WO2023041657A1

WO2023041657A1 - Alkylated haloquinolines for use in epilepsy

Info

Publication number: WO2023041657A1
Application number: PCT/EP2022/075665
Authority: WO
Inventors: Karin Thevissen; Bruno Cammue; Annelii NY; Lieven LAGAE; Peter De Witte
Original assignee: Katholieke Universiteit Leuven
Priority date: 2021-09-15
Filing date: 2022-09-15
Publication date: 2023-03-23

Abstract

The invention relates to alkylated haloquinolines for use in the prevention or treatment of a seizure in epilepsy or in neuroinflammation associated with epilepsy.

Description

ALKYLATED HALOQUINOLINES FOR USE IN EPILEPSY

Field of the invention

The invention relates to compounds activating Phgdh which are suitable in the field of epilepsy.

Background of the invention

Epilepsy is a common neurological disorder, affecting more than 70 million individuals worldwide. This chronic brain disease is characterized by unprovoked, recurrent seizures, which have a negative impact on the patient's cognitive abilities, psychological health and social interactions and which are typically caused by a disrupted balance of inhibitory and excitatory processes. On top, neuro-inflammation is often co-occurring with epileptic seizures: various studies suggest that neuroinflammation can be regarded as both a consequence and a cause of epileptic activity [Vezzani et al. (2011) Nat Rev Neurol 7, 31-40]. The majority of patients uses marketed antiseizure drugs (ASDs) to control epileptic seizures, potentially supplemented with anti-inflammatory drugs. Despite the presence of more than 25 ASDs on the market and the continuous efforts to develop new ones, approximately one third of epilepsy patients is unable to control their seizures. According to the International League Against Epilepsy (ILAE) epilepsy is termed drug-resistant upon "failure of adequate trials of two tolerated and appropriately chosen and used antiepileptic drug schedules (whether as monotherapies or in combination) to achieve sustained seizure freedom" [Kwan et al. (2010) Epilepsia 51, 1069-1077]. Hence, novel therapeutic strategies are required to treat patients suffering from drugresistant epilepsy.

Clioquinol has been used systemically for many years to treat traveller's diarrhoea and certain fungal and protozoal gastrointestinal tract infections [Perez et al. (2019) Pharmacol Ther 199, 155-163]. However, clioquinol was considered a possible cause of subacute myelo-optic neuropathy (SMON), a severe disease that mainly occurred among the Japanese population. Various recent studies report that SMON results from other biologic factors and/or pharmacogenetics primarily associated with the Japanese population (Perez et al. cited above).

Neurological toxicity has been observed in dogs when treated with clioquinol at doses exceeding 200 mg/(kg day) for over a month, and at doses of 400 mg/(kg day) toxicity can be observed in a week (reviewed in Mao (2008) Toxicol Lett. 182, 1-6. In all but one study, a dose of 100 mg/(kg day) for over 30 days did not produce toxicity in animals.

Humans have been treated with Clioquinol at a usual dose of 1.5-2 g/day (-25-30 mg/(kg day)), with reports of patients receiving 3.5g (-50 mg/(kg day))/day without toxicity. In Japanese humans, administration of daily Clioquinol has been associated with neurological side effects. Recently, evidence was gathered that clioquinol inhibits cAMP-transporting ABC pumps (reviewed in Perez et al. (2019) Pharmacol Ther. 199, 155-163). A further analysis revealed the presence of single nucleotide polymorphisms (SNPs) in both ABCC4 and ABCC11, capable of reducing transporter function and at the same time present with a high frequency in the Japanese population. This finding provides a plausible explanation for the SMON phenomenon: patients that carry SNPs in ABC transporters that dramatically affect nucleotide efflux are expected to be more sensitive to clioquinol.

8-hydroxy-7-substituted quinolines are described as anti-viral agents in EP0927164.

Summary of the invention

The activity of human recombinant PHGDH can be increased by various members of the drug class of haloquinolines, including clioquinol. The anticonvulsant activity of clioquinol as well as a methylated derivative of clioquinol (methyl-clioquinol) was investigated in the zebrafish EKP-induced seizure model and mouse 6-Hz psychomotor seizure model, both validated as relevant models for drug-resistant seizures Zhang et al. (2017) Sci Rep 7, 1-13; Leclercq et al. (2014) Epilepsy Res 108, 675-683.]. In contrast to clioquinol which is known to act as an ion chelator, the methylated clioquinol derivative cannot. Hence, potential anti-seizure activity of methyl-clioquinol is independent of ion chelation. Potential anti-inflammatory properties of clioquinol were assessed in the SSSE mouse epilepsy model. In addition, the anticonvulsant mode of action of clioquinol was unravelled by determining its effect on glutamate levels in head homogenates of zebrafish.

The invention is further summarized in the following statements:

1. An alkylated haloquinoline with the structure depicted in formula I or an pharmaceutically acceptable salt thereof

(I) wherein Ri and R₂ are a halogen, or Ri is a halogen and R2 is H, or Ri is H and R2 is a halogen, and wherein R₃ is a linear, branched or cyclic saturated or unsaturated Ci-Ce or C1-C4 chain, optionally substituted with a halogen, for use in the prevention or treatment of a seizure in epilepsy or in neuroinflammation associated with epilepsy.

2. The alkylated haloquinoline or salt thereof for use in accordance with statement 1, wherein R₃ is methyl.

3. The alkylated haloquinoline or salt thereof for use in accordance with statement 1 or 2, wherein Ri is Cl and R2 is I.

4. The alkylated haloquinoline or salt thereof for use in accordance with any one of statements 1 to 3, wherein Ri is Cl and R2 is I, and R₃ is methyl.

5. The alkylated haloquinoline or salt thereof for use in accordance with any one of statements 1 to 4, wherein the epilepsy is a genetic disorder.

6. The alkylated haloquinoline or salt thereof for use in accordance with any one of statements 1 to 5, wherein the epilepsy is Dravet syndrome.

7. The alkylated haloquinoline or salt thereof for use in accordance with any one of statements 1 to 4, wherein the epilepsy is caused by PHGDH deficiency.

8. The alkylated haloquinoline or salt thereof for use in accordance with any one of statements 1 to 7, wherein the epilepsy is a drug resistant epilepsy.

9. The alkylated haloquinoline or salt thereof for use in accordance with statement 8, wherein the drug resistant epilepsy is resistant against two or more selected from the group consisting of valproate, carbamazepine, levetiracetam, lamotrigine, topiramate, briveracetam, lacosamide, perampanel and phenobarbital.

10. The alkylated haloquinoline or salt thereof for use in accordance to any one of statements 1 to 9, wherein the alkylated haloquinoline is administered orally.

11. The alkylated haloquinoline or salt thereof for use according to any one of statements 1 to 10, wherein the alkylated haloquinoline treatment is a monotherapy. 12. The alkylated haloquinoline or salt thereof for use according to any one of statements 1 to 10, in a combination treatment with a further anti-epilepsy drug.

13. The alkylated haloquinoline or salt thereof for use according to any one of statements 12, wherein the further anti-epilepsy drug is fenfluramine.

14. A method of treating or preventing a seizure in epilepsy or neuroinflammation associated with epilepsy in a human individual, comprising the step of administering an effective amount of an alkylated haloquinoline with the structure depicted in formula I or an pharmaceutically acceptable salt thereof

(I) wherein Ri and R₂ are a halogen, or Ri is a halogen and R₂ is H, or Ri is H and R₂ is a halogen, wherein R₃ is a linear, branched or cyclic saturated or unsaturated Ci-Ce or C1-C4 chain, optionally substituted with a halogen.

