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WO2025096549A1 - Composés et méthodes pour le traitement de lésions cérébrales - Google Patents

Composés et méthodes pour le traitement de lésions cérébrales Download PDF

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WO2025096549A1
WO2025096549A1 PCT/US2024/053572 US2024053572W WO2025096549A1 WO 2025096549 A1 WO2025096549 A1 WO 2025096549A1 US 2024053572 W US2024053572 W US 2024053572W WO 2025096549 A1 WO2025096549 A1 WO 2025096549A1
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methyl
trpv4
oxo
alkyl
azaspiro
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Chen Gu
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Ohio State Innovation Foundation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • A61K31/497—Non-condensed pyrazines containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

  • Mild traumatic brain injury (mTBI) or concussion represents a major health problem worldwide.
  • the mTBI is a diagnostic term for TBIs with loss of consciousness less than 30 minutes (min), an initial Glasgow Coma Scale of 13–25 after 30 min, and posttraumatic amnesia less than 24 hours (h).
  • Accurate diagnosis and effective treatment remain difficult because many aspects of mTBI are still poorly understood. For instance, it is unknown why most mTBIs recover but approximately 15% of them develop long ⁇ term deficits.
  • the concussion threshold remains elusive. For instance, in studies of American football players, one of the most puzzling findings is that a given head impact that causes serious injury in some players often appears harmless for hundreds of other players.
  • Neurons and relatively softer glial cells including microglia, astrocytes, and pre ⁇ myelinating oligodendrocytes, display differential viscoelastic properties that depend on their location in the brain, subcellular domains, and developmental and differentiation stages.
  • mTBI which usually does not involve bleeding
  • an external mechanical force likely creates larger deformation in glial cells than their adjacent neurons, but the deformation threshold leading to cellular injury in different cell types may differ and remains poorly understood.
  • both neuronal and glial cell damages are involved in the secondary injury of 1 Attorney Docket No.
  • 103361 ⁇ 611WO1 mTBI which shares some aspects of the secondary injury of moderate ⁇ to ⁇ severe TBIs, such as diffuse axonal injury, excitotoxicity, microglia ⁇ mediated inflammation, demyelination, and astrogliosis. Due to the lack of early biomarkers in mTBI, these interlinked secondary injuries are often independently measured days or even weeks after head impact(s) in various studies, leading to no identifiable causal relationship. Thus, it remains unknown whether neurons and glia within the same brain region simultaneously or sequentially respond to the mechanical stress of a concussive head impact.
  • N ⁇ methyl ⁇ D ⁇ aspartate (NMDA) glutamate receptor could act as an initial sensor for a head impact, especially because it can be directly activated in vitro by mechanical stress in the absence of ligand. Indeed, a concussive head impact can quickly trigger glutamate ⁇ mediated excitotoxicity.
  • TRPV4 transient receptor potential channel represents another mechanosensitive Ca 2+ ⁇ permeable ion channel.
  • Gain ⁇ of ⁇ function missense mutations in the TRPV4 gene are linked to human diseases including two major groups, autosomal dominant neuromuscular disorders (Charcot ⁇ Marie ⁇ Tooth disease type 2C and distal spinal muscular atrophies) and skeletal disorders (skeletal dysplasias and osteoarthropathy).
  • Axonal varicosities (swelling or beading), are enlarged, heterogeneous structures along axonal shafts, can be immediately induced by mechanical stress in vitro and in vivo, and hence represent an early biomarker for axonal injury.
  • TRPV4 axonal varicosity formation in vivo
  • TRPV4 broad expression in multiple types of brain cells, including neurons, microglia, astrocytes, oligodendrocyte progenitor cells, and endothelial cells.
  • microglia were shown to be rapidly activated in brain injury as well.
  • FIG. 1 depicts CHIMERA ⁇ induced axonal varicosities precede cortical demyelination with partial recovery.
  • A YFP+ cortical neurons from Thy1 ⁇ YFP transgenic mice: no impact (Sham, left), 0h (middle), or 60d (right) after one 0.9 ⁇ J head impact in CHIMERA.
  • YFP fluorescence signals are inverted in low ⁇ mag grayscale images (top) and are in green in high ⁇ mag confocal images from EC (bottom).
  • E Summary of dMBP staining levels in the mouse cortex at different time points after CHIMERA. One ⁇ way ANOVA followed by Dunnett’s test: *** p ⁇ 0.001.
  • F TEM images from mouse cortex without head impact (Sham; left) or 24h after CHIMERA (right). High ⁇ mag TEM images are at the bottom. Red arrowheads, damaged myelin sheath. Blue arrowheads, asymmetric synapses.
  • G Summary of percentage of axons with damaged myelin in the cortex, gray matter. Image numbers are provided. Unpaired t ⁇ test: *** p ⁇ 0.001.
  • FIG. 1 depicts memantine inhibits CHIMERA ⁇ induced microglial activation and cortical demyelination but not axonal varicosity formation.
  • FIG. 1 depicts memantine inhibits CHIMERA ⁇ induced microglial activation in the cortex.
  • the anti ⁇ CD68 staining signals are in red, YFP in green, and Hoechst in blue.
  • C Diagram for the memantine experiment. Memantine was injected (via i.p. at 10 mg/kg each dose) 1.5 hours before CHIMERA and 3 hours afterward.
  • D Representative images of induced axonal varicosities at 0h from vehicle (top) and memantine (bottom) treated Thy1 ⁇ YFP transgenic mice. YFP fluorescence signals are in green, the CD68 signals in red, and Hoechst in blue.
  • E Summary of the effect of 3 Attorney Docket No.
  • mice were gavaged with GSK219 (20 mg/kg) or vehicle (as control) once 3 hours before CHIMERA (0.9J).
  • B ⁇ (C) Confocal images of the cortex of Thy1 ⁇ YFP mice 24h after CHIMERA with the pretreatment of vehicle (B) or GSK219 (C). The dMBP staining signals are inverted in grayscale images (right) and in red in merged images (left), YFP in green, and Hoechst in blue.
  • D Summary of YFP+ axons with varicosities in EC 0h after CHIMERA with vehicle or GSK219 pretreatment. The green line, the basal level in Sham. The mouse numbers are provided in the chart.
  • Unpaired t ⁇ test: p 0.0000021.
  • F Summary of CD68+ cell density in the cortex 24h after CHIMERA with vehicle or GSP219 pretreatment. The basal level in Sham is close to 0.
  • Unpaired t ⁇ test: p 3.257 ⁇ 10 ⁇ 14 .
  • G Example traces of mouse movement in the elevated plus maze (EPM) before (cyan) or 3d after CHIMERA (black) with vehicle (left) or GSK219 pretreatment (right).
  • D Protein changes in the brains of TRPV4 ⁇ / ⁇ mice versus age ⁇ and sex ⁇ matched WT mice revealed by genome ⁇ wide proteomics with mass spectrometry analysis.
  • E Upregulated proteins in the TRPV4 ⁇ / ⁇ mouse brain in GO subgroups based on subcellular components.
  • FIG. 5 depicts acute deletion of neuronal TRPV4 prevents CHIMERA ⁇ induced axonal varicosity formation and adjacent glial changes.
  • A Diagram of injecting AAV9 ⁇ hSyn ⁇ Cre ⁇ dTomato into the right cortex of mice.
  • Adult WT and TRPV4 fl/fl mice were used in CHIMERA about 1 month after the viral injection.
