Treatment of neurological disorders caused by allicin and its analogues

Abstract

This invention relates to allicin and related compounds for use in the prevention or treatment of epilepsy.

Description

Technical Field

[0001] The present invention relates to the treatment of neurological disorders such as epilepsy.

Background Art

[0002] Epilepsy is the most common neurological disorder, with over 75 million people affected worldwide. Epilepsy is characterized by the presence of spontaneous, unprovoked, recurrent seizures and is associated with a risk of comorbidities including anxiety, depression, and increased mortality. Although structural lesions or metabolic deficiencies in the brain can cause epilepsy, most patients have an underlying genetic cause. Although there are over 40 approved anti-seizure medicines (ASMs) available commercially to treat epilepsy, 30% of patients are resistant to the effects of these drugs. Although there are various hypotheses regarding the mechanisms, the fact that the underlying pathogenesis is diverse and multifactorial in epilepsy and among patients, and between them, makes the treatment of drug-resistant epilepsy (DRE) particularly difficult (Non-Patent Document 1).

[0003] Currently, there is no treatment that can prevent epilepsy or slow its progression. In fact, with the current treatment schedule, once a seizure occurs, it is only symptomatically suppressed. Fortunately, during the past few decades, the understanding of the molecular and cellular changes that occur during epileptogenesis has improved significantly. Furthermore, the introduction of new epilepsy models and high-resolution research techniques has provided opportunities for the discovery of new targets for anti-epileptogenesis drugs and the development of treatments as a result. Such new disease-modifying treatments act directly on the mechanisms of the underlying disease and have the potential to prevent or modify the onset and / or progression of the disease, and may address the current unmet medical needs including epilepsy prevention / progression, comorbidities, and pharmacoresistance.

[0004] Given the heterogeneity of epilepsy, the Dravet syndrome (DS) model, a prototype of hereditary epilepsy and developmental epilepsy encephalopathy, is of particular interest for studying the potential of compounds to induce anti-epileptic activity. DS is a rare hereditary epilepsy encephalopathy with neurological and psychomotor developmental disorders, presenting within the first year of life. In approximately 80% of patients, it is caused by loss-of-function mutations in SCN1A, which encodes the major Na+ channel of GABAergic neurons. Dravet syndrome is characterized by a high mortality rate, primarily due to sudden, unexpected death in epileptic episodes (Non-Patent Literature 2).

[0005] Patent Document 1 discloses a combination of flavonoids, allicin, anisodamine, and erythritol ester ("red ancient alcohol" ester) for the treatment of epilepsy. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Chinese Patent Application Publication No. 106727496 [Non-patent literature]

[0007] [Non-Patent Document 1] Loscher & Klein (2021) CNS Drugs. 35, 935-963 [Non-Patent Document 2] Lagae (2021). Curr Opin Neurol. 34, 213-218 [Overview of the Initiative]

[0008] In this invention, compounds targeting epilepsy development are identified using a zebrafish model of Dravet syndrome. Zebrafish are lower vertebrates that have many advantages over classical rodent models, including high genetic and physiological homology to humans, high reproductive capacity, external fertilization, transparency through the early larval stage enabling powerful imaging techniques, and ease of various genetic manipulations. These attributes have enabled the development of a large-scale, systematic drug screening to identify small molecules that can suppress the disease phenotype (Patton et al. (2021) Nat Rev Drug Discov. 20, 611-628).

