Saccharomyces boulardii for the treatment of epileptic seizures

Abstract

The present invention relates to Saccharomyces boulardii yeast cell-derived product for use in the prevention or treatment of epileptic seizures in an individual.

Description

[0001] SACCHAROMYCES BOULARDII FOR THE TREATMENT OF EPILEPTIC SEIZURES

[0002] Filed of the invention

[0003] The present invention relates to a medicament for the prevention or treatment of epileptic seizures in an individual.

[0004] Background of the invention

[0005] Epilepsy is a neurological disorder characterized by recurrent, unprovoked seizures, which are transient occurrences of signs and / or symptoms due to abnormal excessive or synchronous neuronal activity in the brain.

[0006] Epileptic seizures significantly impair the quality of life and functionality in affected individuals. These impairments arise due to the unpredictable and recurrent nature of seizures, which can result in frequent interruptions of daily activities and social interactions. Individuals may experience limitations in employment, education, and social engagement due to the constant fear of seizure episodes. The cognitive, psychological, and emotional stress associated with managing epilepsy can further exacerbate these impairments, often leading to depression, anxiety, and reduced self-esteem. Furthermore, the physical consequences of seizures, such as injuries from falls or accidents during episodes, add to the overall burden, making routine tasks and independent living challenging. Consequently, the cumulative effects of epileptic seizures can lead to substantial declines in overall well-being and life satisfaction.

[0007] Standard treatments for epilepsy typically include various antiseizure medications (ASM) such as phenytoin, carbamazepine, valproic acid (Bialer ef al. (2007) Neurotherapeutics, 4: 130-137; kanner et al. (2022) JAMA, 327: 13), sodium valproate, phenobarbital, and newer agents like levetiracetam.

[0008] However, antiseizure medications are associated with a range of side effects, including dizziness, drowsiness, fatigue, and gastrointestinal disturbances such as nausea and vomiting. Certain ASM, such as phenytoin and carbamazepine, carry a risk of serious dermatological reactions, such as Stevens-Johnson syndrome and toxic epidermal necrolysis. Long-term use of some ASM, such as valproic acid, may lead to weight gain, polycystic ovary syndrome, and liver toxicity.In addition, despite the availability of these pharmacological treatments, a significant subset of patients has refractory epilepsy, wherein seizures are not adequately controlled even with the appropriate administration of ASM.

[0009] In light of the aforementioned limitations in current epilepsy treatments, there remains a need for alternative therapeutic options to provide an additional or complementary approach to traditional antiepileptic therapies.

[0010] Summary of the invention

[0011] The present invention arises from the unexpected finding by the inventors that administration of Saccharomyces boulardii CNCM I-745 significantly reduces the duration of epileptic seizures as well as the number of seizure over time. Saccharomyces boulardii CNCM I-745 also increases the time between seizures. Without wishing to be bound to a particular theory, the inventors believe that Saccharomyces boulardii reduces neuroinflammation at the gut level and that this reduction in neuroinflammation extends to the brain level thereby reducing epileptic seizures.

[0012] Thus, the present invention relates to Saccharomyces boulardii yeast cell-derived product for use in the prevention or treatment of epileptic seizures in an individual.

[0013] The present invention also relates to Saccharomyces boulardii yeast cell-derived product for use as defined above, in combination with at least one additional compound useful for the prevention or treatment of epileptic seizures.

[0014] The present invention also relates to a pharmaceutical composition comprising Saccharomyces boulardii yeast cell-derived product as active substance, and optionally at least one pharmaceutically acceptable carrier or excipient, for use in the prevention or treatment of epileptic seizures in an individual.

[0015] The present invention also relates to a pharmaceutical composition comprising: - Saccharomyces boulardii yeast cell-derived product as active substance, and

[0016] - at least one additional compound useful for the prevention or treatment of epileptic seizures, and

[0017] - optionally at least one pharmaceutically acceptable carrier or excipient.

[0018] The present invention also relates to products comprising:

[0019] - Saccharomyces boulardii yeast cell-derived product as active substance, and- at least one additional compound useful for the prevention or treatment of epileptic seizures,

[0020] as a combined preparation for simultaneous, separated or sequential use for preventing or treating epileptic seizures in an individual.

[0021] The present invention also relates to a method for the prevention or treatment of epileptic seizures in an individual comprising administering to the individual a prophylactically or therapeutically acceptable amount of Saccharomyces boulardii yeast cell-derived product.

[0022] In an embodiment of the method as defined above, Saccharomyces boulardii yeast cell-derived product is in combination with at least one additional compound useful for the prevention or treatment of epileptic seizures.

[0023] The present invention also relates to a method for the prevention or treatment of epileptic seizures in an individual comprising administering to the individual a prophylactically or therapeutically acceptable amount of a pharmaceutical composition comprising Saccharomyces boulardii yeast cell-derived product as active substance, and optionally at least one pharmaceutically acceptable carrier or excipient and optionally at least one additional compound useful for the prevention or treatment of epileptic seizures.

[0024] The present invention also relates to a method for the prevention or treatment of epileptic seizures in an individual in need thereof comprising:

[0025] - selecting an individual having epileptic seizures;

[0026] - administering an effective amount of Saccharomyces boulardii yeast cell-derived product to said individual.

[0027] The present invention also relates to the use of Saccharomyces boulardii yeast cell-derived product for the preparation of a medicament for the prevention or treatment of epileptic seizures in an individual.

[0028] Detailed description of the invention

[0029] Definition

[0030] In the specification and in the claims, the terms "including", "comprising" and "containing" can be used interchangeably. These terms are open-ended terms and should be interpreted to mean "including", but not limited to. Thus, when an object"comprises" or "contains" one or several elements, other elements than those mentioned may also be included in the object. These terms encompass the more restrictive terms "consisting essentially of" and "consisting of." When an object is said to "consist of" one or several elements, the object is limited to the listed elements and cannot include other elements than those mentioned.

[0031] Furthermore, as the skilled person will well understand, the expression "substance or composition for use in the prevention or treatment of a disease" is synonymous with the expression "substance or composition for use in a method of preventing or treating a disease".

[0032] Saccharomyces boulardii yeast cell-derived product Saccharomyces boulardii is well known to one of skilled in the art and is notably described in Hennequin et al. (2001) J. Clin. Microbiol. 39:551-559.

