WO2024246162A1 - Procédé et composition pharmaceutique destinés à être utilisés dans le traitement de la dysplasie corticale focale - Google Patents

Procédé et composition pharmaceutique destinés à être utilisés dans le traitement de la dysplasie corticale focale Download PDF

Info

Publication number
WO2024246162A1
WO2024246162A1 PCT/EP2024/064839 EP2024064839W WO2024246162A1 WO 2024246162 A1 WO2024246162 A1 WO 2024246162A1 EP 2024064839 W EP2024064839 W EP 2024064839W WO 2024246162 A1 WO2024246162 A1 WO 2024246162A1
Authority
WO
WIPO (PCT)
Prior art keywords
gluk2
inhibitor
fcd
epilepsy
treatment
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2024/064839
Other languages
English (en)
Inventor
Valérie CREPEL
Christophe MULLE
Céline BOILEAU
Alfonso REPRESA-BERMEJO
Séverine DEFORGES
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Aix Marseille Universite
Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
Universite de Bordeaux
Original Assignee
Aix Marseille Universite
Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
Universite de Bordeaux
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Aix Marseille Universite, Centre National de la Recherche Scientifique CNRS, Institut National de la Sante et de la Recherche Medicale INSERM, Universite de Bordeaux filed Critical Aix Marseille Universite
Publication of WO2024246162A1 publication Critical patent/WO2024246162A1/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system

Definitions

  • the present invention relates to an inhibitor of GluK2-containing kainate receptors (KARs) for use in the treatment of focal cortical dysplasia (FCD) in a subject in need thereof.
  • KARs GluK2-containing kainate receptors
  • FCD Focal Cortical Dysplasia
  • FCDI cortical dyslamination
  • FCDIIA dysmorphic cytomegalic neurons
  • FCDIIB balloon cells
  • FCDII mammalian target of rapamycin
  • GluK2-containing kainate receptors play a key role in epileptiform activities in neocortex in an animal model of type II FCD (FCDII) demonstrating that KARs can constitute a major antiepileptic target to treat chronic epilepsy in FCD.
  • the present invention relates to an inhibitor of GluK2-containing kainate receptors (KARs) for use in the treatment of focal cortical dysplasia (FCD) in a subject in need thereof.
  • KARs GluK2-containing kainate receptors
  • a first object of the invention relates to an inhibitor of GluK2-containing kainate receptors (KARs) for use in the treatment of focal cortical dysplasia (FCD) in a subject in need thereof.
  • FCD focal cortical dysplasia
  • the patient with FCD has an epilepsy.
  • the invention relates to an inhibitor of GluK2-containing KARs for use in the treatment of epilepsy in patient with FCD.
  • the inhibitor of the invention can prevent either the activation, the expression, or the formation of GluK2, or of the heteromeric complex GluK2/GluK5.
  • the inhibitor of the invention can also prevent either the activation, the expression, or the formation of GluK2-containing KARs (i.e heteromeric complexes GluK2/GluKl, GluK2/GluK3 or GluK2/GluK4).
  • the invention relates to an inhibitor of the heteromeric complex GluK2/GluK5 for use in the treatment of focal cortical dysplasia (FCD) in a subject in need thereof.
  • FCD focal cortical dysplasia
  • GluK2-containing kainate receptors denotes all heteromeric receptor containing at least one unit of the subunit GluK2 (including the heteromeric complex GluK2/GluKl, GluK2/GluK3, GluK2/GluK4 or GluK2/GluK5).
  • GluK2 also known as GluR6
  • KAR kainate receptor
  • GluK2/GluK5 receptor denotes a heteromeric complex formed by the kainate receptor subunits GluK2 and GluK5 [see for example Reiner et al., 2012, Ruiz et al, 2005],
  • the IUPHAR-2009-term “GluKl”, “Glut”, GluK3”, “GluK4” or “GluK5” denotes subunits of the kainate receptor family of ionotropic glutamate receptors.
