Selenium nanoparticles for use in preventing, reducing risk of developing or treating epilepsy in a subject in need thereof

Abstract

Selenium nanoparticles for use in preventing, reducing risk of developing or treating epilepsy in a subject in need thereof, wherein selenium nanoparticles are administered parenterally to the subject, together with a transient disruption of the blood-brain barrier (BBB) of the subject.

Description

[0001] Selenium nanoparticles for use in preventing, reducing risk of developing or treating epilepsy in a subject in need thereof

[0002] Technical field

[0003] The present invention relates to the use of selenium nanoparticles in preventing, reducing risk of developing or treating epilepsy in a subject in need thereof, wherein said selenium nanoparticles are administered parenterally to the subject during transient disruption of the blood brain barrier (BBB) of the subject.

[0004] Background

[0005] Epilepsy is a chronic neurodegenerative condition that affects about 0.5% to 1% of the total world population, featured with repeated and unprovoked seizures, with neural cell damage or loss. One of every ten people will have at least one epileptic seizure during a normal lifespan, and a third of these will develop epilepsy. While all age groups can be affected by epileptic seizures, the disorder is most prevalent among the young and elderly.

[0006] Although seizures represent the most dramatic hallmark of epilepsy, many epilepsy patients develop neurological or psychiatric disease (memory or cognitive impairment, depression). For example, mesial temporal lobe epilepsy (the most frequent epileptic form) is usually accompanied by memory deficits probably due to hippocampal system damages and / or brain inflammation.

[0007] Despite the availability of recent antiepileptic drugs (ezogabine, pregabalin, levetiracetam, lamotrigine, topiramate, valproate, rufmamide, gabapentin, carbamazepine, clonazepam, oxcarbazepine, phenobarbital and phenytoin), available treatment options are not efficacious enough to prevent or treat the disease and seizures remain difficult to eradicate completely. Approximately one-third of patients still have uncontrolled seizures and an even larger percentage suffer from at least one anticonvulsant-related side-effect. Indeed, current antiepileptic drugs are frequently associated with numerous adverse impacts, such as mood changes, sleepiness, unsteadiness in gait memory deficits, fatigue, tremors, gastrointestinal symptoms, osteoporosis, depression, dizziness and nausea.

[0008] Although the treatment for epilepsy has evolved in the last decade, available treatment options are focused on preventing seizures once they are underway, and the current medications may not cure or even improve the course of disease. In addition, 20 to 30% of patients remain refractory to current medical treatment. For drug-resistant patients, alternative therapy options are radiosurgery, resective surgery and non-resective neuromodulatory treatments (deep brain stimulation, vagus nerve stimulation). However, resective, thermoablative or neuromodulatory surgery in the treatment of epilepsy are invasive procedures, sometimes requiring long-stay-in for patients, risks of permanent neurological deficit, etc. Furthermore, an important proportion of patients are not eligible for surgery due to seizure originating from an eloquent or deep- seated brain area or diffuse, multifocal in onset.

[0009] Therefore, there is a need in the art for new therapies to prevent and treat epilepsy, including drug-resistant epilepsy.

[0010] Summary

[0011] The present invention provides a novel, safe and efficacious method of preventing, reducing risk of developing, or treating epilepsy in a subject in need thereof. More particularly, the inventors have developed a method using selenium nanoparticles, combined to the transient disruption of the BBB of the subject. Indeed, the inventors have surprisingly discovered that selenium nanoparticles may be efficiently administered parenterally to the subject, whom BBB has been transiently disrupted, and that said selenium nanoparticles reach the central nervous system (CNS) of the subject and positively act on epileptic seizures. The present method may be used to treat diagnosed epilepsies as well as to prevent or reduce the risks of developing epilepsy, e.g. in a subject which already had at least one epileptic seizure.

[0012] It is therefore an object of the present invention to provide selenium nanoparticles (SeNPs) for use in preventing, reducing risk of developing or treating epilepsy in a subject in need thereof, wherein selenium nanoparticles are administered parenterally to the subject, preferably intravenously, together with a transient disruption of the blood-brain barrier (BBB) of the subject.

[0013] It is a further object of the present invention to provide a method for preventing, reducing risk of developing or treating epilepsy in a subject in need thereof, wherein selenium nanoparticles (SeNPs) are administered parenterally to the subject, preferably intravenously, together with a transient disruption of the blood-brain barrier (BBB) of the subject.

[0014] It is a further object of the present invention to provide the use of selenium nanoparticles (SeNPs) in the manufacture of a medicament for preventing, reducing risk of developing or treating epilepsy in a subject in need thereof, wherein selenium nanoparticles are administered parenterally to the subject, preferably intravenously, together with a transient disruption of the blood-brain barrier (BBB) of the subject. In a particular embodiment, the BBB disruption is performed by sonication, preferably by combined use of an ultrasound contrast agent administered before and / or during the application to the central nervous system (CNS) of the subject, of ultrasounds (US). In a preferred embodiment, US are low intensity pulsed ultrasounds.

[0015] In a particular embodiment, the present invention is applied to prevent or reduce risk of developing status epilepticus and / or seizures.

[0016] In another embodiment, the present invention is applied to treat epilepsy in a subject having a diagnosed epilepsy, in particular a subject with epileptic seizures.

[0017] The present invention may be further used to prevent or reduce risk of developing epilepsy in a subject in risk thereof.

[0018] The present invention is particularly suited against cryptogenic focal epilepsy, secondarily generalized epilepsy and lesional focal epilepsy.

[0019] In a particular embodiment, the epilepsy is a temporal lobe epilepsy, preferably a mesial temporal lobe epilepsy, a chronic epilepsy, and / or a drug-resistant epilepsy.

[0020] Preferably, the subject is a human, in particular a patient with epilepsy and / or risk to develop epilepsy or status epilepticus.

[0021] Brief description of the drawings

[0022] Fig. 1. Example of design of longitudinal in vivo experiments in the kainate mouse model. BBB (A to F), blood-brain barrier; GFAP, Glial fibrillary acidic protein; Ibal, ionized calcium- binding adapter molecule 1; iEEG, intracranial electroencephalogram; KA, kainate; LIPU, low-intensity pulsed ultrasound; NeuN, neuronal nuclei; SeNPs, selenium nanoparticles.

[0023] Fig- 2. Detection and dosage of intracerebral selenium (in pg per g of brain tissue) in different groups of mice at DO (30 minutes after treatment) and D7 (7 days after treatment). Comparison between each group at DO and D7 (Figure 2A). For each group, comparison at DO and D7 (Figure 2B). LIPU, low-intensity pulsed ultrasound; Se, selenium; SeNPs, selenium nanoparticles.

[0024] Fig. 3. Monitoring of the weight in two mice groups (sham mice and treated mice) from DO to D42. LIPU, low -intensity pulsed ultrasound; SeNPs, selenium nanoparticles.

[0025] Fig. 4. Monitoring of the frequency of epileptic seizures (in number of seizures per week) was measured in different mice groups (sham, LIPU, SeNPs, SeNPs+LIPU). (Figure 4A) Mean weekly seizure frequency in n=5 individual kainate animals over five iEEG recording sessions (sham group). (Figure 4B) Mean weekly seizure frequency in n=10 individual kainate animals before / after three sessions of LIPU-induced blood-brain barrier opening. (Figure 4C) Mean weekly seizure frequency in n=6 individual kainate animals before / after three sessions of SeNPs administration. (Figure 4D) Mean weekly seizure frequency in n=6 individual kainate animals before / after three sessions of LIPU-induced blood-brain barrier opening combined with SeNPs administration. iEEG, intracranial electroencephalogram; HP, hippocampus; LIPU, low -intensity pulsed ultrasound; SeNPs, selenium nanoparticles. * 0.01 < P < 0.05, statistics: two-tailed Mann-Whitney test.