15. The method according to statement 14, wherein R₃ is methyl.

16. The method according to statement 14 or 15, wherein Ri is Cl and R2 is I.

17. The method according to any one of statements 14 to 16, wherein Ri is Cl and R2 is I, and R₃ is methyl.

Detailed description of the invention

Figure 1. PHGDH activity in the presence of various haloquinolines whether or not combined with a PHGDH inhibitor, CBR-5884. PHGDH activity in the presence of 25 pM chloroxine (CH), 25 pM broxyquinoline (BX) or 25 pM clioquinol (CQ), alone and in combination with 10 pM CBR-5884, was assessed. Mean area under the curve (AUC), representing PHGDH activity during Ih, +/- SEM is shown for at least 3 independent repeats. DMSO background concentration was 0.5%. Normality of the data was assumed and a one-way ANOVA with Dunnett's multiple comparisons test was performed to assign significant differences compared to the control condition (0.5% DMSO). Statistical differences are indicated by: ****p<0.0001, ***p<0.001 and **p<0.01. Figure 2. Activity profile of 1 pM clioquinol after exposure of zebrafish larvae (7 dpf) to 300 pM EKP. Locomotor activity was expressed in mean actinteg units per 5-min +/- SEM during a 30 min recording interval, relative to EKP-only treatment. DMSO background concentration was 1%. A one-way ANOVA with Dunnett's multiple comparison test was performed to assign significant differences compared to EKP- only treated larvae. A statistical difference is indicated by: ****p<0.0001. For each condition n=10 larvae were used and the experiment was performed seven times (n total =70 per condition).

Figure 3. Electrophysiological antiseizure activity of clioquinol (CQ), whether or not combined with a PHGDH inhibitor (CBR-5884) in the zebrafish EKP-induced seizure model. Electrophysiological antiseizure activity (10 min non-invasive local field potential recording) was expressed in mean normalized power spectral density (PSD) within a 10-90 Hz frequency range per larvae +/- SEM, relative to the VHC control. The PSD per larvae represents the power of the signal per larvae within 10-90 Hz. DMSO background concentration was 1%. A one-way ANOVA with Dunnett's multiple comparison test was performed to assign significant differences compared to EKP- only treated larvae. Outliers were removed by means of the ROUT method (Q=l%). Statistical difference is indicated by:*p<0.05, **p<0.01 and ***p <0.001. ns = not significant. Number of recordings analysed were EKP (n = 14), 1 pM CQ (n=12), 0.75 pM CQ (n = 12), 0.5 pM CQ (n=ll), 0.25 pM CQ (n = 10), 0.5 pM CQ + 0.64 pM CBR- 5884 (n=ll), 0.64 pM CBR-5884 (n=9) and VHC (n = ll).

Figure 4. PHGDH activity of ion saturated clioquinol. PHGDH activity in the presence of 20 pM clioquinol and 10 pM ZnSO4 (Zn²⁺) or CuSO4 (Cu²⁺) was assessed. Mean area under the curve (AUC), representing PHGDH activity during Ih, +/- SEM is shown for 4 independent repeats. DMSO background was 0.5%. Normality of data was assumed and a one-way ANOVA with Sidak's multiple comparisons test was performed to assign significant differences between clioquinol in the presence of CuSO4 or ZnSO4 and the corresponding control condition (CuSO4 and ZnSO4 with 0.5% DMSO, respectively). Statistical differences are indicated by: *p<0.05.

Figure 5. (Left panel) PHGDH activity in the presence of methylclioquinol and (right panel) activity profile of 1-12 pM methylclioquinol (MCQ) after exposure of zebrafish larvae (7 dpf) to 300 pM EKP. (Left panel) Mean area under the curve (AUC), representing PHGDH activity during Ih, +/- SEM is shown for at least 3 independent repeats. DMSO background concentration was 0.5%. Normality of the data was assumed and a two-tailed unpaired student t test was performed to demonstrate significant differences between PHGDH activity in the presence of MCQ and the control condition (0.5% DMSO). A statistical difference is indicated by: *p<0.05. (Right panel) Locomotor activity was expressed in mean actinteg units per 5-min +/- SEM during a 30 min recording interval, relative to EKP-only treatment. DMSO background concentration was 1%. A one-way ANOVA with Dunnett's multiple comparison test was performed to assign significant differences compared to EKP- only treated larvae. A statistical difference is indicated by: ****p<0.0001. Conditions tested were EKP (n=5, n_totai=50), 12 pM MCQ (n=3, n_totai=30), 6 pM MCQ (n=3, n_totai=30), 1 pM MCQ (n=4, n_totai=40) and VHC (n = 5, n_totai=49).

Figure 6. Effect of clioquinol on glutamate levels in 7 dpf zebrafish heads. Glutamate levels in 7 dpf zebrafish heads upon treatment with or without 1 pM clioquinol in VHC (embryo medium, 1 % DMSO) in wild-type (panel A), EKP treated (300 pM, 1% DMSO) (panel B), and Dravet syndrome (scnlLab) zebrafish (panel C). Results are expressed as normalised glutamate amount per 10 homogenised heads +/- SD (sample size panel A: VHC n=21, clioquinol n=18; panel B: VHC n=22, clioquinol n = 19; panel C: VHC scnlLab^+/+;+/’ n=12, clioquinol scnlLab^+/+;+/’ n=9, scnlLab⁷’ VHC n= 12, scnlLab⁷’ clioquinol n=9). Unpaired t-tests were performed to assign significant differences in panels A and B, while an one-way ANOVA with Tukey's multiple comparisons test was performed to assign significant differences in panel C. Statistical differences are indicated by: *p<0.05, **p < 0.01, ***p < 0.001 and ***p < 0.0001.

Figure 7. Antiseizure activity analysis of clioquinol (CQ) in the mouse 6-Hz (44 mA) psychomotor seizure model. Drug-resistant psychomotor seizures were induced by electrical stimulation through the cornea, 60 min after i.p. injection of vehicle (VHC, n = 8), clioquinol (5 mg/kg, n = 8) and clioquinol (10 mg/kg, n=7). Mean seizure durations (±SD) are depicted. A one-way ANOVA with Dunnett's multiple comparison test was performed to assign significant differences compared to the VHC control treatment (0 mg/kg clioquinol). Outliers were removed by means of the ROUT method (Q=l%). Statistical difference is indicated by: **p < 0.01.

Figure 8. The hippocampal mRNA expression of PHGDH in SHAM animals and SSSE animals. Data is represented as dot plots with median represented by a line. Clioquinol upregulated the expression of PHGDH in SHAM and SSSE animals compared to vehicle treated animals.

Figure 9. The hippocampal mRNA expression of proinflammatory genes in Clioquinol- treated animals compared to vehicle treated animals. Clioquinol treated animals expressed more anti-inflammatory cytokines than vehicle-treated animals. Figure 10. PHGDH activity over time (60 minutes) in the presence of various haloquinolines whether or not combined with a PHGDH inhibitor, CBR-5884. PHGDH activity in the presence of 25 pM clioquinol, alone and in combination with 10 pM CBR-5884, 25 pM chloroxine and 25 pM broxyquinoline was assessed.

Figure 11. Representative LFP recordings of 7 dpf zebrafish, treated with 1% DMSO (VHC) or 0.25 - 1 pM clioquinol, in the absence or presence of 0.64 pM PHGDH inhibitor CBR-5884, followed by the addition of EKP. DMSO background was 1%.

Figure 12. Study design of the investigation of potential anti-inflammatory activity of clioquinol (Clio) in the mouse epilepsy model of SSSE.