[0009] The present invention can be summarized as follows: 1. Alliin or a derivative thereof, selected from the group consisting of allicin, diallyl sulfide, diallyl disulfide, diallyl trisulfide, 1,4-dithiine, and 1,2-dithiine, and ajoene, for use in the prevention, reduction, or treatment of neurological disorders. 2. Allicin for use as described in Description 1, in the prevention, reduction, or treatment of neurological disorders. 3. Alliin or a derivative thereof as described in description 1 or 2 for use as described in description 1 or 2 in the prevention, reduction, or treatment of seizures in patients with epilepsy. 4. Alliin or a derivative thereof as described in Description 1 or 2 for use as described in Description 3, wherein the epilepsy is Dravet syndrome. 5. Alliin or a derivative thereof as described in Description 1 or 2 for use as described in Description 3, wherein the epilepsy is drug-resistant epilepsy. 6. Alliin or a derivative thereof as described in Description 1 or 2 for use as described in Description 5, wherein the drug-resistant epilepsy is an epilepsy resistant to two or more drugs selected from the group consisting of valproic acid, carbamazepine, levetiracetam, lamotrigine, topiramate, brivalacetam, lacosamide, perampanel, and phenobarbital. 7. A method for preventing, reducing or treating neurological disorders in a patient, comprising the step of administering to the patient a therapeutically effective amount of alliin or a metabolite selected from the group consisting of allicin, diallyl sulfide, diallyl disulfide, diallyl trisulfide, 1,4-dithiin and 1,2-dithiin, and ajoene. 8. The method according to description 7, comprising the step of administering a therapeutically effective amount of allicin to the patient. 9. The method according to description 7 or 8, wherein the neurological disorder is epilepsy. 10. The method according to description 9, wherein the epilepsy is Dravet syndrome. 11. The method according to description 7 or 8, wherein the epilepsy is drug-resistant epilepsy. 12. The method according to description 11, wherein the drug-resistant epilepsy is resistant to two or more drugs selected from the group consisting of valproic acid, carbamazepine, levetiracetam, lamotrigine, topiramate, brivalacetam, lacosamide, perampanel, and phenobarbital. 13. A composition comprising alliin or a derivative thereof, selected from the group consisting of allicin, diallyl sulfide, diallyl disulfide, diallyl trisulfide, 1,4-dithiine, 1,2-dithiine, and ajoene, for use in the prevention, relief, or treatment of seizures in epileptic patients. 14. The composition according to description 13, comprising allicin, for use according to description 13. 15. The composition according to description 13 or 14 for use as described in description 13, wherein the composition does not contain one or more of flavones, flavonoids, erythrol esters, or anisodamines. 16. The composition according to any one of descriptions 13 to 15, wherein the composition does not contain anisodamine, for use as described in description 13. 17. The composition according to any one of descriptions 13 to 16 for use as described in description 13, wherein the composition does not contain any of flavones, flavonoids, erythrol esters, and anisodamines. 18. The composition according to any one of descriptions 13 to 16 for use as described in description 13, wherein the epilepsy is Dravet syndrome. 19. The composition according to any one of descriptions 13 to 15 for use according to description 1 or 6, wherein the epilepsy is drug-resistant epilepsy. 20. The composition according to any one of descriptions 13 to 17 for use according to description 19, wherein the drug-resistant epilepsy is resistant to two or more drugs selected from the group consisting of valproic acid, carbamazepine, levetiracetam, lamotrigine, topiramate, brivalacetam, lacosamide, perampanel, and phenobarbital. 21. A method for preventing, reducing or treating seizures in an epileptic patient, comprising the step of administering a composition comprising alliin or a derivative thereof selected from the group consisting of allicin, diallyl sulfide, diallyl disulfide, diallyl trisulfide, 1,4-dithiine, 1,2-dithiine, and ajoene. [Brief explanation of the drawing]

[0010] [Figure 1] This is a schematic diagram of the timeline of epilepsy onset in a zebrafish scn1lab- / - mutant model of Dravet syndrome. From 3 dpf onwards, subtle epilepsy-like abnormalities can be observed using invasive local potential recording (Baraban et al. (2013) Nat Commun. 4, 2410), suggesting that epilepsy onset occurs between 0 and 3 dpf. At 5 dpf, epilepsy-like events become more pronounced. Scn1lab- / - larvae die prematurely between 10 and 12 dpf. [Figure 2-1]This figure shows that allicin treatment has an anti-epileptic effect. (A) Seizure suppression treatment schedule: scn1lab- / - larvae were exposed to MTC allicin or VHC control at 3 dpf. Two days later, abnormal spontaneous motor behavior and epileptic brain activity in the treated larvae were analyzed using an automated tracking device (Noldus) and local potential recording, respectively. (B) Normalized cumulative activity time (%) is expressed as mean ± SEM. The seizure suppression effect of allicin was investigated in scnLab- / - larvae (n=22) at 5 dpf, and compared with VHC-treated scnLab- / - larvae (n=24). VHC-treated scn1Lab+ / + larvae (n=24) were included as a control. (C) The mean number of epileptic events per 10 minutes of recording is expressed as mean ± SEM. The treatment schedule shown in Panel A was followed. The electrophysiological effects of allicin were investigated in scnLab- / - larvae (n=15) at 5 dpf and compared with those of VHC-treated scnLab- / - larvae (n=15). VHC-treated scn1Lab+ / + larvae (n=8) were included as a control. (D) Early treatment schedule: The egg membranes of scn1lab- / - larvae were removed at 1 dpf and exposed to MTC allicin or VHC control. Two days later, the larval swimming medium was supplemented with the same concentration of allicin or VHC, respectively. At 5 dpf, abnormal spontaneous motor behavior and epileptic brain activity of the treated larvae were analyzed using an automated tracking device (Noldus) and local potential recording, respectively. (Continued on page 6) (Continued from page 5) (E) Normalized cumulative activity time (%) is expressed as mean ± SEM. The long-term effects of allicin were investigated in scnLab- / - larvae (n=14) at 5 dpf according to the treatment schedule shown in Panel D, and compared with VHC-treated scnLab- / - larvae (n=23). VHC-treated scn1Lab+ / + larvae (n=24) were included as a control. (F) The mean number of epileptic-like events per 10-minute recording is expressed as mean ± SEM. The treatment schedule shown in Panel D was followed. Where appropriate, the electrophysiological effects of allicin were investigated in scnLab- / - larvae (n=18) at 5 dpf and compared with VHC-treated scnLab- / - larvae (n=14). VHC-treated scn1Lab+ / + larvae (n=10) were included as a control.(G) Anti-epileptic onset treatment schedule: The egg membranes of 1 dpf scn1lab- / - larvae were removed and exposed to allicin in MTC or VHC control. Two days later, 0.1% DMSO was supplemented to the swimming medium of all larvae. At 5 dpf, the abnormal spontaneous motor behaviors and epileptic-like brain activities of the treated larvae were analyzed using an automated tracking device (Noldus) and local field potential recording, respectively. (H) The normalized cumulative activity time (%) is represented as mean ± SEM. According to the treatment schedule shown in panel G, the anti-epileptic onset effect of allicin was examined in 5 dpf scnLab- / - larvae (n = 8) and compared with VHC-treated scnLab- / - larvae (n = 17). VHC-treated scn1Lab+ / + larvae (n = 23) were included as controls. (I) The average number of epileptic-like events per 10-minute recording is represented as mean value ± SEM. It followed the treatment schedule shown in panel G. Appropriately, the electrophysiological effect of allicin was examined in 5 dpf scnLab- / - larvae (n = 12) and compared with VHC-treated scnLab- / - larvae (n = 25). VHC-treated scn1Lab+ / + larvae (n = 20) were included as controls. For all experiments, statistical analysis was performed using one-way ANOVA. Compared with VHC-treated scnLab- / - larvae, ****p ≤ 0.0001, **p ≤ 0.01, *p ≤ 0.05. [Figure 2-2] Same as above [Figure 2-3] Same as above

