A novel treatment for dravet syndrome

Abstract

Compound for use in a method of treating Dravet Syndrome, wherein the compound is of Formula (I), wherein: X is hydrogen or deuterium or N(CH3)2; W is CH2 or C=O; R is hydrogen or a halogen; A is hydr

Description

Technical Field The present invention relates to methods and compositions for treating Dravet Syndrome (DS). The present invention also relates to a compound of Formula (I), or a composition comprising a compound of Formula (I) for use in a method of treating DS. Background of the Invention Dravet Syndrome (DS), is described by the Dravet Syndrome Foundation (https: / / dravetfoundation.Org / what-is-dravet-svndrome. / ), as an intractable developmental and epileptic encephalopathy that begins in infancy and proceeds with accumulating morbidity that significantly impacts individuals throughout their lifetime. DS is a rare disease, with an estimated incidence rate of 1:15,700, with the majority of patients carrying a mutation in the sodium channel gene SCN1A (sodium channel alpha 1 subunit). DS is classified as a developmental and epileptic encephalopathy (also known as a DEE), which is part of a group of severe epilepsies with frequent and difficult to treat seizures and significant developmental delays. Seizures in DS usually begin during the first 2-15 months of life, often in the presence of fever or high temperatures. Seizures are frequently prolonged and are not well managed with current medications. Patients present with a variety of seizure types that generally evolve with age. As with all developmental and epileptic encephalopathies, DS includes more than just difficult to control seizures. Other comorbidities such as developmental delay and abnormal EEGs often emerge during the second or third year of life. Common issues associated with DS include: prolonged seizures, frequent seizures, behavioral and developmental delays, movement and balance issues, orthopedic conditions, delayed language and speech issues, growth and nutrition issues, sleeping difficulties, chronic infections, sensory integration disorders, dysautonomia, or disruptions of the autonomic nervous system which can lead to difficulty regulating body temperature, heart rate, blood pressure, and other issues. Current treatment options focus mainly on GABA (gamma-aminobutyric acid) activation. GABA is the most common inhibitory neurotransmitter in the developmentally mature mammalian central nervous system. Currently approved drugs act as GABA-A receptor agonists, stimulate biosynthesis of GABA or, inhibit metabolism or re-uptake of GABA. However, there remains a need for more effective treatments for DS. Current treatment options are very limited, and the constant care required for someone suffering from DS can severely impact the patient's and the family’s quality of life. Patients with DS face a 15-20% mortality rate due to SUDEP (Sudden Unexpected Death in Epilepsy), prolonged seizures, seizure-related accidents such as drowning, and infections. Therefore, an object of the present invention is to provide a new treatment for DS. An object of the present invention is to provide an alternative or addition to existing treatments for DS. A further object of the present invention is to provide a treatment option that is superior to the existing available treatment options. As discussed herein, the present inventors have surprisingly and advantageously identified compounds which show activity as a treatment for DS. WO 2020 / 214043, WO 2021 / 210997 and WO 2023 / 096512, the contents of which are incorporated herein by reference, relate to compounds which demonstrate activity as positive allosteric modulators (PAM) of the excitatory amino acid transporter-2 (EAAT2), a major glutamate transporter expressed in the brain. Summary of the Invention In a first aspect the present invention provides a compound for use in a method of treating Dravet Syndrome (DS), wherein the compound is of Formula (I): (I) wherein: X is hydrogen or deuterium or N(CH3)2 WisCH2 orC=O R is hydrogen or a halogen, A is hydrogen or deuterium, B is hydrogen or deuterium, Y is hydrogen or deuterium, or a pharmaceutically acceptable salt thereof. In a second aspect, the present invention provides a method of treating Dravet Syndrome (DS), wherein the method comprises administering a therapeutically effective amount of a compound of Formula (I) to a subject in need thereof, wherein Formula (I) is: A (I) wherein: X is hydrogen or deuterium or N(CH3)2 WisCH2 orC=O R is hydrogen or a halogen, A is hydrogen or deuterium, B is hydrogen or deuterium, Y is hydrogen or deuterium, or a pharmaceutically acceptable salt thereof. In the compound for the use according to the first aspect, or in the method according to the second aspect of the present invention, R can be a halogen, and optionally R can be F. In the compound for the use according to the first aspect, or in the method according to the second aspect of the present invention the compound may be selected from: (2R)- / V-benzyl-2-(2,5-dioxopyrrolidin-1-yl)propanamide, (2R)-2-(2,5-dioxopyrrolidin-1-yl)- / V-(2-fluorobenzyl)propanamide, (2R)- / V-benzyl-2-(2-oxopyrrolidin-1-yl)propanamide, (2R)- / V-(2-fluorobenzyl)-2-(2-oxopyrrolidin-1-yl)propanamide, (2R)- / V-benzyl-2-(3-(dimethylamino)-2,5-dioxopyrrolidin-1-yl)propanamide, and (2R)-2-(3-(dimethylamino)-2,5-dioxopyrrolidin-1-yl)- / \ / -(2-fluorobenzyl)propanamide. In embodiments the compound may be selected from: (2R)- / V-benzyl-2-(2,5-dioxopyrrolidin-1-yl)propanamide, or (2R)-2-(2,5- dioxopyrrolidin-1-yl)- / V-(2-fluorobenzyl)propanamide. Optionally the compound is (2R)- / V-benzyl-2-(2,5-dioxopyrrolidin-1-yl)propanamide. In embodiments the compound can be selected from: (2R)- / V-benzyl-2-(2-oxopyrrolidin-1-yl)propanamide, (2R)- / V-(2-fluorobenzyl)-2-(2-oxopyrrolidin-1-yl)propanamide, (2R)- / V-benzyl-2-(3-(dimethylamino)-2,5-dioxopyrrolidin-1-yl)propanamide, or (2R)-2-(3-(dimethylamino)-2,5-dioxopyrrolidin- 1-yl)-A / -(2-fluorobenzyl)propanamide. Optionally the compound may be (2R)- / V-benzyl-2-(3-(dimethylamino)-2,5-dioxopyrrolidin-1-yl)propanamide, or (2R)-2-(3-(dimethylamino)-2,5- dioxopyrrolidin-1-yl)- / V-(2-fluorobenzyl)propanamide. In the compound for the use according to the first aspect, or in the method according to the second aspect of the present invention at least one A may be deuterium. Optionally each A is deuterium. In the compound for the use according to the first aspect, or in the method according to the second aspect of the present invention the compound may be selected from: (2R)- / V-benzyl-2-(2,5-dioxopyrrolidin-1-yl-3,3,4,4-d4)propanamide, (2R)-2-(2,5-dioxopyrrolidin-1-yl-3,3,4,4-d4)- / V-((phenyl-d5)methyl)propanamide, (2R)-2-(2,5-dioxopyrrolidin-1-yl-3,3,4,4-d4)- / V-(2-fluorobenzyl)propanamide, (2R)-2-(2,5-dioxopyrrolidin-1-yl-3,3,4,4-d4)- / V-(phenylmethyl-d2)propanamide, (2R)-2-(2,5-dioxopyrrolidin-1-yl-3,3,4,4-d4)- / V-((phenyl-d5)methyl-d2)propanamide, or (2R)-2-(2,5-dioxopyrrolidin-1-yl-3,3A4-d4)- / V-((2-fluorophenyl)methyl-cfe)propanamide In the compound for the use according to the first aspect, or in the method according to the second aspect of the present invention the compound may be in the form of a pharmaceutically acceptable salt selected from: hydrochloride, sulfate, methanesulfonate, toluenesulfonate, succinate, fumarate or lactate. Optionally, in the compound for use according to the first aspect, or in the second aspect, X is N(CH3)2 the compound may be in the form of a pharmaceutically acceptable salt selected from: hydrochloride, sulfate, methanesulfonate, toluenesulfonate, succinate, fumarate or lactate. The first and the second aspect of the present invention also embrace i) use of a compound of Formula (I) in the manufacture of a medicament for the treatment of DS, ii) a pharmaceutical composition comprising a compound of Formula (I) for use in treating DS, and iii) use of a compound of Formula (I) for the treatment of DS. The present invention offers the following advantages and surprising new benefits over and above the prior art. • Compounds of Formula (I) for use