for Alzheimer's disease. J Clin Invest 126, 779- 783 (2016).

[00110] Open field and rotor rod performance were analyzed in TBI (n= 17- 18/group) and sham (n=7 -9/group) groups.Two-way ANOVAs were used to assess statistical significance on each day of the open field test, followed by Tukey’s multiple comparisons test.

[00111] The animals’ average per day was used for quantitation of the rotor rod. Two- way ANOVAs were used to assess statistical significance at each time point, followed by Tukey’s multiple comparisons test.

3. RESOURCES TABLE

4. SINGLE-CELL RNA SEQUENCING TO IDENTIFY BRAIN CELL LINEAGES AND CELL SUBTYPES IN ACUTE TBI.

[00112] It has been shown that Ccr2 ^y mice demonstrated improvements in cognitive memory and histopathology following TBI, compared to WT mice. Hsieh, C.L. el al. CCR2 deficiency impairs macrophage infiltration and improves cognitive function after traumatic brain injury. J Neurotrauma 31, 1677-1688 (2014). To better understand the mechanisms associated with this improvement, the differences in immune responses in acute TBI between Ccr2 ^y mice and WT C57BL/6 micedd were determined. Samples were analyzed four days post-TBI, a time point previously demonstrated to have peak infiltration of peripheral monocytes. Id. Single-cell RNA sequencing (scRNA-seq) on 111,717 high quality single cells combined from wildtype (WT) TBI mice (n=3 animals, 1 animal/sample), Ccr2 ^y TBI mice (n=3 animals, 1 animal/sample) and normal control mice (n=2-3 animals/group, 1 animal/sample) from both genotypes isolated from ipsilateral brain hemispheres was performed. Normal mice were chosen for single-cell transcriptomic analysis to generate controls for gene expression analysis. Sham controls can elicit low localized levels of inflammation, and were used in downstream validation experiments. Cells were sorted by flow cytometry, gating for CD45 ⁺ Ly6G live singlets, which includes microglia and macrophages, but excludes neutrophils (FIG. 1A).

[00113] ScRNA-seq was performed, cell doublets were excluded, and data were analyzed from microglia and circulating leukocytes from 35,405 individual transcriptomes of WT cells after TBI, 28,918 Ccr '- cells after TBI, 33,365 WT cells without TBI, and 14,029 Ccr2 ^y cells without TBI expressing an average of 22,701 genes/sample. WT and Ccr2 ^y cells were compared and aligned to each other by using canonical correlation analysis (CCA) with Seurat. Butler, A., Hoffman, P., Smibert, P., Papalexi, E. & Satija, R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat Biot echnol 36, 411-420 (2018). The high dimensional data were visualized by using UMAP (uniform manifold approximation and projection) (Becht, E. et al. Dimensionality reduction for visualizing single-cell data using UMAP. Nat Biotechnol (2018)) (FIG. IB). An analysis of cell lineage markers to define cell clusters identified a very large population of microglia, a cluster comprised of a mix of monocytes/macrophages and dendritic cells (referred to as macrophage/dendritic cells cluster), a separate Ccr7 ^hl dendritic cell subset, lymphocyte clusters, and a subset of cells with unclear immune cell lineage labelled “other” (FIG. 1A). Examples of cell lineage markers used to define clusters include: microglia ( Salll , Tmemll9) (Bennett, M.L. et al. New tools for studying microglia in the mouse and human CNS. Proc Natl Acad Sci USA 113, E1738-1746 (2016); Buttgereit, A. et al. Salll is a transcriptional regulator defining microglia identity and function. Nat Immunol 17, 1397-1406 (2016)), monocyte/macrophages ( Ccr2 , F13al (Hammond, T.R. et al. Single-Cell RNA Sequencing of Microglia throughout the Mouse Lifespan and in the Injured Brain Reveals Complex Cell- State Changes. Immunity 50, 253-271 e256 (2019)), dendritic cells (Flt3, Zbtb46) (Merad,

M., Sathe, P., Helft, L, Miller, J. & Mortha, A. The dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting. Annu Rev Immunol 31, 563-604 (2013)), B cells ( Cdl9 ), T cells ( CD3e ), NK cells (Ncrl), neutrophils (Ly6g), neurons ( Slcl2a5 ), astrocytes ( Aldhlll ), and oligodendrocytes ( Mog ) (FIG. 1C and FIG. 7). In normal brains, a large majority of the cells identified were microglia as these samples lacked infiltrating leukocytes.

