WO2020051624A1 - Methods and compositions for improving recovery from a brain injury - Google Patents

Methods and compositions for improving recovery from a brain injury Download PDF

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Publication number
WO2020051624A1
WO2020051624A1 PCT/AU2019/050015 AU2019050015W WO2020051624A1 WO 2020051624 A1 WO2020051624 A1 WO 2020051624A1 AU 2019050015 W AU2019050015 W AU 2019050015W WO 2020051624 A1 WO2020051624 A1 WO 2020051624A1
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Prior art keywords
inhibitor
brain injury
antagonist
neurotrophin receptor
certain embodiments
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PCT/AU2019/050015
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French (fr)
Inventor
Xin-fu ZHOU
Mehreen Rahman
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Grophin Pty Ltd
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Priority claimed from AU2018903407A external-priority patent/AU2018903407A0/en
Application filed by Grophin Pty Ltd filed Critical Grophin Pty Ltd
Publication of WO2020051624A1 publication Critical patent/WO2020051624A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present disclosure relates, at least in part, to methods and compositions for improving recovery from brain injuries involving neuronal damage.
  • Neuronal damage is a consequence of many types of brain injury. For example, in cases of cerebral ischemia, such as those arise from stroke or cardia arrest, there may be significant neuronal damage, leading to a variety of short term and long tem effects . Other types of brain injury resulting in neuronal damage also arise as a result of trauma and/or inflammation.
  • the present disclosure is based on the recognition that in a rat photothrombotic ischemic model agents that antagonise ligands of p75 neurotrophin receptors are neuroprotective in cerebral ischemia. As such, inhibitors of p75 neurotrophin activity are neuroprotective in brain injuries involving neuronal damage.
  • Certain embodiments of the present disclosure provide a method of improving recovery from brain injury involving neuronal damage in a subject, the method comprising reducing activity of a p75 neurotrophin receptor in the subject and thereby improving recovery from the brain injury.
  • Certain embodiments of the present disclosure provide a method of improving recovery from a brain injury involving neuronal damage in a subject, the method comprising administering an effective amount of an inhibitor of a p75 neurotrophin receptor to the subject and thereby improving recovery from the brain injury.
  • Certain embodiments of the present disclosure provide use of an inhibitor of p75 neurotrophin receptor for improving recovery from a brain injury involving neuronal damage.
  • Certain embodiments of the present disclosure provide use of an inhibitor of p75 neurotrophin receptor in the preparation of a medicament for improving recovery from a brain injury involving neuronal damage.
  • Certain embodiments of the present disclosure provide a method of treating a brain injury involving neuronal damage in a subject, the method comprising administering an effective amount of an inhibitor of a p75 neurotrophin receptor to the subject and thereby treating the brain injury.
  • Certain embodiments of the present disclosure provide a pharmaceutical composition for treating a brain injury involving neuronal damage, the pharmaceutical composition comprising an effective amount of an inhibitor of a p75 neurotrophin receptor.
  • Certain embodiments of the present disclosure provide a method of treating a subject suffering from a brain injury involving neuronal damage, the method comprising administering to the subject a pharmaceutical composition as described herein.
  • Certain embodiments of the present disclosure provide a method of identifying a therapeutic agent for improving recovery from a brain injury involving neuronal damage, the method comprising determining the ability of an inhibitor of a p75 neurotrophin receptor to improve recovery from a brain injury involving neuronal damage and identifying the inhibitor of a p75 neurotrophin receptor agent as a therapeutic agent for improving recovery from a brain injury involving neuronal damage.
  • Certain embodiments of the present disclosure provide a method of identifying a therapeutic agent for improving recovery from a brain injury involving neuronal damage, the method comprising:
  • identifying a candidate agent as an inhibitor of a p75 neurotrophin receptor identifying a candidate agent as an inhibitor of a p75 neurotrophin receptor; and determining the ability of the inhibitor of a p75 neurotrophin receptor to improve recovery from a brain injury involving neuronal damage,
  • Figure 1 shows localization of proBDNF around the ischemic infarct.
  • B. Rat brain cortex of stroke group at 3d. Scale bar 200 pm
  • Figure 2 shows localization of p75 around the ischemic infarct.
  • B. Rat ipsilateral brain cortex of ischemic group at 3d. Scale bar 200 pm
  • Figure 6 shows cylinder testing performed to assess the deficit in the fine motor function after anti-proBDNF treatment on photothrombotic ischemia after 7d compared to the 7d stroke (without treatment).
  • Ordinary one-way ANOVA using (Tukey's multiple comparisons test) was performed, where *P ⁇ 0.05, ***P ⁇ 0.00l versus 7d stroke (without treatment).
  • Figure 7 shows cylinder testing performed to assess the deficit in the fine motor function after p75ECD-Fc treatment on photothrombotic ischemia after (a) 7d and (b) l4d compared to the 7d stroke.
  • Ordinary one-way ANOVA, using (Tukey's multiple comparisons test) was performed, where * P ⁇ 0.05,“P ⁇ 0.00l versus 7d stroke, and l4d stroke.
  • Figure 8 shows rotarod testing performed to assess the deficit in the forced motor function.
  • A. Forced motor test was used to assess the motor function through the running ability on the speeding rod (r.p.m) after anti-proBDNF treatment on photothrombotic ischemia after 7d compared to the 7d stroke (without treatment).
  • B. Duration of running (sec) on the speeding rod was measured after anti-proBDNF treatment on photothrombotic ischemia after 7d compared to the 7d stroke (without treatment).
  • Figure 9 shows rotarod testing to assess the deficit in the forced motor function.
  • A Forced motor test after p75ECD-Fc treatment on photothrombotic ischemia after 7d compared to the 7d stroke.
  • B Duration of running (sec) on the speeding rod was measured after p75ECD-Fc treatment on photothrombotic ischemia after 7d compared to the 7d stroke.
  • C The running ability on the speeding rod (r.p.m) after p75ECD-Fc treatment on photothrombotic ischemia after l4d compared to the l4d stroke.
  • D The running ability on the speeding rod (r.p.m) after p75ECD-Fc treatment on photothrombotic ischemia after l4d compared to the l4d stroke.
  • Duration of running (sec) on the speeding rod was measured after p75ECD-Fc treatment on photothrombotic ischemia after l4d compared to the l4d stroke.
  • Ordinary one-way ANOVA using (Tukey's multiple comparisons test) was performed, where * P ⁇ 0.05, “P ⁇ 0.00l versus 7d stroke, and l4d stroke respectively.
  • Time to contact the adhesive tape (a) was considered to assess the sensory function and time to remove the adhesive tape (b) was considered to assess the motor function after anti- proBDNF treatment on photothrombotic ischemia at 7d compared to the 7d stroke (without treatment).
  • Figure 14 shows adhesive removal testing for both contralateral and ipsilateral forepaws at 7d.
  • A Time to contact the adhesive tape to assess the sensory function.
  • B time to remove the adhesive tape to assess the motor function of the contralateral forepaw.
  • C Time to contact the adhesive tape to assess the sensory function and
  • D time to remove the adhesive tape to assess the motor function of the ipsilateral forepaw after p75ECD-Fc treatment on photothrombotic ischemia after 7d compared to the 7d stroke.
  • Figure 15 shows adhesive removal testing for both contralateral and ipsilateral forepaws at l4d.
  • A. Time to contact the adhesive tape to assess the sensory function
  • Figure 16 shows the results of cresyl violet staining. Necrotic area, where filled with all inflammatory cell-like cells was measured.
  • Figure 17 shows the results of cresyl violet staining. Necrotic area, where filled with inflammatory cell-like cells was measured.
  • B Site of peri-infarct at lOx magnification.
  • Figure 18 shows Hematoxylin and Eosin staining after anti-proBDNF IgG treatment.
  • A Site of infarct at 4x magnification.
  • B Site of peri-infarct at lOx magnification.
  • Figure 19 shows Hematoxylin and Eosin staining after antiproBDNF treatment.
  • A Site of infarct at 4x magnification.
  • B Site of peri-infarct at lOx magnification.
  • Figure 20 shows TUNNEL staining for apoptotic cells after anti-proBDNF IgG treatment.
  • Figure 21 shows TUNNEL staining for apoptotic cells after antiproBDNF treatment.
  • A Site of infarct at 4x magnification.
  • B Site of infarct at 20x magnification.
  • Figure 22 shows immuno staining of Map2.
  • A Site of infarct at 4x magnification.
  • B Site of infarct at 20x magnification.
  • FIG. 23 shows immuno staining of Map2.
  • A Site of infarct at 4x magnification.
  • B Site of infarct at 20x magnification.
  • Figure 24 shows immuno staining of GFAP.
  • A Site of infarct at 4x magnification.
  • B Site of infarct at 20x magnification.
  • Figure 25 shows immuno staining of GFAP.
  • A Site of infarct at 4x magnification.
  • B Site of infarct at 20x magnification.
  • Figure 26 shows immunostaining of IBA1.
  • A Site of infarct at 4x magnification.
  • B Site of infarct at 20x magnification.
  • Figure 27 shows immuno staining of IBA-l.
  • A Site of infarct at 4x magnification.
  • B Site of infarct at 20x magnification.
  • Figure 28 shows immunoblotting of JNK and JNK(P) signaling enzymes.
  • A B Representative immunoblots of JNK and JNK(P) proteins in the ipsilateral hemisphere after anti-proBDNF treatment on photothrombotic ischemia after 7d compared to the sham group.
  • Figure 29 shows immunoblotting of JNK and JNK(P) signaling enzymes.
  • A Representative immunoblots of JNK and JNK(P) proteins in the ipsilateral hemisphere after p75ECD-Fc treatment on photothrombotic ischemia after 7d compared to the sham group.
  • FIG. 30 shows immunoblotting of PARP signaling enzyme.
  • A Representative immunoblots of full length PARP (PARP(FL)) and cleaved PARP (PARP(CL)) proteins in the ipsilateral hemisphere after anti-proBDNF treatment on photothrombotic ischemia after 7d compared to the sham group.
  • FIG. 31 shows immunoblotting of PARP signaling enzyme.
  • A Representative immunoblots of full length PARP (PARP(FL)) and cleaved PARP (PARP(CL)) proteins in the ipsilateral hemisphere after p75ECD-Fc treatment on photothrombotic ischemia after 7d compared to the sham group.
  • Figure 32 shows immunoblotting of PSD95 post-synaptic protein.
  • A Representative immunoblots of PSD95 in the ipsilateral hemisphere after anti-proBDNF treatment on photothrombotic ischemia after 7d compared to the sham group.
  • Figure 33 shows immunoblotting of PSD95 synpatic protein.
  • A Representative immunoblots of PSD95 in the ipsilateral hemisphere after p75ECD-Fc treatment on photothrombotic ischemia after 7d compared to the sham group.
  • the present disclosure relates generally to methods and compositions for improving recovery from brain injuries involving neuronal damage, and for treating a brain injury.
  • Certain embodiments of the present disclosure provide a method of improving recovery from brain injury involving neuronal damage in a subject.
  • One or more embodiments of the present disclosure are directed to methods and products that have one or more combinations of the following advantages: new methods and/or products for treating a brain injury; new methods and/or products for improving recovering from a treating a brain injury; the new use of a class of agents for treating brain injury and/or for improving the recovery from a brain injury; to address one or more problems, to provide one or more advantages, and/or to provide a commercial alternative.
  • the present disclosure provides a method of improving recovery from brain injury involving neuronal damage in a subject, the method comprising reducing activity of a p75 neurotrophin receptor in the subject and thereby improving recovery from the brain injury.
  • the subject is a human subject.
  • Other types of subjects are contemplated, and it will be understood that veterinary applications of the present disclosure in animals are also contemplated.
  • the subject is suffering from a brain injury.
  • the subject is susceptible to developing a brain injury.
  • the present disclosure relates to methods for improving recovery by reducing the activity of a p75 neurotrophin receptor prior to, concomitant with, and/or after a brain injury has occurred, and that treatment after a brain injury has occurred and prophylactic treatment before a brain injury has occurred are contemplated.
  • the brain injury comprises one or more of an ischemic injury, a traumatic injury, a haemorrhagic injury, and an epileptic injury.
  • Other types of brain injury are contemplated. Methods for identifying brain injuries involving neuronal damage are known in the art.
  • the brain injury is associated with a cerebral occlusion.
  • the brain injury comprises a stroke.
  • Methods for identifying cerebral occlusions such as stroke are known in the art.
  • the improvement in recovery comprises an improvement in motor function. Methods for assessing motor function are known in the art.
  • the improvement in recovery comprises reducing neuronal cell death.
  • Methods for assessing neuronal cell death are known in the art.
  • the improvement in recovery comprises a reduction in size of an infarct associated with the stroke.
  • the method comprises administering to the subject an effective amount of an agent that reduces p75 neurotrophin receptor activity.
  • an agent that reduces p75 neurotrophin receptor activity refers to an agent that directly or indirectly results in a reduction in the activity of a p75 neurotrophin activity, for example so as to cause a decrease in the level of activity, an inhibition of activity, a prevention of activity, a downregulation in the level of activity, a reduction in the ability of the receptor to be stimulated, an alteration in the timing and/or location of activity, or otherwise provide some form of negative control over activity or combinations thereof.
  • the agent may (i) act to directly reduce activity, alter the level of expression of a target, alter localisation of a target, alter signalling, and/or alter timing of function, (ii) act to change the activity of a signalling pathway associated with activation, (iii) act to alter the level and/or the activity of another molecule that regulates a target, such as by competitive/non-competitive binding, or by altering the synthesis, breakdown, and/or localisation of another molecule.
  • Other forms of action are contemplated, and combinations of forms of action are contemplated.
  • agents include a drug, a small molecule, a protein, a polypeptide, a lipid, a carbohydrate, a nucleic acid, an oligonucleotide, a ribozyme, a biologic, a recombinant peptide, an aptamer, a cofactor, a ligand, a ligand mimetic, a receptor, a peptidomimetic, an enzyme, a kinase, a phosphatase, a cytokine, a growth factor, a metal ion, a chelate, an antisense nucleic acid, an inhibitor RNA, a microRNA, a siRNA, an antibody or an antigen binding part thereof, an antibody mimetic, or combinations thereof.
  • Other types of agents are contemplated. It will be appreciated than an agent as described herein also includes a prodrug of the agent, and/or a metabolite of the agent.
  • the agent comprises a drug or small molecule, and/or a pro-drug or a metabolite thereof.
  • the agent comprises a nucleic acid.
  • the agent comprises an antibody and/or a part thereof, such as an antigen binding part thereof or a part of a Fc region.
  • the agent that reduces p75 neurotrophin activity comprises an inhibitor of p75 neurotrophin receptor activity.
  • the inhibitor comprises all or part of the ectodomain of the p75 neurotrophin receptor.
  • the inhibitor comprises all or part of the ectodomain of the p75 neurotrophin receptor fused to a Fc region of an antibody.
  • the p75ECD comprises a p75ECD-Fc fusion.
  • the p75ECD-Fc fusion is a recombinant peptide.
  • the inhibitor comprises a p75ECD-Fc.
  • recombinant p75ECD-Fc may be obtained from Fujian Tiantai Medical Technology, China, or the inhibitor may be produced by a method known in the art.
  • P75 ECD fusions may be produced for example as described in US Patent Application No. 20180170996, which is hereby incorporated by reference in its entirety.
  • Other P75 neurotrophin receptor antagonists are described, for example, in US Josephy-Hemandez et al (2017) Neurobiology of Disease 97:139-155.
  • p75 neurotrophin receptor antagonists examples include THX-B (l,3-diisopropyl-l-[2-(l,3-dimethyl- 2,6-dioxo-l,2,3,6-tetrahydro-purin-7-yl)-acetyl]-urea), THX-A and THX-C (also known as LM11A-2426) as described in Yang et al (2008) PLoS ONE 3: e3604.
  • the inhibitor comprises an agent that binds to a p75 neurotrophin receptor.
  • Methods for assessing binding of agents to a p75 neurotrophin receptor are known in the art.
  • the inhibitor of a p75 neurotrophin receptor comprises a ligand antagonist.
  • the ligand antagonist comprises a ligand mimetic or an antibody or an antigen binding part thereof.
  • the ligand antagonist comprises one or more of a proBDNF antagonist, a proNGF antagonist, a proNT3 antagonist, and a proNT4 antagonist.
  • the ligand antagonist comprises a polypeptide/protein encoding all or part of the soluble extracellular domains of the receptors p75 and/or Sortilin/SORCS2, which are commercially available.
  • the proBDNF antagonist comprises PNS-Rbl, which is commercially available.
  • the ligand antagonist comprises an antibody to proBDNF, proNGF, proNT3 antagonist, or proNT4. Examples of such antibodies are available commercially.
  • the ligand antagonist comprises an inhibitor of the formation of the ligand.
  • the proBDNF antagonist comprises PNS-Rbl, which is commercially available.
  • the reducing of the activity of a p75 neurotrophin receptor in the subject comprises administering to the subject an effective amount of an agent.
  • the method comprises administering to the subject an effective amount of an agent that reduces the activity of a p75 neurotrophin receptor.
  • the term "effective amount” as used herein refers to that amount of an agent that is sufficient to effect therapeutic treatment, when administered to a subject.
  • the effective amount will vary depending upon a number of factors, including for example the specific activity of the agent being used, the severity of the condition, the subject, the age, physical condition, existence of other disease states, nutritional status of the subject and genetic background of the subject.
  • treatment refers to obtaining a desired pharmacologic and/or physiologic effect in terms of improving the condition of the subject, ameliorating, arresting, preventing, managing, suppressing, relieving and/or slowing the progression of one or more symptoms in the subject, a partial or complete stabilization of the subject, a regression of one or more symptoms, or a cure in the subject.
  • the agent is administered to a subject to provide a blood or plasma concentration in the range of 1 mM to 100 mM, 5 pM to 100 pM, 10 pM to 100 pM, 50 pM to 100 pM, 1 pM to 50 pM, 5 pM to 50 pM, 10 pM to 50 pM, 1 pM to 10 pM, 5 pM to 10 pM, or 1 pM to 5 pM.
  • Other ranges are contemplated.
  • the agent is administered to the subject in an amount ranging from one of the following selected ranges: 1 pg/kg to 1000 mg/kg, 1 pg/kg to 100 mg/kg; 1 pg/kg to 10 mg/kg; 1 pg/kg to 1 mg/kg; 1 pg/kg to 100 pg/kg; 1 pg/kg to lOpg/kg; 10 pg/kg to 1000 mg/kg, 10 pg/kg to 100 mg/kg; 10 pg/kg to 10 mg/kg; 10 pg/kg to 1 mg/kg; 10 pg/kg to 100 pg/kg; 10 pg/kg to 1000 mg/kg, 100 pg/kg to 100 mg/kg; 100 pg/kg to 10 mg/kg; 100 pg/kg to 10 mg/kg; 100 pg/kg to 1 mg/kg; 1 mg/kg to 10 mg/kg; 10 mg/kg to 1000 mg/kg, 100 pg/kg to 100 mg/kg; 100
  • a suitable administration period may be selected.