in treating DS, and methods of treating DS comprising administering compounds for Formula (I) have not previously been proposed. • The present invention provides new compounds valuable for treating DS, which are the compounds of Formula (I). • The present invention provides alternatives to existing DS treatments. The present invention provides improved DS treatments in comparison with existing treatments. • In health, neurotransmission is a balance between inhibition and stimulation. Whilst existing therapies for DS focus on modulating GABA (the main inhibitory neurotransmitter in the mammalian brain), the compounds of Formula (I) function to cause an increase in glutamate uptake from the synaptic cleft and hence focus on modulating glutamate (the main excitatory neurotransmitter in the mammalian brain). Consequently, the present invention offers a new paradigm in DS treatment. • Compounds of Formula (I) have previously been shown to have anticonvulsant activity and utility in epilepsy treatment proposed. However, DS is a different and distinct condition from other neurological conditions including being distinct from other types of epilepsy in symptoms and underlying causes. • A compound of Formula (I), iQ-007 ((2F?)- / V-benzyl-2-(2,5-dioxopyrrolidin-1-yl)propanamide), has been demonstrated to significantly reduce total moved distance in the zebrafish model for DS. The total moved distance was reduced such that it matched the total moved distance of wild type (WT) zebrafish as shown in Example 1 below. • iQ-007 has been shown to significantly reduce the average number, the cumulative duration and the mean duration of ictal-like events in the zebrafish model for DS during EEG testing as shown in Example 2, confirming the effect of iQ-007 on total moved distance shown in Example 1. • iQ-007 has been demonstrated to show a superior effect in reducing the total moved distance in the zebrafish model for DS in comparison with the effect of valproic acid (VPA), which is the best in class of the existing DS therapies, as well as other compounds (e.g.stiripentol, fenfluramine) approved for the treatment of DS, as shown in Example 3 below. • Twelve compounds demonstrating the structural variation within Formula (I), namely analogs of iQ-007 according to this invention, have been shown to significantly enhance glutamate uptake by EAAT2, confirming their activities match iQ-007, and hence they would all find utility in treating DS. Brief Description of the Drawings Figure 1 shows the results of a locomotor activity analysis of zebrafish by Zefit. The zebrafish were either WT zebrafish (control) or Dravet Syndrome model (scnllab - / -) zebrafish. The figure shows the average Total Moved Distance (mm) in zebrafish WT or scnllab -I- larvae treated with VPA, or iQ-007 at the indicated concentration; * p <0.05, ** p <0.01, *** p <0.001, ****, P<0.0001 when compared to control animals, # p <0.05, ## p <0.01, ### p <0.001, #### p <0.0001 when compared to scnllab - / -treated animals (T-test). Mean ± S.D. Figure 2 shows a representative EEG recording of non-epileptiform activity and ictal-like discharge events. Figure 3 shows the average number of ictal-like discharge events in zebrafish WT (control) or scnllab -I- larvae treated with VPA or iQ-007 at the indicated concentrations, or untreated control; * p <0.05, ** p <0.01, *** p <0.001, ****, P<0.0001 when compared to control animals, # p <0.05, ## p <0.01, ### p <0.001, #### p <0.0001 when compared to scnllab - / - treated animals (T-test). Mean ± S.D. Figure 4 shows the cumulative duration of ictal-like discharge events in zebrafish WT (control) or scnllab -I- larvae treated with VPA or iQ-007 at the indicated concentrations, or untreated control; * p <0.05, ** p <0.01, *** p <0.001, ****, P<0.0001 when compared to control animals, # p <0.05, ## p <0.01, ### p <0.001, #### p <0.0001 when compared to scnllab -I- treated animals (T-test). Mean ± S.D. Figure 5 shows the mean duration of ictal-like discharge events in zebrafish WT (control) or scnllab -I- larvae treated with VPA or iQ-007 at the indicated concentrations, or untreated control; * p <0.05, ** p <0.01, *** p <0.001, ****, P<0.0001 when compared to control animals, # p <0.05, ## p <0.01, ### p <0.001, #### p <0.0001 when compared to scnllab -I- treated animals (T-test). Mean ± S.D. Figure 6 is from Griffin A, Hamling KR, Hong S, Anvar M, Lee LP and Baraban SC (2018) Preclinical Animal Models for Dravet Syndrome: Seizure Phenotypes, Comorbidities and Drug Screening. Front. Pharmacol. 9:573. doi: 10.3389 / and shows 6 dpf scn1labs552 homozygous mutants, which were identified based on pigmentation and arrayed in a 96-well plate. Larvae were treated with 2% DMSO (control) or compound using drug concentrations previously published. After 22-h incubation at 28°C with a 14:10 h light / dark photoperiod, and 30 min of chamber habituation, the swim behavior of 7 dpf larvae was analyzed for 10 min under dark conditions. Total distance moved was analyzed by normalizing the activity of AED treated larvae to vehicle treated controls. Graphs represents mean (±SD) normalized to control treated larvae from three independent experiments using the average from 12 larvae per treatment each time. Statistical significance was determined by oneway ANOVA followed by Dunnet’s multiple comparison test. No statistical significance changes in swim behavior of scn1labs552 homozygous mutants when exposed to known AEDs or putative AEDs. Recommended treatments (blue), contraindicated treatments (red) and recent experimental treatments (yellow) for DS are shown. Since the methodology used by Zefit (Figure 1) and Griffin et al (Figure 6) were the same and the results of VPA in both experiments were similar (60% in Figure 6 and 55% in Figure 1), all results can be compared against each other. Figure 7 shows the head-to-head comparison of iQ-007 results from Zefit with VPA and other GABA-ergic and serotonergic compounds showing an unexpected superior effect of iQ-007 on distance moved in the zebrafish model of DS as presented as percentage of negative or disease control. Detailed Description of the invention Compounds Compounds of use in the present invention are compounds of Formula (I): wherein: X is hydrogen or deuterium or N(CH3)2 Wis CH2 orC=O R is hydrogen or a halogen, A is hydrogen or deuterium, B is hydrogen or deuterium, Y is hydrogen or deuterium, or a pharmaceutically acceptable salt thereof. Exemplary compounds of Formula (I) are shown in Table 1 below. Table 1 Compound Structure 1 (2R)- / V-benzyl-2-(2,5-dioxopyrrolidin-1-yl)propanamide Referred to herein as iQ-007 nVo 2 (2R)-2-(2,5-dioxopyrrolidin-1-yl)- / V-(2-fluorobenzyl)propanamide z—Z — / o / =o IZ 3 (2R)- / V-benzyl-2-(2-oxopyrrolidin-1-yl)propanamide 4 (2R)- / V-(2-fluorobenzyl)-2-(2-oxopyrrolidin-1-yl)propanamide _ / 0 0 F i h d j 5 (2R)- / V-benzyl-2-(3-(dimethylamino)-2,5-dioxopyrrolidin-1-yl)propanamide 6 (2R)-2-(3-(dimethylamino)-2,5-dioxo-pyrrolidin-1-yl)-A / -(2-fluorobenzyl)propanamide U-— ZT °= / °\ / "y— z / 7 (2R)- / V-benzyl-2-(2,5-dioxopyrrolidin-1-yl- 3,3,4,4-d4)propanamide D^° D><r 0 TiVo 8 (2R)-2-(2,5-dioxopyrrolidin-1-yl-3,3,4,4-(^)- / V-((phenyl-cfc)methyl)propanamide Dyr o D V 1 N n AT 0 i H JI JL D 9 (2R)-2-(2,5-dioxopyrrolidin-1-yl-3,3,4,4-d4)- / V-(phenylmethyl-d2)propanamide o o — / o 2=0 Ao 10 (2R)-2-(2,5-dioxopyrrolidin-1-yl-3,3,4,4-d4)- / V-((phenyl-d5)methyl-d2)propanamide o o o K \__ / o D O 11 (2R)-2-(2,5-dioxopyrrolidin-1-yl-3,3,4,4-^)- / V-((2-fluorophenyl)methyl-d2)propanamide Dy.o 9 D D F D N / / I N T O 1 H 12 (2R)-2-(2,5-dioxopyrrolidin-1-yl-3,3,4,4-^)- / \ / -(2-fluorobenzyl)propanamide 14--^ ZI oA O / “ Q O Synthesis and characterisation of compounds of Formula (I) and the exemplary compounds of Table 1 above was previously described in WO 2020 / 214043, WO 2021 / 210997 and WO 2023 / 096512, the contents of which are incorporated by reference. Dravet Syndrome Dravet Syndrome (DS) is classified as a developmental and epileptic encephalopathy which is part of a group of severe epilepsies with frequent and difficult to treat seizures and significant