[00114] A gross analysis of cell numbers of microglia and circulating leukocytes per half brain by genotype and by injury showed that TBI induced significant increases in cell numbers of the macrophage/dendritic cells cluster (ANOVA p val = 0.01, WT TBI = 793 + 315 (mean + SD), WT normal control = 190 + 73, adj p val=0.01), as well as increases in the T and NK cell cluster (ANOVA p=0.007, WT TBI = 345 + 145 (mean + SD), WT normal control = 92 + 46, adj p val=0.04) (FIG. ID). Statistical analysis found a significant difference by genotype in microglia and in the macrophage/dendritic cell cluster (ANOVAs p val=0.01) (FIG. ID). Post hoc Tukey’s analysis identified a significant difference in macrophage/dendritic cell numbers the pairwise comparison between WT TBI and Ccr2 ^_/

TBI mice (WT TBI=793 + 315 (mean + SD), Ccr '- TBI=220 + 51, adj p val=0.02), but not in microglia (FIG. ID). It was next assessed whether further analysis of microglia subclusters would clarity the source of variation.

5. IDENTIFICATION OF MICROGFIA SUBSETS IN NORMAF AND ACUTE TBI

IPSIFATERAF BRAINS

[00115] Joint graph-based clustering with canonical correlation vectors defined 13 distinct subclusters or subsets of microglia that were visualized by using the UMAP dimensionality reduction technique (FIG. 2A). Each microglia subset was designated by a differentially expressed gene (DEG) with the highest fold change that distinguished that subset from all other microglia subclusters. DEGs throughout the analyses were identified by using the Wilcoxon rank sum test, and p-values were then corrected for multiple testing using the Bonferroni correction method. The highest expressed DEGs possibly suggest some phenotype to its assigned subset, but the DEGs were defined based on relative expression to other subclusters and were not necessarily exclusively expressed in its subset. The 13 microglia subsets were named as follows: 1) CxcllC ¹ (C-X-C motif chemokine ligand 10, also known as interferon gamma-induced protein 10, IP-10), 2) Cenpf ¹¹ (centromere protein F), 3) Ccl4 ^h ‘ (chemokine C-C motif ligands 4), 4) Ifi27l2a ^h ‘ (interferon alpha-inducible protein 27-like protein 2A), 5) Sppl ^hl (secreted phosphoprotein 1), 6) Btg2 ^hl (B-cell translocation gene 2), l)Jun ^h ‘ (Jun proto-oncogene, AP-1 transcription factor subunit), )IIba-a I ¹ " (hemoglobin alpha, adult chain 1), 9) Crybbl ^hl (crystallin beta B 1), 10) Smad7 ^hl (SMAD family member 7), 11) Nfkbia ^hl (NFKB inhibitor alpha), 12) S100a9 ^hl (S100 calcium binding protein A9), and 13) Ctsl ^hl (cathepsin L) microglia. To visualize gene expression of a few top DEGs of a few microglia subclusters and to see their expression in normal vs TBI brain microglia, uMAPs of Irf7 were observed, which is a top 10 DEG of Cxcll0 ^hl and Ifi27l2a ^hl microglia, ( 'cl 4 and Clec7a (Dectin 1), which are top 5 DEGS of Ccl4 ^hl microglia, and Tnf which was the top DEG for Xfkbia ¹ " microglia (FIG. 2B). UMAPs show that each DEG highlights a specific cluster and that the expression of Irf7, Ccl4, and Clec7a increase in TBI microglia compared to normal microglia, though there appears to be little to no increase in Tnf expressing microglia (FIG. 2B).

[00116] To better understand the differences between the microglia subsets, a heatmap showing the top 10 DEGs of each subset relative to other microglia is shown (FIG. 8). Gene ontology (GO) analysis of significant DEGs was performed to begin to understand the phenotype of each microglia subset (FIG. 2C, FIG. 2D, and FIG. 3). There were five notable observations from the functional pathway analysis of enriched genes in microglia subsets.