Figure 13. Antiseizure activity analysis of fenfluramine (FA) in the mouse 6-Hz (44 mA) psychomotor seizure model. Drug-resistant psychomotor seizures were induced by electrical stimulation through the cornea, 60 min after i.p. injection of vehicle (VHC, n = 9), FA (20 mg/kg, n = 6) and FA (20 mg/kg, n=6). Mean seizure durations (±SD) are depicted. A one-way ANOVA with Dunnett's multiple comparison test (GraphPad Prism 8) was performed to assign significant differences compared to the VHC control treatment (0 mg/kg FA). Outliers were removed by means of the ROUT method (Q=l%). Statistical difference is indicated by: **p < 0.01 and ***p < 0.001.

The enzyme phosphoglycerate dehydrogenase (PHGDH) is a novel target for treating drug-resistant epilepsy. PHGDH catalyses the conversion of the glycolytic intermediate 3-phosphoglycerate into 3-phosphonooxypyruvate, which is the ratelimiting reaction in the de novo serine biosynthesis [Grant (2018) Front Mol Biosci 5, 110].

Herein disclosed are PHGDH agonists in treating (drug-resistant) epilepsy. Notably, the enzyme following PHGDH for de novo serine biosynthesis, phosphoserine aminotransferase 1 (PSAT 1), utilizes glutamate [Basurko et al. (1999) IUBMB Life 48,525-529], which is a pro-convulsant neurotransmitter [Barker-Haliski & Steve White (2015) Cold Spring Harb Perspect Med 5, a22863]. By increasing activity of PHGDH, which is the rate limiting enzyme for de novo serine biosynthesis, the activity of the downstream enzyme PSAT1 will be increased as well, thereby reducing excess levels of pro-convulsant glutamate and on top, resulting in neuroprotection. The latter is due to the central metabolic checkpoint role that is attributed to PHGDH for steering M2 polarization of macrophages [Wilson et al. (2020) Cell Rep 30, 1542- 1552 e7]. The present invention discloses PHGDH agonists for use in treating (drug-resistant) epilepsy as these compounds may have both anticonvulsant and anti-inflammatory properties, allowing them to combat epileptic seizures as well as the co-occurring neuro-inflammation. Examples hereof are (e.g. clioquinol, chloroxine and broxyquinoline) acting as PHGDH agonists, and methyl-clioquinol, which has lost zinc binding properties by modification of the 8 OH substituents of such haloquinolines. Herein anticonvulsant activity of such modified haloquinolines was assessed in preclinical zebrafish.

Using zebrafish models is advantageous as they allow high throughput drug screening and share high homologies to humans on a genetic, cellular and organ level. In this study, the zebrafish ethyl ketopentanoate (EKP)-induced seizure model was used to investigate the activity of clioquinol and methylclioquinol against drug-resistant seizures. This chemical model uses the lipid-permeable glutamic acid decarboxylase (GAD)-inhibitor, EKP, inducing drug-resistant seizures in zebrafish [Zhang et al. (2017) Sci Rep 7, 1-13]. GAD is an essential enzyme in the dynamic regulation of neural network excitability that catalyses the conversion of glutamate into y- aminobutyric acid (GABA) and various studies expose links between reduced GAD activity and several, often treatment-resistant forms of epilepsy [Errichiello et al. (2009) J Neuroimmunol 211, 120-123]. Thus, chemical GAD inhibition results in reduced GABA levels and increased levels of the most prevalent proconvulsant neurotransmitter, glutamate, as demonstrated in this study. Anticonvulsant activity of various ASDs has been investigated in this model based on locomotion tracking and local field potential recording (LFP) of the zebrafish. Only 1 pM of the recently introduced ASD perampanel (1 pM), which inhibits glutamate receptor signalling and hence interferes with the glutaminergic system, exhibited anticonvulsant activity in this zebrafish epilepsy model based on locomotion tracking and LFP recording. Thus, the zebrafish EKP-induced seizure model allows the identification of novel ASDs with a novel mode of action, primarily focused on restoring glutamate balance and downstream glutamate signalling.

The present invention demonstrates PHGDH-dependent antiseizure activity of clioquinol and methylclioquinol in the zebrafish EKP-induced seizure model and shows that it normalizes glutamate levels, which is one of the most prevalent proconvulsant neurotransmitters. Hence, clioquinol and methylclioquinol acts on the glutaminergic system, like perampanel, to block drug-resistant epileptic seizures.

It was further demonstrated that clioquinol's ability to activate PHGDH or its anticonvulsant properties do not arise from its capacity to chelate metal ions. In this context, it is demonstrated that methylclioquinol, lacking ion-chelating abilities, could still increase PHGDH activity and reduce EKP-induced seizures in zebrafish larvae. Moreover, it was found that ion saturated clioquinol still increased PHGDH activity and hence, that clioquinol's anticonvulsant activity and PHGDH activating properties are not linked to ion chelation.

To further validate the data obtained in the zebrafish EKP-induced seizure model, anticonvulsant activity of clioquinol was investigated in the mouse 6-Hz (44 mA) psychomotor seizure model, which is relevant for mimicking drug-resistant seizures [Leclercq et al. (2014) Epilepsy Res 108, 675-683]. Moreover, activity in this model upon intraperitoneal administration is a standard requirement for a new ASD. A significant reduction in seizure duration was observed upon treatment of mice with 10 mg/kg clioquinol as compared to untreated ones. The observed reduction in seizure duration by clioquinol treatment is comparable to that induced by fenfluramine (5 mg/kg and 20 mg/kg) treatment in the 6-Hz model (Figure 13). Fenfluramine is an anti-obesity drug that is currently being repurposed as a novel ASD against drug-resistant epilepsies such as Dravet Syndrome) Additionally, clioquinol's potential anti-inflammatory activity was assessed in a mouse epilepsy model of SSSE and it was found that PHGDH expression was increased in clioquinol- treated mice. Furthermore, the levels of "anti-inflammatory" mediators were altered, potentially participating in resolving inflammatory processes.

The present invention shows that clioquinol has anti-inflammatory as well as anticonvulsant properties against drug-resistant seizures in vivo by targeting the glutaminergic system. Hence, these compounds have an additional effect on tilting the balance toward anti-inflammation bears high potential as a next generation antiepileptic drug with dual mode of action.

"modified haloquinoline" or "alkylated haloquinoline" as used in the present invention relates to quinolines with depicted in formula I or pharmaceutically acceptable salts thereof

(I)

Herein R₃ is a linear, branched or cyclic saturated or unsaturated Ci-Ce or C1-C4 chain, optionally substituted with a halogen.

In preferred embodiments R₃ is methyl or ethyl.

Herein Ri and R2 are a halogen, or Ri is a halogen and R2 is H, or Ri is H and R2 is a halogen.

In dihalogenated compounds, Ri and R2 are each independently selected from the group consisting of fluorine (F), chlorine (Cl), bromine (Br), iodine (I).

More typically Ri and R2 are each independently selected from the group consisting of chlorine (Cl) iodine (I).

In specific embodiments at least one of Ri or R₂ is chlorine.

In other specific embodiments at Ri is chlorine.

In specific embodiments at least one of Ri or R₂ is bromine.

In a preferred embodiment Ri is Cl, R2 is I and R3 is methyl. The name of this compound is "5-chloro-7-iodo-8-methoxyquinoline" and is referred to in the present application as "methylclioquinol" and abbreviated in examples and figures as MCQ.

The use of alkylated compounds overcomes possible interference with zinc metabolism as could be the case with unmodified haloquinoline such as clioquinol.