[0026] Fig. 5. Histological characterizations of wild-type mice. (Figure 5A): semi-automatic analysis of the fluorescence resulting from the NeuN immunostaining in the hippocampus. (Figure 5B): analysis of the BRDU-positive cells counts in the dentate gyrus. (Figure 5C): analysis of the DCX-positive cells counts in the dentate gyrus. BrDU, bromodeoxyuridine; DCX,

[0027] Fig. 6. Histological characterizations of kainate mice. (Figure 6A): semi-automatic analysis of the fluorescence resulting from the NeuN immunostaining in the hippocampus. (Figure 6B): analysis of the BRDU-positive cells counts in the dentate gyrus. (Figure 6C): analysis of the DCX-positive cells counts in the dentate gyrus. BrDU, bromodeoxyuridine; DCX,

[0028] Fig. 7. Histological characterization in the dentate gyrus of kainate mice. (Figure 7A): semiautomatic analysis of the fluorescence resulting from the GFAP immunostaining in the hippocampus. (Figure 7B): analysis of the fluorescence resulting from the Ibal immunostaining in the hippocampus. (Figure 7C): analysis of the fluorescence resulting from the NeuN immunostaining in the hippocampus. GFAP, Glial fibrillary acidic protein; Ibal, ionized

[0029] Detailed description

[0030] The invention relates to selenium nanoparticles for use for treating or preventing epilepsy in a subject in need thereof, wherein said selenium nanoparticles are administered parenterally, such as intravenously, to the subject together with the transient disruption of the BBB of said subject. The administration of selenium nanoparticles combined to the BBB disruption has a direct and positive impact on epileptic seizures. In particular, a decrease of the frequency and / or of the intensity and / or of the duration of epileptic seizures is observed. Alternatively or in addition, administration of selenium nanoparticles combined to the BBB disruption has a direct and positive impact on status epilepticus, e.g., status epilepticus are prevented. Advantageously, repeated administrations of selenium nanoparticles during repeated transient disruptions of the BBB are provided to the subject.

[0031] The present disclosure will be best understood by reference to the following definitions.

[0032] Definitions

[0033] The term "epilepsy" can be classified according the electroclinical syndromes following the Classification and Terminology of the International League Against Epilepsy (ILAE) [Berg et al., 2010], Over forty types of epileptic seizures have been characterized and these are divided into generalized (seizure onset in both hemispheres of the brain) and partial (focal, seizure onset in one part of the brain). Generalized seizures are further divided into absence, myoclonic, atonic, and tonic seizures, while partial seizures are subdivided into simple and complex. Partial seizures account for approximately sixty percent of all adult cases and temporal lobe epilepsy (TLE) is the most common form of partial seizure. TLE patients often have a history of early risk factors such as febrile seizures, status epilepticus, and infection. A seizure-free period may be present before uncontrolled partial seizures begin. There are some progressive features such as increasing seizure frequency and cognitive decline.

[0034] The terms “convulsive disorder”, “seizure disorder”, and “cerebral seizures” are used synonymously with epilepsy, as they all refer to recurrent paroxysmal episodes of brain dysfunction manifested by stereotyped alterations in behavior.

[0035] An “epileptic seizure” is known as a sudden change in behavior that is the consequence of electrical hypersynchronization of neuronal networks involving the cortex.

[0036] “Status epilepticus” or “status seizure” refers to a medical condition involving a single epileptic seizure lasting more than 5 minutes, or two or more epileptic seizures within a 5-minute period without the subject returning to normal between them.

[0037] The term "refractory epilepsy" or “drug-resistant epilepsy” denotes an epilepsy which is refractory to current pharmaceutical treatment; that is to say that current pharmaceutical treatment does not allow an effective treatment of patients' disease (see for example Dario J. Englot et al., 2013). In the context of the invention, the term “disrupting the blood brain barrier or BBB”, “opening the blood brain barrier or BBB” or “increasing the permeability of the blood brain barrier or BBB” are used to refer to an increased susceptibility of the blood brain barrier to the passage of molecules and agents there through that occurs without detectable damages of the central nervous system.

[0038] In the context of the invention, a “transient” opening refers to a reversible opening occurring preferably for more than 1 hour, the BBB returning after that to its initial state, i.e., state before the application of the first US beam. Generally, the opening occurs for a period of time from 1 to 48 hours, preferably from 5 to 24 hours, more preferably from 6 to 10 hours, such as for approximately 8 hours.

[0039] As used herein, “subject” refers to a “human”, i.e., a person of the species Homo sapiens, including man, woman and child. In one embodiment, a subject may be a “patient” who is awaiting to receive, or is receiving medical care, or was / is / will be the subject of a medical procedure, or is monitored for the diagnosis or the development of a disease.

[0040] In the context of the invention, the terms “treatment”, “treat” or “treating” are used herein to characterize a therapeutic method or process that is aimed at (1) slowing down or stopping the progression, aggravation, or deterioration of the symptoms of the disease state or condition to which such term applies; (2) alleviating or bringing about ameliorations of the symptoms of the disease state or condition to which such term applies; and / or (3) reversing or curing the disease state or condition to which such term applies.

[0041] A “therapeutically effective amount” or “efficient concentration” refers to mean levels or amount of substance that is aimed at, without causing significant negative or adverse side effects to the target, delaying or preventing the onset of a disease, disorder, or condition related to epilepsy; slowing down or stopping the progression, aggravation, or deterioration of one or more symptoms of epilepsy; bringing about ameliorations of the symptoms of the disease, disorder, or condition related to epilepsy; reducing the severity or incidence of epilepsy; or curing epilepsy. A therapeutically effective amount may be administered prior to the onset of epilepsy, for a prophylactic or preventive action. Alternatively, or additionally, the therapeutically effective amount may be administered after onset of the disease, for a therapeutic action.

[0042] Throughout the disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is for convenience and brevity and should not be constructed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range, range values being included.

[0043] Selenium is a fundamental micronutrient that is incorporated in the structure of the active center of selenoproteins as selenocysteine moiety. Selenium is necessary for various physiological functions such as regulating thyroid hormones production, redox homeostasis, and immune response. The deficiency of selenium and selenoproteins was associated with permanent brain injury.

[0044] Recently, selenium nanoparticles (SeNPs) attracted the interest of many researchers due to their biocompatibility, bioavailability, and low toxicity. The minor size of SeNPs allowed them to cross membrane barriers and accumulate in tissues, resulting in more reactivity. Therefore, due to their higher bioactivity selenium nanoparticles are largely being used in various biomedical applications. In particular correlations between selenium deficiency and epilepsy have been reported. Previous studies showed the potential neuroprotective and anticonvulsant properties of selenium’s oral supplementation.

[0045] By working on the development of new therapy against epilepsy, the inventors have discovered that SeNPs may be efficiently injected to an epileptic subject in order to reduce the risk of epileptic seizures.

[0046] In the context of the present invention, any biocompatible SeNP may be used. The SeNPs may be synthesized through different routes, well known by the skilled person, including chemical methods (e.g., template method, chemical reduction, hydrothermal method), biological methods (e.g., by plants, fungi, bacteria, algae) and physical methods (e.g., pulsed laser ablation, microwave irradiation, ultrasonication).