[0033] Example of Saccharomyces boulardii strains include Saccharomyces boulardii CNCM I-745; Saccharomyces boulardii ATCC MYA-796; Saccharomyces boulardii CBS 5926; Saccharomyces boulardii HANSEN CBS 5949; Saccharomyces boulardii MUCL 3792; Saccharomyces boulardii DSM 18734; Saccharomyces boulardii CNCM 1-3799; and Saccharomyces boulardii CNCM 1-1518.

[0034] Particularly preferably, the cells of Saccharomyces boulardii according to the invention are obtained from Ultra-Levure®, Bioflor®, Codex®, Econorm®, Enflor®, Enterol®, Florastor®, Floratil®, Florestor®, Inteflora®, Perenterol®, Perenteryl®, Precosa®, Refloi®, Ultra-Levura®, or from deposits made with the American Type Culture Collection (ATCC, USA) under reference 74012 or with the Collection Nationale de Culture et de Microorganismes (CNCM, France) under reference 1-745.

[0035] As intended herein the expression "yeast cell-derived product" relates to any product which can be obtained from yeast cells in themselves or which contains yeast cells secretions. It is preferred that the yeast cell-derived product according to the invention is selected from the group consisting of yeast cells, a yeast cell culture, a yeast cell extract, a yeast cell conditioned medium and a yeast cell culture supernatant.

[0036] As used herein, the expression "yeast cells" or "yeast cell culture" include viable or dead yeast cells, whole or in the form of debris. In particular, the expression "yeast cells" or "yeast cell culture" include viable cells, dead cells and cells debris withoutaffecting the quality or efficacy of the yeast cells. The term "debris" refers to fragmented or broken-down parts of yeast cells. This can include any pieces or remaining of yeast cells that are not intact or whole.

[0037] Preferably, at least some of the Saccharomyces boulardii yeast cells according to the invention are viable, in particular viable and cultivable, and more preferably a majority of the Saccharomyces boulardii yeast cells according to the invention are viable, in particular viable and cultivable.

[0038] The viability of a yeast cell is defined as the capacity of a yeast cell to multiply. Yeast cell viability can be determined by methylene blue staining and microscopic observation. The number of viable, cultivable cells, which defines vitality, can be estimated by determining the number of Colony Forming Units (CFU) in the sample.

[0039] By way of example, the number of CFU of yeast cells in a liquid sample containing yeast can be determined by spreading a given volume of the sample on a solid medium, e.g. agar, allowing yeast growth, and incubating the solid medium for a time, e.g. 48 h, and at a temperature, e.g.30°C, allowing yeast colonies to grow. The number of colonies per volume spread on the solid medium is used to determine the number of CPUs in the sample. A detailed protocol for CFU determination in accordance with the invention is described in Toothaker & Elmer ( 1984) Antimicrobial Agents and Chemotherapy 26:552-556 under the heading “Assay forS. boulardii". Alternatively, when the yeast sample is in the form of a solid, such as a freeze-dried powder, it is preferable to determine the number of CPUs comprised in the sample after taking up a determined mass of the sample in an aqueous solution, in particular distilled water or a 0.9% NaCI solution at pH 7.

[0040] Preferably, the yeast cells according to the invention are lyophilized, such as Saccharomyces boulardii yeast cells from Ultra-Levure®, Bioflor®, Codex®, Econorm®, Enflor®, Enterol®, Florastor®, Floratil®, Florestor®, Inteflora®, Perenterol®, Perenteryl®, Precosa®, Reflor®, or Ultra-Levura®.

[0041] Advantageously, the viability and vitality of yeast cells obtained from lyophilizates are greater than can be obtained with other methods of preservation of yeast cells.

[0042] As understood here, "lyophilization" is a method of preservation in which the yeast cells are frozen and are then submitted to sublimation of the frozen water that they contain to give a lyophilizate in the form of dry yeast powder preferably containing lessthan 2% of water and more preferably less than 1% of water. Preferably, the lyophilized yeast cells are obtained from concentrates of yeast cells. Any type of method of lyophilization of yeast cells known by one of skilled in the art can be used. However, the yeast cells are preferably lyophilized by means of the following method of lyophilization:

[0043] - cultivate the yeast cells in a liquid nutrient medium until the cells reach a stationary phase;

[0044] - concentrate the cultivated yeast cells and freeze the concentrate;

[0045] - lyophilize the concentrate.

[0046] Freeze-dried Saccharomyces boulardii yeast cells are in powder form.

[0047] Numerous methods for preparing yeast cells, a yeast cell culture, a yeast cell extract, a yeast cell conditioned medium and a yeast cell culture supernatant are well known in the art and are notably described in " Yeast Protocols" (1996) Methods in Cell and Molecular Biology, Ed. Ivor H. Evans, Humana Press.

[0048] The yeast cell culture according to the invention can be obtained by any standard method well known to one of skilled in the art for culturing yeast cells. By way of example the yeast cell culture according to the invention can be obtained by inoculating a complete liquid culture medium such as yeast extract peptone dextrose (YEPD or YPD) and incubating the medium at 30°C - 37°C under agitation and aerobic conditions during at least 24 hours, 36 hours, or 48 hours and preferably less than 96 hours.

[0049] Yeast cells according to the invention can be obtained from a yeast cell culture medium according to the invention through sedimentation or centrifugation of the yeast cells.

[0050] Yeast cell extracts according to the invention can be obtained by any yeast cell fragmentation method known in the art applied to the yeast cell culture or the yeast cells according to the invention, such as autolysis, hydrolysis or autoclaving. In particular, the yeast cells extract according to the invention is selected from the group consisting of a membrane extract, a cytoplasmic extract ora nuclear extract.

[0051] Preferably, the yeast cell-conditioned medium according to the invention relates to any medium, such as a liquid cell culture medium, which has been contacted by yeast cells. Preferably, the medium has been contacted by yeast cells for a time sufficient for the yeast cells to have secreted in the medium. By way of example, the medium has been contacted by yeast cells during 1 to 9 days, preferably 1, 2, 3, 4, 5, 6, 7, 8 or 9 days.The conditioned medium according to the invention preferably contains molecules secreted by the yeast cells such as proteins. Preferably, the conditioned medium according to the invention contains no cellular debris.

