Methods of treating injury to the central nervous system

Abstract

Methods for treating injuries to the central nervous system with a cilia activator are provided. The injury may be a traumatic brain injury (TBI). Related pharmaceutical compositions are provided.

Description

DESCRIPTIONMETHODS OF TREATING INJURY TO THE CENTRAL NERVOUS SYSTEMBACKGROUND

[0001] This application claims the benefit of United States Provisional Patent Application No. 63 / 722,938, filed November 20, 2024, the entirety of which is incorporated herein by reference.1. Field

[0002] The present disclosure relates generally to the field of molecular biology and medicine. More particularly, it concerns methods and compositions for treating injury to the central nervous system.2. Description of Related Art

[0003] Traumatic brain injuries (TBIs) are a major source of morbidity and mortality in the US. The Centers for Disease Control (CDC) estimates that 2.8 million cases of TBI occurred between 2007-2013, resulting in billions of economic and noneconomic losses.1TBIs are a significant financial burden on the healthcare system and affect a broad patient population including active soldiers, veterans, and civilians. Despite years of research in drug development, no effective therapeutics for TBIs have been identified that resolve the acute emergence of secondary pathogenesis, driven, in part, by vasogenic edema and an adverse robust pro-inflammatory response that give rise to neuronal injury / death, axonal degeneration, and an increase risk of chronic neurodegeneration.3SUMMARY

[0004] The present disclosure overcomes limitations in the prior art by, in some aspects providing new treatments for TBI. Data is provided supporting and it is anticipated that increasing cerebrospinal fluid (CSF) flux by stimulating ependymal cilia can be used to expedite the clearance of toxic molecules generated after a brain injury. A variety of molecules that alter the beat frequency of motile airway cilia affect ependymal cilia can be used. In some preferred aspects, the antibiotic clindamycin can be used to increase beat frequency cilia. Administering clindamycin intraventricularly can increase the beat frequency of ependymal cilia and this effect may expedite the clearance of neurotoxic compounds from an injured brain,- 1 -4925-8843-5814, v. 1thus reducing damage or promoting recovery after a traumatic injury to the brain of a mammalian subject. These approaches can be utilized with additional therapies to treat TBI, and it is anticipated that these approaches may synergize with surgical procedures aimed at reducing intracranial pressure (TCP).

[0005] An aspect of the present disclosure relates to a method of treating a traumatic injury to the central nervous system of a mammalian subject, comprising administering a therapeutically relevant amount or an effective amount of a cilia activator to the mammalian subject. The cilia activator may stimulate the motility of ependymal cilia in the mammalian subject. The cilia activator may be an adrenergic drug, a cholinergic agonist, a beta-adrenergic agonist, a methylated xanthine, roxithromycin, a nitric oxide releasing compound, or clindamycin. The cilia activator may be terbutaline or clindamycin. In some aspects, the cilia activator is clindamycin. The nitric oxide releasing compound may be terbutaline, bacitracin, or gramicidin. The method may comprise administering from about 50 mg to about 600 mg to the mammalian subject either once or preferably every 6 hours. The method may comprise administering about 50, 60, 70, 80, 90, 100, 125, 152, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 mg, or any range derivable therein. The method may comprise administering from about 150 mg to about 300 mg to the mammalian subject either once or preferably every 6 hours. The method may comprise administering from about 300 mg to about 450 mg to the mammalian subject either once or preferably every 6 hours. The clindamycin may be administered repeatedly to the mammalian subject over 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14 or more days. In some aspects, the traumatic injury to the central nervous system is a traumatic brain injury (TBI or craniocerebral trauma) or concussion. The cilia activator may be administered to the mammalian subject intraventricularly, orally, intravenously, intrathecally, intraperitoneally, or intracerebrally. The clindamycin or terbutaline may be administered intraventricularly to the subject. The mammalian subject may be a human. The method may comprise administering a second therapy to treat the traumatic injury to the central nervous system. The second therapy may comprise administering a surgery or a second therapeutic to the subject. The medication may be a diuretic, an anti-inflammatory drug, an anti-anxiety drug, an anticonvulsant, an antidepressant, an anticoagulant, an antibiotic, an antimicrobial compound, or a muscle relaxant. The second therapy may reduce intracranial pressure in the mammalian subject. In some aspects, the second therapy is a craniectomy.- 2 -4925-8843-5814, v. 1

[0006] It is anticipated that the compositions and methods provided herein can be used to treat a variety of neuronal injuries. In some embodiments, the neurological injury is TBI. Traumatic brain injury can be classified depending on the severity of the injury. For example, mild TBI may generally refer to injuries resulting in loss of consciousness and / or disorientation is shorter than 30 minutes. For those experiencing mild traumatic brain injury, medical imaging often fails to detect abnormalities. But over time the individual may experience challenges such as headache, difficulty thinking, memory problems, attention deficits, mood swings and frustration, fatigue, visual disturbance, sleep disturbances, dizziness / loss of balance, irritability / emotional disturbances, feelings of depression, seizures, nausea, loss of smell, and / or sensitivity to light and sounds. The initial injury to the brain in mild traumatic brain injury may trigger a cascade of delayed secondary injury responses due to biochemical changes that account for these challenges. This secondary brain injury period may create a window of opportunity for therapeutic intervention to prevent progressive tissue and other damage.

[0007] Moderate TBI may generally refer to a brain injury resulting in a loss of consciousness from 30 minutes to 6 hours. Severe TBI may generally refer to a brain injury resulting in a loss of consciousness of greater than 6 hours. A person experiencing moderate or severe traumatic brain injury may experience receptive aphasia; expressive aphasia; slurred speech; reading problems; writing problems; difficulties interpreting touch, temperature, movement, limb position and fine discrimination; partial or total loss of vision; weakness of eye muscles; double vision; blurred vision; involuntary eye movements; intolerance of light; hearing loss; ringing in the ears; sensitivity to sounds; anosmia; diminished sense of taste; seizures; physical paralysis; chronic pain; sleep disorders; emotional challenges; lack of motivation; irritability; aggression; depression; disinhibition; and / or lack of awareness. In various embodiments, methods of preventing injury as disclosed herein can prevent one or more symptoms associated with moderate or severe traumatic brain injury

[0008] The severity of a traumatic brain injury may be diagnosed based on industry standards. For example, the traumatic brain injury may be diagnosed based on the Glasgow Coma Scale that considers multiple factors when determining the level of severity. The factors may include the individual’ s motor response, verbal response, and eye response. A value of 13 to 15 on the Glasgow Coma Scale generally relates to mild traumatic brain injury. A value of 9-12 on the Glasgow Coma Scale generally relates to moderate traumatic brain injury. A- 3 -4925-8843-5814, v. 1value of 3-8 on the Glasgow Coma Scale generally relates to severe traumatic brain injury. It is anticipated that any of these patients having a TBI as classified herein can be treated with the compositions and methods provided herein.

[0009] TBI sequalae can be divided in to 3 phases: acute, subacute, and chronic. For the purposes of this disclosure, an acute traumatic brain injury may be an injury that has occurred within approximately the previous three months. In some cases, the acute phase of an acute traumatic brain injury may include a subacute phase which may occur between about six weeks and about three months following the injury. For the purposes of this disclosure, a chronic phase may include a traumatic brain injury that occurred in a period over three months prior to time of analysis.

[0010] The mammalian subject may be a human, a livestock animal, a companion animal, a lab animal, or a zoological animal, such as for example a rodent, a mouse, a rat, a guinea pig, dogs, cats, horse, or non-human primate. In some preferred embodiments, the subject is a human.

[0011] In various embodiments, a subject in need can be at high risk for TBI. Nonlimiting examples of subjects that may be at high risk for TBI include those who are in the military or who engage in contact sports, drive motorcycles or all-terrain vehicles, work in construction, or are elderly. The subject may have already had at least one TBI. In some embodiments, the subject has not been diagnosed with a TBI.

[0012] The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of an pharmaceutical composition that contains at least one an inhibitor of DUSP16 and / or an inhibitor of DUSP8 or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington: The Science and Practice of Pharmacy, 2fEd., Lippincott Williams and Wilkins, 2005, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should typically meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.- 4 -4925-8843-5814, v. 1

[0013] As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.

[0014] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” As used herein “another” may mean at least a second or more.

[0015] Throughout this application, the term “about" is used to indicate that a value includes the inherent variation of error for the device, the inherent variation in the method being employed to determine the value, the variation that exists among the study subjects, or a value that is within 10% of a stated value.

[0016] As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and / or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01 %. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.

[0017] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0018] The terms “subject,” “host,” “patient,” and “individual” are used interchangeably herein to refer to any mammalian subject for whom therapy is desired, particularly humans. Other subjects may include cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and so on.

[0019] The term “effective amount” is an amount sufficient to effect beneficial or desired clinical results. An effective amount can be administered in one or more administrations. For purposes of this application, an effective amount is an amount that is sufficient to palliate, ameliorate, stabilize, reverse, slow or delay the progression of the disease- 5 -4925-8843-5814, v. 1state. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease. For example, treatment of cancer may involve, for example, a reduction in the invasiveness of a tumor, reduction in the growth rate of the cancer, or prevention of metastasis. Treatment of cancer may also refer to prolonging survival of a subject with cancer.

[0020] An effective response of a patient or a patient’s “responsiveness” to treatment refers to the clinical or therapeutic benefit imparted to a patient at risk for, or suffering from, a disease or disorder. Such benefit may include cellular or biological responses, a complete response, a partial response, a stable disease (without progression or relapse), or a response with a later relapse. For example, an effective response can be reduced tumor size or progression-free survival in a patient diagnosed with cancer.

[0021] The terms “in operable combination”, “in operable order”, and “operably linked” refer to a linkage wherein the components so described are in a relationship permitting them to function in their intended manner, for example, a linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene or the synthesis of desired protein molecule, or a linkage of amino acid sequences in such a manner so that a fusion protein is produced.

[0022] “Treatment” and “treating” refer to administration or application of a therapeutic agent to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition.

[0023] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.- 6 -4925-8843-5814, v. 1BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0025] FIGS. 1A-B. Administering clindamycin enhances beat frequency of ependymal cilia. A) a mouse is administered clindamycin I.C.V. After 45 minutes the brain is harvested and sliced into 1 mm thick sections. Within 15 minutes, sections are imaged to determine cilia motility. B) Cilia beat frequency increased to 136% + / - 11.8 compared to control (*= p < 0.05 via T test n=3 mice / group, STD).

[0026] FIGS. 2A-E. Stabilizing the disruption of the BBB after stab wound to the cortical mantle via administration of motile cilia stimulators. A) Overall scheme of the figure. Mice undergo a cortical stab wound. Next, saline or a motile cilia stimulator (terbutaline or clindamycin) is administered intraventricularly (I.C.V.) ~30 minutes post-injury. After 24 hours, the brains are harvested, fixed, and stained for murine IgG in the brain as a marker of BBB disruption. B) Representative whole brain image of a mouse administered saline I.C.V. post-injury. Murine IgG signal is rendered white. The white circle marks the site of injury. C) Representative murine brain from a mouse administered terbutaline and stained as in B. D) Representative murine brain from a mouse administered clindamycin and stained as in B. E) Quantification of barrier disruption in close proximity to the injury site 24 hours after treatment with saline or cilia stimulators (* = p < 0.01 via ANOVA, n=3 mice / group, STD).

[0027] FIGS. 3A-E. Enhancing BBB restoration post CCI TBI by administering motile cilia stimulators. A) Mice undergo a CCI TBI. Next, saline (non-treated control), gentamicin (antibiotic control), or clindamycin (ependymal cilia stimulator group), is administered I.C.V. After 24 hours, the brains are harvested, fixed, and stained for murine IgG in the brain as a marker of BBB disruption. B) Representative whole brain image of a mouse administered saline intraventricularly post-TBI. Murine IgG is rendered white. The circle denotes the CCI TBI region. C) Representative murine brain from a mouse administered gentamicin and stained as in B. D) Representative murine brain from a mouse administered clindamycin and stained as in B. E) Quantification of IgG signal from TBI wound area 24 hours- 7 -4925-8843-5814, v. 1after treatment with saline, antibiotic control, or cilia stimulator (* = p < 0.01 via ANOVA, n=4 mice / group, STD).

[0028] FIGS. 4A-D. Clindamycin enhances BBB restoration 72 hours post CCITBI. A) Mice undergo a CCI TBI. Next, saline (non-treated control), or clindamycin (ependymal cilia stimulator group), is administered I.C.V. After 72 hours, the brain is fixed, removed, and stained for murine IgG in the brain as a marker of BBB disruption. B) Representative whole brain image of a mouse administered saline intraventricularly post-TBI. Murine IgG is rendered white. The circle denotes the CCI TBI region. C) Representative murine brain from a mouse administered clindamycin and stained as in B. D) Quantification of IgG signal from TBI wound area 72 hours after treatment with saline or cilia stimulator (* = p < 0.01 via T-test, n=4 mice / group, STD).

[0029] FIG. 5. Mice recover faster from anesthesia following I.C.V. treatment with motile cilia stimulators. The time between ceasing isoflurane to ambulatory is measured in mice that underwent a CCI and I.C.V. administration of saline (non-treated control), gentamicin (antibiotic control), or clindamycin (ependymal cilia stimulator group (* = p < 0.01 via ANOVA, n=4 mice / group, STD).