[0047] According to the present invention, the selenium nanoparticle size is less than 100 nm. In a particular embodiment, the selenium nanoparticle size is between 20 to 80 nm, such as between 25 to 70 nm, between 30 to 65 nm, between 35 to 60 nm, between 40 and 60 nm, between 40 and 55 nm, between 45 and 60 nm, between 45 and 50 nm. In a preferred embodiment, the selenium nanoparticle size is between 40 to 50 nm.

[0048] In a particular embodiment, the selenium nanoparticles are administered at a dosage between 0.1 and 0.5 mg / kg per administration, preferably between 0.1 and 0.4 mg / kg, more preferably between 0.1 and 0.3 mg / kg, even more preferably between 0.1 and 0.2 mg / kg, in particular between 0.1 and 0.15 mg / kg per administration. The effective amount of SeNPs to be given to a particular subject will depend on a variety of factors, several of which will be different from subject to subject. Utilizing ordinary skill, the competent clinician will be able to tailor the dosage of SeNPs in the course of routine clinical trials. The SeNPs may be formulated in therapeutic formulation, comprising said SeNPs and optionally pharmaceutically acceptable carriers, excipients, and / or stabilizers, in the form of aqueous solutions.

[0049] For instance, a solution of SeNP is administered parenterally to the subject, wherein the solution comprises between lOOpg / mL and 200pg / mL of SeNPs, and between 50pL and 150pL of pharmaceutically acceptable excipient in the form of an aqueous solution. For instance, the solution of SeNP comprises 150 pg / mL of SeNPs, + / -10%, and 100 pL of pharmaceutically acceptable excipient in the form of an aqueous solution, + / - 10%.

[0050] According to the invention, the SeNPs may be administered using any medically appropriate procedure, e.g., intravascular (intravenous, intraarterial, intracapillary) administration, injection into the cerebrospinal fluid, intravitreal, intracavity or direct injection in the brain.

[0051] In a particular embodiment, the SeNPs are administered intravenously, in order to reach the systemic circulation of the subject.

[0052] In a particular embodiment, the selenium nanoparticles are labelled with a detectable molecule as an imagery tracer, preferably a fluorescent label or radioactive element, such as a radionuclide selected from Gallium 68 (Ga68), Lutetium 177 (Lul77), Fluor 18 (F 18), Yttrium 90 (Y90), Bismuth 213 (Bi213), Actinium 225 (Ac225), Lead 212 (Pb212), Indium 111 (Ini 11) Technetium 99 (Tc99) and copper 64 (Cu64), preferably, Gallium 68 (Ga68).

[0053] Alternatively or in addition, the selenium nanoparticles may be conjugated to a therapeutic agent, such as a drug, or an immune agent. In a particular embodiment, the selenium nanoparticles are conjugated to an antiepileptic drug.

[0054] Transient BBB opening

[0055] According to the invention, the SeNPs are administered parenterally, while the BBB is transiently disrupted, thereby increasing the passage of SeNPs through the BBB. According to the invention, the BBB may be disrupted by any means known by the skilled person as suitable for safely and temporally opening the BBB of a subject, in particular of a human. For instance, the BBB may be opened by sonication, by intra-arterial injection of hyperosmotic mannitol or leukotrienes, by use of vasoactive substances such as bradykinin, etc.

[0056] Preferably, the BBB is opened by sonication, i.e., with ultrasound. Any ultrasound system, suitable to apply ultrasound(s) to the brain of the subject may be used, in particular low intensity pulsed ultrasounds (LIPU). The term “ultrasound beam”, “ultrasound wave” and “ultrasound” are used indifferently for designating sound waves with frequencies higher than 200 kHz.

[0057] According to the invention, the BBB may be disrupted by both applying ultrasound to the brain of the subject, and administering parenterally (e.g., intravenously) an ultrasound contrast agent.

[0058] The term “ultrasound contrast agent” is used herein to refer to a substance (solid, liquid or gas) that is able to enhance the contrast between the region containing the agent and the surrounding tissue in an ultrasound image. Advantageously, the ultrasound contrast agent corresponds to small bubbles of a gas, termed "microbubbles," with an average diameter between 1 pm and 20pm. Said microbubbles oscillate and vibrate when US is applied and may reflect ultrasound waves. In some embodiments, the ultrasound contrast agent is a microbubble contrast agent, preferably selected from the group consisting of sulphur hexafluoride microbubbles (SonoVue®), microbubbles made of an albumin shell and octafluoropropane gas core (Optison®), perflexane microbubbles encapsulated in an outer lipid shell (Imagent®), microbubbles made of octafluoropropane gas core encapsulated in an outer lipid shell (Definity®), or perfluorobutaine and nitrogen gas encapsulated in a lipid shell (BR38 - Schneider et al., 2011). Preferably, the ultrasound contrast agent consists of sulphur hexafluoride microbubbles.

[0059] The ultrasound contrast agent may be administered by injection, preferably by systemic injection. Systemic administration is a route of administration of an agent into the circulatory system so that the entire body is affected. Examples of systemic injections include intravenous, subcutaneous, intramuscular, intradermal, intravitreal, or perfusion. In some embodiments, the ultrasound contrast agent is injected intravenously into the bloodstream of the subject, wherein it remains for a limited period of time.

[0060] The microbubbles may have a mean diameter in a range from 1 pm to 20pm. In some embodiments, the microbubbles have a mean diameter in a range from 4 pm to 5 pm. In some other embodiments, the microbubbles have a mean diameter in a range from 2 to 6 pm. In some embodiments, the microbubbles have a mean diameter of approximately 7 pm, 6 pm, 5 pm, 4pm, 3 pm or 2pm. In a particular embodiment, the microbubbles have a mean diameter of approximately 2.5 pm.

[0061] In some embodiments, the dose of ultrasound contrast agent ranges between 0.01 and 0.4 ml / kg based on the total weight of the subject, preferably from 0.05 and 0.2 ml / kg. In a particular embodiment, the maximum dose of ultrasound contrast agent is up to 5 ml, up to 6 ml, up to 10ml, up to 15 ml, up to 20 ml, up to 25 ml, or up to 30 ml. More generally, the maximum dose may depend of the dilution of the ultrasound contrast agent. Preferably, the dose of ultrasound contrast agent is approximately the dose used for diagnostic imaging, with a maximum dose corresponding to twice the dose used for diagnostic imaging.

[0062] Preferably, the ultrasound contrast agent is administered as a bolus just before the US application. More preferably, the US contrast agent is administered between 0 and 30 seconds before the US application. Advantageously, the US application and the US contrast agent administration are concomitant. The ultrasound contrast agent is preferably delivered only once per session of US application, though it may be delivered by a continuous infusion through the activation of successive US applications. Preferably, ultrasound contrast agent is administered for each session of US application.

[0063] In a particular embodiment, the sonication is performed by applying low intensity pulsed ultrasounds (LIPU) to the brain of the subject.

[0064] According to the invention, the US are focused or unfocused, preferably unfocused.

[0065] Advantageously, the unfocused US are applied to the brain of the subject, with a pressure level ranging from 0.3 to 2 MPa, preferably from 0.3 to 1.2 MPa. In the context of the invention, the “pressure level” refers to the maximum acoustic pressure measured in the acoustic field of the emitter in water. Advantageously, the unfocused US are applied within a pressure range of 0.7 MPa to 1.25 MPa, preferably within a pressure range of 0.8 MPa to 1.1 MPa, more preferably of about 1 MPa.