[0052] The yeast cell culture supernatant can be obtained by any method well known to one of skilled in the art. The yeast cell culture supernatant according to the invention may notably be obtained by centrifugation of the yeast cell culture according to the invention and taking the supernatant part of the centrifugated culture. The yeast cell culture supernatant according to the invention may also be obtained by filtrating the yeast cell culture according to the invention through a filter retaining yeast cells and recuperating the filtrate.

[0053] The yeast cell culture, the yeast cell extract, the yeast cell-conditioned medium and the yeast cell culture supernatant according to the invention may have been subjected to at least one treatment or processing step such as centrifugation, filtration, purification, chromatography, concentration, decantation, heating, pasteurisation, autoclaving, drying, freeze-drying or distillation, in particular filtration, more particularly sterilizing filtration for example with 0.2 µm filters.

[0054] Epileptic seizure

[0055] Epilepsy, as defined according to the International Classification of Diseases, 11th Revision (ICD-11), comprises a disease of the brain characterized by a predisposition to generate epileptic seizures, and by the neurobiological, cognitive, psychological, and social consequences of this condition. The definition requires the occurrence of at least one epileptic seizure.

[0056] Epilepsy according to the present description encompasses subtypes of epilepsy selected from the group consisting of genetic epilepsy syndrome; structural epilepsy; infectious epilepsy; metabolic epilepsy; immune epilepsy; unknown cause epilepsy; epilepsy of infancy such as Dravet syndrome and West Syndrome; epilepsy of childhood such as Lennox-Gastaut syndrome (LGS), childhood absence epilepsy (CAE) and Panayiotopoulos syndrome; epilepsy of adolescence such as juvenile myoclonic epilepsy (JME); epilepsy of adulthood such as temporal lobe epilepsy (TLE) and progressive myoclonic epilepsies (PME), epilepsy of unknown etiology, focal epilepsy; generalized epilepsy; combined generalized and focal epilepsy; epileptic spasms;absence epilepsy; myoclonic epilepsy; reflex epilepsy; sleep-related epilepsy; photosensitive epilepsy, status epilepticus, refractory epilepsy and progressive myoclonic epilepsies.

[0057] As used herein, the term "epileptic seizure" refers to a transient occurrence of signs and / or symptoms due to abnormal excessive or synchronous neuronal activity in the brain. Epileptic seizures can present as convulsions, loss of consciousness, limb jerking, sensory disturbances, or other neurological disruptions. The diagnosis and classification of epileptic seizures are typically governed by clinical criteria as outlined by the International League Against Epilepsy (ILAE).

[0058] The seizures according to the present description generally fall into two primary categories: focal (partial) seizures and generalized seizures, with several subtypes under each category.

[0059] a. Focal (Partial) Seizures:

[0060] - Focal aware seizures: also known as simple partial seizures, these seizures involve a region of the brain and do not impair consciousness. Symptoms can include motor, sensory, autonomic, or psychological phenomena.

[0061] - Focal impaired awareness seizures: previously called complex partial seizures, these alter or diminish consciousness and may produce motor disturbances, automatisms, and other complex behaviors.

[0062] - Focal to bilateral tonic-clonic seizures: these seizures start in one area of the brain and spread to involve both hemispheres, leading to a generalized tonic-clonic seizure.

[0063] b. Generalized Seizures:

[0064] - Absence seizures: Characterized by brief, sudden lapses in attention, often described as "staring spells." They typically last a few seconds and do not involve convulsive movements.

[0065] - Myoclonic seizures: brief, shock-like jerks of muscle or a group of muscles. They can affect one limb or the entire body.

[0066] - Tonic seizures: these involve a sudden onset of increased muscle tone, leading to stiffness, usually lasting a few seconds.

[0067] - Clonic seizures: characterized by rhythmic, jerking movements of the muscles, typically affecting both sides of the body simultaneously.- Tonic-clonic seizures: known as grand mal seizures, these involve an initial tonic phase (stiffening of the muscles) followed by a clonic phase (rhythmic jerking movements).

[0068] - Atonic seizures: these cause sudden loss of muscle tone, leading to drooping of the head, loss of posture, or sudden collapse.

[0069] Preferably, epilepsy according to the present description is selected among epilepsies that result in frequent and / or severe seizures.

[0070] In an embodiment, epilepsy according to the present description is selected from epilepsy in which seizures are not controlled by at least 1 antiepileptic drug or antiseizure medication, preferably at least 2 antiepileptic drugs or antiseizure medications.

[0071] Preferably, epilepsy according to the present description is a severe epilepsy. Severe epilepsy is preferably characterized by the frequence occurrence of epileptic seizure and / or a short time between two epileptic seizure and / or a long duration of epileptic seizure.

[0072] Severe epilepsy can manifest in several types, including but not limited to:

[0073] Status epilepticus: a condition characterized by prolonged or repetitive epileptic seizures. It can be defined by a single epileptic seizure lasting more than 5 minutes, or two or more distinct episodes of epileptic seizures between which the individual does not return to baseline consciousness, with a cumulative duration exceeding 5 minutes. Subtypes include convulsive status epilepticus involving tonic-clonic movements and non-convulsive status epilepticus.

[0074] Refractory epilepsy (also known as drug-resistant epilepsy): a form of epilepsy where seizures cannot be controlled with medication despite the appropriate choice and adequate usage of at least two antiepileptic drugs (AEDs) or antiseizure medications (ASM).

[0075] Genetic epilepsy syndromes: severe forms of epilepsy with a genetic basis, such as Dravet syndrome, West syndrome, Lennox-Gastaut syndrome (LGS), and temporal lobe epilepsy (TLE)

[0076] Epileptic Encephalopathies: a group of disorders where epilepsy manifests early in life and is accompanied by cognitive and developmental impairments. Examples include Lennox-Gastaut syndrome (LGS) and severe myoclonic epilepsy of infancy.Progressive myoclonic epilepsies (PME): rare, severe epileptic disorders that feature myoclonic seizures (sudden muscle jerks), generalized seizures, and progressive neurologic decline. Examples include Unverricht-Lundborg disease and Lafora disease.