[0030] FIG. 6. MRI of the rodent ventricular system - A mouse is imaged using the 7 T MR imaging machine at the UT Austin Biologic Imaging Core, which is in the same vivarium that holds the Umlauf Lab rodent colony. The ventricular system is clearly visible by T2 scans. The top arrow indicates the lateral ventricle, second from top arrow is the third ventricle, second from bottom arrow is the cerebral aqueduct, and bottom arrow is the fourth ventricle.

[0031] FIGS. 7A-B. Clearing CCI TBI brains. A) Brain from a mouse that underwent CCI TBI. B) Same brain as in A) that has undergone clarity protocol in the Umlauf Lab.

[0032] FIGS. 8A-C. Schematic for behavioral studies proposed in Specific Aim 2.A) Outline of the four groups that will be studied. B) Timeline of the single-dose treatment regimen and behavioral testing of the two independent cohorts. C) Timeline of the continuous treatment regimen and behavioral testing of the two independent cohorts.

[0033] FIGS. 9A-D. CCI mice receiving clindamycin I.C.V. recover activity levels quicker after surgery than control animals. Mice underwent a CCI and then were- 8 -4925-8843-5814, v. 1administered PBS or clindamycin I.C.V. Eighteen hours after surgery, activity levels were measured in an open field test that was quantified using the EthoVision XT software. A) Representative trace from a mouse that underwent a sham surgery. Dark to light shading shift indicates more time spent in one spot. B) Representative trace from a mouse that underwent CCI and PBS I.C.V. C) Representative trace from a mouse that underwent CCI and clindamycin I.C.V. D) Quantification indicates that animals that receive clindamycin I.C.V. recover normal activity levels quicker post-surgery than control mice (*= p < 0.05 using T- tests, n=4

[0034] FIGS. 10A-B. Example of Ki67 staining. A) KI67+IHC staining from a treated mouse. B) KI67+IHC staining from a control mouse. Brown cells are positive for Ki67 (indicated by arrows).

[0035] FIG. 11. Schematic for pilot clinical trial proposed in Specific Aim 3. Patients who qualify for the study (measured by CT scan) will have an EVD placed on Day - 1 . Starting at Day 0, clindamycin will be administered I.C.V. twice a day for 5 days. CSF and blood will be sampled daily for 7 days. At Day 3 the patient will undergo a second CT scan. At Day 7 the patient will undergo an MRI scan. The patient will follow up with Dr. Wang for at least 6 months.

[0036] FIGS. 12A-F. I.C.V. administration of clindamycin enhances ependymal cilia motility which increased parenchymal excretion of a small molecule tracer via the glymphatic system. A) Graphical depiction of the experiment. PBS or Clindamycin was administered I.C.V. and the brain is harvested 45 minutes later. Ependymal cilia motility was quantified via DIC microscopy. B) Quantification of ependymal cilia motility in mice administered I.C.V. PBS or clindamycin (* = p<0.05 via T-test, n=3 mice / group). C) Representative brains from mice administered a far red tracer into the cortex and I.C.V. PBS (left panel) or clindamycin (right panel). Dye signal is rendered white, and the arrow indicates site of dye injection. D) Quantification of dye signal from mice brains depicted in C (* = p < 0.05 via T-test, n= 4 mice / group). E) Cervical lymph nodes were harvested from mice depicted in C. The far red tracer signal was rendered white in mice administered I.C.V. PBS (left panel) or clindamycin (right panel). Individual lymph nodes are circled. F) Quantification of cervical lymph nodes depicted in E indicates clindamycin enhanced dye clearance from the brain via the glymphatic system (* = p< 0.05 via T-test, n= 4 mice / group).- 9 -4925-8843-5814, v. 1

[0037] FIGS. 13A-G. Mice administered Clindamycin LC.V. post focal TBI demonstrate enhanced restoration of the blood-brain barrier. A) Graphical depiction of the experiment. Mice received a focal TBI followed by I.C.V. administration of a motile cilia stimulator or control. Brains were then harvested and IgG extravasation into the brain was quantified to determine blood brain barrier disruption. B) Representative brains from mice subjected to craniotomy followed by stab wound TBI and I.C.V. administration of PBS (left panel), terbutaline (middle panel), or clindamycin (right panel). Twenty four hours post injury brains are harvested and stained for IgG extravasation. Arrow indicates location of the stab wound. C) Quantification of mice depicted in B indicates administration of motile cilia stimulators enhances restoration of the blood brain barrier (* = p<0.05 via ANOVA, n= 4 mice / group). D) Representative brains from mice subjected to craniotomy followed by CO TBI and I.C.V. administration of PBS (left panel), gentamycin (middle panel), or clindamycin (right panel). Twenty four hours post injury brains are harvested and stained for IgG extravasation. Arrow indicates location of the CO wound. E) Quantification of mice depicted in D indicates I.C.V. administration of clindamycin enhances restoration of the blood brain barrier (* = p<0.05 via ANOVA, n= 4 mice / group). F) Representative brains from mice subjected to craniotomy followed by CCI TBI and I.C.V. administration of PBS (left panel), gentamycin (middle panel), or clindamycin (right panel). Seventy two hours post injury brains are harvested and stained for IgG extravasation. Arrow indicates location of the CCI wound. G) Quantification of mice depicted in F indicates I.C.V. administration of clindamycin enhances restoration of the blood brain barrier (* = p<0.05 via ANOVA, n= 4 mice / group).

[0038] FIGS. 14A-E. Mice subjected to Weight Drop with Acceleration (WDA) model of TBI administered I.C.V. clindamycin demonstrated enhanced BBB restoration post injury. A) Graphical depiction of the diffuse, WDA TBI model. Mice placed on slitted aluminum foil were subjected to a closed head weight drop which causes the brain to accelerate into the skull to model diffuse TBI. After 24 hours mice were administered a far red small molecule tracer I.P. to quantify blood brain barrier disruption. B) Representative images of mice subjected to WDA administered I.C.V. PBS, in the left panel, or clindamycin in the right panel. Twenty four hours post injury, brains were bisected coronally and the amount of dye that extravasated into the brain was measured using a Licor scanner. Dye signal is rendered white. C) Quantification of mice depicted in B indicates I.C.V. administration of clindamycin significantly reduces dye extravasation into the brain (* = p<0.05 via T-test, n=4 mice / group. D) Brain sections from mice subjected to WDA were stained for claudin 5, blood vessels, and- 10 -4925-8843-5814, v. 1nuclei. Panel on the left represents a typical section from mice that administered I.C.V. PBS and the right panel represents a typical section from mice administered I.C.V. clindamycin. Mice administered I.C.V. PBS frequently demonstrated vessels devoid of claudin 5 staining while those administered I.C.V. Clindamycin generally demonstrated normal claudin 5 signal surrounding vessels. Representative vessels are indicated by a white arrow. Scale bars = 50um. E) Quantification of vessel length without claudin 5 from mice administered I.C.V. PBS or clindamycin ( * = p < 0.05 via T-test, n= 4 mice / group with 3-5 fields counted / mouse.

[0039] FIGS. 15A-G. I.C.V. clindamycin safely improves biomarkers post CCI TBI. A) Representative H&E staining of the wound area penumbra one week after CCI TBI from a mouse administered I.C.V. PBS. Red blood cells (RBC) are indicated by arrows. Scale bar = 50 um. B) Representative H&E staining of the wound area penumbra one week after CCI TBI from a mouse administered I.C.V. PBS. Red blood cells (RBC) are indicated by arrows. Scale bar = 50 um. C) Quantification of RBC from groups depicted in A and B (* = p< 0.05 via T-test, n=3 mice / group with 5 fields counted / mouse) D) Quantification of serum GFAP levels 24 hours post CCI TBI. I.C.V. administration of clindamycin significantly reduces GFAP levels compared to I.C.V. PBS (*= p < 0.05 via T-test, n= 3 mice / group). E) The amount of water in the brains of mice 24 hours afte a CCI TBI and I.C.V. administration clindamycin or PBS was quantified as a measure of hydrocephalous. Administering I.C.V. clindamycin did not increase hydrocephalus and brain water content was within normal ranges. F,G) Mice subjected to CCI TBI and treatment with I.C.V. clindamycin or PBS had normal ALT and AST serum levels.

[0040] FIGS. 16A-E. I.C.V. Clindamycin improves Murine Behavior Post CCI TBI. A) Mice subjected to a CCI TBI that are administered I.C.V. Clindamycin recover significantly quicker from isoflurane anesthesia (* = p < 0.05 via ANOVA n = 4 animals / group). B) Representative traces of murine activity 18 hours after CCI TBI plus treatment (treatment groups are indicated below the traces). C) Quantification of data in B indicates mice administered I.C.V. clindamycin demonstrate enhanced activity compared to I.C.V. PBS (* = p < 0.05 via ANOVA, n = 8 animals / group). D) Representative traces from novel object recognition test performed one week after mice were subjected to CCI TBI and administered I.C.V. PBS, in the top two panels, or I.C.V. clindamycin in the bottom two panels. The panels on the left depict the training set with objects in the lower left and top right comers. Panels on the right depict the test set where the object in the lower left comer is the same as in- 11 -4925-8843-5814, v. 1the left panels while the object in the top right corner has been replaced. E) Quantification of data depicted in D. Mice administered I.C.V. clindamycin demonstrate a significantly improved cognition during the first two weeks post injury compared to mice administered I.C.V. PBS (** = p< 0.01 via ANOVA. n = 8 mice / group including both old and young as well as male and female mice).

[0041] FIGS. 17A-C. Gross wound area is not significantly different one week after CCI TBI + I.C.V clindamycin. Brains were harvested, sectioned, and stained with H&E one week after receiving a CCI TBI and treatment with ICV clindamycin. Gross wound area in groups administered A) I.C.V. PBS B) I.C.V. clindamycin were not significantly different C), indicating this strategy reduces secondary pathogenesis rather than eliminating the primary wound.

[0042] FIG. 18. Total Claudin 5 signal was not significantly different 24 hours after WDA TBI + I.C.V clindamycin. Brains were harvested, sectioned, and stained with H&E twenty four hours after receiving a WDA TBI and treatment with I.C.V. clindamycin. While there was a trend for reduced claudin 5 signal the groups were not significantly different via T-test with 5% false discovery rate used as a cutoff for significance.

[0043] FIG. 19. Activity of animals receiving CCI TBI + I.C.V. PBS or clindamycin did not differ one week after injury. Post CCI injury, animals were subjected to a battery of behavioral analysis assays. Starting one week post injury, no significant differences in activity (either hyper or hypo) were identified between animals administered I.C.V. PBS or clindamycin using a 2-way ANOVA with 5% false discovery rate as the cutoff for significance.- 12 -4925-8843-5814, v. 1DESCRIPTION OF ILLUSTRATIVE EMBODIMENTSI. Cilia Activators

[0044] Cilia are microtubule-based organelles that extend from the surface of eukaryotic cells and are critical to various biological processes. In the central nervous system (CNS), cilia serve essential functions including cerebrospinal fluid (CSF) circulation, regulation of neurodevelopmental signaling pathways (such as Sonic Hedgehog and Wnt), and maintenance of homeostasis in ventricular and periventricular compartments. Disorders involving ciliary dysfunction, referred to broadly as "ciliopathies", include hydrocephalus, ependymal inflammation, neurodevelopmental disorders, and neurodegenerative conditions. As described below, a variety of cilia activators can be used to treat a traumatic injury to the CNS.

[0045] Pharmaceutical compositions and methods for activating or enhancing the motility and / or functional integrity of cilia within the CNS are provided herein. As shown in the below examples, a cilia activator can be used to treat and / or improve responses after a traumatic CNS injury. Cilia activator is can preferably modulate ciliary motility, structure, or associated signaling pathways through various mechanisms, including increasing intracellular cyclic nucleotides (cAMP or cGMP), modulating calcium signaling, enhancing microtubule stability, and / or exerting anti-inflammatory or membrane-modulatory effects. Cilia inhibitors are provided below and may be used alone or in combination with other therapeutic agents, e.g., in a synergistic or additive fashion, in the treatment of a traumatic CNS injury. Cilia activators include nitric oxide (NO) releasing compounds.

[0046] Nitric oxide (NO) releasing compounds: in some aspects the administration of nitric oxide (NO) releasing compounds have been shown to increase ciliary beat frequency (CBF) via stimulation of soluble guanylyl cyclase and downstream activation of cyclic guanosine monophosphate (cGMP) signaling. Suitable nitric oxide donors include, but are not limited to, sodium nitroprusside, S-nitrosoglutathione, diazeniumdiolates (NONOates), and nitroglycerin. In a preferred aspect, sodium nitroprusside can be administered intravenously at a dose of approximately 0.5 to 3 mg / kg for a period ranging from one to four hours daily, or may be delivered intracerebroventricularly at a concentration of approximately 10 to 100 micromolar in sterile solution. Controlled-release formulations and localized CNS delivery systems may be employed to achieve targeted effects while minimizing systemic side effects such as hypotension.- 13 -4925-8843-5814, v. 1

[0047] Beta-adrenergic agonists are another class of cilia activators that can be used. in some aspects Beta-adrenergic agonists, such as terbutaline, salbutamol, and isoproterenol can be used to elevate intracellular cyclic adenosine monophosphate (cAMP) levels, thereby increasing CBF. Terbutaline sulfate may be administered orally at a dosage of approximately 2.5 to 5 mg every 6 to 8 hours, or subcutaneously at 0.25 mg every 4 to 6 hours, not exceeding 1 mg / day. These agents may also be incorporated into targeted delivery systems such as nanoparticles or liposomes for site-specific delivery to periventricular regions affected by TBI.