The first is the presence of two microglia subsets, Cxcll0 ^hl and Ifi27l2a ^hl , that showed significantly enriched expression of genes from the type I interferon (IFN) pathway, including a 2-3 fold increase in the expression of a type I IFN stimulated gene (ISG) and transcription factor, Irf7 (interferon regulatory factor 7) (FIG. 8 and FIG. 9). Although these two ISG-expressing microglia subsets share some level of markers, a direct comparison of significant DEGs between the two subsets found that there were clear distinctions between them (FIG. 10). The second observation is that the Cenpf' microglia subset was enriched in expression of cell cycle and cell division genes, including Mki67 (marker of proliferation Ki- 67) (FIG. 8 and FIG. 9). A third important observation is that the Ccl4 ^hl microglia subset was remarkably similar to TREM2 -dependent disease-associated microglia (DAM) (Keren-Shaul, H. et al. A Unique Microglia Type Associated with Restricting Development of Alzheimer's Disease. Cell 169, 1276-1290 el217 (2017)) as it is enriched in several DAM marker genes, including Ccl6 (chemokine C-C motif ligand 6), Lpl (lipoprotein lipase), Clec7a (Dectinl), Cd9, Cd63, Ctsb (cathcpsin B), . Ink (progressive ankylosis), Cst7 (cathepsin 7), and Apoe (apo lipoprotein E) (FIG. 8 and FIG. 9). Fourth, multiple microglia subsets, including Ccl4 ^hl and Sppl ^hl , preferentially expressed genes with functions related to leukocyte migration and chemotaxis genes, particularly for monocytes and neutrophils (FIG. 2C, FIG. 2D, and FIG. 9). Finally, three of the microglia subsets, Btg2 ^h ‘, Jun ^h ‘, and Nfkbia ^hl , were enriched in genes associated with the response to IL-1 (FIG. 2D and FIG. 9).

6. TBI INDUCTION OF A RISE IN MICROGLIA SUBSETS RESPONSIVE TO TYPE I IFNs THAT EXPRESS PROLIFERATION OR DAM MARKER GENES.

[00117] The effects of TBI on the cell numbers and gene expression of each microglia subset were determined. In response to acute TBI, five of the thirteen WT microglia subsets, CxcllO ^hl , Cenpf', Ccl4 ^h ‘, Ifi27l2a ^h ‘, and Sppl ^h ‘, increased in number by 3 -fold to 8-fold in WT TBI animals compared to normal WT brain controls (FIG. 2B). The remaining 8 microglia subset cell numbers were unchanged by TBI (FIG. 2C, FIG. 11). Genes elevated following TBI by each WT microglia subset were also evaluated for pathway enrichment analysis (FIG. 12). Following TBI, five WT microglia subsets expressed genes indicative of a response to IFN-b and/or IFN-a during TBI, including the following microglia subsets, proliferating Cenpf', Ccl4 ^hl , Sppl ^hl , Nfkbia ^hl , and Smad7 ^hl (FIG. 12). TBI induced heterogeneous and specific responses from microglia subsets.

7. CCR2 DEFICIENCY ALTERATION OF TBI MICROGLIA CELL NUMBERS AND TRANSCRIPTION.

[00118] To determine the effects CCR2 deficiency on the cell numbers of each microglia subset, cell counts of WT TBI microglia to Ccr2 ^~A TBI microglia from the scRNA seq dataset were compared. Ccr2 deficiency specifically and significantly reduced the cell numbers of CxcllO ^h ‘, Cenpf', and Ccl4 ^h ‘ microglia in Ccr2 ^/ TBI mice by approximately one- third compared to WT TBI brains, while the other microglia were not reduced (FIG. 2B).

One microglia subset was significantly increased in Ccr2 TBI mice, Hba-al ^h ‘ microglia (FIG. 11). The predicted functions for this subset by GO analysis however, did not reveal any unique pathways (FIG. 9).

