Anti-seizure combination therapy

Abstract

The invention relates a composition comprising clioquinol and idebenone as active ingredients for use in treating or preventing of a seizure in epilepsy.

Description

[0001] ANTI-SEIZURE COMBINATION THERAPY

[0002] Field of the invention

[0003] The present invention relates to treatment of epilepsy, in particular drug resistant epilepsy.

[0004] The present invention relates to combination treatments of clioquinol and idebenone.

[0005] Background of the invention

[0006] Drug-resistant epilepsy (DRE) affects over 25 million people worldwide and is often associated with neuroinflammation. Antiseizure medications (ASMs) are commonly used to control seizures, while anti-inflammatory treatment is used in certain epilepsy syndromes, such as infantile spasms [Wanigasinghe (2014) Pediatr Neurol. 51, 24-30]. However, approximately one third of patients are classified as having drug-resistant epilepsy (DRE), as they do not adequately respond to current ASMs despite the availability of more than 25 drugs on the market [Chen et al. (2018) JAMA Neurol. 75, 279],

[0007] Clioquinol and idebenone both activate the enzyme phosphoglycerate dehydrogenase (PHGDH) [WO2021185791; WO2022258629], and have antiseizure activity. PHGDH is rate-limiting for de novo biosynthesis of the neurotrophic factor serine [Grant (2018) Front Mol Biosci. 5, 110] and steers antiinflammatory responses [Wilson et al. (2020) Cell Rep. 30, 1542-1552]. PHGDH malfunctioning has been linked to drug resistant epilepsy.

[0008] Summary of the invention

[0009] The present invention identified novel combinations of clioquinol (CQ) and idebenone (IDE) as more effective in treating drug-resistant seizures as compared to compounds alone. Specifically, a ratio of CQ:IDE of 1 :4 - 1 :8 for administering the combination results in higher antiseizure activity as compared to compounds only. The CQ:IDE combination in a 1 :4 ratio (i.e. 0.5 pM CQ + 2 pM IDE) is significantly more effective than a 2-fold higher CQ dose (1 pM) against drugresistant seizures.

[0010] Clioquinol (CQ) and idebenone (IDE) may be formulated together in a single pill or liquid, or may be formulated separately.

[0011] The invention is summarised in the following statements:

[0012] 1. A composition comprising clioquinol and idebenone as active ingredients for use in treating or preventing of a seizure in epilepsy. 2. The composition for use according to statement 1, wherein the molar ratio between clioquinol to idebenone is between 1 to 2 and 1 to 16.

[0013] 3. The composition for use according to statement 1 or 2, wherein the molar ratio between clioquinol to idebenone is between 1 to 4 and 1 to 8.

[0014] Other envisaged molar ratio between clioquinol to idebenone are between 1 to 2 and 1 to 8, between 1 to 2 and 1 to 4, between 1 to 4 and 1 to 16 between 1 to 8 and 16.

[0015] Alternatively molar ratios between clioquinol to idebenone are 1 to 2, 1 to 4, 1 to 6, 1 to 8, 1 to 10, 1 to 12 or 1 to 16. Herein, the values of 2, 4, 6, 8, 10, 12 and 16 may vary with 10 %. E.g. 1 to 2 means from 1 to 1.18 to 1 to 2.2.

[0016] 4. The composition for use according to any one of statements 1 to 3, wherein the epilepsy is a treatment resistant epilepsy.

[0017] 5. The composition for use according to any one statements 1 to 4, 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.

[0018] Detailed description of the invention

[0019] Brief descriptions of the figures.

[0020] Figure 1. Combination of CQ and IDE reduces locomotor activity more efficiently than treatment of CQ or IDE alone.

[0021] A: Activity profile of 2 pM IDE, 0.25 pM CQ and combinations of 2 pM IDE and 0.25 pM CQ (ratio 8: 1) after 300 pM EKP exposure.

[0022] B: Activity profile of 2 pM IDE, 0.5 pM CQ and combinations of 2 pM IDE and 0.5 pM CQ (ratio 4: 1) after 300 pM EKP exposure. Activity was quantified by assessing locomotion i.e. cumulative duration of the highly active state for 30 minutes normalized to the control EKP treated larvae. Statistical analysis: one-way ANOVA with Dunnett's multiple comparison test. Statistical differences are indicated by: ****p<0.0001, ***p<0.001, **p<0.01 and *p<0.05** by one-way ANOVA with Dunnett's multiple comparisons test. For each condition at least 10 larvae were used, and the experiment was performed three times (n total >34 per condition). Abbreviation used in the figures are: CQ: clioquinol; IDE: idebenone; VHC: vehicle; EKP: ethyl ketopentenoate. The IUPAC name of clioquinol is '5-chloro-7-iodoquinolin-8-ol" and has MW 305.5g / moL

[0023] The IUPAC name of idebenone is '2-(10-hydroxydecyl)-5,6-dimethoxy-3- methylcyclohexa-2,5-diene-l, 4-dione' and has MW 338.4g / mol .