  • the agent is administered to the subject for a period of at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 2 weerks, at least 3 weeks, or at least 4 weeks.
  • the administration is for a continuous period. Other administration periods are contemplated.
  • the agent is administered to the subject in an amount ranging from one of the following selected ranges: 1 mg/kg/day to 1000 mg/kg/day, 1 mg/kg/day to 100 mg/kg/day; 1 mg/kg/day to 10 mg/kg/day; 1 mg/kg/day to 1 mg/kg/day; 1 mg/kg/day to 100 mg/kg/day; 1 mg/kg/day to 10mg/kg/day; 10 mg/kg/day to 1000 mg/kg/day, 10 mg/kg/day to 100 mg/kg/day; 10 mg/kg/day to 10 mg/kg/day; 10 mg/kg/day to 1 mg/kg/day; 10 mg/kg/day to 100 mg/kg/day; 10 mg/kg/day to 1000 mg/kg/day, 100 mg/kg/day to 100 mg/kg/day; 100 mg/kg/day to 10 mg/kg/day; 100 mg/kg/day to 1 mg/kg/day; 1 mg/kg/day to 1000 mg/kg/day,
  • the agent may be administered to the subject in a suitable form.
  • administering or “providing” includes administering the agent, or administering a prodrug, or a derivative, that will form an effective amount of the desired agent within the body of the subject.
  • routes of administration that are systemic (e.g., via injection such as intravenous injection, orally in a tablet, pill, capsule, or other dosage form useful for systemic administration), and topical (e.g., creams, solutions, pastes, ointment, including solutions such as mouthwashes, for topical oral administration). Other routes of administration are contemplated.
  • the agent is administered intraventricularly, intracranialy or by intramuscular injection. In certain embodiments, the agent is administered orally. In certain embodiments, the agent is administered intravenously. In certain embodiments, the agent is administered intraperitoneally. In certain embodiments, the agent is administered intravenously. In certain embodiments, the agent is administered via injection, such as intravenous injection. In certain embodiments, the agent is administered by nebulized administration, by aerosolized administration or by being instilled into the lung. In certain embodiments, the agent is administered by implant. In certain embodiments, the agent is administered by subcutaneous injection, intra-articularly, rectally, intranasally, intraocularly, vaginally, or transdermally. Methods of administration are known in the art.
  • Intravenous administration is the administration of substances directly into a vein.
  • Oral administration is a route of administration where a substance is taken through the mouth, and includes buccal, sublabial and sublingual administration, as well as enteral administration.
  • Typical forms for the oral administration of therapeutic agents includes the use of tablets or capsules.
  • the agent may be administered alone or may be delivered in a mixture with other therapeutic agents and/or agents that, for example, enhance, stabilise or maintain the activity of the agent.
  • an administration vehicle e.g., pill, tablet, implant, injectable solution, etc.
  • the methods as described herein may also include combination therapy.
  • the subject may be treated or given another drug or treatment modality in conjunction with the agent as described herein.
  • This combination therapy can be sequential therapy where the subject is treated first with one and then the other, or the two or more treatment modalities are given simultaneously.
  • Co-administering or “co-administration” refers to the administration of two or more therapeutic agents together at one time.
  • the two or more therapeutic agents can be co-formulated into a single dosage form or “combined dosage unit”, or formulated separately and subsequently combined into a combined dosage unit, typically for intravenous administration or oral administration.
  • the therapeutically effective dosage of an agent may vary depending upon the particular agent utilized, the mode of administration, the condition, and severity thereof, as well as the various physical factors related to the subject being treated.
  • the dosages are expected to vary with route of administration, and the nature of the agent administered and any other agents administered.
  • the methods comprise administering to the subject escalating doses of the agent and/or repeated doses.
  • the agent is administered as a continuous release formulation.
  • the agent is administered as an immediate release formulation.
  • immediate release formulation is a formulation which is designed to quickly release a therapeutic agent in the body over a shortened period of time. Immediate release formulations are known in the art.
  • the agent is administered as a sustained release formulation.
  • sustained release formulation is a formulation which is designed to slowly release a therapeutic agent in the body over an extended period of time. Sustained release formulations are known in the art.
  • the agent is administered as a pharmaceutically acceptable salt.
  • pharmaceutically acceptable salt refers to acid addition salts or metal complexes which are commonly used in the pharmaceutical industry. Metal complexes include zinc, iron, and the like.
  • Suitable acids for use in the preparation of pharmaceutically acceptable salts include, but are not limited to, acetic acid, 2,2-dichloroacetic acid, acylated amino acids, adipic acid, alginic acid, ascorbic acid, L-aspartic acid, benzenesulfonic acid, benzoic acid, A- acetamidobenzoic acid, boric acid, (+)-camphoric acid, camphorsulfonic acid, (+)-(lS)- camphor- lO-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, cyclohexanesulfamic acid, dodecylsulfuric acid, ethane- l,2-disulfonic acid, ethanesulfonic acid, 2-hydroxy-ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid,
  • Suitable bases for use in the preparation of pharmaceutically acceptable salts including, but not limited to, inorganic bases, such as magnesium hydroxide, calcium hydroxide, potassium hydroxide, zinc hydroxide, or sodium hydroxide; and organic bases, such as primary, secondary, tertiary, and quaternary, aliphatic and aromatic amines, including L-arginine, benethamine, benzathine, choline, deanol, diethanolamine, diethylamine, dimethylamine, dipropylamine, diisopropylamine, 2- (diethylamino)-ethanol, ethanolamine, ethylamine, ethylenediamine, isopropylamine, N-methyl-glucamine, hydrabamine, IH- imidazole, L-lysine, morpholine, 4-(2- hydroxyethyl)-morpholine, methylamine, piperidine, piperazine, propylamine, pyrrolidine, l-
  • the administration comprises systemic administration.
  • the administration comprises intraventricular, intracranial or intramuscular administration.
  • the method is used to treat a brain injury in a subject.
  • the method is used to reduce the appearance and/or progression of symptoms associated with a brain injury. [00110] In certain embodiments, the method is used to provide an early intervention in a subject presenting with a brain injury.
  • the present disclosure provides a method of improving recovery from a brain injury involving neuronal damage in a subject, the method comprising administering an effective amount of an inhibitor of a p75 neurotrophin receptor to the subject and thereby improving recovery from the brain injury.
  • Brain injuries are as described herein.
  • the brain injury comprises one or more of an ischemic injury, a traumatic injury, a haemorrhagic injury, and an epileptic injury.
  • the brain injury is associated with a cerebral occlusion.
  • the brain injury comprises a stroke.
  • Improvements in recovery are as described herein.
  • the improvement in recovery comprises an improvement in motor function and/or reducing neuronal cells death.
  • the improvement in recovery comprises a reduction in size of an infarct associated with the stroke.
  • Inhibitors of a p75 neurotrophin receptor are as described herein.
  • the inhibitor comprises all or part of the ectodomain of the p75 neurotrophin receptor.
  • the inhibitor comprises all or part of the ectodomain of the p75 neurotrophin receptor fused to a Fc region of an antibody.
  • the p75ECD comprises a p75ECD-Fc fusion.
  • the p75ECD-Fc fusion is a recombinant peptide.
  • the inhibitor comprises a p75ECD-Fc.
  • recombinant p75ECD-Fc may be obtained from Fujian Tiantai Medical Technology, China.
  • P75 neurotrophin receptor antagonists are described, for example, in Other P75 neurotrophin receptor antagonists are described, for example, in Josephy-Hemandez el al (2017) Neurobiology of Disease 97:139-155.
  • Examples of p75 neurotrophin receptor antagonists include THX-B (l,3-diisopropyl-l-[2-(l,3-dimethyl-2,6-dioxo-l, 2,3,6- tetrahydro-purin-7-yl)-acetyl]-urea), THX-A and THX-C (also known as FM11A-2426) as described in Yang et al (2008) PLoS ONE 3: e3604.
  • the inhibitor comprises an agent that binds to a p75 neurotrophin receptor.
  • Methods for assessing binding of agents to a p75 neurotrophin receptor are known in the art.
  • the inhibitor of a p75 neurotrophin receptor comprises a ligand antagonist.
  • the ligand antagonist comprises a ligand mimetic or an antibody or an antigen binding part thereof.
  • the ligand antagonist comprises a ligand mimetic or an antibody or an antigen binding part thereof.
  • the ligand antagonist comprises one or more of a proBDNF antagonist, a proNGF antagonist, a proNT3 antagonist, and a proNT4 antagonist.
  • the proBDNF antagonist comprises PNS-Rbl, which is commercially available.
  • the ligand antagonist comprises an antibody to proBDNF, proNGF, proNT3 antagonist, or proNT4. Examples of such antibodies are available commercially.
  • the ligand antagonist comprises a polypeptide/protein encoding all or part of the soluble extracellular domains of the receptors p75 and/or Sortilin/SORCS2, which are commercially available.
  • the ligand antagonist comprises an inhibitor of the formation of the ligand.
  • the proBDNF antagonist comprises PNS-Rbl, which is commercially available.
  • the administration comprises systemic administration.
  • Certain embodiments of the present disclosure provide use of an inhibitor of p75 neurotrophin receptor for improving recovery from a brain injury involving neuronal damage.
  • Certain embodiments of the present disclosure provide use of an inhibitor of p75 neurotrophin receptor in the preparation of a medicament for improving recovery from a brain injury involving neuronal damage.
  • Certain embodiments of the present disclosure provide a method of treating a brain injury involving neuronal damage in a subject.
  • the present disclosure provides a method of treating a brain injury involving neuronal damage in a subject, the method comprising administering an effective amount of an inhibitor of a p75 neurotrophin receptor to the subject and thereby treating the brain injury.
  • the administration comprises systemic administration. In certain embodiments, the administration comprises intraventricular, intracranial or intramuscular administration.
  • the method is used to recover from a brain injury.
  • the method is used to reduce the appearance and/or progression of symptoms associated with a brain injury.
  • the method is used to provide an early intervention in a subject presenting with a brain injury.
  • the subject is suffering from a brain injury. In certain embodiments, the subject is recovering from a brain injury.
  • the subject is susceptible to a brain injury.
  • the method is a prophylactic method.
  • Brain injuries are as described herein.
  • the brain injury comprises one or more of an ischemic injury, a traumatic injury, a haemorrhagic injury, and an epileptic injury.
  • the brain injury is associated with a cerebral occlusion.
  • the brain injury comprises a stroke.
  • Inhibitors of a p75 neurotrophin receptor are as described herein.
  • the inhibitor comprises all or part of the ectodomain of the p75 neurotrophin receptor.
  • the inhibitor comprises all or part of the ectodomain of the p75 neurotrophin receptor fused to a Fc region of an antibody.
  • the p75ECD comprises a p75ECD-Fc fusion.
  • the p75ECD-Fc fusion is a recombinant peptide.
  • the inhibitor comprises a p75ECD-Fc.
  • recombinant p75ECD-Fc may be obtained from Fujian Tiantai Medical Technology, China.
  • P75 neurotrophin receptor antagonists are described, for example, in Other P75 neurotrophin receptor antagonists are described, for example, in Josephy-Hemandez el al (2017) Neurobiology of Disease 97:139-155.
  • p75 neurotrophin receptor antagonists examples include THX-B (l,3-diisopropyl-l-[2-(l,3-dimethyl-2,6-dioxo-l, 2,3,6- tetrahydro-purin-7-yl)-acetyl]-urea), THX-A and THX-C (also known as LM11A-2426) as described in Yang et al (2008) PLoS ONE 3: e3604.
  • the inhibitor comprises an agent that binds to a p75 neurotrophin receptor.
  • Methods for assessing binding of agents to a p75 neurotrophin receptor are known in the art.
  • the inhibitor of a p75 neurotrophin receptor comprises a ligand antagonist.
  • the ligand antagonist comprises a ligand mimetic or an antibody or an antigen binding part thereof.
  • the ligand antagonist comprises a ligand mimetic or an antibody or an antigen binding part thereof.
  • the ligand antagonist comprises one or more of a proBDNF antagonist, a proNGF antagonist, a proNT3 antagonist, and a proNT4 antagonist.
  • the proBDNF antagonist comprises PNS-Rbl, which is commercially available.
  • the ligand antagonist comprises an antibody to proBDNF, proNGF, proNT3 antagonist, or proNT4. Examples of such antibodies are available commercially.
  • the ligand antagonist comprises a polypeptide/protein encoding all or part of the soluble extracellular domains of the receptors p75 and/or Sortilin/SORCS2, which are commercially available. [00161] In certain embodiments, the ligand antagonist comprises an inhibitor of the formation of the ligand.
  • the proBDNF antagonist comprises PNS-Rbl, which is commercially available.
  • Certain embodiments of the present disclosure provide a pharmaceutical composition.
  • the present disclosure provides a pharmaceutical composition for treating a brain injury involving neuronal damage, the pharmaceutical composition comprising an effective amount of an inhibitor of a p75 neurotrophin receptor.
  • Inhibitors of a p75 neurotrophin receptor are as described herein.
  • the inhibitor comprises all or part of the ectodomain of the p75 neurotrophin receptor.
  • the inhibitor comprises all or part of the ectodomain of the p75 neurotrophin receptor fused to a Fc region of an antibody.
  • the p75ECD comprises a p75ECD-Fc fusion.
  • the p75ECD-Fc fusion is a recombinant peptide.
  • the inhibitor comprises a p75ECD-Fc.
  • recombinant p75ECD-Fc may be obtained from Fujian Tiantai Medical Technology, China.
  • P75 neurotrophin receptor antagonists are described, for example, in Josephy- Hernandez et al (2017) Neurobiology of Disease 97:139-155.
  • Examples of p75 neurotrophin receptor antagonists include THX-B (l,3-diisopropyl-l-[2-(l, 3-dimethyl- 2,6-dioxo-l,2,3,6-tetrahydro-purin-7-yl)-acetyl]-urea), THX-A and THX-C (also known as LM11A-2426) as described in Yang et al (2008) PLoS ONE 3: e3604.
  • the inhibitor comprises an agent that binds to a p75 neurotrophin receptor.
  • Methods for assessing binding of agents to a p75 neurotrophin receptor are known in the art.
  • the inhibitor of a p75 neurotrophin receptor comprises a ligand antagonist.
  • the ligand antagonist comprises a ligand mimetic or an antibody or an antigen binding part thereof.
  • the ligand antagonist comprises one or more of a proBDNF antagonist, a proNGF antagonist, a proNT3 antagonist, and a proNT4 antagonist.
  • the proBDNF antagonist comprises PNS-Rbl, which is commercially available.
  • the ligand antagonist comprises an antibody to proBDNF, proNGF, proNT3 antagonist, or proNT4. Examples of such antibodies are available commercially.
  • the ligand antagonist comprises a polypeptide/protein encoding all or part of the soluble extracellular domains of the receptors p75 and/or Sortilin/SORCS2, which are commercially available.
  • the ligand antagonist comprises an inhibitor of the formation of the ligand.
  • the proBDNF antagonist comprises PNS-Rbl, which is commercially available.
  • the composition comprises a liquid composition, a semi-solid composition, a suppository, a gel, a solid, a tablet, a capsule, a cream, a solution, a paste, or an ointment.
  • the composition comprises a substrate with a releasable agent that reduces spinal glial activation.
  • compositions for administration by various routes are known in the art.
  • a suitable dosage of the agent may be selected.
  • the composition comprises the agent that reduces spinal glial activation in an amount ranging from 1 pg to 500 mg, 10 pg to 500 mg, 10 pg to 100 mg, 10 pg to 10 mg, 10 pg to 1 mg, 10 pg to 100 pg, 100 pg to 500 mg, 100 pg to 100 mg; 100 pg to 10 mg; 100 pg to 1 mg, 1 mg to 500 mg 1 mg to 100 mg, or 1 mg to 10 mg. Other ranges are contemplated. Other amounts are contemplated.
  • the composition comprises the agent in an amount to provide a dose ranging from one of the following selected ranges: 0.1 pg/kg to 10 mg/kg; 0.1 pg/kg to 1 mg/kg; 0.1 pg/kg to 100 pg/kg; 0.1 pg/kg to lOpg/kg; 1 pg/kg to 10 mg/kg; 1 pg/kg to 1 mg/kg; 1 pg/kg to 100 pg/kg; 1 pg/kg to lOpg/kg; 10 pg/kg to 10 mg/kg; 10 pg/kg to 1 mg/kg; 10 pg/kg to 100 pg/kg; 100 pg/kg to 10 mg/kg; 100 pg/kg to 1 mg/kg; or 1 mg/kg to 10 mg/kg.
  • Other ranges are contemplated.
  • the composition comprises the agent in an amount to provide a dose ranging from one of the following selected ranges: 0.1 pg/kg/day to 10 mg/kg/day; 0.1 pg/kg/day to 1 mg/kg/day; 0.1 pg/kg/day to 100 pg/kg/day; 0.1 pg/kg/day to 10 pg/kg/day; 1 pg/kg/day to 10 mg/kg/day; 1 pg/kg/day to 1 mg/kg/day; 1 pg/kg/day to 100 pg/kg/day; 1 pg/kg/day to 10 pg/kg/day; 10 pg/kg/day to 10 mg/kg/day; 10 mg/kg/day to 1 mg/kg/day; 10 mg/kg/day to 100 mg/kg/day; 100 mg/kg/day to 10 mg/kg/day; 100 mg/kg/day to 1 mg/kg/day; or 1 mg/kg/day to 10 mg.
  • the composition comprises an acceptable carrier suitable for administering the composition to a subject.
  • the carrier may be chosen based on various considerations including the agent(s) being delivered and the time course of delivery of the agents.
  • acceptable carrier refers to a substantially inert solid, semi-solid or liquid filler, diluent, excipient, encapsulating material or suitable auxiliary formulation . Physiologically acceptable carriers and their formulations are known in the art.
  • the composition is suitable for administering the agent to the subject on a regular basis, such as twice daily, daily, weekly, monthly, annually or multi-annually administration.
  • the composition is suitable for continuous administration of the agent.
  • the composition is an immediate release formulation.
  • the composition is a slow/sustained release formulation.
  • the composition provides long-term administration of the agent. In certain embodiments, the composition provides long-term continuous administration of the agent.
  • the composition comprises a substrate with a releasable form of the agent.
  • the composition is an intraventricular composition, an intracranial composition, an intramuscular composition, an oral composition, or a intravenous composition.
  • a suitable composition for intravenous injection includes the agent in a suitable carrier, such as isotonic saline.
  • Certain embodiments of the present disclosure provide a method of treating a subject suffering from a brain injury involving neuronal damage, the method comprise administering a pharmaceutical composition as described herein.
  • Certain embodiments of the present disclosure provide methods for identifying or screening for therapeutic agents for improving recovering from a brain injury involving neuronal damage.
  • the present disclosure provides a method of identifying a therapeutic agent for improving recovery from a brain injury involving neuronal damage, the method comprising determining the ability of an inhibitor of a p75 neurotrophin receptor to improve recovery from a brain injury involving neuronal damage and identifying the inhibitor of a p75 neurotrophin receptor agent as a therapeutic agent for improving recovery from a brain injury involving neuronal damage.