  • the term “inhibitor of GluK2-containing kainate receptors” or “inhibitor of the receptor GluK2” denotes a compound or molecule which prevents the activation of the pathway receptor or a compound or molecule which prevents the formation of the Glutcontaining kainate receptors or a compound or molecule which prevents the synaptic localization of Glut-containing kainate receptors.
  • the inhibitor of the invention may be a molecule which binds to the Glut KAR subunit receptor to prevent the activation or the formation of the Glut-containing KARs (including the heteromeric complex GluK2/GluK5).
  • the term “inhibitor of the receptor GluK2” also denotes an inhibitor of the expression of grik2, the gene coding for the protein GluK2.
  • FCD Fluorescence Cortical Dysplasia
  • epilepsy chronic and recurrent seizures
  • the epilepsy can be classified according to the electroclinical syndromes following the Classification and Terminology of the International League against Epilepsy (ILAE) [Berg et al., 2010], These syndromes can be categorized by age at onset, distinctive constellations, including epileptic syndromes that can benefit from surgery, and structural-metabolic causes: (A) age at onset: (i) neonatal period includes Benign familial neonatal epilepsy (BFNE), Early myoclonic encephalopathy (EME), Ohtahara syndrome, (ii) Infancy period includes Epilepsy of infancy with migrating focal seizures, West syndrome, Myoclonic epilepsy in infancy (MEI), Benign infantile epilepsy, Benign familial infantile epilepsy, Dravet syndrome, Myoclonic encephalopathy in nonprogressive disorders, (iii) Childhood period includes Febrile seizures plus (FS+), Panayiotopoulos syndrome, Epilepsy with myoclonic atonic (previous
  • Distinctive constellations include Mesial Temporal Lobe Epilepsy (MTLE), Rasmussen syndrome, Gelastic seizures with hypothalamic hamartoma, Hemi convulsionhemiplegia-epilepsy.
  • C Epilepsies attributed to and organized by structural-metabolic causes include Malformations of cortical development (Focal cortica dysplasia, Hemimegalencephaly, heterotopias, etc.), Neurocutaneous syndromes (tuberous sclerosis complex,Sturge-Weber, etc.), Tumor, Infection, Trauma, Angioma, Perinatal insults, Stroke, Etc.
  • the epilepsy may be a benign Rolandic epilepsy, a frontal lobe epilepsy, an infantile spasms, a juvenile myoclonic epilepsy, a juvenile absence epilepsy, a childhood absence epilepsy (pyknolepsy), a hot water epilepsy, a Lennox-Gastaut syndrome, a Landau-Kleffner syndrome, a Dravet syndrome, a progressive myoclonus epilepsies, a reflex epilepsy, a Rasmussen’s syndrome, a temporal lobe epilepsy, a limbic epilepsy, a status epilepticus, an abdominal epilepsy, a massive bilateral myoclonus, a catamenial epilepsy, a Jacksonian seizure disorder, a Lafora disease or photosensitive epilepsy.
  • the epilepsy is a chronic epilepsy.
  • the epilepsy can be a refractory epilepsy.
  • refractory epilepsy denotes an epilepsy which is refractory to current pharmaceutical treatment, that is to say that current pharmaceutical treatment doesn’t allow a treatment of patients (see for example Englot et al., 2014).
  • the refractory epilepsy is a chronic refractory epilepsy.
  • treatment refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
  • the treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • therapeutic regimen is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
  • a therapeutic regimen may include an induction regimen and a maintenance regimen.
  • the phrase “induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease.
  • the general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen.
  • An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • maintenance regimen refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years).
  • a maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).
  • a subject denotes a mammal, such as a rodent, a feline, a canine, and a primate.
  • a subject according to the invention is a human. More particularly, the subject is suffering from FCD or from an epilepsy with FCD.
  • the inhibitor according to the invention may be a low molecular weight compound, e. g. a small organic molecule (natural or not).
  • small organic molecule refers to a molecule (natural or not) of a size comparable to those organic molecules generally used in pharmaceuticals.
  • Preferred small organic molecules range in size up to about 10000 Da, more preferably up to 5000 Da, more preferably up to 2000 Da and most preferably up to about 1000 Da.