  • a low ⁇ mag image (bottom) shows the injection sites with dTomato fluorescence in green and Hoechst in blue.
  • the dTomato signals are in green and dMBP signals are in red.
  • FIG. 6 GSK279 markedly inhibits CHIMERA ⁇ induced axon ⁇ glial changes in WT but not TRPV4 ⁇ / ⁇ mice.
  • A Structural diagrams of GSK219 and GSK279.
  • B Diagram for the CHIMERA experiment with GSK279 pretreatment. WT;Thy1 ⁇ YFP or TRPV4 ⁇ / ⁇ ; Thy1 ⁇ YFP mice (3 ⁇ 4 months old) were injected via tail vein with GSK279 (18 ⁇ g/kg) or vehicle (as control) once 1.5 hours before CHIMERA (0.9J). Mice were perfused and fixed either immediately (0h) or 24h after head impact.
  • C Summary of YFP+ axons with varicosities in EC 0h after CHIMERA with vehicle or GSK279 pretreatment. The green line, the basal level in Sham.
  • D Confocal images of the EC of WT (left) and TRPV4 ⁇ / ⁇ (right) mice 24h after CHIMERA with the pretreatment of vehicle (top) or GSK279 (bottom). The CD68 staining signals are inverted in grayscale images (right) and red in merged images (left), YFP in green, and Hoechst in blue.
  • E Summary of YFP+ axons with varicosities in EC 24h after CHIMERA.
  • FIG. 1 Summary of CD68+ cell density in the cortex 24h after CHIMERA.
  • G The dMBP staining signals (inverted in gray ⁇ scale images (right) and in red in merged images (left) in the cortex of WT mice 24h after CHIMERA with vehicle (top) or GSP279 (middle) pretreatment. The summary is at the bottom.
  • FIG. 7 depicts TRPV4 blockers inhibit fluid mechanical stress ⁇ induced axonal varicosities in cultured neurons.
  • A The diagram of the micro biomechanical assay using local fluid puffing.
  • the glass pipette (tip diameter ⁇ 50 ⁇ m) was connected to a syringe via tubing filled with Hank’s buffer. There was about 190 mm in vertical distance between the surface of Hank’s buffer in the syringe and the pipette tip.
  • the pipette tip was positioned about 0.4 mm (vertical distance) above cultured neurons.
  • RhoA proteins are not highly concentrated in puffing ⁇ induced axonal varicosities.
  • Cultured hippocampal neurons expressing YFP were puffed at 8 DIV and then fixed and stained for endogenous RhoA (red in the merged images). Cornered areas are enlarged and shown on the right.
  • Scale bars 10 ⁇ m in (B), 100 ⁇ m in (E) and (F), 50 ⁇ m in (H) left and 25 ⁇ m in (H) right.
  • Figure 8 Gabapentin markedly inhibits CHIMERA ⁇ induced axon ⁇ glial changes in TRPV4 ⁇ / ⁇ mice, as well as WT mice.
  • FIG. 1 Diagram for the CHIMERA experiment with gabapentin pretreatment. WT;Thy1 ⁇ YFP or TRPV4 ⁇ / ⁇ ;Thy1 ⁇ YFP mice were injected via I.P. with gabapentin (50 mg/kg) or vehicle (as control) once 1.5 hours before CHIMERA (0.9J). Mice were perfused and fixed either immediately (0h) or 24h after head impact.
  • B Summary of YFP+ axons with varicosities in EC 0h after CHIMERA with vehicle or gabapentin pretreatment. The green line, the basal level in Sham.
  • C Summary of YFP+ axons with varicosities in EC 24h after CHIMERA.
  • the CD68 staining signals are inverted in grayscale images (right) and in red in merged images (left), YFP in green, and Hoechst in blue.
  • E Summary of CD68+ cell density in the cortex 24h after CHIMERA.
  • F The dMBP staining signals in the cortex of WT (left) or TRPV4 ⁇ / ⁇ (right) mice 24h after CHIMERA.
  • Figure 9 depicts CHIMERA ⁇ induced axonal varicosities in different brain regions are partially reversible. Images from the corpus callosum (CC) (A) or the external capsule (EC) (B) of Thy1 ⁇ YFP transgenic mice that received no impact (Sham, left), or 0h (middle) or 3d (right) after one 0.9 ⁇ J head impact in CHIMERA. YFP signals are in green and Hoechst in blue.
  • CC corpus callosum
  • EC external capsule
  • Figure 11 depicts ultrastructural changes of the mouse cortex 24 hours after CHIMERA.
  • A Example transmission electron microscopy (TEM) images of myelinated axons and synapses in the cortex of sham mice.
  • B Example TEM images of damaged myelin and synapses in the mouse cortex 24h after CHIMERA. Blue arrowheads: asymmetric synapses. Red arrowheads: damaged myelin.
  • Scale bars 300 nm
  • Figure 12 depicts a head impact in 0.9J CHIMERA does not cause bleeding in the mouse brain.
  • FIG. 1 The experimental diagram for assessing the cerebrovascular integrity using Evans Blue (2% in saline; 2 ml/kg mouse body weight) via tail vein injection 1.5 h before the impact.
  • FIG. 1 The experimental diagram for assessing the cerebrovascular integrity using Evans Blue (2% in saline; 2 ml/kg mouse body weight) via tail vein injection 1.5 h before the impact.
  • FIG. 1 The experimental diagram for assessing the cerebrovascular integrity using Evans Blue (2% in saline; 2 ml/kg mouse body weight) via tail vein injection 1.5 h before the impact.
  • FIG. 1 The experimental diagram for assessing the cerebrovascular integrity using Evans Blue (2% in saline; 2 ml/kg mouse body weight) via tail vein injection 1.5 h before the impact.
  • FIG. 1 The experimental diagram for assessing the cerebrovascular integrity using Evans Blue (2% in saline; 2 ml/kg mouse body weight) via tail vein injection 1.5 h before the impact.
  • FIG. 1 The experimental diagram for assessing the cerebrovascular integrity
  • FIG. 13 depicts GSK219 pretreatment markedly reduced cortical demyelination, microglial activation, and behavioral alterations caused by CHIMERA.
  • A GSK219 pretreatment markedly reduced cortical demyelination 24h after CHIMERA.
  • the dMBP staining signals are in red in merged images (left) and inverted in grayscale images (right), YFP in green, and Hoechst in blue.
  • B GSK219 pretreatment eliminated CD68 signals in the cortex and EC 24h after CHIMERA. The CD68 signals are in red in merged images and inverted in grayscale images (middle). Confocal images are on the right.
  • mice The total travel distance of mice in CHIMERA with vehicle or GSK219 pretreatment.
  • F Summary of spontaneous 8 Attorney Docket No. 103361 ⁇ 611WO1 alternation (%) in the Y ⁇ maze test at different time points in CHIMERA with vehicle or GSK219 pretreatment. Unpaired t ⁇ test: *, p ⁇ 0.05. Scale bars, 250 ⁇ m in (A) and (B) left; 30 ⁇ m in (B) right.
  • Figure 14 depicts GSK219 post ⁇ treatment reduced axonal varicosity level and cortical demyelination but not microglial activation 24h after CHIMERA.
  • C Low mag (left) and confocal (right) images of the mouse cortex 24h after CHIMERA with vehicle post ⁇ treatment. The dMBP staining signals are in red in merged images and inverted in gray ⁇ scale images.
  • D Images 24h after CHIMERA with GSK219 post ⁇ treatment.