  • the present disclosure provides a method of identifying a therapeutic agent for improving recovery from a brain injury involving neuronal damage, the method comprising:
  • identifying a candidate agent as an inhibitor of a p75 neurotrophin receptor identifying a candidate agent as an inhibitor of a p75 neurotrophin receptor; and determining the ability of the inhibitor of a p75 neurotrophin receptor to improve recovery from a brain injury involving neuronal damage,
  • the present disclosure provides a method of identifying a therapeutic agent for treating a brain injury involving neuronal damage, the method comprising determining the ability of an inhibitor of a p75 neurotrophin receptor to improve recovery from a brain injury involving neuronal damage and identifying the inhibitor of a p75 neurotrophin receptor agent as a therapeutic agent for treating a brain injury involving neuronal damage.
  • the present disclosure provides a method of identifying a therapeutic agent for treating a brain injury involving neuronal damage, the method comprising:
  • identifying a candidate agent as an inhibitor of a p75 neurotrophin receptor identifying a candidate agent as an inhibitor of a p75 neurotrophin receptor; and determining the ability of the inhibitor of a p75 neurotrophin receptor to treat a brain injury involving neuronal damage
  • the brain injury comprises one or more of an ischemic injury, a traumatic injury, a haemorrhagic injury, and an epileptic injury.
  • Agents so identified are potential therapeutic agents.
  • candidate agents include a drug, a small molecule, a protein, a polypeptide, a lipid, a carbohydrate, a nucleic acid, an oligonucleotide, a ribozyme, a biologic, a recombinant peptide, an aptamer, a cofactor, a ligand, a ligand mimetic, a receptor, a peptidomimetic, an enzyme, a kinase, a phosphatase, a cytokine, a growth factor, a metal ion, a chelate, an antisense nucleic acid, an inhibitor RNA, a microRNA, a siRNA, an antibody or antigen binding part thereof, an antibody mimetic.
  • Other types of agents are contemplated.
  • the method of identifying comprises use of in vitro studies and/or use of an animal model(s).
  • the present disclosure provides a kit for performing a method as described herein.
  • Standard techniques and equipment may be used for recombinant DNA technology, oligonucleotide synthesis, molecular biology and enzymatic reactions.
  • the foregoing techniques and procedures may be generally performed according to methods known in the art and/or as commercially available, and are as described for example in Sambrook et al. Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)) and Ausubel et al Current Protocols in Molecular Biology (2003) John Wiley & Sons, both of which are herein incorporated by reference.
  • the agent drug utilises the extracellular domain sequence of human neurotrophin receptor p75 fused with human immunoglobulin fragment C for the treatment of nerve damage induced by ischemia. Because it can protect brain from ischemic injury, it is expected that the agent may also be used for the treatment of other brain damage, such as trauma and inflammation.
  • the agent is antagonize ligands of p75 receptors, which are regarded as pathological factors.
  • antibodies to proBDNF, a ligand of p75 are also neuroprotective.
  • inhibitors against p75 or p75 ligands are neuroprotective in brain injuries involving neuronal damage, such as ischemic stroke.
  • Neurotrophins are a small family of secreted neurotrophic factors that act via complicated signalling pathways that result in a broad spectrum of actions in the nervous system, including involvement in neurogenesis, neuronal survival, proliferation, differentiation, myelination, axonal growth and synaptic plasticity, and conversely, apoptosis and cell death, at all stages of neuronal development, adult life, neuronal injury and disease.
  • the neurotrophin family includes nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3) and neurotrophin-4/5 (NT4/5).
  • Neurotrophins mediate their effects by binding to at least three different types of receptors, tropomyosin-related kinase (Trk) receptors (TrkA, TrkB and TrkC), sortilin, and the p75 neurotrophin receptor (p75;synonyms include p75NTR, p75(NTR), Gp80-LNGFR, Low-affinity nerve growth factor receptor, Low affinity neurotrophin receptor, NGF receptor, and tumour necrosis factor receptor superfamily member 16 precursor (TNFRSF16)).
  • Prk tropomyosin-related kinase
  • TrkA, TrkB and TrkC the p75 neurotrophin receptor
  • p75 neurotrophin receptor
  • p75 p75
  • Gp80-LNGFR Low-affinity nerve growth factor receptor
  • Low affinity neurotrophin receptor Low affinity neurotrophin receptor
  • NGF receptor tumour necrosis factor receptor superfamily member 16 precursor
  • TNFRSF16 tumour necrosis factor receptor super
  • AD Alzheimer's Disease
  • CAA cerebral amyloid angiopathy
  • ALS amyotrophic lateral sclerosis
  • tauopathies neuronal injury such as spinal cord injury; although no human disease has yet been shown to be caused directly by a defect in neurotrophins or their receptors.
  • Cerebral ischemia instigates multifaceted cellular functions that trigger cell death, inflammation, cognitive and sensorimotor dysfunction. The detrimental effects of ischemia are believed to be due to certain deviations of physiological factors after ischemia. Several studies have showed that different signaling cascades are altered in ischemic condition and responsible for continuing post-ischemic damages.
  • Brain-derived neurotrophic factor is generated from its precursor preproBDNF and proBDNF, which are physiologically processed by removing the signalling peptide and the prodomain, respectively.
  • ProBDNF interacts with p75/sortilin complex with high affinity and initiates apoptosis, inflammation, long term depression (LTD), excitation, persistent cortical neuronal firing and neuronal inhibition.
  • mBDNF has high affinity to tyrosine kinase receptor (TrkB) receptor, and participates in cellular growth, long term potential (LTP) and neuronal survival.
  • TrkB tyrosine kinase receptor
  • the transmembrane p75 receptor has an extracellular ectodomain (p75ECD) and intracellular domain (p75ICD).
  • ICD holds a death domain in the cytoplasmic region.
  • the main signalling pathways for p75 receptor are-cell surviving NF-kB and apoptotic Jun kinase (JNK) pathways.
  • JNK Jun kinase
  • Neurotrophins bind to the ectodomain part of the receptor and transduce signal through ICD inside the cell. Mature neurotrophins have low affinity, and on the contrary, proneurotrophins have higher affinity to bind with this transmembrane receptor.
  • Photothrombotic Ischemic Model Sprague-Dawley rats (250-350 grams) were used at 8-12 weeks of age for the experimental purpose. Focal ischemia was induced on the rat brain by intravenous (i.v.) injection of the photosensitive Rose Bengal (RB) dye (Sigma- Aldrich, USA. Cat#330000-lG) through tail vein (at 20 mg/kg) followed by the skull exposure to the cold light for 20 min (Schmidt, Hoppen et al. 2012, Madinier, Bertrand et al. 2013). The surgical procedure uses is as described in Rahman, Luo et al. (2016). Control group animals did not receive any surgical procedure.
  • RB photosensitive Rose Bengal
  • mice were also treated with sheep antibodies to proBDNF or sheep IgG by intraperitoneal injection at dosage of 5 mg/kg twice a week (Table 2).
  • Table 2 Treatments given to animals
  • B. Rotarod Test The rotarod test assesses the forced motor function to understand the physical state for balance and movements (Hamm, Pike et al. 1994). Animals were positioned on a whirling rod. The rod started rotation from 0 rpm and reached to 30rpm within 2 min by increasing 3 rpm speed every 10 sec. Both the time (sec) and speed (rpm) were recorded as soon as the animal was fallen off the rod.
  • C. Corner Test This test is assessing the sensorimotor function of the animal. A comer board with an angle of 30° approximately of a rat cage height was placed inside the cage. Animal was allowed to move 10 times inside the corner and turn backward to come in the free side. The direction of turning (left or right) backward after each contact with the corner was closely monitored and recorded.
  • D. Conventional Adhesive Tape Test This behavioural test was used to assess the sensorimotor functions of the animal. An adhesive tape of lcm was applied on each limb of the animal, and the time to contact (sensory) and removal (motor) was recorded in 1 min task time (Sughrue, Mocco et al. 2006).
  • Tissue Collection After behavioural studies, animals were killed at 7d and l4d and tissues were collected. A half of the infarcted ipsilateral hemisphere immediately frozen and stored at -80°C is used for biochemical assays. The other half of the infarcted ipsilateral brain section was collected for histological assays, and tissues were fixed in 4% paraformaldehyde followed by the dehydration in 30% sucrose and stored at 4°C until sectioning.
  • A. DAB Staining Paraformaldehyde-fixed infarcted ipsilateral brain tissues were embedded in optical cutting temperature compound (OCT) and were sectioned at l5pm thickness using a cryostat (Leica, Vic, Australia). Brain sections were preserved in anti-freeze solution and stored at -20°C until used. Prior staining, sections were washed in PBS and mounted on gelatin coated slides. After sections were attached on the slides, antigen retrieval step was performed by incubating sections in 1% sodium dodecyl sulphate (SDS) for 5 min at room temperature. 3% hydrogen peroxide (H 2 0 2 ) was applied on the sections for 10 min in order to block the endogenous peroxidase.
  • SDS sodium dodecyl sulphate
  • bovine serum albumin (BSA)-PBST After blocking in 5% bovine serum albumin (BSA)-PBST for lh at room temperature, sections were incubated with the following primary antibodies overnight at 4°C - 1:1000 monoclonal anti-proBDNF 1D3 (China), 1:1000 rabbit anti-microtubule-associated protein 2 (MAP2) (Osenses, Australia), 1:1000 rabbit anti-glial fibrillary acidic protein (GFAP) (Abeam, Vic, Australia) and 1:500 rabbit anti- ionized calcium-binding adapter molecule l(IBA-l) (WAKO, Japan).
  • BSA bovine serum albumin
  • Respective biotin-conjugated secondary antibodies were applied (1:2000) on the sections for 2h at room temperature followed by incubation in ABC reagent (Vectra Laboratories, USA) according to the manufacturer's protocol. All sections were incubated in DAB substrate according to the manufacturer's (Osenses, Australia) instructions. Slides were dehydrated by immersing the slides in graded alcohol and xylene, and coverslipped using Depex mounting medium (Sigma- Aldrich, St Louis, MO, USA). All sections were imaged using Olympus BX53 Light microscope (Olympus, NSW, Australia). Immuno positive cells were measured by measuring the optical density of five different areas in ImageJ software and expressed as % of area fraction.
  • Cy2 conjugated anti-goat and anti-mouse, and Cy3 conjugated anti-rabbit antibodies were used. Sections were counterstained with 4', 6-Diamidino-2-phenylindole (DAPI) and coverslipped using 90% glycerol for imaging under fluorescent microscope (Zeiss LSM 710, Germany).
  • DAPI 6-Diamidino-2-phenylindole
  • H&E Haematoxylin and Eosin Staining. Brain sections were mounted on gelatin coated slides and allowed to be fixed. After a brief wash in distilled water, sections were stained in Ehrlich's haematoxylin for 30 sec-2 min and washed in distilled water. Sections were differentiated briefly in 1% acid-alcohol followed by a wash in distilled water. Slides were immersed in ammonia water for blueing and rinsed again in distilled water. To finish, sections were counterstained in eosin, and dehydrated in graded alcohol and xylene, and coverslipped for imaging.
  • TUNNEL Staining was performed to observe the apoptosis around the infarcted area of the ipsilateral hemisphere before and after the p75ECD-Fc treatment. The complete staining procedure was followed by the manufacturer's (Abeam, Vic, Australia, Cat# ab206386) instructions.
  • HRP horseradish peroxidase
  • B- actin antibody conjugated with HRP (1:10000; Sigma- Aldrich) was used for incubating the membranes at room temperature for 2h.
  • LAS 4000 imager GE Healthcare, UK was used to capture the image of the blots and Image J software (NIH, Bethesda, MD, USA) was used for quantitation.
  • proBDNF staining was observed in neuron-like cells of sham brain sections. However, proBDNF staining was more intense in neuron-like cells around the ischemic infarct after 3d of the ischemia than the sham control.
  • Figure 2 shows localization of p75 around the ischemic infarct.
  • Anti-proBDNF treatment showed improvement after ischemia in rotarod test (Fig. 8).
  • P75ECD-Fc reduces the infiltration of inflammatory cells in the peri-infarcted area
  • H&E staining was performed to understand the morphological changes in different dosage of p75ECD-Fc treatment.
  • We found that at the dose of lOmg of p75ECD-Fc, infarcted area is significantly shrunk (1.5+0.48 mm2, R ⁇ 0.0001, n 5) compared to the 7d stroke group and cortical lesion is mostly covered up by new structure (Fig. 19). This indicates that p75ECD-Fc treatment is promoting the cellular growth and reducing the cortical lesion size.
  • Tunnel staining kit was used to detect the apoptotic cells after the treatment of p75ECD-Fc at different dosages (Fig. 21).
  • Anti-proBDNF IgG reduces the expression of JNK(P) in the ipsilateral brain
  • Table 3 Effects of anti-proBDNF IgG treatment on ischemic recovery.
  • p75ECD-Fc is beneficial after ischemia as it functions as an anti-inflammatory and anti-apoptotic agent, and improves cellular growth and sensorimotor behavioural impairments. Therefore, p75ECD may be used as a therapeutic target after ischemia.

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Abstract

The present disclosure relates to methods and compositions for improving recovery from brain injuries involving neuronal damage, and for treating a brain injury involving neuronal damage. In certain embodiments, the present disclosure provides a method of improving recovery from brain injury involving neuronal damage in a subject, the method comprising reducing activity of a p75 neurotrophin receptor in the subject and thereby improving recovery from the brain injury. Other embodiments are also disclosed.

Description

METHODS AND COMPOSITIONS FOR IMPROVING RECOVERY FROM A
BRAIN INJURY
PRIORITY CLAIM
[001] This application claims priority to Australian Provisional Patent Application 2018903407 filed on 11 September 2018, the content of which is hereby incorporated by reference in its entirety.
FIELD
[002] The present disclosure relates, at least in part, to methods and compositions for improving recovery from brain injuries involving neuronal damage.
BACKGROUND
[003] Neuronal damage is a consequence of many types of brain injury. For example, in cases of cerebral ischemia, such as those arise from stroke or cardia arrest, there may be significant neuronal damage, leading to a variety of short term and long tem effects . Other types of brain injury resulting in neuronal damage also arise as a result of trauma and/or inflammation.
[004] The pharmacological options for treatment of brain injuries, and the recovery from the injuries, remain limited. In the case of stroke, treatment may involve thrombolytic drugs to breakdown the clot. However, in most cases treatment of brain injuries involves ameliorating the effects of the underlying injury, such as the use of anticonvulsants to reduce seizures arising from the damage.
[005] Accordingly, there is a need for new treatment options for improving the recovery from brain injuries involving neuronal damage. The present disclosure is directed to overcoming and/or at least ameliorating one of more disadvantages in the art, and/or providing one or more advantages. SUMMARY
[006] The present disclosure is based on the recognition that in a rat photothrombotic ischemic model agents that antagonise ligands of p75 neurotrophin receptors are neuroprotective in cerebral ischemia. As such, inhibitors of p75 neurotrophin activity are neuroprotective in brain injuries involving neuronal damage.
[007] Certain embodiments of the present disclosure provide a method of improving recovery from brain injury involving neuronal damage in a subject, the method comprising reducing activity of a p75 neurotrophin receptor in the subject and thereby improving recovery from the brain injury.
[008] Certain embodiments of the present disclosure provide a method of improving recovery from a brain injury involving neuronal damage in a subject, the method comprising administering an effective amount of an inhibitor of a p75 neurotrophin receptor to the subject and thereby improving recovery from the brain injury.
[009] Certain embodiments of the present disclosure provide use of an inhibitor of p75 neurotrophin receptor for improving recovery from a brain injury involving neuronal damage.
[0010] Certain embodiments of the present disclosure provide use of an inhibitor of p75 neurotrophin receptor in the preparation of a medicament for improving recovery from a brain injury involving neuronal damage.
[0011] Certain embodiments of the present disclosure provide a method of treating a brain injury involving neuronal damage in a subject, the method comprising administering an effective amount of an inhibitor of a p75 neurotrophin receptor to the subject and thereby treating the brain injury.
[0012] Certain embodiments of the present disclosure provide a pharmaceutical composition for treating a brain injury involving neuronal damage, the pharmaceutical composition comprising an effective amount of an inhibitor of a p75 neurotrophin receptor. [0013] Certain embodiments of the present disclosure provide a method of treating a subject suffering from a brain injury involving neuronal damage, the method comprising administering to the subject a pharmaceutical composition as described herein.
[0014] Certain embodiments of the present disclosure provide a method of identifying a therapeutic agent for improving recovery from a brain injury involving neuronal damage, the method comprising determining the ability of an inhibitor of a p75 neurotrophin receptor to improve recovery from a brain injury involving neuronal damage and identifying the inhibitor of a p75 neurotrophin receptor agent as a therapeutic agent for improving recovery from a brain injury involving neuronal damage.
[0015] Certain embodiments of the present disclosure provide a method of identifying a therapeutic agent for improving recovery from a brain injury involving neuronal damage, the method comprising:
identifying a candidate agent as an inhibitor of a p75 neurotrophin receptor; and determining the ability of the inhibitor of a p75 neurotrophin receptor to improve recovery from a brain injury involving neuronal damage,
thereby identifying the candidate agent as a therapeutic agent for improving recovery from a brain injury involving neuronal damage.
[0016] Other embodiments are described herein.
BRIEF DESCRIPTION OF THE FIGURES
[0017] For a better understanding of the present disclosure, and to show more clearly how the present disclosure may be carried into effect according to one or more embodiments thereof, reference will be made, by way of example, to the accompanying figures.
[0018] Figure 1 shows localization of proBDNF around the ischemic infarct. A. Rat brain cortex of sham group. B. Rat brain cortex of stroke group at 3d. Scale bar=200 pm [0019] Figure 2 shows localization of p75 around the ischemic infarct. A. Rat brain cortex of sham group. B. Rat ipsilateral brain cortex of ischemic group at 3d. Scale bar=200 pm
[0020] Figure 3 shows double labelling of neuron (Tujl) and p75 on ischemic (3d) rat brain. Scale bar=200 pm.
[0021] Figure 4 shows double labelling of astrocyte (GFAP) and p75 on ischemic (3d) rat brain. Scale bar=200 pm.
[0022] Figure 5 shows double labelling of microglia (IBA-l) and p75 on ischemic rat brain. Scale bar=200 pm.
[0023] Figure 6 shows cylinder testing performed to assess the deficit in the fine motor function after anti-proBDNF treatment on photothrombotic ischemia after 7d compared to the 7d stroke (without treatment). Ordinary one-way ANOVA, using (Tukey's multiple comparisons test) was performed, where *P<0.05, ***P<0.00l versus 7d stroke (without treatment). The sample size was n=8.