Mode for Carrying Out the Invention

[0011] The compounds used in the present invention include compounds alliin and its derivatives derived from garlic, such as allicin, diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DAT), dithiin, and ajoene.

[0012] In a specific embodiment, the compound used in the present invention is allicin.

[0013] Alliin (IUPAC name: (2R)-2-amino-3-[(S)-(prop-2-en-1-sulfinyl)]propanoic acid) has the following structure: [Chemical formula]

[0014] Allicin (IUPAC name: S-(prop-2-en-1-yl)prop-2-en-1-sulfinothioate) has the following structure: [Chemical formula]

[0015] Allicin has antioxidant activity and can react with thiol-containing proteins. Allicin has been studied for its potential to treat various types of multidrug-resistant bacterial infections, as well as viral and fungal infections.

[0016] Diallyl sulfide (DAS) (IUPAC name: 3-prop-2-enylsulfanylprop-1-ene) has the formula CH2=CH-CH2-S-CH2-CH=CH2.

[0017] Diallyl disulfide (DADS) (IUPAC name: 4,5-dithia-1,7-octadiene) has the formula CH2=CH-CH2-S-S-CH2-CH=CH2.

[0018] Diallyl trisulfide (DAT), (IUPAC name: di(prop-2-en-1-yl)trisulfane) has the formula CH2=CH-CH2-S-S-S-CH2-CH=CH2.

[0019] Dithiin, 1,4-dithiin and 1,2-dithiin have the following chemical structures: [Chemical formula] 1,4-Dithiin 1,2-Dithiin

[0020] Ajoene (IUPAC name (1E)-3-(propa-2-ene-1-sulfinyl)-1-[(propa-2-ene-1-yl)disulfanyl]propa-1-ene) has the following structure: [ka]

[0021] The inventions described in the claims also envision the use of pharmaceutically acceptable salts, hydrates, etc., of these compounds.

[0022] Reducing agents and / or antioxidants may be optionally added to the pharmaceutical formulation.

[0023] Patent Document 1 discloses a mixture of compounds, including allicin, for the treatment of epilepsy. Other compounds in the mixture are flavones or flavonoids, anisodamine, and erythritol ester.

[0024] This Chinese patent application demonstrates that in mixtures of these compounds, anisodamine is an essential element of the mixture, and that any mixture without anisodamine is invalid.

[0025] It should be noted that the results of this mixture show efficacy that raises questions about its accuracy, even when compared to treatment using valproic acid at a technically advanced level.

[0026] Patent Document 1 neither discloses nor suggests the use of allicin in the treatment of epilepsy.