developmental delays (https: / / dravetfoundation.org / what-is-dravet-syndrome / ). The clinical diagnosis may be supported by electroencephalography (EEG), neuroimaging, and detection of SCN1A gene mutation. EEG is typically normal at onset, but often progresses to generalized spike-and-wave discharges. Like the seizure semiology, a variety of interictal EEG findings is more common. Some patients may have persistently normal interictal records. Neuroimaging is normal. In the United States, testing for SCN1A mutation is commercially available. A high degree of clinical suspicion is needed for this diagnosis, because magnetic resonance imaging (MRI) is typically normal and the electroencephalography (EEG) findings are nonspecific. However, the early clinical presentation of DS is unique, with the onset of recurrent, convulsive seizures, which are often prolonged and triggered by fever, in a developmentally normal infant. More than 80% of people diagnosed with DS have a mutation in the SCN1A gene. (www.dravet.org.uk / about-dravet-syndrome / genetics-dravet-syndrome Genetics of Dravet Syndrome - Dravet Syndrome UK) The SCNIAgene, encodes for the alpha subunit of the voltage-gated sodium channel (VGSC) Nav1.1. 90% of SCN1A mutations are de novo, meaning they are not inherited but appear spontaneously in the genetic make-up of the child / adult with DS. There are over 6,000 places for a mutation to occur on the SCN1A gene and since SCN1A mutations can occur in so many different places of the gene, one child / adult with DS is unlikely to have exactly the same mutation as another child / adult. It is partly for this reason that DS is a spectrum condition, affecting each individual differently. Any type of SCN1A mutation can be seen in DS and the mutation type does not predict the severity of the disease. Around 10-15% of individuals with a clinical diagnosis of DS have no detected SCN1A mutation. It may be that the mutation exists in these individuals, but not yet identified, or it may be that there are modifying factors other than the genetic mutation at play. Mutations in a number of genes other than SCN1A may also be implicated in a clinical diagnosis of DS (https: / / www.dravet.orq.uk / about-dravet-syndrome / qenetics-dravet-syndrome / genetic-changes-beyond-scn 1 a / ). A genetic test can be helpful to clarify or confirm a clinical diagnosis of DS. A diagnosis of DS is generally made by a medical practitioner. DS differs in various ways from other types of epilepsy. Firstly, for more than 80% of patients there is a detected SCN1A mutation. In at least 80% of cases, DS is caused by haploinsufficiency of SCN1A gene, that encodes for the alpha subunit of the voltage-gated sodium channel (VGSC) Nav1.1. Therefore, DS, unlike many other types of epilepsy, has a quite precisely defined genetic cause. The mutation occurs de novo, which means that it is not inherited from the parents, it seems, that many types of epilepsy are genetic-based, but often epilepsy is the result of having a set of abnormal genes, while the presence of single abnormalities causes a low risk of the seizures. Secondly DS has atypical treatment compared with other types of epilepsy. Treatment of DS generally avoids drugs that are effective and safe in other types of epilepsy, such as e.g. carbamazepine, lamotrigine, phenytoin, which function as sodium channel blockers. Often, DS seizures are exacerbated after using sodium channel blockers as SCN1A mutation causes dysfunction of Nay1.1 sodium channels located on GABA-ergic inhibitory interneurons in the brain. Thirdly there is a huge risk of SUDEP (sudden unexpected death in epilepsy). Children with DS are at a higher risk of SUDEP than children with other types of epilepsy. The risk of SUDEP in DS is up to 15 times higher than other childhood-onset epilepsies. It’s the biggest cause of death in DS, responsible for nearly half of all DS deaths. Sadly, SUDEP tends to occur at a younger age (73% before the age of 11) than in other epilepsies. Fourthly temperature as a trigger. The first seizure in DS is often considered a febrile seizure (FS), but shortly thereafter, both FSs and seizures without fever occur, leading to diagnosis. Fever remains a factor that easily precipitates seizures. Some may be triggered by slight changes in body temperature, as from hot weather or a warm bath. Currently used treatments for DS GABA (gamma-aminobutyric acid) is the most common inhibitory neurotransmitter in the mammalian central nervous system. Its principal role is reducing neuronal excitability throughout the nervous system. Inhibitory neurotransmitters prevent or block chemical messages and decrease the stimulation of nerve cells in the brain. GABA binds to GABA receptors (GABA-A or GABA-B) and decreases the responsiveness of the nerve cell. This means that as an inhibitory neurotransmitter, GABA lessens the ability of a nerve cell to receive, create or send chemical messages to other nerve cells. Because the primary effect of mutation of Nav1.1 is failure of action potential generation in inhibitory interneurons, the most direct approach to restoring normal nervous system function would be to enhance GABAergic neurotransmission. Therefore, therapeutic approaches for DS have to date focused on stimulation of the GABAergic system by activation of GABA-A receptors, increasing the level of GABA in the synaptic cleft, or inhibiting the uptake of GABA from the synaptic cleft. Such treatments of DS are only symptomatic, and do not interfere in any way with the mechanism of the disease. To date all primary and secondary compounds approved for the use in DS (valproate (VPA), and clobazam (CLB in some regions), as initial therapy, and stiripentol (STP), cannabidiol (CBD) or fenfluramine (FFA) as escalation therapies or alternatives such as topiramate (TPM) are focused directly or indirectly on inhibitory transmission through the GABA pathway. Furthermore, most of the DS treatments currently in clinical development are GABAergic molecules. First line therapy usually relies on VPA or CLB. Second line therapy may rely on fenfluramine, stiripentol or cannabidiol. Drugs that block sodium channels are a mainstay of antiepileptic therapy, including phenytoin and lamotrigine. However, as would be expected in a disease caused by loss-of-function mutations in a sodium channel, these traditional sodium channel blockers actually exacerbate seizures in DS. New paradigm for DS treatment Therefore, current treatments available for DS are GABA receptor agonists or are aimed at enhancing GABAergic or serotonergic neurotransmission by influencing on its’ biosynthesis, degradation or uptake. To have a properly functioning brain, a delicate balance must be maintained between the inhibitory effects of GABA and the excitatory effects of glutamate. The present invention is based on the surprising finding that modulation of excitatory neurotransmission pathways is beneficial in the treatment of DS. In direct contrast to all of the current treatments for DS and the focus for all other DS therapies in development which are known to the inventors, the present inventors have focused on modulating the amount and effect of glutamate. Glutamate, is the main excitatory neurotransmitter in the brain, permitting the chemical messages to be carried from nerve cell to nerve cell. The present inventors have surprisingly and advantageously identified compounds which show activity as a treatment for DS. These compounds act as positive allosteric modulators (PAMs) of the excitatory amino acid transporter-2 (EAAT2), a major glutamate transporter expressed in the brain. Compounds used in methods of the present invention increase the activity and function of EAAT2 in removing glutamate from the synaptic cleft. Furthermore, as discussed below, the present inventors have surprisingly demonstrated that the compounds used in the methods of the present invention show superior effects in the DS test models, in comparison with known and abovementioned GABAergic and serotonergic therapies. Earlier studies by the present inventors, such as those reported in WO 2020 / 