[0024] Figure 7 shows cylinder testing performed to assess the deficit in the fine motor function after p75ECD-Fc treatment on photothrombotic ischemia after (a) 7d and (b) l4d compared to the 7d stroke. Ordinary one-way ANOVA, using (Tukey's multiple comparisons test) was performed, where *P<0.05,“P<0.00l versus 7d stroke, and l4d stroke. The sample size was n=8.
[0025] Figure 8 shows rotarod testing performed to assess the deficit in the forced motor function. A. Forced motor test was used to assess the motor function through the running ability on the speeding rod (r.p.m) after anti-proBDNF treatment on photothrombotic ischemia after 7d compared to the 7d stroke (without treatment). B. Duration of running (sec) on the speeding rod was measured after anti-proBDNF treatment on photothrombotic ischemia after 7d compared to the 7d stroke (without treatment). Ordinary one-way ANOVA, using (Tukey's multiple comparisons test) was performed, where *P<0.05, ***P<0.00l versus 7d stroke (without treatment). The sample size was n=8. [0026] Figure 9 shows rotarod testing to assess the deficit in the forced motor function. A. Forced motor test after p75ECD-Fc treatment on photothrombotic ischemia after 7d compared to the 7d stroke. B. Duration of running (sec) on the speeding rod was measured after p75ECD-Fc treatment on photothrombotic ischemia after 7d compared to the 7d stroke. C. The running ability on the speeding rod (r.p.m) after p75ECD-Fc treatment on photothrombotic ischemia after l4d compared to the l4d stroke. D. Duration of running (sec) on the speeding rod was measured after p75ECD-Fc treatment on photothrombotic ischemia after l4d compared to the l4d stroke. Ordinary one-way ANOVA, using (Tukey's multiple comparisons test) was performed, where *P<0.05, “P<0.00l versus 7d stroke, and l4d stroke respectively. The sample size was n=8.
[0027] Figure 10 shows the results of comer testing (lesion site on the right). This test was performed to assess the deficit of the sensory function after anti-proBDNF treatment on photothrombotic ischemia after 7d compared to the 7d stroke (without treatment). Ordinary one-way ANOVA, using (Tukey's multiple comparisons test) was performed, where *P<0.05, ***P<0.00l versus 7d stroke (without treatment). The sample size was n=8.
[0028] Figure 11 shows the results of comer testing. This test was performed to assess the deficit of the sensory function after p75ECD-Fc treatment on photothrombotic ischemia after 7d (a) compared to the 7d stroke, and l4d (b) compared to the l4d stroke respectively. Ordinary one-way ANOVA, using (Tukey's multiple comparisons test) was performed, where *P<0.05,”P<0.00l versus 7d, and l4d stroke. The sample size was n=8.
[0029] Figure 12 shows adhesive removal testing for the contralateral forepaw. Time to contact the adhesive tape (a) was considered to assess the sensory function and time to remove the adhesive tape (b) was considered to assess the motor function after anti- proBDNF treatment on photothrombotic ischemia at 7d compared to the 7d stroke (without treatment). Ordinary one-way ANOVA, using (Tukey's multiple comparisons test) was performed, where *P<0.05, ***P<0.00l versus 7d stroke (without treatment). The sample size was n=8. [0030] Figure 13 shows adhesive removal testing for the ipsilateral forepaw. Time to contact the adhesive tape (a) was considered to assess the sensory function and time to remove the adhesive tape (b) was considered to assess the motor function after anti- proBDNF treatment on photothrombotic ischemia at 7d compared to the 7d stroke (without treatment). Ordinary one-way ANOVA, using (Tukey's multiple comparisons test) was performed, where *P<0.05, ***P<0.00l versus 7d stroke (without treatment). The sample size was n=8.
[0031] Figure 14 shows adhesive removal testing for both contralateral and ipsilateral forepaws at 7d. A. Time to contact the adhesive tape to assess the sensory function. B. time to remove the adhesive tape to assess the motor function of the contralateral forepaw. C. Time to contact the adhesive tape to assess the sensory function and D time to remove the adhesive tape to assess the motor function of the ipsilateral forepaw after p75ECD-Fc treatment on photothrombotic ischemia after 7d compared to the 7d stroke. Ordinary one-way ANOVA, using (Tukey's multiple comparisons test) was performed, where *P<0.05,“P<0.00l versus 7d stroke. The sample size was n=8.
[0032] Figure 15 shows adhesive removal testing for both contralateral and ipsilateral forepaws at l4d. A. Time to contact the adhesive tape to assess the sensory function, B time to remove the adhesive tape to assess the motor function of the contralateral forepaw, C Time to contact the adhesive tape to assess the sensory function and D time to remove the adhesive tape to assess the motor function of the ipsilateral forepaw after p75ECD-Fc treatment on photothrombotic ischemia after l4d compared to the l4d stroke. Ordinary one-way ANOVA, using (Tukey's multiple comparisons test) was performed, where *P<0.05,“P<0.00l versus l4d stroke. The sample size was n=8.
[0033] Figure 16 shows the results of cresyl violet staining. Necrotic area, where filled with all inflammatory cell-like cells was measured. A. Site of infarct at lOx magnification. B. Site of peri-infarct at lOx magnification. C. Quantitative analysis of inflammatory cell-like cells in the necrotic area after anti-proBDNF treatment on photothrombotic ischemia after 7d compared to the sham group. Ordinary one-way ANOVA, using (Tukey's multiple comparisons test) was performed, where *P<0.05, ***P<0.00l versus sham group. The sample size was n=6. Scale bar=200pm. [0034] Figure 17 shows the results of cresyl violet staining. Necrotic area, where filled with inflammatory cell-like cells was measured. A. Site of infarct at lOx magnification.
B. Site of peri-infarct at lOx magnification. C. Quantitative analysis of inflammatory cell-like cells in the necrotic area after p75ECD-Fc treatment on photothrombotic ischemia after 7d compared to the sham group. Ordinary one-way ANOVA, using (Tukey's multiple comparisons test) was performed, where *P<0.05, ***P<0.00l versus sham group. The sample size was n=6. Scale bar=200 pm.
[0035] Figure 18 shows Hematoxylin and Eosin staining after anti-proBDNF IgG treatment. A. Site of infarct at 4x magnification. B. Site of peri-infarct at lOx magnification. C. Quantitative analysis of infarcted area after anti-proBDNF treatment on photothrombotic ischemia after 7d compared to the sham group. Ordinary one-way ANOVA, using (Tukey's multiple comparisons test) was performed, where *P<0.05, ***P<0.00l versus sham group. The sample size was n=5. Scale bar=200pm.
[0036] Figure 19 shows Hematoxylin and Eosin staining after antiproBDNF treatment. A. Site of infarct at 4x magnification. B. Site of peri-infarct at lOx magnification. C. Quantitative analysis of infarcted area after p75ECD-Fc treatment on photothrombotic ischemia after 7d compared to the sham group. Ordinary one-way ANOVA, using (Tukey's multiple comparisons test) was performed, where *P<0.05, ***P<0.00l and ****P<0.000l versus sham group. The sample size was n=5. Scale bar=200 pm.
[0037] Figure 20: shows TUNNEL staining for apoptotic cells after anti-proBDNF IgG treatment. A. Site of infarct at 4x magnification. B. Site of infarct at 20x magnification.
C. Quantitative analysis of apoptotic cells after anti-proBDNF treatment on photothrombotic ischemia after 7d compared to the sham group. Ordinary one-way ANOVA, using (Tukey's multiple comparisons test) was performed, where *P<0.05, ***p<0.00l versus sham group. The sample size was n=6. Scale bar=200pm.
[0038] Figure 21 shows TUNNEL staining for apoptotic cells after antiproBDNF treatment. A. Site of infarct at 4x magnification. B. Site of infarct at 20x magnification. C. Quantitative analysis of apoptotic cells after p75ECD-Fc treatment on photothrombotic ischemia after 7d compared to the sham group. Ordinary one-way ANOVA, using (Tukey's multiple comparisons test) was performed, where *P<0.05, ***p<0.00l versus sham group. The sample size was n=6. Scale bar=200 pm.
[0039] Figure 22 shows immuno staining of Map2. A. Site of infarct at 4x magnification. B. Site of infarct at 20x magnification. C. Quantitative analysis of Map2 positive cells after anti-proBDNF treatment on photothrombotic ischemia after 7d compared to the sham group. Ordinary one-way ANOVA, using (Tukey's multiple comparisons test) was performed, where *P<0.05, ***P<0.00l versus sham group. The sample size was n=6. Scale bar=200pm.
[0040] Figure 23 shows immuno staining of Map2. A. Site of infarct at 4x magnification. B. Site of infarct at 20x magnification. C. Quantitative analysis of Map2 positive staining after p75ECD-Fc treatment on photothrombotic ischemia after 7d compared to the sham group. Ordinary one-way ANOVA, using (Tukey's multiple comparisons test) was performed, where *P<0.05, ***P<0.00l versus sham group. The sample size was n=6. Scale bar=200 pm.
[0041] Figure 24 shows immuno staining of GFAP. A. Site of infarct at 4x magnification. B. Site of infarct at 20x magnification. C. Quantitative analysis of GFAP positive cells after anti-proBDNF treatment on photothrombotic ischemia after 7d compared to the sham group. Ordinary one-way ANOVA, using (Tukey's multiple comparisons test) was performed, where P<0.05, P<0.00l versus sham group. The sample size was n=6. Scale bar=200pm.
[0042] Figure 25 shows immuno staining of GFAP. A. Site of infarct at 4x magnification. B. Site of infarct at 20x magnification. C. Quantitative analysis of GFAP positive staining after p75ECD-Fc treatment on photothrombotic ischemia after 7d compared to the sham group. Ordinary one-way ANOVA, using (Tukey's multiple comparisons test) was performed, where *P<0.05, ***P<0.00l versus sham group. The sample size was n=6. Scale bar=200 pm.
[0043] Figure 26 shows immunostaining of IBA1. A. Site of infarct at 4x magnification. B. Site of infarct at 20x magnification. C. Quantitative analysis of IBA1 positive cells after anti-proBDNF treatment on photothrombotic ischemia after 7d compared to the sham group. Ordinary one-way ANOVA, using (Tukey's multiple comparisons test) was performed, where *P<0.05, ****P<0.000l versus sham group. The sample size was n=6. Scale bar=200pm.
[0044] Figure 27 shows immuno staining of IBA-l. A. Site of infarct at 4x magnification. B. Site of infarct at 20x magnification. C. Quantitative analysis of IBA-l positive staining after p75ECD-Fc treatment on photothrombotic ischemia after 7d compared to the sham group. Ordinary one-way ANOVA, using (Tukey's multiple comparisons test) was performed, where *P<0.05, ***P<0.00l and ****P<0.000l versus sham group. The sample size was n=6. Scale bar=200 pm.
[0045] Figure 28 shows immunoblotting of JNK and JNK(P) signaling enzymes. A, B Representative immunoblots of JNK and JNK(P) proteins in the ipsilateral hemisphere after anti-proBDNF treatment on photothrombotic ischemia after 7d compared to the sham group. C, D Quantitative analyses of A, B. Protein loading was calibrated by B actin. Ordinary one-way ANOVA, using (Tukey's multiple comparisons test) was performed, where *P<0.05, ***P<0.00l versus sham group. The sample size was n=4 for control, n=5 for sham, and n=6 for ischemic groups.
[0046] Figure 29 shows immunoblotting of JNK and JNK(P) signaling enzymes. A. Representative immunoblots of JNK and JNK(P) proteins in the ipsilateral hemisphere after p75ECD-Fc treatment on photothrombotic ischemia after 7d compared to the sham group. B, C Quantitative analyses of A. Protein loading was calibrated by B actin. Ordinary one-way ANOVA, using (Tukey's multiple comparisons test) was performed, where *P<0.05, ***P<0.00l versus sham group. The sample size was n=4 for control, n=5 for sham, and n=6 for ischemic groups.
[0047] Figure 30 shows immunoblotting of PARP signaling enzyme. A. Representative immunoblots of full length PARP (PARP(FL)) and cleaved PARP (PARP(CL)) proteins in the ipsilateral hemisphere after anti-proBDNF treatment on photothrombotic ischemia after 7d compared to the sham group. B, C Quantitative analyses of A. Protein loading was calibrated by B actin. Ordinary one-way ANOVA, using (Tukey's multiple comparisons test) was performed, where *P<0.05, ***P<0.00l versus sham group. The sample size was n=4 for control, n=5 for sham, and n=6 for ischemic groups. [0048] Figure 31 shows immunoblotting of PARP signaling enzyme. A. Representative immunoblots of full length PARP (PARP(FL)) and cleaved PARP (PARP(CL)) proteins in the ipsilateral hemisphere after p75ECD-Fc treatment on photothrombotic ischemia after 7d compared to the sham group. B, C Quantitative analyses of A. Protein loading was calibrated by b actin. Ordinary one-way ANOVA, using (Tukey's multiple comparisons test) was performed, where *P<0.05, ***P<0.00l and ****P<0.000l versus sham group. The sample size was n=4 for control, n=5 for sham, and n=6 for ischemic groups.
[0049] Figure 32 shows immunoblotting of PSD95 post-synaptic protein. A. Representative immunoblots of PSD95 in the ipsilateral hemisphere after anti-proBDNF treatment on photothrombotic ischemia after 7d compared to the sham group. B. Quantitative analyses of A. Protein loading was calibrated by b actin. Ordinary one-way ANOVA, using (Tukey's multiple comparisons test) was performed, where *P<0.05, ****P<0.000l versus sham group. The sample size was n=4 for control, n=5 for sham, and n=6 for ischemic groups.
[0050] Figure 33 shows immunoblotting of PSD95 synpatic protein. A. Representative immunoblots of PSD95 in the ipsilateral hemisphere after p75ECD-Fc treatment on photothrombotic ischemia after 7d compared to the sham group. B. Quantitative analyses of A. Protein loading was calibrated by b actin. Ordinary one-way ANOVA, using (Tukey's multiple comparisons test) was performed, where *P<0.05, ****P<0.000l versus sham group. The sample size was n=4 for control, n=5 for sham, and n=6 for ischemic groups.
DETAILED DESCRIPTION
[0051] The present disclosure relates generally to methods and compositions for improving recovery from brain injuries involving neuronal damage, and for treating a brain injury.
[0052] Certain embodiments of the present disclosure provide a method of improving recovery from brain injury involving neuronal damage in a subject. [0053] One or more embodiments of the present disclosure are directed to methods and products that have one or more combinations of the following advantages: new methods and/or products for treating a brain injury; new methods and/or products for improving recovering from a treating a brain injury; the new use of a class of agents for treating brain injury and/or for improving the recovery from a brain injury; to address one or more problems, to provide one or more advantages, and/or to provide a commercial alternative.
[0054] In certain embodiments, the present disclosure provides a method of improving recovery from brain injury involving neuronal damage in a subject, the method comprising reducing activity of a p75 neurotrophin receptor in the subject and thereby improving recovery from the brain injury.
[0055] In certain embodiments the subject is a human subject. Other types of subjects are contemplated, and it will be understood that veterinary applications of the present disclosure in animals are also contemplated.
[0056] In certain embodiments, the subject is suffering from a brain injury. In certain embodiments, the subject is susceptible to developing a brain injury. In this regard, it will be appreciated that the present disclosure relates to methods for improving recovery by reducing the activity of a p75 neurotrophin receptor prior to, concomitant with, and/or after a brain injury has occurred, and that treatment after a brain injury has occurred and prophylactic treatment before a brain injury has occurred are contemplated.
[0057] In certain embodiments, the brain injury comprises one or more of an ischemic injury, a traumatic injury, a haemorrhagic injury, and an epileptic injury. Other types of brain injury are contemplated. Methods for identifying brain injuries involving neuronal damage are known in the art.
[0058] In certain embodiments, the brain injury is associated with a cerebral occlusion. In certain embodiments, the brain injury comprises a stroke. Methods for identifying cerebral occlusions such as stroke are known in the art. [0059] In certain embodiments, the improvement in recovery comprises an improvement in motor function. Methods for assessing motor function are known in the art.
[0060] In certain embodiments, the improvement in recovery comprises reducing neuronal cell death. Methods for assessing neuronal cell death are known in the art.
[0061] In certain embodiments, the improvement in recovery comprises a reduction in size of an infarct associated with the stroke.
[0062] Other types of improvement in recovery are contemplated.
[0063] In certain embodiments, the method comprises administering to the subject an effective amount of an agent that reduces p75 neurotrophin receptor activity.
[0064] The term“an agent that reduces p75 neurotrophin receptor activity” as used herein refers to an agent that directly or indirectly results in a reduction in the activity of a p75 neurotrophin activity, for example so as to cause a decrease in the level of activity, an inhibition of activity, a prevention of activity, a downregulation in the level of activity, a reduction in the ability of the receptor to be stimulated, an alteration in the timing and/or location of activity, or otherwise provide some form of negative control over activity or combinations thereof.
[0065] For example, the agent may (i) act to directly reduce activity, alter the level of expression of a target, alter localisation of a target, alter signalling, and/or alter timing of function, (ii) act to change the activity of a signalling pathway associated with activation, (iii) act to alter the level and/or the activity of another molecule that regulates a target, such as by competitive/non-competitive binding, or by altering the synthesis, breakdown, and/or localisation of another molecule. Other forms of action are contemplated, and combinations of forms of action are contemplated.
[0066] Examples of agents include a drug, a small molecule, a protein, a polypeptide, a lipid, a carbohydrate, a nucleic acid, an oligonucleotide, a ribozyme, a biologic, a recombinant peptide, an aptamer, a cofactor, a ligand, a ligand mimetic, a receptor, a peptidomimetic, an enzyme, a kinase, a phosphatase, a cytokine, a growth factor, a metal ion, a chelate, an antisense nucleic acid, an inhibitor RNA, a microRNA, a siRNA, an antibody or an antigen binding part thereof, an antibody mimetic, or combinations thereof. Other types of agents are contemplated. It will be appreciated than an agent as described herein also includes a prodrug of the agent, and/or a metabolite of the agent.
[0067] In certain embodiments, the agent comprises a drug or small molecule, and/or a pro-drug or a metabolite thereof.
[0068] In certain embodiments, the agent comprises a nucleic acid. In certain embodiments, the agent comprises an antibody and/or a part thereof, such as an antigen binding part thereof or a part of a Fc region.
[0069] Methods for identify agents that reduce the activity of a p75 neurotrophin receptor are known in the art.
[0070] In certain embodiments, the agent that reduces p75 neurotrophin activity comprises an inhibitor of p75 neurotrophin receptor activity.
[0071] In certain embodiments, the inhibitor comprises all or part of the ectodomain of the p75 neurotrophin receptor.
[0072] In certain embodiments, the inhibitor comprises all or part of the ectodomain of the p75 neurotrophin receptor fused to a Fc region of an antibody.
[0073] In certain embodiments, the p75ECD comprises a p75ECD-Fc fusion. In one embodiment, the p75ECD-Fc fusion is a recombinant peptide.