[0027] Embodiments of the present invention exclude the use of anisodamine in the claims for medical use described in the patent claims.

[0028] A more specific embodiment further excludes the use of one or more anisodamines, erythrol esters, and flavones or flavonoids in the claims for medical use described in the patent claims.

[0029] Neurological disorders, though not limited to these, include Parkinson's disease, Alzheimer's disease, Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis, AIDS-induced dementia, epilepsy, alcoholism, alcohol withdrawal, drug-induced seizures, viral / bacterial / fever-induced seizures, head trauma (traumatic brain injury), spinal cord injury, hypoglycemia, hypoxia, cerebral vascular occlusion, cerebral hemorrhage, hemorrhage, environmental excitotoxicity, dementia, trauma, drug-induced brain injury, stroke / ischemia, and aging.

[0030] A "seizure" refers to a short episode of signs or symptoms resulting from abnormally excessive or synchronous neural activity in the brain. The apparent effects can range from uncontrollable convulsive movements (tonic-clonic seizures) to milder ones such as momentary loss of consciousness (absence seizures).

[0031] Seizure types are typically classified by observation (clinical and EEG) rather than by underlying pathophysiology or anatomy. I. Focal seizures (formerly known as partial seizures) IA (Incomplete Seizure) - Consciousness is not impaired. IA1 accompanied by motor signs IA2 accompanied by sensory symptoms IA3: With autonomic nervous system symptoms or signs IA4 accompanied by psychiatric symptoms IB Complex partial seizures - impaired consciousness (formerly known as temporal lobe seizures or psychomotor seizures) IB1 manifests as a simple partial seizure, followed by impaired consciousness. IB2 is accompanied by impaired consciousness. Partial seizures that progress to secondary generalized seizures (IC) IC1 Simple partial seizures that progress to generalized seizures IC2 Complex partial seizures that progress to generalized seizures IC3: Progression from simple partial seizures to complex partial seizures and then to generalized seizures. II. Generalized seizures IIA Absence Seizure (formerly known as Petit Seizure) IIA1 Typical Absence Seizure IIA2 Atypical Absence Seizures IIB myoclonic seizures IIC (Clonic Illness) IID Tonic seizure IIE (Type Ieno-Conic Seizure) (formerly known as Grand Seizure) IIF (Involuntary Incapacitation) III. Unclassifiable epileptic seizures A more recent classification was published in Fisher et al. (2017) Epilepsia 58(4), 522-530.

[0032] Epilepsy is a brain condition characterized by susceptibility to recurrent seizures. The causes of epilepsy are numerous, but not limited to birth trauma, perinatal infections, anoxia, infectious diseases, toxin ingestion, brain tumors, genetic or degenerative disorders, head injury or trauma, metabolic disorders, sudden cerebrovascular disorders, and alcohol withdrawal.

[0033] Many subtypes of epilepsy have been characterized and classified. The classification and categorization system widely accepted in this field is that adopted by the Classification and Terminology Committee of the International League Against Epilepsy's ("ILAE") (see, for example, Berg et al. (2010), "Revised terminology and concepts for organization of seizures," Epilepsia, 51(4), 676-685).

[0034] I. Electrochemical Syndromes (Classified by Age of Onset): IA (Neonatal Period): Benign familial neonatal epilepsy (BFNE), Early myoclonic encephalopathy (EME); Ohtahara syndrome IB infant: Infant epilepsy with migratory focal seizures; West syndrome; Myoclonic epilepsy in infancy (MEI); Benign infant epilepsy; Benign familial infant epilepsy; Dravet syndrome; Myoclonic encephalopathy in non-progressive disorders IC in childhood: fever-plus (FS+) (can develop in infancy); Panayeotopoulos syndrome; epilepsy with myoclonic atonic seizures (formerly known as dysarthrodysesthesia); benign epilepsy with centrotemporal spikes (BECTS); autosomal-dominant nocturnal frontal lobe epilepsy (ADNFLE); late-onset childhood occipital lobe epilepsy (Gastaut type); epilepsy with myoclonic absence seizures; Lennox-Gastaut syndrome; epileptic encephalopathy with continuous spike-and-wave during sleep (CSWS); electrical status epilepticus during slow sleep (ESES). Also known as Sleep); Landau-Kleffner syndrome (LKS); Childhood absence epilepsy (CAE) ID Adolescent-Adult: Juvenile absence epilepsy (JAE); Juvenile myoclonic epilepsy (JME); Epilepsy with generalized tonic-clonic seizures only; Progressive myoclonus epilepsies (PME); Autosomal dominant epilepsy with auditory features (ADEAF); Other familial temporal lobe epilepsies The association with IE age is not specific: Familial focal epilepsy (childhood to adulthood) exhibiting diverse focal points; reflex epilepsy.