[0077] Frequent epileptic seizures, as referenced in the context of the specification, are typically defined by seizure frequency metrics such as the occurrence of multiple seizures within a specified time period. An example definition of frequent epileptic seizures refers to the manifestation of at least 2 seizure within one year period, more preferably at least two seizures within a six-month period. This definition is used to categorize individuals who experience recurrent seizure episodes, which necessitate the prevention or treatment regimens as described in the present description. By way of example, frequent epileptic seizures refers to at least 3, at least 4, at least 5, at least 6 seizures within a six-month period.

[0078] In another embodiment, frequent epileptic seizures refer to an individual experiencing between 1 to 5 seizure per month, such as for example from 1 to 4 seizure episodes per month, from 1 to 3 seizure episodes per month, or from 1 to 2 seizure episodes per month or from 2 to 5 seizure episodes per month, from 3 to 5 seizure episodes per month, or from 4 to 5 seizure episodes per month or from 2 to 4 seizure episodes per month, or from 2 to 3 seizure episodes per month.

[0079] In another embodiment, frequent epileptic seizures refer to an individual experiencing at least 2 seizure episodes per year, at least 3 seizure episodes per year, at least 4 seizure episodes per year, at least 5 seizure episodes per year, at least 6 seizure episodes per year, at least 7 seizure episodes per year, at least 8 seizure episodes per year, at least 9 seizure episodes per year, at least 10 seizure episodes per year, at least 11 seizure episodes peryear, at least 12 seizure episodes peryearat least 13 seizure episodes per year, at least 14 seizure episodes peryear, or at least 15 seizure episodes peryear.

[0080] In another embodiment, frequent epileptic seizures refer to an individual experiencing multiple seizure episodes per week such as from 2 to 6 seizure episodes per week, from 3 to 6 seizure episodes per week, from 4 to 6 seizure episodes per week, or from 2 to 5 seizure episodes per week, from 2 to 4 seizure episodes per week, or from 2 to 3 seizure episodes per week.

[0081] In another embodiment, frequent epileptic seizures refer to an individual experiencing multiple seizure episodes per day, such as for example from 2 to 5 seizureepisodes per day, from 2 to 4 seizure episodes per day, from 2 to 3 seizure episodes per day or from 3 to 5 seizure episodes per day, from 4 to 5 seizure episodes per day.

[0082] The duration of an epileptic seizure can vary widely, typically ranging from a few seconds to several minutes.

[0083] Preferably, seizures according to the present described last at least 10 seconds, at least 15 seconds, at least 20 seconds, at least 30 seconds such as from 30 seconds to 40 seconds, from 30 seconds to 50 seconds, from 30 seconds to 1 minute, from 1 minute to 2 minutes, from approximately 2 minutes to approximately 5 minutes, from approximately 2 minutes to approximately 4 minutes, or from approximately 2 minutes to approximately 3 minutes. In the context of the present description, seizures preferably last from 10 seconds to 5 minutes, preferably from 30 seconds to 2 minutes.

[0084] In an embodiment, seizures as described herein last at least 5 minutes such as 5 minutes to 24 hours, 5 minutes to 20 hours, 5 minutes to 15 hours, 5 minutes to 10 hours, 5 minutes to 5 hours, 5 minutes to 1 hour, 5 minutes to 30 minutes, 5 minutes to 10 minutes or from 10 minutes to 30 minutes, from 10 minutes to 1 hour, from 30 minutes to 1 hour, from 30 minutes to 5 hours, from 30 minutes to 10 hours, from 30 minutes to 15 hours, from 30 minutes to 20 hours, or from 30 minutes to 24 hours.

[0085] The length of time between epileptic seizures, also referred to as the interictal period, is the duration from the end of one seizure to the onset of the next seizure.

[0086] Interictal period according to the invention is selected from the group consisting of short interictal periods, moderate interictal periods, and long interictal periods.

[0087] In short interictal periods, time between seizures is less than 24 hours, preferably it ranges from minutes to hours such as for example from 5 minutes to 24 hours, from 5 minutes to 20 hours, from 5 minutes to 15 hours, from 5 minutes to 10 hours, from 5 minutes to 5 hours, or from 5 minutes to 2 hours.

[0088] In moderate interictal periods, time between seizures is approximately 24 hours. Preferably, the time between seizures is between 24 hours and 120 hours, such as for example from 24 hours to 96 hours, from 24 hours to 72 hours, from 24 hours to 48 hours, from 48 hours to 120 hours, from 48 hours to 96 hours, or from 48 hours to 72 hours.

[0089] In long interictal periods, time between seizures preferably ranges from 7 days to 12 months, such as from example from 7 days to 8 months, from 7 days to six months, from 7 days to 2 months, from 7 days to 30 days, from 7 days to 14 days, from 14 days to about30 days, from 30 days to about 2 months, from 30 days to 3 months, from 30 days to 4 months, from 30 days to 5 months, from 30 days to 6 months, or from 2 months to 6 months.

[0090] Diagnosis of epileptic seizures is a multi-step process that typically involves a combination of clinical assessment, diagnostic tests, and detailed patient history, as outlined below:

[0091] Patient history and symptomatology: a thorough patient history is taken to document the frequency, duration, and characteristics of the seizures. Information on any aura (pre-seizure sensation), postictal state (recovery phase), and triggers is collected. Consideration of family history of epilepsy or related conditions.

[0092] Neurological examination: a comprehensive neurological examination is conducted to assess mental status, motor skills, and sensory perception.

[0093] Diagnostic tests: electroencephalogram (EEG): this is the primary diagnostic tool for epilepsy. It measures electrical activity in the brain and can identify abnormal electrical patterns characteristic of epilepsy.

[0094] Magnetic Resonance Imaging (MRI): an MRI scan is used to detect structural abnormalities or lesions in the brain that could be causing seizures.

[0095] Computed Tomography (CT) Scan: in some cases, a CT scan may be used to identify any acute abnormalities.

[0096] Positron Emission Tomography (PET) Scan: a PET scan may help identify areas of the brain with abnormal metabolism that could correlate with seizure activity. Single Photon Emission Computed Tomography (SPECT) Scan: this scan may be used to localize the region of seizure onset by showing blood flow changes during seizure activity.