  • the inhibitor according to the invention is selected from the group consisting of UBP310, UBP302, UBP316 and NBQX.
  • the inhibitor according to the invention is UBP310 an antagonist of the heteromeric kainate GluK2/GluK5 receptor [Pinheiro et al., 2013],
  • the inhibitor according to the invention is (2S,4R)-4- Methylglutamate (SYM2081), a inhibitor that is a potent broad-spectrum agonist of kainate receptors; it binds to homomeric and heteromeric kainate receptors including homomeric GluKl, and GluK2 receptors and heteromeric GluKl/GluK2, GluKl/GluK5 and GluK2/GluK5 receptors [Jane et al., 2009],
  • This inhibitor is currently used to selectively block kainate receptor signaling via desensitization of the receptors in control and epileptic conditions [Jane et al., 2009] [Li et al., 1999; Cossart et al., 2002; Epsztein et al., 2005; Epsztein et al., 2010; Joseph et al., 2011],
  • the inhibitor according to the invention is an antibody.
  • Antibodies directed against GluK2-containing kainate receptors can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others.
  • a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others.
  • Various adjuvants known in the art can be used to enhance antibody production.
  • antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred.
  • Monoclonal antibodies against GluK2-containing kainate receptors can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture.
  • Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975); the human B-cell hybridoma technique (Cote et al., 1983); and the EBV-hybridoma technique (Cole et al. 1985).
  • techniques described for the production of single chain antibodies can be adapted to produce anti- Glutcontaining kainate receptors single chain antibodies.
  • Coumpounds useful in practicing the present invention also include anti- Glut-containing kainate receptors antibody fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to GluK2-containing kainate receptors.
  • Humanized anti- GluK2-containing kainate receptors antibodies and antibody fragments therefrom can also be prepared according to known techniques.
  • “Humanized antibodies” are forms of non-human (e.g., rodent) chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDRs) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity.
  • donor antibody such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity.
  • framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the inhibitor according to the invention is an aptamer.
  • Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition.
  • Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.
  • Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990.
  • the random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence.
  • Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996).
  • the inhibitor according to the invention is a polypeptide.
  • the polypeptide is an antagonist of GluK2-containing kainate receptors and is capable to prevent the function of GluK2-containing kainate receptors and particularly the recruitment or stabilisation of synaptic GluK2/GluK5 receptors or KARs.
  • the polypeptide can be a mutated GluK2 subunit protein or a similar protein without the function of GluK2 subunit.
  • the polypeptides of the invention may be produced by any suitable means, as will be apparent to those of skill in the art.
  • polypeptide or functional equivalents thereof for use in accordance with the present invention, expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide of the invention.
  • the polypeptide is produced by recombinant means, by expression from an encoding nucleic acid molecule.
  • Systems for cloning and expression of a polypeptide in a variety of different host cells are well known.
  • the polypeptide is preferably generated by expression from an encoding nucleic acid in a host cell. Any host cell may be used, depending upon the individual requirements of a particular system. Suitable host cells include bacteria mammalian cells, plant cells, yeast and baculovirus systems.
  • Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells. HeLa cells, baby hamster kidney cells and many others. Bacteria are also preferred hosts for the production of recombinant protein, due to the ease with which bacteria may be manipulated and grown. A common, preferred bacterial host is E coli.
  • polypeptides used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy. Such modification of therapeutic inhibitors may be used to decrease toxicity, increase circulatory time, or modify biodistribution. For example, the toxicity of potentially important therapeutic inhibitors can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution. In example adding dipeptides can improve the penetration of a circulating agent in the eye through the blood retinal barrier by using endogenous transporters.
  • a strategy for improving drug viability is the utilization of water-soluble polymers.
  • Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body.
  • water- soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain.
  • Polyethylene glycol (PEG) has been widely used as a drug carrier, given its high degree of biocompatibility and ease of modification. Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity.
  • PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule.
  • copolymers of PEG and amino acids were explored as novel biomaterials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications.