[0035] II. Characteristic Disease Groups II.A. Medial temporal lobe epilepsy with hippocampal sclerosis (MTLE with HS) II.B. Rasmussen Syndrome II.C. Laughter seizures accompanied by hypothalamic hamartoma II.D. Hemiconvulsions - hemiplegia - epilepsy E. Epilepsy that does not fit into any of these diagnostic categories and is distinguished based on the presumed cause (presence or absence of a known structural or metabolic condition) or on the dominant seizure pattern (generalized seizure versus focal seizure).

[0036] III. Epilepsy caused by and systematized by structural-metabolic factors III.A. Cerebral cortical developmental disorders (hemispheric megalencephaly, ectopic gray matter, etc.) III.B. Neurocutaneous syndromes (tuberous sclerosis complex, Sturge-Weber syndrome, etc.) Tumors III.D. Infection III.E.Trauma

[0037] IV. Hemangioma IV.A. Perinatal Injuries IV.B. Stroke IV.C. Other Causes

[0038] V. Epilepsy of unknown cause VI. Pathophysiology of epileptic seizures that have not been traditionally diagnosed as a subtype of epilepsy itself. VI.A. Benign neonatal seizures (BNS) VI.B.Febrile seizures (FS)

[0039] A more recent classification can be found in Scheffer et al. (2017) Epilepsia. 58, 512-521.

[0040] "Prevention, reduction, and / or treatment of epileptic seizures" encompasses any improvement in conditions associated with epilepsy, such as delayed seizure onset, milder or shorter seizure duration, lower seizure frequency, and lower susceptibility to seizure triggers.

[0041] Dravet syndrome is a severe form of childhood epilepsy characterized by drug-resistant seizures and numerous physical, behavioral, and intellectual comorbidities. Nearly 90% of all patients with Dravet syndrome carry a mutation in the SCN1A gene (voltage-gated sodium channel type 1 α subunit), which codes for the major Na+ channel in GABAergic neurons. Typically, the first seizure (often triggered by fever) occurs in infancy (around 3-6 months of age) in otherwise healthy children. Subsequently, seizures slow down, and pharmaco-resistant tonic-clonic, myoclonic, and absence seizures become more frequent. Patients with DS have severe comorbidities, including developmental, cognitive, and behavioral deficits, and have a high mortality rate, which is primarily due to sudden unexpected death in epilepsy (SUDEP).

[0042] Drug-resistant epilepsy (DRE) is defined by Kwan et al. (2010) Epilepsia 52, 1069-1077 as "the failure to achieve sustained seizure cessation despite sufficient trials of two well-tolerated and appropriately selected and used antiepileptic drugs (AED schedule) (whether monotherapy or combination therapy)."

[0043] Non-exclusive examples of antiepileptic compounds include: paraaldehyde; stiripentol; barbiturates (phenobarbital, methylphenobarbital, verbexacron, etc.); benzodiazepines (clobazam, clonazepam, chlorazepam, diazepam, midazolam, and lorazepam, etc.); potassium bromide; ferbamate; carboxamides (carbamazepine, oxcarbazepine, and eslicarbazepine acetate, etc.); fatty acids (valproic acid, sodium valproate, divalproex sodium, vigabatrin, progavid, and thiagavin, etc.); topiramate; hydantoin (E Examples include totoin, phenytoin, mephenytoin, and fosphenytoin; oxazolidinedione (paramethadione, trimetadione, and etadione); beclamide; primidone; pyrrolidine (brivalacetam, etilacetam, levetiracetam, etc.); celetracetam; succinimide (ethosuximide, fenxuximide, and mesxuximide, etc.); sulfonamide (acetazolamide, sultium, metazolamide, and zonisamide, etc.); lamotrigine; phenetulide; phenasemide; valbromide; valnoctamide; perampanel; stiripentol; and pyridoxine.

[0044] The specific type of "drug-resistant epilepsy" is epilepsy that is resistant to two or more drugs selected from the group consisting of valproic acid, carbamazepine, levetiracetam, lamotrigine, topiramate, brivalacetam, lacosamide, perampanel, and phenobarbital.

[0045] Previously, scn1lab - / - It has been shown that larvae begin to exhibit mild epileptic-like events at 3 dpf, and these become more pronounced from 5 dpf onward (Figure 1) (Baraban et al. (2013) Nat Commun. 4, 2410). This theoretically means that, in order to claim an anti-epileptic effect, the treatment should be administered early (i.e., before 3 dpf).