  • GSK219 post ⁇ treatment did not inhibit microglial activation revealed by increased CD68 staining signals.
  • the CD68 staining signals are in red in the merged image and inverted in the gray ⁇ scale image. Scale bars, 200 ⁇ m in (A), (C) left, (D) left, and (E); 20 ⁇ m in (B), (C) right, and (D) right.
  • F The effect of GSK219 post ⁇ treatment on the EPM result, the percentage of open ⁇ arm time, in CHIMERA. Mouse numbers are provided in the chart.
  • G The effect of GSK219 post ⁇ treatment on the rotarod test (the 5th and last trial), Latency to fall, in CHIMERA.
  • FIG. 16 depicts upregulated proteins in the membrane protein complex and mitochondrial protein complex. Upregulated proteins in the top two GO categories are the membrane protein complex and mitochondrial protein complex. Individual proteins are shown in pink ovals and GO categories are in green rectangles.
  • FIG. 17 depicts upregulation of Cav2.1 proteins in the brain of TRPV4 ⁇ / ⁇ mice revealed by immunostaining.
  • A Anti ⁇ Cav2.1 staining signals in hippocampal CA3 and dentate gyrus, the cortex, and the corpus callosum of WT (TRPV4 +/+ ;Thy1 ⁇ YFP) mice.
  • Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes ⁇ from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. [0012] “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
  • salts are acid addition salts formed with inorganic acids, for example, hydrochloric, hydrobromic, sulfuric, phosphoric, and nitric acids and the like; salts formed with organic acids such as acetic, oxalic, tartaric, succinic, maleic, fumaric, gluconic, citric, malic, methanesulfonic, p ⁇ toluenesulfonic, napthalenesulfonic, and polygalacturonic acids, and the like; salts formed from elemental anions such as chloride, bromide, and iodide; salts formed from metal hydroxides, for example, sodium hydroxide, potassium hydroxide, calcium hydroxide, lithium hydroxide, and magnesium hydroxide; salts formed from metal carbonates, for example, sodium carbonate, potassium carbonate, calcium carbonate, and magnesium carbonate; salts formed from metal bicarbonates, for example, sodium bicarbonate and potassium bicarbonate; salts formed from metal sulfates,
  • alkyl refers to a radical of a straight ⁇ chain or branched hydrocarbon group having a specified range of carbon atoms (e.g., a "C 1 ⁇ 16 alkyl” can have from 1 to 16 carbon atoms).
  • An alkyl group can be a saturated alkyl group or an unsaturated alkyl group, i.e., an alkyl group having one or more carbon ⁇ carbon double/triple bonds, i.e., an alkenyl or alkynyl group.
  • an “alkyl” group includes both saturated alkyl groups and unsaturated alkyl groups.
  • heteroalkyl refers to an alkyl group, which further includes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, nitrogen, or sulfur within (i.e., inserted between adjacent carbon atoms of) and/or placed at one or more terminal position(s) of the parent chain.
  • a heteroC 1 ⁇ 6 alkyl (which may also be designated a C 1 ⁇ 6 heteroalkyl) group includes, but is not limited to, the following structures: . bonded through the specified heteroatom.
  • a OC 1 ⁇ 6 heteroalkyl group includes, but it not limited to, the following structures: 11 Attorney Docket No. 103361 ⁇ 611WO1 .
  • Alkylene is the polyvalent moiety of alkyl
  • alkenylene is the divalent moiety of alkenyl
  • alkynylene is the divalent moiety of alkynyl
  • heteroalkylene is the divalent moiety of heteroalkyl
  • heteroalkenylene is the divalent moiety of heteroalkenyl
  • heteroalkynylene is the divalent moiety of heteroalkynyl
  • carbocyclylene is the divalent moiety of carbocyclyl
  • heterocyclylene is the divalent moiety of heterocyclyl
  • arylene is the divalent moiety of aryl
  • heteroarylene is the divalent moiety of heteroaryl (each of which parent groups as defined herein).
  • alkoxy refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom.
  • aryl refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 ⁇ electrons shared in a cyclic array) having 6 ⁇ 14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“C 6 ⁇ 14 aryl").
  • Aryl also includes ring systems wherein the aryl ring, as defined above, is fused with one or more carbocyclyl or heterocyclyl groups wherein the radical or point of attachment is on the aryl ring, and in such instances, the number of carbon atoms continue to designate the number of carbon atoms in the aryl ring system.
  • each instance of an aryl group is independently unsubstituted (an “unsubstituted aryl") or substituted (a "substituted aryl”) with one or more substituents.
  • alkyl is a subset of “alkyl” and refers to an alkyl group substituted by an aryl group, wherein the point of attachment is on the alkyl moiety.
  • heteroaryl refers to a radical of a 5 ⁇ 14 membered monocyclic or polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 ⁇ electrons shared in a cyclic array) having ring carbon atoms and 1 ⁇ 4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5 ⁇ 14 membered heteroaryl").
  • Heteroaryl also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused polycyclic (aryl/heteroaryl) ring system.
  • Polycyclic heteroaryl groups wherein one ring does not contain a heteroatom e.g., indolyl, quinolinyl, carbazolyl, and the like
  • the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2 ⁇ indolyl) or the ring that does not contain a heteroatom (e.g., 5 ⁇ indolyl).
  • heteroaryl and heterocyclyl rings include: benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, chromenyL cirrnolinyl, decahydroquinolinyl, 2H,6H ⁇ 1,5,2 ⁇ dithiazinyl, dihydrofuro[2,3 b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, lH ⁇ indazolyl, indolenyl, indolinyl, ind
  • alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl groups defined herein (and the “ene” versions of said groups) may be substituted or unsubstituted.
  • a substituted group includes a non ⁇ hydrogen substituent at a position where in the unsubstituted version a hydrogen atom would be found.
  • Substituents include, but are not limited to, halogen, hydroxy, alkyl, alkoxy, nitro, cyano, oxo, carbocyclyl, carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl, 13 Attorney Docket No.
  • traumatic brain injury is a mild traumatic brain injury, moderate traumatic brain injury, or severe traumatic brain injury.
  • the traumatic brain injury is a concussive brain injury.
  • methods of reducing physical symptoms following a traumatic brain injury in a subject can be used to reduce nausea, vomiting, dizziness, balance problems, headaches, light sensitivity, impaired memory, sleep abnormalities, impaired concentration, impaired vision, or a combination thereof in a subject following traumatic brain injury.
  • the TRPV4 antagonist may be administered until the traumatic brain injury symptoms have resolved.
  • the TRPV4 antagonist is administered to the subject prior to the subject undertaking an activity at risk of receiving a traumatic brain injury.
  • the TRPV4 antagonist can be administered to a subject prior to participating in a contact sport (football, American football, basketball, boxing, martial arts, wrestling, water polo, baseball/softball, lacrosse, hockey (field or ice) rugby, volleyball, handball, gymnastics, cheerleading, etc).
  • the TRPV4 antagonist can be administered to a warfighter prior to engaging in combat or combat training.
  • the TRPV4 antagonist can be administered to a subject expected to be in proximity to a blast or other shockwave (for example in demolitions, mining, rocketry, etc.). 15 Attorney Docket No. 103361 ⁇ 611WO1 [0020] In some implementations, the TRPV4 antagonist is administered to a subject experiencing post ⁇ concussion syndrome, defined herein as the persistence of concussion symptoms three months or more following the original concussive event. [0021] In some implementations the TRPV4 antagonist can be administered in combination with one or more additional therapies for traumatic brain injury to the subject. In some implementations the TRPV4 antagonist can be administered in combination with acetaminophen.
