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WO2025051360A1 - Trkb-t1 agonist for the treatment of absence seizure and/or comorbidities - Google Patents

Trkb-t1 agonist for the treatment of absence seizure and/or comorbidities Download PDF

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Publication number
WO2025051360A1
WO2025051360A1 PCT/EP2023/074506 EP2023074506W WO2025051360A1 WO 2025051360 A1 WO2025051360 A1 WO 2025051360A1 EP 2023074506 W EP2023074506 W EP 2023074506W WO 2025051360 A1 WO2025051360 A1 WO 2025051360A1
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WIPO (PCT)
Prior art keywords
trkb
human test
test animal
absence
absence seizures
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PCT/EP2023/074506
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French (fr)
Inventor
Vincenzo CRUNELLI
Tatiana MORAIS
Sandra Henriques VAZ
Ana Maria SEBASTIÃO
Giuseppe Di Giovanni
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Cardiff University
Universita ta Malta UM
Faculdade De Medicina Da Universidade De Lisboa
Instituto de Medicina Molecular Joao Lobo Antunes
Original Assignee
Cardiff University
Universita ta Malta UM
Faculdade De Medicina Da Universidade De Lisboa
Instituto de Medicina Molecular Joao Lobo Antunes
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Priority to PCT/EP2023/074506 priority Critical patent/WO2025051360A1/en
Publication of WO2025051360A1 publication Critical patent/WO2025051360A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/30—Psychoses; Psychiatry

Definitions

  • Anti-Seizure Agents Field The present invention relates to agents for use in the treatment of absence seizures and their comorbidities.
  • Background Absence seizures are genetic, generalised, non-convulsive seizures that consist of sudden, relatively brief (few seconds to half a minute) lapses of consciousness that are invariably associated with generalized 2.5 – 4Hz spike/polyspike-wave discharges (SWDs), as measured via electroencephalography (EEG). Absence seizures are believed to have a complex polygenic background involving a combination of rare genetic variants and polymorphisms and are highly prevalent in paediatric and juvenile populations.
  • Absence seizures are the only clinical symptom in Childhood Absence Epilepsy (CAE), though they may often be present with other seizure types in various age-dependent and age- independent epilepsies having different severities and clinical outcomes (Crunelli et al.2020).
  • Ethosuximide and valproate are typical first-line drugs for use in the treatment of absence seizures.
  • Canonical anti-seizure medications e.g., that suppress focal and generalized convulsive seizures
  • Absence seizures are therefore believed to be fundamentally different from other seizure types, and to have a unique pharmacological profile (Crunelli et al, 2020).
  • absence seizures were considered relatively benign seizures, because of their non-convulsive nature and high remittance rate in early adulthood.
  • recent studies in large CAE cohorts now demonstrate that 30% of children with CAE exhibit pharmaco-resistant seizures (Glauser et al.2013; Cnaan et al.2017), a rate similar to that of more severe convulsive seizures. Polytherapy is therefore common and is associated with a marked increase in drug adverse effects.
  • Recent studies also show that ⁇ 60% of children with absence seizures suffer various neuropsychiatric comorbidities. Attention deficit disorders and memory and learning impairments were observed to be the most common comorbidities of absence seizures (35–40%), followed by mood disorders (Caplan et al.
  • comorbidities may precede the first absence seizure and an epilepsy diagnosis (Hermann et al.2007; Jones et al.2007) and may persist even after full pharmacological control of the seizures has been achieved (Glauser et al.2013). Moreover, comorbidities may be aggravated via the use of existing anti- seizure therapies including valproate.
  • neuropsychiatric comorbidities are currently controlled by classical pro-cognitive drugs: that is, juvenile patients receive both an anti-absence medication and a cognitive enhancer, which in turn leads to an unwanted increase in adverse effects. Consequently, there is an urgent and unmet clinical need for a new therapeutic approach to treat absence seizures and their comorbidities.
  • the present invention has been devised in light of the above considerations.
  • TrkB isoform TrkB-T1 regulates signalling at GABAergic synapses and that activation of TrkB-T1 reduces the levels of extracellular GABA by increasing the function of GAT1, one of the GABA transporters. Agonists of TrkB-T1 may therefore be useful in the treatment of absence seizures and/or their co-morbidities.
  • a first aspect of the invention provides a method of treating absence seizures and/or a comorbidity thereof, the method comprising: administering a therapeutically effective amount of a TrkB-T1 agonist to a subject in need thereof.
  • a second aspect of the invention provides a TrkB-T1 agonist for use in a method of treating absence seizures and/or a comorbidity thereof in a subject.
  • a third aspect of the invention provides the use of a TrkB-T1 agonist for the manufacture of a medicament for use in a method of treating absence seizures and/or a comorbidity thereof.
  • the method of treatment may be a method of the first aspect of the invention.
  • the method of treatment may be a method of treating absence seizures and a comorbidity thereof.
  • a fourth aspect of the invention provides a method of screening for a compound useful in treating absence seizures and/or a comorbidity thereof, comprising: determining the activity of TrkB-T1 in the presence or absence of a test compound, wherein an increase in the activity of TrkB-T1 in the presence relative to the absence of the test compound is indicative that the test compound is a candidate compound useful in treating absence seizures and/or a comorbidity thereof.
  • Methods of the fourth aspect may comprise determining the activity of isolated TrkB-T1, or determining the activity of TrkB-T1 in a mammalian cell, tissue or non-human test animal.
  • methods of the fourth aspect may be in vitro or in vivo methods.
  • Suitable non-human test animals include animal models of absence seizures.
  • a fifth aspect of the invention provides a method of screening for a compound useful in treating absence seizures and/or a comorbidity thereof, the method comprising: (i) administering a test compound to a first and a second non-human test animal; wherein the first and second non-human test animals are animal models of absence seizures; and wherein the second non-human test animal exhibits reduced expression of TrkB-T1 and/or reduced TrkB-T1 function relative to the first non-human test animal, (ii) measuring the frequency of occurrence and/or duration of absence seizures in the first and second non-human test animals, and (iii) comparing the frequency of occurrence and/or duration of absence seizures as measured in step (ii) to a control sample.
  • a reduction in the frequency of occurrence and/or duration of absence seizures in the first non-human test animal relative to the control sample, and no change or substantially no change in the frequency of occurrence and/or duration of absence seizures in the second non-human test animal relative to the control sample may be indicative that the test compound is a candidate compound useful in treating absence seizures and/or a comorbidity thereof.
  • Suitable non-human test animals (exhibiting reduced expression of TrkB-T1 and/or expressing a non-functional variant of TrkB-T1) include animals administered with a TrkB-T1 antagonist.
  • Suitable animal models of absence seizures include stargazer (STG) mice and Genetic Absence Epilepsy Rats from France (GAERS).
  • Methods of the fifth aspect of the invention may further comprise assessing a co-morbidity of an absence seizure in the first and second non-human test animals.
  • the cognitive behaviour of the first and second non-human test animals may be determined following administration of the test compound.
  • Cognitive behaviour may include learning and/or memory or the presentation of an attention- deficit/hyperactivity disorder, a cognitive impairment, a memory or learning deficit, an autism spectrum disorder, schizophrenia, depression and/or an anxiety disorder.
  • An improvement in the cognitive behaviour of the first non-human test animal as compared to the second non-human test animal may be indicative that the test compound is useful in treating a comorbidity of absence seizures.
  • a sixth aspect of the invention provides a method of screening for a compound useful in treating absence seizures and/or a comorbidity thereof, the method comprising: (i) administering a test compound to a first and a second non-human test animal; wherein the first and second non-human test animals are animal models of absence seizures, and wherein the second non-human test animal exhibits reduced expression of TrkB-T1 and/or reduced TrkB-T1 function relative to the first non-human test animal; and (ii) measuring the cognitive behaviour of the first and second non-human test animals.
  • Figure 1 shows a schematic diagram of a GABA synapse and illustrates the role of TrkB-T1 receptors in regulating the plasma membrane expression and function of GAT1, one of the GABA transporters, in astrocytes.
  • GAT1 removes GABA from the extracellular space.
  • TrkB-T1 receptors are exclusively located on astrocytes, and act as negative regulators of GAT1 recycling from the plasma membrane to the cytoplasm.
  • mice carry a single spontaneous recessive mutation in the “Stargazin” (calcium voltage-gated channel auxiliary subunit gamma 2, CACNG2) gene and exhibit head tossing, ataxia, impaired vestibular function, increased mossy fibre sprouting in hippocampus, and hyperactivity.
  • Stargazin calcium voltage-gated channel auxiliary subunit gamma 2, CACNG2
  • GAERS rats exhibit absence seizures with a frequency of SWDs of 7-11 Hz and their ontogeny is >30 days (with all animals exhibiting seizures at 3 months).
  • These rats carry unknown polygenic mutations that follow a pattern of autosomal dominant transmission and exhibit astrocytic alterations but no neuropathological abnormalities. In both animal models, absence seizures may be blocked by ethosuximide and valproic acid.
  • FIG. 3 shows a decrease in GAT1-mediated GABA uptake in brain slices derived from treatment-na ⁇ ve GAERS and Non-Epileptic Control (NEC) rats (that have no absence seizures), but no change in the GABA uptake mediated by GAT3, the other main GABA transporter in the brain.
  • NEC Non-Epileptic Control
  • FIG. 5 shows that BDNF (a TrkB-T1 agonist) increases GAT1 expression at the plasma membrane and GAT1-mediated GABA uptake in GAERS rats.
  • GAT1 67 kDa
  • expression was measured in homogenates derived from thalamic slices in the presence and absence of BDNF (30min incubation, 20ng/ml). Cytoplasmic and plasma membrane homogenates show the exclusive GAT1 expression in the plasma membrane.
  • ACSF Artificial Cerebrospinal Fluid
  • FIG. 8 shows that BDNF administration rescues absence seizures in vivo.
  • A A schematic representation of the study protocol.
  • B Systemic (i.e., intraperitoneal) administration of BDN or Vehicle 1 (Veh1) occurred 20 min after intravenous administration of Vehicle 2 (Veh2) or Elacridar (Ela) (a phosphoglycoprotein inhibitor) to allow brain penetration of BDNF.
  • FIG. 9 shows that the anti-seizure effect of BDNF is mediated via TrkB-T1 receptors.
  • A A schematic representation of the study protocol.
  • B Targeting TrkB-T1 in the cortex.
  • Total seizure duration was measured in GAERS rats administered intrathalamically with an astrocyte-specific scrambled TrkB-T1 siRNA (6.86E+10 viral particles) (closed circles) or an astrocyte-specific TrkB-T1 knockdown siRNA (6.23E+10 viral particles) (open circles) and BDNF (500ng) administrated intrathalamically to both groups of rats.
  • an astrocyte-specific scrambled TrkB-T1 siRNA (6.86E+10 viral particles) (closed circles) or an astrocyte-specific TrkB-T1 knockdown siRNA (6.23E+10 viral particles) (open circles) and BDNF (500ng) administrated intrathalamically to both groups of rats.
  • the measurement of seizure duration was normalised to pre-injection levels for each animal. Representative EEG traces can be found on the bottom of the graph.
  • Figure 10 shows that BDNF rescues the deficiencies in novel object recognition observed in GAERS rats.
  • (A) A schematic representation of the study protocol.
  • Figure 11 shows that the BDNF-mediated rescue of the impaired LTP and deficient novel object recognition of GAERS rats occurs via TrkB-T1.
  • (B) LTP magnitude was increases by ⁇ 75% in BDNF-treated compared to ACSF-treated GAERS hippocampal slices (*, P 0,0161; unpaired t-test).
  • (D) LTP magnitude was not different between ACSF- and BDNF-treated GAERS hippocampal slices (P 0.114, unpaired t-test).
  • G Graphical representation of the percentage of time each GAERS rat spends exploring the familiar (black) and the novel (white) object for astrocyte-specific scrambled TrkB-T1 siRNA- and astrocyte-specific TrkB-T1 knockdown siRNA-injected animals.
  • the invention relates to the treatment of absence seizures and their comorbidities by administering a therapeutically effective amount of a TrkB-T1 agonist to a subject in need thereof.
  • TrkB-T1 agonists Activation of TrkB-T1 receptors by TrkB-T1 agonists are shown herein to act as negative regulators of GAT1 recycling from the plasma membrane to the cytoplasm: this decreases the levels of extracellular GABA which in turn rescues both absence seizures and their memory comorbidity.
  • TrkB-T1 agonists significantly reduce the frequency of occurrence and duration of absence seizures in vivo and are effective in alleviating the memory/learning comorbidities, such as deficiencies in novel object recognition that are typical of animal models of absence seizures.
  • TrkB-T1 agonists may be useful in the treatment of absence seizures and their comorbidities.
  • TrkB-T1 is a truncated variant of Tropomyosin receptor kinase B (TrkB). TrkB is also known as tyrosine receptor kinase B, BDNF/NT-3 growth factors receptor, or neurotrophic tyrosine kinase receptor type, and may also be termed TrkB full-length (“Trkb-FL”). TrkB-FL is encoded by the gene NTRK2 in humans and mice (Homo sapiens gene ID 4915; Mus musculus gene ID 18212).
  • TrkB-T1 is located exclusively on astrocytes and is the major isoform of TrkB-FL that is expressed in the adult mammalian brain.
  • TrkB-T1 as described herein may be present in thalamic, cortical or hippocampal astrocytes.
  • An overview of the role of the astrocytic TrkB-T1 in regulating the expression of GAT1 at GABAergic synapses is shown in Figure 1.
  • TrkB-FL may be human TrkB-FL and may have the amino acid sequence of UniProt Q16620-1 (Homo sapiens) (SEQ ID NO: 3) or a variant thereof.
  • TrkB-T1 may be human TrkB-T1 and may have the amino acid sequence of UniProt Q16620-2 (Homo sapiens) (SEQ ID NO: 4) or a variant thereof.
  • TrkB-FL may be mouse TrkB-FL and may have the amino acid sequence of UniProt P15209-1 (Mus musculus) (SEQ ID NO: 1) or a variant thereof.
  • TrkB-T1 may be mouse TrkB-T1 and may have the amino acid sequence of UniProt P15209-2 (Mus musculus) (SEQ ID NO: 2) or a variant thereof.
  • a TrkB-T1 agonist is an agent that is capable of stimulating or activating a TrkB-T1 receptor.
  • TrkB-T1 may enhance the acidic metabolite release from a cell (Baxter et al., 1997) and elicit calcium transients, i.e. calcium release from intracellular stores, in astrocytes (Rose et al., 2003).
  • a “full agonist” may achieve the maximum possible biological response, whilst a “partial agonist” may generate a partial biological response with equivalent receptor occupancy.
  • a TrkB-T1 agonist as described herein may be a selective agonist of TrkB-T1.
  • a selective agonist is an agonist that is selective for a particular species of receptor over one or more other species of receptor.
  • a selective agonist of TrkB-T1 may exhibit a higher binding affinity for TrkB-T1 than for TrkB-FL.
  • a selective agonist of TrkB-T1 may exhibit a binding affinity (Kd) for TrkB-T1 that is lower than its binding affinity (Kd) for TrkB-FL.
  • a TrkB-T1 agonist disclosed herein may for example exhibit a Kd value in the range of picomolar (10 -12 M) to micromolar (10 -6 M) concentrations, such as a Kd value between 1, 10 or 100 pM and 1, 10 or 100 ⁇ M.
  • a selective agonist of TrkB-T1 may also exhibit higher efficacy (in eliciting a biological response) on binding to TrkB-T1 compared to binding to TrkB-FL.
  • a selective agonist of TrkB-T1 may exhibit a half effective concentration (EC50) for activation of TrkB-T1 that is lower than its half effective concentration (EC 50 ) for activation of TrkB-FL.
  • a non-selective agonist of TrkB may activate both TrkB-T1 and TrkB-FL.
  • BDNF brain-derived neurotrophic factor
  • a TrkB-T1 agonist disclosed herein may exhibit an EC50 value in the range of picomolar (10 -12 M) to micromolar (10 -6 M) concentrations, for example, an EC50 between 1 pM, 10 pM or 100 pM and 1 ⁇ M, 10 uM or 100 ⁇ M.
  • Methods suitable for assessing the binding affinity (Kd) and half effective concentration (EC50) of an agonist are well known in the art (see e.g.
  • TrkB-T1 agonists include an organic compound having a molecular weight of 900 Da or less; a protein or peptide that specifically binds TrkB-T1, for example, an antibody molecule that specifically binds TrkB-T1, or a peptide that binds TrkB-T1 and enhances its activity (an agonist peptide); and a nucleic acid that specifically binds TrkB-T1, for example, an aptamer that specifically binds TrkB-T1.
  • TrkB-T1 agonists may include brain derived neurotrophic factor (BDNF; Gene ID 627; reference sequence NP_001137277.1) and variants and derivatives thereof.
  • BDNF brain derived neurotrophic factor
  • a TrkB-T1 agonist as described above may be administered alone or may be formulated into a pharmaceutical composition.
  • a pharmaceutical composition is a formulation comprising one or more active agents and one or more pharmaceutically acceptable excipients. The pharmaceutical composition may be capable of eliciting a therapeutic effect.
  • a suitable pharmaceutical composition for use as described herein may comprise an agent described above and a pharmaceutically acceptable excipient.
  • a pharmaceutical composition may comprise a TrkB-T1 agonist as described herein, and a pharmaceutically acceptable excipient, carrier, diluent, or adjuvant.
  • pharmaceutically acceptable relates to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound veterinary or medical judgement, suitable for use in contact with the tissues of a subject (e.g. human or other mammal) without excessive toxicity, irritation, allergic response, or other problem or complication, and that are commensurate with a reasonable benefit/risk ratio.
  • a subject e.g. human or other mammal
  • Each carrier, excipient, etc. must be “acceptable” in the sense of being compatible with the other ingredients of the formulation.
  • Suitable excipients and carriers include, without limitation: water, saline, buffered saline, phosphate buffer, alcoholic/aqueous solutions, emulsions or suspensions.
  • Such carriers can include ethanol, polyols, and suitable mixtures thereof, vegetable oils, and injectable organic esters.