[0024] "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."

[0025] 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.

[0026] The compounds as claimed and their use includes pharmaceutically accepted derivatives thereof. These may be used as a free acid or base, and / or in the form of a pharmaceutically acceptable acid-addition and / or base-addition salt (e.g. obtained with non-toxic organic or inorganic acid or base), in the form of a hydrate, solvate and / or complex, and / or in the form of a pro-drug or pre-drug, such as an ester. As used herein and unless otherwise stated, the term "solvate" includes any combination which may be formed by a pharmaceutical composition of this invention with a suitable inorganic solvent (e.g. hydrates) or organic solvent, such as but not limited to alcohols, ketones, esters, and the like. Such salts, hydrates, solvates, etc. and the preparation thereof will be clear to the skilled person.

[0027] "Treatment" relates to any medical benefit and in the context of epilepsy to less severe seizures, shorter seizure periods or a reduced frequency of seizures. The effectiveness of a compound for use in the treatment and prevention of a drug resistant epilepsy is assessed in a Zebrafish ethyl ketopentenoate (EKP)-induced seizure model.

[0028] Zebrafish have emerged as a promising new animal model for epileptic seizure disorders, with particular relevance for genetic and developmental epilepsies [Burrows et al. (2020) Eur J Paediatr Neurol. 24, 70-80]. Although it underwent a whole genome duplication, the zebrafish genome is highly homologous to the human genome, with over 80% conservation of disease-causing genes, whilst also being genetically tractable. A recent review of this literature indicates that zebrafish models of epilepsy featuring spontaneous seizures can be more reliable in terms of clinical relevance and pharmacological predictability than their mammalian counterparts [Griffin et al. (2018) Front Pharmacol. 9, 573]. Consequently, over recent years several chemical and genetic zebrafish models of acute seizures or epilepsy have been generated either by immersion of larvae in chemical proconvulsants like pentylenetetrazol (PTZ) [Afrikanova et al. (2013) PLoS One. 8, e54166] or allylglycine (AG) [Leclercq et al. (2015) Epilepsy Behav. 45, 53-63], or by knocking-down or introducing mutations in epilepsy susceptible genes including scnllab [Dinday et al. (2015) eNeuro. 2: ENEURO.0068-15.2015; Zhang et al. (2015) PLoS one 10, e0125898]

[0029] More specifically, in the present invention a zebrafish EKP-induced seizure model is used. In this model, the lipid-permeable glutamic acid decarboxylase (GAD)- inhibitor, Ethyl ketopentenoate (EKP), is used that induces drug-resistant seizures in zebrafish [Zhang etal. (2017) Sci Rep. 7(1), 7195]. GAD, converting glutamate into y-aminobutyric acid (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 [Lloyd et al. (1986) Adv Neurol. 44, 1033-1044]. In this respect, reduced GAD activity has been found in epileptic foci from patients with intractable epilepsy indicating that failure to synthetize GABA and loss of inhibitory synaptic activity may lead to epilepsy [Lloyd et al. (1986) Adv Neurol 44, 1033-44]. Furthermore, in so-called autoimmune epilepsies GAD antibodies have been detected especially in patients with focal epilepsies like drug-resistant temporal lobe epilepsy (TLE) [Errichiello et al. (2009) J. Neuroimmunol 211, 120-123; Errichiello et al. (2011 Neurol Sci 32, 547-550]. Hence, chemical inhibition of GAD is relevant to induce drug resistant seizures, resulting in reduced levels of GABA and increased levels of glutamate, which is the most important proconvulsant neurotransmitter. Allylglycine (AG) is a known GAD inhibitor and was previously used to develop a zebrafish seizure model [Leclercq, cited above], showing that AG reduced GABA content and as a consequence induced epileptiform activity in zebrafish larvae and mice. However, AG-induced seizures in zebrafish were often asynchronous with long latency onset. As the oxidative metabolite of AG, i.e. 2-keto-4-pentenoic acid (KPA), was proven to be a far more potent inhibitor of GAD as compared to AG, KU Leuven explored the possibility to use ethyl ketopentenoate (EKP), a lipid- permeable form of KPA to induce refractory seizures in zebrafish larvae. This zebrafish EKP-induced seizure model was validated as a reliable model for drugresistant epilepsy [Zhang et al. cited above].

[0030] The EKP-induced zebrafish epilepsy model is a validated epilepsy model and allows to identify novel AEDs with a novel mode of action, primarily focused on restoring glutamate balance and downstream glutamate signaling, thus targeting the glutaminergic system.