[0097] Blood Tests: blood tests are conducted to rule out metabolic disorders, infections, electrolyte imbalances, and other conditions that may mimic or exacerbate seizures.

[0098] Neuropsychological Testing: these tests assess cognitive function and can help differentiate epilepsy from other conditions affecting mental status. They may also help localize the region of the brain affected by seizures.

[0099] Video EEG Monitoring: long-term video EEG monitoring may be employed to capture seizures while recording brain activity and to correlate clinical events with EEG findings.The diagnosis process is guided by evaluating diagnostic criteria explained above in concert with clinical judgment.

[0100] In an embodiment of the invention, the Saccharomyces boulardii yeast cell-derived product further improves learning and / or memory in the individual, or improves a deficit of memory in the individual.

[0101] As such, the present invention also relates to the Saccharomyces boulardii yeast cell-derived product for use in preventing or treating a memory deficit or a memory disorder, in particular an epilepsy- or a seizure-induced memory deficit or memory disorder, in an individual, in particular an epileptic individual.

[0102] Individual

[0103] The individual according to the invention is an animal, preferably a mammal, and more preferably a human.

[0104] Preferably, the individual is at least one year old, such as for example at least two years old, at least three years old, at least four years old, at least five years old, at least six years old.

[0105] In a particular embodiment, the individual according to the present description is a child or an adolescent, preferably, between 1 and 18 years old, such as between 1 and 10 years old, between 1 and 6 years old, between 2 and 18 years old, between 6 and 18 years old, between 8 and 18 years old, between 10 and 18 years old. Preferably, children and adolescents according to the present invention are suffering from specific childhood epilepsy syndromes, such as Dravet syndrome, West Syndrome, juvenile myoclonic epilepsy, Childhood Absence Epilepsy (CAE), Panayiotopoulos Syndrome, or Lennox-Gastaut syndrome.

[0106] In another embodiment, the individual according to the present description is an adult.

[0107] In an embodiment, the individual according to the present description is an elderly person, such as for example of at least 65 years old.

[0108] Preferably, the individual according to the invention is diagnosed with epilepsy. More preferably, individual according to the invention suffers from severe epilepsy, in particular selected from status epilepticus, refractory epilepsy, a genetic epilepsysyndrome, Lennox-Gastaut Syndrome, temporal lobe epilepsy, an epileptic encephalopathies, ora progressive myoclonic epilepsies.

[0109] The individual in the current context may experience frequent, recurrent, and / or prolonged epileptic seizures. Frequence of the seizures are as defined above.

[0110] In an embodiment, the individual according to the invention have more than one type of seizure as defined above.

[0111] In an embodiment, the individual does not recover of consciousness between two seizures.

[0112] In an embodiment, the individual according to the invention has other neurological conditions that can lead to seizures and requires a treatment of the seizures, such as brain tumors, stroke, head trauma, central nervous system infections (meningitis, encephalitis), Alzheimer's disease, and cerebral or congenital malformations.

[0113] In an embodiment, the individual according to the invention has one or multiple co-morbidities, such as intellectual disability, developmental delay, behavioral issues, and psychiatric disorders.

[0114] In an embodiment, the individual according to the description does not have a another medical condition, such as a neurological conditions or infections. In particular, the individual according to the description does not suffer from rotavirus infection.

[0115] In an embodiment, the individual according to the invention has epileptic seizure in response to reversible external factors, such as electrolyte imbalances, acute infections, or alcohol / drug withdrawal.

[0116] In an embodiment, the individual suffers or is at risk of suffering from a memory deficit or a memory disorder linked, associated, or due to epilepsy or epileptic seizures.

[0117] Additional compound

[0118] In an embodiment, Saccharomyces boulardii yeast cell-derived product, the pharmaceutical composition or the products according to the present description are administered with at least one additional compound useful for the prevention or treatment of epileptic seizure.

[0119] Preferably, the at least one additional compound useful for the prevention or treatment of epileptic seizure is an antiepileptic drug also referred to antiseizure medication. In the context of the present specification, the term "antiepileptic drug" and"anticonvulsant drug" can be used interchangeably. Both terms refer to pharmaceutical compounds that are administered to individuals for the purpose of controlling, reducing, or eliminating epileptic seizures.

[0120] Example of antiepileptic drug, or antiseizure medication, according to the present description include acetazolamide, brivaracetam, carbamazepine, clobazam, clonazepam, diazepam, eslicarbazepine, ethosuximide, everolimus felbamate, fenfluramine, fosphenytoin, gabapentin, lacosamide, lamotrigine, levetiracetam, lorazepam, mephobarbital, methsuximide, midazolam, oxcarbazepine, perampanel, phenobarbital, phenytoin, pregabalin, primidone, rufinamide, stiripentol, sodium valproate, tiagabine, topiramate, valproic acid, vigabatrin, zonisamide, cannabidiol.

[0121] In an embodiment of the present description, Saccharomyces boulardii yeast cell-derived product are not administered in combination with another antiepileptic, antiseizure or anticonvulsant compound.

[0122] In an embodiment of the present description, Saccharomyces boulardii yeast cell-derived product are not administered with another probiotic and the pharmaceutical composition, medicament or products according to the present description do not comprises probiotic other than Saccharomyces boulardii. In particular, in an embodiment Saccharomyces boulardii yeast cell-derived product are not administered with another probiotic selected from the group consisting of Lactobacillus, such as L. casei, L acidophilus, L rhamnosus, L reuteri, L plantarum, L helveticus, L brevis, Bifidobacterium such as B. bifidum, B. infantis, B. lactis, B. longum and Streptococcus such as S. salivarius.

[0123] Administration

[0124] Preferably, Saccharomyces boulardii yeast cell-derived product according to the invention is administered in a prophylactically or therapeutically effective amount for preventing or treating epileptic seizure. More preferably, Saccharomyces boulardii yeast cell-derived product according to the invention is administered in a prophylactically or therapeutically effective amount for reducing the number of epileptic seizures and / or for increasing the time between epileptic seizures and / or for reducing the duration of epileptic seizures and / or for preventing the onset of epileptic seizure and / or forpreventing deterioration of the disease, in an individual according to the present description.