  • Buffers and pH-adjusting agents may also be employed, and include, without limitation, salts prepared from an organic acid or base.
  • Representative buffers include, without limitation: organic acid salts, such as salts of citric acid (e.g., citrates), ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, phthalic acid, Tris, trimethylamine hydrochloride, or phosphate buffers.
  • Parenteral carriers can include sodium chloride solution, Ringer's dextrose, dextrose, trehalose, sucrose, lactated Ringer's, or fixed oils.
  • Intravenous carriers can include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like.
  • Preservatives and other additives such as, for example, antimicrobials, antioxidants, chelating agents (e.g., EGTA; EDTA), inert gases, and the like may also be provided in the pharmaceutical carriers.
  • the pharmaceutical compositions described herein are not limited by the selection of the carrier.
  • compositions from the above-described components, having appropriate pH, isotonicity, stability and other conventional characteristics, is within the skill of the art.
  • Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical texts. See, for example, Handbook of Pharmaceutical Additives, 2 nd Edition, 2001, Remington's Pharmaceutical Sciences, 20 th Edition, 2000; and Handbook of Pharmaceutical Excipients, 2 nd Edition, 1994.
  • a pharmaceutical composition may conveniently be presented in unit dosage form and may be prepared by any methods well-known in the art of pharmacy. Such methods include the step of bringing the one or more TrkB-T1 agonists into association with a carrier or excipient as described above which may constitute one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both.
  • Pharmaceutical compositions described herein may be produced in various forms, depending upon the route of administration. Routes may include parenteral, intravenous, intraarterial, intramuscular, oral and nasal routes.
  • the pharmaceutical compositions may include liquid or solid forms and may be prepared for administration to subjects in the form of, for example, liquids, powders, aerosols, tablets, capsules, enteric-coated tablets or capsules, or suppositories.
  • Pharmaceutical compositions may also be in the form of suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained- release or biodegradable formulations.
  • compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials, such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
  • Pharmaceutical compositions may be made in the form of sterile aqueous solutions or dispersions, suitable for injectable use, or made in lyophilized forms using freeze-drying techniques. Lyophilized pharmaceutical compositions are typically maintained at about 4°C, and can be reconstituted in a stabilizing solution, e.g., saline or HEPES, with or without adjuvant.
  • Pharmaceutical compositions can also be made in the form of suspensions or emulsions.
  • the carrier or other material will depend on the route of administration, which may be any convenient route, for example by injection, e.g. cutaneous, subcutaneous, or intravenous.
  • the agent is administered systemically, e.g. intravenously.
  • the pharmaceutical compositions comprising the active compounds may be formulated in a dosage unit formulation that is appropriate for the intended route of administration.
  • Pharmaceutical compositions may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections immediately prior to use.
  • Methods of determining the most effective means and dosage of administration are well known in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the physician. Administration in vivo can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals). Multiple doses of the composition may be administered, for example 2, 3, 4, 5 or more than 5 doses may be administered. The administration of the composition may continue for sustained periods of time. For example, treatment with the composition may be continued for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month or at least 2 months.
  • a TrkB-T1 agonist may be useful in treating an absence seizure and/or one or more of their comorbidities in a patient.
  • An absence seizure is a genetic, generalised, non-convulsive seizure that consists of sudden, relatively brief (few seconds to half a minute) lapses of consciousness that are associated with generalized 2.5 – 4Hz spike/polyspike-wave discharges (SWDs), as measured via electroencephalography (EEG).
  • SWDs spike/polyspike-wave discharges
  • EEG electroencephalography
  • absence seizures may be accompanied by convulsive seizures.
  • a comorbidity of an absence seizure is a clinical manifestation that accompanies or is associated with an absence seizure.
  • a comorbidity may occur before the first absence seizure or the first diagnosis of an absence seizure in a patient and may persist even after full pharmacological control of the absence seizures in a patient.
  • Comorbidities of absence seizures may include neuropsychiatric comorbidities such as attention-deficit/hyperactivity disorder, cognitive impairment, memory and learning deficits, autism spectrum disorder, schizophrenia, depression and anxiety disorders (such as agoraphobia, selective mutism, generalized anxiety disorder (GAD), social anxiety disorder, obsessive-compulsive disorder (OCD) and panic disorder).
  • Treatment may be any treatment and therapy, whether of a human or an animal (e.g. in veterinary applications), in which some desired therapeutic effect is achieved, for example, improving or ameliorating one or more symptoms of an absence seizure or comorbidity. Treatment may be preventative or curative.
  • An subject suitable for treatment as described above may be a mammal, such as a rodent (e.g. a guinea pig, a hamster, a rat), murine (e.g. a mouse), canine (e.g. a dog), feline (e.g.
  • a cat equine
  • a primate e.g. a monkey or ape
  • a monkey e.g. marmoset, baboon
  • an ape e.g. gorilla, chimpanzee, orang-utan, gibbon
  • the subject is a human.
  • non-human mammals especially mammals that are conventionally used as models for demonstrating therapeutic efficacy in humans (e.g. murine, rodent, primate, porcine, canine, or leporid) may be employed.
  • Suitable subjects for treatment as described here include a human paediatric subject having Childhood Absence Epilepsy (CAE), a teenage human subject having Juvenile Absence Epilepsy (JAE), and a young or adult subject having absence as well as convulsive seizures.
  • a subject may carry a dysfunctional or non-functional sodium- and chloride-dependent GABA transporter 1 (GAT1), for example, caused by a loss-of-functional SLC6A1 gene variant.
  • SLC6A1 Homo sapiens gene ID 6529; Mus musculus gene ID 232333 encodes GABA transporter 1 (GAT1).
  • GAT1 is expressed on astrocytes and is responsible for removing GABA from the synaptic cleft at GABAergic synapses.
  • TrkB-T1 agonists are shown herein to reduce the internalisation of GAT1, thereby increasing the expression of GAT1 in the plasma membrane and thus facilitating an increase in GAT1-mediated GABA uptake at the synapse.
  • An individual with an absence seizure or comorbidity may display at least one identifiable sign, symptom, or laboratory finding that is sufficient to make a diagnosis of absence seizure or comorbidity in accordance with clinical standards known in the art. Examples of such clinical standards can be found in textbooks of medicine. It will be appreciated that appropriate dosages of a TrkB-T1 agonist may vary from patient to patient.
  • Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the treatments of the present invention.
  • the selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular agent, the route of administration, the time of administration, the rate of loss or inactivation of the agent, the duration of the treatment, other drugs, compounds, and/or materials used in combination, and the age, sex, weight, condition, general health, and prior medical history of the patient.
  • the dosage of agent and the route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve local concentrations at the site of injury which achieve the desired effect without causing substantial harmful or deleterious side-effects.
  • Prescription of treatment e.g.
  • a therapeutically effective amount or suitable dose of a agent described herein may be determined by comparing its in vitro activity and in vivo activity in an animal model. Methods for extrapolation of effective dosages in mice and other test animals to humans are known. Treatment may comprise the administration of a therapeutically effective amount of the agent or pharmaceutical composition to the individual. “Therapeutically effective amount” relates to the amount of a agent or pharmaceutical composition that is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio.
  • a suitable amount of a agent or pharmaceutical composition for administration to an individual may be an amount that generates a therapeutic effect in the individual.
  • a therapeutic effect may be at least amelioration of at least one symptom.
  • a treatment as described herein may have a duration of up to 3 weeks, up to 6 weeks, up to 3 months, up to 6 months or up to 12 months.
  • the treatment schedule for an individual may be dependent on the pharmacokinetic and pharmacodynamic properties of the agent, the route of administration and the nature of the condition being treated. Treatment may be in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals).
  • Treatment may be periodic, and the period between administrations may be about 12 hours or more, 24 hours or more, 36 hours or more, 48 hours or more, 96 hours or more, or one week or more. Suitable formulations and routes of administration are described above and may be readily determined by a physician for any individual patient.
  • the TrkB-T1 agonist may be administered for the treatment absence seizures and/or co-morbidities without the co-administration of other therapies.
  • a TrkB-T1 agonist may be administered in combination with one or more other therapies, either simultaneously or sequentially dependent upon the circumstances of the individual to be treated.
  • the second pharmaceutical agent may be an anti-seizure agent. Appropriate anti-seizure agents will be readily appreciated by those skilled in the art.
  • TrkB-T1 agonists When TrkB-T1 agonists are used in combination with additional therapeutic agents, the compounds may be administered either sequentially or simultaneously by any convenient route. When TrkB-T1 agonists are used in combination with an additional therapeutic agent active against the same disease, the dose of each agent in the combination may differ from that when the therapeutic agents are used alone. Appropriate doses will be readily appreciated by those skilled in the art.
  • TrkB-T1 may be useful in screening for compounds that may be useful in the development of therapeutics for treating absence seizures and/or co-morbidities of absence seizures. A method of screening for a compound useful in treating absence seizures and/or a comorbidity thereof may comprise determining the activity of TrkB-T1 in the presence or absence of a test compound.
  • TrkB-T1 An increase in the activity of TrkB-T1 in the presence relative to the absence of the test compound is indicative that the test compound is a TrkB-T1 agonist and is a candidate compound useful in treating absence seizures and/or a comorbidity thereof.
  • Methods described herein may comprise determining the activity of isolated TrkB-T1, or the activity of TrkB-T1 in a mammalian cell, a mammalian tissue or non-human mammal.
  • a method of screening for a compound useful in treating absence seizures and/or a comorbidity thereof may comprise determining the activity of TrkB-T1 and TrkB-FL in the presence or absence of a test compound.
  • TrkB-T1 An increase in the activity of TrkB-T1 in the presence relative to the absence of the test compound that is greater than the increase in the activity of TrkB-FL in the presence relative to the absence of the test compound may be indicative that the test compound is a selective TrkB-T1 agonist and is a candidate compound useful in treating absence seizures and/or a comorbidity thereof.
  • the activity of TrkB-T1 may increase in the presence relative to the absence of the test compound and the activity of TrkB-FL may not increase or may decrease in the presence relative to the absence of the test compound.
  • Methods suitable for determining the activity of TrkB-T1, TrkB-FL and GAT1 are well known in the art and are readily available to those skilled in the art (see e.g.
  • suitable methods may involve assessing the expression of TrkB-T1, TrkB-FL and GAT1 at the plasma membrane, assessing modifications thereof (e.g. dimerization, phosphorylation), and/or assessing biological responses produced by cells or organisms in which the TrkB-T1, TrkB-FL or GAT1 are expressed.
  • Biophysical techniques for example X-ray crystallography, cryo-electron microscopy, and nuclear magnetic resonance spectroscopy may also be used in order to determine the location of TrkB-T1, TrkB-FL and GAT1 and to characterise any conformational changes that occur upon ligand binding and/or activation.
  • determining the activity of TrkB-T1 comprises measuring the expression of GABA transporter 1 (GAT1) at the plasma membrane or measuring the trafficking of GAT1 between the cytoplasm and the plasma membrane.
  • GAT1 GABA transporter 1
  • Suitable methods include enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), or immunofluorescence (IF).
  • ELISA enzyme-linked immunosorbent assay
  • IHC immunohistochemistry
  • IF immunofluorescence
  • the expression profile may also be quantified using flow cytometry and associated techniques (e.g. time-of-flight (TOF) cytometry or mass spectrometry, CyTOF).
  • TOF time-of-flight
  • CyTOF mass spectrometry
  • Methods described herein may comprise determining the activity of isolated TrkB-T1, or the activity of TrkB-T1 in a mammalian cell, a mammalian tissue or non-human mammal.
  • a test compound showing activity in an in vitro method described herein may be subsequently tested in one or more in vivo methods described herein.
  • a method of screening for a compound useful in treating absence seizures and/or a comorbidity thereof comprising: (i) administering a test compound to a first and a second non-human test animal; in which the first and second non-human test animals are animal models of absence seizures, and in which the second non-human test animal exhibits reduced expression of TrkB-T1 and/or reduced TrkB-T1 function as compared to the first non-human test animal; (ii) measuring the frequency of occurrence and/or duration of absence seizures in the first and second non-human test animals, and (iii) comparing the frequency of occurrence and/or duration of absence seizures as measured in step (ii) to a control sample; in which a reduction in the frequency of occurrence and/or duration of absence seizures in the first non-human test animal relative to the control sample, and substantially no change in the frequency of occurrence and/or duration of absence seizures in the second non-human test animal relative to the control sample are indicative that the test compound is a candidate
  • Suitable animal models of absence seizures include GAERS rat and Stargazer mouse as described herein.
  • the non-human test animal exhibiting reduced expression of TrkB-T1 or expressing a non- functional variant of TrkB-T1 may be an animal administered with a TrkB-T1 antagonist.
  • a TrkB-T1 antagonist is agent that is capable of binding to and inhibiting the activation of TrkB-T1.
  • the antagonist may bind to the agonist binding site, or may achieve inhibition by allosteric modulation of TrkB-T1.
  • An antagonist may exhibit affinity for the agonist binding site of the TrkB-T1, but without exhibiting efficacy (i.e., without stimulating receptor signalling).
  • a suitable TrkB-T1 antagonist may be a competitive antagonist of a TrkB-T1 agonist.
  • Antagonists may also include agents that are capable of inhibiting the expression of a signalling receptor.
  • a suitable TrkB-T1 antagonists may include competitive antagonists of the test compound, an allosteric modulator of TrkB- T1, a TrkB-T1 small interfering RNA (siRNA), and a TrkB-T1 microRNA (miRNA).
  • Suitable TrkB-T1 antagonists may also include selective antagonists of TrkB-T1 on astrocytes.
  • a selective antagonist of TrkB-T1 may exhibit a half inhibitory concentration (IC50) for TrkB-T1 that is lower than its half inhibitory concentration (IC50) for TrkB-FL.
  • IC50 half inhibitory concentration
  • a TrkB-T1 antagonist suitable for use as described may exhibit an IC50 value in the range of picomolar (10 -12 M) to micromolar (10 -6 M) concentrations, for example, between 1, 10 or 100 pM and 1, 10 or 100 ⁇ M.
  • SEQ ID NO: 5 is a suitable TrkB-T1 antagonist.
  • test compound may be an isolated molecule or may be comprised in a sample, mixture or extract, for example, a biological sample.
  • Compounds which may be screened using the methods described herein may be natural or synthetic chemical compounds used in conventional drug screening programmes.
  • Extracts of plants, microbes or other organisms, which contain several characterised or uncharacterised components may also be used.
  • Suitable test compounds may include analogues, derivatives, variants and mimetics of known TrkB-T1 agonists, such as BDNF.
  • compounds produced using rational drug design to provide test candidate compounds with particular molecular shape, size and charge characteristics suitable for the selective agonism of TrkB-T1 may be tested.
  • a combinatorial library technology provides an efficient way of testing a potentially vast number of different compounds for their ability to act as agonists of TrkB-T1, preferably selective agonists of TrkB- T1.
  • Such libraries and their use are known in the art, for all manner of natural products, small molecules and peptides, among others.
  • test compound which may be added to a screening assay of the invention will normally be determined by trial and error depending upon the type of compound used. Typically, from about 0.001 nM to 1 mM or more concentrations of putative agonist compound may be used, for example from 0.01 nM to 100 ⁇ M, e.g.0.1 to 50 ⁇ M, such as about 10 ⁇ M. Even a compound having a weak agonist effect may be a useful lead compound for further investigation and development.
  • the precise format of any of the screening or assay methods disclosed herein may be varied by those of skill in the art using routine skill and knowledge. The skilled person is well aware of the need to employ appropriate control experiments.
  • suitable control samples include, for example, reference values, treatment-na ⁇ ve non-human test animals, and non-human test animals administered a control compound (e.g. a vehicle control), in which the control compound does not exhibit anti-seizure activity, and in which the control compound is not a TrkB-T1 agonist nor a TrkB-T1 antagonist.
  • a control compound e.g. a vehicle control
  • Non-human test animals may be species of rodent (e.g., mouse, rat, guinea pig, hamster, gerbil), fly (e.g., Drosophila) fish (e.g., zebrafish), livestock (e.g.
  • the non-human test animal is an animal model of absence seizures.
  • the non-human test animal may be an inbred laboratory rodent, such as a GAERS rat (Vergnes et al.1982; Micheletti et al.1985; Marescaux et al.1992).
  • the non-human test animal may be a genetically modified animal or an animal with a single spontaneous gene mutation, such an STG mouse.
  • suitable methods may involve DNA recombination, gene knockout (e.g., via homologous recombination or through the use of nucleases), gene insertion (e.g., cisgenesis or transgenesis), RNA interference (RNAi), genome editing (for example using CRISPR-Cas9 and associated techniques) or via mutagenesis (which may be random or targeted).
  • gene knockout e.g., via homologous recombination or through the use of nucleases
  • gene insertion e.g., cisgenesis or transgenesis
  • RNAi RNA interference
  • genome editing for example using CRISPR-Cas9 and associated techniques
  • mutagenesis which may be random or targeted.
  • the non-human test animal may be a genetically modified animal comprising a calcium voltage-gated channel auxiliary subunit gamma 2 (CACNG2) gene variant (Homo sapiens gene ID 10369; Mus musculus gene ID 12300).
  • CACNG2 gene variant in which the altered gene product lacks the molecular function of the wild-type gene product.
  • the gene variant may comprise a partial or total deletion of the CACNG2 gene.
  • the non-human test animal may exhibit a dysfunctional or non-functional CACNG2 protein. For example, Stargazer mice (Letts et al., 1997; Letts et al., 1998; Seo and Leitch, 2014).
  • TrkB-T1 A candidate compound identified as a selective agonist of TrkB-T1 may be investigated further.
  • the selectivity of a compound for TrkB-FL and/or TrkB-T1 in astrocytes may be determined in animal models. Suitable methods for determining the effect of a compound on TrkB-T1 are disclosed herein and alternative methods are well known in the art.