214043, WO 2021 / 210997 and WO 2023 / 096512 demonstrate that compounds of Formula (I), including iQ-007, perform well in a range of different test models for epilepsy. The test models used in previous studies focused on seizure models driven by external factors. The test models used in previous studies concern chemically induced seizures or electrically induced seizures. The information from epilepsy test models triggered by external chemical or electrical factors could not predict an effect in DS. This is because, as discussed above, DS is a considerably different condition from other types of epilepsy. DS is different from any neurological or neurodegenerative condition considered in WO 2020 / 214043, WO 2021 / 210997 and WO 2023 / 096512. Therefore, WO 2020 / 214043, WO 2021 / 210997 and WO 2023 / 096512 provide no teaching or suggestion of utility of the compounds of Formula (I) in DS. To analyse and confirm the utility of compounds of Formula (I), including iQ-007, in treating DS, the present inventors selected a test model driven by a genetic factor, namely a mutated SCN1a gene. In contrast to disease test models used to test compounds for use in treating other types of epilepsy, which rely on external factors such as chemically induced seizures or electrically induced seizures, the disease test model used to test compounds for use in treating DS relies on an internal factor. The present inventors selected the zebrafish model for DS to demonstrate therapeutic efficacy instead of DS rodent models. The zebrafish model for DS used in the Examples below is scnllab knock out zebrafish, i.e. scnllab -I- zebrafish. Notably, there is a much higher correlation between successfully demonstrating a positive effect in zebrafish model (-95%) over mouse models (-50%) and showing positive effects in human (Griffin, A., et al, June 2018, vol 9, Article 573, Frontiers in Pharmacology https: / / www.frontiersin.org / iournals / pharmacology / articles / 10.3389 / fphar.2018.00573 / fu II). Therefore zebrafish models are superior to rodent models for testing for therapeutics for DS. As noted above, the present inventors have surprisingly and advantageously identified compounds which show activity as a treatment for DS. These compounds act as positive allosteric modulators (PAMs) of the excitatory amino acid transporter-2 (EAAT2), a major glutamate transporter expressed in the brain. Compounds used in methods of the present invention increase the activity and function of EAAT2 in removing glutamate from the synaptic cleft. Furthermore, as discussed below, the present inventors have surprisingly demonstrated that the compounds used in the methods of the present invention show superior effects in the DS test models, in comparison with known and abovementioned GABAergic and serotonergic therapies. iQ-007 has been used in the Examples below as an exemplary compound of Formula (I). Example 1 and 2 shows that iQ-007 can treat DS. Furthermore, Example 3 shows iQ-007 is superior to currently used treatments for DS. It is understood that the primary reason for the beneficial effects demonstrated for iQ-007 is that it increases glutamate uptake by EAAT2. Compounds of Formula (I) are positive and selective allosteric modulators of the EAAT2 glutamate transporter (Abram, M. etal. J. Med. Chem. 2022, 65, 11703-11725). As such the compounds of Formula (I) cause increased uptake of glutamate from the synaptic cleft which in turn reduces excitatory neurotransmission. Compounds of Formula (I) act on excitatory neurotransmission in the brain in contrast to existing treatments for DS which act on the inhibitory neurotransmission of the brain. Example 4 demonstrates that the beneficial increase in glutamate uptake achieved with iQ-007 is also achieved by analogs of iQ-007. The analogs tested in Example 4 exemplify the range of iQ-007 analogs of Formula (I). Examples Example 1 - Therapeutic effect in zebrafish model of Dravet syndrome (DS) Test compound The compound tested in Example 1 was: Compound Structure Name used herein (2F?)- / V-benzyl-2-(2,5-dioxopyrrolidin-1-yl)propanamide iQ-007 Synthesis and characterisation of compounds of (2R)- / V-benzyl-2-(2,5-dioxopyrrolidin-1-yl)propanamide (iQ-007) Formula (I) was previously described in WO 2020 / 214043, WO 2021 / 210997 and WO 2023 / 096512, the contents of which are incorporated by reference. Summary This example investigated the therapeutic effects of iQ-007 through behavioural analysis in a zebrafish model of DS. Results showed that iQ-007 demonstrated efficacy in improving behavior in zebrafish model of DS. Behavioral analysis results of DS model: The locomotor activity (LMA), measured as total moved distance (mm), significantly increased in scnllab - / - zebrafish (a model for Dravet syndrome). The total moved distance (mm) showed a statistically significant decrease when scnllab - / - zebrafish were treated with iQ-007 at concentrations of 50 pM to 1000 pM. Methods and conditions The experimental conditions for zebrafish were managed as follows. Zebrafish used in Example • Zebrafish DS model = The zebrafish model for DS used in the Examples is scnllab knock out zebrafish, i.e. scnllab -I- zebrafish. (The scnllab - / -zebrafish is a known test model.) • Wild type = wild type (WT) zebrafish (Danio rerid) of the AB strain. Zebrafish Facility Management • The housing tank was cleaned daily to prevent the growth of algae, and ensure that exposed facilities such as pipes and lights are always kept in a clean state. • Zebrafish were maintained at the optimal breeding temperature of 27±1 °C, and the internal temperature of the laboratory was regulated accordingly. • The facility temperature was maintained at the optimal breeding temperature of 27±1°C, and the humidity regulated to within the range of 30-70%. Water Quality Management for Zebrafish Rearing • The reverse osmosis method was utilized for obtaining purified water. • The pH of the water was maintained at 6.5-7.5. Zebrafish Rearing Conditions • A light-dark cycle was implemented, with 14 hours of light and 10 hours of darkness each day to synchronize the circadian rhythm. Elimination of Surrounding Noise ■ To ensure stable behavioral measurements, analyses were conducted in a dedicated soundproof space. ■ Only the entry of measuring personnel was allowed once measurements commenced, and external noise was minimized throughout the measurement duration. Treatment protocol: • The positive control drug, valproic acid sodium salt (VPA) (Sigma-Aldrich, P4543) was prepared as a 1000 mM stock solution using DMSO. It was then diluted to a concentration of 1 mM in E3 medium. • The drug candidate, iQ-007 was dissolved in E3 medium at concentrations of 50 pM, 100 pM, 500 pM, and 1 mM. • 6 days post fertilization (dpf) zebrafish larvae (scnllab - / -) were randomly distributed into a 6-well plate, with 20 larvae per well. • The wells contained E3 medium treated with VPA or iQ-007 or E3 medium only for control as set out in Table 2 below. As shown in Table 2: AB larvae (WT zebrafish larvae) were placed in E3 medium only and did not receive any VPA or iQ-007. DS model (sen 1 lab - / -) zebrafish of the negative or disease control group were placed in E3 medium only and did not receive any VPA or iQ-007. DS model (sen 1 lab - / -) zebrafish of the positive control group were placed in E3 medium with 1mM VPA. DS model (scnllab - / -) zebrafish of the test groups were placed in E3 medium with iQ-007 at concentrations of 50 pM, 100 pM, 500 pM, or 1000 pm (i.e. 1mM). • The zebrafish larvae were held in the wells for 24 hours. Table 2. Group setting for behavior analysis in Dravet syndrome model Sample number Drug measurement Group name Animal for concentration stage behaviour analysis 1 Vehicle + Vehicle AB larvae 7dpf N=20 Vehicle + Vehicle sen 1lab -1- larvae 7dpf N=20 Vehicle + 1mM VPA sen 1lab -1- larvae 7dpf N=20 2 iQ-007 50 qM sen 1lab -1- larvae 7dpf N=20 100 qM sen 1lab -1- larvae 7dpf N=20 500 qM sen 1lab -1- larvae 7dpf N=20 1 mM sen 1lab - / - larvae 7dpf N=20 * Behavior analysis (AB(n=140)) Total moved distance (mm) -1 (Positive control