[0125] As used herein, the deterioration of the disease refers to a worsening of the frequency, severity, or duration of seizures. This deterioration can occur due to several factors, including the natural course of certain epileptic syndromes such as Lennox-Gastaut Syndrome (LGS) and Dravet syndrome, inadequate control with antiepileptic drugs (AEDs) or antiseizure medication (ASM), progression of an underlying neurological or systemic disease.

[0126] Preferably, Saccharomyces boulardii yeast cell-derived product, the pharmaceutical composition, the medicament or the products according to the invention are in a form suitable for an administration or is administered by the oral route, the rectal route, the nasal route, the intradermal route, the intravenous route, the arterial route, the intramuscular route, or the subcutaneous route. More preferably, Saccharomyces boulardii yeast cell-derived product are administered by the oral route.

[0127] Preferably, Saccharomyces boulardii yeast cell-derived product, the pharmaceutical composition, the medicament or the products according to the invention are, or are presented, preferably in the form of capsule, tablet, powder sachets, patch, aerosol, granules or microgranules, solution, suspension, drops, gum, lozenge, suppository, or ovule.

[0128] Preferably, Saccharomyces boulardii yeast cell-derived product according to the invention are administered at a dose of 0.5 x 108to 100 x 1010CFU / kg / d or at a dose of 0.00125 g / kg / d to 25 g / kg / d.

[0129] Saccharomyces boulardii yeast cell-derived product according to the invention are preferably administered at a dose of 0.5 g / kg to 10 g / kg, more preferably at a dose of 1 to 6 g / kg and even more preferably at a dose of around 3 g / kg, in particular 1, 2, 3, 4 or 5 times a day.

[0130] As the skilled person will understand, the quantity of yeast cell-derived product to be administered per unit of mass (kg) refers to the mass of the individual for whom the yeast cell-derived product are intended to be administered. Furthermore, when the quantity of yeast cell-derived product to be administered is expressed in units of mass (g), the yeast cell-derived product are preferably in freeze-dried form, more preferably under lyophilized form. When the yeast cell-derived product is administered in liquid form,it is preferably administered in a volume of from 1µL to 10 mL, more preferably 100 µL to 2 mL, optionally after prior concentration or dilution.

[0131] Preferably, the pharmaceutical composition, medicament or products according to the invention comprise Saccharomyces boulardii yeast cell-derived product in a dose of 50 mg to 500 mg. Furthermore, the Saccharomyces boulardii yeast cell-derived product for their use as defined above are preferably administered in a unit dose of 50 mg to 500 mg.

[0132] As intended herein "pharmaceutically acceptable carrier or excipient "refers to any material suitable with a pharmaceutical composition. Preferably, the pharmaceutically acceptable carrier or excipient according to the invention is suitable for a yeast cell-derived product according to the invention in the form of a solid, a liquid or an aerosol.

[0133] Preferably, the pharmaceutically acceptable carrier or excipient according to the invention, includes but is not limited to any of the standard carrier or excipient known to one of skilled in the art such as water, glycerin, alcohol, oil emulsion, water emulsion, buffered saline solution, preservative, stabilizer and wetting agents.

[0134] As intended herein, "combined" or "in combination" means that the yeast cell-derived product, as defined above, is administered at the same time than another compound or product, either together, i.e. at the same administration site, or separately, or at different times, provided that the time period during which the yeast cell-derived product, as defined above, exerts its effects on the individual and the time period during which the additional agent or product exerts its pharmacological effects on the individual, at least partially intersect.

[0135] The invention will be further explained with the following Figures and non-limiting Examples.of the figures

[0136] Figure 1

[0137] Figure 1 depicts the onset time of spontaneous seizures in days post-SE as a function of the group of mice receiving vehicle and the group of mice receiving Saccharomyces boulardii (Sb), each group being divided into 2 subgroups of mice:

[0138] - one group of mice, surrounded by solid black lines, with a long delay between induction of SE and onset of seizures. Data are mean ± SEM; *p≤0.05 vs vehicle by Mann Whitney; - one group of mice, surrounded by a dotted black line, with a shorter delay between induction of SE and onset of seizures. Data are mean ± SEM.

[0139] Figure 2

[0140] Figure 2 depicts the minimum inter-seizure interval, in days, in function of the group of mice receiving vehicle and the group of mice receiving Sb. Data are mean ± SEM. *p<0.05 vs vehicle by Mann Whitney.

[0141] Figure 3

[0142] Figure 3 depicts the total number of seizure measured over 66 days as a function of the group of mice receiving vehicle and the group of mice receiving Sb, each group being divided into 2 subgroups of mice:

[0143] - one group of mice, surrounded by solid black lines, with high seizure number. Data are mean ± SEM. *p<0.05 vs vehicle by Mann Whitney;

[0144] - one group of mice, surrounded by a dotted black line, with a low seizure number. Data are mean ± SEM.

[0145] Figure 4

[0146] Figure 4 depicts the total time in seizure, in hour, as a function of the group of mice receiving vehicle and the group of mice receiving Sb, each group being divided into 2 subgroups of mice:

[0147] - one group of mice, surrounded by solid black lines, with longer time in seizure. Data are mean ± SEM. *p<0.05 vs vehicle by Mann Whitney.- one group of mice, surrounded by a dotted black line, with shorter time of seizure. Data are mean ± SEM.

[0148] Figure 5

[0149] Figure 5 depicts the cumulative number of seizure as a function of days post-SE induction for the group of mice receiving vehicle and the group of mice receiving Sb.

[0150] Figure 6

[0151] Figure 6 depicts the total number of seizures during 60 days or during days 1-30 and 31-60.

[0152] Figure 7

[0153] Figure 7 depicts the data of daily seizures.

[0154] Figure 8

[0155] Figure 8 shows the number of mice with seizure clusters (≥3 seizures / 24 h).

[0156] Figure 9

[0157] Figure 9 shows the results related to Barnes Maze to test spatial learning and memory.Example: the inventors have evaluated the therapeutic effect of Saccharomyces boulardii (CNCM I 745) in a murine model of acquired epilepsy.