[0046] In this study, allicin's seizure-suppressing effect was merely symptomatic (observed). Therefore, abnormal hyperactivity and epileptic brain activity were evaluated by administering allicin from 3 dpf (i.e., after seizure onset, Figure 2A). At 5 dpf, neither behavioral nor electrophysiological experiments demonstrated a significant reduction in abnormal spontaneous motor behavior or epileptic brain activity compared to the VHC-treated control group (Figures 2B and 2C).

[0047] Next, the effects of early allicin treatment were evaluated. For this purpose, the egg membrane of 1 dpf embryos was removed and exposed to allicin for 4 days (i.e., before and during seizure onset, Figure 2D). At 5 dpf, behavioral analysis was performed comparing allicin-treated embryos with VHC controls. - / - We demonstrated a significant reduction in abnormal hyperactivity behavior in larvae (Figure 2E). These results were confirmed by electrophysiological experiments, which showed that treatment with allicin significantly reduced epileptic events to a similar degree, as observed in wild-type and heterozygous littermates (Figure 2F).

[0048] These results indicate that allicin must be administered early in the seizure cycle, more specifically before the onset of seizure, to induce a significant effect; however, a truly prophylactic anti-epileptic effect can only be claimed if treatment is administered only during the epileptic period (i.e., before the onset of an epileptic episode). Therefore, embryos with the ovarian membrane removed at 1 dpf were exposed to allicin for only 2 days (Figure 2G), followed by a washout with 0.1% DMSO. Behavioral and electrophysiological experiments at 5 dpf demonstrated a similar reduction in abnormal hyperactivity and epileptic-like brain activity observed with the early treatment schedule, highlighting the limited potential of allicin's anti-epileptic effect.

[0049] The experimental results demonstrate that allicin and its analogs, including alliin, diallyl sulfide, diallyl disulfide, diallyl trisulfide, dithiin, and ajoene, are neuroprotective, pathomodulatory, and anti-epileptic. Since neurological diseases share common pathophysiological mechanisms, the compounds described herein may be used similarly in other neurological diseases. [Examples]

[0050] Materials and methods Zebrafish model The larval zebrafish scn1lab, which carries a missense loss-of-function mutation in one of the SCN1A orthologs, accurately mimics the epileptic phenotype observed in patients with Dravet syndrome. In fact, homozygous scn1lab- / - mutants have been shown to exhibit spontaneous abnormal electroencephalogram activity, hyperactivity, seizure behavior, increased anxiety, sleep disturbances, and decreased metabolic function compared to wild-type (WT) and heterozygous littermates.

[0051] Mutant larval fish typically exhibit signs of epilepsy (excessive spontaneous motor activity, abnormal brain electrical discharges assessed by local potential (LFP) measurements) at 4-5 dpf (days post-fertilization). This suggests that epilepsy onset occurs earlier in this timeframe, particularly during the 0-3 dpf period.

[0052] Measurement of the effect of allicin in a zebrafish model When mutant larval fish were exposed to non-toxic concentrations of allicin for a period of 0 dpf to 3 dpf, typical epileptic symptoms such as excessive motor activity (measured at 5 dpf) and abnormal brain electrical discharges (data not shown) were absent. However, this rescue effect was not present when the fish were exposed to the compound for a period of 3 dpf to 5 dpf. This suggests that allicin possesses potent anti-epileptic or pathological modifying activity.

[0053] Measurement of the effects of allicin-related compounds in a zebrafish model The effects of a series of allicin-related compounds (alliin, diallyl sulfide, diallyl disulfide, diallyl trisulfide, 1,4-dithiine and 1,2-dithiine, and ajoene), disulfiram (a disulfide used to support the treatment of chronic alcoholism), and garlic oil will be tested in this zebrafish model. Mutant larval fish will be exposed to non-toxic concentrations of the compounds between 0 dpf and 3 dpf and between 3 dpf and 5 dpf, followed by an assessment of excessive motility by LFP measurement at 5 dpf.

[0054] Keeping zebrafish Adult AB wild-type (WT) zebrafish (Danio rerio) and zebrafish heterozygous for the scn1Lab mutation (scn1Lab+ / -) and backcrossed with Tupfel longfin WT (scn1Lab+ / +) were reared at 28.0°C with a 14 / 10 hour light / dark cycle under standard aquaculture conditions. Fertilized eggs were collected by natural spawning. Embryos and larvae were selected and cultured at 28.0°C with a 14 / 10 hour light / dark cycle in Petri dishes containing embryo medium (1.5 mM HEPES, pH 7.6, 17.4 mM NaCl, 0.21 mM KCl, 0.12 mM MgSO4, and 0.18 mM Ca(NO3)2) in a Peltier-cooled incubator (IPP 260, Memmert, Schwabach, Germany). 5 dpf homozygous scn1Lab - / - Larvae were selected from their heterozygotes and WT littermates based on their darker coloration, lack of swim bladder, and slight curvature. All animal experiments were approved by the KU Leuven Ethics Committee (Approval No. P055 / 2020) and the Belgian Federal Ministry of Public Health, Food Safety and Environment (Approval No. LA1210199).