  • the TRPV4 antagonist can be administered in combination with an antihistamine, for example diphenhydramine, cyclizine, dimenhydrinate, doxylamine, mirtazapine, meclizine, promethazine, or hydroxyzine.
  • an antihistamine for example diphenhydramine, cyclizine, dimenhydrinate, doxylamine, mirtazapine, meclizine, promethazine, or hydroxyzine.
  • the TRPV4 antagonist can be administered in combination with medicine for headache, for example botulinum toxin (BOTOX®), topiramate, triptans such as sumatriptan, rizatriptan, naratriptan, eletriptan, donitriptan, almotriptan, frovatriptan, avitriptan, zolmitriptan, LY ⁇ 334370, or L ⁇ 694247.
  • BOTOX® botulinum toxin
  • triptans such as sumatrip
  • the TRPV4 antagonist can be administered in combination with an anti ⁇ nausea medication (anti ⁇ emetic), for example a 5 ⁇ HT 3 antagonist list dolasetron, granisetron, ondansetron, tropisetron, palonosetron, a dopamine antagonist like amisulpride, domperidone, droperidol, olanzapine, haloperidol, alizapride, prochlorperazine, chlorpromazine, metoclopramide, an NK1 receptor antagonist like aprepitant, casopitant, or rolapitant.
  • an anti ⁇ nausea medication for example a 5 ⁇ HT 3 antagonist list dolasetron, granisetron, ondansetron, tropisetron, palonosetron, a dopamine antagonist like amisulpride, domperidone, droperidol, olanzapine, haloperidol, alizapride, prochlorperazine, chlorpromazine, metoclopr
  • the TRPV4 antagonist comprises GSK3395879, GSK3527497, GSK205, GSK3491943, GSK1016790A, GSK2798745, GSK2193874, HC ⁇ 067047, RN ⁇ 1734, RN ⁇ 1747, RN ⁇ 9893, PF ⁇ 05214030, rosmarinic acid or rosmarinic acid derivative, or a combination thereof.
  • the TRPV4 antagonist has the formula: 16 Attorney Docket No. 103361 ⁇ 611WO1 Attorney Docket No.
  • the TRPV4 antagonist is a compound having the formula: , L 1 is CR a R b ; L 2 is CR c R d ; 18 Attorney Docket No.
  • R a is H, halo, cyano, (C 1 ⁇ C 5 )alkyl, (C 1 ⁇ C 5 )alkoxy, (C 3 ⁇ C 5 )cycloalkyloxy, or (C 3 ⁇ C 5 )cycloalkyl, wherein any (C 1 ⁇ C 5 )alkyl, (C 1 ⁇ C 5 )alkoxy, (C 3 ⁇ C 5 )cycloalkyloxy, and (C 3 ⁇ C 5 )cycloalkyl is optionally substituted with one or more fluoro;
  • R b is H, halo, cyano, (C 1 ⁇ C 5 )alkyl, (C 1 ⁇ C 5 )alkoxy, or (C 3 ⁇ C 5 )cycloalkyl, wherein any (C 1 ⁇ C 5 )alkyl, (C 1 ⁇ C 5 )alkoxy, and (C 3 ⁇ C 5 )cycloalkyl is optional
  • the TRPV4 antagonist is a compound having the formula: 19 Attorney Docket No. 103361 ⁇ 611WO1 , R 1 is hydrogen, C 1 ⁇ 3 alkyl, CH 2 OH, CH 2 —O—CH 3 , CH 2 OCH 2 Ph, CH 2 CN, CN, halo or C(O)OCH 3 ; R 2 is independently hydrogen, CN, CF 3 , halo, SO 2 C 1 ⁇ 3 alkyl, C 1 ⁇ 3 alkyl or C ⁇ CH; R 3 is hydrogen, C 1 ⁇ 2 alkyl, CF 3 or OH; R 4 is hydrogen, halo or C 1 ⁇ 3 alkyl; X is CR 4 or N; A is (CH 2 ) n ⁇ Het; or A is (CH 2 ) n —(CR a R b )—(CH 2 ) m ⁇ Het; R a is hydrogen or C 1 ⁇ 3 alkyl, wherein the C 1 ⁇ 3 alkyl may
  • the TRPV4 antagonist is 1 ⁇ ( ⁇ (5S,7S) ⁇ 3 ⁇ [3 ⁇ (1,1 ⁇ dimethylethyl) ⁇ 5 ⁇ isoxazolyl] ⁇ 7 ⁇ methyl ⁇ 2 ⁇ oxo ⁇ 1 ⁇ oxa ⁇ 3 ⁇ azaspiro[4.5]dec ⁇ 7 ⁇ yl ⁇ methyl) ⁇ 1H ⁇ benzimidazole ⁇ 6 ⁇ carbonitrile; 1 ⁇ (((5S,7S) ⁇ 3 ⁇ (3 ⁇ (2 ⁇ cyanopropan ⁇ 2 ⁇ yl)isoxazol ⁇ 5 ⁇ yl) ⁇ 7 ⁇ methyl ⁇ 2 ⁇ oxo ⁇ 1 ⁇ oxa ⁇ 3 ⁇ azaspiro[4.5]decan ⁇ 7 ⁇ yl)methyl) ⁇ 1H ⁇ benzo[d]imidazole ⁇ 6 ⁇ carbonitrile; 1 ⁇ ( ⁇ (5S,7S) ⁇ 3 ⁇ [5 ⁇ (1,1 ⁇ dimethylethyl) ⁇ 3 ⁇ isoxazolyl] ⁇ 7 ⁇ methyl ⁇ 2 ⁇ oxo ⁇ 1
  • R 4 is C 1 ⁇ C 3 alkyl or C 1 ⁇ C 3 haloalkyl
  • R 3a and each R 3b are independently selected from hydroxy, C 1 ⁇ C 8 alkyl, C 3 ⁇ C 6 cycloalkyl, a 5 ⁇ to 6 ⁇ membered heterocycle, ⁇ N(R 5 )(S(O) 2 R 6 ), ⁇ N(R 5 )C(O)OR 6 , and C 1 ⁇ C 6 alkoxy
  • the C 1 ⁇ C 8 alkyl is optionally substituted with up to three substituents selected from hydroxy, halo, oxo, carboxy, C 1 ⁇ C 3 alkoxy, ⁇ S(O) 2 R 6 , ⁇ S(O) 2 NH 2 , ⁇ C(O)OR 6 , and ⁇ NR 5 R 6
  • the 5 ⁇ to 6 ⁇ membered heterocycle is optionally substituted with up to three substituents selected from oxo
  • 103361 ⁇ 611WO1 which they are attached, may form a 3 to 8 membered ring which may contain 0 to 4 heteroatoms independently selected from oxygen, sulfur, and nitrogen; wherein the said 3 to 8 membered ring is optionally substituted with 1 to 6 substituents independently selected from the group consisting of: halogen, hydroxyl, (C 1 ⁇ C 6 )alkyl, (C 1 ⁇ C 6 )haloalkyl, —SO 2 (C 1 ⁇ C 6 )alkyl, —SO 2 (C 1 ⁇ C 6 )haloalkyl, —C( ⁇ O)(C 1 ⁇ C 6 )alkyl, and —C( ⁇ O)(C 1 ⁇ C 6 )haloalkyl;
  • R 3 is independently selected from the group consisting of: hydrogen, fluoride, methyl, ethyl, and (C 1 ⁇ C 6 )haloalkyl;
  • X is selected from the group consisting of: a chemical
  • R 4 is independently CF 3 , halo, C 1 ⁇ 3 alkyl or OC 1 ⁇ 3 alkyl;
  • R 5 is independently SO 2 R 1 , NH 2 , NHSO 2 R 1 , NR 1 SO 2 R 1 , C(O) piperazinyl, pyrimidinyl, OH, OCH 2 CH 2 OH, OCH 2 CH 2 OR 1 , OCF 3 , OCH 2 CF 3 , OCH 2 CN, OR 1 or CH 2 R 7 ; wherein or pyrimidinyl may be unsubstituted or substituted with one or two halo, OH, OR 1 or R 1 ; or two adjacent R 5 groups may be combined to form
  • R 6 is independently halo, methyl, or OMe;