[0074] In certain embodiments, the inhibitor comprises a p75ECD-Fc. For example, recombinant p75ECD-Fc may be obtained from Fujian Tiantai Medical Technology, China, or the inhibitor may be produced by a method known in the art. P75 ECD fusions may be produced for example as described in US Patent Application No. 20180170996, which is hereby incorporated by reference in its entirety. [0075] Other P75 neurotrophin receptor antagonists are described, for example, in US Josephy-Hemandez et al (2017) Neurobiology of Disease 97:139-155. Examples of p75 neurotrophin receptor antagonists include THX-B (l,3-diisopropyl-l-[2-(l,3-dimethyl- 2,6-dioxo-l,2,3,6-tetrahydro-purin-7-yl)-acetyl]-urea), THX-A and THX-C (also known as LM11A-2426) as described in Yang et al (2008) PLoS ONE 3: e3604.
[0076] In certain embodiments, the inhibitor comprises an agent that binds to a p75 neurotrophin receptor. Methods for assessing binding of agents to a p75 neurotrophin receptor are known in the art.
[0077] In certain embodiments, the inhibitor of a p75 neurotrophin receptor comprises a ligand antagonist. In certain embodiments, the ligand antagonist comprises a ligand mimetic or an antibody or an antigen binding part thereof.
[0078] In certain embodiments, the ligand antagonist comprises one or more of a proBDNF antagonist, a proNGF antagonist, a proNT3 antagonist, and a proNT4 antagonist.
[0079] In certain embodiments, the ligand antagonist comprises a polypeptide/protein encoding all or part of the soluble extracellular domains of the receptors p75 and/or Sortilin/SORCS2, which are commercially available.
[0080] In certain embodiments, the proBDNF antagonist comprises PNS-Rbl, which is commercially available.
[0081] In certain embodiments, the ligand antagonist comprises an antibody to proBDNF, proNGF, proNT3 antagonist, or proNT4. Examples of such antibodies are available commercially.
[0082] In certain embodiments, the ligand antagonist comprises an inhibitor of the formation of the ligand.
[0083] In certain embodiments, the proBDNF antagonist comprises PNS-Rbl, which is commercially available. [0084] In certain embodiments, the reducing of the activity of a p75 neurotrophin receptor in the subject comprises administering to the subject an effective amount of an agent. In certain embodiments, the method comprises administering to the subject an effective amount of an agent that reduces the activity of a p75 neurotrophin receptor.
[0085] The term "effective amount" as used herein refers to that amount of an agent that is sufficient to effect therapeutic treatment, when administered to a subject. The effective amount will vary depending upon a number of factors, including for example the specific activity of the agent being used, the severity of the condition, the subject, the age, physical condition, existence of other disease states, nutritional status of the subject and genetic background of the subject.
[0086] The term“treatment”, and related terms such as“treating” and“treat” as used herein, refer to obtaining a desired pharmacologic and/or physiologic effect in terms of improving the condition of the subject, ameliorating, arresting, preventing, managing, suppressing, relieving and/or slowing the progression of one or more symptoms in the subject, a partial or complete stabilization of the subject, a regression of one or more symptoms, or a cure in the subject.
[0087] In certain embodiments, the agent is administered to a subject to provide a blood or plasma concentration in the range of 1 mM to 100 mM, 5 pM to 100 pM, 10 pM to 100 pM, 50 pM to 100 pM, 1 pM to 50 pM, 5 pM to 50 pM, 10 pM to 50 pM, 1 pM to 10 pM, 5 pM to 10 pM, or 1 pM to 5 pM. Other ranges are contemplated.
[0088] In certain embodiments, the agent is administered to the subject in an amount ranging from one of the following selected ranges: 1 pg/kg to 1000 mg/kg, 1 pg/kg to 100 mg/kg; 1 pg/kg to 10 mg/kg; 1 pg/kg to 1 mg/kg; 1 pg/kg to 100 pg/kg; 1 pg/kg to lOpg/kg; 10 pg/kg to 1000 mg/kg, 10 pg/kg to 100 mg/kg; 10 pg/kg to 10 mg/kg; 10 pg/kg to 1 mg/kg; 10 pg/kg to 100 pg/kg; 10 pg/kg to 1000 mg/kg, 100 pg/kg to 100 mg/kg; 100 pg/kg to 10 mg/kg; 100 pg/kg to 1 mg/kg; 1 mg/kg to 1000 mg/kg, 1 mg/kg to 100 mg/kg; 1 mg/kg to 10 mg/kg; 10 mg/kg to 1000 mg/kg; 10 mg/kg to 100 mg/kg; and 100 mg/kg to 1000 mg/kg body weight. Other ranges are contemplated. [0089] In certain embodiments, the agent is administered once a day, twice, multiple times a day, or continuously. Other administration regimes are contemplated.
[0090] A suitable administration period may be selected. In certain embodiments, the agent is administered to the subject for a period of at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 2 weerks, at least 3 weeks, or at least 4 weeks. In certain embodiments, the administration is for a continuous period. Other administration periods are contemplated.
[0091] In certain embodiments, the agent is administered to the subject in an amount ranging from one of the following selected ranges: 1 mg/kg/day to 1000 mg/kg/day, 1 mg/kg/day to 100 mg/kg/day; 1 mg/kg/day to 10 mg/kg/day; 1 mg/kg/day to 1 mg/kg/day; 1 mg/kg/day to 100 mg/kg/day; 1 mg/kg/day to 10mg/kg/day; 10 mg/kg/day to 1000 mg/kg/day, 10 mg/kg/day to 100 mg/kg/day; 10 mg/kg/day to 10 mg/kg/day; 10 mg/kg/day to 1 mg/kg/day; 10 mg/kg/day to 100 mg/kg/day; 10 mg/kg/day to 1000 mg/kg/day, 100 mg/kg/day to 100 mg/kg/day; 100 mg/kg/day to 10 mg/kg/day; 100 mg/kg/day to 1 mg/kg/day; 1 mg/kg/day to 1000 mg/kg/day, 1 mg/kg/day to 100 mg/kg/day; 1 mg/kg/day to 10 mg/kg/day; 10 mg/kg/day to 1000 mg/kg/day; 10 mg/kg/day to 100 mg/kg/day; and 100 mg/kg/day to 1000 mg/kg/day body weight. Other ranges are contemplated.
[0092] The agent may be administered to the subject in a suitable form. In this regard, the terms “administering” or “providing” includes administering the agent, or administering a prodrug, or a derivative, that will form an effective amount of the desired agent within the body of the subject. The terms include routes of administration that are systemic (e.g., via injection such as intravenous injection, orally in a tablet, pill, capsule, or other dosage form useful for systemic administration), and topical (e.g., creams, solutions, pastes, ointment, including solutions such as mouthwashes, for topical oral administration). Other routes of administration are contemplated.
[0093] In certain embodiments, the agent is administered intraventricularly, intracranialy or by intramuscular injection. In certain embodiments, the agent is administered orally. In certain embodiments, the agent is administered intravenously. In certain embodiments, the agent is administered intraperitoneally. In certain embodiments, the agent is administered intravenously. In certain embodiments, the agent is administered via injection, such as intravenous injection. In certain embodiments, the agent is administered by nebulized administration, by aerosolized administration or by being instilled into the lung. In certain embodiments, the agent is administered by implant. In certain embodiments, the agent is administered by subcutaneous injection, intra-articularly, rectally, intranasally, intraocularly, vaginally, or transdermally. Methods of administration are known in the art.
[0094] Other forms/routes of administration are contemplated.
[0095] "Intravenous administration" is the administration of substances directly into a vein.
[0096] "Oral administration" is a route of administration where a substance is taken through the mouth, and includes buccal, sublabial and sublingual administration, as well as enteral administration. Typical forms for the oral administration of therapeutic agents includes the use of tablets or capsules.
[0097] The agent may be administered alone or may be delivered in a mixture with other therapeutic agents and/or agents that, for example, enhance, stabilise or maintain the activity of the agent. In certain embodiments, an administration vehicle is used (e.g., pill, tablet, implant, injectable solution, etc.) and would contain both the agent and additional agent(s).
[0098] The methods as described herein may also include combination therapy. In this regard, the subject may be treated or given another drug or treatment modality in conjunction with the agent as described herein. This combination therapy can be sequential therapy where the subject is treated first with one and then the other, or the two or more treatment modalities are given simultaneously.
[0099] "Co-administering" or "co-administration" refers to the administration of two or more therapeutic agents together at one time. The two or more therapeutic agents can be co-formulated into a single dosage form or "combined dosage unit", or formulated separately and subsequently combined into a combined dosage unit, typically for intravenous administration or oral administration.
[00100] When administered to a subject the therapeutically effective dosage of an agent may vary depending upon the particular agent utilized, the mode of administration, the condition, and severity thereof, as well as the various physical factors related to the subject being treated. The dosages are expected to vary with route of administration, and the nature of the agent administered and any other agents administered. In certain embodiments, the methods comprise administering to the subject escalating doses of the agent and/or repeated doses.
[00101] In certain embodiments, the agent is administered as a continuous release formulation.
[00102] In certain embodiments, the agent is administered as an immediate release formulation. The term "immediate release formulation" is a formulation which is designed to quickly release a therapeutic agent in the body over a shortened period of time. Immediate release formulations are known in the art.
[00103] In certain embodiments, the agent is administered as a sustained release formulation. The term "sustained release formulation" is a formulation which is designed to slowly release a therapeutic agent in the body over an extended period of time. Sustained release formulations are known in the art.
[00104] In certain embodiments, the agent is administered as a pharmaceutically acceptable salt. In this regard, the term "pharmaceutically acceptable salt" refers to acid addition salts or metal complexes which are commonly used in the pharmaceutical industry. Metal complexes include zinc, iron, and the like. Suitable acids for use in the preparation of pharmaceutically acceptable salts include, but are not limited to, acetic acid, 2,2-dichloroacetic acid, acylated amino acids, adipic acid, alginic acid, ascorbic acid, L-aspartic acid, benzenesulfonic acid, benzoic acid, A- acetamidobenzoic acid, boric acid, (+)-camphoric acid, camphorsulfonic acid, (+)-(lS)- camphor- lO-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, cyclohexanesulfamic acid, dodecylsulfuric acid, ethane- l,2-disulfonic acid, ethanesulfonic acid, 2-hydroxy-ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid, D-glucuronic acid, L-glutamic acid, oxo-glutaric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, hydroiodic acid, (+)-L-lactic acid, (±)-DL-lactic acid, lactobionic acid, lauric acid, maleic acid, (-)-L-malic acid, malonic acid, (±)-DL-mandelic acid, methanesulfonic acid, naphthalene-2 -sulfonic acid, naphthalene- 1, 5 -disulfonic acid, 1 -hydroxy -2 -naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, perchloric acid, phosphoric acid, L-pyroglutamic acid, saccharic acid, salicylic acid, 4-amino- salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, suberic acid, valeric acid and the like.
[00105] Suitable bases for use in the preparation of pharmaceutically acceptable salts, including, but not limited to, inorganic bases, such as magnesium hydroxide, calcium hydroxide, potassium hydroxide, zinc hydroxide, or sodium hydroxide; and organic bases, such as primary, secondary, tertiary, and quaternary, aliphatic and aromatic amines, including L-arginine, benethamine, benzathine, choline, deanol, diethanolamine, diethylamine, dimethylamine, dipropylamine, diisopropylamine, 2- (diethylamino)-ethanol, ethanolamine, ethylamine, ethylenediamine, isopropylamine, N-methyl-glucamine, hydrabamine, IH- imidazole, L-lysine, morpholine, 4-(2- hydroxyethyl)-morpholine, methylamine, piperidine, piperazine, propylamine, pyrrolidine, l-(2-hydroxyethyl)-pyrrolidine, pyridine, quinuclidine, quinoline, isoquinoline, secondary amines, triethanolamine, trimethylamine, triethylamine, N- methyl-D-glucamine, 2-amino-2-(hydroxymethyl)- 1,3 -propanediol, tromethamine, and the like.
[00106] In certain embodiments, the administration comprises systemic administration.
[00107] In certain embodiments, the administration comprises intraventricular, intracranial or intramuscular administration.
[00108] In certain embodiments, the method is used to treat a brain injury in a subject.
[00109] In certain embodiments, the method is used to reduce the appearance and/or progression of symptoms associated with a brain injury. [00110] In certain embodiments, the method is used to provide an early intervention in a subject presenting with a brain injury.
[00111] In certain embodiments, the present disclosure provides a method of improving recovery from a brain injury involving neuronal damage in a subject, the method comprising administering an effective amount of an inhibitor of a p75 neurotrophin receptor to the subject and thereby improving recovery from the brain injury.
[00112] Brain injuries are as described herein. In certain embodiments, the brain injury comprises one or more of an ischemic injury, a traumatic injury, a haemorrhagic injury, and an epileptic injury.
[00113] In certain embodiments, the brain injury is associated with a cerebral occlusion.
[00114] In certain embodiments, the brain injury comprises a stroke.
[00115] Improvements in recovery are as described herein. In certain embodiments, the improvement in recovery comprises an improvement in motor function and/or reducing neuronal cells death.
[00116] In certain embodiments, the improvement in recovery comprises a reduction in size of an infarct associated with the stroke.
[00117] Inhibitors of a p75 neurotrophin receptor are as described herein.
[00118] In certain embodiments of the present disclosure, the inhibitor comprises all or part of the ectodomain of the p75 neurotrophin receptor.
[00119] In certain embodiments, the inhibitor comprises all or part of the ectodomain of the p75 neurotrophin receptor fused to a Fc region of an antibody.
[00120] In certain embodiments, the p75ECD comprises a p75ECD-Fc fusion. In an embodiment, the p75ECD-Fc fusion is a recombinant peptide. [00121] In certain embodiments, the inhibitor comprises a p75ECD-Fc. For example, recombinant p75ECD-Fc may be obtained from Fujian Tiantai Medical Technology, China.
[00122] P75 neurotrophin receptor antagonists are described, for example, in Other P75 neurotrophin receptor antagonists are described, for example, in Josephy-Hemandez el al (2017) Neurobiology of Disease 97:139-155. Examples of p75 neurotrophin receptor antagonists include THX-B (l,3-diisopropyl-l-[2-(l,3-dimethyl-2,6-dioxo-l, 2,3,6- tetrahydro-purin-7-yl)-acetyl]-urea), THX-A and THX-C (also known as FM11A-2426) as described in Yang et al (2008) PLoS ONE 3: e3604.
[00123] In certain embodiments, the inhibitor comprises an agent that binds to a p75 neurotrophin receptor. Methods for assessing binding of agents to a p75 neurotrophin receptor are known in the art.
[00124] In certain embodiments, the inhibitor of a p75 neurotrophin receptor comprises a ligand antagonist.
[00125] In certain embodiments, the ligand antagonist comprises a ligand mimetic or an antibody or an antigen binding part thereof.
[00126] ] In certain embodiments, the ligand antagonist comprises a ligand mimetic or an antibody or an antigen binding part thereof.
[00127] In certain embodiments, the ligand antagonist comprises one or more of a proBDNF antagonist, a proNGF antagonist, a proNT3 antagonist, and a proNT4 antagonist.
[00128] In certain embodiments, the proBDNF antagonist comprises PNS-Rbl, which is commercially available.
[00129] In certain embodiments, the ligand antagonist comprises an antibody to proBDNF, proNGF, proNT3 antagonist, or proNT4. Examples of such antibodies are available commercially. [00130] In certain embodiments, the ligand antagonist comprises a polypeptide/protein encoding all or part of the soluble extracellular domains of the receptors p75 and/or Sortilin/SORCS2, which are commercially available.
[00131] In certain embodiments, the ligand antagonist comprises an inhibitor of the formation of the ligand.
[00132] In certain embodiments, the proBDNF antagonist comprises PNS-Rbl, which is commercially available.
[00133] Methods of administration are as described herein. In certain embodiments, the administration comprises systemic administration.
[00134] Certain embodiments of the present disclosure provide use of an inhibitor of p75 neurotrophin receptor for improving recovery from a brain injury involving neuronal damage.
[00135] Certain embodiments of the present disclosure provide use of an inhibitor of p75 neurotrophin receptor in the preparation of a medicament for improving recovery from a brain injury involving neuronal damage.
[00136] Certain embodiments of the present disclosure provide a method of treating a brain injury involving neuronal damage in a subject.
[00137] In certain embodiments, the present disclosure provides a method of treating a brain injury involving neuronal damage in a subject, the method comprising administering an effective amount of an inhibitor of a p75 neurotrophin receptor to the subject and thereby treating the brain injury.
[00138] Methods of administration are as described herein. In certain embodiments, the administration comprises systemic administration. In certain embodiments, the administration comprises intraventricular, intracranial or intramuscular administration.
[00139] In certain embodiments, the method is used to recover from a brain injury. [00140] In certain embodiments, the method is used to reduce the appearance and/or progression of symptoms associated with a brain injury.
[00141] In certain embodiments, the method is used to provide an early intervention in a subject presenting with a brain injury.
[00142] In certain embodiments, the subject is suffering from a brain injury. In certain embodiments, the subject is recovering from a brain injury.
[00143] In certain embodiments, the subject is susceptible to a brain injury. In certain embodiments, the method is a prophylactic method.
[00144] Brain injuries are as described herein. In certain embodiments, the brain injury comprises one or more of an ischemic injury, a traumatic injury, a haemorrhagic injury, and an epileptic injury.
[00145] In certain embodiments, the brain injury is associated with a cerebral occlusion.
[00146] In certain embodiments, the brain injury comprises a stroke.
[00147] Inhibitors of a p75 neurotrophin receptor are as described herein.
[00148] In certain embodiments of the present disclosure, the inhibitor comprises all or part of the ectodomain of the p75 neurotrophin receptor.
[00149] In certain embodiments, the inhibitor comprises all or part of the ectodomain of the p75 neurotrophin receptor fused to a Fc region of an antibody.
[00150] In certain embodiments, the p75ECD comprises a p75ECD-Fc fusion. In an embodiment, the p75ECD-Fc fusion is a recombinant peptide.
[00151] In certain embodiments, the inhibitor comprises a p75ECD-Fc. For example, recombinant p75ECD-Fc may be obtained from Fujian Tiantai Medical Technology, China. [00152] P75 neurotrophin receptor antagonists are described, for example, in Other P75 neurotrophin receptor antagonists are described, for example, in Josephy-Hemandez el al (2017) Neurobiology of Disease 97:139-155. Examples of p75 neurotrophin receptor antagonists include THX-B (l,3-diisopropyl-l-[2-(l,3-dimethyl-2,6-dioxo-l, 2,3,6- tetrahydro-purin-7-yl)-acetyl]-urea), THX-A and THX-C (also known as LM11A-2426) as described in Yang et al (2008) PLoS ONE 3: e3604.
[00153] In certain embodiments, the inhibitor comprises an agent that binds to a p75 neurotrophin receptor. Methods for assessing binding of agents to a p75 neurotrophin receptor are known in the art.
[00154] In certain embodiments, the inhibitor of a p75 neurotrophin receptor comprises a ligand antagonist.
[00155] In certain embodiments, the ligand antagonist comprises a ligand mimetic or an antibody or an antigen binding part thereof.