[0055] compound Allicin, ajoene, S-allyl cysteine, diallyl disulfide, diallyl sulfide, alliin, diethyl disulfide, 3-vinyl-4H-1,2-dithiine), as well as disulfiram and normalized garlic oil, are purchased from Selleck Chemicals, Sigma-Aldrich, or BenChem. A stock solution is prepared in DMSO and maintained at -20°C. For the experiment, the stock solution is diluted in Danio medium to achieve a final DMSO concentration of 0.1% v / v. 0.1% DMSO in Danio medium is used as a vehicle (VHC) control.

[0056] Toxicity assessment (zebrafish) To evaluate the maximum tolerable concentration (MTC) of the compound, 4 dpf WT (AB strain) larvae were individually transferred to 96-well plates containing 100 μl volume / well and exposed to a certain concentration (a 2-fold dilution series starting from the highest soluble concentration). After 30 hours of exposure, the following parameters were examined: contact reaction, morphology, posture, edema, signs of necrosis, presence of swim bladder, and heart rate. The MTC is defined as the highest soluble concentration at which the larvae do not die and show no signs of toxicity or motor impairment at 5 dpf. If the MTC determined in AB larvae shows signs of toxicity in scn1lab- / - larvae, a lower concentration (2-fold dilution) should be tested.

[0057] Pharmacological evaluation Before conducting pharmacological experiments, the maximum tolerable concentration (MTC) was determined as previously described (Zhang et al., 2017). Briefly, the egg membranes of 12 larvae at 1 dpf were removed, and each was individually transferred to a 96-well plate containing 100 μl volume / well and exposed to a certain concentration (a 2-fold dilution series starting from the highest soluble concentration). The medium was refilled to 3 dpf and 5 dpf, and the following parameters were examined: contact response, morphology, posture, edema, signs of necrosis, presence of swim bladder, and heart rate. The MTC was defined as the highest soluble concentration at which larvae did not die and showed no signs of toxicity or motility impairment compared to VHC-treated control larvae. For pharmacological evaluation, larvae were treated with the respective MTC VHC or compound. A clear treatment schedule was followed (Figures 2A, 2D, and 2G).

[0058] Assessment of physical activity 5 dpf zebrafish larvae were individually transferred to 96-well plates containing 100 μl of treatment medium per well. After placing the plates in a DanioVision box (Noldus, Netherlands), larval behavior was recorded for 10 minutes in the dark following a 30-minute acclimatization period. Spontaneous motor behavior was quantified as cumulative time (seconds) spent in a high-activity state using Ethovision XT16 (Noldus) and normalized to the VHC control (%). The activity state parameter quantified typical seizure behavior ("high activity") according to the following settings: mean interval of 30 samples, numerical setting 2, exclusion of inactivity <0.60%, and <0.20 seconds.

[0059] Non-invasive local potential (LFP) recording Electroencephalographic activity of 5 dpf larvae was assessed by non-invasive local potential (LFP) recording from the tectum. A blunt-end glass electrode (soda-lime glass, Hilgenberg, Germany) was stretched using a DMZ Universal Puller (Zeitz, Germany) to an opening of approximately 15-20 microns, connected to a high-impedance amplifier, and placed on the skin above the tectum of larvae embedded in 2% low-melting-point agarose (Thermo Scientific) filled with artificial cerebrospinal fluid (124 mM NaCl, 2 mM KCl, 2 mM MgSO4, 2 mM CaCl2, 1.25 mM KH2PO4, 26 mM NaHCO3, and 10 mM glucose). A differential extracellular amplifier (DAGAN 2400, Minneapolis, Minnesota, USA) amplified the voltage difference between the signal (measured at the signal electrode) and the reference electrode by a factor of 10,000. The differential signal was passed through a 0.3 Hz to 300 Hz band-pass filter, digitized at 2 kHz using a PCI-6251 interface (National Instruments, UK), and recorded using WinEDR software (John Dempster, University of Strathclyde, UK). The electrical system was grounded using a ground electrode. All three electrodes remained connected during ACSF recording. Each recording was continued for 10 minutes and performed at room temperature. Epileptic activity was quantified, and the electrophoresis was visually analyzed using Clampfit 10.2 software (Molecular Devices Corporation, USA60) and MatLab v8.3 (The Mathworks, Inc.). Spontaneous epileptic events were considered if the amplitude exceeded three times the background noise and lasted longer than 50 ms.