  • a candidate compound identified as a TrkB-T1 agonist through one or more of the screening methods disclosed herein may be isolated and/or purified. Alternatively, it may be synthesised using conventional techniques of recombinant expression or chemical synthesis. Furthermore, it may be manufactured and/or used in preparation, i.e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition or drug. Methods described herein may thus comprise formulating the test compound in a pharmaceutical composition with a pharmaceutically acceptable excipient, vehicle or carrier for therapeutic application. Following identification of a TrkB-T1 agonist that is potentially useful in the treatment of absence seizures and/or their comorbidities as described herein, a method may further comprise modifying the compound to optimise its pharmaceutical properties.
  • Suitable methods of optimisation for example by structural modelling, are well known in the art. Further optimisation or modification can then be carried out to arrive at one or more final compounds for in vivo or clinical testing.
  • Other aspects and embodiments of the invention provide the aspects and embodiments described above with the term “comprising” replaced by the term “consisting of” and the aspects and embodiments described above with the term “comprising” replaced by the term ”consisting essentially of”.
  • the term “in vitro” relates to actions performed in isolated laboratory conditions or in cell culture, for example using materials, biological substances, cells and/or tissues that are isolated from an organism.
  • the term “in vivo” relates to actions performed in intact multi-cellular organisms, such as humans or other mammals.
  • ex vivo relates to actions performed outside a multi-cellular organisms e.g. outside a human or mammalian body, which may be on tissue (e.g. whole organs) or cells obtained or derived from a multi-cellular organism. It is to be understood that the application discloses all combinations of any of the above aspects and embodiments described above with each other, unless the context demands otherwise. Similarly, the application discloses all combinations of the preferred and/or optional features either singly or together with any of the other aspects, unless the context demands otherwise. Modifications of the above embodiments, further embodiments and modifications thereof will be apparent to the skilled person on reading this disclosure, and as such, these are within the scope of the present invention.
  • Figure 3 shows a decrease in GAT1-mediated GABA uptake in brain slices derived from treatment-na ⁇ ve GAERS and Non-Epileptic Control (NEC) rats (that have no absence seizures), but no change in the GABA uptake mediated by GAT3, the other main GABA transporter in the brain.
  • NEC Non-Epileptic Control
  • MC motor cortex
  • SC visual cortex
  • VC visual cortex
  • HIPPO hippocampus
  • Transverse 300 ⁇ m slices were then cut from each region using a McIlwain tissue chopper and allowed to recover for 1 hour (Fredholm et al., 1984) in small chambers filled with Krebs solution saturated with 5% CO2 - 95% O2.
  • drugs of interest namely 20 ⁇ M SKF-8996A (a GAT1 blocker) and 20 ⁇ M SNAP-5411 (a GAT3 blocker) were added to the ACSF solution.
  • the uptake assay was then started by immersion of the slices for 10 min in a solution containing 2.5, 5, 10, 25, 50 and 100 ⁇ M of GABA (10% of which was [3H]GABA with specific activity of 0.141 Ci/mmol, PerkinElmer, Boston, MA, USA) with or without the drugs of interest in DMEM medium (DMEM, Gibco, Paisley, UK),) at 37oC.
  • DMEM medium DMEM, Gibco, Paisley, UK
  • the slices were washed twice in an ice-cold PBS solution (in mM: 140 NaCl, 2.7 KCl, 1.5 KH2PO4 and 8.1 NaHPO4, pH 7.40), and then homogenized in 200 ⁇ l of lysis buffer (in mM: 100 NaOH and 0.1% SDS).
  • Figure 4 shows a decrease in GAT1-mediated, but not GAT3-mediated, GABA uptake in primary cultures of astrocytes derived from treatment-na ⁇ ve GAERS and NEC rats.
  • GABA uptake assays were carried out in primary cultures of astrocytes.
  • Primary cultures of astrocytes were prepared from the cerebral cortex and the thalamus of neonatal GAERS and NEC rat pups (0–3 days old) (Morais et al, 2018). Briefly, rat pups were sacrificed by decapitation and the brains were dissected in ice cold phosphate buffered saline solution (PBS) containing 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4.2H2O and 1.5 mM KH2PO4 (pH 7.4).
  • PBS ice cold phosphate buffered saline solution
  • DMEM Modified Eagles Medium
  • the pellet was resuspended in 4.5 g/l glucose DMEM, sorted again through a 70- ⁇ m cell strainer (BD Falcon, NJ, USA) and then centrifuged. The final pellet was resuspended in DMEM and seeded at the density of 2x105 or 5x105 cells/ml (for thalamic cultures) and 5x105 cells/ml (for cortical cultures) in 24 wells plates. Thalamic and cortical astrocyte cultures were grown for 3 weeks in an incubator at 37oC with a humidified atmosphere of 95% O2 and 5% CO2. The culture medium was changed twice a week.
  • astrocytes were shaken for 5 hours at 300 rpm to remove any contaminating microglia cells.
  • GABA uptake assays were performed in 21 days-old primary cultures of astrocytes following the procedures described by Vaz et al. (2011). Briefly, 3h before the uptake assay, the cell culture medium was changed to serum-free 1g/L DMEM medium, and the cells were kept at 37oC with 95% O2 - 5% CO2.
  • a solution made of the drugs of interest diluted in the culture medium was added to the culture medium (10 ⁇ l/well), namely, 20 ⁇ M SKF-8996A (a GAT1 antagonist) or 20 ⁇ M SNAP-5411 (a GAT3 antagonist), and to some culture wells 10 ⁇ l of culture medium (without any drugs) was added for these wells to serve as controls.
  • GABA uptake assay was performed by removing the culture medium from the wells and then incubating the cells for 60 s at 37oC with 1, 2.5, 5, 10, 25 and 50 ⁇ M of GABA, (from a stock solution that contained 10% of [3H]GABA ([3H]GABA specific activity: 0.141 Ci/mmol) (PerkinElmer, Boston, MA, USA) and drugs of interest, namely SKF-8996A and SNAP-5411, in a solution of (in mM) 137 NaCl, 5.4 KCl, 1.8 CaCl2 ⁇ 2H2O, 1.2 MgSO4 and 10 HEPES (pH adjusted with NaOH to 7.40).
  • the GABA solutions were aspirated from the wells and an ice-cold STOP solution (in mM: 137 NaCl and 10 HEPES, pH adjusted with Tris-base to 7.40) was added to the wells, twice, and the cells were then washed in PBS. Cellular solubilization was then performed for 1 hour at room temperature by adding 200 ⁇ L lysis buffer (in mM: 100 NaOH and 0.1% SDS (Sodium Dodecyl Sulfate)) per well.
  • lysis buffer in mM: 100 NaOH and 0.1% SDS (Sodium Dodecyl Sulfate)
  • slices were incubated for 30min in 250 ⁇ l of RIPA buffer [50mM Tris pH 8.0, 1mM EDTA, 150mM NaCl, 1% NP40 (Nonyl phenoxlpoylethanol, from Fluka Biochemika, Switzerland), 1% SDS and 10% glycerol] with 25x protease inhibitors (Complete Mini-EDTA free, Roche, Germany) and 1mM PMSF to perform cell lysis.
  • RIPA buffer 50mM Tris pH 8.0, 1mM EDTA, 150mM NaCl, 1% NP40 (Nonyl phenoxlpoylethanol, from Fluka Biochemika, Switzerland), 1% SDS and 10% glycerol
  • 25x protease inhibitors Complete Mini-EDTA free, Roche, Germany
  • PMSF 1mM PMSF
  • Lysates were then centrifuged at 4oC for 10min and at 11000g. The supernatant was stored at -20 oC until further use.
  • Total protein in lysates obtained from primary cultures and brain slices was quantified with Bio-Rad DC reagent (Hercules, CA, USA) Bradford’s Assay, using BSA (Bovine Serum Albumin) as the standard to establish the calibration curves. Concentrations of 0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1 ⁇ g/ ⁇ l BSA were used to correlate protein concentration and absorbance, allowing the quantification of proteins.
  • Membranes were then blocked at room-temperature with 3% BSA in TBS-T (20 mM Tris base, 137 mM NaCl and 0, 1% Tween- 20) for 1 h and incubated with the primary antibody overnight at 4oC. On the following day, membranes were washed twice for 10 minutes with TBS-T and incubated for 1 h with the secondary antibody at room temperature. Image acquisition was performed by ECL Plus Western Blotting Detection System (Amersham-ECL Western Blotting Detection Reagents from GE Healthcare, Buckinghamshire, UK) and visualized with the ChemiDocTM XRS+Imager system (Hercules, CA, USA).
  • GAT1 glyceraldehyde 3-phosphate dehydrogenase
  • Figure 6 shows a decrease in GAT1-mediated GABA uptake in brain slices derived from treatment-na ⁇ ve STG compared to wildtype (WT) control mice.
  • GABA uptake in 12 weeks-old STG mice and their WT littermates was assessed, and the effect of BDNF upon the increase on GAT1-mediated GABA uptake was also measured using the methods described above for GAERS uptake.
  • TrkB-T1 agonists improve the deficient synaptic correlate of memory
  • Figure 7 shows that BDNF rescues the deficient hippocampal long-term potentiation (LTP) of GAERS rats. Twelve to 16 weeks old animals were used to evaluate synaptic plasticity using extracellular recordings of field excitatory postsynaptic potentials (fEPSPs) in GAERS and NEC as the cellular correlate of memory disfunction and the effect of BDNF upon LTP was evaluated. To obtain hippocampal slices, animals were deeply anesthetized with isoflurane, and dissection of the two hippocampi was rapidly made in ice-cold Krebs solution.
  • fEPSPs field excitatory postsynaptic potentials
  • Hippocampal slices 400 ⁇ m thick were cut perpendicularly using a McIlwain tissue chopper and recovered for 1h upon incubation at room temperature in oxygenated Krebs solution (5% CO2, 95% O2). After the recovery period, one slice at a time was placed in the recording chamber and continuously superfused with oxygenated Krebs solution at a constant flow of 3 ml/min and 32 °C.
  • a microelectrode filled with Krebs solution (2–6 M ⁇ resistance) was placed in the stratum radiatum of the CA1 region of the hippocampus, coupled to an Axoclamp 2B Amplifier (Axon Instruments) and the traces digitized with a BNC-2110 (National Instruments).
  • Stimulation (rectangular, 0.1 ms pulses every 20 seconds) was delivered through a concentric electrode placed on the Schaffer collateral-commissural fibres, in the stratum radiatum, near the CA3-CA1 border (P0) and near CA1 (P1). Stimulation alternately of two pathways of the Schaffer collateral-commissural fibres was performed. Individual responses were monitored, and averages of eight consecutive responses were continuously stored using WinLTP Software. LTP was induced by ⁇ -burst stimulation (TBS) since this pattern of stimulation is similar to what occurs physiologically in the hippocampus during episodes of learning and memory.
  • TBS ⁇ -burst stimulation
  • LTP was induced using TBS delivered at basal stimulus intensity, comprising of 5 bursts (200 ms interburst interval) at 5 Hz, with each burst composed of 5 pulses at 100Hz.
  • fEPSPs slope was recorded for 60 or 120 min after LTP induction, and each slope value corresponding to an average of 8 responses of 20 s.
  • BDNF (20ng/ml) was perfused in the bath for 30 minutes. After BDNF 30 min incubation, LTP was induced in the second pathway (P1 electrode). LTP was quantified as the percentage of change in the average slope of the fEPSP.
  • FIG 8 shows that BDNF administration rescues absence seizures in vivo.
  • Adult (3-5 month old) male GAERS rats were anesthetized with 2-5% inhalation isoflurane and implanted with gold plated epidural screws (Svenska Dentorama AB, Sweden) (to record the electroencephalogram (EEG), which were positioned bilaterally in the frontal, temporal and cerebellar cortices.
  • EEG electroencephalogram
  • Two different groups of GAERS rats were also bilaterally implanted with guide cannulae in either the primary somatosensory cortex or the thalamic ventrobasal nucleus for the subsequent intracerebral injection of Artificial Cerebrospinal Fluid (ACSF) or BDNF.
  • ACSF Artificial Cerebrospinal Fluid
  • BDNF Since BDNF does not easily penetrate the brain following systemic administration, it was injected intraperitoneally 20 min after intravenous injection of Elacridar (Ela), a phosphoglycoprotein inhibitor (5mg/kg) which is known to increase the permeability of the blood-brain barrier (Kallen et al, 2012). BDNF was administered (at the time indicated by the arrow) at three different doses (1 ⁇ g/, 10 ⁇ g/ and 30 ⁇ g/animal) and its effect was recorded for 2 hours in freely moving GAERS rats previously implanted with EEG electrodes (Figure 8A). The results are presented in 20 min bins as data normalized to the control period, i.e. pre-BDNF injection.
  • Veh1 vehicle 1
  • Veh2 vehicle 2
  • Figure 8B vehicle 8B
  • Ela alone i.e. the group injected with Elacridar+Veh2
  • Ela alone had no effect on the total time spent in absence seizures (Figure 8B), as previously shown (Yacone et al., 2021).
  • the same lack of effect was observed when BDNF was injected alone without pre-treatment with Elacridar (i.e. the group injected with Veh1+BDNF 10 ⁇ g).
  • the smallest dose of BDNF (1 ⁇ g/rat) had no effect on the total time spent in absence seizures.
  • BDNF at 10 and 30 ⁇ g/rat markedly decreased the total time spent in seizures (Two-Way ANOVA, p ⁇ 0.01 and 0.001, respectively) during the entire course of the 2 hours of recordings after BDNF injection compared to the control period ( Figure 8B).
  • Some representative EEG traces showing the effect of BDNF on the SWDs following the illustrated injections are shown below the plot in Figure 8B.
  • BDNF bilaterally injected (at the time indicated by the arrow) in the thalamic ventrobasal nucleus of another group of freely moving rats also led to a decrease in the total time spent in seizures compared to administration of ACSF (Figure 8C, right panels). However, this effect took longer to develop (since it was first observed in the 60-80 min time bin) but reached the same percentage decrease as the intracortical injection 2 hour after BDNF injection.
  • Figure 9 shows that the anti-seizure effect of BDNF is mediated via TrkB-T1 receptors.
  • Cannulae were bilaterally lowered into the previously implanted guide-cannulae and ACSF injected via a micropump (at ??/min flow rate) either the primary somatosensory cortex or the thalamic ventrobasal nucleus. Following a 1-hour (control) period, the ACSF solution was changed to one containing BDNF (20ng/ml) and recording continued for another 2-hours (drug-effect period) (Figure 9A). EEG data were analysed using the SeizureDetect routine of SPIKE2 (Cambridge Electronic Design, Cambridge, UK) that allows automatic extraction of spike-and-wave discharges from the raw EEG data, as described by McCafferty e al. (2018).
  • TrkB-T1 agonists mitigate the neuropsychiatric comorbidities of absence seizures in vivo.
  • Figure 10 shows that BDNF rescues the deficiencies in novel object recognition observed in GAERS rats.
  • the Novel Object Recognition (NOR) test was performed in adult GAERS and NEC rats and in GAERS rats bilaterally injected (via a pre-implanted cannula) with ACSF or BDNF (500ng) in the CA1 regions of the hippocampus. The animals were handled for 5 days (10 min/day) and on day 6 were habituated to the open field twice, for 10 min each time.
  • mice were 1) placed in the maze with two similar objects for 5 minutes or 2) injected with 500ng/side of BDNF or ACSF and after 1h hour were placed in the maze and allowed to freely explore two similar objects for 5 minutes. Animal order, injection drug, object place and objects were randomized using the Excel randomization tool.
  • the test day one object was replaced with a novel object, and the animal was allowed to explore the objects for 5 min. Exploration of the object for less than 10 seconds was deemed to be an invalid trial. Higher time exploring novel object compared with similar object is a measure of intact memory (Ennaceur, 2010). The percentage of time exploring an object and the difference in time exploring the familiar and the novel object (i.e. the novelty index) were analysed.
  • Figure 11 shows that the BDNF-mediated rescue of the impaired LTP and deficient novel object recognition of GAERS rats occurs via TrkB-T1.
  • the methods of injection of siRNA viral construct, 3 weeks before the behavioural experiments) and the cannula implantation are similar to those described in Figure 8, except that cannulae were inserted bilaterally in the hippocampus.
  • the results are shown in Figure 11.
  • Hippocampal slices of GAERS rats pre-injected with a viral vector containing an astrocyte-selective (GFAP promoter) scramble siRNA for TrkB-T1 were used for LTP measurements in the presence of ACSF or BDNF.
  • GFAP promoter astrocyte-selective
  • GFAP promoter astrocyte-selective
  • TrkB-T1 agonists are efficacious agents against absence seizures and their comorbidities.
  • a single therapeutic agent e.g., a TrkB-T1 agonist
  • BDNF Brain- derived neurotrophic factor
  • GABA transporter-1 GABA transporter-1
  • ivabradine a hyperpolarization-activated cyclic nucleotide-gated channel inhibitor, blocks spontaneous absence seizures.
  • Crispr-Cas A Laboratory Manual, 1 st edition, 2016, Cold Spring Harbor Laboratory Press, New York, USA. Diagnostic and Statistical Manual of Mental Disorders, 5 th Edition, 2013, American Psychiatric Association (APA), Washington D.C., USA. Handbook of Pharmaceutical Additives, 2 nd Edition, 2001, Endicott, New York, USA.

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Abstract

The invention relates to novel therapeutic agents and uses thereof. Methods of treating absence seizures and/or their comorbidities are provided herein, the methods comprising administering a TrkB-T1 agonist to a subject in need thereof. Methods of screening test compounds for TrkB-T1 agonist activity are also disclosed.