[00156] ] In certain embodiments, the ligand antagonist comprises a ligand mimetic or an antibody or an antigen binding part thereof.
[00157] In certain embodiments, the ligand antagonist comprises one or more of a proBDNF antagonist, a proNGF antagonist, a proNT3 antagonist, and a proNT4 antagonist.
[00158] In certain embodiments, the proBDNF antagonist comprises PNS-Rbl, which is commercially available.
[00159] In certain embodiments, the ligand antagonist comprises an antibody to proBDNF, proNGF, proNT3 antagonist, or proNT4. Examples of such antibodies are available commercially.
[00160] In certain embodiments, the ligand antagonist comprises a polypeptide/protein encoding all or part of the soluble extracellular domains of the receptors p75 and/or Sortilin/SORCS2, which are commercially available. [00161] In certain embodiments, the ligand antagonist comprises an inhibitor of the formation of the ligand.
[00162] In certain embodiments, the proBDNF antagonist comprises PNS-Rbl, which is commercially available.
[00163]
[00164] Certain embodiments of the present disclosure provide a pharmaceutical composition.
[00165] In certain embodiments, the present disclosure provides a pharmaceutical composition for treating a brain injury involving neuronal damage, the pharmaceutical composition comprising an effective amount of an inhibitor of a p75 neurotrophin receptor.
[00166] Inhibitors of a p75 neurotrophin receptor are as described herein.
[00167] In certain embodiments of the present disclosure, the inhibitor comprises all or part of the ectodomain of the p75 neurotrophin receptor.
[00168] In certain embodiments, the inhibitor comprises all or part of the ectodomain of the p75 neurotrophin receptor fused to a Fc region of an antibody.
[00169] In certain embodiments, the p75ECD comprises a p75ECD-Fc fusion. In an embodiment, the p75ECD-Fc fusion is a recombinant peptide.
[00170] In certain embodiments, the inhibitor comprises a p75ECD-Fc. For example, recombinant p75ECD-Fc may be obtained from Fujian Tiantai Medical Technology, China.
[00171] P75 neurotrophin receptor antagonists are described, for example, in Josephy- Hernandez et al (2017) Neurobiology of Disease 97:139-155. Examples of p75 neurotrophin receptor antagonists include THX-B (l,3-diisopropyl-l-[2-(l, 3-dimethyl- 2,6-dioxo-l,2,3,6-tetrahydro-purin-7-yl)-acetyl]-urea), THX-A and THX-C (also known as LM11A-2426) as described in Yang et al (2008) PLoS ONE 3: e3604.
[00172] In certain embodiments, the inhibitor comprises an agent that binds to a p75 neurotrophin receptor. Methods for assessing binding of agents to a p75 neurotrophin receptor are known in the art.
[00173] In certain embodiments, the inhibitor of a p75 neurotrophin receptor comprises a ligand antagonist.
[00174] In certain embodiments, the ligand antagonist comprises a ligand mimetic or an antibody or an antigen binding part thereof.
[00175] In certain embodiments, the ligand antagonist comprises one or more of a proBDNF antagonist, a proNGF antagonist, a proNT3 antagonist, and a proNT4 antagonist.
[00176] In certain embodiments, the proBDNF antagonist comprises PNS-Rbl, which is commercially available.
[00177] In certain embodiments, the ligand antagonist comprises an antibody to proBDNF, proNGF, proNT3 antagonist, or proNT4. Examples of such antibodies are available commercially.
[00178] In certain embodiments, the ligand antagonist comprises a polypeptide/protein encoding all or part of the soluble extracellular domains of the receptors p75 and/or Sortilin/SORCS2, which are commercially available.
[00179] In certain embodiments, the ligand antagonist comprises an inhibitor of the formation of the ligand.
[00180] In certain embodiments, the proBDNF antagonist comprises PNS-Rbl, which is commercially available. [00181] In certain embodiments, the composition comprises a liquid composition, a semi-solid composition, a suppository, a gel, a solid, a tablet, a capsule, a cream, a solution, a paste, or an ointment. In certain embodiments, the composition comprises a substrate with a releasable agent that reduces spinal glial activation.
[00182] Compositions for administration by various routes are known in the art.
[00183] Additional numerous various excipients, dosage forms, dispersing agents and the like that are suitable for use in connection with administration and/or the formulation into medicaments or compositions are known and described in, for example, Remington's Pharmaceutical Sciences, l7th ed., Mack Publishing Company, Easton, Pa., 1985, which is incorporated herein by reference in its entirety. Methods for formulating compositions are known in the art.
[00184] A suitable dosage of the agent may be selected. In certain embodiments, the composition comprises the agent that reduces spinal glial activation in an amount ranging from 1 pg to 500 mg, 10 pg to 500 mg, 10 pg to 100 mg, 10 pg to 10 mg, 10 pg to 1 mg, 10 pg to 100 pg, 100 pg to 500 mg, 100 pg to 100 mg; 100 pg to 10 mg; 100 pg to 1 mg, 1 mg to 500 mg 1 mg to 100 mg, or 1 mg to 10 mg. Other ranges are contemplated. Other amounts are contemplated.
[00185] In certain embodiments, the composition comprises the agent in an amount to provide a dose ranging from one of the following selected ranges: 0.1 pg/kg to 10 mg/kg; 0.1 pg/kg to 1 mg/kg; 0.1 pg/kg to 100 pg/kg; 0.1 pg/kg to lOpg/kg; 1 pg/kg to 10 mg/kg; 1 pg/kg to 1 mg/kg; 1 pg/kg to 100 pg/kg; 1 pg/kg to lOpg/kg; 10 pg/kg to 10 mg/kg; 10 pg/kg to 1 mg/kg; 10 pg/kg to 100 pg/kg; 100 pg/kg to 10 mg/kg; 100 pg/kg to 1 mg/kg; or 1 mg/kg to 10 mg/kg. Other ranges are contemplated.
[00186] In certain embodiments, the composition comprises the agent in an amount to provide a dose ranging from one of the following selected ranges: 0.1 pg/kg/day to 10 mg/kg/day; 0.1 pg/kg/day to 1 mg/kg/day; 0.1 pg/kg/day to 100 pg/kg/day; 0.1 pg/kg/day to 10 pg/kg/day; 1 pg/kg/day to 10 mg/kg/day; 1 pg/kg/day to 1 mg/kg/day; 1 pg/kg/day to 100 pg/kg/day; 1 pg/kg/day to 10 pg/kg/day; 10 pg/kg/day to 10 mg/kg/day; 10 mg/kg/day to 1 mg/kg/day; 10 mg/kg/day to 100 mg/kg/day; 100 mg/kg/day to 10 mg/kg/day; 100 mg/kg/day to 1 mg/kg/day; or 1 mg/kg/day to 10 mg/kg/day. Other ranges are contemplated.
[00187] In certain embodiments, the composition comprises an acceptable carrier suitable for administering the composition to a subject. The carrier may be chosen based on various considerations including the agent(s) being delivered and the time course of delivery of the agents. The term "acceptable carrier" as used herein refers to a substantially inert solid, semi-solid or liquid filler, diluent, excipient, encapsulating material or suitable auxiliary formulation . Physiologically acceptable carriers and their formulations are known in the art.
[00188] In certain embodiments, the composition is suitable for administering the agent to the subject on a regular basis, such as twice daily, daily, weekly, monthly, annually or multi-annually administration.
[00189] In certain embodiments, the composition is suitable for continuous administration of the agent.
[00190] In certain embodiments, the composition is an immediate release formulation.
[00191] In certain embodiments, the composition is a slow/sustained release formulation.
[00192] In certain embodiments, the composition provides long-term administration of the agent. In certain embodiments, the composition provides long-term continuous administration of the agent.
[00193] In certain embodiments, the composition comprises a substrate with a releasable form of the agent.
[00194] In certain embodiments, the composition is an intraventricular composition, an intracranial composition, an intramuscular composition, an oral composition, or a intravenous composition. [00195] An example of a suitable composition for intravenous injection includes the agent in a suitable carrier, such as isotonic saline.
[00196] Certain embodiments of the present disclosure provide a method of treating a subject suffering from a brain injury involving neuronal damage, the method comprise administering a pharmaceutical composition as described herein.
[00197] Certain embodiments of the present disclosure provide methods for identifying or screening for therapeutic agents for improving recovering from a brain injury involving neuronal damage.
[00198] In certain embodiments, the present disclosure provides a method of identifying a therapeutic agent for improving recovery from a brain injury involving neuronal damage, the method comprising determining the ability of an inhibitor of a p75 neurotrophin receptor to improve recovery from a brain injury involving neuronal damage and identifying the inhibitor of a p75 neurotrophin receptor agent as a therapeutic agent for improving recovery from a brain injury involving neuronal damage.
[00199] In certain embodiments, the present disclosure provides a method of identifying a therapeutic agent for improving recovery from a brain injury involving neuronal damage, the method comprising:
identifying a candidate agent as an inhibitor of a p75 neurotrophin receptor; and determining the ability of the inhibitor of a p75 neurotrophin receptor to improve recovery from a brain injury involving neuronal damage,
thereby identifying the candidate agent as a therapeutic agent for improving recovery from a brain injury involving neuronal damage.
[00200] In certain embodiments, the present disclosure provides a method of identifying a therapeutic agent for treating a brain injury involving neuronal damage, the method comprising determining the ability of an inhibitor of a p75 neurotrophin receptor to improve recovery from a brain injury involving neuronal damage and identifying the inhibitor of a p75 neurotrophin receptor agent as a therapeutic agent for treating a brain injury involving neuronal damage.
[00201] In certain embodiments, the present disclosure provides a method of identifying a therapeutic agent for treating a brain injury involving neuronal damage, the method comprising:
identifying a candidate agent as an inhibitor of a p75 neurotrophin receptor; and determining the ability of the inhibitor of a p75 neurotrophin receptor to treat a brain injury involving neuronal damage,
thereby identifying the candidate agent as a therapeutic agent for treating a brain injury involving neuronal damage.
[00202] In certain embodiments, the brain injury comprises one or more of an ischemic injury, a traumatic injury, a haemorrhagic injury, and an epileptic injury.
[00203] Agents so identified are potential therapeutic agents.
[00204] Examples of candidate agents include a drug, a small molecule, a protein, a polypeptide, a lipid, a carbohydrate, a nucleic acid, an oligonucleotide, a ribozyme, a biologic, a recombinant peptide, an aptamer, a cofactor, a ligand, a ligand mimetic, a receptor, a peptidomimetic, an enzyme, a kinase, a phosphatase, a cytokine, a growth factor, a metal ion, a chelate, an antisense nucleic acid, an inhibitor RNA, a microRNA, a siRNA, an antibody or antigen binding part thereof, an antibody mimetic. Other types of agents are contemplated.
[00205] Methods for determining the ability of an agent to improve recovery from a brain injury involving neuronal damage are as described herein.
[00206] In certain embodiments, the method of identifying comprises use of in vitro studies and/or use of an animal model(s).
[00207] In certain embodiments, the present disclosure provides a kit for performing a method as described herein. [00208] Standard techniques and equipment may be used for recombinant DNA technology, oligonucleotide synthesis, molecular biology and enzymatic reactions. The foregoing techniques and procedures may be generally performed according to methods known in the art and/or as commercially available, and are as described for example in Sambrook et al. Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)) and Ausubel et al Current Protocols in Molecular Biology (2003) John Wiley & Sons, both of which are herein incorporated by reference.
[00209] The present disclosure is further described by the following examples. It is to be understood that the following description is for the purpose of describing particular embodiments only and is not intended to be limiting with respect to the above description.
EXAMPLE 1 - IMPROVING RECOVERY FROM ISCHEMIC BRAIN DAMAGE
USING P75 INHIBITORS
[00210] This present studies s related to the medicinal field in which an agent has been identified for the treatment of brain injuries such as ischemic stroke as a neuroprotection agent. In one embodiment, the agent drug utilises the extracellular domain sequence of human neurotrophin receptor p75 fused with human immunoglobulin fragment C for the treatment of nerve damage induced by ischemia. Because it can protect brain from ischemic injury, it is expected that the agent may also be used for the treatment of other brain damage, such as trauma and inflammation. The agent is antagonize ligands of p75 receptors, which are regarded as pathological factors. In addition, antibodies to proBDNF, a ligand of p75, are also neuroprotective. Thus the present studies demonstrate that inhibitors against p75 or p75 ligands are neuroprotective in brain injuries involving neuronal damage, such as ischemic stroke.
[00211] INTRODUCTION
[00212] Neurotrophins are a small family of secreted neurotrophic factors that act via complicated signalling pathways that result in a broad spectrum of actions in the nervous system, including involvement in neurogenesis, neuronal survival, proliferation, differentiation, myelination, axonal growth and synaptic plasticity, and conversely, apoptosis and cell death, at all stages of neuronal development, adult life, neuronal injury and disease. The neurotrophin family includes nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3) and neurotrophin-4/5 (NT4/5). Neurotrophins mediate their effects by binding to at least three different types of receptors, tropomyosin-related kinase (Trk) receptors (TrkA, TrkB and TrkC), sortilin, and the p75 neurotrophin receptor (p75;synonyms include p75NTR, p75(NTR), Gp80-LNGFR, Low-affinity nerve growth factor receptor, Low affinity neurotrophin receptor, NGF receptor, and tumour necrosis factor receptor superfamily member 16 precursor (TNFRSF16)). High affinity binding of neurotrophins frequently involves two of these receptor types interacting as co-receptors. p75 mediates survival or apoptosis of neurons depending on relative expression levels of high affinity neurotrophin Trk receptors.
[00213] Alteration in neurotrophin levels and signalling pathways have been implicated with the neuronal survival and pathophysiology in a wide variety of neurodegenerative and psychiatric disorders including Alzheimer's Disease (AD), cerebral amyloid angiopathy (CAA), Huntington's Disease, Parkinson's Disease and amyotrophic lateral sclerosis (ALS) and tauopathies, as well as neuronal injury such as spinal cord injury; although no human disease has yet been shown to be caused directly by a defect in neurotrophins or their receptors.
[00214] Cerebral ischemia instigates multifaceted cellular functions that trigger cell death, inflammation, cognitive and sensorimotor dysfunction. The detrimental effects of ischemia are believed to be due to certain deviations of physiological factors after ischemia. Several studies have showed that different signaling cascades are altered in ischemic condition and responsible for continuing post-ischemic damages.
[00215] Brain-derived neurotrophic factor (BDNF) is generated from its precursor preproBDNF and proBDNF, which are physiologically processed by removing the signalling peptide and the prodomain, respectively. ProBDNF interacts with p75/sortilin complex with high affinity and initiates apoptosis, inflammation, long term depression (LTD), excitation, persistent cortical neuronal firing and neuronal inhibition. On the contrary to proBDNF function, mBDNF has high affinity to tyrosine kinase receptor (TrkB) receptor, and participates in cellular growth, long term potential (LTP) and neuronal survival.
[00216] Structurally, the transmembrane p75 receptor has an extracellular ectodomain (p75ECD) and intracellular domain (p75ICD). ICD holds a death domain in the cytoplasmic region. The main signalling pathways for p75 receptor are-cell surviving NF-kB and apoptotic Jun kinase (JNK) pathways. Neurotrophins bind to the ectodomain part of the receptor and transduce signal through ICD inside the cell. Mature neurotrophins have low affinity, and on the contrary, proneurotrophins have higher affinity to bind with this transmembrane receptor. Interaction with mature neurotrophins aids in the survival functions of the p75 receptor, such as neuronal survival, differentiation, synaptic plasticity. On the contrary, interactions between p75 receptor and proneurotrophins leads to the damaging events, such as apoptosis, inflammation, neurite outgrowth inhibition and long term depression (LTD).
[00217] METHODS
[00218] Photothrombotic Ischemic Model: Sprague-Dawley rats (250-350 grams) were used at 8-12 weeks of age for the experimental purpose. Focal ischemia was induced on the rat brain by intravenous (i.v.) injection of the photosensitive Rose Bengal (RB) dye (Sigma- Aldrich, USA. Cat#330000-lG) through tail vein (at 20 mg/kg) followed by the skull exposure to the cold light for 20 min (Schmidt, Hoppen et al. 2012, Madinier, Bertrand et al. 2013). The surgical procedure uses is as described in Rahman, Luo et al. (2018). Control group animals did not receive any surgical procedure.
[00219] All ischemic rats were grouped and received p75ECD-Fc and Human Fc for intervention through the intraperitoneal (i.p.) route (Table 1). Recombinant protein p75ECD-Fc was obtained from Fujian Tiantai Medical Technology, China, and Human Fc was extracted from BL21 bacteria following IPTG induction. Table 1: Treatments received by animals
Groups Treatment Dosage No. of Euthanizing animals time point
7d stroke No No 8 7d
1 mg/kg once at 6h
7d stroke+p75ECD- p75ECD- after ischemic 8 7d Fc lmg Fc
induction
3 mg/kg once at 6h
7d stroke+p75ECD- p75ECD- after ischemic 8 7d Fc 3mg Fc
induction
lOmg/kg once at
7d stroke+p75ECD- p75ECD- 6h after ischemic 8 7d
Fc lOmg Fc
induction
lOmg/kg once at
7d stroke+Human Fc Human Fc 6h after ischemic 8 7d
induction
l4d stroke No No 8 l4d
1 mg/kg once at 6h
l4d stroke+p75ECD- p75ECD- after ischemic 8 l4d Fc lmg Fc
induction
3 mg/kg once at 6h
l4d stroke+p75ECD- p75ECD- after ischemic 8 l4d Fc 3mg Fc
induction
lOmg/kg once at
l4d stroke+p75ECD- p75ECD- 6h after ischemic 8 l4d Fc lOmg Fc
induction
lOmg/kg once at
l4d stroke+p75ECD- p75ECD- 6h and 7d after 8 l4d Fc lOmg (lxwk) Fc
ischemic induction
lOmg/kg once at
l4d stroke+Human
Human Fc 6h after ischemic 8 l4d Fc
induction
[00220] In a separate study, rats were also treated with sheep antibodies to proBDNF or sheep IgG by intraperitoneal injection at dosage of 5 mg/kg twice a week (Table 2). Table 2: Treatments given to animals
Groups Treatment Dosage No. of Euthanizing animals time point
7d stroke No No 8 7d anti-
7d stroke+anti-
5mg/kg once at 6h
proBDNF 8 7d
and on 3d after
proBDNF IgG
ischemic induction
IgG
7d stroke+Sheep
5mg/kg once at 6h
Sheep IgG 8 7d
and on 3d after
IgG ischemic induction
[00221] Behavioural Studies: Four behavioural tests were performed in order to understand the sensorimotor function- the cylinder, rotarod, corner and conventional adhesive tape tests. Rats were under training for rotarod and conventional adhesive tape tests approximately for 1 week before surgery. The baseline data was collected on the day before surgery and considered as control data. Later, all behavioural tests were performed again at 7 day (7d) and l4d after surgery.