[0048] Adrenergic drugs: Non-selective adrenergic agents, such as epinephrine and norepinephrine, can be used to stimulate both alpha- and beta-adrenergic receptors and may indirectly enhance ciliary motility. While generally used in acute settings, these agents may be reformulated for controlled, localized CNS delivery to stimulate ependymal cilia.

[0049] Methylated Xanthines: Methylated xanthines, including theophylline, aminophylline and caffeine, can also be employed as cilia activators. These compounds inhibit phosphodiesterase enzymes, increasing cAMP and stimulating ciliary motility. In one embodiment, theophylline is administered orally at about 5 to about 10 mg / kg / day to maintain plasma concentrations of about 10 to about 20 pg / mL. Xanthine derivatives may offer dual benefits in TBI by enhancing arousal and supporting ciliary activity.

[0050] Cholinergic Agonists: Additional embodiments include cholinergic agonists, such as methacholine and carbachol, which increase intracellular calcium via muscarinic receptor activation. These agents may be administered intranasally, intravenously, or intracerebroventricularly at concentrations ranging from 0.1 to 10 pM in sterile buffered solution.

[0051] Purinergic Receptor Agonists: Purinergic receptor agonists, such as adenosine triphosphate (ATP) and uridine triphosphate (UTP), activate P2Y receptors and similarly promote calcium-mediated ciliary stimulation. These compounds may be administered locally via local ventricular perfusion at concentrations ranging from about 1 to about 100 pM or encapsulation for sustained release, optionally in conjunction with agents that stabilize extracellular ATP.

[0052] HDAC6 Inhibitors: Histone deacetylase 6 (HDAC6) inhibitors, such as tubastatin A and ACY-1215, can be used to promote the acetylation and stabilization of microtubules, supporting primary cilia integrity following injury. These compounds may be- 14 -4925-8843-5814, v. 1administered systemically or locally in concentrations ranging from about 0.1 to about 10 mg / kg, depending on formulation and route of administration.

[0053] Melanin-concentrating hormone (MCH) modulators: Melanin-concentrating hormone (MCH) modulators, known to influence hypothalamic ciliary dynamics, may be used in post-TBI settings to support neuroendocrine regulation and circadian rhythm recovery. MCH receptor agonists or antagonists may be administered in a dosage range of from about 0.1 to about 10 mg / kg.

[0054] SHH / Wnt Pathway Modulators: Sonic Hedgehog (SHH) and Wnt pathway modulators, including SHH agonists such as SAG and Wnt inhibitors, can be used to restore cilia-dependent signaling impaired by TBI. These agents may be administered systemically or via direct CNS delivery at doses ranging from about 0.01 to about 10 mg / kg.

[0055] Antimicrobial Agents with Ciliary Effects: In a particularly preferred embodiment, clindamycin, a lincosamide antibiotic can be used to cause secondary cilia- stimulating effects through anti-inflammatory and membrane-stabilizing mechanisms. Clindamycin may be administered to a mammalian subject at a dosage ranging from about 50 mg to about 600 mg, either as a single dose or, preferably, every 6 hours. In specific embodiments, the method may comprise administering about 50, 60, 70, 80, 90, 100, 125, 152, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 mg, or any range derivable therein. In some embodiments, the method may comprise administering from about 150 mg to about 300 mg either once or preferably every 6 hours. Clindamycin may be administered repeatedly over a treatment period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days, depending on the severity of the injury and therapeutic response. Intrathecal or intracerebroventricular administration may also be employed in doses of about 1 to about 10 pg per administration, using sterile, preservative-free solutions suitable for CNS delivery.

[0056] Other antimicrobial agents with cilia-stimulating effects include roxithromycin, bacitracin, and gramicidin. Roxithromycin may be administered orally at about 150 to about 300 mg twice daily. Bacitracin may be delivered locally via localized perfusion in sterile irrigation solutions at concentrations ranging from about 10 to 100 pg / mL for use in intraventricular or subarachnoid space. Gramicidin may be administered in localized CNS formulations at concentrations of about 0.01 to 1.0 pg / mL, providing ciliary stimulation through modulation of membrane potential and ion flux.- 15 -4925-8843-5814, v. 1

[0057] In certain embodiments, cilia activators from multiple mechanistic classes may be co-administered for synergistic benefit. For example, a nitric oxide donor may be combined with clindamycin to simultaneously stimulate motility and reduce inflammation. Combination therapy may be adjusted based on injury severity, patient status, and route of administration.

[0058] Pharmaceutical compositions comprising the cilia activators disclosed herein may be formulated in a variety of dosage forms, including solutions, suspensions, liposomes, nanoparticles, or biodegradable polymers. Administration routes that can be used include oral, parenteral, intranasal, intrathecal, or intracerebroventricular delivery. Controlled-release systems may be employed to extend duration of action and enable sustained ciliary stimulation.IL Traumatic Injuries to the CNS

[0059] Therapeutic methods for the treatment of traumatic injury to the central nervous system (CNS) can be achieved due to administering a cilia activator. Traumatic injury to the CNS encompasses a range of acute and chronic pathological conditions typically resulting from mechanical, biochemical, or penetrating insult to CNS structures, including but not limited to the brain, spinal cord, and associated meningeal or ventricular systems. Such injuries are frequently accompanied by neuroinflammatory responses, edema, blood-brain barrier disruption, and secondary cellular injury that contribute to long-term neurological dysfunction.

[0060] In one preferred embodiment, the invention is directed to the treatment of traumatic brain injury (TBI). As used herein, “traumatic brain injury” refers to any structural injury or functional impairment of the brain caused by an external mechanical force. TBI may result from direct impact, rapid acceleration or deceleration, blast exposure, or penetration of the skull, and may be classified as mild, moderate, or severe based on clinical criteria including Glasgow Coma Scale (GCS) score, duration of loss of consciousness, and radiological findings. TBI is further subdivided into closed (non-penetrating) and open (penetrating) injuries. Common clinical manifestations include loss of consciousness, confusion, headache, memory deficits, seizures, mood disturbances, and motor impairment. Secondary complications such as hydrocephalus, diffuse axonal injury, intracranial hemorrhage, and chronic neuroinflammation may further exacerbate neuronal loss and functional decline.

[0061] The pathophysiology of TBI involves a complex cascade of primary and secondary injury mechanisms. Primary injury occurs at the moment of trauma and includes neuronal and glial disruption, contusion, axonal shearing, and mechanical deformation of CNS- 16 -4925-8843-5814, v. 1tissues. Secondary injury develops over minutes to days following the primary insult and includes oxidative stress, glutamate excitotoxicity, microvascular dysfunction, mitochondrial failure, and neuroimmune activation. Importantly, emerging evidence indicates that disruption of ependymal and neuronal cilia following TBI contributes to impaired cerebrospinal fluid (CSF) dynamics, altered neurodevelopmental signaling, and decreased clearance of toxic metabolites.

[0062] In addition to TBI, traumatic CNS injury includes spinal cord injury (SCI), which involves mechanical or ischemic damage to the spinal cord that may result in partial or complete loss of sensory, motor, and autonomic function below the level of injury. SCI may result from fracture, dislocation, compression, or transection of vertebral elements and is frequently associated with hemorrhage, edema, and inflammatory cell infiltration (Hellenbrand et al., 2021).

[0063] Other forms of traumatic CNS injury that may be treated with a cilia activator include penetrating brain injuries, such as gunshot wounds or shrapnel injury, blast-induced neurotrauma, typically observed in military personnel exposed to explosive devices, and iatrogenic CNS injury, such as that resulting from neurosurgical intervention or invasive diagnostic procedures. While these forms of trauma may differ in mechanism and anatomical distribution, they share common downstream consequences including cellular injury, inflammation, and impairment of cilia-based CSF regulation (Xiong et al., 2014).III. Treatment of CNS injuries

[0064] Described herein is a method of enhancing fluid flux within the brain as an innovative strategy to improve outcomes after a severe TBI. Cerebrospinal fluid (CSF), an ultrafiltrate of plasma is primarily produced in the choroid plexus and occupies the ventricles, subarachnoid space, and the central canal of the spinal cord.4,5This ultrafiltrate is vital to the central nervous system (CNS), providing nourishment, protection, and waste removal.4,5Recent studies of CSF demonstrate that the glymphatic system circulates CSF throughout the brain parenchyma.6,7While most studies have focused on periarterial channels involved in shuttling nutrients to the parenchyma,7-23this invention utilizes an understudied key function of the glymphatic system, namely the removal of toxic metabolites from the brain.8

[0065] In studying waste removal from the brain, it has been found that motile ependymal cilia, which mechanically mix CSF in the ventricles as part of the physical forces- 17 -4925-8843-5814, v. 1that influence the flux of CSF,6play an important role in the glymphatic system in addition to the well-characterized perivascular movement of fluid. Modulating motile ependymal cilia could provide a new target for altering CSF dynamics that are known to be altered in severe TBIs.24

[0066] One strategy described herein is to increase CSF flux by stimulating ependymal cilia to expedite the clearance of toxic molecules generated after a brain injury. It has been recently demonstrated that molecules known to alter the beat frequency of motile airway cilia affect ependymal cilia similarly.25Clindamycin has been identified as a drag that increases the beat frequency of airway cilia, in addition to its on-label use as an antibiotic.26,27In a new context described herein, it was determined that administering clindamycin intraventricularly increases the beat frequency of ependymal cilia and propose that this will expedite the clearance of neurotoxic compounds from an injured brain. Importantly, this approach synergizes with surgical management aimed at reducing intracranial pressure (ICP).

[0067] By increasing CSF flux the goal is to reduce secondary pathogenesis by enhancing the clearance of neurotoxic molecules, including inflammatory mediators and products of excitotoxicity, as well as potentially reducing vasogenic edema. This early neuroprotection resulting from enhancing CSF flux will result in improved neurological function.

[0068] Traumatic Brain Injury: TBIs are characterized by heterogeneity that is in part attributed to the nature and severity of the injury.28-31A report on military service members from 2000-2019 found that nearly 414,000 troops experienced TBIs, and more than 185,000 veterans using the VA system have been diagnosed with at least one TBI.32,33TBI was declared the “signature” injury among military personnel involved in the Iraq and Afghanistan conflicts (2001-2021).2During the first 12 years, nearly 250,000 service members were diagnosed with TBIs. Given the difficulties of diagnosing and reporting TBIs in the field, this incidence rate likely underestimates the total number of injuries during these conflicts2Importantly, there are long-term adverse consequences of TBIs, including mental and physical health issues that can significantly impact quality of life ,30'32’34’35Moreover, it has become increasingly clear that acute exposure to a TBI may culminate in chronic neurodegeneration.36Unfortunately, there are no pharmacological interventions that substantially improve the quality of lite after a severe TBI.37This unmet need will be addressed by reducing the concentration of neurotoxic molecules that have been released extracellularly and give rise to secondary pathogenesis. To- 18 -4925-8843-5814, v. 1accomplish this objective, a motile ependymal cilia stimulator will be repurposed as a pharmacological treatment for TBI. This treatment could improve combat readiness by reducing recovery time post-TBI for active soldiers and reduce VA costs associated with longterm care of TBI patients.

[0069] Repurposing an existing FDA-approved drug to treat TBI: One significant aspect of this proposal is the repurposing of an existing FDA-approved drug as cilia stimulator to treat TBI. Repurposed drugs have established safety profiles, have already received FDA approval for a different use, and are commercially available as pharmaceutical-grade products. For drug delivery, an external ventricle drain (EVD) will be utilized that is placed into a lateral ventricle as a standard-of-care procedure that is regularly required to treat severe TBIs.38-40Clindamycin is an FDA-approved antimicrobial that is used to treat diverse infections. In the context described herein, its ability to stimulate motile cilia (26, 27) is utilized to provide a new pharmacological treatment. Administering clindamycin into a ventricle will stimulate motile ependymal cilia to treat acute TBIs by enhancing the removal of toxic molecules such as heme from the injury site. Importantly, one widely used EVD is coated with clindamycin as an antimicrobial, indicating that at least trace amounts of clindamycin (which leach from the coating) are tolerated in the central nervous system (CNS).39,41This novel approach will attenuate secondary pathogenesis by reducing the acute toxic burden in the damaged brain, thereby shortening the recovery time post-severe TBI, so soldiers can return to active service quicker as well as reduce VA expenditures associated with long-term care of brain-injured patients.

[0070] Pharmacological modulation of motile ependymal cilia: No studies have previously attempted to modulate the beat frequency of ependymal cilia as a strategy to remove the accumulation of toxic metabolites in the injured brain by enhancing CSF fluid flux. While there have been some attempts to alter CSF flow to treat severe TBIs, this currently involves complex and invasive neurosurgical procedures42,43Current standard of care after a TBI focuses on CSF diversion strategies to treat (not prevent) hydrocephalus. Proposed herein is a method for stimulating ependymal cilia to improve CSF flux thereby increasing the clearance of neurotoxins resulting from a brain injury. The treatment is clinically feasible (in terms of using an FDA-approved compound with minimal known adverse events in the brain). The medication will be administered intraventricularly using an EVD that is placed to monitor intracranial pressure (ICP) as part of the standard of care for treating severe TBIs. An EVD is- 19 -4925-8843-5814, v. 1the most commonly used method to monitor ICP in the US; thus, the ventricle is accessible without any additional invasive procedures in the targeted patient population. Taken together, targeting the beat frequency of ependymal cilia to enhance CSF flux is an ideal target for pharmacological intervention to treat severe TBI.