[00119] Differential gene expression analysis between WT TBI and Ccr2 ^~/~ TBI microglia subsets revealed immune response pathways promoted by Ccr2 in WT TBI mice. Compared to their corresponding Ccr2 ^~/~ microglia subsets from TBI mice, four different WT microglia subsets from WT TBI mice were found to be preferentially expressing elevated levels of the ISG, CxcllO. The four microglia subsets were Cenpf', Ifi27l2a ^h ‘, Btg2 ^h ‘, and Jun ^h ‘ microglia subsets with robust statistical significance (adj p values=3E-14, 4E-118, 5E- 37, 2E-71, respectively) (FIG. 2B, FIG. 2C, FIG. 11). Lgals3 (Galectin-3) was increased in multiple WT TBI microglia subsets compared to Ccr2 ^~/~ TBI microglia subsets, including in CxcllOf", Btg2 ^h ', Crybbl ^hl , Smad7 ^h ‘, and S100a9 ^h ' microglia subsets (adj p value= 3E-20 and 7E-111, respectively) (FIG. 2B, FIG. 2C, FIG. 11). Chemokines, ( 'cl 3, Ccl4, and ( 'cl 5. were elevated in four WT TBI microglia subsets, Cenpf ¹ , Ifi27l2a ^h ‘, Btg2 ^h ‘, and Jun ^h ‘ cells, compared to Ccr2 ^_/ TBI microglia (all adj p val >2xE-8, FIG. 2B and FIG. 2C). Although the increase of gene expression in WT TBI mice was mild, ranging from 10-45%, they were statistically highly significant. [00120] In summary, Ccr2 deficiency reduced the cell numbers of multiple type I IFN responding microglia, and reduced the gene expression of CxcllO, Lgals3, and chemokines in several microglia subsets.

8. TYPE I IFN RESPONDING MICROGLIA AND A PROLIFERATING MICROGLIA SUBSET LOCALIZATION TO THE TBI LESION SITE.

[00121] To validate the identification and localization of at least two microglia subsets at the protein level, immunohistochemistry was performed. Histological analysis for expression of CXCL10, IBA1, NEUN, and DAPI showed co-expression of CXCL10 and IBA-1 in cells localized in the ipsilateral (side of injury) hippocampus and thalamus (FIG.

3A and FIG. 3B). Quantification of double-positive P3A1 ⁺ CXCL10 ⁺ microglia/macrophages showed that ipsilateral TBI tissue had a significant increase in the mean number of IBA1 ⁺ CXCL10 ⁺ cells compared to sham brain tissue (mean+SD; 141+73 vs 18+15, unpaired t-test with Holm-Sidak correction p val=0.017) (FIG. 3C). No significant increase in CXCL10 ⁺ cells was observed between contralateral TBI and sham tissue (mean+SD; 50+36 vs 11+8, p val— 0.08) (FIG. 3C)

[00122] Immunohistochemistry for the cell proliferation marker, KI67, revealed robust expression of Ki67 in a subset of IBA1 ⁺ cells that resembled ramified, activated microglia localized in the thalamus of TBI animals (FIG. 3D). Quantification demonstrated a significant increase of double-positive Ki67 ⁺ IB Al ⁺ cells in the ipsilateral thalamus, but not the contralateral or sham control thalami (mean+SD; 49+18 vs 2+1, unpaired t test with Benjamini, Krieger, Yekutieli correction p val p=0.003) (FIG. 3E). We also observed a marked increase in the number of non-proliferative IB A1+ cells localized to the thalamus in acute TBI (mean+SD; 398+95 vs 20+1, unpaired t-test with FDR approach of Benjamini, Krieger, Yekutieli, p val p=0.0004) (FIG. 3E).

[00123] Thus, acute TBI induces the rise of CXCL10 ⁺ and KI67 ⁺ subsets of IB A1 expressing cells in the thalamus near the site of brain injury corroborating the scRNA seq findings on microglia subsets.

9. IDENTIFICATION OF MONOCYTE/MACROPHAGE AND DENDRITIC CELL SUBSETS IN NORMAL AND ACUTE TBI IPSILATERAL BRAINS

[00124] The cell cluster expressing both monocyte and dendritic cell markers in FIG.