  • R 7 is pyrrolidinyl, morpholinyl, or piperidinyl; n is independently 0, 1, or 2; X is N or C; and y is independently 0, 1 or 2 [0031]
  • n
  • the TRPV4 antagonist has the formula: , R 1 is selected from: aryl, aryl substituted from 1 to 4 times by R a , heteroaryl, heteroaryl substituted from 1 to 4 times by R a , bicycloheteroaryl, and bicycloheteroaryl substituted from 1 to 4 times by R a ; R 2 is selected from: aryl, aryl substituted from 1 to 4 times by R b , heteroaryl, heteroaryl substituted from 1 to 4 times by R b , bicycloheteroaryl, and bicycloheteroaryl substituted from 1 to 4 times by R b , and Y 1 is selected from: C 1 ⁇ 6 alkyl, and C 1 ⁇ 6 alkyl substituted with from: 1 to 9 substitutents independently selected from: fluoro, chloro, bromo, iodo, —OC 1 ⁇ 6 alkyl, —OC 1 ⁇ 6 alkyl substituted with from
  • each R a is independently selected from: fluoro, chloro, bromo, iodo, —OH, C 1 ⁇ 6 alkyl, C 1 ⁇ 6 alkyl substituted with from 1 to 5 substituents independently selected from: fluoro, chloro, bromo, iodo, C 1 ⁇ 4 alkoxy, — OH, C 1 ⁇ 4 alkyl, phenyl, oxo, —NO 2 , —NH 2 and —CN, cyano, —OC 1 ⁇ 6 alkyl, —OC 1 ⁇ 6 alkyl substituted with from 1 to 5 substituents independently selected from: fluoro, chloro, bromo, iodo, C 1 ⁇ 4 alkoxy, —OH, C 1 ⁇ 4 alkyl, phenyl, oxo, —NO 2 , —NH 2 and —CN, cyano, —OC 1 ⁇ 6 alkyl, —OC 1 ⁇ 6 alkyl substituted with from 1 to 5 substituents
  • R 1 is selected from: aryl, aryl substituted from 1 to 4 times by R a , heteroaryl, heteroaryl substituted from 1 to 4 times by R a , bicycloheteroaryl, and bicycloheteroaryl substituted from 1 to 4 times by R a .
  • R 2 is selected from: aryl, aryl substituted from 1 to 4 times by R b heteroaryl, heteroaryl substituted from 1 to 4 times by R b bicycloheteroaryl, and bicycloheteroaryl substituted from 1 to 4 times by R b .
  • Y 1 is selected from: C 1 ⁇ 6 alkyl, and C 1 ⁇ 6 alkyl substituted with from: 1 to 9 substitutents independently selected from: fluoro, chloro, bromo, iodo, —OC 1 ⁇ 6 alkyl, —OC 1 ⁇ 6 alkyl substituted with from 1 to 6 substituents independently selected from: fluoro, oxo, —OH, —NH 2 , and —CN, mercapto, —S(O)H, —S(O) 2 H, oxo, hydroxy, amino, —NHR x11 , where R x11 is selected from C 1 ⁇ 6 alkyl, and C 1 ⁇ 6 alkyl substituted with from 1 to 6 substituents independently selected from: fluoro, oxo, —OH, —NH 2 , —CN, —OC 1 ⁇ 5 alkyl, —OC 1 ⁇ 5 alkyl substituted from 1 to 6 times by fluoro and —NH 2
  • 103361 ⁇ 611WO1 Y 1 is taken together with the adjacent —OH to form a heterocyclic ring selected from: morpholinyl, morpholinyl substituted by —CH 3 , and oxazolidin ⁇ 2 ⁇ one.
  • R a is selected from: fluoro, chloro, bromo, iodo, —OH, C 1 ⁇ 6 alkyl, cyano, —CF 3 , —C 1 ⁇ 5 alkylCF 3 , —CHF 2 , — CH 2 F, —OC 1 ⁇ 5 alkyl, —OCF 3 , —OC 1 ⁇ 5 alkylCF 3 , —Ophenyl, —Obenzyl, —C 1 ⁇ 5 alkylCN, —C(O)OC 1 ⁇ 5 alkyl, — C(O)OH, and —Ocycloalkyl; and R b is selected from: fluoro, chloro, bromo, iodo, —OH, C 1 ⁇ 6 alkyl, cyano, —CF 3 , —C 1 ⁇ 5 alkylCF 3 , —CHF 2 , —CH 2 F, —OC 1 ⁇ 5 alkyl, —OCF 3 , —OC 1 ⁇
  • R 1 is selected from: aryl, aryl substituted from 1 to 4 times by R a , heteroaryl, heteroaryl substituted from 1 to 4 times by R a , bicycloheteroaryl, and bicycloheteroaryl substituted from 1 to 4 times by R a .
  • the TRPV4 is a rosmarinic acid derivative having the formula: . additional active ingredients) enterally, parenterally, intranasally, vaginally, by inhalation, or a combination thereof.
  • the TRPV4 antagonist When the TRPV4 antagonist is administered enterally, it may be administered by oral administration, sublingual administration, buccal administration, rectal administration, or a combination thereof.
  • the TRPV4 antagonist may be provided in tablets, capsules (filled with powders, pellets, beads, mini ⁇ tablets, pills, micro ⁇ pellets, small tablet units, multiple unit pellet systems (MUPS), disintegrating tablets, or dispersible tablets.
  • MUPS multiple unit pellet systems
  • Suitable excipients may be used for formulating the dosage forms according to the present invention such as, but not limited to, surface stabilizers or surfactants, viscosity modifying agents, polymers including extended release polymers, stabilizers, disintegrants or super disintegrants, diluents, plasticizers, binders, glidants, lubricants, sweeteners, flavoring agents, anti ⁇ caking agents, opacifiers, anti ⁇ microbial agents, antifoaming agents, emulsifiers, buffering agents, coloring agents, carriers, fillers, anti ⁇ adherents, solvents, taste ⁇ masking agents, preservatives, antioxidants, texture enhancers, channeling agents, coating agents or combinations thereof.
  • the TRPV4 antagonist is administered at a dosage from 1 ⁇ 5,000 mg, from 1 ⁇ 2,500 mg, from 1 ⁇ 1,000 mg, from 1 ⁇ 500 mg, from 1 ⁇ 250 mg, from 1 ⁇ 100 mg, from 1 ⁇ 50 mg, from 10 ⁇ 100 mg, from 25 ⁇ 100 mg, from 50 ⁇ 250 mg, from 100 ⁇ 500 mg, from 100 ⁇ 1,000 mg, from 500 ⁇ 1,000 mg, from 500 ⁇ 2,500 mg, from 1,000 ⁇ 5,000 mg, from 1,000 ⁇ 2,500 mg, or from 2,500 ⁇ 5,000 mg. In some implementations the TRPV4 antagonist is administered at a dosage from 5,000 ⁇ 10,000.