[0066] In the context of the invention, the value of the pressure level corresponds to the value of the pressure level onto the brain. In particular embodiment, the pressure coming out of the emitter may be higher, in order to take into account attenuation due to intervening tissues. Generally speaking, such attenuation may be at most of 30%.

[0067] According to the invention, the resonance frequency of the unfocused US preferably ranges from 0.5 to 3 MHz, such as from 1 to 1.1 MHz, particularly at 1.05 MHz. In a particular embodiment, the frequency of the unfocused US is approximately 1 MHz. In another particular embodiment, the frequency of the unfocused US is approximately 2 MHz.

[0068] In an embodiment, the unfocused US is applied in pulses of duration ranging from 10 to 300 ms and with a pulse repetition frequency ranging from 0.3 to 3 Hz, preferably from 0.5 to 1 Hz.

[0069] Unfocused US may be applied in pulses of duration ranging from 10 to 50 msec, preferably from 20 to 30 msec, more preferably about 25 msec, and with a pulse repetition frequency ranging from 0.3 to 1.2 Hz, preferably at 1 Hz. Preferably, the US are applied to the brain of the subject. Particularly, the US may target a portion of the mesial temporal lobe of the subject, preferably the hippocampus.

[0070] Advantageously, the US are applied by use of a US transducer that has been previously implanted within a burr hole in the skull of the subject. Examples of suitable implantable US transducer for brain are disclosed in US 13 / 577,938, WO2016 / 097867, WO2018 / 234280, and Carpentier, A. et al. 2016 (Clinical trial of blood-brain barrier disruption by pulsed ultrasound. Set. Transl. Med. 8, 343re2). Of course, an external US transducer may be used either. Examples of suitable external US transducer for brain are disclosed in Abrahao, A. et al. 2019 (First-inhuman trial of blood-brain barrier opening in amyotrophic lateral sclerosis using MR-guided focused ultrasound. Nat. Commun. 10, 4373)

[0071] In some embodiments, the BBB disruption is delimited, i.e., occurs solely in a target region of the BBB. In particular, at least 5 cm3, preferably at least 10 cm3, more preferably at least 20 cm3 of the BBB is disrupted. In other embodiment, the BBB disruption is generalized.

[0072] In some embodiments, the BBB opening occurs for a period from 1 to 24 hours, preferably from 5 to 12 hours, more preferably from 6 to 10 hours. In some embodiments, the BBB opening occurs for approximately 8 hours.

[0073] The disruption may be confirmed and / or evaluated by magnetic resonance imaging (MRI), blood biomarkers, electrophysiology or any other mean. For example, a gadolinium -based magnetic resonance (MR) contrast agent such as Dotarem® (gadoterate meglumine, Guerbet USA), which does not normally cross the BBB, can be used to visualize the region of the neurovascular barrier disruption. When the agent is injected in a patient, a Tlw MR sequence can be used to visualize regions of hypersignal and therefore visualize the effect of neurovascular barrier disruption by US. BBB disruption typically leads to a change of 5-10% or more in MR signal enhancement after contrast agent administration. In addition, dynamic contrast enhanced (DCE) MR imaging techniques can be used to calculate the permeability of the neurovascular barrier and to quantify the magnitude of the permeability enhancement after ultrasound treatment.

[0074] It is the purpose of the present invention to provide a method of preventing, reducing risk of developing, and / or treating epilepsy in a subject in need thereof. The method of the present invention is particularly suited against cryptogenic focal epilepsy, secondarily generalized epilepsy and / or lesional focal epilepsy. In a particular embodiment, the epilepsy is a temporal lobe epilepsy, preferably a mesial temporal lobe epilepsy. In an embodiment, the epilepsy is a chronic epilepsy. In another embodiment, the epilepsy can be a drug-resistant (i.e., refractory) epilepsy. In a particular embodiment, the refractory epilepsy is a chronic refractory epilepsy. In an embodiment, the epilepsy is a traumatic brain injury induced epilepsy.

[0075] According to the present invention, a therapeutically effective amount of SeNPs is administered to a subject, in particular a patient, in need thereof.

[0076] Advantageously, the treatment allows to reduce frequency, duration and / or intensity of epileptic seizure in the subject.

[0077] When the BBB is disrupted by sonication, selenium nanoparticles may be administered to the subject before, after and / or during the administration of the ultrasound contrast agent. For instance, the selenium nanoparticles and the ultrasound contrast agent may be administered concomitantly, or simultaneously, (e.g., by way of a same solution). In a preferred embodiment, the selenium nanoparticles are administered at first, then the ultrasound contrast agent, before to perform the sonication.

[0078] In a particular embodiment, SeNPs may be administered to a subject which presents a diagnosed epilepsy, i.e., to a subject which already occurred epileptic seizures and / or an inaugural status epilepticus, in order to treat epilepsy and / or to prevent or reduce the risk of developing epileptic seizure and / or status epilepticus.

[0079] In another embodiment, the SeNPs may be administered to a subject at risk, in order to prevent or to reduce the risk of developing epileptic seizure and / or status epilepticus. Such preventing treatment may be particularly useful with subject having brain injury or trauma.

[0080] In an embodiment, the selenium nanoparticles are administered during or within the first 4 weeks after an epileptic seizure or an inaugural status epilepticus, preferably during or within the first week after even more preferably during or within 24 hours after an epileptic seizure or inaugural status epilepticus.

[0081] In an embodiment, at least two doses of selenium nanoparticles are administered to the subject, with a latency period between two successive doses of selenium nanoparticles, preferably of at least one week and at most 9 weeks, preferably between 1 to 4 weeks, more preferably between 1 to 2 weeks. For instance, the latency period between the administration of two successive doses of selenium nanoparticles is 7 days, + / - 2 days.

[0082] According to the invention, the BBB may be disrupted before, after and / or during each administration of selenium nanoparticles. In a particular embodiment, three doses of selenium nanoparticles are administered successively to the subject. Preferably, the latency period between two doses is about 7 days, + / - 2 days. The BBB is disrupted before and / or during each administration of SeNPs to the subject.

[0083] EXAMPLES

[0084] The kainic acid (KA) model, because of its high level of similarity with human epilepsy, is used in the following experimentations (Ben- Ari and Lagowska, 1978 “Epileptogenic action of intraamygdaloid injection of kainic acid ; Ben-Ari et al., 1979a “A new model of focal status epilepticus: intra-amygdaloid application of kainic acid elicits repetitive secondarily generalized convulsive seizures” ; Bouilleret et al. Neuroscience. 1999 “Recurrent seizures and hippocampal sclerosis following intrahippocampal kainate injection in adult mice: electroencephalography, histopathology and synaptic reorganization similar to mesial temporal lobe epilepsy »).

[0085] MATERIAL AND METHODS

[0086] Animals

[0087] All experimental studies were performed in accordance with the European Committee Council Directive (2010 / 63 / UE) and approved by the local ethics committee (A75-13-19; 20102-2019). Experiments were performed on C57BL / 6 J male mice, aged of 2 months of age and weighing 25-30 g. The manuscript was prepared in accordance with the ARRIVE guidelines 2.0.

[0088] Design of longitudinal in vivo experiments in the kainate mouse model.