[0071] Increasing fluid movement to enhance healing of an internal CNS wound: Longstanding data demonstrate that increasing fluid flow through an external surgical wound results in improved healing and reduced scarring.44-47Increased blood flow through a wound is thought to aid healing by reducing infection, clearing toxic factors, and increasing factors that remodel an injury site.47-49While this technique is commonly used for external wounds and incisions, described herein is the first method to apply this concept to an internal CNS wound resulting from a TBI. Reducing infection is not the mechanism of action for enhanced TBI recovery because administering gentamicin intraventricularly, which does not stimulate ependymal cilia, does not enhance re-establishment of the blood-brain barrier post-TBI (FIGS. 3A-D). However, increasing fluid flow through a TBI may still accelerate the clearance of toxic metabolites resulting in enhanced wound healing.30Thus, this proposal is innovatively applying historical lessons to develop a pharmacological treatment for TBI.

[0072] Increasing the motility of ependymal cilia may expedite the removal of toxic molecules released into the extracellular compartment after a brain injury, enhancing the resolution of TBI symptoms. Clindamycin was identified herein as a promising pharmacological candidate to treat severe TBI because it is known to enhance the motility of airway cilia.26,51Recent studies and preliminary data (FIGS. 1 A-B) indicate that motile cilia in the airway and motile ependymal cilia respond similarly to drugs that increase or decrease cilia motility.23Clindamycin is FDA-approved, has a longstanding history of clinical use with a good safety profile, and is well-tolerated by patients. Mice that received a stab wound to the cerebral cortex and I.C.V. clindamycin (or the alternative drug terbutaline) approximately 30 minutes post-TBI exhibited a significant reduction in blood-brain barrier (BBB) disruption (measured by IgG in the brain) compared to saline controls (3-fold and 6-fold decrease in IgG signal in the TBI area, respectively, p<0.05 via ANOVA: FIGS. 2A-E). These initial findings are encouraging as stabilization of the BBB reduces exposure of the brain to toxic molecules circulating in the bloodstream, implying that stimulating ependymal cilia could benefit patients presenting with a severe TBI and adverse events resulting from TBI. These results were replicated with clindamycin in a controlled cortical impact (CGI) model of TBI (FIGS. 3 A-E).- 20 -4925-8843-5814, v. 1Additionally, a significant reduction in BBB disruption (measured by IgG in the brain) was observed 72 hours post-CCI (FIGS. 4A-D). Finally, mice that receive ependymal cilia inhibitors I.C.V. recover from anesthesia 38% quicker than groups receiving placebo (2.635 versus 7 minutes, p<0.01 via T-test, FIG. 5), indicating that increasing ependymal cilia motility enhances clearance of isoflurane from the brain.

[0073] Studying the utility of I.C.V. administered cilia stimulators for treating TBI in a murine model of TBI and performing a pilot clinical trial is described herein. The impact of enhancing ependymal cilia motility on mice that receive a CCI TBI will be examined using in vivo studies to investigate pathological and behavioral changes. A method of increasing ependymal cilia motility in the acute phase post-injury to reduce early secondary pathogenesis and improve performance in behavioral assays of cognition, anxiety, and hyperactivity will be tested. A pilot clinical study by administering cilia stimulators to a cohort of patients presenting with severe TBI is also described herein. The outcomes of the pilot study will be compared to historical data.

[0074] First, the effect of stimulating ependymal cilia will be determined on the degree of tissue injury, restoring CSF dynamics, clearance of neurotoxic metabolites, and restoration of the blood-brain barrier a murine model of TBI. The neuropathology of brain-injured mice administered motile cilia stimulators will be quantified versus placebo I.C.V. Indices of neuronal injury / death will be analyzed using immunocytochemistry to identify NeuN+, caspase3+, an indicator of neuronal programmed cell death, TunneF cells to identify DNA fragmentation, Flurojade-C to detect degenerating neurons, and Aquaporin 4 to monitor changes in the glymphatic system at both acute (days 1-7) and late stages (>1 month) of TBI resolution. Changes in CSF metabolites will be quantified, including amino acids, sugars, and neurotransmitters, as well as neurotoxic metabolites such as heme in TBI mice treated with cilia stimulators or placebo. CSF dynamics and cerebral edema will be analyzed using MR imaging in treatment and placebo groups with a focus on ventricular flux, cytotoxicity, and vasogenic edema using DWI sequence post-TBI. BBB integrity will be quantified using IgG leakage, vascular tracers, and MR imaging at multiple time points during acute and late phases post brain injury. These data will determine the optimal dosing regimen of cilia stimulators to restore BBB integrity in mice.

[0075] Second, the benefits and tolerability of administering ependymal cilia stimulators post-TBI will be measured using behavioral assays in mice to quantify changes in- 21 -4925-8843-5814, v. 1cognition, anxiety, and hyperactivity-like behaviors. Brain-injured mice will be treated with a single dose of cilia stimulators or placebo and assess cognition, anxiety, and activity levels (hyper and hypo) in two cohorts, studied one week (acute) or one month (late-stage) after treatment to determine the benefits and tolerability of this regimen. Next, brain-injured mice will be treated with a continuous dose of cilia stimulators or placebo for one week and evaluate learning and memory, anxiety, and activity levels (hyper and hypo) in two cohorts, studied at one week (acute) or one month (late-stage) after treatment to determine the benefits and tolerability of this regimen.

[0076] A clinical trial may be performed to determine the feasibility, safety, and potential benefits of using motile ependymal cilia stimulators to treat patients with severe TBI. Cilia stimulators I.C.V. will be administered to a cohort of patients (n=4-10) who present with severe TBI and have had an external ventricular drain placed as the standard of care treatment. Analyze clinical and radiographic data collected as part of the current standard of care protocol to determine the safety and potential benefits of treating patients presenting with a severe TBI with motile cilia stimulators. CSF and serum collected daily post-TBI from patients who received cilia stimulators will be analyzed for changes in inflammatory cytokines / chemokines, neurofilament light chain, and toxic metabolites such as heme.52Serum levels of GFAP, UCHL1, and neurofilament light chain will be quantified as systemic biomarkers to determine the degree of brain injury.25’36,53These data will be compared to matched historical controls using specimens from the University of Texas at Austin Department of Neurosurgery tissue biobank.

[0077] Additional Therapies to treat TBI or Brain Injuries: A cilia activator can be administered to a mammalian to treat a traumatic CNS injury in combination with one or more therapeutic intervention, for examples as follows. TBI and other CNS injuries are can additionally be treated with a range of pharmacological and surgical interventions that address the complex and evolving consequences of trauma. These therapeutic modalities target specific secondary effects of injury, including inflammation, infection, coagulopathy, seizure activity, neuropsychiatric symptoms, and intracranial hypertension.

[0078] Anti-inflammatory drugs can be administered following CNS trauma to suppress the post-injury inflammatory response. After TBI, activation of microglia and astrocytes, release of pro-inflammatory cytokines, and recruitment of peripheral immune cells contribute to secondary damage. Anti-inflammatory agents are administered to reduce cerebral- 22 -4925-8843-5814, v. 1edema, protect neural tissue, and stabilize the blood-brain barrier. Non-limiting examples include corticosteroids such as dexamethasone or methylprednisolone, and non-steroidal antiinflammatory drugs (NSAIDs) such as ibuprofen or ketorolac.

[0079] Anti-anxiety drugs can be administered to manage acute or chronic anxiety symptoms that may follow TBI. Trauma to regions of the brain involved in emotional regulation, such as the amygdala and prefrontal cortex, can result in persistent agitation, fear, and restlessness. Pharmacological anxiolytics are used to reduce these symptoms and improve patient tolerance of rehabilitation and recovery processes. Examples include benzodiazepines such as lorazepam or diazepam, and non-benzodiazepine agents such as buspirone or hydroxyzine.

[0080] Anticonvulsants can also be used in the management of TBI-related seizure activity. Seizures may occur in the immediate aftermath of trauma or may emerge as a delayed complication, often resulting from cortical contusion, hemorrhage, or scar formation. Antiepileptic drugs are used to prevent or control both early and late-onset post-traumatic seizures. Examples of anticonvulsants include phenytoin, levetiracetam, valproic acid, and carbamazepine.

[0081] Antidepressants may be administered during the subacute and / or chronic phases of recovery from TBI. Disruption of monoaminergic pathways and structural injury to moodregulating regions frequently result in depressive symptoms, apathy, or affective instability. Antidepressants can be used to support psychological stabilization, enhance engagement with therapy, and improve long-term functional outcomes. Non-limiting examples include selective serotonin reuptake inhibitors (SSRIs) such as sertraline or fluoxetine, serotonin-norepinephrine reuptake inhibitors (SNRIs) such as venlafaxine, and tricyclic antidepressants such as amitriptyline.

[0082] Anticoagulants may be used in some clinical situations following brain injury, particularly in patients at increased risk for thromboembolic events. Prolonged immobilization, systemic inflammation, and vascular damage can predispose patients to deep vein thrombosis or pulmonary embolism. When deemed clinically safe, anticoagulant therapy is initiated to prevent these complications. Examples include low molecular weight heparin (e.g., enoxaparin), unfractionated heparin, and direct oral anticoagulants such as apixaban or rivaroxaban.- 23 -4925-8843-5814, v. 1

[0083] Antibiotics can be administered following penetrating brain injuries, open skull fractures, or neurosurgical procedures. In these settings, the protective barriers of the brain may be compromised, allowing for bacterial invasion. Antibiotics are used to prevent or treat infections involving the meninges, brain parenchyma, or surgical sites. Examples include ceftriaxone, vancomycin, metronidazole, and meropenem.

[0084] Antimicrobial compounds, including antiviral, antifungal, or antiseptic agents, can be used in CNS trauma cases where there is risk or presence of non-bacterial infections. These agents may be administered prophylactically or therapeutically depending on exposure history, immune status, and clinical findings. Examples include acyclovir for herpes simplex virus, amphotericin B for fungal infections, and chlorhexidine or povidone-iodine as topical antiseptics.

[0085] Muscle relaxants may be administered in patients who develop spasticity or increased muscle tone following TBI. Injury to descending motor pathways may result in exaggerated reflexes and involuntary muscle contractions. Muscle relaxants are prescribed to reduce rigidity, enhance mobility, and improve the effectiveness of physical rehabilitation. Examples include baclofen, tizanidine, dantrolene, and diazepam.

[0086] Surgical intervention(s), including decompressive craniectomy, is a recognized treatment for elevated intracranial pressure or mass effect following TBI. When intracranial hypertension does not respond to medical management, decompressive surgery is performed to allow for cerebral expansion, reduce the risk of herniation, and preserve cerebral perfusion. Other surgical procedures may include hematoma evacuation, insertion of intracranial pressure monitors, or cerebrospinal fluid diversion via ventriculostomy.

[0087] These established therapeutic approaches may be included in the standard of care for treatment of TBI and CNS trauma. In certain applications, one or more of these treatments may be used in conjunction with the administration of cilia-activating agents. The combined use of pharmacological and surgical interventions with cilia-targeted therapies may support enhanced clearance of inflammatory mediators, normalization of cerebrospinal fluid dynamics, and improved neurological recovery.- 24 -4925-8843-5814, v. 1III. Pharmaceutical Preparations

[0088] Pharmaceutical formulations provided herein preferably include a cilia activator. The pharmaceutical formulation may include, as optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, and salts of the type that are available in the art. Examples of such substances include normal saline solutions such as physiologically buffered saline solutions and water. Specific non-limiting examples of the carriers and / or diluents that are useful in the pharmaceutical formulations of the present invention include water and physiologically acceptable buffered saline solutions such as phosphate buffered saline solutions pH 7.0-8.0. Suitable pharmaceutical carriers include, but are not limited to, sterile water, salt solutions (such as Ringer's solution), alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, etc. The pharmaceutical preparations can be mixed with auxiliary agents, e.g., lubricants, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and / or aromatic substances and the like which do not deleteriously react with the active compounds. They can also be combined where desired with other active substances, such as anti-inflammatory agents or neuroactive compounds useful in the treatment of traumatic brain injury. The therapeutic provided herein may be formulated in a pharmaceutical composition, which may include pharmaceutically acceptable carriers, thickeners, diluents, buffers, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients and the like.

[0089] The pharmaceutical compositions provided herein may include pharmaceutically acceptable carriers, thickeners, diluents, buffers, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or excipients and the like. The formulations may be optimized for delivery to the central nervous system, particularly in patients suffering from traumatic brain injury.

[0090] Pharmaceutical compositions may also include one or more active ingredients such as anti-inflammatory agents, anesthetics, neuroprotective agents, and the like. Formulations for oral, parenteral, or intrathecal administration may include buffers, liposomes, diluents, and other suitable additives. The compositions provided herein may additionally- 25 -4925-8843-5814, v. 1contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional compatible pharmaceutically active materials such as anti-inflammatory agents, antibiotics, statins, neuromodulators, or may contain additional materials useful in physically formulating various dosage forms of the composition of the present invention, such as dyes, flavoring agents, antioxidants, opacifiers, thickening agents, and stabilizers. Depending on the particular active ingredients, the formulations may be administered in the same pill or tablet or as a distinct pill or tablet as part of a co-admini strati on protocol. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions provided herein.