1 was broken down further to identity what subsets comprised the mixture of cells. Reclustering analysis with Seurat identified 9 monocyte/macrophage clusters designated as Rgsl ^hl (regulator of G protein signaling 1), Argl ^hl (arginase 1), Chil3 ^hl (chitinase-like 3), Sppl ^h ‘ (secreted phosphoprotein 1), Tmeml76b ^h ‘ (transmembrane protein 176B), Ear2 ^h ‘ (eosinophil-associated, ribonuclease A family, member 2), Apoe ^hl (apolipoprotein E), Ccl8 ^hl (C-C motif chemokine ligand 8), and S100a9 ^hl (S100 calcium binding protein A9) (FIG. 4A). In addition to the Ccr7 ^hl dendritic cells identified in FIG. 1, reclustering found two additional dendritic cell clusters designated as Ciita ^hl (class II major histocompatibility complex transactivator), and Cd209a ^hl (also known as DC-SIGN), both of which co-express Flt3 (fms related receptor tyrosine kinase 3), Itgax (integrin subunit alpha X, CD1 lc), and Zbtb46 (zinc finger and BTB domain containing 46) (FIG. 4A). The top 10 genes that were significantly and differentially expressed, and that allow the definition of each monocyte and dendritic cell subset were visualized with a heatmap (FIG. 12).

[00125] To begin to understand the phenotypes of these monocyte/macrophage and dendritic cell clusters, differential gene expression was analyzed and gene ontology analysis of the subsets was performed. Ly6c2 was highly co-expressed in ( 'hits'" monocytes (FIG.

13), suggesting that these might be pro -inflammatory LyOC ¹ " monocytes. GO analyses found two subclusters, Chil3 ^hl monocytes and Cd209a ^hl dendritic cells, expressing ISGs, suggesting the presence of active type I IFN pathways (FIG. 4B, FIG. 13). Four of the monocyte/macrophage subsets, Argl ^hl , Rgsl ^hl , Apoe ^h ‘, and Ccl8 ^hl , expressed genes enriched in wound healing processes (FIG. 4B, FIG. 13). The Apoe ^hl macrophage subset expressed other DEG similar to DAM (Keren-Shaul, H. et al. A Unique Microglia Type Associated with Restricting Development of Alzheimer's Disease. Cell 169, 1276-1290 el217 (2017)), and lipid-associated macrophages (LAMs) (Jaitin, D. A. et al. Lipid-Associated Macrophages Control Metabolic Homeostasis in a Trem2 -Dependent Manner. Cell 178, 686-698 e614 (2019)), and are enriched in pathways for remodeling protein-lipid complexes (FIG. 4B,

FIG. 13). It was observed that Ccl8 ^hl macrophages expressed little to no Ccr2, but expressed relatively high levels of Mrcl and Lyvel, which are markers of brain resident CNS-associated macrophages (CAMs). Jordao, M.J.C. et al. Single-cell profiling identifies myeloid cell subsets with distinct fates during neuroinflammation. Science 363 (2019). Another recurring biological pathway found among macrophages involved leukocyte chemotaxis, which was found in Chil3 ^hl , Argl ^h ‘, Rgsl ^hl , and S100a9hi macrophages (FIG. 4B, FIG. 13). All three dendritic cells clusters, Ciita ^hl , Cd209a ^h ‘, and Ccr7 ^hl , were enriched in genes associated with T cell activation (FIG. 13).

10. CCR2 DEFICIENCY EFFECTS ON SPECIFIC MONOCYTE/MACROPHAGE AND DENDRITIC CELL SUBSETS ELEVATED IN THE CNS BY TBI.

[00126] TBI resulted in infiltration and/or expansion of cell numbers in six out of the nine identified monocyte/macrophage subsets, Chil3 ^h ‘, Argl ^h ‘, Rgsl ^h ‘, Sppl ^h ‘, Ear2 ^h ‘, and Apoe ^hl macrophages, and two out of the three dendritic cell subsets, Cd209 ^hl and Ciita ^hl dendritic cells were also increased (Two way ANOVA with Tukey’s multiple comparison tests, FIG. 4C and FIG. 4D). In five of the six monocyte/macrophage subsets, the increases in cell numbers induced by TBI were significantly attenuated in mice deficient in Ccr2 Chil3 ^h ‘, Argl ^hl , Rgsl ^hl , Sppl ^hl , and Ear2 ^hl macrophage subsets showed 53-86% reduction in mean cell numbers (adj p values=0.002 to 0.04) in the ipsilateral brain of Ccr2 ^/ TBI mice compared to WT TBI mice (FIG. 4C). The Chil3 ^hl monocytes/macrophages in particular are enriched in ISGs, and these cells were significantly reduced (FIG. 4C). There was also a lesser reduction of Cd209a ^hl dendritic cells, which are also enriched in ISGs (FIG. 4D).