[0089] (Figure 1A) First, the mice were placed in a stereotaxic frame under general anesthesia, injected with kainate, and implanted with depth stainless-steel electrodes. (Figure IB) The wires of the electrodes were soldered to a connector. The electrodes and connectors were fixed on the mouse skull with cyanoacrylate and dental acrylic cement. (Figure 1C) EEG activity from the cortex and hippocampus and mouse behavior were recorded from mice moving freely in individual plexiglass cages. The mice were recorded on a computer-based digital acquisition system. EEG- video recordings were obtained before the first treatment session (baseline recordings starting 28 days after the kainate injection and electrode implantation), after each treatment session, and 7 days after the last treatment session. Mice that had at least one seizure during the baseline iEEG-video were randomly assigned to one of the four treatment groups: LIPU-induced BBB opening, SeNPs, SeNPs associated with LIPU-induced BBB opening, or Sham. A weekly treatment session was performed for three consecutive weeks in implanted KA mice under general anesthesia. Concerning the mice allocated to the LIPU and SeNPs + LIPU groups, a 100-microliter bolus of microbubbles was administered via the right retro-orbital sinus prior to initiating sonication. (Figure ID) The mice were subsequently positioned on the ultrasound platform with their heads in contact with demineralized and degassed water, and the target region (the right hippocampus) was aligned with the transducer. Ultrasound was applied for a duration of 120 s, resulting in a 10-millimeter diameter pulsed ultrasound beam from the transducer, which facilitated the opening of the blood-brain barrier. (Figure IE) After the final iEEG-video recordings, the seizure outcome of each mouse group was assessed based on the frequency, duration, and severity of the seizures. (Figure IF) Six months after the final treatment session, the mice were euthanized and subjected to histological examination to evaluate any long-term histological changes, including microglial activation, astrogliosis, and neuronal loss, using Ibal, GFAP, and NeuN immunostaining.

[0090] Synthesis of selenium nanoparticles

[0091] Selenium nanoparticles (SeNPs) were synthesized by the following protocol: a sodium selenite aqueous solution (5mL, 6 mM) is mixed with a Tween-20 (0.25 mL, 10 g / L in water). Ascorbic acid (4 mL, 30 mM in water) is added dropwise, and the solution is stirred at ambient temperature in the dark for 12h. SeNPs are then purified by ultrafiltration with a 100 kDa cutoff and resuspended in phosphate buffer saline. SeNPs have a typical diameter of 40-50 nm, as characterized by transmission electron microscopy and dynamic light scattering.

[0092] Low intensity pulsed ultrasound-induced BBB opening procedures

[0093] The LIPU preclinical platform (SonoCloud® Technology, Car Thera, Lyon, France) consisted of a 12 mm diameter ultrasound transducer surrounded by a cylinder of degassed and demineralized water to ensure acoustic transmission. The transducer used a center frequency of 1-MHz, pulse-repetition frequency of 1 Hz, pulse length of 25,000 cycles, and in situ acoustic pressure level of 0.3 MPa.

[0094] For all BBB opening procedures, mice’s heads hairs were shaved, and depilatory cream was applied on the skin of the sonicated area. The mouse skull bone thickness allowed LIPU- induced BBB opening without performing craniotomy. Sonications were performed under general anesthesia through intraperitoneal injection of 200 pL of a mixture of 10 mg / kg xylazine and 100 mg / kg ketamine. A 100-pL bolus of microbubbles (MB) (Sonovue®, Bracco, Milan, Italy) was injected through the right retro-orbital sinus just before starting sonications. Mice were then placed on the ultrasound platform and the head was positioned in contact with water and the targeted area (right hippocampus) was aligned with the transducer. Sonication was then launched for a duration of 120 seconds. A lOmm-diameter pulsed ultrasound beam was therefore provided by the transducer for BBB opening. When relevant, a solution of lOOpL of SeNPs (150pg / mL) or Se (150pg / mL) was injected through the left retro-orbital sinus just before injecting microbubbles (Figure 1).

[0095] Detection and dosage of intracerebral selenium

[0096] To assess the selenium concentration in the brain under different experimental conditions, thirty-six mice were allocated to several treatment groups: Sham, Se, SeNPs, LIPU+Se and LIPU+SeNPs.

[0097] The mice were euthanized either on DO (30 min after treatment) or D7, i.e., 7 days after treatment. The brains were extracted, dissolved, and diluted 100 times. Intracerebral selenium assays were performed on the solution obtained using inductively coupled plasma mass spectrometry (ICP-MS). Each group included 8 mice (4 euthanized on DO and 4 on D7), except for the sham group, which included 4 mice euthanized on DO.

[0098] Intrahippocampal injection

[0099] Mice were anesthetized with 2-4% isoflurane under analgesia (O. lmg / kg buprecare for 48 hours) and placed in a stereotaxic frame. A solution of 50 nl of kainate (KA, 20 nM in PBS) or PBS for control mice was injected (lOnl / min) into the right dorsal hippocampus (anterior- posterior, -1.8 mm; medio-lateral, -1.8 mm; dor so- ventral, -1.8 mm, from the bregma) according to the methodology described in Mathon et al. 2015 (Mathon B, Nassar M, Simonnet J, Le Duigou C, Clemenceau S, Miles R, et al. Increasing the effectiveness of intracerebral injections in adult and neonatal mice: a neurosurgical point of view. Neurosci Bull 2015;31 :685-96. https: / / doi.org / 10.1007 / sl2264-015-1558-0.). Coordinates were derived and adjusted from the Franklin and Paxinos atlas (Franklin K, Paxinos G. The mouse brain in stereotaxic coordinates . compact. 3rd ed. Waltham, MA: Academic Press. 2008).

[0100] Safety profile of LIPU-induced BBB opening combined with SeNPs administration

[0101] Four weeks after the intra hippocampal injection of KA, 12 KA mice were weighed and then separated into two groups: Sham and LIPU+SeNPs.

[0102] The mice then underwent a treatment session once per week for 3 consecutive weeks.

[0103] To assess the safety and tolerance of LIPU-induced BBB opening combined with SeNPs administration, the following endpoints were recorded three times a week from the first treatment session and until one month after the last treatment session: animal survival, animal weight, and general appearance and behavior of the animal. The qualitative and quantitative criteria were compared between the two groups. Depth electrode implantation and video-EEG recordings

[0104] To assess the epileptic behavior and seizure frequency of KA mice, they were implanted with stainless-steel electrodes (#791400; A-M Systems, Sequim, WA) inserted respectively into the right hippocampus (bipolar electrode) and the right and left motor cortex (monopolar electrodes) just after kainate injection. A reference electrode was placed on the cerebellum of each mouse. After four weeks consisting in the epileptogenesis period, implanted mice were freely moving and connected to a digital converter amplifier (Brainbox EEG- 1166), as part of a video-EEG acquisition system (Deltamed®, Natus Neuro, Paris, France). EEG signals were acquired at 4096 Hz and band-pass filtered (0.5-70 Hz). Continuous EEG and video acquisition were then performed for 8 days (baseline video- intracranial EEG (iEEG)) under infrared light conditions at night (12 / 12 hours, light on at 8:00 a.m.). Signal analyses were performed using the Deltamed® software and the mouse seizure frequency per week was determined. Focal hippocampal discharges limited to right hippocampal electrodes were not considered as seizures. The severity of seizures during video iEEG-recordings was ranked by the modified Racine scoring system as follows:

[0105] 1 : stereotypic mounting, eye blinking, and / or mild facial clonus;

[0106] 2: head nodding and / or multiple facial clonus;

[0107] 3: myoclonic jerks in the forelimbs;

[0108] 4: clonic convulsions in the forelimbs with rearing;

[0109] 5: clonic convulsions with loss of balance or jump, and

[0110] 6: generalized clonic convulsions.