  • Those of skill in the art are aware of PEGylation techniques for the effective modification of drugs. For example, drug delivery polymers that consist of alternating polymers of PEG and tri -functional monomers such as lysine have been used by VectraMed (Plainsboro, N.J.).
  • the PEG chains (typically 2000 daltons or less) are linked to the a- and e-amino groups of lysine through stable urethane linkages.
  • Such copolymers retain the desirable properties of PEG, while providing reactive pendent groups (the carboxylic acid groups of lysine) at strictly controlled and predetermined intervals along the polymer chain.
  • the reactive pendent groups can be used for derivatization, cross-linking, or conjugation with other molecules.
  • These polymers are useful in producing stable, long-circulating pro-drugs by varying the molecular weight of the polymer, the molecular weight of the PEG segments, and the cleavable linkage between the drug and the polymer.
  • the molecular weight of the PEG segments affects the spacing of the drug/linking group complex and the amount of drug per molecular weight of conjugate (smaller PEG segments provides greater drug loading).
  • increasing the overall molecular weight of the block co-polymer conjugate will increase the circulatory half-life of the conjugate.
  • the conjugate must either be readily degradable or have a molecular weight below the threshold-limiting glomular filtration (e.g., less than 60 kDa).
  • linkers may be used to maintain the therapeutic agent in a pro-drug form until released from the backbone polymer by a specific trigger, typically enzyme activity in the targeted tissue.
  • tissue activated drug delivery is particularly useful where delivery to a specific site of biodistribution is required and the therapeutic agent is released at or near the site of pathology.
  • Linking group libraries for use in activated drug delivery are known to those of skill in the art and may be based on enzyme kinetics, prevalence of active enzyme, and cleavage specificity of the selected disease-specific enzymes. Such linkers may be used in modifying the protein or fragment of the protein described herein for therapeutic delivery.
  • the inhibitor GluK2-containing kainate receptors according to the invention is an inhibitor of GluK2 subunit gene expression.
  • Small inhibitory RNAs can also function as inhibitors of GluK2 subunit expression for use in the present invention.
  • GluK2 subunit gene expression can be reduced by a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that GluK2 subunit gene expression is specifically inhibited (i.e. RNA interference or RNAi).
  • dsRNA small double stranded RNA
  • RNAi RNA interference
  • Methods for selecting an appropriate dsRNA or dsRNA- encoding vector are well known in the art for genes whose sequence is known (e.g. see for example Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ.
  • Ribozymes can also function as inhibitors of GluK2 subunit gene expression for use in the present invention.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of GluK2 mRNA sequences are thereby useful within the scope of the present invention.
  • ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.
  • antisense oligonucleotides and ribozymes useful as inhibitors of G-CSF gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, antisense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life.
  • Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-O-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.
  • Antisense oligonucleotides, siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector.
  • a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably cells expressing GluK2.
  • the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector.
  • the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences.
  • Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40- type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus.
  • retrovirus such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus
  • adenovirus adeno-associated virus
  • SV40- type viruses polyoma viruses
  • Epstein-Barr viruses Epstein-Barr viruses
  • papilloma viruses herpes virus
  • Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA.
  • Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle).
  • retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo.
  • viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy.
  • the adeno- associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species.
  • the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection.
  • wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event.
  • the adeno-associated virus can also function in an extrachromosomal fashion.
  • Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al., 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigenencoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid.
  • Plasmids may be delivered by a variety of parenteral, mucosal and topical routes.
  • the DNA plasmid can be injected by intramuscular, eye, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally.
  • the plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and mi croencap sul ati on .
  • the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter.
  • the promoter can also be, e.g., a viral promoter, such as CMV promoter or any synthetic promoters.
  • an endonuclease can be used to abolish the expression of the gene, transcript or protein variants of GluK2 subunit.
  • the endonuclease is CRISPR-cas.
  • CRISPR-cas has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences.
  • the endonuclease is CRISPR-cas9 which is from Streptococcus pyogenes.