[00222] A: Cylinder Test: The cylinder test was performed to obtain the fine motor function (Takamatsu, Tamakoshi et al. 2016). The method for this behavioural test was described in chapter 2. Animals were positioned in a plastic cylinder and the contact numbers on the cylinder wall with the forelimbs by expanding themselves above their shoulder height were counted for 5 min.
[00223] B. Rotarod Test: The rotarod test assesses the forced motor function to understand the physical state for balance and movements (Hamm, Pike et al. 1994). Animals were positioned on a whirling rod. The rod started rotation from 0 rpm and reached to 30rpm within 2 min by increasing 3 rpm speed every 10 sec. Both the time (sec) and speed (rpm) were recorded as soon as the animal was fallen off the rod. [00224] C. Corner Test: This test is assessing the sensorimotor function of the animal. A comer board with an angle of 30° approximately of a rat cage height was placed inside the cage. Animal was allowed to move 10 times inside the corner and turn backward to come in the free side. The direction of turning (left or right) backward after each contact with the corner was closely monitored and recorded.
[00225] D. Conventional Adhesive Tape Test This behavioural test was used to assess the sensorimotor functions of the animal. An adhesive tape of lcm was applied on each limb of the animal, and the time to contact (sensory) and removal (motor) was recorded in 1 min task time (Sughrue, Mocco et al. 2006).
[00226] Tissue Collection: After behavioural studies, animals were killed at 7d and l4d and tissues were collected. A half of the infarcted ipsilateral hemisphere immediately frozen and stored at -80°C is used for biochemical assays. The other half of the infarcted ipsilateral brain section was collected for histological assays, and tissues were fixed in 4% paraformaldehyde followed by the dehydration in 30% sucrose and stored at 4°C until sectioning.
[00227] Immunohistochemistry
[00228] A. DAB Staining. Paraformaldehyde-fixed infarcted ipsilateral brain tissues were embedded in optical cutting temperature compound (OCT) and were sectioned at l5pm thickness using a cryostat (Leica, Vic, Australia). Brain sections were preserved in anti-freeze solution and stored at -20°C until used. Prior staining, sections were washed in PBS and mounted on gelatin coated slides. After sections were attached on the slides, antigen retrieval step was performed by incubating sections in 1% sodium dodecyl sulphate (SDS) for 5 min at room temperature. 3% hydrogen peroxide (H202) was applied on the sections for 10 min in order to block the endogenous peroxidase. After blocking in 5% bovine serum albumin (BSA)-PBST for lh at room temperature, sections were incubated with the following primary antibodies overnight at 4°C - 1:1000 monoclonal anti-proBDNF 1D3 (China), 1:1000 rabbit anti-microtubule-associated protein 2 (MAP2) (Osenses, Australia), 1:1000 rabbit anti-glial fibrillary acidic protein (GFAP) (Abeam, Vic, Australia) and 1:500 rabbit anti- ionized calcium-binding adapter molecule l(IBA-l) (WAKO, Japan). Respective biotin-conjugated secondary antibodies were applied (1:2000) on the sections for 2h at room temperature followed by incubation in ABC reagent (Vectra Laboratories, USA) according to the manufacturer's protocol. All sections were incubated in DAB substrate according to the manufacturer's (Osenses, Australia) instructions. Slides were dehydrated by immersing the slides in graded alcohol and xylene, and coverslipped using Depex mounting medium (Sigma- Aldrich, St Louis, MO, USA). All sections were imaged using Olympus BX53 Light microscope (Olympus, NSW, Australia). Immuno positive cells were measured by measuring the optical density of five different areas in ImageJ software and expressed as % of area fraction.
[00229] B. Immunofluorescent Staining. Prior primary antibody incubation, all steps were similar to the 'DAB staining' except immersion of sections into 3% hydrogen peroxide. After blocking, section were incubated with the following primary antibodies overnight at 4°C - 1:1000 rabbit anti-p75 (Department of Cell Biology, Skirball Institute, New York, USA), 1:1000 anti-Tujl (Merck Millipore, Vic, Australia), 1:500 goat anti-GFAP (Santa Cruz Biotechnology, Inc., US A) and 1:500 mouse anti-IB A- 1 (Santa Cruz Biotechnology, Inc., USA). For secondary antibody incubations, Cy2 conjugated anti-goat and anti-mouse, and Cy3 conjugated anti-rabbit antibodies were used. Sections were counterstained with 4', 6-Diamidino-2-phenylindole (DAPI) and coverslipped using 90% glycerol for imaging under fluorescent microscope (Zeiss LSM 710, Germany).
[00230] C. Haematoxylin and Eosin (H&E) Staining. Brain sections were mounted on gelatin coated slides and allowed to be fixed. After a brief wash in distilled water, sections were stained in Ehrlich's haematoxylin for 30 sec-2 min and washed in distilled water. Sections were differentiated briefly in 1% acid-alcohol followed by a wash in distilled water. Slides were immersed in ammonia water for blueing and rinsed again in distilled water. To finish, sections were counterstained in eosin, and dehydrated in graded alcohol and xylene, and coverslipped for imaging.
[00231] D. Cresyl Violet Staining. Brain sections were mounted on gelatin coated slides and allowed to be fixed. Slides were immersed into 1:1 alcohol/chloroform for a few hours/ overnight and then hydrate back in 95% ethanol. Slides were kept in 0.1% Luxol fast blue solution (0.1 gm Luxol fast blue, 0.5 ml Glacial acetic acid and 100 ml 95% ethanol) for 24h at room temperature followed by rinsing off in 95% ethanol and distilled water. Sections were differentiated in the lithium carbonate solution (0.05 gm Lithium carbonate and 100 ml distilled water) for 30 sec or until clear gray matter and sharp white matter was observed under the microscope. After differentiation slides were washed in distilled water and counterstained in cresyl violet solution (0.1 gm Cresyl fast violet, 100 ml distilled water and 10 drops glacial acetic acid prior filter) for 30-40 sec. Slides were rinsed in distilled water followed by the differentiation in 95% ethanol for 5 min, and dehydrated in graded alcohol and xylene, and coverslipped for imaging.
[00232] TUNNEL Staining. TUNNEL staining was performed to observe the apoptosis around the infarcted area of the ipsilateral hemisphere before and after the p75ECD-Fc treatment. The complete staining procedure was followed by the manufacturer's (Abeam, Vic, Australia, Cat# ab206386) instructions.
[00233] Western Blot RIPA buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% SDS, 2 mM EDTA, 1% Na-deoxycholate and 1% NP-40) along with cocktail inhibitors (protease and phosphatase) (1:500; Roche, Castle Hill, NSW, Australia) were used for homogenizing the frozen ipsilateral infarcted brain tissues. Later, centrifugation of the homogenates was performed at 15000 RPM for 30 min at 4°C and the supernatants were collected for BCA assay (Pierce™ BCA, Thermo Fisher Scientific, Rockford, IL, USA, Protein Assay Kit Cat# 23227) to obtain the total protein concentration. 20 pg of protein were loaded in 10% SDS-polyacrylamide gels and gels were transferred to the nitrocellulose membranes by wet transfer method. After blocking the membranes in 5% milk-TBST (0.1% Tween 20 in TBS) at room temperature for lh, primary antibody incubation step was performed with the following antibodies at 4°C overnight - 1:1000 rabbit anti-JNK (Cell signalling Technology, Danvers Massachusetts), 1:1000 rabbit anti-phospho JNK (JNK(P)) (Cell signalling Technology, Danvers Massachusetts), 1:500 rabbit anti-Poly(ADP-ribose) polymerase-l (PARP-l) (Santa Cruz Biotechnology, Inc., US A), 1:1000 mouse anti-postsynaptic density protein 95 (PSD95) (Sigma- Aldrich, St Louis, MO, US), 1:1000 rabbit anti-synaptosomal-associated protein 25 (SNAP25) (Osenses, Australia), 1:1000 rabbit anti-neuronal nuclei antigen (NeuN) (Abeam, Vic, Australia) and rabbit anti tissue plasminogen inhibitor- 1 (PAI-l) (Abeam, Vic, Australia). After this step, membranes were incubated with the horseradish peroxidase (HRP)-conjugated respective secondary antibodies at 1:2000 dilutions in TBST at room temperature for lh. Finally, to normalize the protein loading per well, B- actin antibody conjugated with HRP (1:10000; Sigma- Aldrich) was used for incubating the membranes at room temperature for 2h. LAS 4000 imager (GE Healthcare, UK) was used to capture the image of the blots and Image J software (NIH, Bethesda, MD, USA) was used for quantitation.
[00234] Statistical studies were completed by Graphpad Prism 7 software (USA). For all analyses, ischemic groups with different treatments were compared against the respective non-treatment stroke groups. Data were presented as mean ± standard error of the mean (SEM). Ordinary one-way ANOVA was done using Tukey's multiple comparisons with the variances. All statistical analyses were done considering the significance threshold among means at P < 0.05.
[00235] RESULTS
[00236] (i) ProBDNF is localized around the ischemic infarct after ischemia
[00237] We investigated the area of up-regulated proBDNF in ischemic brain section after ischemia. In this study we have performed the immunostaining in the ipsilateral brain section with the proBDNF antibody after 3d of the ischemic insult (Fig. 1).
[00238] proBDNF staining was observed in neuron-like cells of sham brain sections. However, proBDNF staining was more intense in neuron-like cells around the ischemic infarct after 3d of the ischemia than the sham control.
[00239] (ii) Localization of p75 in neurons, astrocytes and microglia
[00240] Figure 2 shows localization of p75 around the ischemic infarct.
[00241] We used 15 pm thick brain sections for double labelling of p75 along with Tujl (neuron), GFAP (astrocyte) and IBA-l (microglia). In this experiment we observed that p75 is co-localized with Tujl, GFAP and IBA-l (Fig. 3, 4, 5), which indicated that both neuron and glia cells are expressing p75 around the infarcted area after ischemic insult. [00242] (iii) Effect of anti-proBDNF IgG on the behavioural Study after ischemia - cylinder test
[00243] The treatment groups for anti-proBDNF IgG are provided in Table 2.
[00244] Cylinder Test: Motor functional deficit has been observed in animals at 7d after ischemic insult. In a cylinder test, less contact to the cylinder wall by ischemic rats was noticed at 7d compared to the control group (Fig. 6). After anti-proBDNF IgG treatment a significant improvement in motor function was observed compared to the 7d stroke group. More contact on the cylinder wall was observed in the 7d stroke+anti-proBDNF IgG group (16+2.1 no. of contacts, P=0.006l, n=8) compared to the 7d stroke group.
[00245] (iv) Effect of p75ECD-Fc on behavioural function - cylinder test
[00246] Cylinder Test: After surgery animals were injected (i.p.) p75ECD-Fc at different dosage (Table 2). Human Fc injected group was considered as a vehicle group. In cylinder test we observed that no. of contacts on the cylinder wall was improved in the 7d stroke+p75ECD-Fc lOmg group (16+1.8 no. of contacts, P=0.0059, n=8) compared to the 7d stroke group (Fig. 7). A similar trend was found at l4d after p75ECD-Fc treatment. Fine motor function was improved in the l4d stroke+p75ECD- Fc lOmg(lxwk) group (12+1.1 no. of contacts, P=0.0007, n=8) compared to the l4d stroke group. In both cases, the dose of p75ECD-Fc was similar.
[00247] (v) Effect of anti-proBDNF IgG on the behavioural Study after ischemia - rotarod test
[00248] Anti-proBDNF treatment showed improvement after ischemia in rotarod test (Fig. 8). We found that animals had impairment in running on speedy rod (20+2.6 rpm, P=0.0030, n=8) and finishing the rotarod test on time (75+9.8 sec, P=0.003l, n=8) at 7d after ischemia compared to the control animals. However, after anti-proBDNF IgG treatment animals were able to improve significantly in running on speedy rod (29+0.98 rpm, P=0.0l37, n=8) and completing the task on time (105+3.4 sec, P=0.0l58, n=8) at 7d compared to the 7d stroke group.
[00249] (iv) Effect of p75ECD-Fc on behavioural function - cylinder test [00250] (vi) Effect of p75ECD-Fc on behavioural function - rotarod test
[00251] After p75ECD-Fc treatment, 7d stroke+p75ECD-Fc 3mg group showed improvement in their motor function compared to the other ischemic groups (Fig. 9). This group was performing in this behavioural test better than other ischemic groups. In comparison to the 7d stroke group, this group was showing improvement both in managing the speed of the rod (29+0.49 rpm, P=0.0l22, n=8) and finishing the task on time (107+2.5 sec, P=0.0223, n=8) compared to the 7d stroke group.
[00252] (vii) Effect of anti-proBDNF IgG on the behavioural Study after ischemia - corner test
[00253] Corner Test: In comer test we identified that 7d stroke group had remarkably less right turns (3.625+0.68 no. of turns, P=0.02l5, n=8) compared to the left turns after ischemic insult (Fig. 10). This is suggestive of impaired sensory function after ischemia. After anti-proBDNF IgG treatment we observed increased right turns by the ischemic animals compared to the 7d stroke group, which indicates the betterment in sensory function. However, the normal-sheep IgG treated rats showed more left turn than right turn (p=?), being similar to the control rats. This suggests that anti-proBDNF treatment is beneficial for improving the sensory function after ischemia.
[00254] (viii) Effect of p75ECD-Fc on behavioural function - corner test
[00255] Corner Test: We observed that 7d stroke group had significantly less right turns (3.625+ 0.68 no. of turns, P=0.0088, n=8) compared to the left turns after ischemic insult (Fig. 11). This is indicative to poor sensory function after ischemia. After p75ECD-Fc treatment, number of right and left turns after ischemia is almost equal in all treatment groups. This indicates that p75ECD-Fc treatment is improving the sensory function after ischemia.
[00256] Similar to the rotarod test, l4d stroke group showed self-recovery by themselves after l4d of the post-ischemia and there were no significant changes after p75ECD-Fc treatment for all l4d groups. [00257] (ix) Effect of anti-proBDNF IgG on the behavioural Study after ischemia - conventional adhesive tape test
[00258] .Conventional Adhesive Tape Test: Animals required more time to contact (sensory) and remove (motor) the adhesive tape from their contralateral forepaw after ischemia at 7d compared to the control group (24+6.5 sec, P=0.0085, n=8) (Fig. 12). We found that anti-proBDNF IgG treatment aided animals in sensory function in this test. After treatment animals required significantly less time to contact the adhesive tape at 7d (8.9+1.6 sec, P=0.0498, n=8) on the contralateral forepaw compared to the 7d stroke animals.
[00259] Similar to the contralateral forepaw we observed that animals had a trend to require more time to contact and remove the adhesive tape from the ipsilateral forepaw after ischemia (Fig. 13). We also observed the similar pattern of improvement in both contacting and removing the adhesive tape at 7d after anti-proBDNF IgG treatment than the 7d ischemic group. However, there were no significant changes both in 7d stroke and anti-proBDNF IgG treatment groups compared to the control group.
[00260] (x) Effect of p75ECD-Fc on behavioural function - conventional adhesive tape test
[00261] Conventional Adhesive Tape Test: Different dosage of p75ECD-Fc is improving the sensory and motor function after ischemia. In this test, animals required less time to contact the adhesive tape after p75ECD-Fc treatment proving the betterment in the sensory function after treatment. 7d stroke+p75ECD-Fc lOmg group (4.6+1.4 sec, P=0.0450, n=8) was requiring less time to contact the adhesive tape on their contralateral forepaw compared to the 7d stroke group (Fig. 14). Motor function of the ipsilateral forepaw was improved as they required less time to remove the adhesive tape from the ipsilateral forepaw in the 7d stroke+p75ECD-Fc 3mg group (14+2.6 sec, P=0.0325, n=8) compared to the 7d stroke group.
[00262] Similar to the rotarod and comer test, l4d stroke group showed self recovery by themselves after l4d of the post-ischemia and there were no significant changes after the p75ECD-Fc treatment for all l4d groups (Fig. 15). Therefore, we have focused on the 7d treatment groups only for the histological and biochemical analyses.
[00263] (xi) Anti-proBDNF IgG reduces the infiltration of inflammatory cells in the peri-infarcted area
[00264] In this cresyl violet staining we found significant infiltration of inflammatory cells around the ischemic infarct of the brain sections at 7d (76+11% positive staining as % of area fraction, P=0.000l, n=6) compared to the control group (Fig. 17). Interestingly, anti-proBDNF IgG treatment reduces the infiltration of inflammatory cells significantly at 7d (21+4.1% positive staining as % of area fraction, P=0.0002, n=6) compared to the 7d stroke group.
[00265] P75ECD-Fc reduces the infiltration of inflammatory cells in the peri-infarcted area
[00266] In cresyl violet staining, higher infiltration of inflammatory cells was observed in the 7d stroke group (46+2.2% positive staining as % of area fraction, R=0.0171, n=6) (Fig. 17). Interestingly, at higher dose of p75ECD-Fc (p75ECD-Fc lOmg), less inflammatory cells were observed in the brain sections of the 7d stroke+p75ECD-Fc lOmg group (22+3.6% positive staining as % of area fraction, P=O.Ol38, n=6) compared to the 7d stroke group.
[00267] (xii) Effect of anti-proBDNF IgG on the infarcted area
[00268] H&E stained images were used to understand the changes of the infarction in the brain sections after anti-proBDNF IgG treatment (Fig. 18). Significant reduction of infarcted area was observed after anti-proBDNF IgG treatment at 7d (1.3+0.43 mm2, P=0.0003, n=5) compared to the 7d stroke group.
[00269] (xiii) Effect of p75ECD-Fc on the infarcted area
[00270] H&E staining was performed to understand the morphological changes in different dosage of p75ECD-Fc treatment. We also used the same images to identify the infarcted area by ImageJ software. We found that at the dose of lOmg of p75ECD-Fc, infarcted area is significantly shrunk (1.5+0.48 mm2, R<0.0001, n=5) compared to the 7d stroke group and cortical lesion is mostly covered up by new structure (Fig. 19). This indicates that p75ECD-Fc treatment is promoting the cellular growth and reducing the cortical lesion size.
[00271] (xiv) Effect of anti-proBDNF IgG on the apoptosis around the infarcted area
[00272] Significant increment of apoptotic cells was detected around the ischemic infarct of the brain sections after ischemia at 7d (23+4.7% positive staining as % of area fraction, P=0.00l l, n=6) compared to the Sham group (Fig. 20). We found that anti- proBDNF IgG treatment was able to reduce the apoptosis level after ischemia. Apoptotic cells were significantly reduced around the ischemic infarct after anti- proBDNF IgG treatment at 7d (8+1% positive staining as % of area fraction, P=0.0086, n=6) compared to the 7d ischemic group.
[00273] (xv) Effect of p75ECD-Fc on the apoptosis around the infarcted area
[00274] Tunnel staining kit was used to detect the apoptotic cells after the treatment of p75ECD-Fc at different dosages (Fig. 21). We found higher number of apoptotic cells in the 7d stroke brain sections (29+4.1% positive staining as % of area fraction, P=0.0033, n=6). After treatment, apoptotic cells are significantly less observable in the 7d stroke+p75ECD-Fc lOmg group (10+1.8% positive staining as % of area fraction, P=0.0083, n=6) compared to the 7d stroke group.