[0111] Seizure outcome assessment in KA mice

[0112] Mice that experienced at least one seizure during the baseline video-iEEG were randomly assigned to a treatment group. All mice were anaesthetized (general anesthesia). Treatment groups were defined as follows:

[0113] - Sham (Control): microbubbles (MB), n=5

[0114] - LIPU (LIPU-induced BBB opening): MB + LIPU, n=10

[0115] - SeNP (Intravenous selenium nanoparticles): SeNPs (lOOpl (150pg / mL)), n=6 - SeNP + LIPU (LIPU-induced BBB opening combined with intravenous selenium nanoparticles ): SeNPs (lOOpl (150pg / mL)) + MB + LIPU. n=6

[0116] Implanted mice then underwent a treatment session once per week for 3 consecutive weeks and were recorded on the video-EEG platform for 96 hours after each session and 7 days after the third session (Figure 4).

[0117] Thus, implanted mice were clinically and electrophysiologically analyzed for 25 days from the first treatment session. On each video-iEEG recordings the number of seizures, the duration of seizures and the severity of seizures were recorded by an examiner who was blind to the mouse treatment group. The data analysis was carried out as an “intention-to treat analysis” in all mice that have completed at least the video-iEEG recordings following the first treatment session (sham, n=5; LIPU, n=10; SeNPs, n=6; LIPU+SeNPs, n=6).

[0118] Analysis of neurogenesis in the hippocampus

[0119] To assess hippocampal neurogenesis as potentially implicated in epileptogenic mechanisms, 18 KA mice and 18 wildtype (WT) mice were allocated to six treatment groups, including six mice each:

[0120] - WT mice, control (WT-sham): GA (general anesthesia),

[0121] - KA mice, control (KA-sham): GA,

[0122] - WT mice, LIPU-induced BBB opening (WT-LIPU): GA + MB + LIPU,

[0123] - KA mice, LIPU-induced BBB opening (KA-LIPU): GA + MB + LIPU,

[0124] - WT mice, LIPU-induced BBB opening combined with intravenous selenium nanoparticles (WT-LIPU+ SeNPs): GA + SeNPs (lOOpl (150pg / mL)) + MB + LIPU,

[0125] - KA mice, LIPU-induced BBB opening combined with intravenous selenium nanoparticles (KA-LIPU+ SeNPs): GA + SeNPs (lOOpl (150pg / mL)) + MB + LIPU.

[0126] Beginning 48h following the treatment session, animals were injected intraperitoneally with 5- Bromo-20-deoxyuridine (BrdU, abl42567 Abeam) at a dose of lOOmg / kg once a day for 6 days. In this study, it was decided to start BrdU inj ections 2 days after BBB opening in order to ensure that the BBB had completely closed before BrdU injections began as recommended by Mooney et al. 2016 (Mooney SJ, Shah K, Yeung S, Burgess A, Aubert I, Hynynen K. Focused Ultrasound-Induced Neurogenesis Requires an Increase in Blood-Brain Barrier Permeability. PLoS One 2016;l l :e0159892. https: / / doi.org / 10.1371 / journal.pone.0159892.) Animals survived for 12 days post BrdU treatment in order to capture the greatest time period when neurogenesis might be stimulated after treatment. Mice were deeply anaesthetized and sacrificed by an intraperitoneal overdose of pentobarbital (> 50mg / kg). Mice were perfused intracardially, and their brains were postfixed overnight into 4% paraformaldehyde. After cryoprotection with sucrose 30%, brains were frozen in isopentane (50°± 5°C). 20 pm thick coronal sections were cut on a cryostat and slides were frozen until immunocytochemistry. Systematic series of 1 in 6 sections throughout the hippocampus were immunostained. Immunohistochemical analysis was performed. Antigen retrieval was performed with citrate buffer 0.01 M pH6. Then, slices were permeabilized with Triton 0.1% 30 min. Primary antibodies were applied overnight at 4°C (DCX, 1 : 100, chicken ab 153668 Abeam; NeuN 1 :500, mouse MAB77 Merck Millipore; BrDU, 1 / 200, rabbit ab 152095 Abeam). After washes, secondary antibodies were applied for Ih at room temperature (Alexa fluor anti chicken 488, 1 :500, A-11039 Thermo Fischer; Alex fluor anti mouse 647, 1 :500, A-21240 Thermo Fischer; Alexa fluor anti rabbit 555, 1 :500, A-78954 Thermo Fischer). Then, slides were mounted with Fluoromount (F4680, Sigma Aldrich) after washes and let dry out of the light at room temperature.

[0127] The analysis of mature and immature neurons in the dentate gyrus was performed in duplicate. Following immunohistochemical analyses for DCX and BrDU, which were used to identify newborn neurons in the dentate gyrus of the hippocampus, Z-stacks of the entire dentate gyrus were obtained by using the Apotome microscope (Zeiss). Z-stacks of the images were obtained from four equally spaced sections from the hippocampus of each animal. The neuronal cell bodies were counted by using ImageJ software (NIH). Measurements of hippocampal NeuN immunostaining, which identified mature neurons in the hippocampus, were performed on five different slices through the hippocampus for each mouse and were then averaged.

[0128] Long-term histological assessment

[0129] To assess long-term histological changes, such as microglial activation, astrogliosis and neuronal loss, KA mice implanted with iEEG recording electrodes and subjected to three treatment sessions were sacrificed six months after the last treatment session. By month 6, five mice per treatment group had survived and were histologically examined. The slices chosen for immunohistochemical analyses were located between the two right hippocampal electrodes (body of the hippocampus). Animal sacrifice and brain preparation were performed as previously described. Primary antibodies were applied overnight at 4°C (GFAP, 1 :250, chicken AB5541 Merck-Millipore; Ibal 1 :250, mouse MABN92 Merck-Millipore; NeuN 1 :500, mouse MAB77 Merck Millipore). After washes, secondary antibodies were applied for Ih at room temperature (Alexa fluor anti chicken 488, anti-mouse 555 or anti rabbit 647, Thermo Fischer). Images acquisition and measurements of hippocampal immunostaining were performed as described above.

[0130] The number of mice included in each treatment group was:

[0131] - Sham: n=5

[0132] - LIPU: n=5

[0133] - SeNPs: n=5

[0134] - SeNPs + LIPU: n=5

[0135] Statistical analyses

[0136] Prism (Version 8.0.1, GraphPad Software Inc) and SPSS Statistics v22 (IBM Corp., Armonk, NY, USA) were used to perform the following statistical analyses: Wilcoxon matched-pairs signed-rank test (Paired, non-parametric test, two-tailed, confidence level 95%), Mann Whitney test (Unpaired, non-parametric test, comparing ranks, two-tailed, confidence level 95%), and repeated measures one-way ANOVA (not assuming sphericity, using the Geisser-Greenhouse correction). Statistical significance for the trend analysis for ordered alternatives was assessed using the Jonckheere-Terpstra test performed using the Matlab function from Cardillo G. (2008) Jonckheere-Terpstra test: A non-parametric test for Trend

[0137] (http: / / www.mathworks.com / matlabcentral / fileexchange / 22159). All data are presented as mean ± SEM. Results were considered significant when P < 0.05.

[0138] RESULTS

[0139] Detection and dosage of intracerebral selenium after treatment.