[0091] The pharmaceutical composition of the invention may be administered orally, mucosally, parenterally, sublingually, rectally, intrathecally, intranasally, or intracerebroventricularly. Dosing can be dependent on a number of factors, including severity and responsiveness of the TBI to be treated, and with the course of treatment lasting from several days to several months, or until a diminution of neurological dysfunction is achieved. Toxicity and therapeutic efficacy of compounds provided herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals.

[0092] "Pharmaceutically acceptable salt" refers to salts of the compounds of the present invention derived from the combination of such compounds and an organic or inorganic acid (acid addition salts) or an organic or inorganic base (base addition salts). The compounds of the present invention may be used in either the free base or salt forms, with both forms being considered as being within the scope of the present invention.

[0093] Pharmaceutical compositions provided herein may be in any form which allows for administration to a patient via clinically appropriate routes. The pharmaceutical composition is formulated so as to allow the active ingredients contained therein to be bioavailable at the targeted CNS site upon administration to the patient. Compositions that will be administered to a patient take the form of one or more dosage units, where a tablet or capsule may be a single dosage unit, and a container of one or more compounds of the invention in oral form may hold a plurality of dosage units.

[0094] For oral administration, an excipient and / or binder may be present. Examples are sucrose, kaolin, glycerin, starch dextrins, sodium alginate, carboxymethylcellulose and- 26 -4925-8843-5814, v. 1ethyl cellulose. Coloring and / or flavoring agents may be present. A coating shell may be employed, applying common membranes used for microencapsulation and suitable for the microencapsulation of live, killed or attenuated probiotic organisms include biodegradable synthetic "polymers" such as poly-lactide, polyglycolic acid, and polyanhydride. Established "polymers" for live encapsulation and enzyme encapsulation include alginate-polylysine- alginate (APA), alginate-polymethylene-co-guanidine-alginate (A-PMCG-A), hydroymethylacrylate-methyl methacrylate (HEMA-MMA), Multilayered HEMA-MMA- MAA, polyacrylonitrilevinylchloride (PAN-PVC), acrylonitrile / sodium methally-Isulfonate (AN-69), polyethylene glycol / poly pentamethylcyclopentasiloxane / polydimethylsiloxane (PEG / PD5 / PDMS), poly N,N-dimethyl acrylamide (PD-MAAm), Siliceous encapsulates and cellulose sulphate / Sodium alginate / polymethylene-co-guanidine (CS / A / PMCG). Other materials that are useful include, without limitation, cellulose acetate phthalate, calcium alginate and k-carrageenan-Locust bean gum gel beads, gellan-xanthan beads, poly(lactide-co- glycolides), carrageenan, starch poly-anhydrides, starch poly methacrylates, polyamino acids, enteric coating polymers.

[0095] A liquid pharmaceutical composition, whether in the form of a solution, suspension, or other like form, may include one or more of the following adjuvants: diluents such as water, preferably fixed oils such as synthetic mono- or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. Such formulations may be optimized for use in central nervous system delivery, including via intrathecal, intracerebroventricular or intranasal routes.

[0096] In addition to conventional formulations, further pharmaceutical approaches may be utilized to enhance the delivery, efficacy, and tolerability of cilia-activating agents in the treatment of traumatic brain injury. Intranasal administration may be employed to bypass the blood-brain barrier and enable direct delivery of active agents to the central nervous system via olfactory and trigeminal pathways. Nanoparticle-based formulations, including liposomes, micelles, and polymer-based delivery systems, may be used to improve pharmacokinetics, enhance tissue targeting, and enable controlled release of the therapeutic compound. In certain embodiments, cilia activators may be co-administered with neuroprotective peptides,- 27 -4925-8843-5814, v. 1mitochondrial stabilizers, or agents that modulate aquaporin channels to support glymphatic clearance and reduce neuronal injury. In clinical settings requiring cerebrospinal fluid diversion or pressure regulation, localized delivery through external ventricular drains, intraventricular catheters, or shunt systems may be used to administer the pharmaceutical composition directly into the ventricular system. Adjunctive modalities such as pulsed electromagnetic field therapy or low-intensity focused ultrasound may be employed to stimulate endogenous ciliary motion and enhance CSF flow. Additionally, formulation or dosing schedules may be adapted based on circadian rhythms or biomarker-guided parameters to optimize therapeutic outcomes and patient-specific responses. These additional approaches are compatible with the pharmaceutical compositions described herein and may be used to facilitate targeted and sustained therapeutic delivery in patients with TBI or brain injuries.IV. Examples

[0097] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0098] Data are presented as means with standard deviation. All tracing experiments with fluorescent molecules are repeated with at least three biological replicates per experiment and three independent experiments. Age-matched samples between treatment and control groups are used in each experiment. For murine assays, T-tests are used to determine differences between a treatment and a control group. A 5% false discovery rate is used as the cut-off for significance. For mass spectrometry experiments, assays consist of at least three biological replicates per experiment and at least three technical replicates per biological replicate. ANOVA with Tukey correction for multiple comparisons will be used to determine significant differences between groups. For MR imaging experiments, eight animals per group are used to provide sufficient power to determine a 15% difference in signal accumulation. MRI data is quantified by a blinded reviewer using Image J software. Similarly, all behavioral studies are analyzed by a blinded reviewer. Further statistical details for each aim are included- 28 -4925-8843-5814, v. 1below. Studies are performed in female mice and then repeated in male mice to identify sex- associated differences in response to the administration of motile ependymal cilia stimulators. For the pilot clinical trial enrollment is not biased toward any gender, race, ethnicity, or age.Example 1 - The effect of stimulating ependymal cilia on restoring CSF dynamics, clearance of neurotoxic metabolites, and restoration of the blood-brain barrier (BBB) in a murine model of focal cortical injury.

[0099] The method of increasing the motility of ependymal cilia to attenuate disruption of the BBB and reduce secondary damage after TBI will be tested. Previous studies demonstrate that compounds known to alter the motility of airway cilia will similarly alter the motility of ependymal cilia (FIGS. lA-B).23Based on literature regarding airway cilia, two compounds were identified that enhance the motility of ependymal cilia: clindamycin and terbutaline.26,51To test the feasibility of this approach, female mice first underwent a stab wound to the brain. The skull of an anesthetized mouse was exposed, and a borehole craniotomy was performed. A 27 gauge needle was plunged 2.5 mm into the cortical mantle, and a motile cilia stimulator (clindamycin or terbutaline) was administered I.C.V. ~30 minutes after the initial wound. After twenty-four hours, brains were harvested and immunostained for murine IgG as a marker of BBB permeability. Administration of either clindamycin or terbutaline significantly restored the BBB compared to animals receiving PBS control (FIGS. 2A-E). While group sizes were relatively small, these positive findings across two independent drug-treated groups are encouraging.

[0100] To further validate these results, the experiment was repeated using a CCI model. After injury, mice were administered PBS as a negative control, gentamicin as an antibiotic control, or clindamycin I.C.V. Only the clindamycin group demonstrated a significant increase in BBB recovery (FIGS. 3A-E). Additionally, a significant increase in BBB re-establishment was observed 72 hours after injury in animals that received clindamycin compared to PBS (FIGS. 4A-D). Finally, animals administered clindamycin recover significantly faster from isoflurane anesthesia compared to animals administered PBS or gentamicin, indicating more rapid excretion of isoflurane from the brain (FIG. 5). Taken together, this data indicates that I.C.V. administration of clindamycin may serve as a pharmacological treatment for patients with severe TBI.- 29 -4925-8843-5814, v. 1

[0101] The murine model of controlled cortical impact (CO) will be used to generate a focal brain injury.54 56This model was reproduced and utilized to generated the preliminary findings described herein (FIGS. 1 A-3E). Both directly after the injury and treatment as well as one month post -injury and treatment, brains will be harvested and processed for histology to measure both acute and chronic changes associated with administering motile ependymal cilia stimulators. Additionally, the degree of CCI TBI injury will be varied to simulate the range of severe TBI injuries observed in both military and civilian hospitals. To track the degree of injury in groups treated I.C.V. with motile cilia stimulators or controls, a modified H&E assays (Expredia) will be used. This staining technique can identify necrotic regions, blood infiltrates, and live / dead cells in cut tissue sections.25Standard IHC techniques will be used to track immune infiltration, similar to previous studies conducted in the laboratory.70Brains will also be stained for T cell subsets, including regulatory T cells, Th 17, NK, and macrophages (both Ml and M2 polarization) as well as microglia (TMEM119). Additionally, inflammatory cytokine (including IL-ip, IL-6, 11-18, and TNFa) and chemokine (including CXCL8, CCL2, CCL3, CCL4, CCL5, and CX3CL1) levels will be assessed to determine differences in inflammation post-TBl. Brain sections will be stained for caspase3+, as an indicator of neuronal programmed cell death, NeuN+and Tunnel4to identify DNA fragmentation, and Flurojade-C to detect degenerating neurons. In addition to the wound region, parts of the brain distal to the focal wound, such as the contralateral brain and cerebellum, will be quantified for damage that could result from pressure waves propagating through the brain. Finally, brain sections will be stained for Aquaporin-4 and P-cam to determine changes in the glymphatic system resulting from treatment with ependymal cilia stimulators post-CCI injury.7-9’11-13’1’’16’20-22’71

[0102] Mass spectrometry studies will be used to determine the impact of ependymal cilia stimulators on the presence of neurotoxic metabolites in the brain. Briefly, either CSF or whole brain homogenates will be extracted and fragmented with trypsin. The resulting peptides will analyzed using an orbitrap mass spectrometry instrument. Peptides are aligned to murine proteins, and the presence of neurometabolites is determined. The amount of each metabolite is determined by correlating the area of the chromatography peak between samples. These studies will be performed at the UT Austin mass spectrometry core in the Chemistry department.

[0103] MR imaging will be used to quantify CSF dynamics in real-time. Animals will undergo a T2 DWI imaging series to establish the baseline measurements. The mouse will then- 30 -4925-8843-5814, v. 1undergo TBI and treatment with a cilia stimulator or control. The mouse will undergo the same imaging series at 30 minutes, 60 minutes, 120 minutes, and 24 hours post-injury and treatment. This approach allows for real-time measurement of fluid dynamics in the brain. Additionally, changes in ventricle size, cytotoxicity, and vasogenic edema will be quantified. These studies will be performed using a T7 Brunker small animal MRI located in the same vivarium as the Umlauf and Noble lab murine colonies. An example of the MRI data that will be collected is presented in FIG. 6.

[0104] Additionally, ependymal cilia beat frequency and presence will be directly monitored post-injury using DIC microscopy using methods demonstrated in Umlauf et al. 2023 (25, FIGS. 1A-B). Brains that have undergone CCI and treatment with a cilia stimulator or control will be removed and sliced using a brain mold to get 1 mm thick sections. As previously described, brains will be imaged to directly determine the impact of CCI and treatment with ependymal cilia inhibitors on the ependymal cilia motility.

[0105] As demonstrated in FIGS. 2A-4D, treatment with cilia stimulators restores the BBB faster than control-treated animals. In addition to the murine IgG studies present as preliminary data, these studies will be expanded by measuring BBB integrity using injectable tracers (Carboxy late-IRsoo, BSA-IRsoo, Dextran-IRsoo) that can be quantified using ex vivo assays or via MR imaging assays by quantifying the extravasation of gadovist in the TBI site. Finally, IHC imaging of cut histological brain sections stained with tight junction proteins claudin-5 and Zonula occludens-1 (ZO-1) will be used to ensure the integrity of the BBB physical barrier and p-glycoprotein levels and subcellular location used to quantify the energetic portion of the BBB.25,72’73

[0106] Finally, dose dependence and optimal dosing of motile ependymal cilia inhibitors for treating TBI will be determined. Given the complex outcomes of this study, restoration of the BBB measured by the amount of IgG in the brain will be the variable used to determine the optimal dose of cilia inhibitor (FIGS. 2A-4D). Mice will be treated with differing doses of clindamycin ranging from 5uL of 1-100 ug / mL I.C.V. At 24, 72, and 96 hours post CCI and treatment, brains are harvested, immunostained for murine IgG, and imaged. In addition to the single dose studies, mice will undergo CCI and be treated with a continuous infusion of0.1-5 ug / mL at 0.5-luL / hour of clindamycin for 24, 72, or 96 hours. Brains are then harvested and stained for IgG as a marker of BBB integrity. IgG data will be plotted against clindamycin concentration to determine a dose-dependent relationship between BBB integrity- 31 -4925-8843-5814, v. 1and clindamycin treatment, as well as the maximum concentration and route of administration that demonstrates the greatest BBB restoration.