While Apoe ^hl macrophages also trended towards a significant reduction in Ccr2 ^/ TBI mice (adj p value=0.05), there were no changes in Tmeml76b ^h ‘, Ccl8 ^h ‘, and S100a9 ^h ‘ macrophage numbers (FIG. 4C). Ciita ^hl dendritic cells were significantly diminished by a mean of 76% (adj p value=0.01) in Ccr2 ^/ TBI mice compared to WT TBI mice. While the reduction in these subsets is associated with benefit in TBI, many of them, like Argl ^hl , Rgsl ^hl , Apoe ^hl macrophages, are enriched in wound healing processes (Fig. 13), so a reduction in the cell numbers may also limit recovery processes.

11. VALIDATION OF LY6C ^m AND CHIL3 CO-EXPRESSION ON TBI MONOCYTE/MACROPHAGES

[00127] Ly6c2 was identified as a top 20 differentially expressed gene in the Chil3 ^hl TBI macrophage subset with nearly 3 -fold higher expression compared to other subsets (FIG. 13). This was unexpected, since high expression of Ly6C is established as a classical marker for proinflammatory monocytes (Shi, C. & Pamer, E.G. Monocyte recruitment during infection and inflammation. Nat Rev Immunol 11, 762-774 (2011); Swirski, F.K. & Nahrendorf, M. Cardioimmunology: the immune system in cardiac homeostasis and disease. Nat Rev Immunol 18, 733-744 (2018)), while Chil3 (Yml) is described as an antiinflammatory M(IL-4) macrophage marker. Murray, P.J. el al. Macrophage Activation and Polarization: Nomenclature and Experimental Guidelines. Immunity 41, 14-20 (2014); Chawla, A. Control of macrophage activation and function by PPARs. CircRes 106, 1559- 1569 (2010); Loke, P. el al. IL-4 dependent alternatively-activated macrophages have a distinctive in vivo gene expression phenotype. BMC Immunol 3, 7 (2002). Further, the data in FIG. 4 revealed that Chil3 and Argl were designated as significant markers of different TBI macrophage subsets. By using ARG1 reporter mice, it has been previously confirmed at the protein level by histology and by flow cytometry that ARG1 clearly marked a subset of infiltrating F4/80 ⁺ monocyte/macrophages. Hsieh, C.L. el al. Traumatic brain injury induces macrophage subsets in the brain. Eur J Immunol 43, 2010-2022 (2013).

[00128] To follow up on our datasets, it was determined whether LybC ¹ " classical and Ly6C ^b nonclassical macrophages at the protein level in acute TBI could be identified and whether their co-expression of Chil3 and . rig/ RNA by RNA flow cytometry could be examined (FIG. 5A and FIG. 5B). Four days after TBI, brain leukocytes were isolated and flow cytometry was performed to examine CD45 ¹¹¹ Ly6G CD 1 lb ⁺ TBI macrophages for RNA expression of Chil3 and Argl (FIG. 5 A and FIG. 5B, data are representative of three independent experiments). Chil3 ⁺ or. rig/ TBI macrophages were gated and their Ly6C expression was analyzed (FIG. 5A and FIG. 5B). As indicated by the scRNA seq subclustering analysis in FIG. 4 and differential expression analysis between subclusters (FIG. 12 and FIG. 13), it was confirmed that Ly6C RNA and protein expression correlated with each other and that Ly6C was preferentially highly expressed on 60% of Chil3 ⁺ TBI macrophages by flow cytometry (FIG. 5B). In contrast, Argl expression was distinct from Chil3 expression as Argl was predominantly found in Ly6C ^l0 monocyte/macrophages (FIG. 5B). It was found that Gpnmb (glycoprotein nmb, or osteoactivin) served as a robust coexpression marker for Argl (FIG. 5B). The control RNA probe, Dapb , which detects a bacterial gene did not bind TBI macrophages (FIG. 5A) or show correlation with Ly6C (FIG. 5B). The RNA flow cytometry data (FIG. 5B) and uMAP visualization of all cells in the scRNA samples (FIG. 5C) indicate some overlap between Chil3 and Argl expression in the monocytes/macrophages. However, overall the flow cytometry, uMAP and differential expression analysis data (FIG. 12) demonstrate that Chil3 and Argl are differentially expressed on distinct subsets with high statistical significance (FIG. 5B, FIG. 5C, and FIG. 12).