[0140] The selenium concentrations within the brains of mice in various treatment groups were measured using inductively coupled plasma mass spectrometry on both day 0 (30 min after treatment) and day 7 of treatment, expressed in micrograms per gram of brain tissue. (Figure 2A) The concentration of selenium (Se) in the brain was found to be significantly lower in the sham group than in the other treatment groups 30 min after treatment, whereas there was a nonsignificant trend towards higher intracerebral Se concentrations in mice treated with selenium nanoparticles and low-intensity pulsed ultrasound (SeNPs+LIPU) than in those treated with Se or SeNPs (P = 0.11). Seven days after treatment, the intracerebral Se concentration was significantly higher in the SeNPs+LIPU group than in the other groups. (Figure 2B) Furthermore, a significant decrease in the intracerebral Se concentration was observed in each mouse group between days 0 and 7. These results imply that the concurrent administration of SeNPs and LIPU-induced blood-brain barrier opening is the most effective approach for achieving both immediate and sustained delivery of Se to the brain.

[0141] LIPU, low -intensity pulsed ultrasound; Se, selenium; SeNPs, selenium nanoparticles.

[0142] * 0.01 < P < 0.05, statistics: two-tailed Mann-Whitney test.

[0143] Safety profile of LIPU-induced BBB opening combined with SeNPs administration.

[0144] Twelve kainate mice that were administered kainic acid were weighed and divided into two groups. The control group (Sham) consisted of six mice that received general anesthesia and microbubbles. The treatment group (SeNPs+LIPU), consisting of six mice, received low- intensity pulsed ultrasound-induced blood-brain barrier opening, in addition to intravenous administration of selenium nanoparticles. The mice in the treatment group received a single treatment session per week for three consecutive weeks to evaluate the safety and tolerability of low-intensity pulsed ultrasound to open the blood-brain barrier in combination with the administration of selenium nanoparticles. (Figure 3) The weight curves of mice in the treatment group did not differ significantly from those in the control group (statistics: two-tailed Mann- Whitney test). The findings suggest that the intravenous administration of selenium nanoparticles using ultrasound-induced blood-brain barrier opening at the tested dosage was safe and well tolerated.

[0145] Beneficial effect of low-intensity pulsed ultrasound-induced blood-brain barrier opening in combination with selenium nanoparticle administration on epileptic seizures in kainate mice.

[0146] 433 generalized seizures were recorded during the video-iEEG in 27 mice (Figure 4).

[0147] In Sham mice, there was no significant variation of the seizure frequency during the experiment (Figure 4A, Friedman test, P = 0.83).

[0148] In LIPU mice, there was no significant variation of the seizure frequency during the experiment (Figure 4B, Friedman test, P = 0.60).

[0149] In SeNPs mice, there was a significant variation of the seizure frequency during the experiment (Figure 4C, Friedman test, P = 0.02). There was a significant (56%) decrease of the seizure frequency between the baseline recording (before treatment) and the distant recording (Wilcoxon test, P = 0.04). A significant decrease of the seizure frequency since the second treatment session was observed (Wilcoxon test, P = 0.03). In SeNPs+LIPU mice, there was a significant variation of the seizure frequency during the experiment (Figure 4D, Friedman test, P = 0.001). There was a significant (90%) decrease of the seizure frequency between the baseline recording (before treatment) and the distant recording (Wilcoxon test, P = 0.03). A significant decrease of the seizure frequency since the first treatment session was observed (Wilcoxon test, P = 0.03).

[0150] Overall, the generalized seizure frequency variations between the baseline recording (before treatment) and the distant recording were significantly different between treatment groups (Kruskall -Wallis test, P = 0.02). There was a decrease of the seizure frequency that was significantly more important in the SeNPs+LIPU group compared to the Sham group (Mann- Whitney-Wilcoxon test, P = 0.004), and non-significant trends compared to the SeNPs (Mann- Whitney-Wilcoxon test, P = 0.25) and the LIPU (Mann-Whitney-Wilcoxon test, P = 0.06) groups.

[0151] Conclusions

[0152] The main result of this experiment is the significant (-90%) and prolonged reduction in the frequency of generalized seizures from the first session of opening the BBB coupled with the intravenous administration of SeNPs. This experiment shows that the transitory opening of the BBB in the hippocampal area tends to decrease the generalized epileptic seizures in kainate mice. The results further indicate that repeated opening of the BBB decrease the clinical severity of the seizures.

[0153] Low-intensity pulsed ultrasound-induced blood-brain barrier opening promotes newborn neuron formation and limits mature neuron apoptosis in the dentate gyrus of wild-type mice.

[0154] The results indicated a notable increase in the fluorescence intensity of NeuN immunolabeling within the hippocampus of mice that underwent LIPU-mediated blood-brain barrier opening, both with and without SeNP administration, in contrast to the sham group (Figure 5A). This finding suggests a decrease in apoptosis of mature neurons. Immunohistochemical analysis of wild-type mice treated with LIPU in combination with or without SeNPs revealed a significant increase in the number of BrDU-expressing immature neurons in the dentate gyrus compared with the control group (Figure 5B). Similarly, an increase in the number of DCX-positive cells was observed, indicating enhanced neurogenesis (Figure 5C).

[0155] The adjunction of selenium nanoparticles with low-intensity pulsed ultrasound-induced blood-brain barrier opening leads to a decrease in neurogenesis in the dentate gyrus and minimizes hippocampal neuronal death in kainate mice. Hippocampal NeuN immunostaining fluorescence intensity was found to be notably higher in mice from the LIPU and SeNPs + LIPU groups than in those from the sham group, suggesting a decrease in neuronal loss (Figure 6A). There was no statistically significant difference in the number of BrdU-positive cells in the dentate gyrus between the various groups (Figure 6B). Conversely, the number of DCX-positive cells in the dentate gyrus was significantly lower in the SeNPs + LIPU group than in the sham and LIPU groups (Figure 6C), which suggests a decrease in spontaneous neurogenesis in the hilus of kainate mice induced by the combination of LIPU and SeNPs.

[0156] Long-term beneficial effect of low-intensity pulsed ultrasound-induced blood-brain barrier opening in combination with selenium nanoparticle administration on hippocampal inflammation in kainate mice.

[0157] Six months after the end of the different treatment regimens, the kainate mice were euthanized to assess the extent of hippocampal microglial activation, astrogliosis, and neuronal loss (Figure 7). No significant differences in fluorescence intensity were observed between the treatment groups, as evaluated by immunostaining with GFAP (Figure 7A) and NeuN (Figure 7C) antibodies. However, the SeNPs+LIPU group displayed a significantly lower Ibal immunostaining intensity in the hippocampus than all other groups (Figure 7B), suggesting a long-term reduction in hippocampal microglial activation.

[0158] GFAP, Glial fibrillary acidic protein; Ibal, ionized calcium-binding adapter molecule 1; LIPU, low-intensity pulsed ultrasound; NeuN, neuronal nuclei; SeNPs, selenium nanoparticles.

[0159] Long-term histological assessment

[0160] The fluorescence intensity of the GFAP immunostaining in the hippocampus did not differ significantly between treatment groups (Kruskall-Wallis test, P = 0.73) (Figure 7A).

[0161] The fluorescence intensity of the NeuN immunostaining in the hippocampus did not differ significantly between treatment groups (Kruskall-Wallis test, P = 0.19). However, the intensity of the immunostaining is significantly higher in the SeNPs group than in the Sham group (P = 0.03) (Figure 7C).