group): Control, scnllab - / - and VPA groups (AB(n=20), scnllab - / - (n=40)) - 2 (Experimental group): Drug candidate (iQ-007) groups (scnllab - / - (n=80)) Locomotor activity (LMA) analysis was performed as follows: • Zebrafish larvae were individually transferred to 96-well plates for locomotor activity measurement. The Total Moved Distance (mm) was 5 analyzed by recording their movements for 10 minutes. • Movements were captured using a high-quality camera (16mm Telephoto Lens for Raspberry Pi High-Quality Camera) with a frame rate of 30 / s. An infrared (IR) light source (LV-BL-R 150 / 120 IR24) that is invisible to zebrafish, and an IR filter (850nm narrow bandpass filter) were used to 10 track movements in the dark. • The distance from the 96-well plate to the camera was 35cm. A Fresnel lens (IYF-240-1) was used to eliminate image distortion at the plate edges and capture all wells in the vertical direction. • During the 10-minute video analysis, if the speed of zebrafish larvae was 15 below 2mm / s for 1 frame, it was considered non-movement, such as twisting or floating in the water due to trembling. Only movements with a speed above 2mm / s were included in the Total Moved Distance (mm) calculation. • Prism (9.2.0) Software was used for automated analysis of larval locomotion activity. Analysis was performed using t-test in a statistical program. • Results obtained included a Total Distance Moved in mm for each 5 zebrafish. Results of locomotor activity analysis for DS model (scnllab - / -) zebrafish The results of Example 1 are shown in Table 3 below and in Figure 1. Table 3. Locomotor activity analysis for DS model (scnllab - / -) Total moved distance (mm) Control scnllab - / - scnllab - / - 100 qM VPA iQ-007 50 qM 100 qM 500 qM 1000 qM 1 386.6 750.2 67.7 93.1 4.9 329.4 46.5 2 111.9 296.8 58.1 198.8 86.9 177.5 334.8 3 174.3 417.8 312.2 526.8 69.9 22.6 146.2 4 271.2 370.3 163.0 53.2 218.0 223.6 86.2 5 1.5 82.1 58.9 403.2 90.5 36.1 42.4 6 127.8 175.7 13.5 245.7 87.3 167.8 18.6 7 18.7 163.0 208.8 0.0 23.8 612.9 112.1 8 152.9 165.4 336.5 320.5 345.1 0.0 11.3 9 201.5 34.1 55.2 309.3 49.2 22.5 180.4 10 218.1 86.1 34.5 243.4 226.4 138.1 74.6 11 446.9 170.9 289.8 0.0 94.3 111.1 79.7 12 0.0 54.8 523.5 455.6 0.0 137.8 22.6 13 65.9 860.1 25.7 272.1 184.3 91.0 158.9 14 55.5 378.3 34.5 252.7 73.2 33.6 72.2 15 13.5 272.8 303.2 116.2 23.5 1.8 157.9 16 11.4 21.7 346.4 345.4 7.4 49.5 298.3 17 73.0 830.2 130.3 364.8 0.0 23.1 222.4 18 305.6 623.5 316.8 402.0 83.1 114.6 85.4 19 232.5 666.2 330.3 488.7 119.1 408.9 114.6 20 494.3 948.1 289.8 503.3 169.7 331.4 174.1 Average 168.2 368.4 194.9 279.7 97.8 151.7 122.0 S.D 151.0 304.3 148.5 163.7 90.7 160.4 88.8 As shown in Figure 1, the total moved distance (mm) for the untreated DS model scnllab - / - zebrafish larvae was significantly greater than the total moved distance (mm) for the WT zebrafish larvae (compare bar 1 (control) with bar 2). The results show a statistically significant decrease in total moved distance (mm) when DS model scnllab - / - zebrafish larvae were treated with iQ-007 at concentrations of between 100 pM and 1000 pM (compare bars 5, 6 and 7 with bar 2). The results also show that the total moved distance (mm) when DS model scnllab - / -zebrafish larvae were treated with iQ-007 at concentrations of between 100 pM and 1000 pM, was more decreased in comparison with DS model scnllab - / - zebrafish larvae treated with 1000 pM VPA, the positive control drug. Example 1 demonstrates that: • iQ-007 is effective to significantly reduce locomotor activity in the DS model scnllab - / - zebrafish, • iQ-007 reduces locomotor activity in the DS model scnllab - / - zebrafish to a level comparable with the locomotor activity seem from WT zebrafish, and • iQ-007 reduces locomotor activity in the DS model scnllab - / - zebrafish to a level comparable with, or better than, the reduction in locomotor activity achieved using VPA, which is a standard treatment for DS. Conclusions The above results indicate that the new mechanism of action of positive allosteric modulation of the EAAT2 transporter, which normalizes glutamate levels, and which had not been tested so far by any research group in DS, may provide similar or even better efficacy in the treatment of DS compared to VPA the current first line treatment of DS, which is based on stimulation of inhibitory GABAergic neurotransmission. Example 2 - Demonstration of efficacy in inhibiting seizure-like EEG activity observed in the zebrafish Dravet Syndrome (DS) Model {scnllab - / -) Following the positive results in Example 1, and because in some cases locomotor activity may show false positive results, unless confirmed by EEG activity, the inventors wanted to validate and confirm the therapeutic effects of iQ-007 through seizure-like EEG analysis in a zebrafish DS model (scnllab - / -). Summary This Example aimed to validate the therapeutic effects of iQ-007 through seizurelike EEG analysis in a zebrafish DS model (scnllab - / -). The analysis of seizures through zebrafish larval EEG recordings was conducted based on the average number, cumulative duration, and mean duration of ictal-like discharge events following a 10-minute EEG recording session. In comparison with the control group, the DS model scnllab - / - group exhibited a significant increase in the average number, cumulative duration, and mean duration of ictal-like discharge events. Furthermore, treatment with iQ-007 at concentrations of 100 pM, 500 pM, and 1000 pM resulted in a statistically significant decrease in these parameters. Methods and conditions The test compound and the experimental conditions for zebrafish including the facility management, water quality management, rearing conditions and elimination of surrounding noise were as in Example 1. The zebrafish model for DS used in the Example is scnllab knock out zebrafish, i.e. scnllab - / - zebrafish. Wild type = wild type (WT) zebrafish (Danio rerid) of the AB strain. Treatment protocol: • The positive control, valproic acid sodium salt (VPA) (Sigma-Aldrich, P4543) was prepared as a 1000 mM stock solution using DMSO. It was then diluted to a concentration of 100 pM in E3 medium. • The drug candidate, iQ-007 was prepared as a 4000 mM stock solution using DMSO. It was then diluted to a concentration of 100 pM, 500 pM, and 1 mM in E3 medium. • 6 dpf scnllab - / - zebrafish larvae were identified by their “black” appearance, which is indicative of a defect in pigment aggregation, as reported previously. Identified larvae were randomly distributed into a 6-well plate, with 20 larvae per well, containing E3 medium treated with VPAor iQ-007 for 24 hours. Table 4. Group setting for EEG analysis in Dravet syndrome model Sample Drug measurement Group name Animal number concentration stage a for EEG 1 Vehicle + Vehicle AB larvae 7dpf N=10 Vehicle + Vehicle sen 1lab - / - larvae 7dpf N=10 Vehicle + 100 pM VPA sen 1lab - / - larvae 7dpf N=10 iQ-007 100 pM sen 1lab - / - larvae 7dpf N=10 500 pM sen 1lab - / - larvae 7dpf N=10 1 mM sen 1lab - / - larvae 7dpf N=10 * EEG analysis (AB(n=10), scnllab KO F0 (n=60)) Average number of ictal-like discharge events (nr / 10min), Cumulative duration of ictal-like discharge events (s / 10min), Mean Duration of ictal-like discharge events(s / 10min) -1 (Positive control group): Control, scnllab -1- and VPA groups (AB(n=10), scnllab -1- (n=20)) - 2 (Experimental group): Drug candidate (iQ-007) groups (scnllab- / - (n=30)) Electroencephalogram (EEG) analysis for DS model • To measure the EEG of zebrafish larvae, they were injected into the EEG recording chamber using an 18-gauge needle. 10 • The EEG of zebrafish larvae was recorded for 10 minutes using invasive electrodes, as per Lee et al, A Microfluidic System for Stable and Continuous EEG Monitoring from Multiple Larval Zebrafish, Sensors 2020, 2020 Oct 19;20(20):5903. doi: 10.3390 / s20205903 • Data analysis was performed using Biopac Systems. 