[0158] A. Materials and methods

[0159] 1. Animals and housing conditions

[0160] C57BL6 / N adult male mice (8 week-old mice, ~25 g; Charles River, Calco, Italy) were used. Mice were housed (5 mice / cage) in the SPF facility at constant room temperature (23°C) and relative humidity (60 ± 5%) with free access to food and water, and with a fixed 12 h light / dark cycle. After mice were exposed to surgery for EEG (electroencephalography) set up implantation, they were individually housed with environmental enrichment in their cage. Experimental procedures were conducted in conformity with institutional guidelines, in compliance with national (D. L. n.26, G. U. March 4, 2014) and international guidelines and laws (EEC Council Directive 86 / 609, OJ L 358, 1, December 12, 1987, Guide for the Care and Use of Laboratory Animals, U. S. National Research Council, 1996), and reviewed and approved by intramural and extramural ethical committees.

[0161] Loss of EEG implant, failure to record high-quality EEG, mortality rate after SE, >20% weight loss during the week after surgery, were compensated by prospectively increasing by 30% the number of animals to be used in the experiment. Mice (n=41) and their sham controls (n=10) were surgically implanted with cortical recording electrodes, ground and reference electrodes and injection cannula under general gas anesthesia (1.4% isoflurane in a mixture of 70% N2O - 30%O2) and stereotaxic guidance. The injection cannula was unilaterally positioned on top of the dura mater for the intra-amygdala injection of kainic acid (KA; Di Sapia et al, Neurobiol Dis, 2021). The electrodes were connected to a multipin socket and, together with the injection cannula secured to the skull by acrylic dental cement. At the end of stereotaxic surgery, a perioperative analgesia was applied by single injection of buprenorphine (Bupaq, 0.1 mg / kg, s.c Alcyon Italia).Saccharomyces boulardii (Sb) CNCM I-745 or vehicle (drinking water) were administered once daily by gavage at a dose of 3 g / kg. The probiotic was freshly prepared daily from lyophilized cells.

[0162] 2. Induction of status epilepticus to trigger epilepsy

[0163] One week after surgery, kainic acid (KA, 0.3 pg) was injected in the right basolateral amygdala in freely moving mice using a needle protruding of 4.1 mm below the implanted cannula. This procedure induces status epilepticus (SE, i.e., prolonged and uninterrupted seizures lasting for >2 hours) which represents the brain injury leading to epilepsy in this model (Di Sapia et al, (2021) Neurobiol Dis.). SE arises after ~10 min from KA injection, as previously described (Di Sapia et al, (2021) Neurobiol Dis.). Thereafter, 40 min from SE onset, mice were injected with diazepam (10 mg / kg, intraperitoneally, i.p.) to improve their survival by suppressing motor seizures, although EEG SE is not interrupted. SE development was continuously EEG monitored in each mouse until it elapses spontaneously. SE is defined by high amplitude uninterrupted spiking activity with an average frequency of 12-18 Hz. The end of SE was defined when spike frequency fell below 1 Hz (inter-spike interval >1 sec).

[0164] Before inducing SE, mice were randomized in two groups based on their body weight (simple randomization with 1:1 allocation ratio) and treated with either placebo (drinking water) or Sb (3 g / kg) for 5 days to allow attainment of steady state level in the gut. The treatment continued after SE until end of experiment during which time EEG was continuously recorded (24 / 7) in order to monitor seizures.

[0165] 3. Chronic seizures (epilepsy)

[0166] Spontaneous motor seizures develop 5.4 ± 0.5 days after SE (epilepsy onset; two unprovoked spontaneous seizures occurring at least 48 h from the end of SE). Spontaneous seizures are EEG paroxysmal events lasting 30-60 sec on average, always accompanied by generalized motor convulsions. Mice were EEG recorded continuously (24 / 7) from the onset of SE until the onset of epilepsy. EEG activity was recorded using the Twin EEG Recoding System connected with a Comet AS-4032 / 8 Amplifier (sampling rate 400 Hz, high-pass filter 0.3 Hz, low-pass filter 70 Hz, sensitivity 2000 mV / cm; Grass-Telefactor,West Warwick, R. I., USA). Digitized EEG data were processed using the Twin record and review software. EEG analysis was done by two independent investigators blinded to the treatment, who visually reviewed all the EEG tracings. Deviation of ≤5% from concordance was considered acceptable, otherwise EEG tracing was additionally analyzed by a third person.

[0167] Mice were clustered for analysis in two groups based on their number of seizures above (high seizures) or below (low seizures) the median value of the respective experimental group (6.5 days in vehicle; 7.5 days in Sb). Seizure burden (total time in seizures=number of seizures x average seizure duration) was similarly used to cluster mice in two groups.

[0168] 4. Statistical analysis

[0169] Raw data were statistically analysed by GraphPad Prism 8 (GraphPad Software, USA) for Windows. Data are presented in figures as bargrams with individual values or mean ± S.E.M. Choice between parametric and non-parametric tests depended on passing Shapiro-Wilk normality test. Differences between groups are considered significant for values of p<0.05.

[0170] Blinding was applied to outcome measures assessment and data analysis. Sample size (mice / experimental group) was determined by GPower 3.1 software for Windows to detect an "effect size" (50% seizure reduction) with 80% power and a= 5%.

[0171] B. Results

[0172] 1. Effect of Saccharomyces boulardii on epilepsy onset

[0173] A significant delay in seizure onset by Sb was detected vs vehicle (p<0.05) in the group of mice with longer delay from SE induction to seizure onset (50% of mice in Sb or vehicle group) (see Figure 1).

[0174] 2. Effect of Saccharomyces boulardii on seizures

[0175] The minimum inter-seizure interval between seizures was longer in Sb vs vehicle mice (p<0.05, inset) (see Figure 2). Sb significantly reduced seizure number (p<0.05 vs vehicle) by comparing mice with higher number of seizures (see Figure 3). Similarly, the totalseizure duration in mice with longest seizure durations was significantly reduced by Sb (p<0.05 vs vehicle) (Figure 4).