  • the dosage refers to the total amount of TRPV4 antagonist (calculated as the free base in instances where the antagonist is formulated as a salt) that is administered to the subject in a 24 ⁇ hour period.
  • TRPV4 antagonist calculated as the free base in instances where the antagonist is formulated as a salt
  • the dosage refers to the total amount of TRPV4 antagonist (calculated as the free base in instances where the antagonist is formulated as a salt) that is administered to the subject in a 24 ⁇ hour period.
  • EXAMPLES [0037] The following examples are for the purpose of illustration of the invention only and are not intended to limit the scope of the present invention in any manner whatsoever.
  • Mouse line crossing and maintenance [0038] C57BL/6L Thy1 ⁇ YFP ⁇ H transgenic mice with the expression of the yellow fluorescent protein (YFP) in a subset of projection neurons were used in the present and our earlier studies (The Jackson Laboratory Stock # 003782).
  • TRPV4 global KO (TRPV4 ⁇ / ⁇ ) and floxed (TRPV4 fl/fl ) mouse lines were kindly provided by Dr. Hongzhen Hu at Washington University at St Louis.
  • TRPV4 ⁇ / ⁇ mice and TRPV4 fl/fl mice were genotyped.
  • the PCR ⁇ based genotyping strategy was used for maintaining and crossing these mouse lines. All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Ohio State University Institutional Animal Care and Use Committee (IACUC).
  • CHIMERA Closed ⁇ Head Impact Model of Engineered Rotational Acceleration
  • mice are anesthetized with 4% Isoflurane in oxygen and placed in a supine position on a foam/plastic cradle, with a 50 g free ⁇ floating chrome ⁇ coated 40 Attorney Docket No. 103361 ⁇ 611WO1 steel piston under the dorsal surface of the mouse head.
  • the pressure regulator of the accumulator air tank was adjusted to 5.36 psi, resulting in a piston impact on top of the mouse's head (the midpoint between bregma and lambda) at a speed of approximately 5.8 m/s and with an impact energy of 0.9 joules (0.9J).
  • mice were administered subcutaneously with meloxicam (1 mg/kg) and saline (1 mL/100 g body weight) for pain control and hydration. After the head impact, the mice were placed in a warm cage and monitored for recovery of right reflexes. Any mice that did not properly recover in their heart beating and breathing or that exhibited any sign of bleeding were excluded from the analysis.
  • Cardiac perfusion and brain fixation are performed. In brief, anesthesia was induced through intraperitoneal injection of 10 mg/ml Euthasol at 0.5 ml per mouse, and it was ensured the mouse was unresponsive to tail/toe pinches before proceeding. The mouse was then secured in the supine position and an incision was made through the brain to expose the thoracic field. The beating heart was secured with a hemostat, and a needle was immediately inserted into the left ventricle. Next, the right atrium was cut with scissors, and 1x PBS was infused until the fluid exiting the right atrium was entirely clear. The perfusion fluid was then switched to 4% formaldehyde in PBS.
  • the nuclear dye Hoechst was purchased from Vector Laboratories (Catalog #: FL ⁇ 1351–2; Burlingame, CA, USA). Immunofluorescence staining [0048] The immunofluorescence staining protocol was as follows. Each brain slide was incubated in PBS/1% Triton X ⁇ 100 for 1 h at room temperature (RT), then washed and blocked with 2.5% normal goat 43 Attorney Docket No. 103361 ⁇ 611WO1 or donkey serum in washing buffer (PBS/0.02% Triton X ⁇ 100) for another hour at RT. The origin of the blocking serum matches with the secondary antibody host. Primary antibodies were diluted in blocking solution (1:100 or 1:200 depending on the antibody) and applied to each slide, covering each brain slide with parafilm.
  • the slides were incubated overnight at 4°C, then rinsed with washing buffer 7 times for 5 min each at RT.
  • Secondary antibodies were also diluted in blocking solution (1:100 or 1:200 depending on the antibody) and added to each slide, followed by aluminum foil to protect from light.
  • the slides were incubated for 3 h at RT, then washed once with washing buffer. Hoechst was added to the washing buffer (1:10000) and applied to each slide for 10 min at RT, then rinsed 7 times for 5 min each at RT.
  • Tris ⁇ buffered Fluoro ⁇ Gel mounting media Electron Microscopy Sciences, Hatfield, PA, USA) was added and the slides were cover ⁇ slipped with Fisherbrand Microscope Cover Glass.
  • the brain tissue was first homogenized and solubilized in a buffer containing 50 mM Tris ⁇ Cl, pH 7.4, 150 mM NaCl, 1% Triton X ⁇ 100, and complete protease inhibitors at 4°C for 2 h. The resulting mixture was then centrifuged at 10350 rpm for 30 min at 4°C to remove insolubilized materials. The supernatant was resolved in SDS ⁇ PAGE electrophoresis, transferred onto a PVDF membrane, and blotted with a specific antibody, followed by an HRP ⁇ conjugated secondary antibody. An ECL kit was used to detect protein bands.
  • GSK2193874 (GSK219) (Catalog #: 5106; Tocris Bioscience, Bristol, UK) was administered to mice via gavage at a dosage of 20 mg/kg body weight.
  • mice were gavaged with one dose of GSK219 3 h before the head impact in CHIMERA (0.9J). Then the pretreated mice are analyzed using anatomical or behavioral assays at different time points after the impact.
  • mice were gavaged with one dose of GSK219 3 h after the head impact and analyzed at different time points.
  • memantine hydrochloride Sigma Aldrich, M9292, St Louis, MO
  • mice were gavaged with one dose of GSK219 3 h after the head impact and analyzed at different time points.
  • memantine hydrochloride Sigma Aldrich, M9292, St Louis, MO
  • memantine hydrochloride Sigma Aldrich, M9292, St Louis, MO
  • Protein extraction was performed by adding 3 mg of brain tissue to a test tube and digesting it in a solution containing RapiGest or ProteaseMax. The supernatant was then transferred to a new LoBind tube, and the protein concentration was measured using a Thermo Fisher Qubit Fluorometer. Alkylation of 30 ⁇ g of extracted proteins was performed with dithiothreitol (DTT) and iodoacetamide (IAA) and reconstituted with trypsin (Promega). Brain samples were analyzed on a Thermo Scientific Q Exactive Plus High Resolution Accurate Mass (HRAM) mass spectrometer with a C18 Easy ⁇ Spray column (Thermo Scientific).
  • DTT dithiothreitol
  • IAA iodoacetamide
  • the mass spectrometer acquired full ⁇ scan mass spectra from m/z 375 to 1575 at a resolution of 70,000. The top 15 most abundant precursor ions per full MS1 spectrum were selected for fragmentation, and an isolation window of 1.6 m/z was used for fragmentation with a normalized collision energy of 30. Tandem mass spectra (MS2) were acquired at a resolution of 17,500. Peptide and protein identifications were obtained using Thermo Proteome Discoverer software and the Sequest search algorithm against the UniProt human database. Gene Ontology term enrichment is conducted using GOnet(95). 45 Attorney Docket No.