In addition, methylclioquinol has a minimal toxic dose of (12.5 pM) in zebrafish whereas the minimal toxic dose of clioquinol is (1 pM).

Accordingly the present invention allows to uncouple the effects of zinc chelation of haloquinolines from the effects obtained with their analogues wherein zinc chelation is abolished by modification with the R3 groups. Apart from methyl, any of the envisaged R3 groups abolishes zinc chelation.

The examples of the present application show that the methylated clioquinol retains the anti-epileptic properties of its non-methylated counterpart with the free OH group. Accordingly it is more than plausible that methylation or alkylation of haloquinolines other than clioquinol, can be extrapolated based on the present examples on clioquinol and methylclioquinol.

One aspect of the present invention relates to alkylated, especially methylated haloquinolines for neuro protection in a subject, whereby the alkylated haloquinoline activates Phgdh activity. Conditions wherein "neuroprotection" prevents or treats a disorder are for example stroke, Parkinson's disease, Alzheimer's disease, Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis, AIDS-induced dementia, epilepsy, alcoholism, alcohol withdrawal, drug-induced seizure, viral/bacterial/fever-induced seizure, trauma to the head (traumatic brain injury), spinal cord injury, hypoglycaemia, hypoxia, myocardial infarction, cerebral vascular occlusion, cerebral vascular haemorrhage, haemorrhage, an environmental excitotoxin, dementia, trauma, drug-induced brain damage, stroke/ischemia, and aging.

"Seizure" refers to a brief episode of signs or symptoms due to abnormal excessive or synchronous neuronal activity in the brain. The outward effect can vary from uncontrolled jerking movement (tonic-clonic seizure) to as subtle as a momentary loss of awareness (absence seizure).

Seizure types are typically classified on observation (clinical and EEG) rather than the underlying pathophysiology or anatomy.

I Focal seizures (Older term: partial seizures)

IA Simple partial seizures - consciousness is not impaired

IA1 With motor signs

IA2 With sensory symptoms

IA3 With autonomic symptoms or signs

IA4 With psychic symptoms

IB Complex partial seizures - consciousness is impaired (Older terms: temporal lobe or psychomotor seizures)

IB1 Simple partial onset, followed by impairment of consciousness

IB2 With impairment of consciousness at onset

IC Partial seizures evolving to secondarily generalized seizures

IC1 Simple partial seizures evolving to generalized seizures

IC2 Complex partial seizures evolving to generalized seizures

IC3 Simple partial seizures evolving to complex partial seizures evolving to generalized seizures

II Generalized seizures

IIA Absence seizures (Older term: petit mal)

IIA1 Typical absence seizures

IIA2 Atypical absence seizures

IIB Myoclonic seizures

IIC Clonic seizures IID Tonic seizures,

HE Tonic-clonic seizures (Older term: grand mal)

IIF Atonic seizures

III Unclassified epileptic seizures

A more recent classification is published in Fisher et al. (2017) Epilepsia 58, 522- 530.

"Epilepsy" is a condition of the brain marked by a susceptibility to recurrent seizures. There are numerous causes of epilepsy including, but not limited to birth trauma, perinatal infection, anoxia, infectious diseases, ingestion of toxins, tumours of the brain, inherited disorders or degenerative disease, head injury or trauma, metabolic disorders, cerebrovascular accident and alcohol withdrawal.

A large number of subtypes of epilepsy have been characterized and categorized. The classification and categorization system, that is widely accepted in the art, is that adopted by the International League Against Epilepsy's ("ILAE") Commission on Classification and Terminology [See e.g., Berg et al. (2010), Epilepsia, 51(4), 676- 685]:

I. Electrochemical syndromes (arranged by age of onset):

I. A. Neonatal period: Benign familial neonatal epilepsy (BFNE), Early myoclonic encephalopathy (EME); Ohtahara syndrome

I.B. Infancy: Epilepsy of infancy with migrating focal seizures; West syndrome; Myoclonic epilepsy in infancy (MEI); Benign infantile epilepsy; Benign familial infantile epilepsy; Dravet syndrome; Myoclonic encephalopathy in non-progressive disorders I.C. Childhood: Febrile seizures plus (FS+) (can start in infancy); Panayiotopoulos syndrome; Epilepsy with myoclonic atonic (previously astatic) seizures; Benign epilepsy with centrotemporal spikes (BECTS); Autosomal-dominant nocturnal frontal lobe epilepsy (ADNFLE); Late onset childhood occipital epilepsy (Gastaut type); Epilepsy with myoclonic absences; Lennox-Gastaut syndrome; Epileptic encephalopathy with continuous spike-and-wave during sleep (CSWS), also known as Electrical Status Epilepticus during Slow Sleep (ESES); Landau-Kleffner syndrome (LKS); Childhood absence epilepsy (CAE)

I.D. Adolescence-Adult: Juvenile absence epilepsy (JAE);Juvenile myoclonic epilepsy (JME); Epilepsy with generalized tonic-clonic seizures alone; Progressive myoclonus epilepsies (PME); Autosomal dominant epilepsy with auditory features (ADEAF); Other familial temporal lobe epilepsies

I.E. Less specific age relationship: Familial focal epilepsy with variable foci (childhood to adult); Reflex epilepsies

II. Distinctive constellations

ILA. Mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE with

II. B. Rasmussen syndrome

II. C. Gelastic seizures with hypothalamic hamartoma

II. D. Hemiconvulsion-hemiplegia-epilepsy

E. Epilepsies that do not fit into any of these diagnostic categories, distinguished on the basis of presumed cause (presence or absence of a known structural or metabolic condition) or on the basis of Primary mode of seizure onset (generalized vs. focal)

III. Epilepsies attributed to and organized by structural-metabolic causes

III. A. Malformations of cortical development (hemimegalencephaly, heterotopias, etc.)

III. B. Neurocutaneous syndromes (tuberous sclerosis complex, Sturge-Weber, etc.)

III. C. Tumour

III. D. Infection

III. E. Trauma

IV. Angioma

IV. A. Perinatal insults

IV. B. Stroke

IV. C. Other causes

V. Epilepsies of unknown cause

Vi. Conditions with epileptic seizures not traditionally diagnosed as forms of epilepsy per se

VI. A. Benign neonatal seizures (BNS)

VLB. Febrile seizures (FS)

A more recent classification can be found in Scheffer et al. (2017) Epilepsia 58, 512- 521.

"Drug-resistant epilepsy (DRE)" is defined by Kwan et al. (2010) Epilepsia 52, 1069- 1077, as "failure of adequate trials of two tolerated and appropriately chosen and used antiepileptic drugs (AED schedules) (whether as monotherapies or in combination) to achieve sustained seizure freedom."

A non-exhaustive list of anti-epileptic compounds includes Paraldehyde; Stiripentol; Barbiturates (such as Phenobarbital, Methylphenobarbital, Barbexaclone; Benzodiazepines (such as Clobazam, Clonazepam, Clorazepate, Diazepam Midazolam and Lorazepam); Potassium bromide; Felbamate; Carboxamides (such as Carbamazepine Oxcarbazepine and Eslicarbazepine acetate); fatty-acids (such as valproic acid, sodium valproate, divalproex sodium, Vigabatrin, Progabide and Tiagabine); Topiramate; Hydantoins (such as Ethotoin, Phenytoin, Mephenytoin and Fosphenytoin); Oxazolidinediones (such as Paramethadione Trimethadione and Ethadione); Beclamide; Primidone; Pyrrolidines such as Brivaracetam Etiracetam Levetiracetam; Seletracetam; Succinimides (such as Ethosuximide, Phensuximide and Mesuximide); Sulfonamides (such as Acetazolamide, Sultiame Methazolamide and Zonisamide); Lamotrigine; Pheneturide; Phenacemide; Valpromide; Valnoctamide; Perampanel; Stiripentol; Pyridoxine.