15 Seizure-like EEG activities were comprehensively assessed based on criteria including the average number, cumulative duration, and mean duration of Ictal-like discharge events, characterized by amplitudes exceeding three times the baseline, multiple spike waveforms, and durations exceeding 0.1 seconds. Figure 2 shows a representative EEG recording of non-epileptiform activity and ictal-like discharge events. Results The analysis of seizures through zebrafish larval EEG recordings was conducted based on the average number, cumulative duration, and mean duration of ictal-like discharge events following a 10-minute EEG recording session. Tables 5, 6 and 7 along with Figures 3, 4, and 5 show the results. In comparison to the control group, the scnllab KO - / - group exhibited a significant increase in the average number, cumulative duration, and mean duration of ictal-like discharge events. Furthermore, treatment with iQ-700 at concentrations of 100 pM, 500 pM, and 1000 pM resulted in a statistically significant decrease in these parameters (Figure 3, 4 and 5). Table 5. Average number of ictal-like discharge events in zebrafish WT or scnllab -I- larvae treated with VPA and iQ-007 at the indicated concentration Average number (nr / 10 min) Test no. Control scnllab -1- scnllab - / - 100 pM VPA 100 pM 500 pM 1000 pM 1 0 3 1 0 0 3 2 0 5 1 4 0 1 3 0 3 1 0 0 0 4 0 2 0 0 0 0 5 1 1 0 0 0 0 6 0 0 0 0 0 2 7 0 1 0 0 1 0 8 0 5 0 0 0 0 9 0 2 0 0 0 0 10 0 4 0 0 1 0 Average 0.1000 2.6000 0.3000 0.4000 0.2000 0.6000 S.D 0.3162 1.7127 0.4830 1.2649 0.4216 1.0750 Figure 3 shows the average number of ictal-like discharge events (number / 10 min) As shown by bar 1 the control group of WT zebrafish had almost zero ictal-like discharge events within the 10 minute period of observation. 5 The DS model (sen 1 lab - / -) zebrafish had significantly more ictal-like discharge events within the 10-minute period of observation as shown by bar 2 Treatment of the DS model (scnllab - / -) zebrafish with positive control drug VPA led to a significant reduction in the number of ictal-like discharge events within the 10 minute period of observation as shown by bar 3. 10 Treatment of the of the DS model (sen 1 lab - / -) zebrafish with iQ-007 also led to a significant reduction in the number of ictal-like discharge events within the 10 minute period of observation at all tested concentrations of iQ-007 as shown by bars 4, 5 and 6. Furthermore, the number of ictal-like discharge events within the 10 minute period of observation for the DS model (sen 1 lab - / -) zebrafish treated with iQ-007 15 was comparable to VPA treated DS model (scnllab - / -) zebrafish at all tested concentrations of iQ-007. Table 6. Cumulative duration of ictal-like discharge events in zebrafish WT or scnllab -I- larvae treated with VPA and iQ-007 at the indicated concentration Cumulative duration (s / 10min) Test no. Control scnllab -1- scnllab - / - 100 pM VPA 100 pM 500 pM 1000 pM 1 0 2.902852 0.758026 0 0 1.778952 2 0 10.91623 0.239374 7.33785 0 0.851221 3 0 5.837523 0.348669 0 0 0 4 0 1.289426 0 0 0 0 5 0.849023 1.07204 0 0 0 0 6 0 0 0 0 0 1.573429 7 0 1.15088 0 0 0.660088 0 8 0 10.6141 0 0 0 0 9 0 3.979705 0 0 0 0 10 0 13.578 0 0 1.23575 0 Ave rag e 0.085 5.134 0.135 0.734 0.190 0.420 S.D 0.268 4.888 0.252 2.320 0.422 0.715 Figure 4 shows the cumulative duration of ictal-like discharge events (seconds / 10 min) As shown by bar 1 the control group of WT zebrafish had almost zero seconds / 10 minute period of observation. The DS model (scnllab - / -) zebrafish of the had significantly higher cumulative duration of ictal-like discharge events within the 10 minute period of observation as shown by bar 2. Treatment of the DS model (scnllab - / -) zebrafish with positive control drug VPA led to a significant reduction in the cumulative duration of the ictal-like discharge events within the 10-minute period of observation as shown by bar 3. Treatment of the DS model (scnllab - / -) zebrafish with iQ-007 also led to a significant reduction in the cumulative duration of ictal-like discharge events within the 10 minute period of observation at all tested concentrations of iQ-007 as shown by bars 4, 5 and 6. Furthermore, the cumulative duration of ictal-like discharge events within the 10 minute period of observation for the DS model (scnllab - / -) zebrafish treated with iQ-007 was comparable to VPA treated DS model (scnllab - / -) zebrafish at all tested concentrations of iQ-007. Table 7. Mean duration of ictal-like discharge events in zebrafish WT or scnllab - / -larvae treated with VPA and iQ-007 at the indicated concentration Mean duration (s / 10min) Test no. Control scnllab - 1- scnllab - / - 100 pM VPA 100 pM 500 pM 1000 pM 1 0 0.967617 0.758026 0 0 0.592984 2 0 2.183246 0.239374 1.834463 0 0.851221 3 0 1.945841 0.348669 0 0 0 4 0 0.644713 0 0 0 0 5 0.849023 1.07204 0 0 0 0 6 0 0 0 0 0 0.786714 7 0 1.15088 0 0 0.660088 0 8 0 2.12282 0 0 0 0 9 0 1.989852 0 0 0 0 10 0 3.3945 0 0 1.23575 0 Ave rag e 0.085 1.547 0.135 0.183 0.190 0.223 S.D 0.268 0.968 0.252 0.580 0.422 0.365 Figure 5 shows the mean duration of ictal-like discharge events (seconds / 10 min) As shown by bar 1 the control group of WT zebrafish had almost zero for the mean duration of ictal-like discharge events within the 10 minute period of observation. The DS model (sen 1 lab - / -) zebrafish of had a significantly greater mean duration of ictal-like discharge events within the 10-minute period of observation as shown by bar 2. Treatment of the DS model (scnllab - / -) zebrafish with positive control drug VPA led to a significant reduction in the mean duration of ictal-like discharge events within the 10-minute period of observation as shown by bar 3. Treatment of the of the DS model (sen 1 lab - / -) zebrafish with iQ-007 also led to a significant reduction in the mean duration of ictal-like discharge events within the 10 minute period of observation at all tested concentrations of iQ-007 as shown by bars 4, 5 and 6. Furthermore, the mean duration of ictal-like discharge events within the 10 minute period of observation for the DS model (scnllab - / -) zebrafish treated with iQ-007 was comparable to VPA treated DS model (scnllab - / -) zebrafish at all tested concentrations of iQ-007. Example 2 shows that: • In the zebrafish DS model, treatment with iQ-007 at all tested concentrations provides a statistically significant decrease in i) the average number, ii) the cumulative duration, and iii) the mean duration of ictal-like discharge events. Conclusions The above results indicate that the new mechanism of action of positive allosteric modulation of the EAAT2 transporter, which normalizes glutamate levels, and which had not been tested so far by any research group in DS, provides similar or even better efficacy in the treatment of DS compared to VPA the current first line treatment of DS, which is based on stimulation of inhibitory GABA neurons. The positive results in the EEG analysis confirm the validity of the results in the behavioral analysis (Example 1). Example 3 - Inter-laboratory comparison of iQ-007 versus known primary and secondary therapeutic drugs for treating Dravet Syndrome (DS). All currently approved treatments for DS are GABAergic or serotonergic agents.. GABA (gamma-aminobutyric acid) is the main inhibitory neurotransmitter in the developmentally mature mammalian central nervous system. Its principal role is reducing neuronal excitability throughout the nervous system. To date all primary and secondary compounds approved for the use in DS (valproate (VPA), and clobazam (CLB in some regions), as initial therapy, and stiripentol (STP), cannabidiol (CBD) or fenfluramine (FFA) as escalation therapies; alternatives include topiramate (TPM)) are focused directly or indirectly on inhibitory transmission through the GABA pathway. Furthermore, DS treatments currently in clinical development are predominantly GABAergic or serotonergic molecules (Klein et al, 2024). Figure 6 shows data obtained from a literature search showing the relative effects of a number of known DS drugs in the scn1 Lab' / _ zebrafish model. The results are shown as the cumulative distance in large movements (cm / 100 sec) by test groups of zebrafish larvae, normalized against vehicle and therefore this is a study following the same methodology and testing the same activity and effects as in Example 1 above. Literature used • Griffin A, Hamling KR, Hong S, Anvar M, Lee LP and Baraban SC (2018) Preclinical Animal Models for Dravet Syndrome: Seizure Phenotypes, Comorbidities and Drug Screening. Front. Pharmacol. 