Description

Anti-Seizure Agents Field The present invention relates to agents for use in the treatment of absence seizures and their comorbidities. Background Absence seizures are genetic, generalised, non-convulsive seizures that consist of sudden, relatively brief (few seconds to half a minute) lapses of consciousness that are invariably associated with generalized 2.5 – 4Hz spike/polyspike-wave discharges (SWDs), as measured via electroencephalography (EEG). Absence seizures are believed to have a complex polygenic background involving a combination of rare genetic variants and polymorphisms and are highly prevalent in paediatric and juvenile populations. Absence seizures are the only clinical symptom in Childhood Absence Epilepsy (CAE), though they may often be present with other seizure types in various age-dependent and age- independent epilepsies having different severities and clinical outcomes (Crunelli et al.2020). Ethosuximide and valproate are typical first-line drugs for use in the treatment of absence seizures. Canonical anti-seizure medications (e.g., that suppress focal and generalized convulsive seizures) are generally ineffective for the treatment of (or aggravate) absence seizures. Absence seizures are therefore believed to be fundamentally different from other seizure types, and to have a unique pharmacological profile (Crunelli et al, 2020). Previously, absence seizures were considered relatively benign seizures, because of their non-convulsive nature and high remittance rate in early adulthood. However, recent studies in large CAE cohorts now demonstrate that 30% of children with CAE exhibit pharmaco-resistant seizures (Glauser et al.2013; Cnaan et al.2017), a rate similar to that of more severe convulsive seizures. Polytherapy is therefore common and is associated with a marked increase in drug adverse effects. Recent studies also show that ∼60% of children with absence seizures suffer various neuropsychiatric comorbidities. Attention deficit disorders and memory and learning impairments were observed to be the most common comorbidities of absence seizures (35–40%), followed by mood disorders (Caplan et al. 2008; Glauser et al.2010; Masur et al.2013; Gencpinar et al.2016; Lee et al.2018). Notably, comorbidities may precede the first absence seizure and an epilepsy diagnosis (Hermann et al.2007; Jones et al.2007) and may persist even after full pharmacological control of the seizures has been achieved (Glauser et al.2013). Moreover, comorbidities may be aggravated via the use of existing anti- seizure therapies including valproate. The large incidence of neuropsychiatric comorbidities and the marked increase in attention deficit symptoms observed with valproate treatment in CAE cohorts (Masur et al.2013) advise against the use of this drug as first-line monotherapy in paediatric patients. Thus, neuropsychiatric comorbidities are currently controlled by classical pro-cognitive drugs: that is, juvenile patients receive both an anti-absence medication and a cognitive enhancer, which in turn leads to an unwanted increase in adverse effects. Consequently, there is an urgent and unmet clinical need for a new therapeutic approach to treat absence seizures and their comorbidities. The present invention has been devised in light of the above considerations. Summary The present inventors have recognised that the TrkB isoform TrkB-T1 regulates signalling at GABAergic synapses and that activation of TrkB-T1 reduces the levels of extracellular GABA by increasing the function of GAT1, one of the GABA transporters. Agonists of TrkB-T1 may therefore be useful in the treatment of absence seizures and/or their co-morbidities. A first aspect of the invention provides a method of treating absence seizures and/or a comorbidity thereof, the method comprising: administering a therapeutically effective amount of a TrkB-T1 agonist to a subject in need thereof. A second aspect of the invention provides a TrkB-T1 agonist for use in a method of treating absence seizures and/or a comorbidity thereof in a subject. A third aspect of the invention provides the use of a TrkB-T1 agonist for the manufacture of a medicament for use in a method of treating absence seizures and/or a comorbidity thereof. In some embodiments of the second and third aspects of the invention, the method of treatment may be a method of the first aspect of the invention. In some preferred embodiments of the first, second and third aspects of the invention, the method of treatment may be a method of treating absence seizures and a comorbidity thereof. A fourth aspect of the invention provides a method of screening for a compound useful in treating absence seizures and/or a comorbidity thereof, comprising: determining the activity of TrkB-T1 in the presence or absence of a test compound, wherein an increase in the activity of TrkB-T1 in the presence relative to the absence of the test compound is indicative that the test compound is a candidate compound useful in treating absence seizures and/or a comorbidity thereof. Methods of the fourth aspect may comprise determining the activity of isolated TrkB-T1, or determining the activity of TrkB-T1 in a mammalian cell, tissue or non-human test animal. For example, methods of the fourth aspect may be in vitro or in vivo methods. Suitable non-human test animals include animal models of absence seizures. A fifth aspect of the invention provides a method of screening for a compound useful in treating absence seizures and/or a comorbidity thereof, the method comprising: (i) administering a test compound to a first and a second non-human test animal; wherein the first and second non-human test animals are animal models of absence seizures; and wherein the second non-human test animal exhibits reduced expression of TrkB-T1 and/or reduced TrkB-T1 function relative to the first non-human test animal, (ii) measuring the frequency of occurrence and/or duration of absence seizures in the first and second non-human test animals, and (iii) comparing the frequency of occurrence and/or duration of absence seizures as measured in step (ii) to a control sample. A reduction in the frequency of occurrence and/or duration of absence seizures in the first non-human test animal relative to the control sample, and no change or substantially no change in the frequency of occurrence and/or duration of absence seizures in the second non-human test animal relative to the control sample may be indicative that the test compound is a candidate compound useful in treating absence seizures and/or a comorbidity thereof. Suitable non-human test animals (exhibiting reduced expression of TrkB-T1 and/or expressing a non-functional variant of TrkB-T1) include animals administered with a TrkB-T1 antagonist. Suitable animal models of absence seizures include stargazer (STG) mice and Genetic Absence Epilepsy Rats from Strasbourg (GAERS). Methods of the fifth aspect of the invention may further comprise assessing a co-morbidity of an absence seizure in the first and second non-human test animals. For example, the cognitive behaviour of the first and second non-human test animals may be determined following administration of the test compound. Cognitive behaviour may include learning and/or memory or the presentation of an attention- deficit/hyperactivity disorder, a cognitive impairment, a memory or learning deficit, an autism spectrum disorder, schizophrenia, depression and/or an anxiety disorder. An improvement in the cognitive behaviour of the first non-human test animal as compared to the second non-human test animal may be indicative that the test compound is useful in treating a comorbidity of absence seizures. A sixth aspect of the invention provides a method of screening for a compound useful in treating absence seizures and/or a comorbidity thereof, the method comprising: (i) administering a test compound to a first and a second non-human test animal; wherein the first and second non-human test animals are animal models of absence seizures, and wherein the second non-human test animal exhibits reduced expression of TrkB-T1 and/or reduced TrkB-T1 function relative to the first non-human test animal; and (ii) measuring the cognitive behaviour of the first and second non-human test animals. An improvement in the learning and/or memory of the first non-human test animal as compared to the second non-human test animal; or an improvement in the presentation of an attention-deficit/hyperactivity disorder, a cognitive impairment, a memory or learning deficit, an autism spectrum disorder, schizophrenia, depression and/or an anxiety disorder in the first non-human test animal as compared to the second non-human test animal are indicative that the test compound is a candidate compound useful in treating absence seizures and/or a comorbidity thereof. Suitable non-human test animals (exhibiting reduced expression of TrkB-T1 and/or expressing a non-functional variant of TrkB-T1) include animals administered with a TrkB-T1 antagonist. Suitable animal models of absence seizures include STG mice and GAERS rats. Other aspects and embodiments of the invention are described in more detail below. Brief Description of the Figures Figure 1 shows a schematic diagram of a GABA synapse and illustrates the role of TrkB-T1 receptors in regulating the plasma membrane expression and function of GAT1, one of the GABA transporters, in astrocytes. GAT1 removes GABA from the extracellular space. TrkB-T1 receptors are exclusively located on astrocytes, and act as negative regulators of GAT1 recycling from the plasma membrane to the cytoplasm. Activation of TrkB-T1 by the endogenous agonist brain-derived neurotrophic factor (BDNF) thereby increases the expression of GAT1 at the plasma membrane, leading to decreased extracellular levels of GABA and decreased tonic GABAA receptor-mediated inhibition (which is mediated by extrasynaptic GABAA receptors). Figure 2 shows a summary of the main features of two well-established animal models of absence seizures: the (STG mice and the GAERS rats. The absence seizures observed in STG mice exhibit a frequency of spike-and-wave discharges (SWDs) of 5-6 Hz and their ontogeny is >2 weeks. These mice carry a single spontaneous recessive mutation in the “Stargazin” (calcium voltage-gated channel auxiliary subunit gamma 2, CACNG2) gene and exhibit head tossing, ataxia, impaired vestibular function, increased mossy fibre sprouting in hippocampus, and hyperactivity. In contrast, GAERS rats exhibit absence seizures with a frequency of SWDs of 7-11 Hz and their ontogeny is >30 days (with all animals exhibiting seizures at 3 months). These rats carry unknown polygenic mutations that follow a pattern of autosomal dominant transmission and exhibit astrocytic alterations but no neuropathological abnormalities. In both animal models, absence seizures may be blocked by ethosuximide and valproic acid. Moreover, both STG mice and GAERS rats have an enhanced thalamic tonic GABAA inhibition that is developmentally regulated and due to GAT1 loss-of-function. Figure 3 shows a decrease in GAT1-mediated GABA uptake in brain slices derived from treatment-naïve GAERS and Non-Epileptic Control (NEC) rats (that have no absence seizures), but no change in the GABA uptake mediated by GAT3, the other main GABA transporter in the brain. A reduction in the total (i.e., GAT1-mediated + GAT3-mediated) GABA uptake was identified in slices of the somatosensory cortex (*, P= 0,0389), thalamus (****, P <0,0001) and hippocampus (*** P <0,0001) of GAERS compared to NEC animals (GAERS n=8, NEC n=7, extra sum of squares f test). Consequently, levels of GAT1- mediated and GAT3-mediated GABA uptake were assessed independently. A reduction in GAT1- mediated GABA uptake was observed in slices of somatosensory cortex (***, P= 0,0004), thalamus (***, P= 0,0002) and hippocampus (*** P= 0,0009) of GAERS compared to NEC rats (GAERS n=6 vs. NEC n=6, extra sum of squares f test). However, no change in GAT3-mediated uptake was identified in any of the tested brain areas (somatosensory cortex, P= 0,0659; thalamus, P= 0,0534, hippocampus, P= 0,8636; GAERS n=6 vs. NEC n=7, extra sum of squares f test). Figure 4 shows a decrease in GAT1-mediated, but not GAT3-mediated, GABA uptake in primary cultures of astrocytes derived from treatment-naïve GAERS and NEC rats. A reduction in total (i.e., GAT1- mediated + GAT3-mediated) GABA uptake was identified in astrocytes derived from the cortex (*, P= 0,0233) or thalamus (*, P= 0,0071) of GAERS compared to NEC animals (GAERS n=7, NEC n=6, 7 extra sum of squares f test). Consequently, levels of GAT1-mediated and GAT3-mediated GABA uptake were assessed independently. A reduction in GAT1-mediated GABA uptake was observed in cortical (*, P= 0,0318) and thalamic astrocytic cultures (****, P<0,0001) derived from GAERS compared to NEC rats (GAERS n=7 vs. NEC n=6, extra sum of squares f test ). However, no change in GAT3-mediated uptake was identified (cortical astrocytes, P= 0,9559; thalamic astrocytes; P= 0,5921; GAERS n=7 vs. NEC n=7, extra sum of squares f test). Figure 5 shows that BDNF (a TrkB-T1 agonist) increases GAT1 expression at the plasma membrane and GAT1-mediated GABA uptake in GAERS rats. (A) GAT1 (67 kDa) expression was measured in homogenates derived from thalamic slices in the presence and absence of BDNF (30min incubation, 20ng/ml). Cytoplasmic and plasma membrane homogenates show the exclusive GAT1 expression in the plasma membrane. (B) A large increase in total GAT1 expression was observed in BDNF-treated samples as compared to control samples treated with Artificial Cerebrospinal Fluid (ACSF) (GAERS n= 5 vs. GAERS+BDNF n= 5, *, P= 0,0259, unpaired t-test). (C) GAT1-mediated GABA uptake was increased in slices of the motor cortex (*, P= 0,0033), thalamus (**, P= 0,0449) and hippocampus (**, P= 0,0078), but not of somatosensory cortex (P= 0,1612) and visual cortex (P= 0,1411) of GAERS compared to NEC animals (GAERS n= 5-6 vs. NEC n=5-6, unpaired t-test). Figure 6 shows a decrease in GAT1-mediated GABA uptake in brain slices derived from treatment-naïve STG compared to wildtype (WT) control mice. (A) A reduction in total (i.e., GAT1-mediated + GAT3- mediated) GABA uptake was identified in slices of the thalamus (**, P=0,0037) and hippocampus (* P=0,0107) of STG compared to WT mice (STG n=8, WT n=7/8, extra sum of squares f test). GAT1- mediated GABA uptake was subsequently assessed, and a similar decrease was noted in slices of the thalamus (*, P=0,0166) and hippocampus (* P=0.0326) of STG compared to WT animals (STG n=7/8, WT n=8/9, extra sum of squares f test). (B) An increase in GAT-1 mediated GABA uptake was observed in slices of the motor cortex (*, P=0,0124) somatosensory cortex (*, P=0,0437), and thalamus (**, P=0,0069) but not visual cortex (ns, P=0,4062) and hippocampus (ns, P=0,3069) of STG mice incubated with BDNF (30min incubation, 20ng/ml) compared to slices incubated with ACSF (STG n=4-6 vs. STG+BDNF n=4-6, unpaired t-test). Figure 7 shows that BDNF rescues the deficient hippocampal long-term potentiation (LTP) of GAERS rats. (A) Slope of field excitatory postsynaptic potentials (fEPSP) recorded in CA1 hippocampal neurons in response to stimulation of the Schaffer collaterals CA3 are presented as measured in ACSF treated NEC (n=13) and GAERS (n=15) hippocampal slices, alongside slopes measured in GAERS slices treated with BDNF (20ng/ml, 30min incubation) (n=5). (B) LTP magnitude was assessed 2 hours after LTP induction, and an increase of ~25% was observed in BDNF-treated GAERS slices compared to ACSF- treated GAERS slices (**, P=0,0041). Note that the LTP magnitude measured in NEC and BDNF-treated GAERS slices was similar (P=0,3418) but differences were found between ACSF-treated NEC and GAERS slices (*, P=0,0109). Figure 8 shows that BDNF administration rescues absence seizures in vivo. (A) A schematic representation of the study protocol. (B) Systemic (i.e., intraperitoneal) administration of BDN or Vehicle 1 (Veh1) occurred 20 min after intravenous administration of Vehicle 2 (Veh2) or Elacridar (Ela) (a phosphoglycoprotein inhibitor) to allow brain penetration of BDNF. Administration of BDNF (10 or 30 ^g) was observed to significantly reduced the total time spent in seizures across all time points measured (20, 40, 60, 80, 100 and 120 minutes), compared to control animals (Veh1+Veh2, n=8, Ela+Veh2, n=10; Veh1+BDNF, n=7, Ela+BDNF 1 ^g, n= 7, Ela+BDNF 10 ^g, n=8, Ela+BDNF 30 ^g, n=7) (Veh1+Veh2 vs Ela+BDNF 10, *** P=0,0002; Veh1+Veh2 vs Ela+BDNF 30, *** P= 0,0011) (Two-Way ANOVA). Note that the measurement of the total seizure duration was normalised to pre-injection levels (control period) for each animal. On the bottom of the figure, representative EEG traces for Veh1+Veh2 and Ela+BDNF 30 ^g can be seen. (C) Targeting the cortex (left) and the thalamus (right) with intra-cortical and intra- thalamic injection of BDNF, respectively, decreases the time spent in seizures. Cortex, ACSF n=5 , BDNF n=6 , ****P<0.0001. Thalamus, ACSF n=7, BDNF n=8, ***P=0.0002; Two-Way ANOVA. Note that the measurement of the total time spent in seizures was normalised to pre-injection levels (control period) for each animal. Representative EEG traces can be found on the bottom of each graph. Figure 9 shows that the anti-seizure effect of BDNF is mediated via TrkB-T1 receptors. (A) A schematic representation of the study protocol. (B) Targeting TrkB-T1 in the cortex. Total time spent in seizures was measured in GAERS rats administered intracortically with an astrocyte-specific scrambled TrkB-T1 siRNA (6.86E+10 viral particles) (closed circles) or an astrocyte-specific TrkB-T1 knockdown siRNA (6.23E+10 viral particles) (open circles) and BDNF (500ng) administrated intracortically to both groups of rats. BNDF was observed to significantly reduce seizure duration in GAERS injected with the scramble siRNA (across all time points measured: 20, 40, 60, 80, 100 and 120 min, ****, P<0.0001; Two-Way ANOVA), but had no effect in the GAERS treated intracortically with the TrkB-T1 knockdown siRNA (scrambled siRNA, n=7, knockdown siRNA, n=8). Note that the measurement of seizure duration was normalised to pre-injection levels for each animal. Representative EEG traces can be found on the bottom of the graph. (C) Targeting TrkB-T1 in the thalamus. Total seizure duration was measured in GAERS rats administered intrathalamically with an astrocyte-specific scrambled TrkB-T1 siRNA (6.86E+10 viral particles) (closed circles) or an astrocyte-specific TrkB-T1 knockdown siRNA (6.23E+10 viral particles) (open circles) and BDNF (500ng) administrated intrathalamically to both groups of rats. BNDF was observed to significantly reduce the total time spent in seizures in GAERS injected with the scramble siRNA (across all time points measured: 20, 40, 60, 80, 100 and 120 min; *, P<0.05; **, P<0.01; ****, P<0.0001; Two-Way ANOVA), but had no effect in the GAERS treated intrathalamically with the TrkB-T1 knockdown siRNA (scrambled siRNA, n=7, knockdown siRNA, n=7 ). Note that the measurement of seizure duration was normalised to pre-injection levels for each animal. Representative EEG traces can be found on the bottom of the graph. Figure 10 shows that BDNF rescues the deficiencies