[00129] Previous scRNA seq on ~40 ex vivo TBI macrophages using a different technology platform (Fluidigm Cl methods) also found a lack of correlation between Argl, Chil3, Mrcl, Tnfl and Illb expression and refuted the clear presence of the in vitro derived M(IL4) and M1(LPS, IFNy) phenotype for macrophages in the setting of in vivo acute CNS injury. Kim, C.C., Nakamura, M.C. & Hsieh, C.L. Brain trauma elicits non-canonical macrophage activation states. J Neuroinflammation 13, 117 (2016); Ransohoff, R.M. A polarizing question: do Ml and M2 microglia exist? Nat Neurosci 19, 987-991 (2016). In the current greatly expanded scRNA seq dataset of 101,916 microglia and 5,157 monocyte/macrophage/dendritic cells, a glance at expression of signature M(IL-4) genes, Argl, Chil3, and Mrcl found that each gene was preferentially expressed by distinct macrophage subsets (FIG. SC). Argl, Chil3, and Mrcl were not observed to significantly correlate with any identified microglia subclusters, unlike those genes highlighted in FIG. 2B for example.

12. TARGETING CCR2 PHARMACOLOGICALLY ALTER TBI WITH THE COMPOUND OL FORMULA I.

[00130] In order to test the potential clinical use of the compound of Formula I in TBI, the hCCR2 inhibitor was tested for efficacy when given two hours after trauma, and its ability to target the relevant human receptor in vivo by using hCCR2 knock-in mice, in which human Ccr2 replaced the mouse gene, was tested. Sullivan, T. el al. CCR2 antagonist CCX140-B provides renal and glycemic benefits in diabetic transgenic human CCR2 knockin mice. Am J Physiol Renal Physiol 305, F1288-1297 (2013). Using human receptor transgenic mice was important because the affinity of the compound is 100-fold greater for hCCR2 than its affinity for the mouse receptor. CD45 ^lu CDl lb ⁺ macrophages infiltrating the brain early post- TBI in hCCR2 knock-in mice treated subcutaneously with 100 mg/kg (mpk) or 30 mpk of CCX872, or vehicle alone beginning two hours post-TBI (n=5-10 per TBI group, n=3 per sham group) were quantified.

[00131] Representative flow cytometry data of ipsilateral brain leukocytes one day after surgery showed that CCR2 blockade with 30 and 100 mpk of drug reduced the proportion of macrophages infiltrating the brain by 46+13% (mean+SEM) and by 57+ 8%, respectively (ANOVA ***p<0.001, followed by Kruskal-Wallis test with Dunn’s multiple comparisons between TBI vehicle and TBI 30 mpk */?<0.05 and TBI 100 mpk ***/?<0.001 ) (FIG. 6A and FIG. 6B). Treatment with the compound of Formula I post-TBI significantly reduced the absolute cell numbers of LyOC ¹ " macrophages in the ipsilateral brain by 56+14% (30 mpk) and 71+9% (100 mpk) (ANOVA ****p<0.0001, followed by Kruskal-Wallis test with Dunn’s multiple comparisons between TBI vehicle and TBI 30 mpk */?<0.05. and TBI vehicle vs TBI 100 mpk **/?«) 005) (FIG. 6B). While Ly6C ^b macrophages were reduced by 30+11% in 100 mpk treated TBI animals, this difference did not reach statistical significance (ANOVA p=0.08) (FIG. 6B). Additionally, no differences were observed at this early time point in absolute cell numbers of microglia, T cells and neutrophils with drug treatment.