9:573. doi: 10.3389 / fphar.2018.00573 • Li J, Nelis M, Sourbron, J, Copmans D, Lagae L, Cabooter D and de Witte PAM (2021) Efficacy of Fenfluramine and Norfenfluramine Enantiomers and Various Antiepileptic Drugs in a Zebrafish Model of Dravet Syndrome. Neurchemical Research 46:2249-2261 doi: In Figure 6 Bar 1 shows the cumulative distance in large movements in cm / 100 seconds as percentage from vehicle for DS model (scnllab - / -) zebrafish placed in vehicle only (VHC) Bar 2 shows the cumulative distance in large movements in cm / 100 seconds as percentage from vehicle for DS model (sen 1 lab - / -) zebrafish placed in valproate (VPA) at a concentration of 100 pM. Bar 3 shows the cumulative distance in large movements in cm / 100 seconds as percentage from vehicle for DS model (sen 1 lab - / -) zebrafish placed in stiripentol (STP) at a concentration of 12.5 pM. Bar 4 shows the cumulative distance in large movements in cm / 100 seconds as percentage from vehicle for DS model (scnllab - / -) zebrafish placed in diazepam (DZM) at a concentration of about 16 pM. Bar 5 shows the cumulative distance in large movements in cm / 100 seconds as percentage from vehicle for DS model (scnllab - / -) zebrafish placed in topiramate (TPM) at a concentration of 200 pM. Bar 6 shows the cumulative distance in large movements in cm / 100 seconds for DS model (scnllab - / -) zebrafish placed in fenfluramine (FFA) at a concentration of 50 mM. The conclusion from Figure 6 is that the most effective compounds of the GABA agonist (GABAergic) drugs tested in the zebrafish DS model are DZM and VPA. In the zebrafish study VPA was tested at a concentration of 1 mM. Both VPA and iQ-007 were tested in Example 1 and a comparison is shown in Figure 7, whereby the total distance moved for the Example 1 results was also expressed as percentage from disease control (scnla bar). Figure 7 shows the inter-laboratory comparison of the results obtained for iQ-007 and VPA in Example 1 above, versus the results obtained for VPA and other known DS drugs as shown in Figure 6. Figure 7 shows that iQ-007 gives better results than VPA. Importantly, placing zebrafish in 100 pM iQ-007 outperforms 100 pM VPA i.e. 2x less iQ-007 was needed to achieve a superior result. Conclusion Example 3 leads to the conclusion that in the zebrafish DS model, iQ-007 provides superior results in comparison with any of the current treatments for DS. Example 4 - In vitro glutamate uptake studies in COS-7 cell line transfected with EAAT2 Summary This Example tests the effect of iQ-007 and its analogs according to Formula (I) to enhance glutamate uptake mediated by EAAT2 in transfected COS-7 cells. Methods and conditions Compounds Synthesis and characterisation of compounds of Formula (I) and the exemplary compounds of Table 1 above was previously described in WO 2020 / 214043, WO 2021 / 210997 and WO 2023 / 096512, the contents of which are incorporated by reference. Cells and transfection COS-7 cells (Kidney Fibroblast; African Green Monkey, ATCC-CRL-1651, ATCC, Manassas, VA, USA) were seeded at a density of 50,000 cells per well in a 24-well plate and transiently transfected with CMV vectors containing either no insert (background control) or the EAAT2 gene CMV-hEAAT2 (Addgene, Watertown, MA, USA) at a concentration of 100 ng cDNA per well in a final volume of 510 pl of: DM EM (ATCC, Manassas, VA, USA) supplemented with 10% Fetal Bovine Serum (ATCC, Manassas, VA, USA) and 5% 100* penicillin / streptomycin (Thermo Fisher Scientific, Waltham, MA USA), and 100 pl Opti-MEM (Thermo Fisher Scientific, Waltham, MA USA). Transfection was performed using TurboFect transfection reagent, according to the manufacturer's protocol (Thermo Fisher Scientific, Waltham, MA USA). Following transfection, the cells were incubated for 48 hours at 37°C in a 5% CO2 atmosphere. Glutamate uptake assays 48 hours post-transfection, a glutamate uptake assay was performed using [3H] glutamic acid (Revvity, Boston, USA, specific activity 47 Ci / mmol) at a final concentration = 50 nM, according to procedure described by Fontana et al. (Fontana ACK, et al Protocols for Measuring Glutamate Uptake: Dose-Response and Kinetic Assays in In Vitro and Ex Vivo Systems. Curr Protoc Pharmacol. 2018;82(1):e45) with modifications. Transfected cells, after a single wash with the phosphate-buffered saline with calcium and magnesium (PBS-CM) buffer, pH=7.4, were incubated with 200 pl of compounds tested at specified concentrations (ranging from 10-4 M to 10-12 M) for 10 minutes at 37°C. Compounds were dissolved in 100% DMSO, with a maximum DMSO content of 0.01% at tested concentrations. After incubation, 100 pl of the radioligand was added and incubated for 5 minutes at 37°C. Radioactivity counting After incubation with the radioligand, following two washes with PBS-CM buffer, the cells were lysed in lysis buffer (1% SDS / 0.1 N NaOH; 20 min / RT). Radioactivity, measured as disintegrations per minute (DPM), was counted using a liquid scintillation counting Beckman LS 650 (Beckman, USA). Statistical analysis Results were analyzed using GraphPad Prism 10.2.3 software. A one-way ANOVA with post-hoc Dunnett’s multiple comparison test was used to analyze differences between various concentrations of compounds tested vs. control. Results In vitro studies performed in the COS-7 cell line overexpressing the EAAT2 transporter showed that example compounds representing all structural variants according to Formula (I) significantly enhance glutamate uptake in this experimental system. ECso values (i.e. half of the maximal effective concentration required to obtain 50% of the effect after a given exposure time) were 0.05-62.4 nM, while the glutamate uptake efficiency ranged from 116 to 152.5% (for details see Table 8). Importantly, the results obtained prove that despite of structural variations, all compounds listed below share the same mechanism of action and are effective glutamate uptake enhancers at nanomolar concentrations, therefore they are potentially effective in the treatment of DS. Table 8. ECso values and glutamate uptake efficacy for example compounds described by general formula (I) mediated by EAAT2 in COS-7 cells. Cmpd No. Structure Glutamate uptake mediated by EAAT2a ECso value [nM]b Efficacy vs. control [%]c 1 0.28 ±0.1 140 ±8.0 2 4.5 ±2.1 149.7 ±7.1 3 _ / ,0 0 Atq 9.8 ±0.8 116.0 ±4.0 4 ,0 W’s'xi 2.3 ± 1.1 119.0 ±2.2 5 —N7 0.57 ±1.8 117 ±5.0 6 "N 0.61 ±0.7 118±2.3 7 DD 4¾^ 0.82 ±0.8 116.0 ±3.4 8 n D 0^^° 0 D D 4.5 ±4.2 134.3 ± 1.6 9 dd :>0 1.0 ±2.8 116 ±4.4 10 Dy( \ 0 D D D D N ^J^ m D 7 I N ]| nF q / i H JI kL D 0.1 ±0.04 137 ±4.8 11 ll—y Q 2 Q ZI o=2 o / “ q o 62.4 ± 56.9 152.5 ± 13.8 12 dY° 0 F 7 1 N II 0 i H III 0.05 ± 0.03 118.0 ±5.0 a Studies were performed in COS-7 cell line transfected with EAAT2. The results were normalized and described as % of control (EAAT2). Data shows mean ± S.E.M. from 3-6 independent experiments performed in triplicate; bHalf the maximum effective concentration required to obtain 50% effect; c Glutamate uptake efficacy expressed as 5 % of control (EAAT2). One-way analysis of variance (ANOVA) with Dunnett's post-hoc multiple comparisons test was used to analyze differences in glutamate uptake at various concentrations of the tested compounds compared to the control group. Conclusion 10 A wide range of compounds exploring the structural variation within Formula (I) were all shown to be effective glutamate uptake enhancers at nanomolar concentrations. Enhancing glutamate uptake via EAAT2 is understood as the primary mechanism by which compounds of Formula (I) show positive results in the test model for DS. 15 Therefore, the compounds of Formula (I) may be effective in the treatment of DS, and compounds of Formula (I) and compositions comprising such compounds can be used in methods of treating DS.