[0176] Figure 5 depicts the cumulative number of seizures over time, an index of the rate of disease progression. There is a significantly decreased seizure progression in Sb treated vs vehicle mice (see Figure 5). Figure 6 depicts the total number of seizures during 60 days or during days 1-30 and 31-60 showing a significant reduction of seizures by Sb (by 50% on average, p<0.05 vs vehicle) during the various monitoring periods. Figure 7 depicts the data of daily seizures. The AUC (Area Under the Curve) shows a statistically significant reduction in seizures (p<0.01; 50% on average) in mice treated with Sb compared to vehicle mice during 60 days of EEG monitoring from epilepsy onset (day 1 represents epilepsy onset in each mouse).

[0177] Figure 8 shows the number of mice with seizure clusters (≥3 seizures / 24 h). This number is reduced by Sb during days 31-60 of treatment (33%, p<0.01) vs days 1-30 (67%) while the number of mice with seizure clusters was unchanged in vehicle mice (25-29%) when comparing the same time periods.

[0178] 3. Behavioral tests

[0179] Figure 9 shows the results related to Barnes Maze to test spatial learning and memory. The epileptic mice treated with vehicle showed impairment in their learning ability to find the escape hole, as shown by the increased primary latency during days 1-3 of training vs respective sham mice (p<0.01). Sb-treated mice showed improved learning since the latency time to find the escape hole was significantly reduced as compared to epileptic mice treated with vehicle (p<0.05, p<0.01). Similar results were obtained by comparing the respective AUC. The day after the last training trial, mice were tested for their ability to remember the location of the escape hole (probe trial). Epileptic mice treated with vehicle were impaired since they showed a significantly longer primary latency than respective sham controls (p<0.01). Sb-treated epileptic mice did not differ from their respective sham mice and showed a significantly reduced primary latency compared to epileptic mice treated with vehicle (p<0.01) showing improved memory.C. Conclusion

[0180] Sb is very effective in mice in particular with higher seizure burden (seizure number x duration) and Sb treatment significantly increases the time elapsed between seizures. Thus, Sb helps reducing the frequency of seizure. This is a clinically relevant endpoint. Sb also reduces the rate of disease progression and the occurrence of seizure clusters. Thus, Sb is not only controlling seizures in the short term but also slowing down the overall disease associated with seizures over time. There is also a delay in the onset of spontaneous seizures in those mice with a later disease onset. Sb is an effective treatment for reducing both the severity and frequency of seizures. Sb showed benefits in delaying disease onset and progression, making Sb a promising therapeutic option for seizure management and for modifying the disease course.

[0181] Besides, the therapeutic effects of Sb extend to neurological comorbidities such as cognitive deficits. Sb-treated epileptic mice showed improved learning and memory in a spatial memory task, reflecting improvement of a deficit of memory that is observed in human epilepsy.

Claims

Claims1. Saccharomyces boulardii yeast cell-derived product for use in the prevention or treatment of epileptic seizures in an individual.

2. Saccharomyces boulardii yeast cell-derived product for use according to claim 1, for reducing the number of epileptic seizures and / or reducing the duration of epileptic seizures.

3. Saccharomyces boulardii yeast cell-derived product for use according to claim 1 or 2, wherein the individual is at least 1 year old.

4. Saccharomyces boulardii yeast cell-derived product for use according to any one of claims 1 to 3, wherein the individual has a seizure frequency greater than or equal to 2 seizures per one year period.

5. Saccharomyces boulardii yeast cell-derived product for use according to any one of claims 1 to 4, wherein epileptic seizures last at least 10 seconds.

6. Saccharomyces boulardii yeast cell-derived product for use according to any one of claims 1 to 5, for use in preventing or treating a memory deficit or a memory disorder, in particular an epilepsy- or a seizure-induced memory deficit or memory disorder, in the individual.

7. Saccharomyces boulardii yeast cell-derived product for use according to any one of claims 1 to 6, wherein Saccharomyces boulardii yeast cell-derived product are lyophilized.

8. Saccharomyces boulardii yeast cell-derived product for use according to any one of claims 1 to 7, wherein Saccharomyces boulardii yeast cell-derived product are in a form suitable for an administration oris administered by the oral route.

9. Saccharomyces boulardii yeast cell-derived product for use according to any one of claims 1 to 8, wherein Saccharomyces boulardii yeast cell-derived product are administered at a unit dose of 50 mg to 500 mg.

10. Saccharomyces boulardii yeast cell-derived product for use according to any one of claims 1 to 9, wherein Saccharomyces boulardii yeast cell-derived product are Saccharomyces boulardii CNCM I-745 yeast cell-derived product.

11. Saccharomyces boulardii yeast cell-derived product for use according to any one of claims 1 to 10, where the yeast cell-derived product is selected from the group consisting of yeast cells, a yeast cell culture, a yeast cell extract, a yeast cell conditioned medium and a yeast cell culture supernatant.

12. Saccharomyces boulardii yeast cell-derived product for use according to any one of claims 1 to 11, wherein Saccharomyces boulardii yeast cell-derived product are in combination with at least one additional compound useful for the prevention or treatment of epileptic seizure.

13. Saccharomyces boulardii yeast cell-derived product for use according to claim 12, wherein the at least one additional compound useful for the prevention or treatment of epileptic seizure is selected from the group consisting of stiripentol, phenytoin, carbamazepine, valproic acid, sodium valproate, phenobarbital, primidone, lamotrigine, levetiracetam, topiramate, gabapentine, oxcarbazepine, zonisamide, tiagabine, vigabatrine, lacosamide, rufinamide, brivaracetam, and cannabidiol.

14. A pharmaceutical composition comprising Saccharomyces boulardii yeast cell-derived product as active substance, and optionally at least one pharmaceutically acceptable carrier or excipient, for use in the prevention or treatment of epileptic seizures in an individual according to any one of claims 2 to 6.

15. A pharmaceutical composition comprising:- Saccharomyces boulardii yeast cell-derived product as active substance, andat least one additional compound useful for the prevention or treatment of epileptic seizure, andoptionally at least one pharmaceutically acceptable carrier or excipient.

16. The pharmaceutical composition according to claim 15, for use in the prevention or treatment of epileptic seizures in an individual according to any one of claims 2 to 6.

17. Products comprising:- Saccharomyces boulardii yeast cell-derived product as active substance, and - at least one additional compound useful for the prevention or treatment of epileptic seizure,as a combined preparation for simultaneous, separated or sequential use for preventing or treating epileptic seizures in an individual according to any one of claims 2 to 6.

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