[00132] To determine if hCCR2 blockade after TBI improved cognitive outcomes, the effect of CCX872 treatment on behavior, as assessed by the cued platform version of Morris Water Maze testing at four weeks after injury was examined (FIG. 6C). Hsieh, C.L. el al. CCR2 deficiency impairs macrophage infiltration and improves cognitive function after traumatic brain injury . J Neurotrauma 31, 1677-1688 (2014); Patil, S.S., Sunyer, B., Hoger, H. & Lubec, G. Evaluation of spatial memory of C57BL/6J and CD1 mice in the Barnes maze, the Multiple T-maze and in the Morris water maze. Behav Brain Res 198, 58-68 (2009); Vorhees, C.V. & Williams, M.T. Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nature protocols 1, 848-858 (2006); Possin, K.L. et al. Cross-species translation of the Morris maze for Alzheimer's disease. J Clin Invest 126, 779-783 (2016). Mice were given TBI or sham surgery followed by administration of vehicle or hCCR2 inhibitor at 100 mpk beginning 2h post-injury and then daily thereafter for 5 days (n=17 per TBI group, n=4-9 per sham group).

[00133] Mice were assessed for their ability to learn the location of an escape platform with a visible cue on top. Mice treated with 100 mpk of the hCCR2 inhibitor (the compound of Formula I) performed better than mice treated with vehicle, both in escape latency (time to platform) and total distance from the platform (FIG. 6C) (Rank summary scores over both sessions: ANOVA ****/?«) 0001 for both latency and distance, TBI vehicle vs TBI 100 mpk */?=0.0.3.3 for latency and *p= 0.023 for distance). The swim velocities for all groups were equivalent, ruling out potential confounding factors related to swimming ability (FIG. 6C). Animal behavior was also examined using open field testing to assess spontaneous locomotor activity and baseline anxiety behaviors at three weeks post-surgery and no significant differences between drug and vehicle-treated TBI animals were found. Rotarod analysis also found no gross motor function differences. Thus, the reduction in brain macrophages post- TBI in mice treated with CCX872 was accompanied by significant recovery of cognitive function.

[00134] The studies revealed specific responses to type I IFN in microglial subtypes both in cell number and gene expression, and they suggest that these responses may be induced by the Ccr2- dependent expansion of brain macrophages in TBI. Studies by Karve et al have shown that IFNp protein is elevated in human acute TBI brain tissue, and that mice with a genetic deletion of the type I IFN receptor, Ifnarl, or with blockade of IFN signaling even after TBI significantly improved outcomes. Karve, I.P. et al. Ablation of Type-1 IFN Signaling in Hematopoietic Cells Confers Protection Following Traumatic Brain Injury. eNeuro 3 (2016). Thus, whether pharmacologic blockade of CCR2 reduced the type I IFN response was tested. Relative quantitative reverse-transcription (RT)-PCR was performed using fluorescent TaqMan probes on total RNA isolated from the ipsilateral hemisphere of injured brain tissues four days after TBI (n=9-10 per TBI group, n=3-5 per sham group).

[00135] TBI increased expression of a key ISG, Irf7, by an average threefold change (sham vehicle, mean relative quantification (RQ) to GAPDH=0.004505; TBI vehicle, mean RQ=0.001551; SEM=0.001228; ANOVA **/?<() 001 ; Tukey’s multiple comparisons test */?=0.02.3). HCCR2 blockade reduced the increase in Irf7 expression by 52% compared to the TBI vehicle-treated group (TBI vehicle RQ= 0.004505; TBI 100 mpk drug RQ= 0.002183; SEM=0.0007229; ANOVA **p<().()() 1 ; Tukey’s multiple comparisons test **p=().()()5) (FIG. 6D). In contrast, there was no difference observed in the expression of Tnf, which is not an ISG, between dmg and vehicle treated animals (FIG. 6D). These data support findings from Ccr2 ^/ mice that the type I IFN response in TBI is dependent on Ccr2.

[00136] It will be apparent to those skilled in the art that various modifications and variations can be made without departing from the scope or spirit of this disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification and practice disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit being indicated by the following claims.