In the present invention a zebrafish model is used as model for drug resistant epilepsy. The lipid-permeable glutamic acid decarboxylase (GAD)-inhibitor, Ethyl ketopentenoate (EKP), is used that induces drug-resistant seizures in zebrafish. GAD, converting glutamate into GABA, is a key enzyme in the dynamic regulation of neural network excitability. Clinical evidence has shown that lowered GAD activity is associated with several forms of epilepsy that are often treatment resistant. This EKP-induced epilepsy zebrafish model has been validated as a model for drugresistant epilepsy and was used to demonstrate anticonvulsant activity of various anti-epileptic drugs (AEDs).

"Dravet syndrome" is a severe form of childhood epilepsy characterized by drugresistant seizures and numerous physical, behavioural and intellectual comorbidities. Nearly 90% of all patients with Dravet syndrome carries a mutation in the SCN1A gene (sodium channel, voltage gated, type 1 alpha subunit).

The present invention uses a zebrafish model with a mutation in the orthologous SCN1A gene (scnlLab) which has been validated in the epilepsy field to for understanding the pathogenesis and anti-epileptic drug (AED) discovery. The present invention envisages the effect of alkylated, especially methylated haloquinolines, as exemplified by methylclioquinol using this zebrafish line indicative of a beneficial effect on patients suffering from Dravet syndrome.

EXAMPLES

Example 1 Material and methods

Compound preparations

Clioquinol, chloroxine, broxyquinoline and CBR-5884 were purchased from Sigma- Aldrich, St Louis, USA; compound stock solutions were prepared in dimethyl sulfoxide (DMSO; VWR International, Belgium), stock solutions were prepared in milliQ water. Fenfluramine was purchased from.

Methylclioquinol [5-Chloro-7-iodo-8-methoxyquinoline] was synthesized as follows: To a solution of K2CO3 (905 mg, 6.55 mmol, 2.00 equiv.) in dry DMF (10 mL) in a flame-dried round bottomed flask was added clioquinol (1.00 g, 3.27 mmol, 1.00 equiv.) under N₂ atmosphere. While stirring, methyl iodide (0.41 mL, 6.55 mmol, 2.00 equiv.) was added dropwise to this solution at room temperature. Upon completion of addition, the temperature was raised to 100 °C and stirring was continued for 18 h. The reaction mixture was then partitioned between EtOAc and H2O. The organic phase was washed two times with a 10% aqueous LiCI solution, followed by brine. The organic phase was dried over anhydrous MgSC and filtered. The crude filtrate was concentrated under reduced pressure and purified by solidphase flash column chromatography on silica gel (heptane/ EtOAc 7/3). The methylated clioquinol was obtained as an off-white solid in 66 % yield. Characterization of methylclioquinol and chromatography are described in supplementary material and methods.

^XH NMR (400 MHz, DMSO-cfe): 6 9.02 (dd, J = 4.2, 1.6 Hz, 1H), 8.58 (dd, J = 8.6, 1.6 Hz, 1H), 8.12 (s, 1H), 7.75 (dd, J = 8.6, 4.2 Hz, 1H), 4.05 (s, 3H). ¹³C NMR (100 MHz, DMSO-cfe): 6 156.17, 151.52, 142.12, 135.04, 133.60, 127.19, 126.08, 123.63, 91.64, 62.29. IR (neat): v 2972.26 (C-H stretch), 2928.86 (C-H stretch), 2833.78 (C-H stretch). LR-MS (ASAP): m/z calculated for [M + H]⁺ 319.92608, found 319.9.

Characterization of methylclioquinol

NMR.¹^ and ¹³C nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance III HD 400 spectrometer with a Bruker Ascend™ 400 magnet system (^XH basic frequency of 400.17 MHz) and a 5 mm PABBO BB/19F-1H/D probe with z- gradients. ¹³C-detected experiments were ^-decoupled using power-gated broadband decoupling. All samples were dissolved in DMSO-c/e. Data were recorded at room temperature using Bruker TopSpin 3.6.x and processed and analysed using Bruker TopSpin 4.1.3. All data were calibrated using the deuterated solvent as internal reference (a 1 :2: 3: 2: 1 quintet at 2.50 ppm for ^XH and a 1:3:6:7:6:3: 1 septet at 39.52 ppm for ¹³C). The 6-values are expressed in parts per million (ppm). The following acronyms were used: s (singlet), d (doublet), t (triplet), q (quartet), quint (quintet), sext (sextet), sept (septet), ABq (AB quartet), m (multiplet), br (broadened).

ATR-FT-IR. Attenuated total reflection (ATR) Fourier-transformed infrared (FT-IR) spectra were recorded on a Bruker Alpha-P FT-IR spectrometer with single reflection Platinum ATR accessory. Samples were analysed neat in solid or liquid state without any further manipulations. The data were recorded at room temperature using Bruker OPUS 7.5 and processed and analysed using ACD/Spectrus Processor 2019.1.2. The v-values are reported in units of reciprocal centimetres (cm ¹).

LR-MS. Low resolution mass spectra (LR-MS) were recorded using a Waters Radian ASAP direct mass detector with an atmospheric pressure solids analysis probe (ASAP). Samples were loaded onto the probe in 100% methanol. Data were acquired and processed using Waters MassLynx™ software.

Flash column chromatography

Flash column chromatography (medium pressure liquid chromatography, MPLC) was performed using a Buchi Sepacore® flash system, consisting of a Buchi C-660 Fraction Collector, a Buchi C-615 Pump Manager controlling two Buchi C-605 Pump Modules, a Knauer WellChrom K-2501 spectrophotometer (operating at 254 nm), and a Linseis D120S plotter. Buchi PP cartridges (40/150 mm) were filled with 90 g of Acros ultra-pure silica gel for column chromatography (article number 360050300, particle size 40-60 pm, average pore diameter 60 A) using a Buchi C-670 Cartridger. Unless stated otherwise, the eluent flow rate was set to 25 mL/min.

PHGDH enzymatic activity assay

To investigate PHGDH enzymatic activity upon drug treatment, human PHGDH (BPS bioscience, San Diego, USA) was used in combination with a specific colorimetric PHGDH activity kit (PromoCell GmbH, Heidelberg, Germany), containing PHGDH assay buffer and PHGDH reaction mix. Prior to the assay, compound stock solutions were diluted in PHGDH assay buffer to obtain a 5% DMSO solution. Briefly, human PHGDH enzyme was diluted in assay buffer at a concentration of 0.38 mg/mL. Next, 5 pL of this PHGDH enzyme solution, 5 pL of the compounds in PHGDH assay buffer (5% DMSO background), 35 pL PHGDH assay buffer and 50 pL PHGDH reaction mix were added to the wells of a flat bottom 96-well plate (resulting in final DMSO background of 0.5%). Absorbance at 450 nm (OD 450 nm) over time was measured as a readout for the amount of NADH generated through PHGDH activity. GraphPad Prism (version 6) software was used to calculate the area under the curve (OD 450 nm plotted against time (min)), representing PHGDH activity during Ih, and to perform statistical analysis.