in novel object recognition observed in GAERS rats. (A) A schematic representation of the study protocol. (B) GAERS spend less percentage of time exploring the new object compared to NEC rats (left plot) (NEC, n=13; GAERS, n=13; ** P= 0,002). GAERS rats have a negative Novelty Index, contrary to NEC, showing their inability to identify the new object (right plot) (NEC, n=13 vs GAERS, n=13, ** P= 0,0028). (C) Graphical representation of the percentage of time each GAERS and NEC rat spends exploring the familiar object (black) and the novel object (white) (NEC, n=11; GAERS, n=7). (D) In GAERS rats injected with BDNF (500ng) in the CA1 region of the hippocampus the percentage of time exploring the new object is increased compared to ACSF-injected GAERS rats (ACSF, n=6 vs BDNF, n=10, ** P= 0,0011) (left plot). In the same GAERS rats, the negative Novelty Index is rescued by intrahippocampally injected BDNF (ACSF, n=6 vs BDNF, n=10, ** P= 0,0011) (right plot). (E) Graphical representation of the percentage of time each animal spends exploring the familiar object (black) and the novel object (white) in ACSF and BDNF injected GAERS rats (ACSF, n=7; BDNF, n=11). Figure 11 shows that the BDNF-mediated rescue of the impaired LTP and deficient novel object recognition of GAERS rats occurs via TrkB-T1. (A) fEPSP slopes of CA1 neurons in hippocampal slices derived from GAERS rats pre-treated (3 weeks earlier) with an astrocyte-specific, scrambled TrkB-T1 siRNA ((6.86E+10 viral particles), in the presence (n=7) or absence (n=7) of BDNF (20ng/ml, 30min incubation). (B) LTP magnitude was increases by ~75% in BDNF-treated compared to ACSF-treated GAERS hippocampal slices (*, P=0,0161; unpaired t-test). (C) fEPSP slopes of CA1 neurons in hippocampal slices derived from GAERS rats pre-treated (3 weeks earlier) with an astrocyte-specific, TrkB-T1 knockdown siRNA (6.23E+10 viral particles) in the presence (n=6) or absence (n=6) of BDNF (20ng/ml, 30min incubation. (D) LTP magnitude was not different between ACSF- and BDNF-treated GAERS hippocampal slices (P=0.114, unpaired t-test). (E) A schematic representation of the protocol of the Novel Object Recognition study shown in F and G, where all GAERS rats received intrahippocampal BDNF (500ng). (F) GAERS injected with astrocyte-specific TrkB-T1 knockdown siRNA spend less percentage of time exploring the novel object compared to GAERS injected with an astrocyte-specific scrambled TrkB-T1 siRNA (left plot) (scramble, n=8; siRNA, n=7, ** P= 0,0215). GAERS rats injected with an astrocyte-specific TrkB-T1 knockdown siRNA have a smaller Novelty Index, compared to GAERS rats injected with an astrocyte-specific scrambled TrkB-T1 siRNA (right plot) (scramble, n=8 vs siRNA, n=7, ** P= 0,0215). (G) Graphical representation of the percentage of time each GAERS rat spends exploring the familiar (black) and the novel (white) object for astrocyte-specific scrambled TrkB-T1 siRNA- and astrocyte-specific TrkB-T1 knockdown siRNA-injected animals. Detailed Description The invention relates to the treatment of absence seizures and their comorbidities by administering a therapeutically effective amount of a TrkB-T1 agonist to a subject in need thereof. Activation of TrkB-T1 receptors by TrkB-T1 agonists are shown herein to act as negative regulators of GAT1 recycling from the plasma membrane to the cytoplasm: this decreases the levels of extracellular GABA which in turn rescues both absence seizures and their memory comorbidity. The experimental data below demonstrate that TrkB-T1 agonists significantly reduce the frequency of occurrence and duration of absence seizures in vivo and are effective in alleviating the memory/learning comorbidities, such as deficiencies in novel object recognition that are typical of animal models of absence seizures. Consequently, TrkB-T1 agonists may be useful in the treatment of absence seizures and their comorbidities. TrkB-T1 is a truncated variant of Tropomyosin receptor kinase B (TrkB). TrkB is also known as tyrosine receptor kinase B, BDNF/NT-3 growth factors receptor, or neurotrophic tyrosine kinase receptor type, and may also be termed TrkB full-length (“Trkb-FL”). TrkB-FL is encoded by the gene NTRK2 in humans and mice (Homo sapiens gene ID 4915; Mus musculus gene ID 18212). TrkB-T1 is located exclusively on astrocytes and is the major isoform of TrkB-FL that is expressed in the adult mammalian brain. For example, TrkB-T1 as described herein may be present in thalamic, cortical or hippocampal astrocytes. An overview of the role of the astrocytic TrkB-T1 in regulating the expression of GAT1 at GABAergic synapses is shown in Figure 1. In some preferred embodiments, TrkB-FL may be human TrkB-FL and may have the amino acid sequence of UniProt Q16620-1 (Homo sapiens) (SEQ ID NO: 3) or a variant thereof. TrkB-T1 may be human TrkB-T1 and may have the amino acid sequence of UniProt Q16620-2 (Homo sapiens) (SEQ ID NO: 4) or a variant thereof. In other embodiments, TrkB-FL may be mouse TrkB-FL and may have the amino acid sequence of UniProt P15209-1 (Mus musculus) (SEQ ID NO: 1) or a variant thereof. TrkB-T1 may be mouse TrkB-T1 and may have the amino acid sequence of UniProt P15209-2 (Mus musculus) (SEQ ID NO: 2) or a variant thereof. A TrkB-T1 agonist is an agent that is capable of stimulating or activating a TrkB-T1 receptor. This may for example, elicit a biological effect, such as inhibited or reduced recycling of the GAT1 transporter (via inhibition of dynamin/clathrin-dependent constitutive internalization of GAT-1) (Vaz et al, 2011) and the subsequent reduction in extracellular GABA levels at GABAergic synapses. BDNF-induced activation of TrkB-T1 may enhance the acidic metabolite release from a cell (Baxter et al., 1997) and elicit calcium transients, i.e. calcium release from intracellular stores, in astrocytes (Rose et al., 2003). A “full agonist” may achieve the maximum possible biological response, whilst a “partial agonist” may generate a partial biological response with equivalent receptor occupancy. A TrkB-T1 agonist as described herein may be a selective agonist of TrkB-T1. A selective agonist is an agonist that is selective for a particular species of receptor over one or more other species of receptor. A selective agonist of TrkB-T1 may exhibit a higher binding affinity for TrkB-T1 than for TrkB-FL. For example a selective agonist of TrkB-T1 may exhibit a binding affinity (Kd) for TrkB-T1 that is lower than its binding affinity (Kd) for TrkB-FL. A TrkB-T1 agonist disclosed herein may for example exhibit a Kd value in the range of picomolar (10-12 M) to micromolar (10-6 M) concentrations, such as a Kd value between 1, 10 or 100 pM and 1, 10 or 100 µM. A selective agonist of TrkB-T1 may also exhibit higher efficacy (in eliciting a biological response) on binding to TrkB-T1 compared to binding to TrkB-FL. A selective agonist of TrkB-T1 may exhibit a half effective concentration (EC50) for activation of TrkB-T1 that is lower than its half effective concentration (EC50) for activation of TrkB-FL. A non-selective agonist of TrkB may activate both TrkB-T1 and TrkB-FL. For reference, brain-derived neurotrophic factor (BDNF) is a non-selective agonist of TrkB species. A TrkB-T1 agonist disclosed herein may exhibit an EC50 value in the range of picomolar (10-12 M) to micromolar (10-6 M) concentrations, for example, an EC50 between 1 pM, 10 pM or 100 pM and 1 µM, 10 uM or 100 µM. Methods suitable for assessing the binding affinity (Kd) and half effective concentration (EC50) of an agonist are well known in the art (see e.g. Rang & Dale’s Pharmacology 9th Edition, Elsevier Churchill Livingstone 2019, England, UK). Suitable TrkB-T1 agonists include an organic compound having a molecular weight of 900 Da or less; a protein or peptide that specifically binds TrkB-T1, for example, an antibody molecule that specifically binds TrkB-T1, or a peptide that binds TrkB-T1 and enhances its activity (an agonist peptide); and a nucleic acid that specifically binds TrkB-T1, for example, an aptamer that specifically binds TrkB-T1. For example, suitable TrkB-T1 agonists may include brain derived neurotrophic factor (BDNF; Gene ID 627; reference sequence NP_001137277.1) and variants and derivatives thereof. A TrkB-T1 agonist as described above may be administered alone or may be formulated into a pharmaceutical composition. A pharmaceutical composition is a formulation comprising one or more active agents and one or more pharmaceutically acceptable excipients. The pharmaceutical composition may be capable of eliciting a therapeutic effect. A suitable pharmaceutical composition for use as described herein may comprise an agent described above and a pharmaceutically acceptable excipient. For example, a pharmaceutical composition may comprise a TrkB-T1 agonist as described herein, and a pharmaceutically acceptable excipient, carrier, diluent, or adjuvant. The term “pharmaceutically acceptable” relates to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound veterinary or medical judgement, suitable for use in contact with the tissues of a subject (e.g. human or other mammal) without excessive toxicity, irritation, allergic response, or other problem or complication, and that are commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must be “acceptable” in the sense of being compatible with the other ingredients of the formulation. Suitable excipients and carriers include, without limitation: water, saline, buffered saline, phosphate buffer, alcoholic/aqueous solutions, emulsions or suspensions. Other conventionally employed diluents, adjuvants, and excipients may be added in accordance with conventional techniques. Such carriers can include ethanol, polyols, and suitable mixtures thereof, vegetable oils, and injectable organic esters. Buffers and pH-adjusting agents may also be employed, and include, without limitation, salts prepared from an organic acid or base. Representative buffers include, without limitation: organic acid salts, such as salts of citric acid (e.g., citrates), ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, phthalic acid, Tris, trimethylamine hydrochloride, or phosphate buffers. Parenteral carriers can include sodium chloride solution, Ringer's dextrose, dextrose, trehalose, sucrose, lactated Ringer's, or fixed oils. Intravenous carriers can include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives such as, for example, antimicrobials, antioxidants, chelating agents (e.g., EGTA; EDTA), inert gases, and the like may also be provided in the pharmaceutical carriers. The pharmaceutical compositions described herein are not limited by the selection of the carrier. The preparation of these pharmaceutically acceptable compositions, from the above-described components, having appropriate pH, isotonicity, stability and other conventional characteristics, is within the skill of the art. Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical texts. See, for example, Handbook of Pharmaceutical Additives, 2nd Edition, 2001, Remington's Pharmaceutical Sciences, 20th Edition, 2000; and Handbook of Pharmaceutical Excipients, 2nd Edition, 1994. A pharmaceutical composition may conveniently be presented in unit dosage form and may be prepared by any methods well-known in the art of pharmacy. Such methods include the step of bringing the one or more TrkB-T1 agonists into association with a carrier or excipient as described above which may constitute one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both. Pharmaceutical compositions described herein may be produced in various forms, depending upon the route of administration. Routes may include parenteral, intravenous, intraarterial, intramuscular, oral and nasal routes. The pharmaceutical compositions may include liquid or solid forms and may be prepared for administration to subjects in the form of, for example, liquids, powders, aerosols, tablets, capsules, enteric-coated tablets or capsules, or suppositories. Pharmaceutical compositions may also be in the form of suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained- release or biodegradable formulations. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials, such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt. Pharmaceutical compositions may be made in the form of sterile aqueous solutions or dispersions, suitable for injectable use, or made in lyophilized forms using freeze-drying techniques. Lyophilized pharmaceutical compositions are typically maintained at about 4°C, and can be reconstituted in a stabilizing solution, e.g., saline or HEPES, with or without adjuvant. Pharmaceutical compositions can also be made in the form of suspensions or emulsions. The precise nature of the carrier or other material will depend on the route of administration, which may be any convenient route, for example by injection, e.g. cutaneous, subcutaneous, or intravenous. Preferably, the agent is administered systemically, e.g. intravenously. The pharmaceutical compositions comprising the active compounds may be formulated in a dosage unit formulation that is appropriate for the intended route of administration. Pharmaceutical compositions may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections immediately prior to use. Methods of determining the most effective means and dosage of administration are well known in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the physician. Administration in vivo can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals). Multiple doses of the composition may be administered, for example 2, 3, 4, 5 or more than 5 doses may be administered. The administration of the composition may continue for sustained periods of time. For example, treatment with the composition may be continued for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month or at least 2 months. Treatment with the composition may be continued for as long as is necessary to reduce symptoms. A TrkB-T1 agonist may be useful in treating an absence seizure and/or one or more of their comorbidities in a patient. An absence seizure is a genetic, generalised, non-convulsive seizure that consists of sudden, relatively brief (few seconds to half a minute) lapses of consciousness that are associated with generalized 2.5 – 4Hz spike/polyspike-wave discharges (SWDs), as measured via electroencephalography (EEG). In some patients, absence seizures may be accompanied by convulsive seizures. A comorbidity of an absence seizure is a clinical manifestation that accompanies or is associated with an absence seizure. A comorbidity may occur before the first absence seizure or the first diagnosis of an absence seizure in a patient and may persist even after full pharmacological control of the absence seizures in a patient. Comorbidities of absence seizures may include neuropsychiatric comorbidities such as attention-deficit/hyperactivity disorder, cognitive impairment, memory and learning deficits, autism spectrum disorder, schizophrenia, depression and anxiety disorders (such as agoraphobia, selective mutism, generalized anxiety disorder (GAD), social anxiety disorder, obsessive-compulsive disorder (OCD) and panic disorder). Suitable criteria for the diagnosis of comorbidity of absence seizure are well known in the art (see for example Diagnostic and Statistical Manual of Mental Disorders, 5th Edition, 2013) Treatment may be any treatment and therapy, whether of a human or an animal (e.g. in veterinary applications), in which some desired therapeutic effect is achieved, for example, improving or ameliorating one or more symptoms of an absence seizure or comorbidity. Treatment may be preventative or curative. An subject suitable for treatment as described above may be a mammal, such as a rodent (e.g. a guinea pig, a hamster, a rat), murine (e.g. a mouse), canine (e.g. a dog), feline (e.g. a cat), equine (e.g. a horse), a primate, simian (e.g. a monkey or ape), a monkey (e.g. marmoset, baboon), an ape (e.g. gorilla, chimpanzee, orang-utan, gibbon), or a human. In some preferred embodiments, the subject is a human. In other preferred embodiments, non-human mammals, especially mammals that are conventionally used as models for demonstrating therapeutic efficacy in humans (e.g. murine, rodent, primate, porcine, canine, or leporid) may be employed. Suitable subjects for treatment as described here include a human paediatric subject having Childhood Absence Epilepsy (CAE), a teenage human subject having Juvenile Absence Epilepsy (JAE), and a young or adult subject having absence as well as convulsive seizures. A subject may carry a dysfunctional or non-functional sodium- and chloride-dependent GABA transporter 1 (GAT1), for example, caused by a loss-of-functional SLC6A1 gene variant. SLC6A1 (Homo sapiens gene ID 6529; Mus musculus gene ID 232333) encodes GABA transporter 1 (GAT1). GAT1 is expressed on astrocytes and is responsible for removing GABA from the synaptic cleft at GABAergic synapses. TrkB-T1 agonists are shown herein to reduce the internalisation of GAT1, thereby increasing the expression of GAT1 in the plasma membrane and thus facilitating an increase in GAT1-mediated GABA uptake at the synapse. An individual with an absence seizure or comorbidity may display at least one identifiable sign, symptom, or laboratory finding that is sufficient to make a diagnosis of absence seizure or comorbidity in accordance with clinical standards known in the art. Examples of such clinical standards can be found in textbooks of medicine. It will be appreciated that appropriate dosages of a TrkB-T1 agonist may vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the treatments of the present invention. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular agent, the route of administration, the time of administration, the rate of loss or inactivation of the agent, the duration of the treatment, other drugs, compounds, and/or materials used in combination, and the age, sex, weight, condition, general health, and prior medical history of the patient. The dosage of agent and the route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve local concentrations at the site of injury which achieve the desired effect without causing substantial harmful or deleterious side-effects. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors and may depend on the severity of the symptoms and/or progression of a disease being treated. Appropriate doses of therapeutic polypeptides are well known in the art (Ledermann J.A. et al. (1991) Int. J. Cancer 47: 659-664; Bagshawe K.D. et al. (1991) Antibody, Immunoconjugates and Radiopharmaceuticals 4: 915-922). Specific dosages may be indicated herein or in the Physicians’ Desk Reference, 57th Edition, 2003, PDR Network LLC, New Jersey, USA as appropriate for the type of medicament being administered may be used. A therapeutically effective amount or suitable dose of a agent described herein may be determined by comparing its in vitro activity and in vivo activity in an animal model. Methods for extrapolation of effective dosages in mice and other test animals to humans are known. Treatment may comprise the administration of a therapeutically effective amount of the agent or pharmaceutical composition to the individual. “Therapeutically effective amount" relates to the amount of a agent or pharmaceutical composition that is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio. For example, a suitable amount of a agent or pharmaceutical composition for administration to an individual may be an amount that generates a therapeutic effect in the individual. A therapeutic effect may be at least amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the composition, the method of administration, the scheduling of administration and other factors known to medical practitioners. In some embodiments, a treatment as described herein may have a duration of up to 3 weeks, up to 6 weeks, up to 3 months, up to 6 months or up to 12 months. The treatment schedule for an individual may be dependent on the pharmacokinetic and pharmacodynamic properties of the agent, the route of administration and the nature of the condition being treated. Treatment may be in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals). Treatment may be periodic, and the period between administrations may be about 12 hours or more, 24 hours or more, 36 hours or more, 48 hours or more, 96 hours or more, or one week or more. Suitable formulations and routes of administration are described above and may be readily determined by a physician for any individual patient. In some embodiments, the TrkB-T1 agonist may be administered for the treatment absence seizures and/or co-morbidities without the co-administration of other therapies. In other embodiments, a TrkB-T1 agonist may be administered in combination with one or more other therapies, either simultaneously or sequentially dependent upon the circumstances of the individual to be treated. The second pharmaceutical agent may be an anti-seizure agent. Appropriate anti-seizure agents will be readily appreciated by those skilled in the art. When TrkB-T1 agonists are used in combination with additional therapeutic agents, the compounds may be administered either sequentially or simultaneously by any convenient route. When TrkB-T1 agonists are used in combination with an additional therapeutic agent active against the same disease, the dose of each agent in the combination may differ from that when the therapeutic agents are used alone. Appropriate doses will be readily appreciated by those skilled in the art. In other embodiments, TrkB-T1 may be useful in screening for compounds that may be useful in the development of therapeutics for treating absence seizures and/or co-morbidities of absence seizures. A method of screening for a compound useful in treating absence seizures and/or a comorbidity thereof may comprise determining the activity of TrkB-T1 in the presence or absence of a test compound. An increase in the activity of TrkB-T1 in the presence relative to the absence of the test compound is indicative that the test compound is a TrkB-T1 agonist and is a candidate compound useful in treating absence seizures and/or a comorbidity thereof. Methods described herein may comprise determining the activity of isolated TrkB-T1, or the activity of TrkB-T1 in a mammalian cell, a mammalian tissue or non-human mammal. In some embodiments, a method of screening for a compound useful in treating absence seizures and/or a comorbidity thereof may comprise determining the activity of TrkB-T1 and TrkB-FL in the presence or absence of a test compound. An increase in the activity of TrkB-T1 in the presence relative to the absence of the test compound that is greater than the increase in the activity of TrkB-FL in the presence relative to the absence of the test compound may be indicative that the test compound is a selective TrkB-T1 agonist and is a candidate compound useful in treating absence seizures and/or a comorbidity thereof. For example, the activity of TrkB-T1 may increase in the presence relative to the absence of the test compound and the activity of TrkB-FL may not increase or may decrease in the presence relative to the absence of the test compound. Methods suitable for determining the activity of TrkB-T1, TrkB-FL and GAT1 are well known in the art and are readily available to those skilled in the art (see e.g. Rang & Dale’s Pharmacology 9th Edition, Elsevier Churchill Livingstone 2019, England, UK). For example, suitable methods may involve assessing the expression of TrkB-T1, TrkB-FL and GAT1 at the plasma membrane, assessing modifications thereof (e.g. dimerization, phosphorylation), and/or assessing biological responses produced by cells or organisms in which the TrkB-T1, TrkB-FL or GAT1 are expressed. Biophysical techniques, for example X-ray crystallography, cryo-electron microscopy, and nuclear magnetic resonance spectroscopy may also be used in order to determine the location of TrkB-T1, TrkB-FL and GAT1 and to characterise any conformational changes that occur upon ligand binding and/or activation. In preferred embodiments, determining the activity of TrkB-T1 comprises measuring the expression of GABA transporter 1 (GAT1) at the plasma membrane or measuring the trafficking of GAT1 between the cytoplasm and the plasma membrane. Suitable methods include enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), or immunofluorescence (IF). The expression profile may also be quantified using flow cytometry and associated techniques (e.g. time-of-flight (TOF) cytometry or mass spectrometry, CyTOF). For standard molecular biology techniques, see Molecular Cloning, A Laboratory Manual.3rd Edition, 2001. Methods described herein may comprise determining the activity of isolated TrkB-T1, or the activity of TrkB-T1 in a mammalian cell, a mammalian tissue or non-human mammal. In some embodiments, a test compound showing activity in an in vitro method described herein may be subsequently tested in one or more in vivo methods described herein. In addition, a method of screening for a compound useful in treating absence seizures and/or a comorbidity thereof is disclosed herein, comprising: (i) administering a test compound to a first and a second non-human test animal; in which the first and second non-human test animals are animal models of absence seizures, and in which the second non-human test animal exhibits reduced expression of TrkB-T1 and/or reduced TrkB-T1 function as compared to the first non-human test animal; (ii) measuring the frequency of occurrence and/or duration of absence seizures in the first and second non-human test animals, and (iii) comparing the frequency of occurrence and/or duration of absence seizures as measured in step (ii) to a control sample; in which a reduction in the frequency of occurrence and/or duration of absence seizures in the first non-human test animal relative to the control sample, and substantially no change in the frequency of occurrence and/or duration of absence seizures in the second non-human test animal relative to the control sample are indicative that the test compound is a candidate compound useful in treating absence seizures and/or a comorbidity thereof. Suitable animal models of absence seizures include GAERS rat and Stargazer mouse as described herein. The non-human test animal exhibiting reduced expression of TrkB-T1 or expressing a non- functional variant of TrkB-T1 may be an animal administered with a TrkB-T1 antagonist. A TrkB-T1 antagonist is agent that is capable of binding to and inhibiting the activation of TrkB-T1. For example, the antagonist may bind to the agonist binding site, or may achieve inhibition by allosteric modulation of TrkB-T1. An antagonist may exhibit affinity for the agonist binding site of the TrkB-T1, but without exhibiting efficacy (i.e., without stimulating receptor signalling). For example, a suitable TrkB-T1 antagonist may be a competitive antagonist of a TrkB-T1 agonist. Antagonists may also include agents that are capable of inhibiting the expression of a signalling receptor. For example, a suitable TrkB-T1 antagonists may include competitive antagonists of the test compound, an allosteric modulator of TrkB- T1, a TrkB-T1 small interfering RNA (siRNA), and a TrkB-T1 microRNA (miRNA). Suitable TrkB-T1 antagonists may also include selective antagonists of TrkB-T1 on astrocytes. A selective antagonist of TrkB-T1 may exhibit a half inhibitory concentration (IC50) for TrkB-T1 that is lower than its half inhibitory concentration (IC50) for TrkB-FL. A TrkB-T1 antagonist suitable for use as described may exhibit an IC50 value in the range of picomolar (10-12 M) to micromolar (10-6 M) concentrations, for example, between 1, 10 or 100 pM and 1, 10 or 100 µM. For example, SEQ ID NO: 5 is a suitable TrkB-T1 antagonist. Methods suitable for assessing the half inhibitory concentration (IC50) of an antagonist are well known in the art and are readily available to those skilled in the art (see Rang & Dale’s Pharmacology 9th Edition, Elsevier Churchill Livingstone 2019, England, UK). The precise format of any of the screening or assay methods of the present invention may be varied by those of skill in the art using routine skill and knowledge. The skilled person is well aware of the need to employ appropriate control experiments. A test compound may be an isolated molecule or may be comprised in a sample, mixture or extract, for example, a biological sample. Compounds which may be screened using the methods described herein may be natural or synthetic chemical compounds used in conventional drug screening programmes. Extracts of plants, microbes or other organisms, which contain several characterised or uncharacterised components may also be used. Suitable test compounds may include analogues, derivatives, variants and mimetics of known TrkB-T1 agonists, such as BDNF. Alternatively, compounds produced using rational drug design to provide test candidate compounds with particular molecular shape, size and charge characteristics suitable for the selective agonism of TrkB-T1 may be tested. A combinatorial library technology provides an efficient way of testing a potentially vast number of different compounds for their ability to act as agonists of TrkB-T1, preferably selective agonists of TrkB- T1. Such libraries and their use are known in the art, for all manner of natural products, small molecules and peptides, among others. The use of peptide libraries may be preferred in certain circumstances. The amount of test compound which may be added to a screening assay of the invention will normally be determined by trial and error depending upon the type of compound used. Typically, from about 0.001 nM to 1 mM or more concentrations of putative agonist compound may be used, for example from 0.01 nM to 100 μM, e.g.0.1 to 50 μM, such as about 10 μM. Even a compound having a weak agonist effect may be a useful lead compound for further investigation and development. The precise format of any of the screening or assay methods disclosed herein may be varied by those of skill in the art using routine skill and knowledge. The skilled person is well aware of the need to employ appropriate control experiments. For methods of screening disclosed herein, suitable control samples include, for example, reference values, treatment-naïve non-human test animals, and non-human test animals administered a control compound (e.g. a vehicle control), in which the control compound does not exhibit anti-seizure activity, and in which the control compound is not a TrkB-T1 agonist nor a TrkB-T1 antagonist. Non-human test animals may be species of rodent (e.g., mouse, rat, guinea pig, hamster, gerbil), fly (e.g., Drosophila) fish (e.g., zebrafish), livestock (e.g. sheep, cattle, pigs), or Equidae, Canine, Feline, Leporine or non-human primate species that are used as model organisms for research purposes. In embodiments of the invention, the non-human test animal is an animal model of absence seizures. For example, the non-human test animal may be an inbred laboratory rodent, such as a GAERS rat (Vergnes et al.1982; Micheletti et al.1985; Marescaux et al.1992). Alternatively, the non-human test animal may be a genetically modified animal or an animal with a single spontaneous gene mutation, such an STG mouse. Methods suitable for introducing genetic modifications into organisms and cells are readily available to those skilled in the art (for example, see Principles of Gene Manipulation and Genomics, 7th Edition, 2006 and Crispr-Cas: A Laboratory Manual, 1st edition, 2016). For example, suitable methods may involve DNA recombination, gene knockout (e.g., via homologous recombination or through the use of nucleases), gene insertion (e.g., cisgenesis or transgenesis), RNA interference (RNAi), genome editing (for example using CRISPR-Cas9 and associated techniques) or via mutagenesis (which may be random or targeted). In some embodiments, the non-human test animal may be a genetically modified animal comprising a calcium voltage-gated channel auxiliary subunit gamma 2 (CACNG2) gene variant (Homo sapiens gene ID 10369; Mus musculus gene ID 12300). Preferably, a CACNG2 gene variant in which the altered gene product lacks the molecular function of the wild-type gene product. The gene variant may comprise a partial or total deletion of the CACNG2 gene. The non-human test animal may exhibit a dysfunctional or non-functional CACNG2 protein. For example, Stargazer mice (Letts et al., 1997; Letts et al., 1998; Seo and Leitch, 2014). Albino Glaxo/from Rijswijk (WAG/Rij) rats, lethargic mice and tottering mice are also suitable non-human test animals for use in methods of screening as disclosed herein. Importantly though pharmacological animal models of absence seizures have been used in the past, their construct- and face-validity has been recently questioned (Crunelli & Leresche, 2002). A candidate compound identified as a selective agonist of TrkB-T1 may be investigated further. For example, the selectivity of a compound for TrkB-FL and/or TrkB-T1 in astrocytes may be determined in animal models. Suitable methods for determining the effect of a compound on TrkB-T1 are disclosed herein and alternative methods are well known in the art. A candidate compound identified as a TrkB-T1 agonist through one or more of the screening methods disclosed herein may be isolated and/or purified. Alternatively, it may be synthesised using conventional techniques of recombinant expression or chemical synthesis. Furthermore, it may be manufactured and/or used in preparation, i.e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition or drug. Methods described herein may thus comprise formulating the test compound in a pharmaceutical composition with a pharmaceutically acceptable excipient, vehicle or carrier for therapeutic application. Following identification of a TrkB-T1 agonist that is potentially useful in the treatment of absence seizures and/or their comorbidities as described herein, a method may further comprise modifying the compound to optimise its pharmaceutical properties. Suitable methods of optimisation, for example by structural modelling, are well known in the art. Further optimisation or modification can then be carried out to arrive at one or more final compounds for in vivo or clinical testing. Other aspects and embodiments of the invention provide the aspects and embodiments described above with the term “comprising” replaced by the term “consisting of” and the aspects and embodiments described above with the term “comprising” replaced by the term ”consisting essentially of”. The term “in vitro” relates to actions performed in isolated laboratory conditions or in cell culture, for example using materials, biological substances, cells and/or tissues that are isolated from an organism. The term “in vivo” relates to actions performed in intact multi-cellular organisms, such as humans or other mammals. The term “ex vivo” relates to actions performed outside a multi-cellular organisms e.g. outside a human or mammalian body, which may be on tissue (e.g. whole organs) or cells obtained or derived from a multi-cellular organism. It is to be understood that the application discloses all combinations of any of the above aspects and embodiments described above with each other, unless the context demands otherwise. Similarly, the application discloses all combinations of the preferred and/or optional features either singly or together with any of the other aspects, unless the context demands otherwise. Modifications of the above embodiments, further embodiments and modifications thereof will be apparent to the skilled person on reading this disclosure, and as such, these are within the scope of the present invention. All documents and sequence database entries mentioned in this specification are incorporated herein by reference in their entirety for all purposes. The term “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Experimental TrkB-T1 agonists modulate GABA uptake in vitro by controlling GAT1 recycling. Figure 3 shows a decrease in GAT1-mediated GABA uptake in brain slices derived from treatment-naïve GAERS and Non-Epileptic Control (NEC) rats (that have no absence seizures), but no change in the GABA uptake mediated by GAT3, the other main GABA transporter in the brain. To study GABA uptake in GAERS compared to their control, non-epileptic NEC rats, GABA uptake assays in brain slices were performed. Twelve weeks old male rats were anesthetized by isoflurane overdose and decapitated. The brain was quickly removed and regions of interest, i.e. motor cortex (MC) somatosensory cortex (SC), visual cortex (VC) thalamus (TH) and hippocampus (HIPPO) isolated in ice-cold Artificial Cerebrospinal Fluid (ACSF) solution. Transverse 300 μm slices were then cut from each region using a McIlwain tissue chopper and allowed to recover for 1 hour (Fredholm et al., 1984) in small chambers filled with Krebs solution saturated with 5% CO2 - 95% O2. After 1 h, drugs of interest, namely 20 μM SKF-8996A (a GAT1 blocker) and 20 μM SNAP-5411 (a GAT3 blocker) were added to the ACSF solution. After 30 min incubation, the uptake assay was then started by immersion of the slices for 10 min in a solution containing 2.5, 5, 10, 25, 50 and 100 μM of GABA (10% of which was [3H]GABA with specific activity of 0.141 Ci/mmol, PerkinElmer, Boston, MA, USA) with or without the drugs of interest in DMEM medium (DMEM, Gibco, Paisley, UK),) at 37ºC. After 10min, the slices were washed twice in an ice-cold PBS solution (in mM: 140 NaCl, 2.7 KCl, 1.5 KH2PO4 and 8.1 NaHPO4, pH 7.40), and then homogenized in 200μl of lysis buffer (in mM: 100 NaOH and 0.1% SDS). Ten μl of each homogenized slice was collected for protein quantification and the remaining content of each well was transferred to PET 24-well flexible microplate (Part Number 1450-402, PerkenElmer, Boston, MA, USA) and 500μl of Ultima Gold Scintillation liquid (PerkenElmer, Boston, MA, USA) added to each well. Determination of GABA transport in each well was performed using a liquid scintillation counter (MicroBeta®, PerkenElmer, Boston, MA, USA) and normalized to each well protein concentration. Specific GABA uptake mediated by GAT1 and GAT3 was measured by subtracting uptake in the presence of the respective blocker to the uptake in control conditions. Data analysis was carried out with GraphPad 8 (San Diego, CA, USA) Prism software. One Way-Anova followed by Turkey correction post-test was used to