  • AAV9 particles prepared from pAAV ⁇ hSyn ⁇ Cre ⁇ P2A ⁇ dTomato (107738 ⁇ AAV9, Addgene) were injected into the TRPV4 fl/fl (or WT as control) mouse brain with the manufacturer's original Titer ( ⁇ 7 ⁇ 1012 vg/mL) on a stereotaxic apparatus. This virus targets and infects neurons in the mouse brain.
  • the Cre ⁇ Lox system allowed for the conditional knockout of TRPV4 in the infected neurons. The day before the operation, the mice were fed with 30 mg/kg/day ibuprofen drinking water and continued for 3 ⁇ 7 days after the operation.
  • mice were weighed, and ophthalmic lubricant was applied to their eyes to prevent dryness.
  • the mice were anesthetized with a mixture of ketamine (50 ⁇ 100 mg/kg) and xylazine (5 ⁇ 20 mg/kg), and received a first dose of buprenorphine (0.05 ⁇ 0.1 mg/kg).
  • the mouse was mounted on a stereotaxic apparatus. A midline incision (approximately 1 cm) was made in the scalp to expose the skull. The bregma and lambda points were identified, and the left and right sides of the mouse were leveled using a stereotaxic measurement.
  • the target injection point (AP: ⁇ 2.2 mm; ML: ⁇ 1.0 mm; DV: ⁇ 1.0 mm) was determined directly above the skull, and a 1 ⁇ mm ⁇ diameter hole was drilled in the skull at this point.
  • a guide cannula was then implanted through the burr hole into the selected brain region to determine the depth of the injection point.
  • pAAV ⁇ hSyn ⁇ Cre ⁇ P2A ⁇ dTomato ( ⁇ 1 ⁇ 10 ⁇ l) was injected slowly at 0.25 ⁇ L/min using a syringe connected to the guide cannula.
  • TEM Transmission electron microscopy
  • the gray and white matter samples were prepared at the Campus Microscopy and Imaging Facility at The Ohio State University. After fixation, the brain tissue was post ⁇ fixed in 1% osmium tetroxide (Ted Pella, Inc., Redding, CA, USA) and then en bloc stained with 1% uranyl acetate (Ted Pella Inc.). A series of graded ethanol and then 100% acetone were used to fully dehydrate the brain tissue. The fully dehydrated brain tissue was embedded in Eponate 12 epoxy resin (Ted Pella Inc.) to make resin blocks, which were then cut with a Leica EM UC6 ultramicrotome (Leica microsystems, Inc., Deerfield, IL, USA) into thin sections of about 80 nm thickness.
  • Eponate 12 epoxy resin (Ted Pella Inc.)
  • axonal varicosity is defined when its diameter is ⁇ 300% of the diameter of its adjacent axonal shafts.
  • axon diameters are not perfectly uniform with the presence of a low level of varicosities.
  • the baseline of varicosity density is not absolute zero.
  • GSK219 or GSK279 or vehicle was added 20 min before puffing with a 5 ⁇ sec interval in image capture.
  • Sample sizes were chosen based on our previous experiments and publications, as well as related literature in the field. Mice of matching sex and age were allocated into various groups. No formal randomization was conducted. Investigators were blinded to genotype and treatment groups during behavioral analysis.
  • dMBP+ signals in internodes started to increase at 4h after impact, reached the peak level at 24h, slowly declined after 3d, but did not return to the sham level even after 2 months (60d) (Fig. 1D, 1E).
  • TEM transmission electron microscope
  • Microglial activation has been implicated in axonal injury and demyelination in mTBI and other neurological disorders.
  • CHIMERA cortical demyelination induced by CHIMERA.
  • the CD68+ cell density and signal intensity sustained 24h after CHIMERA, and significantly reduced but not completely disappeared after 3 days or 2 months (Fig. 48 Attorney Docket No. 103361 ⁇ 611WO1 2A, 2B).
  • Our results are consistent with a recent meta ⁇ analysis for the activation time course of microglia in various mTBI animal models, in which the earliest time for microglial activation in white matter is 2 hours after a concussive head impact.
  • the activated microglia shown by CD68 staining signals were distributed evenly throughout these brain regions.
  • ⁇ GFAP Glial fibrillary acidic protein, an established marker of astrocytes
  • CD4+ cells were observed in the hippocampus of EAE (experimental autoimmune encephalomyelitis; a well ⁇ established murine model for multiple sclerosis) mice but not the sham or 0.9J CHIMERA mice (Fig. 12F ⁇ H). Therefore, the mTBI model in the present study is unlikely to involve the potential damage caused by innate and adaptive immune responses from the blood.
  • Memantine inhibits microglial activation and cortical demyelination, not axonal varicosities [0063]
  • memantine a noncompetitive blocker for NMDA ⁇ type glutamate receptors to combat glutamate excitotoxicity, which was approved by the FDA for treating Alzheimer’s disease.
  • Memantine treatment was recently shown to inhibit synaptic changes and cognitive dysfunction in a mouse model for high ⁇ frequency subthreshold head impact. This effect is consistent with the notion that NMDA receptor ⁇ mediated excitotoxicity is involved in mTBI.
  • mice were perfused and fixed either 0h (receiving only one dose of memantine) or 24h after a head impact.
  • Memantine pretreatment did not reduce the level of axonal varicosities induced by CHIMERA in the cortex, CC, or EC (Fig. 2C ⁇ 2E).
  • 49 Attorney Docket No. 103361 ⁇ 611WO1 [0064] Interestingly, memantine treatment markedly suppressed microglial activation in both the cortex and white matter CC and EC 24h after head impact (Fig. 2F ⁇ 2H).
  • TRPV4 channel blocker GSK219 was shown orally active and highly selective for the TRPV4 channel and its half ⁇ life in the body is approximately 10 hours.
  • TRPV4 regulates hypotonic morphological changes of retinal microglia and temperature ⁇ dependent motility of microglia in the brain. TRPV4 activity also regulates the proliferation of oligodendrocyte precursor cells (OPCs) but not their differentiation into mature oligodendrocytes.
  • mice with GSK219 posttreatment displayed less axonal varicosities compared to vehicle treatment at 24h, as well as reduced cortical demyelination (Figs. 3K ⁇ 3M and 14A ⁇ 14D).
  • GSK219 posttreatment did not inhibit microglial activation at 24h (Figs. 3N and 14E).
  • GSK219 posttreatment did not reduce time spent in the open arms of EPM at all time points but did significantly increase the latency to fall in rotarod 20d post ⁇ impact and decrease the time to cross a balance beam, suggesting that the posttreatment improved motor coordination, balance, and mobility (Fig. 14F ⁇ H).
  • GSK219 posttreatment did not change the results in the open ⁇ field, NOR, and Y maze tests. Therefore, blocking TRPV4 activation after a concussive head impact increases the recovery of axonal injury and some behavior disturbances.
  • GSK219 has striking beneficial effects in protecting and mitigating brain injuries induced by CHIMERA (0.9 J), it is of paramount importance to verify its target(s).
  • TRPV4 KO mice do not respond to GSK219 and remain vulnerable in mTBI [0069]
  • TRPV4 global KO mice TRPV4 ⁇ / ⁇ mice.
  • TRPV4 ⁇ / ⁇ mice TRPV4 global KO mice.
  • Thy1 ⁇ YFP transgenic mice TRPV4 ⁇ / ⁇ mice with Thy1 ⁇ YFP transgenic mice to label the axons of a subset of projection neurons, to generate TRPV4 ⁇ / ⁇ ;Thy1 ⁇ YFP mice.