Zebrafish EKP-induced seizure model

Adult zebrafish Danio rerio) of AB strain (Zebrafish International Resource Center, Oregon, WA, USA) and scnlLab strain (Dravet syndrome) (Max Planck Institute of Neurobiology, Germany) were kept at 28°C, pH 6.5-7.8 and in a 14/10h light/dark regimen (standard aquaculture conditions). Upon natural spawning, fertilized eggs were selected and raised in embryo medium at 28°C in continuous light until 7 days after fertilization (7 dpf). The approval numbers for zebrafish experiments are 027/2017 (Ethics Committee of the University of Leuven) and LA1210261 (Belgian Federal Department of Public Health, Food Safety, and Environment). Compound stocks were diluted in embryo medium (0.3X Danieau's solution: 1.5 mM HEPES, pH 7.6, 17.4 mM NaCI, 0.21 mM KCI, 0.12 mM MgSO₄, and 0.18 mM Ca(NO₃)₂) in a 96- well plate, containing 1 larva of 7 dpf per well (resulting in 1% final DMSO background).

Toxicity evaluation. The maximum tolerated concentration (MTC) of the compounds for the zebrafish larvae (in 1% DMSO background) was determined as described previously [Copmans et al. (2018a) Neurochem Int 112; 124-133]. To determine MTC of clioquinol in combination with the PHGDH-inhibitor CBR-5884, larvae were treated with a sub-MTC concentration of clioquinol and a concentration range of CBR- 5884; MTC of the combination was determined according to the criteria in. Locomotor activity. To assess potential antiseizure activity of the compounds, larvae were placed in the wells of a 96-well plate (1 larva/well) and treated with the appropriate compound in 100 pL embryo medium (1% DMSO). After 2h of treatment at 28°C in the dark, 100 pL VHC (embryo medium, 1% DMSO) or ethyl ketopenta noate (600 pM EKP in embryo medium, 1% DMSO) was added, resulting in a final EKP concentration of 300 pM. Locomotor activity was subsequently measured in an enclosed tracking device (ZebraBox Viewpoint, France) and expressed in "actinteg" values, previously described as "the sum of all image pixel changes detected during the time window". ZebraLab software (Software Viewpoint, France) was used to plot the actinteg units/5 minutes and mean actinteg units/5 minutes (relative to EKP-only treatment) during the 30 min recording interval were calculated. Electrophysiology. Noninvasive local field potential (LFP) recording of the optic tectum (midbrain) of 7 dpf zebrafish larvae was performed to measure epileptiform brain activities upon treatment with the compounds of interest (e.g. clioquinol, CBR- 5884 or a combination). Larvae were treated with the compounds as described above. Next, 100 pL VHC or EKP (300 pM final concentration) was added, followed by 12 minutes of incubation in the dark, immobilization in 2% low-melting-point agarose (Invitrogen) at room temperature and positioning of a single glass electrode containing artificial cerebrospinal fluid (ACSF; 124 mM NaCI, 2 mM KCI, 2 mM MgSO4, 2 mM CaCI2, 1.25 mM KH2PO4, 26 mM NaHCO3, and 10 mM glucose) above the optic tectum on the skin. Local field potential recordings of 10 minutes were performed as described previously [Zhang et al. cite above; Copmans et al. 2018a. cited above37. Li et al. cited above . Clampfit 10.2 software (Molecular Devices Corporation, USA) and Matlab R.2018 (MATrix LABoratory USA) software were used for visual inspection and execution of a power spectral density (PSD) analysis of the LFP recordings, respectively. The PSD analysis was followed by normalization of the PSD estimates against the VHC control and the mean PSD per larva over a 10-90 Hz frequency range was calculated for each condition. The ROUT test (Q= l%) was performed to identify outliers and the mean PSD per larva (+/- SEM) was subsequently plotted for all conditions. GraphPad Prism (version 6) software was used to plot the data and perform the statistical analysis.

Determination of glutamate levels in zebrafish heads

To determine the amount of glutamate in zebrafish heads, 7 dpf larvae (12/condition) were treated with or without 1 pM clioquinol in 1 mL of VHC (embryo medium, 1 % DMSO) for 2h at 28°C in the dark, followed by the addition of 1 mL VHC or 1 mL EKP solution (600 pM in embryo medium, 1% DMSO). After 6 minutes, larvae were washed in embryo medium, and heads were isolated using a scalpel under a dissecting microscope. Further sample preparation was performed by homogenising 10 heads/condition in 120 pl of antioxidant medium (0.27 mM Na₂EDTA.2H₂O, 0,1 M acetic acid, 3.3 mM L-cystine, 12.5 pM ascorbic acid) by twisting and moving a microtube homogeniser up and down 50 times at 4°C. After centrifugation (12,000 g, 15 min, 4°C), 100 pl of the supernatant was kept at -80°C until analysis. Glutamate concentration in the samples was analysed by LC-MS/MS.

Mouse 6-Hz psychomotor seizure model

Male Naval Medical Research Institute (NMRI) mice, weighting 18-20 g and provided by Charles River Laboratories, were housed (5 mice/cage) and maintained as described [Copmans et al. (2018b) ACS Chem Neurosci 9, 1652-1662] until the experiment was conducted.

Antiseizure activity of clioquinol and fenfluramine was assessed in the mouse 6-Hz psychomotor seizure model as described [Copmans et al. 2018a cited above; Li et al. cited above . Briefly, NMRI mice were randomly divided into different treatment groups and 500 pL (adjusted to the individual weight) of VHC (0.5% sodiumcarboxymethylcellulose (NaCMC)/Tween80 in 0.9% NaCI for clioquinol experiments and DMSO/PEG200 (50:50) for fenfluramine experiments) or treatment (clioquinol or fenfluramine dissolved in VHC) was injected intraperitoneally (i.p.). lh after injection, cornea were moisturized by an ocular anaesthetic (lidocaine, 0.5%) and psychomotor seizures were induced by corneal electrical stimulation (6 Hz, 0.2 ms rectangular pulse width, 3s duration, 44 mA) using an ECT Unit 5780 (Ugo Basile, Comerio, Italy). Typical characteristics of psychomotor seizures were observed by experienced researchers. Initially observed seizure durations were confirmed or corrected upon blinded video analysis. GraphPad Prism (version 6) software was used to plot the mean seizure durations per condition (+/- SD) and to apply the appropriate statistical tests.

SSSE mouse epilepsy model

C57bl/6/j mice were surgically implanted with three extradural screw electrodes (two served as ground/ reference and one over contralateral parietal cortex as active electrode) and one bipolar stimulating electrode (E363/3-2TW/SPC, PlasticsOne, USA) into the right ventral hippocampus at the following coordinates from bregma (anteroposterior: -3.00; mediolateral: -3.00; and dorsoventral: 2.80). Surgery was conducted under isoflurane anaesthesia (5% for induction; and 1-2 % for maintenance) with provision of presurgical analgesia to alleviate pain (Carprofen 5 mg/kg, s.c.; Rimadyl, Pfizer, Australia) and (Buprenorphine 0.5mg/kg, s.c; Tasmanian Alkaloids, Pty Ltd). The electrode assembly was held in place with dental acrylic (Vertex Dental, The Netherlands). The bipolar electrode was connected to an Accupulser Pulse Stimulator (A310, World Precision Instruments, USA). An after-discharge threshold (ADT), defined as the minimum electrical current needed to induce an electrographic seizure exceeding ten seconds, was established by applying electrical stimulations of increasing electrical current (50 Hz, 1-second duration, 1-ms alternating current pulses) to the ventral hippocampus via the bipolar electrode. Subsequently, mice received electrical stimulation through the bipolar electrode for (90 minutes duration, 100-ms trains of 1-ms alternating current pulses (50 Hz) at a suprathreshold current intensity (typically 10 pA above ADT). The current was interrupted every 9 minutes for a minute to confirm development of SSSE on EEG traces. At the end of 90-minute stimulation, mice were monitored for another 150 minutes following which SSSE was terminated with diazepam. From next morning after the termination of SSSE, mice received either clioquinol (5mg/kg twice daily i.p.) or vehicle injections for one week. Clioquinol was suspended in 5% dimethyl sulfoxide and 20% Kolliphor RH40 in 0.01MPBS.