determine statistical significance in the Michaelis-Menten curves. As shown in Figure 3, slices obtained from 12 weeks old GAERS and NEC, i.e. at a time when seizures are fully developed in all GAERS rats, were used to measure total GABA uptake in the somatosensory cortex, thalamus and hippocampus. Total GABA uptake was smaller in somatosensory cortex (p=0.0389), Thalamus (p<0.0001) and Hippocampus (p=0.0001) of GAERS compared to NEC. GAT1-mediated GABA uptake was decreased in all GAERS brain areas examined (somatosensory cortex: p=0.0004; Thalamus: p=0.0002; Hippocampus: p=0.0009) compared to NEC rats. GAT3-mediated GABA uptake was similar in GAERS somatosensory cortex (p=0.0659), thalamus (p=0.0534) and hippocampus (p=0.8636) compared to that in NEC rats. Figure 4 shows a decrease in GAT1-mediated, but not GAT3-mediated, GABA uptake in primary cultures of astrocytes derived from treatment-naïve GAERS and NEC rats. To study GABA uptake in astrocytes of GAERS compared to NEC rats, GABA uptake assays were carried out in primary cultures of astrocytes. Primary cultures of astrocytes were prepared from the cerebral cortex and the thalamus of neonatal GAERS and NEC rat pups (0–3 days old) (Morais et al, 2018). Briefly, rat pups were sacrificed by decapitation and the brains were dissected in ice cold phosphate buffered saline solution (PBS) containing 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4.2H2O and 1.5 mM KH2PO4 (pH 7.4). The meninges were removed with forceps and the thalamus and cortex were isolated and then immersed in 4.5 g/l glucose Dulbecco’s Modified Eagles Medium (DMEM) (Gibco, Paisley, UK). Both the thalamic and cortical tissue were then vigorously dissociated in DMEM, supplemented with 10% fetal bovine serum (FBS) (Gibco), 1% antibiotic/antimycotic (Gibco, Paisley, UK) and glutamine (Gibco, Paisley, UK). Following dissociation, cells were sorted with a 120-μm cell strainer and centrifuged at 1200 rpm for 10 min at room temperature. The pellet was resuspended in 4.5 g/l glucose DMEM, sorted again through a 70-μm cell strainer (BD Falcon, NJ, USA) and then centrifuged. The final pellet was resuspended in DMEM and seeded at the density of 2x105 or 5x105 cells/ml (for thalamic cultures) and 5x105 cells/ml (for cortical cultures) in 24 wells plates. Thalamic and cortical astrocyte cultures were grown for 3 weeks in an incubator at 37ºC with a humidified atmosphere of 95% O2 and 5% CO2. The culture medium was changed twice a week. To purify astrocytes, at 10 days in vitro (DIV), cells were shaken for 5 hours at 300 rpm to remove any contaminating microglia cells. GABA uptake assays were performed in 21 days-old primary cultures of astrocytes following the procedures described by Vaz et al. (2011). Briefly, 3h before the uptake assay, the cell culture medium was changed to serum-free 1g/L DMEM medium, and the cells were kept at 37ºC with 95% O2 - 5% CO2. Thirty minutes before beginning the uptake assay, a solution made of the drugs of interest diluted in the culture medium was added to the culture medium (10μl/well), namely, 20 μM SKF-8996A (a GAT1 antagonist) or 20 μM SNAP-5411 (a GAT3 antagonist), and to some culture wells 10μl of culture medium (without any drugs) was added for these wells to serve as controls. GABA uptake assay was performed by removing the culture medium from the wells and then incubating the cells for 60 s at 37ºC with 1, 2.5, 5, 10, 25 and 50 μM of GABA, (from a stock solution that contained 10% of [3H]GABA ([3H]GABA specific activity: 0.141 Ci/mmol) (PerkinElmer, Boston, MA, USA) and drugs of interest, namely SKF-8996A and SNAP-5411, in a solution of (in mM) 137 NaCl, 5.4 KCl, 1.8 CaCl2·2H2O, 1.2 MgSO4 and 10 HEPES (pH adjusted with NaOH to 7.40). After 60 s, the GABA solutions were aspirated from the wells and an ice-cold STOP solution (in mM: 137 NaCl and 10 HEPES, pH adjusted with Tris-base to 7.40) was added to the wells, twice, and the cells were then washed in PBS. Cellular solubilization was then performed for 1 hour at room temperature by adding 200μL lysis buffer (in mM: 100 NaOH and 0.1% SDS (Sodium Dodecyl Sulfate)) per well. After 1 hour, 10μl of each well were collected for protein quantification and the remaining content of each well was transferred to PET 24-well flexible microplates (Part Number 1450-402, PerkinElmer, Boston, MA, USA) and 500μL of Ultima Gold Scintillation liquid (PerkinElmer, Boston, MA, USA) was added to each well. Determination of GABA uptake in each well was performed by radioactivity count (CPM: counts per minute) in a liquid scintillation counter (MicroBeta®, PerkenElmer, Boston, MA, USA) and normalized to the well protein concentration. GABA uptake mediated by GAT1 and GAT3 was calculated as the difference between wells incubated with SKF 89976a and SNAP 5114, respectively, and wells without drug incubation (CTL wells). Data analysis was done with GraphPad 8 (San Diego, CA, USA) Prism software. One way-Anova followed by Turkey correction post-test was used to determine statistical significance in the Michaelis- Menten curves and unpaired t-test was used to determine statistical significance between the two strains at the same GABA concentration. As shown in figure 4, in primary cultures of cortical astrocytes, total GABA uptake was decreased in GAERS compared to NEC (p=0.0233). This effect due to a decreased GAT1-mediated GABA uptake (p=0.0318) since GAT3-mediated GABA uptake was similar between the two strains (p= 0,9559). In thalamic pure astrocyte cultures, both total GABA uptake (p= 0.0071) and GAT1-mediated GABA uptake (p<0.0001) were smaller in GAERS than in NEC rats whereas GAT3-mediated GABA uptake was similar (p=0.5921) between the two strains. Figure 5 shows that BDNF (a TrkB-T1 agonist) increases GAT1 expression at the plasma membrane and GAT1-mediated GABA uptake in GAERS rats. Measurement of GAT1 expression levels and changes in expression levels upon incubation with BDNF (20ng/ml, 30min incubation) were assessed by Western blotting in thalamic slices obtained from 12 weeks old treatment-naïve rats. For the isolation of the plasma membrane and cytosol fractions a Plasma Membrane Protein Extraction Kit (ab65400, Abcam, UK) was used using the manufacturer instruction. After extraction, lysates were stored at -20 ºC until further use. For obtaining the total protein lysates, slices were incubated for 30min in 250 μl of RIPA buffer [50mM Tris pH 8.0, 1mM EDTA, 150mM NaCl, 1% NP40 (Nonyl phenoxlpoylethanol, from Fluka Biochemika, Switzerland), 1% SDS and 10% glycerol] with 25x protease inhibitors (Complete Mini-EDTA free, Roche, Germany) and 1mM PMSF to perform cell lysis. After 30min, mechanic homogenization of the slices was performed with a P200 pipette and lysates were incubated for further 15min in an orbital shaker (VWR, USA) at 4ºC. Lysates were then centrifuged at 4ºC for 10min and at 11000g. The supernatant was stored at -20 ºC until further use. Total protein in lysates obtained from primary cultures and brain slices was quantified with Bio-Rad DC reagent (Hercules, CA, USA) Bradford’s Assay, using BSA (Bovine Serum Albumin) as the standard to establish the calibration curves. Concentrations of 0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1 μg/μl BSA were used to correlate protein concentration and absorbance, allowing the quantification of proteins. On the day of experiment 20μg of proteins were dissolved with 5x SDS sample buffer (50 mM Tris-Cl, pH 6.8, 2% SDS, 100 mM Dithiothreitol (DTT), 0.1% bromophenol blue, 10% glycerol) to a final volume of 40μl. Proteins were then denatured by heating the samples to 37ºC for 30 min. A 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was utilized to separate samples and protein size marker (Precision Plus Protein Standards, Bio-Rad). Proteins were then transferred to a Polyvinylidene Difluoride (PVDF) membrane (Millipore) for 90 minutes at 150V. Membranes were then blocked at room-temperature with 3% BSA in TBS-T (20 mM Tris base, 137 mM NaCl and 0, 1% Tween- 20) for 1 h and incubated with the primary antibody overnight at 4ºC. On the following day, membranes were washed twice for 10 minutes with TBS-T and incubated for 1 h with the secondary antibody at room temperature. Image acquisition was performed by ECL Plus Western Blotting Detection System (Amersham-ECL Western Blotting Detection Reagents from GE Healthcare, Buckinghamshire, UK) and visualized with the ChemiDocTM XRS+Imager system (Hercules, CA, USA). Protein bands were analyzed with Image J software and standardized for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) levels. Protein levels of GAERS rats were normalized to those of NEC rats. Unpaired t-test was used to measure differences in protein levels between strains or cellular location within the same strain. The effect of BDNF on GAT1 function was measured by performing GABA uptake in brain slices (see previous methodology) incubated with either ACSF or BDNF (20ng/ml, 30 min incubation). As shown in Figure 5, GAT1 showed an almost exclusive location in the plasma membrane both when incubated with ACSF or 20ng/ml BDNF (A). Moreover, BDNF incubation increased GAT1 band intensity (B). Quantification of GAT1 expression in the plasma membrane revealed a 70% increase of GAT1 expression in thalamic slices incubated with BDNF (ACSF: 1±0.1, BDNF: 1.7±0.2, p= 0.0259). In contrast, BDNF was unable to increase GABA uptake when slices were incubated with the GAT1 blocker SKF 89976-A, indicating that the BDNF-induced increase in GABA uptake was mediated by GAT1 (BDNF vs SKF+BDNF: Motor Cortex p=0.0024, Somatosensory Cortex p=0.0019, Visual Cortex p=0.0169, Thalamus p=0.0022, Hippocampus p=0.0004) (C). Figure 6 shows a decrease in GAT1-mediated GABA uptake in brain slices derived from treatment-naïve STG compared to wildtype (WT) control mice. GABA uptake in 12 weeks-old STG mice and their WT littermates was assessed, and the effect of BDNF upon the increase on GAT1-mediated GABA uptake was also measured using the methods described above for GAERS uptake. As shown in Figure 6, saturation curve of total GABA uptake in STG mice were smaller in the thalamus (p=0.0004) and Hippocampus (p=0.0025) compared to WT littermates. The same was observed for STG GAT1-mediated GABA uptake curves that showed deficits in the thalamus (p=0.0337) and hippocampus (p <0,0001) (A). In STG mice, the BDNF-induced increase in GABA uptake was mediated by GAT1 since incubation with SKF 89976-A prevented BDNF effect in all brain areas (motor cortex, p=0.0003; somatosensory cortex, p=0.0064; visual cortex, p=0.0147; thalamus, p<0,0001; and hippocampus (p=0.0014) (B). TrkB-T1 agonists improve the deficient synaptic correlate of memory Figure 7 shows that BDNF rescues the deficient hippocampal long-term potentiation (LTP) of GAERS rats. Twelve to 16 weeks old animals were used to evaluate synaptic plasticity using extracellular recordings of field excitatory postsynaptic potentials (fEPSPs) in GAERS and NEC as the cellular correlate of memory disfunction and the effect of BDNF upon LTP was evaluated. To obtain hippocampal slices, animals were deeply anesthetized with isoflurane, and dissection of the two hippocampi was rapidly made in ice-cold Krebs solution. Hippocampal slices (400 μm thick) were cut perpendicularly using a McIlwain tissue chopper and recovered for 1h upon incubation at room temperature in oxygenated Krebs solution (5% CO2, 95% O2). After the recovery period, one slice at a time was placed in the recording chamber and continuously superfused with oxygenated Krebs solution at a constant flow of 3 ml/min and 32 °C. To obtain the fEPSP recordings, a microelectrode filled with Krebs solution (2–6 MΩ resistance), was placed in the stratum radiatum of the CA1 region of the hippocampus, coupled to an Axoclamp 2B Amplifier (Axon Instruments) and the traces digitized with a BNC-2110 (National Instruments). Stimulation (rectangular, 0.1 ms pulses every 20 seconds) was delivered through a concentric electrode placed on the Schaffer collateral-commissural fibres, in the stratum radiatum, near the CA3-CA1 border (P0) and near CA1 (P1). Stimulation alternately of two pathways of the Schaffer collateral-commissural fibres was performed. Individual responses were monitored, and averages of eight consecutive responses were continuously stored using WinLTP Software. LTP was induced by θ-burst stimulation (TBS) since this pattern of stimulation is similar to what occurs physiologically in the hippocampus during episodes of learning and memory. After a baseline of approximately 20 min was collected, LTP was induced using TBS delivered at basal stimulus intensity, comprising of 5 bursts (200 ms interburst interval) at 5 Hz, with each burst composed of 5 pulses at 100Hz. fEPSPs slope was recorded for 60 or 120 min after LTP induction, and each slope value corresponding to an average of 8 responses of 20 s. To assess the effect of BDNF on LTP, BDNF (20ng/ml) was perfused in the bath for 30 minutes. After BDNF 30 min incubation, LTP was induced in the second pathway (P1 electrode). LTP was quantified as the percentage of change in the average slope of the fEPSP. The effect of BDNF upon LTP was estimated by comparison of the magnitude of LTP in the first pathway (control pathway) with the magnitude of LTP in the second pathway, in the presence of BDNF. The results are shown in Figure 7. To study the in vitro effect of BDNF on memory, its effect on LTP, one of the accepted synaptic mechanisms underlying learning and memory (Albensi et al., 2007), was investigated at the CA3-CA1 neuron synapses by measuring the slope of the field excitatory synaptic potentials (fEPSP) in hippocampal slices of GAERS rats and Non-Epileptic Control (NEC) rats (an inbred, Wistar rat-derived strain) that do not show absence seizures. LTP (induced by TBS at time 0) in GAERS hippocampal slices was smaller than that in NEC rats (p < 0.05; Student t-test; n =15 GAERS and 13 NEC slices) (Figure 7A). In GAERS slices (n =6) perfused with BDNF (20ng/ml), LTP was increased compared to that in GAERS slices not treated with BDNF (p < 0.01; Student t-test) (Figure 7B). Moreover, LTP in GAERS slices perfused with BDNF was similar to that of NEC slices (p > 0.05) (Figure 7B). TrkB-T1 agonists are efficacious anti-seizure agents in vivo. Figure 8 shows that BDNF administration rescues absence seizures in vivo. Adult (3-5 month old) male GAERS rats were anesthetized with 2-5% inhalation isoflurane and implanted with gold plated epidural screws (Svenska Dentorama AB, Sweden) (to record the electroencephalogram (EEG), which were positioned bilaterally in the frontal, temporal and cerebellar cortices. Two different groups of GAERS rats were also bilaterally implanted with guide cannulae in either the primary somatosensory cortex or the thalamic ventrobasal nucleus for the subsequent intracerebral injection of Artificial Cerebrospinal Fluid (ACSF) or BDNF. Animals were allowed to recover from the implantation surgery for at least 5 days before the experiments commenced. To habituate the GAERS rats to the recording conditions, they were positioned in a Plexiglas 40x40x40cm recording cages (positioned within a home-made Faraday cage) and connected to the EEG amplifier. They stayed there for a period of 30 min in 3 consecutive days. On the day of the experiment, following a 30 min-habituation period, absence seizures of GAERS rats were recorded by monitoring the EEG and their behaviour. Absence seizures were identified by the concomitant presence of SWDs in the EEG and associated lack of locomotion. Both EEG and video recordings were stored on a PC for later analysis. Following a 1-hour (control) period, GAERS rats were injected with the drug(s) of interest (either intraperitoneally, intracortically or intrathalamically) and recording then continued for another 2-hours (drug-effect period) (Figure 8A). EEG data were analysed using the SeizureDetect routine of SPIKE2 (Cambridge Electronic Design, Cambridge, UK) that allows automatic extraction of spike-and-wave discharges (SWDs) (the electrographic signature of an absence seizure) from the raw EEG data, as described by McCafferty e al. (2018). Automatically detected SWDs that were not accompanied by motor arrest were not considered absence seizures. The results are shown in Figure 8. Since BDNF does not easily penetrate the brain following systemic administration, it was injected intraperitoneally 20 min after intravenous injection of Elacridar (Ela), a phosphoglycoprotein inhibitor (5mg/kg) which is known to increase the permeability of the blood-brain barrier (Kallen et al, 2012). BDNF was administered (at the time indicated by the arrow) at three different doses (1μg/, 10μg/ and 30μg/animal) and its effect was recorded for 2 hours in freely moving GAERS rats previously implanted with EEG electrodes (Figure 8A). The results are presented in 20 min bins as data normalized to the control period, i.e. pre-BDNF injection. Note that the vehicle of Elacridar is called vehicle 1 (Veh1) and that of BDNF is referred to as vehicle 2 (Veh2) (Figure 8B). Ela alone (i.e. the group injected with Elacridar+Veh2) had no effect on the total time spent in absence seizures (Figure 8B), as previously shown (Yacone et al., 2021). The same lack of effect was observed when BDNF was injected alone without pre-treatment with Elacridar (i.e. the group injected with Veh1+BDNF 10μg). Following pre- treatment with ELA, the smallest dose of BDNF (1 μg/rat) had no effect on the total time spent in absence seizures. In contrast, BDNF at 10 and 30 μg/rat markedly decreased the total time spent in seizures (Two-Way ANOVA, p < 0.01 and 0.001, respectively) during the entire course of the 2 hours of recordings after BDNF injection compared to the control period (Figure 8B). Some representative EEG traces showing the effect of BDNF on the SWDs following the illustrated injections are shown below the plot in Figure 8B. Overall, these results show that systemic administration of BDNF to freely moving GAERS elicits a dose-dependent reduction in spontaneous absence seizures. Having established that the systemic injection of BDNF markedly reduced absence seizures, the anti- absence effect of BDNF injected either the somatosensory cortex or the thalamus, the two key brain areas that are essential for the generation of absence seizures (Crunelli et al, 2020) was investigated. BDNF bilaterally injected (at the time indicated by the arrow) in the cortex of freely moving GAERS rats led to a marked decrease in the total time spent in seizures compared to administration of ACSF (Figure 8C, left panels). This effect was almost immediate since it was observed already in the first 20 min after the injection (0-20min: p < 0.001) and was maintained throughout the entire 2 hour post-injection period. BDNF bilaterally injected (at the time indicated by the arrow) in the thalamic ventrobasal nucleus of another group of freely moving rats also led to a decrease in the total time spent in seizures compared to administration of ACSF (Figure 8C, right panels). However, this effect took longer to develop (since it was first observed in the 60-80 min time bin) but reached the same percentage decrease as the intracortical injection 2 hour after BDNF injection. Some representative EEG traces showing the effect of BDNF on SWDs following intracortical and intrathalamic injection are shown below the plots in the left and right panel of Figure 8C, respectively. Figure 9 shows that the anti-seizure effect of BDNF is mediated via TrkB-T1 receptors. Adult (3-5 month old) male GAERS rats were anesthetized with 2% inhalation isoflurane and then bilaterally implanted with gold plated epidural screws (Svenska Dentorama AB, Sweden) positioned bilaterally in the frontal, temporal and cerebellar cortices to record the EEG. The rats were also bilaterally implanted with guide cannulae in either the primary somatosensory cortex or the thalamic ventrobasal nucleus and were injected with a viral vector containing an astrocyte-selective (GFAP promoter) siRNA that knocks down TrkB-T1 or a scramble siRNA in either one of the above brain areas. Before experiments commenced, 3 weeks were allowed to elapse to allow the transfection by the viral vector to occur (Figure 9A). To habituate the GAERS rats to the recording conditions, GAERS rats were positioned in a Plexiglas 40x40x40cm recording cages (positioned within a home-made Faraday cage) and connected to the EEG amplifier. They stayed there for a period of 30min in 3 consecutive days. On the day of the experiment, following a 30 min-habituation period, absence seizures of GAERS rats were recorded by monitoring the EEG and their behaviour. Absence seizures were identified by the concomitant presence of SWDs in the EEG and associated lack of locomotion. Both EEG and video recordings were stored on a PC for later analysis. Cannulae were bilaterally lowered into the previously implanted guide-cannulae and ACSF injected via a micropump (at ??