Claims

1. A compound for use in a method of treating Dravet Syndrome, wherein the compound is of Formula (I):wherein:X is hydrogen or deuterium or N(CH3)2Wis CH2 or C=OR is hydrogen or a halogen,A is hydrogen or deuterium,B is hydrogen or deuterium,Y is hydrogen or deuterium,or a pharmaceutically acceptable salt thereof.

2. The compound for the use according to claim 1, wherein R is a halogen.

3. The compound for the use according to claim 2, wherein R is F.

4. The compound for the use according to any one of claims 1 to 3, wherein thecompound is selected from:(2R)- / V-benzyl-2-(2,5-dioxopyrrolidin-1-yl)propanamide, (2R)-2-(2,5-dioxopyrrolidin-1-yl)- / V-(2-fluorobenzyl)propanamide,(2R)- / V-benzyl-2-(2-oxopyrrolidin-1-yl)propanamide,(2R)- / V-(2-fluorobenzyl)-2-(2-oxopyrrolidin-1-yl)propanamide,(2R)- / V-benzyl-2-(3-(dimethylamino)-2,5-dioxopyrrolidin-1-yl)propanamide,or(2R)-2-(3-(dimethylamino)-2,5-dioxopyrrolidin-1-yl)- / \ / -(2-fluorobenzyl)propanamide.

5. The compound for the use according to any one of claims 1 to 4, wherein the compound is selected from:(2R)- / V-benzyl-2-(2,5-dioxopyrrolidin-1-yl)propanamide, or (2R)-2-(2,5- dioxopyrrolidin-1-yl)- / V-(2-fluorobenzyl)propanamide.

6. The compound for the use according to any one of claims 1 to 5, wherein the compound is (2R)- / V-benzyl-2-(2,5-dioxopyrrolidin-1-yl)propanamide.

7. The compound for the use according to any one of claims 1 to 4, wherein the compound is selected from:(2R)- / V-benzyl-2-(2-oxopyrrolidin-1-yl)propanamide, (2R)- / V-(2-fluorobenzyl)-2-(2-oxopyrrolidin-1-yl)propanamide, (2R)- / V-benzyl-2-(3-(dimethylamino)-2,5-dioxopyrrolidin-1-yl)propanamide, or (2R)-2-(3-(dimethylamino)-2,5-dioxopyrrolidin-1 -yl)-A / -(2-fluorobenzyl)propanamide.

8. The compound for the use according to any one of claims 1 to 4, or 7, wherein the compound is selected from:(2R)- / V-benzyl-2-(3-(dimethylamino)- 2,5-dioxopyrrolidin-1-yl)propanamide, or (2R)-2-(3-(dimethylamino)-2,5- dioxopyrrolidin-1-yl)- / V-(2-fluorobenzyl)propanamide.

9. The compound for the use according to any one of claims 1 to 3, wherein at least one A is deuterium.

10. The compound for the use according to claim 9, wherein each A is deuterium.

11. The compound for the use according to any one of claims 1 to 3, or 9 to 10, wherein the compound is selected from:(2R)- / V-benzyl-2-(2,5-dioxopyrrolidin-1-yl-3,3,4,4-d4)propanamide, (2R)-2-(2,5-dioxopyrrolidin-1-yl-3,3,4,4-d4)- / V-((phenyl-d5)methyl)propanamide, (2R)-2-(2,5-dioxopyrrolidin-1-yl-3,3,4,4-d4)- / V-(2-fluorobenzyl)propanamide,(2R)-2-(2,5-dioxopyrrolidin-1-yl-3,3A4-d4)- / V-(phenylmethyl-cfe)propanamide, (2R)-2-(2,5-dioxopyrrolidin-1-yl-3,3,4,4-d4)- / V-((phenyl-d5)methyl-cypropanamide, or(2R)-2-(2,5-dioxopyrrolidin-1-yl-3,3,4,4-d4)- / V-((2-fluorophenyl)methyl-cypropenamide.

12. The compound for the use according to any one of claims 1 to 11, wherein the compound is in the form of a pharmaceutically acceptable salt selected from: hydrochloride, sulfate, methanesulfonate, toluenesulfonate, succinate, fumarate or lactate.

13. A method of treating Dravet Syndrome, wherein the method comprises administering a therapeutically effective amount of a compound of Formula (I) to a subject in need thereof, wherein Formula (I) is:A(I)wherein:X is hydrogen or deuterium or N(CH3)2 Wis CH2 or C=OR is hydrogen or a halogen, A is hydrogen or deuterium, B is hydrogen or deuterium, Y is hydrogen or deuterium, or a pharmaceutically acceptable salt thereof.

14. The method according to claim 13, wherein R is a halogen.

15. The method according to claim 14, wherein R is F.

16. The method according to claim 13, wherein the compound is selected from the group consisting of:(2R)- / V-benzyl-2-(2,5-dioxopyrrolidin-1-yl)propanamide,(2R)-2-(2,5-dioxopyrrolidin-1-yl)- / V-(2-fluorobenzyl)propanamide, (2R)- / V-benzyl-2-(2-oxopyrrolidin-1-yl)propanamide, (2R)- / V-(2-fluorobenzyl)-2-(2-oxopyrrolidin-1-yl)propanamide, (2R)- / V-benzyl-2-(3-(dimethylamino)-2,5-dioxopyrrolidin-1-yl)propanamide, and(2R)-2-(3-(dimethylamino)-2,5-dioxopyrrolidin-1-yl)- / \ / -(2-fluorobenzyl)propanamide.

17. The method according to claim 16, wherein the compound is selected from the group consisting of:(2R)- / V-benzyl-2-(2,5-dioxopyrrolidin-1-yl)propanamide, and (2R)-2-(2,5-dioxopyrrolidin-1-yl)- / V-(2-fluorobenzyl)propanamide.

18. The method according to claim 17, wherein the compound is (2R)- / V-benzyl-2-(2,5-dioxopyrrolidin-1-yl)propanamide.

19. The method according to claim 16, wherein the compound is selected from the group consisting of:(2R)- / V-benzyl-2-(2-oxopyrrolidin-1-yl)propanamide,(2R)- / V-(2-fluorobenzyl)-2-(2-oxopyrrolidin-1-yl)propanamide,(2R)- / V-benzyl-2-(3-(dimethylamino)-2,5-dioxopyrrolidin-1-yl)propanamide, and (2R)-2-(3-(dimethylamino)-2,5-dioxopyrrolidin-1-yl)- / \ / -(2-fluorobenzyl)propanamide.

20. The method according to claim 16, wherein the compound is selected from the group consisting of:(2R)- / V-benzyl-2-(3-(dimethylamino)-2,5-dioxopyrrolidin-1-yl)propanamide, and (2R)-2-(3-(dimethylamino)-2,5- dioxopyrrolidin-1-yl)- / V-(2-fluorobenzyl)propanamide.

21. The method according to claim 13, wherein at least one A is deuterium.

22. The method according to claim 21, wherein each A is deuterium.

23. The method according to claim 13, wherein the compound is selected from the group consisting of:(2R)-N-benzyl-2-(2,5-dioxopyrrolidin-1-yl-3,3,4,4-d4)propanamide,(2R)-2-(2,5-dioxopyrrolidin-1-yl-3,3,4,4-d4)- / V-((phenyl-d5)methyl)propanamide, (2R)-2-(2,5-dioxopyrrolidin-1-yl-3,3,4,4-d4)- / V-(2-fluorobenzyl)propanamide, (2R)-2-(2,5-dioxopyrrolidin-1-yl-3,3,4,4-d4)- / V-(phenylmethyl-d2)propanamide, (2R)-2-(2,5-dioxopyrrolidin-1-yl-3,3,4,4-d4)- / V-((phenyl-d5)methyl-cypropanamide, and(2R)-2-(2,5-dioxopyrrolidin-1-yl-3,3,4,4-d4)- / V-((2-fluorophenyl)methyl-d2)propenamide24. The method according to claim 13, wherein the compound is in the form of a pharmaceutically acceptable salt selected from: hydrochloride, sulfate, methanesulfonate, toluenesulfonate, succinate, fumarate or lactate.