[00275] (xvi) Effect of anti-proBDNF IgG on the Map2 staining around the infarcted area
[00276] Using Map2 staining we found significant reduction of Map2 expression around the infarct of the brain sections of the 7d stroke group (12+1.4% positive staining area as % of area fraction, r<0.0001, n=6) compared to the sham animals (Fig 22). A small increment of Map2 expression was observed around the ischemic infarct of the 7d stroke+ anti-proBDNF IgG group. However, there was no significant change after treatment compared to the 7d stroke group. [00277] (xvii) Effect of p75ECD-Fc on the Map2 staining around the infarcted area
[00278] Significant reduction of Map2 expression was observed in the brain sections of the 7d stroke group (8.4+2.2% positive staining as % of area fraction, P=0.0082, n=6) compared to the sham animals (Fig 23). A pattern of increased Map2 expression was observed in the brain section of the 7d stroke+p75ECD-Fc 3mg and 7d stroke+p75ECD-Fc lOmg groups. However, none of the treatment groups showed significant changes compared to the 7d stroke group.
[00279] (xviii) Effect of anti-proBDNF IgG on the GFAP -1 staining around the infarcted area
[00280] GFAP expression was considerably up-regulated in the 7d stroke brain section (63+2.5% positive staining area as % of area fraction, R<0.0001, n=6) compared to the sham group (Fig. 24). After anti-proBDNF IgG treatment we found that GFAP expression was reduced remarkably in the 7d stroke+anti-proBDNF IgG treatment group (37+2.6% positive staining area as % of area fraction, R<0.0001, n=6) compared to the 7d stroke group.
[00281] (xiv) Effect of p75ECD-Fc on the GFAP staining around the infarcted area
[00282] GFAP was found to be up-regulated in the 7d stroke brain section (43+4.5% positive staining % of area fraction, P=0.00l, n=6) compared to the sham group (Fig. 25). Interestingly, after the p75ECD-Fc treatment at different dosages, GFAP expression was significantly reduced in the 7d stroke+p75ECD-Fc lOmg group (14+1.9% positive staining as % of area fraction, P=0.0052, n=6) compared to the 7d stroke group.
[00283] (xv) Effect of anti-proBDNF IgG on IBA-l staining around the infarcted area
[00284] Similar to the GFAP, IBAI-l expression was up-regulated in the brain section of the 7d stroke group (58+5.3% positive staining area as % of area fraction, P<0.000l, n=6) (Fig. 26) and down-regulated in the brain section of the 7d stroke+anti-proBDNF IgG treatment group (30+1.5% positive staining area as % of area fraction, p<0.000l, n=6) compared to the 7d stroke group. [00285] (xv) Effect of p75ECD-Fc on IBA-l staining around the infarcted area
[00286] Similar to the GFAP, IBAI-l expression was up-regulated in the brain section of the 7d stroke group (39+3.5% positive staining as % of area fraction, R<0.0001, n=6) (Fig. 27) and down-regulated in the brain section of the 7d stroke+p75ECD-Fc lOmg group (5+1.3% positive staining as % of area fraction, R<0.0001, n=6) compared to the 7d stroke group.
[00287] (xvi) Anti-proBDNF IgG reduces the expression of JNK(P) in the ipsilateral brain
[00288] We did not obtain any significant changes of the total JNK expression at 7d in the ipsilateral brain homogenates after ischemia (Fig. 28). However, JNK(P) expression was significantly increased at 7d (578+99% vs control taken as 100%, R=0.0169, n=6) compared to the sham group after ischemia. After anti-proBDNF treatment, JNK(P) was down-regulated at 7d (419+60% vs control taken as 100%, P=0.0364, n=6) compared to the sheep IgG treatment group.
[00289] (xvii) p75ECD-Fc treatment reduces the expression of JNK(P)in the ipsilateral brain
[00290] There were no significant changes in the expression of JNK in any p75ECD-Fc treatment groups compared to the 7d stroke group (Fig. 29). However 7d stroke+p75ECD-Fc lOmg group (93+11% vs control taken as 100%, P=0.005l, n=6) showed significant reduction of the JNK(P) expression compared to the 7d stroke+p75ECD-Fc lmg group.
[00291] (xviii) Anti-proBDNF IgG reduces the expression PARP(CF) in the ipsilateral brain
[00292] Total PARP (PARP(FF)) expression was not changed significantly after ischemic insult in the ipsilateral brain homogenates (Fig.30). Cleaved PARP (PARP(CF)) expression was significantly up-regulated at 7d (1363+143% vs control taken as 100%, P=0.0002, n=6) compared to the sham animals after ischemia, and this expression was significantly reduced at 7d (723+97% vs control taken as 100%, P=0.0484, n=6) after anti-proBDNF IgG treatment compared to the 7d ischemic group.
[00293] (xix) p75ECD-Fc treatment reduces the expression of JNK(P) and PARP(CL) in the ipsilateral brain
[00294] Unlike JNK, the expression of PARP(FL) showed significant up-regulation in the 7d stroke group (187+11% vs control taken as 100%, R<0.0001, n=6) compared to the sham group and significant reduction in the 7d stroke+p75ECD-Fc lOmg group compared (130+13, P=0.0007) to the 7d stroke (Fig. 31). Similar significant up- regulation was observed for the expression of PARP(CL) in the 7d stroke group (3073+357% vs control taken as 100%, R<0.0001, n=6) compared to the sham group and significant reduction in the 7d stroke+p75ECD-Fc lOmg group (388+17% vs control taken as 100%, R<0.0001, n=6) compared to the 7d stroke.
[00295] (xx) Effect of anti-proBDNF IgG on the expression of synaptic protein PSD 95 in the ipsilateral brain
[00296] Synaptic marker PSD95 expression was reduced significantly after ischemic insult at 7d (13+3.7% vs control taken as 100%, P<0.000l, n=6) in the ipsilateral brain homogenates compared to the sham group (Fig. 32). The anti-proBDNF IgG treatment after ischemia resulted a small increment of the PSD95 expression; however, this did not give any significant change.
[00297] (xxi) Effect of p75ECD-Fc on the expression of synaptic protein PSD95 and in the ipsilateral brain
[00298] Significant reduction of the expression of PSD95 protein was observed in the 7d stroke group (15+2.8% vs control taken as 100%, R<0.0001, n=6) compared to the sham group (Fig. 33). After the treatment of p75ECD-Fc at different dosages, the psd95 expression showed a trend to be increased compared to the 7d stroke group; however, did not show any significance. [00299] SUMMARY
[00300] These studies show that the anti-proBDNF IgG treatment is useful for ischemic rescue. The intervention strategy improves the ischemic condition by promoting cellular growth and reforming the infarcted area and reducing the inflammation and apoptosis level after ischemia. Moreover, the sensorimotor functional loss is rescued by the anti- proBDNF treatment after ischemia. Therefore, anti-proBDNF is supportive for ischemic recovery.
[00301] The results for the anti-proBDNF treatment are summarised in Table 3.
Table 3: Effects of anti-proBDNF IgG treatment on ischemic recovery.
Post-ischemic Changes after
Factors Treatment
changes treatment
Inflammatory cellular Anti-proBDNF IgG
infiltration 5 mg/kg
Anti-proBDNF IgG
Astrocyte activation
5 mg/kg
Anti-proBDNF IgG
Microglia infiltration
5 mg/kg
Anti-proBDNF IgG
Infarcted area
5 mg/kg
Anti-proBDNF IgG
Apoptosis
5 mg/kg
Anti-proBDNF IgG
Neural protein expression No change
5 mg/kg
Synaptic protein Anti-proBDNF IgG
No change expression 5 mg/kg
Inhibitor for mBDNF Anti-proBDNF IgG
No change expression 5 mg/kg
[00302] In addition, these studies also show that p75ECD-Fc is beneficial after ischemia as it functions as an anti-inflammatory and anti-apoptotic agent, and improves cellular growth and sensorimotor behavioural impairments. Therefore, p75ECD may be used as a therapeutic target after ischemia.
[00303] The results for the p75ECD-Fc treatment are summarised in Table 4. Table 4: Effect of p75ECD-Fc after ischemia.
Post-ischemic Changes after
Factors Treatment
changes treatment
Inflammatory cellular p75ECD-Fc
infiltration lOmg
p75ECD-Fc
Astrocyte infiltration
lOmg
p75ECD-Fc
Microglia infiltration
lOmg
p75ECD-Fc
Infarcted area
lOmg
p75ECD-Fc
Apoptosis
lOmg
Neural protein expression p75ECD-Fc Pattern to increase
Synaptic protein expression p75ECD-Fc No change
Inhibitor for mBDNF
p75ECD-Fc
expression
[00304] REFERENCES
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Madinier, A., N. Bertrand, M. Rodier, A. Quirie, C. Mossiat, A. Prigent-Tessier, C. Marie and P. Gamier (2013). "Ipsilateral versus contralateral spontaneous post-stroke neuroplastic changes: involvement of BDNF?" Neuroscience 231: 169-181.
Rahman, M., H. Luo, N. R. Sims, L. Bobrovskaya and X. F. Zhou (2018). "Investigation of Mature BDNF and proBDNF Signaling in a Rat Photothrombotic Ischemic Model." Neurochem Res 43(3): 637-649. Schmidt, A., M. Hoppen, J. K. Strecker, K. Diederich, W. R. Schabitz, M. Schilling and J. Minnerup (2012). "Photochemically induced ischemic stroke in rats." Exp Transl Stroke Med 4(1): 13.
Sughrue, M. E., J. Mocco, R. J. Komotar, A. Mehra, A. L. D'Ambrosio, B. T. Grobelny, D. L. Penn and E. S. Connolly, Jr. (2006). "An improved test of neurological dysfunction following transient focal cerebral ischemia in rats." J Neurosci Methods 151(2): 83-89.
Takamatsu, Y., K. Tamakoshi, Y. Waseda and K. Ishida (2016). "Running exercise enhances motor functional recovery with inhibition of dendritic regression in the motor cortex after collagenase-induced intracerebral hemorrhage in rats." Behav Brain Res 300: 56-64.
[00305] The above references are each in their entirety incorporated herein by reference.
[00306] Although the present disclosure has been described with reference to particular embodiments, it will be appreciated that the disclosure may be embodied in many other forms. It will also be appreciated that the disclosure described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to, or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the steps or features.
[00307] Also, it is to be noted that, as used herein, the singular forms“a”,“an” and “the” include plural aspects unless the context already dictates otherwise.
[00308] Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as“comprises” or“comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
[00309] Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.
[00310] The subject headings used herein are included only for the ease of reference of the reader and should not be used to limit the subject matter found throughout the disclosure or the claims. The subject headings should not be used in construing the scope of the claims or the claim limitations.
[00311] The description provided herein is in relation to several embodiments which may share common characteristics and features. It is to be understood that one or more features of one embodiment may be combinable with one or more features of the other embodiments. In addition, a single feature or combination of features of the embodiments may constitute additional embodiments.
[00312] The methods described herein can be performed in one or more suitable orders unless indicated otherwise herein or clearly contradicted by context. The use of examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the example embodiments and does not pose a limitation on the scope of the claimed invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential.
[00313] Future patent applications may be filed on the basis of the present application, for example by claiming priority from the present application, by claiming a divisional status and/or by claiming a continuation status. It is to be understood that the following claims are provided by way of example only, and are not intended to limit the scope of what may be claimed in any such future application. Nor should the claims be considered to limit the understanding of (or exclude other understandings of) the present disclosure. Features may be added to or omitted from the example claims at a later date.

Claims

1. A method of improving recovery from brain injury involving neuronal damage in a subject, the method comprising reducing activity of a p75 neurotrophin receptor in the subject and thereby improving recovery from the brain injury.
2. The method according to claim 1, wherein the brain injury comprises one or more of an ischemic injury, a traumatic injury, a haemorrhagic injury, and an epileptic injury.
3. The method according to claims 1 or 2, wherein the brain injury is associated with a cerebral occlusion.
4. The method according to any one of claims 1 to 3, wherein the brain injury comprises a stroke.
5. The method according to any one of claims 1 to 4, wherein the improvement in recovery comprises an improvement in motor function and/or reducing neuronal cell death.
6. The method according to claim 4, wherein the improvement in recovery comprises an reduction in size of an infarct associated with the stroke.
7. The method according to any one of claims 1 to 6, wherein the method comprises administering to the subject an effective amount of an inhibitor of p75 neurotrophin receptor activity.
8. The method according to claim 7, wherein the inhibitor comprises all or part of the ectodomain of the p75 neurotrophin receptor.
9. The method according to claims 7 or 8, wherein the inhibitor comprises all or part of the ectodomain of the p75 neurotrophin receptor fused to a Fc region of an antibody.
10. The method according to any one of claims 7 to 9, wherein the inhibitor comprises a p75ECD-Fc.
11. The method according to claim 7, wherein the inhibitor comprises an agent that binds to a p75 neurotrophin receptor.
12. The method according to any one of claims 7 or 11, wherein the inhibitor of a p75 neurotrophin receptor comprises a ligand antagonist.
13. The method according to claim 12, wherein the ligand antagonist comprises one or more of a proBDNF antagonist, a proNGF antagonist, a proNT3 antagonist, and a proNT4 antagonist.
14. The method according to claim 12, wherein the ligand antagonist comprises a polypeptide/protein encoding all or part of the soluble extracellular domains of the receptors p75 and/or Sortilin/SORCS2.
15. The method according to claims 12 or 13, wherein the ligand antagonist comprises an antibody to the ligand.
16. The method according to claim 13, wherein the proBDNF antagonist comprises PNS-Rbl.
17. The method according to any one of claims 7 to 17, wherein the administration comprises systemic administration.
18. A method of improving recovery from a brain injury involving neuronal damage in a subject, the method comprising administering an effective amount of an inhibitor of a p75 neurotrophin receptor to the subject and thereby improving recovery from the brain injury.
19. The method according to claim 18, wherein the brain injury comprises one or more of an ischemic injury, a traumatic injury, a haemorrhagic injury, and an epileptic injury.
20. The method according to claims 18 or 19, wherein the brain injury is associated with a cerebral occlusion.
21. The method according to any one of claims 18 to 20, wherein the brain injury comprises a stroke.
22. The method according to any one of claims 18 to 21, wherein the improvement in recovery comprises an improvement in motor function and/or reducing neuronal cells death.
23. The method according to claim 21, wherein the improvement in recovery comprises an reduction in size of an infarct associated with the stroke.
24. The method according to any one of claims 18 to 23, wherein the inhibitor comprises all or part of the ectodomain of the p75 neurotrophin receptor.
25. The method according to any one of claims 18 to 24, wherein the inhibitor comprises all or part of the ectodomain of the p75 neurotrophin receptor fused to a Fc region of an antibody.
26. The method according to any one of claims 18 to 25, wherein the inhibitor comprises a p75ECD-Fc.
27. The method according to any one of claims 18 to 23, wherein the inhibitor comprises an agent that binds to a p75 neurotrophin receptor.
28. The method according to any one of claims 18 to 23 or 27, wherein the inhibitor of a p75 neurotrophin receptor comprises a ligand antagonist.
29. The method according to claim 28, wherein the ligand antagonist comprises one or more of a proBDNF antagonist, a proNGF antagonist, a proNT3 antagonist, and a proNT4 antagonist.
30. The method according to claim 28, wherein the ligand antagonist comprises a polypeptide/protein encoding all or part of the soluble extracellular domains of the receptors p75 and/or Sortilin/SORCS2.
31. The method according to claims 28 or 29, wherein the ligand antagonist comprises an antibody to the ligand.
32. The method according to claim 29, wherein the proBDNF antagonist comprises PNS-Rbl.
33. The method according to any one of claims 18 to 32, wherein the administration comprises systemic administration.
34. Use of an inhibitor of p75 neurotrophin receptor for improving recovery from a brain injury involving neuronal damage.
35. Use of an inhibitor of p75 neurotrophin receptor in the preparation of a medicament for improving recovery from a brain injury involving neuronal damage.
36. A method of treating a brain injury involving neuronal damage in a subject, the method comprising administering an effective amount of an inhibitor of a p75 neurotrophin receptor to the subject and thereby treating the brain injury.
37. A pharmaceutical composition for treating a brain injury involving neuronal damage, the pharmaceutical composition comprising an effective amount of an inhibitor of a p75 neurotrophin receptor.
38. The pharmaceutical composition according to claim 37, wherein the inhibitor comprises all or part of the ectodomain of the p75 neurotrophin receptor.
39. The pharmaceutical composition according to claims 37 or 38, wherein the inhibitor comprises all or part of the ectodomain of the p75 neurotrophin receptor fused to a Fc region of an antibody.
40. The pharmaceutical composition according to any one of claims 37 to 39, wherein the inhibitor comprises a p75ECD-Fc.
41. The pharmaceutical composition according to claims 37, wherein the inhibitor comprises an agent that binds to a p75 neurotrophin receptor.
42. The pharmaceutical composition according to any one of claims 37 or 41, wherein the inhibitor of a p75 neurotrophin receptor comprises a ligand antagonist.
43. The pharmaceutical composition according to claim 42, wherein the ligand antagonist comprises one or more of a proBDNF antagonist, a proNGF antagonist, a proNT3 antagonist, and a proNT4 antagonist.
44. The method according to claim 42, wherein the ligand antagonist comprises a polypeptide/protein encoding all or part of the soluble extracellular domains of the receptors p75 and/or Sortilin/SORCS2.
45. The pharmaceutical composition according to claims 42 or 43, wherein the ligand antagonist comprises an antibody to the ligand.
46. The pharmaceutical composition according to claim 43, wherein the proBDNF antagonist comprises PNS-Rbl.
47. A method of treating a subject suffering from a brain injury involving neuronal damage, the method comprise administering a pharmaceutical composition according to any one of claims 37 to 46 to the subject.
48. A method of identifying a therapeutic agent for improving recovery from a brain injury involving neuronal damage, the method comprising determining the ability of an inhibitor of a p75 neurotrophin receptor to improve recovery from a brain injury involving neuronal damage and identifying the inhibitor of a p75 neurotrophin receptor agent as a therapeutic agent for improving recovery from a brain injury involving neuronal damage.
49. A method of identifying a therapeutic agent for improving recovery from a brain injury involving neuronal damage, the method comprising: identifying a candidate agent as an inhibitor of a p75 neurotrophin receptor; and determining the ability of the inhibitor of a p75 neurotrophin receptor to improve recovery from a brain injury involving neuronal damage,
thereby identifying the candidate agent as a therapeutic agent for improving recovery from a brain injury involving neuronal damage.
50. The method according to claims 48 or 49, wherein the brain injury comprises one or more of an ischemic injury, a traumatic injury, a haemorrhagic injury, and an epileptic injury.
PCT/AU2019/050015 2018-09-11 2019-01-11 Methods and compositions for improving recovery from a brain injury WO2020051624A1 (en)

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