  • the GSK219 pretreatment failed to prevent CHIMERA ⁇ induced axon ⁇ glial and behavioral changes in TRPV4 ⁇ / ⁇ mice, confirming that GSK219’s effects on WT mice in CHIMERA were indeed mediated by TRPV4 channels.
  • TRPV4 global deletion did not render significant mTBI resistance in 0.9J CHIMERA. Axonal varicosity formation and behavioral alterations were still clearly found in TRPV4 ⁇ / ⁇ mice after CHIMERA, similar to WT mice. We hypothesized that there might be a compensatory mechanism during development in the absence of TRPV4 channels.
  • Acute deletion of neuronal TRPV4 inhibits mTBI ⁇ induced axonal varicosities and adjacent glial changes
  • CHIMERA To determine whether acute deletion of neuronal TRPV4 channels can prevent axonal varicosity formation and glial changes in CHIMERA, we injected AAV9 ⁇ hSyn ⁇ Cre ⁇ dTomato into the right cortex of TRPV4 fl/fl mice to induce the conditional deletion of TRPV4 channels in infected neurons, or into the right cortex of WT mice as control. One month after injection, these mice were used in CHIMERA (0.9 J). Immediately (0h) or 24h after head impact, axonal varicosity induction was significantly reduced in 52 Attorney Docket No.
  • GSK2798745 inhibits axon ⁇ glial mechanotranduction in WT but not TRPV4 ⁇ / ⁇ mice
  • GSK279 Another blocker, GSK279, which structurally differs from GSK219 (Fig. 6A).
  • Fig. 6A We injected GSK279 into the tail vein of Thy1 ⁇ YFP transgenic mice on the background of WT or TRPV4 ⁇ / ⁇ 1.5 hours before CHIMERA (0.9 J).
  • mice were perfused and fixed either immediately (0h) or 24h after head impact (Fig. 6B).
  • the GSK279 pretreatment markedly decreased the levels of axonal varicosities 0h or 24h after CHIMERA in WT but not TRPV4 ⁇ / ⁇ mouse brains (Fig. 6C ⁇ 6E).
  • Twenty ⁇ four hours after CHIMERA GSK279 pretreatment markedly reduced microglial activation and demyelination reflected by the staining intensities of CD68 and dMBP in the cortex, EC, and CC of WT but not TRPV4 ⁇ / ⁇ mouse brains (Fig. 6D, 6F ⁇ 6H). Therefore, similar to the effects of GSK219 pretreatment (Figs.
  • GSK279 pretreatment also significantly reduces CHIMERA ⁇ induced axon ⁇ glial changes in WT mouse brain, whereas the protective effects are absent when TRPV4 is deleted.
  • our findings provide compelling evidence supporting the key role of TRPV4 in the axon ⁇ glial mechanosensation of the CNS.
  • TRPV4 channel is known to bind multiple signaling and structural molecules, such as protein kinase C and casein kinase substrate in neurons protein 3 (PACSIN3), microtubule ⁇ associated protein 7 (MAP7), inositol triphosphate, ⁇ 2 integrin, and Src tyrosine kinase (63 ⁇ 67).
  • PPSIN3 neurons protein 3
  • MAP7 microtubule ⁇ associated protein 7
  • inositol triphosphate ⁇ 2 integrin
  • Src tyrosine kinase 63 ⁇ 67.
  • the cytoskeleton remodeling of small GTPase RhoA may negatively regulate the activity of TRPV4 channels in axons.
  • RhoA RhoA in the TRPV4 ⁇ mediated formation of axonal varicosities.
  • TRPV4 and RhoA colocalized in their cell bodies and densely intertwined neurites (Fig. 7E).
  • Fig. 7F TRPV4+ and isolated axons were observed and some of them had a low level of RhoA proteins (Fig. 7F).
  • RhoA and RhoB protein levels moderately and markedly increased in TRPV4 ⁇ / ⁇ mouse brains, respectively (Fig.
  • TRPV4 channel proteins were concentrated in puffing ⁇ induced axonal varicosities in cultured CNS neurons.
  • puffing ⁇ induced axonal varicosities did not contain a high level of RhoA proteins (Fig. 7H). Therefore, our in vitro results suggest that TRPV4+/RhoA ⁇ axons are more likely to form varicosities after puffing than TRPV4+/RhoA+ ones, consistent with the heterogeneity of varicosity formation observed in different axons.
  • Gabapentin is an FDA ⁇ approved drug to treat seizures, nerve pain, and restless legs syndrome, despite some side effects including respiratory depression.
  • gabapentin significantly inhibited axonal varicosity induction at both time points in TRPV4 ⁇ / ⁇ mice, as well as in WT mice (Fig. 8B, 8C).
  • Twenty ⁇ four hours (24h) after CHIMERA gabapentin pretreatment markedly reduced microglial activation and cortical demyelination in both TRPV4 ⁇ / ⁇ and WT brains (Fig. 8D ⁇ 8G).
  • gabapentin appeared to have less protective effects for axonal varicosity induction and cortical demyelination at 24h in WT mice than those in TRPV4 ⁇ / ⁇ mice (Fig. 8C, 8G).
  • compositions and methods of the appended claims are not limited in scope by the specific compositions and methods described herein, which are intended as illustrations of a few aspects of the claims and any compositions and methods that are functionally equivalent are intended to fall within the scope of the claims.
  • Various modifications of the compositions and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims.

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Abstract

L'invention concerne des méthodes de traitement de lésions cérébrales traumatiques, notamment des lésions cérébrales traumatiques légères par administration à un sujet en ayant besoin d'un antagoniste de TRPV4. Les méthodes de l'invention peuvent être utilisées pour traiter la commotion et le syndrome post-commotionel.
PCT/US2024/053572 2023-10-30 2024-10-30 Composés et méthodes pour le traitement de lésions cérébrales Pending WO2025096549A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130184316A1 (en) * 2010-07-15 2013-07-18 Andrew Hornstein Methods for diagnosing and treating concussive disorders
US20190091206A1 (en) * 2016-04-07 2019-03-28 Duke University Small molecule dual-inhibitors of trpv4 and trpa1 for sanitizing and anesthetizing
US20190262350A1 (en) * 2012-12-03 2019-08-29 Indiana University Research And Technology Corporation Use of trpv4 antagonists to ameliorate hydrocephalus and related materials and methods
US20210052702A1 (en) * 2014-02-05 2021-02-25 The Regents Of The University Of California Methods of treating mild brain injury
CN111808909B (zh) * 2020-07-14 2021-09-17 中国人民解放军军事科学院军事医学研究院 Trpv4离子通道作为药物靶点在有机磷化合物中毒中的应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130184316A1 (en) * 2010-07-15 2013-07-18 Andrew Hornstein Methods for diagnosing and treating concussive disorders
US20190262350A1 (en) * 2012-12-03 2019-08-29 Indiana University Research And Technology Corporation Use of trpv4 antagonists to ameliorate hydrocephalus and related materials and methods
US20210052702A1 (en) * 2014-02-05 2021-02-25 The Regents Of The University Of California Methods of treating mild brain injury
US20190091206A1 (en) * 2016-04-07 2019-03-28 Duke University Small molecule dual-inhibitors of trpv4 and trpa1 for sanitizing and anesthetizing
CN111808909B (zh) * 2020-07-14 2021-09-17 中国人民解放军军事科学院军事医学研究院 Trpv4离子通道作为药物靶点在有机磷化合物中毒中的应用

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