[0060] Mouse model Allicin and related compounds (alliin, diallyl sulfide, diallyl disulfide, diallyl trisulfide, 1,4-dithiine and 1,2-dithiine, and ajoene), disulfiram (disulfide, used to support the treatment of chronic alcoholism), and garlic oil compounds will be similarly tested in a mouse model of Dravet syndrome. This model reflects various characteristics of Dravet syndrome, including spontaneous seizures, seizures induced by hyperthermia, and premature death.

[0061] Mouse breeding Mice are housed together in a pathogen-free mouse facility under standard laboratory conditions of a 14-hour light-period and 10-hour dark-period cycle. Access to food and water is unrestricted, except during experiments to induce hyperthermia-induced seizures.

[0062] Pharmacological evaluation (mouse) To evaluate the anti-epileptic or pathological modification effects, heterozygous Dravet mice (Scn1atm1Kea 50%C57BL / 6J, 50%129S6 / SvEvTac background strain) (Jackson Laboratory, Bar Harbor, Maine, USA) were used. These mice received a daily ip dose of one compound selected from P3 at three different doses (1 mg / kg, 10 mg / kg, and 50 mg / kg). Hyperthermia-induced seizures between P25 and P28 were examined. For this purpose, a rectal probe was inserted, and the mice were given a 10-minute acclimatization period to both the probe and the test chamber. Core body temperature was then gradually increased by 0.5°C every 2 minutes using a heating lamp controlled by a feedback temperature controller, until it reached a maximum of 42.5°C 60 minutes after treatment. Seizure activity typically begins with myoclonic seizures, followed by generalized tonic-clonic (GTC) seizures. Video recordings from each experiment were used for reviewing mouse behavior.

[0063] statistical analysis GraphPad Prism 9 software (Graphpad Software Inc., San Diego, USA) was used for all statistical analyses. Dunnett's multiple comparison test was used to compare the means between groups, following a one-way ANOVA. A p-value less than 0.05 (p ≤ 0.05) was considered statistically significant for the difference between the treatment group and the control group. [Explanation of Symbols]

[0064] Drawing translation Figure 1 epileptogenesis onset expression progression progression lethality Figure 2B Normalized cumulative duration of activity (%) + / + and + / - + DMSO + / + and + / - +DMSO - / - + allicin 75 μM - / - + allicin 75 μM Figure 2C Frequency of epileptiform events / 10-minute recording + / + and + / - + DMSO + / + and + / - +DMSO - / - + allicin 75 μM - / - + allicin 75 μM Figure 2E Normalized cumulative duration of activity (%) + / + and + / - + DMSO + / + and + / - + DMSO - / - + allicin 75 μM - / - + allicin 75 μM Figure 2F Frequency of epileptiform events / 10-minute recording + / + and + / - + DMSO + / + and + / - +DMSO - / - + allicin 75 μM - / - + allicin 75 μM Figure 2H Normalized cumulative duration of activity (%) + / + and + / - + DMSO + / + and + / - +DMSO - / - + allicin 75 μM - / - + allicin 75 μM Figure 2I Frequency of epileptiform events / 10-minute recording + / + and + / - + DMSO + / + and + / - +DMSO - / - + allicin 75 μM - / - + allicin 75 μM

Claims

1. A composition comprising alliin or a derivative thereof, selected from the group consisting of allicin, diallyl sulfide, diallyl disulfide, diallyl trisulfide, 1,4-dithiine, 1,2-dithiine, and ajoene, for use in the prevention, alleviation, or treatment of seizures in epileptic patients.

2. The composition according to claim 1 for the use according to claim 1, comprising allicin.

3. The composition according to claim 1 or 2 for use according to claim 1, wherein the composition does not contain one or more of flavones, flavonoids, erythrol esters, or anisodamines.

4. The composition according to any one of claims 1 to 3 for use according to claim 1, wherein the composition does not contain anisodamine.

5. The composition according to any one of claims 1 to 4 for use according to claim 1, wherein the composition does not contain any of flavones, flavonoids, erythrol esters, and anisodamines.

6. The composition according to any one of claims 1 to 5 for use according to claim 1, wherein the epilepsy is Dravet syndrome.

7. The composition according to any one of claims 1 to 5 for use according to claim 1 or 6, wherein the epilepsy is drug-resistant epilepsy.

8. The composition according to any one of claims 1 to 5 for use according to claim 7, wherein the drug-resistant epilepsy is an epilepsy resistant to two or more drugs selected from the group consisting of valproic acid, carbamazepine, levetiracetam, lamotrigine, topiramate, brivalacetam, lacosamide, perampanel, and phenobarbital.

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