[0107] Terbutaline will be utilized as a back-up to if clindamycin cannot be used to complete these studies. Terbutaline is also FDA-approved and demonstrated promise in preliminary studies (FIGS. 2A-E). If the degree of CO wound area cannot be quantified due to the processing required for traditional histology, the entire brain will undergo the clarity protocol and staining post-CCI injury (FIGS. 7A-B). This procedure streamlines and enhances the quantification and staining of brain tissue samples compared to traditional histology, particularly in damaged tissues such as brains from CCI studies.74,75If IHC staining cannot quantify the amount of inflammatory cytokines and chemokines in the brain and CSF, an ELISA-based assay will be employed. R&D ELISA kits have previously been used to quantify the level of Thl -associated cytokines in the brain. If this approach is also unsuccessful, singlecell extracts will be made from the brain, and cytokine levels will be measured using flow cytometry. If ependymal cilia motility cannot be quantified using DIC microscopy, a beadbased assay will be used to quantify the motility of ependymal cilia via fluorescence signal. If differences in neurotoxic metabolites in the brain and CSF of mice that undergo CCI cannot be identified using whole brain and CSF extracts, mass spectrometry imaging will be used to measure differences in metabolites at the CCI wound site. Post-injury, the mouse will be anesthetized, perfused, the brain harvested, and flash-frozen in liquid nitrogen. Frozen samples will be mounted in OCT, cut into 8um thick sections, and mounted on a MALDI plate. A serial section is mounted on a typical charged glass slide. The section on the MALDI plate is sprayed with 2,5-dihydroxybenzoic acid (30 mg / mL in 50:50 H20:Methanol + 0.2% TFA) as the matrix and then subjected to mass spectrometry imaging to identify metabolites.76 78Initial studies will focus on heme because heme is readily detectable by mass spectrometry imaging. Serial sections will be fixed in 4% PFA and then stained with Hoechst 33342 and isolectin GS-IB4 Alexa Fluor 488 to identify nuclei and blood vessels, respectively. The image generated from the MALDI mass spectrometer will be overlaid onto the fluorescent image to determine the distribution and amount of metabolites in the wound area, parenchyma, and ventricles. If changes in CSF flux cannot be measured by MRI using T2 DWI series, then T1 imaging via gadolinium tracers will be employed. Briefly, the gadolinium (Gadovist) will be injected I.C.V. in the ventricle along with the clindamycin or control saline, and changes in signal washout will be quantified over time as a surrogate measure of CSF flux. While this approach does not allow for pre-imaging mice before CCI, it will allow for comparisons within the studies and- 32 -4925-8843-5814, v. 1groups. If BBB integrity cannot be used as a measure for optimizing the dose of clindamycin, MR imaging data will be used, including changes in ventricle size, vasogenic edema, and cytotoxicity, as optimizable variables. Each variable will be assigned a numeric value from 1- 5 and summed together to generate a score that can be used to identify the optimal dose for treating severe TBI with clindamycin.

[0108] Statistics and Data Analysis - Generally, data are presented as means with standard deviation. For fluorescent imaging and IHC studies, each assay will contain a minimum of 16 animals (8 female and 8 male), and at least three independent assays will be conducted to ensure the data is representative. A blinded individual will count all fields, including in the cortex surrounding the impact site as well as on the contralateral cortex and ipsilateral hippocampus of the same brain. ANOVA with Tukey correction will be used to determine significant differences between multiple groups (such as assays conducted over time). T-tests with a 5% false discovery rate will be used to determine differences between matched treatment and control groups. For MR imaging assays, the DCE-MRI data will be investigated at both the level of the whole volume within the CSF, as well as the distributions of various metrics at the voxel level within that volume. The metrics of interest include washin slope, wash-out slope, and the signal-enhancement-ratio. The pre-injection T2 maps will be used to quantify the flux within the CSF cavities and the size of the cavities over time by relating the change in the T2 DWI signal. For each MR imaging measurement, statistical differences between the control group (injection of saline) and the treatment group (injection of clindamycin) will be tested. If the data are normally distributed (as determined by the Kolmogorov-Smimov test), the student’s t-test will be utilized. Otherwise, Wilcoxon rank sum test will be used. This comparison will be performed at the level of each ventricle (lateral ventricles, third ventricle, cerebral aqueduct, and fourth ventricle, FIGS. 7A-B) to identify differences between treatment groups at each imaging time point. To determine the optimal amount and route of clindamycin dosing, the signal derived from IgG will be plotted, and a function will be fitted using a simple a linear regression. To determine the optimal dose, data will be fitted to a log-curve, and the IC50 and IC90 doses for the amount of BBB restoration mediated by I.C.V. clindamycin will be calculated.Example 2 - Measuring the benefits and effects of administering motile ependymal cilia stimulators post-TBI on brain function as determined by behavioral assays that address cognition as well as anxiety-like, and hyperactivity-like behaviors.- 33 -4925-8843-5814, v. 1

[0109] Altering the motility of ependymal cilia could affect higher-order neurological function. While some of these behaviors may be desirable, such as improved cognition compared to non-treated controls, enhancing CSF flux could also result in changes in necessary neural metabolites, causing changes in normal brain function. To test these potential outcomes, a battery of behavioral tests will be performed to determine the benefit, tolerability, and potential adverse events of treating severe TBI with motile ependymal cilia stimulators.

[0110] The approach for using behavior studies to determine the benefit and tolerance of motile ependymal cilia stimulators to treat TBI is as follows: each study contains an acute (one week) and late (one month) cohort of animals. Each cohort contains four groups of mice to identify changes in behavior resulting from administering ependymal cilia stimulators: stimulator + sham TBI, stimulators + TBI, control + TBI, and control + sham TBI (FIGS. 10A- B). Each group will be assessed for changes in cognition, anxiety, and both hyper and hypo - activity levels.56,79Finally, all these assays will be repeated in animals administered clindamycin I.C.V. continuously for one week.

[0111] Mice will undergo CCI or sham injury and then be administered clindamycin or saline control injection I.C.V. The list of groups is: clindamycin I.C.V.+CCI (treatment), clindamycin I.C.V. (cilia stimulator only control), PBS I.C.V.+CCI (injury only control), or PBS I.C.V. (non-treated control).

[0112] After a single cycle of injury and treatment, the mice are divided into two cohorts. An acute cohort where behavior will be measured by a battery of tests one week postinjury, and a completely independent late cohort where behavior will be measured using the same battery of tests one month post -injury. The first cohort will test for acute treatment effects (1 week), while the late cohort (1 month) will test for the resolution of long-term symptoms following treatment. Mice will first be tested using open-field tests to determine both activity levels and anxiety. Mice will be placed in an open arena 18-24 inches square under moderate lighting conditions. Mice will be allowed to move freely for the duration of the test, usually 5 minutes. An overhead camera records their movement. The overall movement of each mouse will be recorded. The overall rate of movement will be compared between treatment and control groups to determine hyper or hypo activity resulting from TBI and treatment with a cilia stimulator. Preliminary data for this assay is demonstrated in FIGS. 9A-D.- 34 -4925-8843-5814, v. 1

[0113] Anxiety levels of treated and control mice will also be measured using openfield tests. A normal mouse prefers the edge of the open field cage but will also explore the middle. In contrast, an anxious mouse will not explore the middle of the cage and remain on the peripheral edges of the open field. Similar to above, mice will be placed in an open arena 18-24 inches square under moderate lighting conditions. Mice will be allowed to move freely for the duration of the 5-minute test. An overhead camera will record their movement and the degree to which a mouse stays in the periphery versus the center of the arena. Anxiety level will be determined by quantifying the time treated or controlled mice explore the center of the open field.

[0114] Next, a Barnes maze will be used to assess cognition. The maze consists of an elevated circular platform (92 cm diameter) with twenty, five centimeter holes equally spaced along the perimeter. One of the holes leads to a small dark recessed chamber under the platform (target box). Gentle aversive stimuli may be employed to motivate mice to find the target box. Visual cues placed on the walls serve as unique reference points for locating the target box. To start each trial, mice will be placed in a cylindrical black start chamber in the center of the maze for 10 seconds. Mice will be initially trained to use the maze by gently guiding them by hand to the target box. If a mouse does not enter the target box on its own, it will be gently placed inside. In subsequent acquisition training, mice can explore the maze independently until they enter the target box or 3 minutes have elapsed. Mice repeat acquisition training four times daily for four days, returning to their home cage between trials, with at least 15 minutes between probes. On day 5, the probe trial will be conducted without a target box. Mice are allowed to explore the maze for 90 seconds. The probe trial assesses the mouse's memory for the location of the target box. How long it takes a mouse that underwent CCI and treatment with motile cilia stimulators versus controls to find the target box will be evaluated to determine changes in cognition. In addition to the Barnes maze, cognition will also be tested using a novel object recognition test.80In this assay, mice will be first introduced to an open field box on day one. On the second day, mice will be introduced to the same box containing two objects. Finally, on the third day, one of the objects will be the same as on day two, and the other will be different. Mice will naturally explore new objects; thus, mice with normal cognition will spend more time with the new object than the object from day two. The amount of time the mouse spends with each object will be recorded. This assay will be repeated with mice treated with motile cilia stimulators or control groups described in FIG. 9A-D to determine cognitive benefits associated with CCI and treatment with motile cilia stimulators.- 35 -4925-8843-5814, v. 1

[0115] Finally, at the end of the study, mice will be euthanized, and their brains will be harvested, sectioned, mounted, stained, and imaged to evaluate the neuropathology in treated and non-treated mice. In particular, hippocampal neurogenesis will be examined as impaired neurogenesis has been previously observed in animals that undergo CCI by staining brains with NeuN and Ki67 (Immunohistochemistry (IHC) images of sections stained for Ki67 are presented in FIG. 10A-B).79IHC staining for cytokines including TNFa, IL-6, IL-8, IL-10, and MCP-1, which were previously associated with TBI, will also be performed.81

[0116] Next, animals will be administered clindamycin or PBS control continuously I.C.V. for one week. This approach is clinically relevant as patients with severe TBI will have an EVD placed that could be attached to an infusion pump. The groups for this study are outlined in FIGS. 9A-D and are similar to those described above: clindamycin I.C.V.+CCI (treatment), clindamycin I.C.V. (cilia stimulator only control), PBS I.C.V.+CCI (injury only control), or PBS I.C.V. (non-treated control). After CCI injury or sham, a cannula will be placed in the right ventricle and attached to an osmotic pump to administer 0.5-1 uL of 1-5 ug / mL of clindamycin or PBS for one week. Next, mice will be randomly split into two cohorts. After one week of treatment, the first cohort will undergo a battery of tests to quantify acute changes in activity levels, anxiety, and cognition using the assays described above. The second cohort will undergo the same battery of tests one month after treatment to measure long-term changes resulting from I.C.V. administration of motile cilia stimulators. This scheme will allow for monitoring acute changes in behavior associated with the administration of cilia stimulators and the resolution of TBI symptoms. At the end of the study, mice will be euthanized, and the brains will be harvested, sectioned, mounted, stained, and imaged to examine changes in histopathology.81

[0117] If activity levels cannot be quantified in an open field due to enhanced anxiety, activity levels will be measured via home cage monitoring. Briefly, treated or control mice will be housed in a Noldus Phenotyper, a square box with a 12.5"x 12.5" floor and a nest enclosure in one corner, and serves as the animal's home cage. This cage will have an infrared videotracking system on the ceiling and food and water sensors. This setup allows observation and tracking of animals within their home cage in a controlled environment. The advantages of home-cage testing include minimal handling, long-term recording, and automated analyses that are useful for detecting subtle changes in patterns of activity and consumption. If anxiety cannot be measured with an open-field test, a light / dark box assay will be used. The apparatus- 36 -4925-8843-5814, v. 1consists of two chambers: a smaller, dark, safe chamber and a larger, open, brightly lit aversive chamber. The animal will initially be placed on the brightly lit side and then allowed to explore both sides freely for the duration of the test. The degree of exploration before moving to the dark box and the amount of time in the dark box are used to measure anxiety. If a Barnes maze or novel object recognition test cannot be used to measure cognition, a Morris water maze will be employed instead. If changes in hippocampal neurogenesis cannot be measured using NeuN and Ki67 markers, NeuN and BrdU staining will be used. If cytokine levels in brain sections cannot be measured accurately due to inconsistent tissue staining or processing issues, an ELISA assay will be used to determine TNFa, IL-6, IL-8, IL-10, and MCP-1 cytokine levels in brain extracts (R&D systems). If acute symptoms of TBI are not observed using the CCI protocol employed to generate the preliminary data for this proposal, the CCI depth will be adjusted to increase the initial injury. If TBI symptoms are not resolved after one month of treatment, additional cohorts will be added to the study to extend testing beyond one-month post-treatment.

[0118] Statistics and Data Analysis - Generally, data will be presented as means and standard deviation. All behavioral testing assays will be performed with at least 16 animals per group (8 female and 8 male), and ANOVA with Tukey correction for multiple comparisons will be used to determine significant differences. Video from the behavior studies will be analyzed using the EthoVision XT software. For histology analysis, a minimum of five fields will be quantified from at least two independent replicates. A blinded lab member will count fields. ANOVA with Tukey correction for multiple comparisons will be used to determine significant differences. All studies are conducted in female mice and then repeated in males to assess sex differences in response to treatment.Example 3 - Pilot clinical trial to determine the safety and potential benefits of using motile ependymal cilia stimulators for treating patients with severe TBI.

[0119] Currently, severe TBI treatment is designed to prevent secondary injuries. Multiple previous pharmacological treatment trials for severe TBI have failed, so a novel pharmacological treatment to improve the outcome of severe TBI patients is desperately needed. To begin addressing this issue, a pilot clinical trial will be performed to demonstrate the feasibility of using motile ependymal cilia stimulators to adjuvant current surgical interventions. Administering clindamycin intraventricularly or intrathecally is feasible given the low adverse events profile, affordability of the drug, and accessibility of the ventricles using- 37 -4925-8843-5814, v. 1an external ventricular drain that is placed to manage hydrocephalus and monitor intracranial pressure. Thus, administering clindamycin intraventricularly to patients presenting with severe TBI is feasible, and the pilot trial will lay the foundation for developing the first pharmacological intervention for treating TBI.