/min flow rate) either the primary somatosensory cortex or the thalamic ventrobasal nucleus. Following a 1-hour (control) period, the ACSF solution was changed to one containing BDNF (20ng/ml) and recording continued for another 2-hours (drug-effect period) (Figure 9A). EEG data were analysed using the SeizureDetect routine of SPIKE2 (Cambridge Electronic Design, Cambridge, UK) that allows automatic extraction of spike-and-wave discharges from the raw EEG data, as described by McCafferty e al. (2018). Automatically detected SWDs that were not accompanied by motor arrest were not considered absence seizures. The results are shown in Figure 9. Since BDNF modulate the function of the GABA transporter GAT1 via TrkB-T1 (Vaz et al, 2011) and TrkB-T1 is preferentially expressed in astrocytes (De Biasi et al., 1998), we next investigated whether the anti-absence effect of this neurotrophin was mediated by TrkB-T1 by knocking down this isoform with an astrocyte-selective siRNA or a scramble siRNA 3 weeks before the injection of BDNF (Figure 9A). The intracortical injection of the scramble siRNA had no action on the anti- absence effect of intracortically injected BDNF (at the time indicated by the arrow) since it was still able to block absence seizures (Figure 9B, black symbols) (p > 0.05). In contrast, intracortically injected BDNF did not exert any action on absence seizures in GAERS rats that had been pretreated with the astrocyte- selective siRNA that knocked down TrkB-T1 (Figure 9B, grey symbols). Representative traces of the SWDs observed before and after intracortical BDNF injection in GAERS rats pre-injected with a scramble or a TrkB-T1 knocking down siRNA are shown below the plot. In a similar manner, the intrathalamic injection of the scramble siRNA had no effect on the anti-absence effect of intrathalamically injected BDNF (at the time indicated by the arrow)(Figure 9C, black symbols) (p > 0.05), whereas the ability of this neurotrophin to block absence seizures was abolished in GAERS rats that had been pre-treated with the astrocyte-selective siRNA that knocked down TrkB-T1 (Figure 9C, grey symbols). Representative traces of the SWDs observed before and after intrathalamic BDNF injection in GAERS rats pre-injected with a scramble and a TrkB-T1 knocking down siRNA are shown below the plot. TrkB-T1 agonists mitigate the neuropsychiatric comorbidities of absence seizures in vivo. Figure 10 shows that BDNF rescues the deficiencies in novel object recognition observed in GAERS rats. To investigate recognition memory, the Novel Object Recognition (NOR) test was performed in adult GAERS and NEC rats and in GAERS rats bilaterally injected (via a pre-implanted cannula) with ACSF or BDNF (500ng) in the CA1 regions of the hippocampus. The animals were handled for 5 days (10 min/day) and on day 6 were habituated to the open field twice, for 10 min each time. On day 7, the learning day, animals were 1) placed in the maze with two similar objects for 5 minutes or 2) injected with 500ng/side of BDNF or ACSF and after 1h hour were placed in the maze and allowed to freely explore two similar objects for 5 minutes. Animal order, injection drug, object place and objects were randomized using the Excel randomization tool. On day 8, the test day, one object was replaced with a novel object, and the animal was allowed to explore the objects for 5 min. Exploration of the object for less than 10 seconds was deemed to be an invalid trial. Higher time exploring novel object compared with similar object is a measure of intact memory (Ennaceur, 2010). The percentage of time exploring an object and the difference in time exploring the familiar and the novel object (i.e. the novelty index) were analysed. Post- analysis was performed using Solomon Coder (V. beta 9.08.02). Unpaired t-test was used to account for statistical significance between GAERS and NEC rats or ACSF-injected and BDNF-injected GAERS. The NOR results showed that the percentage of time spent exploring the new object was <50% in GAERS rats, while the NEC rats spent 60% of their exploration time analysing the new object (NEC: 60.31±2.17, GAERS: 48.58±2.58, p=0.0020) (Figure 10B, left plot). In agreement with this result, the novelty index (i.e. the ratio of time spent exploring the new object with respect to the familiar one) for NEC was positive while GAERS index was negative (NEC: 0.19±0.04, GAERS: -0.03±0.05, p=0.0028), demonstrating that contrary to NEC, the GAERS rats cannot discriminate between the familiar and the novel object (Figure 10B, right plot). The percentage of time that each animal spends exploring the familiar and the novel object are shown in Figure 10C. The same parameters were evaluated in GAERS rats receiving either ACSF or BDNF bilaterally in the hippocampus. BDNF doubled the time of exploration of the new object, that is, BDNF-injected GAERS rats spend 66.73±4.09% of their time exploring the new object while ACSF-injected GAERS only spend 30.25±9.50% (p=0.0011) (Figure 10D, left plot). This result was reflected in the novelty index that showed a positive and negative value for the GAERS injected with BDNF (0.33±0.08) and ACSF (-0.40±0.19, p=0.0011), respectively, indicative of a failure to discriminate between the familiar and novel object (Figure 10D, right plot). The percentage of time that ACSF or BDNF-injected animals spends exploring the familiar and the novel object are represented in Figure 10E. Figure 11 shows that the BDNF-mediated rescue of the impaired LTP and deficient novel object recognition of GAERS rats occurs via TrkB-T1. The methods of injection of siRNA viral construct, 3 weeks before the behavioural experiments) and the cannula implantation are similar to those described in Figure 8, except that cannulae were inserted bilaterally in the hippocampus. The results are shown in Figure 11. Hippocampal slices of GAERS rats pre-injected with a viral vector containing an astrocyte-selective (GFAP promoter) scramble siRNA for TrkB-T1 were used for LTP measurements in the presence of ACSF or BDNF. BDNF was shown to rescue the deficient LTP in GAERS rats injected with scramble siRNA for TrkB-T1 (p < 0.05; Student t-test; n =7 for each condition) (Figure 11A, B). Hippocampal slices of GAERS rats pre-injected with a viral vector containing an astrocyte-selective (GFAP promoter) knockdown siRNA for TrkB-T1 were used for LTP measurements in the presence of ACSF or BDNF. BDNF was unable to rescue the deficient LTP in GAERS rats injected with siRNA knockdown for TrkB-T1 (P=0,8069; Student t-test; n=6 for each condition) (Figure 11C,D). NOR results showed that upon 500ng of BDNF administration the percentage of time spent exploring the new object was higher in the scramble siRNA-injected animal than in the siRNA knockdown animals (scramble T1 + BDNF vs siRNA T1+ BDNF, p=0,0215) (Figure 11F, left plot). Similarly, the novelty index in scramble siRNA BDNF-injected GAERS was higher than in siRNA knockdown T1 BDNF-injected GAERS (scramble T1+ BDNF vs siRNA T1+ BDNF, p=0,0215) (Figure 11F, right plot). The percentage of time that each animal spends exploring the familiar and the novel object are represented in Figure 11G. Summary In combination, these findings demonstrate that TrkB-T1 agonists are efficacious agents against absence seizures and their comorbidities. These findings also demonstrate for the first time that a single therapeutic agent (e.g., a TrkB-T1 agonist) may be used to concurrently treat both absence seizures and comorbidities thereof. Sequences SEQ ID NO: 1 TrkB-FL UniProt P15209-1 (Mus musculus) (821aa) MSPWLKWHGPAMARLWGLCLLVLGFWRASLACPTSCKCSSARIWCTEPSPGIVAFPRLEPNSVDPENITEILIANQK RLEIINEDDVEAYVGLRNLTIVDSGLKFVAYKAFLKNSNLRHINFTRNKLTSLSRRHFRHLDLSDLILTGNPFTCSC DIMWLKTLQETKSSPDTQDLYCLNESSKNMPLANLQIPNCGLPSARLAAPNLTVEEGKSVTLSCSVGGDPLPTLYWD VGNLVSKHMNETSHTQGSLRITNISSDDSGKQISCVAENLVGEDQDSVNLTVHFAPTITFLESPTSDHHWCIPFTVR GNPKPALQWFYNGAILNESKYICTKIHVTNHTEYHGCLQLDNPTHMNNGDYTLMAKNEYGKDERQISAHFMGRPGVD YETNPNYPEVLYEDWTTPTDIGDTTNKSNEIPSTDVADQSNREHLSVYAVVVIASVVGFCLLVMLLLLKLARHSKFG MKGPASVISNDDDSASPLHHISNGSNTPSSSEGGPDAVIIGMTKIPVIENPQYFGITNSQLKPDTFVQHIKRHNIVL KRELGEGAFGKVFLAECYNLCPEQDKILVAVKTLKDASDNARKDFHREAELLTNLQHEHIVKFYGVCVEGDPLIMVF EYMKHGDLNKFLRAHGPDAVLMAEGNPPTELTQSQMLHIAQQIAAGMVYLASQHFVHRDLATRNCLVGENLLVKIGD FGMSRDVYSTDYYRVGGHTMLPIRWMPPESIMYRKFTTESDVWSLGVVLWEIFTYGKQPWYQLSNNEVIECITQGRV LQRPRTCPQEVYELMLGCWQREPHTRKNIKSIHTLLQNLAKASPVYLDILG SEQ ID NO: 2 TrkB-T1, UniProt P15209-2 (Mus musculus) (476aa) MSPWLKWHGPAMARLWGLCLLVLGFWRASLACPTSCKCSSARIWCTEPSPGIVAFPRLEPNSVDPENITEILIANQK RLEIINEDDVEAYVGLRNLTIVDSGLKFVAYKAFLKNSNLRHINFTRNKLTSLSRRHFRHLDLSDLILTGNPFTCSC DIMWLKTLQETKSSPDTQDLYCLNESSKNMPLANLQIPNCGLPSARLAAPNLTVEEGKSVTLSCSVGGDPLPTLYWD VGNLVSKHMNETSHTQGSLRITNISSDDSGKQISCVAENLVGEDQDSVNLTVHFAPTITFLESPTSDHHWCIPFTVR GNPKPALQWFYNGAILNESKYICTKIHVTNHTEYHGCLQLDNPTHMNNGDYTLMAKNEYGKDERQISAHFMGRPGVD YETNPNYPEVLYEDWTTPTDIGDTTNKSNEIPSTDVADQSNREHLSVYAVVVIASVVGFCLLVMLLLLKLARHSKFG MKGFVLFHKIPLDG SEQ ID NO: 3 TrkB-FL, UniProt Q16620-1 sapiens) (822aa)
Figure imgf000028_0001
RLEIINEDDVEAYVGLRNLTIVDSGLKFVAHKAFLKNSNLQHINFTRNKLTSLSRKHFRHLDLSELILVGNPFTCSC DIMWIKTLQEAKSSPDTQDLYCLNESSKNIPLANLQIPNCGLPSANLAAPNLTVEEGKSITLSCSVAGDPVPNMYWD VGNLVSKHMNETSHTQGSLRITNISSDDSGKQISCVAENLVGEDQDSVNLTVHFAPTITFLESPTSDHHWCIPFTVK GNPKPALQWFYNGAILNESKYICTKIHVTNHTEYHGCLQLDNPTHMNNGDYTLIAKNEYGKDEKQISAHFMGWPGID DGANPNYPDVIYEDYGTAANDIGDTTNRSNEIPSTDVTDKTGREHLSVYAVVVIASVVGFCLLVMLFLLKLARHSKF GMKGPASVISNDDDSASPLHHISNGSNTPSSSEGGPDAVIIGMTKIPVIENPQYFGITNSQLKPDTFVQHIKRHNIV LKRELGEGAFGKVFLAECYNLCPEQDKILVAVKTLKDASDNARKDFHREAELLTNLQHEHIVKFYGVCVEGDPLIMV FEYMKHGDLNKFLRAHGPDAVLMAEGNPPTELTQSQMLHIAQQIAAGMVYLASQHFVHRDLATRNCLVGENLLVKIG DFGMSRDVYSTDYYRVGGHTMLPIRWMPPESIMYRKFTTESDVWSLGVVLWEIFTYGKQPWYQLSNNEVIECITQGR VLQRPRTCPQEVYELMLGCWQREPHMRKNIKGIHTLLQNLAKASPVYLDILG SEQ ID NO: 4 TrkB-T1, UniProt Q16620-2 (Homo sapiens) (477aa) MSSWIRWHGPAMARLWGFCWLVVGFWRAAFACPTSCKCSASRIWCSDPSPGIVAFPRLEPNSVDPENITEIFIANQK RLEIINEDDVEAYVGLRNLTIVDSGLKFVAHKAFLKNSNLQHINFTRNKLTSLSRKHFRHLDLSELILVGNPFTCSC DIMWIKTLQEAKSSPDTQDLYCLNESSKNIPLANLQIPNCGLPSANLAAPNLTVEEGKSITLSCSVAGDPVPNMYWD VGNLVSKHMNETSHTQGSLRITNISSDDSGKQISCVAENLVGEDQDSVNLTVHFAPTITFLESPTSDHHWCIPFTVK GNPKPALQWFYNGAILNESKYICTKIHVTNHTEYHGCLQLDNPTHMNNGDYTLIAKNEYGKDEKQISAHFMGWPGID DGANPNYPDVIYEDYGTAANDIGDTTNRSNEIPSTDVTDKTGREHLSVYAVVVIASVVGFCLLVMLFLLKLARHSKF GMKGFVLFHKIPLDG SEQ ID NO: 5 TrkB-T1[shRNA#1] (Synthetic) (22nt) GGCTAACACTCTTAAGTATTGG SEQ ID NO: 6 Scramble[miR30-shRNA#1] (Synthetic) (22nt): ACCTAAGGTTAAGTCGCCCTCG
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Claims

Claims: 1. A method of treating absence seizures and/or a comorbidity thereof, the method comprising: administering a therapeutically effective amount of a TrkB-T1 agonist to a subject in need thereof. 2. The method of claim 1, wherein the comorbidity is selected from the group consisting of: attention-deficit/hyperactivity disorder, cognitive impairment, memory/learning deficits, autism spectrum disorders, schizophrenia, depression and anxiety disorders. 3. The method of claim 2, wherein the anxiety disorder is selected from the group consisting of: agoraphobia, selective mutism, generalized anxiety disorder (GAD), social anxiety disorder, obsessive- compulsive disorder (OCD) and panic disorder. 4. The method of any one of claims 1 to 3, wherein the agonist is a selective agonist of TrkB-T1. 5. The method of claim 4, wherein the selective agonist exhibits a binding affinity (Kd) for TrkB-T1 that is lower than its Kd for TrkB-FL. 6. The method of claim 4 or claim 5, wherein the selective agonist exhibits a half effective concentration (EC50) for activation of TrkB-T1 that is lower than its EC50 for activation of TrkB-FL. 7. The method of any one of claims 1 to 6, wherein the subject is a human subject. 8. The method claim 7, wherein the subject is: (i) a paediatric subject having Childhood Absence Epilepsy (CAE) (ii) a paediatric or teenage subject having Juvenile Absence Epilepsy (JAE); or (iii) a paediatric, teenage or adult subject having absence seizures. 9. A TrkB-T1 agonist for use in method of treating absence seizures and/or a comorbidity thereof in a subject. 10. The TrkB-T1 agonist for use of claim 9, wherein the method is a method of any one of claims 1 to 8. 11. Use of a TrkB-T1 agonist for the manufacture of a medicament for use in a method of treating absence seizures and/or a comorbidity thereof in a subject. 12. The use of claim 11, wherein the method is a method of any one of claims 1 to 8. 13. A method of screening for a compound useful in treating absence seizures and/or a comorbidity thereof, the method comprising: determining the activity of TrkB-T1 in the presence or absence of a test compound, wherein an increase in the activity of TrkB-T1 in the presence relative to the absence of the test compound is indicative that the test compound is a candidate compound useful in treating absence seizures and/or a comorbidity thereof. 14. The method of claim 13, comprising determining the activity of TrkB-T1 in a mammalian cell or determining the activity of TrkB-T1 in a non-human test animal. 15. The method of claim 13 or 14, wherein determining the activity of TrkB-T1 comprises measuring the cell-surface expression of GABA transporter 1 (GAT1). 16. A method of screening for a compound useful in treating absence seizures and/or a comorbidity thereof, the method comprising: (i) administering a test compound to a first and a second non-human test animal; wherein the first and second non-human test animals are animal models of absence seizures, and wherein the second non-human test animal exhibits reduced expression of TrkB-T1 and/or reduced TrkB-T1 function relative to the first non-human test animal; (ii) measuring the frequency of occurrence and/or duration of absence seizures in the first and second non-human test animals, and (iii) comparing the frequency of occurrence and/or duration of absence seizures as measured in step (ii) to a control sample; wherein a reduction in the frequency of occurrence and/or duration of absence seizures in the first non-human test animal relative to the control sample, and wherein substantially no change in the frequency of occurrence and/or duration of absence seizures in the second non-human test animal relative to the control sample are indicative that the test compound is a candidate compound useful in treating absence seizures and/or a comorbidity thereof. 17. A method of screening for a compound useful in treating absence seizures and/or a comorbidity thereof, the method comprising: (i) administering a test compound to a first and a second non-human test animal; wherein the first and second non-human test animals are animal models of absence seizures, and wherein the second non-human test animal exhibits reduced expression of TrkB-T1 and/or reduced TrkB-T1 function relative to the first non-human test animal; and (ii) measuring the cognitive behaviour of the first and second non-human test animals, wherein an improvement in the learning and/or memory of the first non-human test animal as compared to the second non-human test animal; or an improvement in the presentation of an attention- deficit/hyperactivity disorder, a cognitive impairment, a memory or learning deficit, an autism spectrum disorder, schizophrenia, depression and/or an anxiety disorder in the first non-human test animal as compared to the second non-human test animal is indicative that the test compound is a candidate compound useful in treating absence seizures and/or a comorbidity thereof. 18. The method of claim 16 or claim 17, wherein the control sample is selected from the group consisting of: (i) a reference value; (ii) a treatment-naïve non-human test animal; and (iii) a non-human test animal administered a control compound, wherein the control compound does not exhibit anti-seizure activity; and wherein the control compound is not a TrkB-T1 agonist; and wherein the control compound is not a TrkB-T1 antagonist. 19 The method of any one of claims 16 to 18, wherein the non-human test animal is an inbred laboratory rodent. 20. The method of claim 18, wherein the inbred laboratory rodent is a “Genetic Absence Epilepsy Rat from Strasbourg” (“GAERS”) rat. 21. The method of claims 16 to 18, wherein the non-human test animal comprises a dysfunctional or non-functional CACNG2 variant. 22. The method of claim 21, wherein the non-human test animal is a “Stargazer” mouse. 23. The method of any one of claims 16 to 18 wherein the non-human test animal is an Albino Glaxo/from Rijswijk (WAG/Rij) rat, a lethargic mouse or a tottering mouse. 24. The method of any one of claims 16 to 23, wherein the second non-human test animal is genetically modified and/or is administered a TrkB-T1 antagonist causing reduced expression of TrkB-T1 and/or reduced TrkB-T1 function. 25. The method of claim 16, further comprising: measuring the cognitive behaviour of the first and second non-human test animals; wherein an improvement in the learning and/or memory of the first non-human test animal as compared to the second non-human test animal; or an improvement in the presentation of an attention- deficit/hyperactivity disorder, a cognitive impairment, a memory or learning deficit, an autism spectrum disorder, schizophrenia, depression and/or an anxiety disorder in the first non-human test animal as compared to the second non-human test animal is indicative that the test compound is a candidate compound useful in treating absence seizures and/or a comorbidity thereof.
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