At the completion of treatment, brains were removed and placed in ice-cold 0.1M phosphate-buffered saline (pH 7.4). Hippocampi were dissected and immediately frozen on dry ice and stored at -80°C. mRNA was extracted using a Nucleospin RIMA Plus kit (Machery-Nagel) according to the manufacturer's instructions. cDNA synthesis was performed using the Omniscript RT Kit (QIAGEN). The real-time quantitative PCR was completed using high throughput gene expression platform based on microfluidic dynamic arrays (Dynamic array IFC). Taqman gene expression assays are provided as 20x forward and reverse primer and probe mixes. Each primer was at a concentration of 18 pM and probe at concentration of 4 pM. The final concentration of each assay was 0.2X (180nM). Gene expression levels for each sample were normalized to the geometric means of housekeeping genes including GAPDH, ACTB, HPRT 1 and PPIA. All mRNA expression levels were reported as levels relative to housekeeping gene and were normalised to the values in control animals.

Example 2. Various haloquinolines increase activity of the human enzyme phosphoglycerate dehydrogenase

The effect of all previously identified survival-promoting drugs [Spincemaille et al. (2014) Microb Cell 1, 352-364] on the activity of recombinant human PHGDH was investigated and it was found that the haloquinolines clioquinol, chloroxine and broxyquinoline resulted in the most pronounced increase of PHGDH activity (i.e. 70- 80% increase in PHGDH activity as compared to control treatment; Figure 1, Figure 10). The PHGDH activating ability was most pronounced for clioquinol (AUC for clioquinol of 71 compared to 64 and 68 for chloroxine and broxyquinoline, respectively). Co-administration of PHGDH inhibitor CBR-5884 [Mullarky etal. (2016) Proc Natl Acad Sci USA 113, 1778-1783] with clioquinol completely blocked the clioquinol-induced PHGDH activation (Figure 1, Figure 10), pointing to PHGDH specificity of clioquinol's readout in the assay.

Example 3. Clioquinol shows antiseizure activity in the zebrafish EKP- induced seizure model

Prior to investigating clioquinol's potential antiseizure activity, its maximum tolerated concentration (MTC) was determined (/.e. 1 pM or 0.3 pg/mL). Consistent with previous studies [Zhang et al. (2017) Sci Rep 7, 1-13; Li et al. (2020) ACS Chem Neurosci 11, 730-742; Sourbron et al. (2019) Epilepsia 60, e8-el3], a significant increase in locomotor activity was observed upon single compound treatment of 7 dpf larvae with EKP (300 pM, 1% DMSO) as compared to VHC (1% DMSO) control treatment (Figure 2). Furthermore, the present results show that 1 pM clioquinol significantly reduced EKP-induced seizures (Figure 2). Locomotor activity upon treatment with 1 pM clioquinol, without EKP addition, did not differ significantly from VHC-only treated larvae.

Example 4. Clioquinol alleviates hyperexcitable state of the brain in the zebrafish EKP-induced seizure model through PHGDH-activation

Besides locomotion tracking, electrophysiological recordings [Hunyadi et al. (2017) J Neurosci Methods 287, 13-24], such as non-invasive local field potential (LFP) recordings, can be used to observe seizures as their occurrence among epilepsy patients results from abnormal activity of neuronal populations. Such LFP recordings have already been used in numerous studies for validation of novel zebrafish epilepsy models and to evaluate new drug candidates' efficacy [Zhang et al. (2017) Sci Rep 7, 1-13; Copmans et al. (2018) Neurochem Int 112, 124-133; Copmans et al. (2018) ACS Chem Neurosci 9, 1652-1662;Sourbron et al. (2019) Epilepsia 60, e8- el3; Afrikanova etal. (2013) PLoS One 8, e54166]. To investigate whether clioquinol can decrease EKP-induced epileptiform discharges, indicative for a hyperexcitable state of the brain upon EKP treatment, 7 dpf zebrafish larvae were treated with a concentration series of clioquinol (0.25 pM - 1 pM), followed by the addition of EKP (300 pM final concentration, 1% DMSO) or VHC (1% DMSO) and non-invasive LFP measurement from the optic tectum. Consistent with previous studies [Zhang et al. cited above; Li et al cited above], a significant increase in epileptiform activity was observed upon single compound treatment of 7 dpf larvae with EKP as compared to VHC treatment (Figure 3, Figure 11). Treatment of the larvae with 0.5 pM up to 1 pM clioquinol significantly reduced EKP-induced epileptiform activity, expressed as the mean normalized power spectral density (PSD) per larvae within a 10-90 Hz frequency interval (Figure 3, Figure 11). This activity is comparable with the antiseizure activity of 1 pM of the ASD Perampanel in this model [Zhang et al. cited above;]. Furthermore, 0.5 pM clioquinol was combined with PHGDH inhibitor CBR- 5884 to demonstrate PHGDH-dependent antiseizure activity of clioquinol. First, the MTC of the CBR-5884 - clioquinol combination was determined, which was set to 0.64 pM CBR-5884 in combination with 0.5 pM clioquinol. In contrast to 0.5 pM clioquinol alone, co-administration of clioquinol and CBR-5884 did not result in a significant decrease of EKP-induced epileptiform activity compared to EKP-only treated larvae (Figure 3, Figure 11), pointing to PHGDH-dependent anticonvulsant action of clioquinol. The level of epileptiform activity upon treatment with 0.64 pM CBR-5884, followed by the addition of EKP, did not differ significantly from that of larvae treated with EKP alone (Figure 3). Furthermore, none of the compounds or combinations (at concentrations used in Figure 3) resulted in a significant increase or decrease in epileptiform activity in the absence of EKP as compared to the VHC control treatment.

Example 5 Anticonvulsant activity of clioquinol does not depend on ion chelation

Clioquinol is known for its metal ion chelating capacities with a high affinity towards copper and zinc cations (Cu²⁺ and Zn²⁺, respectively) , which is thought to be responsible for its anti-Alzheimer activity [Di Vaira et al. (2004) Inorg Chem 43 3795-3797; Rodnguez-Santiago et al. (2015) Phys Chem Chem Phys 17, 13582- 13589; Cherny et al. (2001) Neuron 30, 665-676; Regland et al. (2001) Dement Geriatr Cogn Disord 12, 408-414].

To uncouple clioquinol's antiseizure activity from ion chelation, PHGDH agonist activity of ion saturated clioquinol was investigated. To this end, clioquinol was incubated with excess Cu²⁺ or Zn²⁺ in a 2: 1 ratio. As shown in Figure 4, 20 pM clioquinol still increases PHGDH activity in the presence of 10 pM CuSC or 10 pM ZnSC (an excess metal ions), indicating that clioquinol activates PHGDH independent of its metal ion chelating capacity. Furthermore, methylclioquinol was synthesized, a methylated form of clioquinol lacking metal ion chelating abilities, to further confirm that the anticonvulsant activity of clioquinol does not depend on ion chelation. First, the capacity of methylclioquinol to activate human PHGDH was evaluated by performing a PHGDH enzymatic activity assay. As shown in Figure 5, 500 pM methylclioquinol significantly increased PHGDH activity as compared to the DMSO control (Figure 5, left panel), which confirms its PHGDH activation to be independent of ion chelation, although higher doses as compared to non-methylated clioquinol are needed. Hence, enzyme affinity seems lower for methyl-clioquinol. Subsequently, potential antiseizure activity of methylclioquinol at its MTC (12 pM) was assessed using the zebrafish EKP-induced seizure model with locomotion tracking. Methylclioquinol concentrations as high as 6 pM and 12 pM significantly decreased EKP-induced locomotor activity as compared to larvae treated with EKP alone (Figure 5, right panel). In conclusion, these data show that clioquinol's antiseizure activity is independent of metal ion chelation.