[0120] This study proposes repurposing clindamycin to treat TBI. Given that clindamycin is a well-tolerated therapeutic, rather than a new molecule, a pilot study is feasible to perform during the four-year duration of this proposal. Years one and two will be devoted to seeking guidance from the FDA on the necessity of filing an IND and receiving IRB approval to administer clindamycin intraventricularly (I.C.V.). Years two through four of this proposal will focus on patient recruitment, administering clindamycin, patient follow-up, and analysis of patient samples. The pilot study will be an open-labeled study, and data gathered from study participants will be compared to radiographic / clinical data from historical control and CSF / blood samples previously collected from head-injured patients that is stored in the UT Austin Department of Neurosurgery Biobank (Table 1).

[0121] Patients who present with severe TBI at a level 1 trauma emergency room may be recruited. Inclusion criteria included patients presented with a non-penetrating head injury with a Glasgow Coma Scale less than 9 who have an external ventricular drain (EVD) placed as part of the standard management protocol (FIG. 11). Recruitment will not be biased for gender. Generally, 10-15 patients a year who fit these criteria are seen in the ER. As mentioned above, CSF and blood samples from -15 similar patients (Table 1) have been collected in less than two years; thus, reaching the enrollment described in the Human Subjects sections will be feasible.

[0122] Table 1. CSF and blood samples were collected from patients who presented at the emergency room with severe TBI.Current CSF and Blood Sample inventory# of Patients Mates Females Age Range Mean Age15 4 11 25-76 54

[0123] Patients will receive 10-15 mg of clindamycin I.C.V. over the course of five days. This dosing is based on dosing of other antibiotics currently used in the CNS, such as gentamicin. Clindamycin will be formulated by the research clinical pharmacists here at Dell Med and administered via infusion through an EVD. All patients will be managed according- 38 -4925-8843-5814, v. 1to institutional protocol during the hospitalization and be followed in the Neurosurgery clinic after discharge. As part of the standard protocol, Glasgow Outcome Score -Extended (GOS-E) will be collected at 2 weeks, 3 months, and 6 months. GOS-E scores will be compared to historical controls with similar injury characteristics from the trauma registry. In addition, data will be abstracted from the medical records to evaluate the need for osmotherapy for intracranial pressure management, need for a CSF diversion procedure, ventilator days, duration of EVD placement, need for tracheostomy, length of stay in the ICU, length of stay in hospital, and discharge disposition.

[0124] As part of the standardized protocol for the management of severe TBI, serial CT scans (2 in the first 24 hour, one between day 3-5) and MRI (between day 5-7 of injury) of the brain will be obtained. These radiological images will be downloaded for further analysis in terms of contusion progression, semiquantitative analysis of cerebral edema, and semiquantitative analysis of ventricular and sulci size.

[0125] In addition to imaging, CSF and serum from TBI patients will also be analyzed for differences derived from treatment with motile ependymal cilia stimulators. CSF samples will be analyzed for inflammatory markers, including cytokines IL-ip, IL-6, IL- 18, and TNFa and chemokines CXCL8, CCL2, CCL3, CCL4, CCL5, and CX3CL1 by ELISA. Additionally, levels of neurofilament light chain will be quantified by ELISA as a measure of neuronal damage. Samples of study participants will be compared to historical controls using samples from patients with similar characteristics from the University of Texas at Austin Department of Neurosurgery Biobank.

[0126] Proteomic mass spectrometry experiments will be performed on the CSF of patients treated with cilia stimulators.50Proteins from CSF samples will be extracted and fragmented with trypsin. The resulting peptides will analyzed using an orbitrap mass spectrometry instrument. Peptides are aligned to CNS proteins, and the presence of neurometabolites will be determined. The amount of each metabolite will be quantified by correlating the area of the chromatography peak between samples. These studies will be performed at the UT Austin mass spectrometry core in the Chemistry Department. The laboratory has an ongoing relationship with this core, including a project to measure drug accumulation in the brain (please see recently published study reference 25), and does not anticipate any issues conducting these studies or analyzing the data. The primary analysis will be levels of metabolites in CSF samples over time in patients treated with cilia stimulators. A- 39 -4925-8843-5814, v. 1similar analysis will be performed on matched non-treated controls. These data will also be compared to historical data sets0

[0127] Finally, serum from TBI patients treated with I.C.V. cilia stimulators or control will be analyzed. Serum levels of GFAP, UCHL1, and neurofilament light chain will be measured in serum daily for one week by ELISA (R&D Systems) to determine the role of ependymal cilia motility on neuronal damage post brain injury. The primary analysis will be levels of biomarkers in CSF samples over time treated with cilia stimulators. A similar analysis will be performed on and non-treated controls. These data will also be compared to historical data sets.

[0128] Potential Pitfalls and Alternatives - if cytokines and chemokines levels in the CSF and serum of treated patients cannot be quantified using traditional ELISA assays, the Luminex platform will be employed rather than a typical ELISA. This instrument is available on the UT Austin campus in the College of Nursing and allows for picogram detection of cytokines and chemokines. If patient recruitment, as described in the human subjects section, cannot be completed within the four years of this award, other academic trauma centers will be enlisted to increase the number of patients that meet enrollment criteria. UT-Austin is part of the TRACK-TBI network, and collaborations with other study sites are common and feasible and will be used to complete this study if necessary. If changes in neurometabolites cannot be measured by mass spectrometry, NMR will be used.82NMR will be performed at the UT Austin Chemistry Department Core facility, which has a 9T instrument capable of performing1H- NMR previously used to characterize neurometabolites in CSF samples by other groups.30

[0129] Statistical Plan and Data Analysis - Generally, data will be presented as means and standard deviation. For MR images, all data will be deidentified and randomized before analysis. A blinded reviewer will quantify the size of each ventricle using ImageJ software from the NIH. A blinded reviewer will also rank changes in cerebral edema on an improvement scale from 1-5. Given the heterogeneity of patients that present with severe TBI and the limited sample size for the pilot study, data will be compared within a single patient’s samples (i.e. differences in a single patient’s score over time) rather than grouping patients together. These data will be compared to matched historical data / samples for analysis. ANOVA with Tukey correction will be used to identify differences in a patient’s data over time. Similar schemes will be used to analyze the serum and CSF neurotoxicity, cytokine, and chemokine data. To analyze mass spectrometry data, metabolite fragments are identified from the M / Z+ ratios using- 40 -4925-8843-5814, v. 1MASCOT. Using code written in the lab, peptide metabolite fragments are automatically processed through the NCBI database to identify the protein of origin, subcellular location, and function. To determine significant differences between metabolites, the area under the curve derived from the liquid chromatography column will be calculated for protein fragments. Changes in proteomic data over time will be determined using ANOVA with Tukey correction for multiple comparisons. A similar analysis will be performed on historical controls to determine the potential benefits of treatment with motile cilia stimulators. For abstracted clinical data from the medical records, variables from patients treated with motile cilia stimulators will be compared to historical controls. Based on the data distribution, significant differences between control and treatment groups are determined using T-tests or Wilcoxon rank sum tests.Example 4 - Pharmacological Enhancement of Cerebrospinal Fluid Flux Reduces Secondary Pathogenesis Post Traumatic Brain Injury

[0130] A pharmacologic treatment for TBI is proposed herein that is designed to reduce secondary pathogenesis post injury by promoting the clearance of neurotoxic metabolites from the brain by enhancing cerebrospinal fluid flux via stimulating ependymal cilia motility. To enhance excretion of toxic molecules from the brain after an injury, the motility of ependymal cilia was increased which enhances cerebrospinal fluid (CSF) flux. To stimulate motile ependymal cilia, clindamycin was repurposed. Clindamycin is an FDA-approved second line antibiotic that previous studies indicated enhances the motility of airway cilia. Intracerebroventricular (I.C.V.) administration of clindamycin enhanced the motility of ependymal cilia resulting in increased brain clearance of tracer administered in the cortex via the glymphatic system. Enhanced clearance was independent of the antibiotic activity of clindamycin. Further, I.C.V. administration of clindamycin resulted in enhanced restoration of the BBB following focal or diffuse TBI. In terms of molecular markers, I.C.V. administration of clindamycin significantly reduced red blood cells in the wound penumbra and safely lowered serum glial fibrillary acidic protein (GFAP) post injury. Finally, mice administered I.C.V. clindamycin recovered quicker from anesthesia, demonstrate enhanced activity 18 hours post injury, and exhibit enhanced cognition up to two weeks post brain injury compared to controls.

[0131] I.C.V. administration of clindamycin is clinically feasible as patients afflicted with a severe TBI will receive an external ventricular drain (EVD) to manage intracranial pressure as standard of care treatment. Therefore, we present a novel, feasible method for- 41 -4925-8843-5814, v. 1treating TBI by enhancing clearance of toxic molecules from the brain which reduces secondary pathogenesis associated with TBI.

[0132] A literature search identified multiple compounds potentially capable of enhancing the motility of motile cilia. Criteria was added to exclude any non-FDA approved compounds, compounds with known off target effects, and non-commercially availability compounds. Applying these criteria, two lead compounds were identified. First, clindamycin sulfate, a well-tolerated compound that is FDA approved as a second line antibiotic (albeit for systemic rather than intracranial / intrathecal use), and second, terbutaline which is FDA approved as a bronchodilator. Given the low cost, wide availably, and no known targets outside of bacteria, the studies described in this example primarily focused on clindamycin.Results

[0133] LC.V. administration of clindamycin enhances ependymal cilia motility - clindamycin does not cross the blood-brain barrier (BBB); thus, our first study examined if intracerebroventricular (I.C.V.) administration of clindamycin could enhance the motility of ependymal cilia similarly to previous studies of motile airway cilia. In FIGS. 12A-B, Mice administered I.C.V. clindamycin demonstrated a -30% increase in ependymal cilia motility one hour post injection compared to mice administered I.C.V. PBS (p<0.05 via T test, n=3 mice / group).

[0134] LC.V. administration of clindamycin enhances tracer extravasation from the cortex via the cervical lymph nodes - To determine if enhancing cerebrospinal fluid flux can alter the extravasation of molecules from the brain, a far-red tracer was administered into the cortex of mice followed by I.C.V. clindamycin (to stimulate ependymal cilia motility) or PBS (control). As demonstrated in FIGS. 12C-D, a significant reduction of brain dye signal was observed in animals receiving I.C.V. clindamycin compared to control mice (p<0.05 via T-test, n=4 mice / group). To determine if enhancing CSF Flux (via stimulating motility of ependymal cilia) alters brain dye clearance via the glymphatic system, the levels of dye in the cervical lymph nodes were quantified in FIGS. 12E-F. A significant increase in dye signal was observed in the cervical lymph nodes of mice administered I.C.V. clindamycin compared to PBS implying enhancing CSF flux enhances clearance of molecules from the cortex via the glymphatic system.- 42 -4925-8843-5814, v. 1

[0135] LC.V. administration of clindamycin or terbutaline reduces brain IgG levels 24 hours post stab wound TBI - to determine the effect of enhancing the motility of ependymal cilia on focal TBI a craniotomy was first performed followed by a stab wound with a 33 gauge needle that penetrated the cortex (FIG. 13A). Next 5 uL of PBS, terbutaline (XX ug / mL), or clindamycin (20ug / mL) was administered I.C.V. Twenty-four hours post injury the amount of murine IgG was quantified as a measure of blood-brain barrier (BBB) restoration. FIGS. 13B-C demonstrate that administering motile cilia stimulators (either terbutaline or clindamycin) results in a significant reduction in brain levels of murine IgG (p<0.05 via ANOVA, n=4mice / group).

[0136] LC.V. administration of clindamycin reduces brain levels of IgG 24 and 72 hours post controlled cortical impact (CCI) TBI that is not associated with the antibiotic function of clindamycin - Next mice were subjected to a controlled cortical impact as a second model of focal TBI then administered PBS, gentamycin, or clindamycin I.C.V. After either 24 (FIGS. 13D-E) or 72 hours (FIGS. 13F-G) the brains were harvested and levels of murine IgG in the brain were quantified. Only mice administered I.C.V. clindamycin demonstrate a significant reduction in brain IgG levels post injury (p<0.05 via ANOVA, n=4 mice / group).

[0137] I.C.V administration of clindamycin reduces brain extravasation of a systemically administered tracer in a weight drop with acceleration diffuse TBI model - While subjecting mice to focal TBIs is a convenient laboratory model, diffuse TBIs are more commonly seen in the clinic. Thus, a weight drop was employed with acceleration (WDA) TBI to model (FIG. 14A) to test the benefit of administering motile ependymal cilia enhancers post injury. Twenty-four hours post injury and treatment, mice were administered IRsoo carboxylate via intraperitoneal injection. Brains were harvested and the amount of dye that extravasated into the brain was quantified as another measure of BBB disruption. As demonstrated in FIGS. 14B-C, treating mice with I.C.V. clindamycin post injury significantly reduces extravasation of dye from the bloodstream into the brain (p<0.05 via T test, n=4 mice / group).