[0031] Examples

[0032] Example 1. Combinations of clioquinol and idebenone reduce locomotor activity in a zebrafish EKP-model for drug resistant epilepsy to a greater extent than clioquinol (even at a 2 fold higher concentration) or idebenone alone

[0033] The EKP-induced epilepsy zebrafish model is a suitable model to investigate drug resistant epilepsy. Ethyl ketopentenoate (EKP) is a lipid-permeable GAD-inhibitor that results in increased glutamate levels and drug resistant seizures in zebrafish [Zhang et al. (2017) Sci Rep. 7, 7195]. Glutamic acid decarboxylase (GAD) which converts glutamate into GABA is a key enzyme in the dynamic regulation of neural network excitability. Clinical evidence has shown that lowered GAD activity (resulting in increased glutamate levels) is associated with several forms of epilepsy that are often treatment resistant [Lloyd et al. (1986) Adv Neurol. 44, 1033-1044]. The EKP-induced epilepsy zebrafish model has been validated as a model to identify drugs that can be used to treat drug resistant epilepsy [Sourbron et al. (2019) Epilepsia 60, e8-el3].

[0034] Larvae (7dpf) were arrayed individually in a 96-well plate (tissue culture plate, at bottom, Falcon, USA) in 100 pl VHC (Danieau's) and clioquinol, Idebenone or combinations of clioquinol and idebenone were added. Afterwards, the 96-well plates were placed in darkness at 28 °C for 2 hours. Just prior to tracking 100 pl of VHC or EKP (600 pM in Danieau's) solution was added to each well to obtain an EKP concentration of 300 pM. The plates were placed in an automated video tracking device (Daniovision apparatus, Noldus, The Netherlands). The locomotor behavior of the larvae was monitored for 40 min in the dark at 28 °C and quantified using Ethovision software (Noldus, The Netherlands). Locomotor activity per 5 min were quantified as cumulative duration (seconds) of activity lasting longer than 0.6 seconds. The experiment was performed in triplicate. After each tracking, larvae were checked for toxicity signs (death, loss of posture etc). VHC treated larvae with toxicity signs or EKP treated larvae that were dead or sedated were removed from the data set.

[0035] The results show that 1 pM, 0.5 pM and 0.25 pM of clioquinol reduce the locomotor activity in a dose dependent manner with 51.3%, 41.6% and 25.8%, respectively, and that 2 pM of idebenone significantly reduces the locomotor activity by 35%. By combining treatment consisting of 0.5 pM or 0.25 pM of clioquinol with 2 pM of idebenone, locomotor activity is further reduced by 72.1% and 58.6%, respectively, which is significantly more than the reduction obtained by compounds only. In case of the combination of 0.5 pM clioquinol with 2 pM Idebenone, locomotor activity is even more reduced than in case of locomotor activity reduction by a 2- fold higher dose of clioquinol.

[0036] Example 2. Combinations of clioquinol and idebenone show anti-seizure activity in the 6 Hz psychomotor seizure mouse model. Male Naval Medical Research Institute (NMRI) mice, weighting 18-20 g and provided by Charles River Laboratories, are housed (5 mice / cage) and maintained as described in (42) until the experiment was conducted.

[0037] Antiseizure activity of clioquinol, idebenone and combinations thereof is assessed in the mouse 6-Hz psychomotor seizure model as described in [Copmans et al. (2018) Neurochem Int 112,124-133; Li et al. (2020) ACS Chem Neurosci 11, 730-742]. Briefly, NMRI mice are randomly divided into different treatment groups and 500 pL (adjusted to the individual weight) of VHC (0.5% sodium carboxymethylcellulose (NaCMC) / Tween80 in 0.9% NaCI) or treatment (clioquinol, idebenone or combinations thereof dissolved in VHC) are injected intraperitoneally (i.p.). lh after injection, cornea is moisturized by an ocular anaesthetic (lidocaine, 0.5%) and psychomotor seizures are 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 are assessed by experienced researchers. Initially observed seizure durations are confirmed or corrected upon blinded video analysis. GraphPad Prism (version 6) software is used to plot the mean seizure durations per condition (+ / - SD) and to apply the appropriate statistical tests.

Claims

CLAIMS1. Clioquinol and idebenone for use in treating or preventing a seizure in epilepsy.

2. Clioquinol and idebenone for use according to claim 1, wherein the molar ratio between clioquinol to idebenone is between 1 to 2 and 1 to 16.

3. Clioquinol and idebenone for use according to claim 1 or 2, wherein the molar ratio between clioquinol to idebenone is between 1 to 4 and 1 to 8.

4. Clioquinol and idebenone for use according to any one of claims 1 to 3, wherein the epilepsy is a treatment resistant epilepsy.

5. Clioquinol and idebenone for use according to any one of claims 1 to 4, wherein the epilepsy is Dravet syndrome.

6. Clioquinol and idebenone for use according to any one claims 1 to 5, 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.

7. A pharmaceutical composition comprising clioquinol and idebenone.

8. The composition according to claim 7, wherein the molar ratio between clioquinol to idebenone is between 1 to 2 and 1 to 16.

9. The composition according to claim 7, wherein the molar ratio between clioquinol to idebenone is between 1 to 4 and 1 to 8.

10. A method of treating or preventing a seizure in epilepsy in a subject, the method comprising the step of administering an effective amount of clioquinol and idebenone to said subject.

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