Example 6 Clioquinol reduces glutamate in wild-type, EKP-treated and Dravet syndrome zebrafish brains.

PHGDG facilitates the conversion of 3-phosphoglycerate to phosphohydroxypyruvate. Phosphohydroxy-pyruvate is then converted to phospho-serine by PSAT1 which consumes glutamate as a co-factor. Since clioquinol activates PHGDH, a greater amount of phosphohydroxy-pyruvate will be generated, which in turn will need more of glutamate in order to be converted to phospho-serine. It is postulated that glutamate levels in the brain will be reduced after clioquinol treatment. To investigate this, glutamate levels in zebrafish heads were assessed in wild-type (panel A), EKP-treated-(panel B) and spontaneous seizing Dravet syndrome (scnlLab^7-, panel C) zebrafish brains. Seven dpf larvae were treated with or without 1 pM clioquinol in VHC (embryo medium, 1% DMSO) (panel A and C), and with an addition of EKP (300 pM, 1% DMSO) (panel B). Heads were subsequently isolated, followed by tissue homogenization and glutamate concentration was analysed by LC- MS/MS. Results show that the amount of glutamate per 10 homogenised heads were significantly reduced in clioquinol as compared to VHC controls in both wild-type, EKP-treated and Dravet syndrome zebrafish (Figure 6, panels A, B and C). These findings shows that clioquinol can reduce one of the most important proconvulsant neurotransmitters, glutamate, in the brain, which contributes to its anticonvulsant activity. Example 7. Clioquinol reduces seizure duration in a mouse 6-Hz psychomotor seizure model

The antiseizure activity of clioquinol was further evaluated in the mouse 6-Hz (44 mA) psychomotor seizure model as described [Copmans et al. cited above; Li et al. cited above . Focal seizures initiated in the animals using 6 Hz corneal stimulation, as intense as two times the convulsive current in 50% of the mice (CC50), are very hard to prevent by currently available antiseizure drugs and are generally considered pharmaco-resistant. The present results show significantly reduced seizure duration in 10 mg/kg (but not 5 mg/kg) clioquinol treated mice as compared to untreated ones (Figure 7).

Example 8. Clioquinol significantly increases anti-inflammatory markers/ markers for neuro-protection in the SSSE mouse epilepsy model

Clioquinol acts as zinc and copper chelator. Metal chelation is a potential therapeutic strategy for Alzheimer's disease (AD) because zinc and copper are involved in the deposition and stabilization of amyloid plaques, and chelating agents can dissolve amyloid deposits in vitro and in vivo. In general, the ability of clioquinol to chelate and redistribute metals plays an important role in diseases characterised by Zn, Cu, Fe dyshomeostasis, such as AD and Parkinson's disease, as it reduces oxidation and the amyloid burden. Zinc chelators may also act as anticancer agents.

In addition the anti-inflammatory activity of clioquinol was investigated in a mouse epilepsy model of SSSE by assessing the expression of various anti- and pro- inflammatory markers after 1 week of clioquinol treatment (administered at 10 mg/kg/day) upon induction of status epilepticus (SE) via electrical stimulation (SSSE). Eight week old male mice (n=35) were allocated into four treatment groups which includes SHAM+ vehicle (n=8), SHAM+Clioquinol (n=9), SSSE+Vehicle (n=9), and SSSE + Clioquinol (n=10). All the mice received Clioquinol 5mg/kg or vehicle twice daily for 7-days. At the end of the experiments, unilateral hippocampi were collected for gene expression analysis. A scheme of the study design is shown in Figure 12.

Clioquinol-treated animals displayed a significant increase in the expression of PHGDH (Figure 8) as well as several anti-inflammatory cytokines and neuroprotective genes such as TGFp, IL lOra, Dual-specificity phosphatase 1/ (MAPK) phosphatase-1 (Duspl), PPAR gamma, Gdnf (p=0.07) and Yml (p=0.06) when compared to the vehicle-treated animals (Figure 9). The levels of TGFp, ILlO-ra were also upregulated by SSSE induction, while the level of DUSP1 that is involved in resolution of inflammation was significantly reduced by induction of SSSE (Figure 9). These data show that clioquinol may potentially alter the levels of "anti-inflammatory" mediators that could participate in the resolution of the inflammatory processes and promote repair mechanisms apart from modulating brain inflammation from "Ml" to "M2" phenotype.

Claims

(I) wherein Ri and R₂ are a halogen, or Ri is a halogen and R₂ is H, or Ri is H and

R2 is a halogen, and wherein R₃ is a linear, branched or cyclic saturated or unsaturated Ci-Ce or C1-C4 chain, optionally substituted with a halogen, for use in the prevention or treatment of a seizure in epilepsy or in neuroinflammation associated with epilepsy.

2. The alkylated haloquinoline or salt thereof for use in accordance with claim 1, wherein R₃ is methyl.

3. The alkylated haloquinoline or salt thereof for use in accordance with claim 1 or 2, wherein Ri is Cl and R2 is I.

4. The alkylated haloquinoline or salt thereof for use in accordance with any one of claims 1 to 3, wherein Ri is Cl and R2 is I, and R₃ is methyl.

5. The alkylated haloquinoline or salt thereof for use in accordance with any one of claims 1 to 4, wherein the epilepsy is a genetic disorder.

6. The alkylated haloquinoline or salt thereof for use in accordance with any one of claims 1 to 5, wherein the epilepsy is Dravet syndrome.

7. The alkylated haloquinoline or salt thereof for use in accordance with any one of claims 1 to 4, wherein the epilepsy is caused by PHGDH deficiency.

8. The alkylated haloquinoline or salt thereof for use in accordance with any one of claims 1 to 7, wherein the epilepsy is a drug resistant epilepsy.

9. The alkylated haloquinoline or salt thereof for use in accordance with claim 8, wherein the drug resistant epilepsy is resistant against two or more selected from the group consisting of valproate, carbamazepine, levetiracetam, lamotrigine, topiramate, briveracetam, lacosamide, perampanel and phenobarbital.

10. The alkylated haloquinoline or salt thereof for use in accordance to any one of claims 1 to 9, wherein the alkylated haloquinoline is administered orally.

11. The alkylated haloquinoline or salt thereof for use according to any one of claims 1 to 10, wherein the alkylated haloquinoline treatment is a monotherapy.

12. The alkylated haloquinoline or salt thereof for use according to any one of claims 1 to 10, in a combination treatment with a further anti-epilepsy drug.

13. The alkylated haloquinoline or salt thereof for use according to any one of claims 12, wherein the further anti-epilepsy drug is fenfluramine.

R2 is a halogen, wherein R₃ is a linear, branched or cyclic saturated or unsaturated Ci-Ce or C1-C4 chain, optionally substituted with a halogen. The method according to claim 14, wherein R₃ is methyl. The method according to 14 or 15, wherein Ri is Cl and R2 is I, and R₃ is methyl.