[0162] The fluorescence intensity of the Ibal immunostaining in the hippocampus did differ significantly between the highest treatment groups (Kruskall-Wallis test, P = 0.006). Notably, the intensity of the immunostaining is significantly lower in the LIPU+SeNPs group than in the Sham group (4.2 times lower, P = 0.008), than in the LIPU group (3.7 times lower, P = 0.008), than in the SeNPs group (2.2 times lower, P = 0.03) (Figure 7B). Conclusions

[0163] A lower microglial activation was observed in mice treated with both ultrasound and selenium nanoparticles than the other mice groups. This confirms that anti-inflammatory effect - the reduction of the microglial activation - is caused by the selenium nanoparticles.

[0164] Astrogliosis was not changed, on the long term, by the different treatments administered. As a reminder, astrogliosis was strongly increased in KA mice (82,265,266).

[0165] Regarding the assessment of the neuronal population by the NeuN immunostaining, the analysis showed that the treatments consisting in BBB opening by ultrasound, the administration of selenium nanoparticles, or both lead to a lower neuronal mortality than in KA Sham mice.

[0166] CONCLUSIONS

[0167] A previous study (Mathon B, et al. “Safety Profile of Low-Intensity Pulsed Ultrasound-Induced Blood-Brain Barrier Opening in Non-epileptic Mice and in a Mouse Model of Mesial Temporal Lobe Epilepsy.” Ultrasound Med Biol. 2023 May;49(5): 1327-1336.) demonstrated that LIPU, administered using the SonoCLOUD® preclinical platform, open the BBB in a transitory and reproductible manner in KA model mice. This occurs also when the mice are implanted with electrodes for intracerebral EEG recording.

[0168] It has now been demonstrated that repeated opening of the BBB associated with the IV administration of selenium nanoparticles is safe, well tolerated and allows a reduction in generalized epileptic seizures of about 90%.

[0169] In particular, the experimental data above confirmed that the intravenous administration of selenium nanoparticles associated with the BBB opening by ultrasound is the most effective technique to obtain a high and persisting concentration of selenium in the brain, and that this method is safe for the subject. The data further show that this method allows to significantly reduce the generalized epileptic seizures in the subject. Importantly, the data show the existence of short-term histological changes (e.g., reduction in aberrant neurogenesis in the hippocampal hilum, decrease in neuron mortality in the entire hippocampus) as well as long-term histological changes (e.g., decrease of the microglial activation in the hippocampus).

Claims

CLAIMS1. Selenium nanoparticles for use in preventing, reducing risk of developing or treating epilepsy in a subject in need thereof, wherein selenium nanoparticles are administered parenterally to the subject, together with a transient disruption of the blood-brain barrier (BBB) of the subject.

2. Selenium nanoparticles for use in preventing, reducing risk of developing or treating epilepsy in a subject in need thereof, according to claim 1, wherein selenium nanoparticles are administered intravenously.

3. Selenium nanoparticles for use in preventing, reducing risk of developing or treating epilepsy in a subject in need thereof, according to claim 1 or 2, wherein the BBB disruption is performed by sonication.

4. Selenium nanoparticles for use in preventing, reducing risk of developing or treating epilepsy in a subject in need thereof, according to claim 3, wherein an ultrasound contrast agent is administered before and / or during the application to the central nervous system (CNS) of the subject, of ultrasounds (US), preferably low intensity pulsed ultrasounds in order to transiently disrupt the BBB of the subject.

5. Selenium nanoparticles for use in preventing, reducing risk of developing or treating epilepsy in a subject in need thereof according to claim 4, wherein the ultrasound contrast agent is administered parenterally, preferably intravenously.

6. Selenium nanoparticles for use in preventing, reducing risk of developing or treating epilepsy in a subject in need thereof, according to claim 4 or 5, wherein the selenium nanoparticles are administered to the subject before, after and / or during the administration of the ultrasound contrast agent.

7. Selenium nanoparticles for use in preventing, reducing risk of developing or treating epilepsy in a subject in need thereof according to any one of claims 1 to 6, wherein the selenium nanoparticles are administered at a dosage between 0.1 and 0.5mg / kg per administration, preferably between 0.1 and 0.15mg / kg per administration.

8. Selenium nanoparticles for use in preventing, reducing risk of developing or treating epilepsy in a subject in need thereof according to any one of claims 1 to 7, wherein the selenium nanoparticle size is between 20 to 80 nm, preferably between 40 to 50 nm.

9. Selenium nanoparticles for use in preventing, reducing risk of developing or treating epilepsy in a subject in need thereof according to any one of claims 1 to 8, wherein the selenium nanoparticles are administered to the subject before, after and / or during disruption of the BBB.

10. Selenium nanoparticles for use in preventing, reducing risk of developing or treating epilepsy in a subject in need thereof according to any one of claims 1 to 9, wherein at least two doses of selenium nanoparticles are administered to the subject, and wherein a latency period between two successive doses of selenium nanoparticles is preferably of at least one week and at most 9 weeks, preferably between 1 to 4 weeks, more preferably between 1 to 2 weeks.

11. Selenium nanoparticles for use in preventing, reducing risk of developing or treating epilepsy in a subject in need thereof according to claim 10, wherein the BBB is disrupted after and / or during each administration of selenium nanoparticles.

12. Selenium nanoparticles for use in preventing, reducing risk of developing or treating epilepsy in a subject in need thereof according to claim 10 or 11, wherein three doses of selenium nanoparticles are administered successively to the subject.

13. Selenium nanoparticles for use in preventing, reducing risk of developing or treating epilepsy in a subject in need thereof according to any one of claims 10 to 12, wherein the latency period between the administration of two successive doses of selenium nanoparticles is 7 days, + / - 2 days.

14. Selenium nanoparticles for use in preventing, reducing risk of developing or treating epilepsy in a subject in need thereof according to any one of claims 1 to 13, wherein the subject is a subject having a diagnosed epilepsy, in particular a subject with epileptic seizures.

15. Selenium nanoparticles for use in preventing, reducing risk of developing or treating epilepsy in a subject in need thereof, according to any one of claims 1 to 14, wherein the epilepsy is a cryptogenic focal epilepsy, secondarily generalized epilepsy or lesional focal epilepsy.

16. Selenium nanoparticles for use in preventing, reducing risk of developing or treating epilepsy in a subject in need thereof, according to any one of claims 1 to 15, wherein the epilepsy is a temporal lobe epilepsy, preferably a mesial temporal lobe epilepsy, a chronic epilepsy, and / or a drug-resistant epilepsy.

17. Selenium nanoparticles for use in preventing, reducing risk of developing or treating epilepsy in a subject in need thereof according to any one of claims 1 to 16, wherein the subject is a human.

18. A method for preventing, reducing risk of developing or treating epilepsy in a subject in need thereof, wherein selenium nanoparticles (SeNPs) are administered parenterally to the subject, together with a transient disruption of the blood-brain barrier (BBB) of the subject.

19. The method of claim 18, wherein the BBB disruption is performed by sonication.

20. The method of claim 19, wherein an ultrasound contrast agent is administered before and / or during the application to the central nervous system (CNS) of the subject, of ultrasounds (US), preferably low intensity pulsed ultrasounds in order to transiently disrupt the BBB of the subject.

21. Use of selenium nanoparticles (SeNPs) in the manufacture of a medicament for preventing, reducing risk of developing or treating epilepsy in a subject in need thereof, wherein selenium nanoparticles are administered parenterally to the subject, together with a transient disruption of the blood-brain barrier (BBB) of the subject.

22. The use of claim 21, wherein the BBB disruption is performed by sonication.

23. The use of claim 22, wherein an ultrasound contrast agent is administered before and / or during the application to the central nervous system (CNS) of the subject, of ultrasounds (US), preferably low intensity pulsed ultrasounds in order to transiently disrupt the BBB of the subject.

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