[0138] I.C.V administration of clindamycin reduces the number of brain vessels without claudin 5 after mice are subjected weight drop with acceleration TBI - The total amount of claudin 5 and the number of brain vessels with proximal claudin 5 staining was quantified in FIGS.17-C and 14D-E, respectively, twenty-four hours post WDA TBI plus treatment. The total amount of claudin 5 in the brain demonstrated a trend but did not significantly differ between mice administered I.C.V. PBS or clindamycin (p=0.06 via T test,- 43 -4925-8843-5814, v. 1n=3 mice per group with at least 3 fields quantified / mouse). However, the length of brain vessels that did not demonstrate proximal claudin 5 staining was significantly higher in mice administered I.C.V PBS compared to clindamycin (p<0.05 via T test, n=3 mice per group with at least 3 fields quantified / mouse).

[0139] I.C.V. administration of clindamycin resulted in lower number of red blood cells (RBCs) in the CCI wound penumbra one week post injury - To characterize if stimulating motile ependymal cilia effects healing of the primary wound or serves to reduce secondary pathogenesis associated wound healing, the wound area of mice subjected to CCI TBI and administered I.C.V. PBS or clindamycin was measured one week post injury by modified H&E staining. FIG. 18 demonstrates a trend but no significant differences in wound area between groups (p=0.08 via T-test, n=4 mice / group with at least 5 sections quantified / mouse). However, as demonstrated in FIGS. 15A-C, there was a significant reduction in red blood cells (RBCs) in the wound penumbra from an average of 23.7 ± 75 and 257.0 ± 124 cells in mice administered I.C.V. clindamycin compared to PBS respectively (p<0.05 via T-test, n=4 mice / group with at least 5 sections quantified / mouse).

[0140] I.C.V. administration of clindamycin reduces serum levels of glial fibular acid protein (GFAP) 24 hours post CCI injury - Serum levels of GFAP were measured 24 hours after mice were subjected to CCI TBI and administered either I.C.V. PBS or clindamycin. FIG. 15D demonstrates a significant reduction from 24.07 to 67.73 pg / mL (2.8 fold) in serum GFAP levels in mice administered I.C.V. clindamycin compared to PBS (p<0.05 T test, n=3 mice / group).

[0141] Hydrocephalus as well as serum AST, and ALT levels are within normal range after being subjected to CCI TBI and administration of I.C.V. clindamycin - In terms of safety, FIG. 15E demonstrates the amount of water in the brain 24 hours post treatment was within the normal range and did not differ between I.C.V. clindamycin or PBS groups (p=0.8 T test, n=3 mice / group). Further, FIGS. 15F-G demonstrate serum levels of AST and ALT were within normal ranges following CCI TBI and administration of I.C.V. clindamycin.

[0142] Animals administered I.C.V. clindamycin recover faster from isoflurane anesthesia - As demonstrated in FIG. 16A, mice administered I.C.V. clindamycin recover approximately twice as fast from isoflurane anesthesia compared to I.C.V. administration of- 44 -4925-8843-5814, v. 1either PBS or gentamycin (means, STD = 2.63 ± 0.87, 8.00 ± 0.94, and 9.25 ± 1.25 mins, respectively, p<0.05 via ANOVA, n=4 mice / group).

[0143] Animals administered LC.V. clindamycin demonstrate greater activity 18 hours post injury - In addition to recovery from isoflurane, FIGS. 16B-C demonstrate mice subjected to CO TBI and administered I.C. V. clindamycin have significantly increased activity 18 hours post injury compared to mice administered I.C.V. PBS (p<0.05 via T-test, n=6 mice / group). Importantly, activity was below mice that received a sham TBI indicating treated mice were not hyperactive. FIG. 19 demonstrates that this benefit was abrogated one week post injury and treatment.

[0144] Animals administered I.C.V. clindamycin demonstrate improved cognition for 2 weeks post injury - to measure changes in cognition post injury and treatment, cohorts of mice were subjected to a novel object recognition test. As demonstrated in FIGS. 16D-E, mice subjected to a CCI TBI and administered I.C.V. clindamycin have improved cognition at one and two weeks post injury and treatment compared to mice administered I.C.V. PBS (n=8 mice / group via ANOVA). These studies were performed in both male and female mice as well as young (6 weeks old) and aged (>10 months) mice. The cognition benefit was abrogated one month post injury.Discussion

[0145] Proposed herein is a new method for treating traumatic brain injury by enhancing the motility of ependymal cilia to increase the extravasation of toxic molecules from the brain post injury. In addition to enhancing extravasation, it was also observed to enhance restoration of the BBB which further shields the brain from molecules extravasating from the bloodstream that could cause secondary pathogenesis as the brain heals. Thus, stimulating the motility of ependymal cilia represents a distinct break from the current approaches for treating TBI which have primarily focused on mitigating inflammation or enhancing growth factors to address the primary wound.

[0146] To identify molecules capable of enhancing ependymal cilia motility, previous literature describing motility of cilia in airways was utilized. Previous studies, along with the data presented here, demonstrate molecules that alter the beat frequency of motile airway cilia will function similarly in motile ependymal cilia provided the compounds are administered behind, or penetrate, the BBB. This observation allows for the repurposing of FDA approved- 45 -4925-8843-5814, v. 1drugs to potentially speed the transition of this approach to the clinic (although for clindamycin given the change in route of administration, a multitude of safety and tolerance studies are needed before use in patients). In cases of severe TBI, it is standard practice to place an EVD for monitoring and controlling intracranial pressure. This EVD could also be used to administer motile cilia stimulators I.C.V. Thus, the transition of this approach to the clinic appears feasible in the near future.

[0147] During the research described herein it became natural to examine how increasing the motility of ependymal cilia (thereby increasing CSF flux) results in enhanced clearance of molecules from the brain cortex. Increased dye clearance through cervical lymph nodes implies that increasing ependymal cilia motility functions by enhancing excretion of molecules from the cortex via the glymphatic system. This is the first report that altering CSF flux can alter the glymphatic system. Thus, in addition to identifying a pharmacologic treatment for TBI, a novel mechanism for altering the glymphatic system has also been demonstrated. Future studies will demonstrate the utility of this approach in larger brains that have a different ratio of CSF to motile ependymal cilia as well as define the types of molecules that can be cleared via this mechanism such as protein aggregates that are associated with neurodegenerative disease or particles used in drug delivery systems.

[0148] In addition to enhancing the clearance of toxic molecules from the brain, stimulating motile ependymal cilia also increases restoration of the BBB. It was demonstrated that administering clindamycin I.C.V. reduces extravasation of both large proteins (IgG) and small molecules (IR800 dye) from the blood stream into the brain. Additionally, the loss of claudin 5 proximity to medium sized brain blood vessels provides a potential mechanism for changes in BBB permeability post injury. However, it is unclear if BBB restoration results from enhanced clearance of toxic molecules in the brain or is a direct effect of administering clindamycin I.C.V. Future studies using genetic murine models of enhanced and impaired ependymal cilia motility might define this relationship.

[0149] Importantly, enhanced BBB restoration was observed in both focal and diffuse models of TBI. TBI is a heterogenous disease, with focal TBIs being relatively rare and associated with military conflicts while diffuse TBIs from vehicle accidents, sports injuries, and falls are considerably more common. One caveat of this study is experiments were limited to a single TBI; however, in reality individuals my undergo multiple TBIs that may alter the benefits provided by enhancing ependymal cilia motility.- 46 -4925-8843-5814, v. 1

[0150] Another form of heterogeneity in TBI is the wide age range of patients afflicted with TBIs. This study attempted to capture this variably by performing cognition studies in both male and female as well as young and old mice. The cognition benefits associated with stimulating motile ependymal cilia were independent of sex or age, implying stimulating motile ependymal cilia is a robust method for reducing secondary pathogenesis from TBI.

[0151] Currently, the treatment of severe TBI is restricted to managing acute symptoms, namely intracranial pressure. While TBIs are associated with changes in personality and cognition, these symptoms have been difficult to treat and are often secondary considerations. Enhancing ependymal cilia motility appears to be a robust strategy for managing acute symptoms, such as removing neurotoxins (RBCs and heme) and increase acute activity, which we propose as a surrogate for reducing hospitalization time, as well as providing long term benefits associated with enhanced cognition. Future studies will address more complicated TBI symptoms such as changes in personality and aggression. Finally, we observe a reduction in serum GFAP levels associated with stimulating motile ependymal cilia. While it is unclear if the reduction in serum GFAP is derived from restoration of the BBB or enhanced neuronal healing from removal of toxin molecules generated during wound healing, reducing serum GFAP is considered a valid clinical test to determine the benefit of a TBI treatment and further implies stimulating motile ependymal cilia could benefit patients afflicted with a TBI in the future.

[0152] This study focused only on severe TBI that would result in the placement of an EVD. This caveat is necessary to due to the requirement of administering clindamycin I.C.V. Future studies identifying motile cilia stimulators that cross the BBB may allow for broader application of this strategy to models of multiple / moderate TBI. Additionally, future studies are needed to define the safety of administering clindamycin I.C.V. because currently gentamicin is the only antibiotic approved for intracranial / intrathecal use. Further, this study was limited to a single dose of clindamycin. Multiple dose may further improve outcomes post TBI. Finally, given the differential rates of CSF production, secretion, and flux between brain sizes these studies should be repeated in animal models with larger brains, such as porcine, to accurately determine the benefits of administering I.C.V. clindamycin to treat severe TBI.

[0153] In conclusion, this data demonstrates that enhancing CSF flux by stimulating motile ependymal cilia reduces secondary pathogenesis associated with TBI. This occurs by enhancing restoration of the BBB, and enhancing clearance of toxic molecules from the cortex- 47 -4925-8843-5814, v. 1via a novel mechanism for altering the glymphatic system. Importantly, I.C.V administration of clindamycin is clinically feasible and improves both serum and functional biomarkers after focal and diffuse TBIs, implying stimulating motile ependymal cilia is a viable pharmacological treatment for TBI.

[0154] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. 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Inflammation after spinal cord injury: a review of the critical timeline of signaling cues and cellular infiltration. J Neuroinflammation 18, 284 (2021). https: / / doi.org / 10.1186 / sl2974-021-02337-2.84 Xiong G, Elkind JA, Kundu S, Smith CJ, Antunes MB, Tamashiro E, Kofonow JM, Mitala CM, Cole J, Stein SC, Grady MS, Einhorn E, Cohen NA, Cohen AS. Traumatic brain injury-induced ependymal ciliary loss decreases cerebral spinal fluid flow. J Neurotrauma. 2014 Aug 15;31(16): 1396-404. doi: 10.1089 / neu.2013.3110. Epub 2014 Jun 20. Erratum in: J Neurotrauma. 2015 Feb 15 ;32(4):287. doi: 10. 1089 / neu.2015.0001.32.4.. Cole, Jeffrey [added], PMID: 24749541; PMCID: PMC4132584.- 53 -4925-8843-5814, v. 1

Claims

WHAT IS CLAIMED IS:

1. A method of treating a traumatic injury to the central nervous system of a mammalian subject, comprising administering a therapeutically relevant amount or an effective amount of a cilia activator to the mammalian subject.

2. The method of claim 1, wherein the cilia activator stimulates the motility of ependymal cilia in the mammalian subject.

3. The method of claim 2, wherein the cilia activator is an adrenergic drug, a cholinergic agonist, a beta-adrenergic agonist, a methylated xanthine, roxithromycin, a nitric oxide releasing compound, or clindamycin.

4. The method of claim 2, wherein the cilia activator is terbutaline or clindamycin.

5. The method of claim 4, wherein the cilia activator is clindamycin.

6. The method of claim 3, wherein the nitric oxide releasing compound is terbutaline, bacitracin, or gramicidin.

7. The method of claim 5, wherein the method comprises administering from about 50 mg to about 600 mg to the mammalian subject either once or preferably every 6 hours.

8. The method of claim 7, wherein the method comprises administering about 50, 60, 70, 80, 90, 100, 125, 152, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 mg, or any range derivable therein.

9. The method of claim 7, wherein the method comprises administering from about 150 mg to about 300 mg to the mammalian subject either once or preferably every 6 hours.

10. The method of claim 7, wherein the method comprises administering from about 300 mg to about 450 mg to the mammalian subject either once or preferably every 6 hours.

11. The method of any one of claims 5-10, wherein the clindamycin is administered repeatedly to the mammalian subject over 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days.

12. The method of any one of claims 1-11, wherein the traumatic injury to the central nervous system is a traumatic brain injury (TBI or craniocerebral trauma) or concussion.- 54 -4925-8843-5814, v.

113. The method of any one of claims 1-12. wherein the cilia activator is administered to the mammalian subject intraventricularly, orally, intravenously, intrathecally, intraperitoneally, or intracerebrally.

14. The method of claim 13, wherein clindamycin or terbutaline is administered intraventricularly to the subject.

15. The method of any one of claims 1-13, wherein the mammalian subject is a human.

16. The method of any one of claims 1-13, wherein the method comprises administering a second therapy to treat the traumatic injury to the central nervous system.

17. The method of claim 16, wherein the second therapy comprises administering a surgery or a second therapeutic to the subject.

18. The method of claim 17, wherein the medication is a diuretic, an anti-inflammatory drug, an anti-anxiety drug, an anticonvulsant, an antidepressant, an anticoagulant, an antibiotic, an antimicrobial compound, or a muscle relaxant.

19. The method of claim 18, wherein the second therapy reduces intracranial pressure in the mammalian subject.

20. The method of claim 18, wherein the second therapy is a craniectomy.- 55 -4925-8843-5814, v. 1

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