  • the CRISPR/Cas9 system has been described in US 8697359 Bl and US 2014/0068797. Originally an adaptive immune system in prokaryotes (Barrangou and Marraffini, 2014), CRISPR has been recently engineered into a new powerful tool for genome editing. It has already been successfully used to target important genes in many cell lines and organisms, including human (Mali et al., 2013, Science, Vol. 339 : 823-826), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol.
  • the endonuclease is CRISPR-Cpfl which is the more recently characterized CRISPR from Provotella and Francisella 1 (Cpfl) in Zetsche et al. (“Cpfl is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).
  • one object of the present invention relates to a method of treating a focal cortical dysplasia (FCD) in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an inhibitor of the GluK2-containing KARs.
  • FCD focal cortical dysplasia
  • both in vitro and/or in vivo systems can be used, such as cell lines, primary neuronal cultures, a recombinant system (Pinheiro et al., 2013), acute or organotypic cortical slices for in vitro systems, and rodent models of FCD as an in vivo system.
  • the efficacy can be determined using different methods.
  • Inhibitor of the invention may be administered in the form of a pharmaceutical composition, as defined below.
  • the invention in another aspect, relates to a therapeutic composition comprising an inhibitor of the GluK2-containing KARs for use in the treatment of focal cortical dysplasia (FCD) in a subject in need thereof.
  • FCD focal cortical dysplasia
  • the inhibitor of the GluK2-containing KARs or the therapeutic composition of the invention are administrated in a therapeutically effective amount.
  • Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.
  • the term "therapeutically effective amount” or “effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result.
  • a therapeutically effective amount of the inhibitor or the composition of the present invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the inhibitor or the composition of the present invention to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the inhibitor or the composition are outweighed by the therapeutically beneficial effects.
  • the efficient dosages and dosage regimens for the inhibitor or the composition of the present invention depend on the disease or condition to be treated and may be determined by the persons skilled in the art.
  • a physician having ordinary skill in the art may readily determine and prescribe the effective amount of the inhibitor or the composition of the invention required.
  • the physician could start doses of the inhibitor or the composition of the present invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • a suitable dose of the inhibitor or the composition of the present invention will be that amount of the inhibitor which is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen.
  • Such an effective dose will generally depend upon the factors described above.
  • a therapeutically effective amount for therapeutic use may be measured by its ability to stabilize the progression of disease.
  • the ability of a inhibitor to inhibit cancer may, for example, be evaluated in an animal model system predictive of efficacy in human tumors.
  • this property of a composition may be evaluated by examining the ability of the inhibitor to induce cytotoxicity by in vitro assays known to the skilled practitioner.
  • a therapeutically effective amount of a therapeutic inhibitor may decrease tumor size, or otherwise ameliorate symptoms in a subject.
  • One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.
  • An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present invention is about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example about 0.1-20 mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, about such as 0.3, about 1, about 3 mg/kg, about 5 mg/kg or about 8 mg/kg.
  • An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the present invention is 0.02-100 mg/kg, such as about 0.02-30 mg/kg, such as about 0.05-10 mg/kg or 0.1-3 mg/kg, for example about 0.5-2 mg/kg. Administration may e.g.
  • the efficacy of the treatment is monitored during the therapy, e.g. at predefined points in time. In some embodiments, the efficacy may be monitored by visualization of the disease area, or by other diagnostic methods described further herein, e.g.
  • an effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • the monoclonal antibodies of the present invention are administered by slow continuous infusion over a long period, such as more than 24 hours, in order to minimize any unwanted side effects.
  • An effective dose of an antibody of the present invention may also be administered using a weekly, biweekly or triweekly dosing period.
  • the dosing period may be restricted to, e.g., 8 weeks, 12 weeks or until clinical progression has been established.
  • treatment according to the present invention may be provided as a daily dosage of an antibody of the present invention in an amount of about 0.1-100 mg/kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45,
  • Administration may be intravenous, intramuscular, intraperitoneal, intratumoral, intracerebral injection (also known as intraparenchymal injection) or subcutaneous, and for instance administered proximal to the site of the target.
  • Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • the efficacy of the treatment is monitored during the therapy, e.g. at predefined points in time.
  • the efficacy may be monitored by visualization of the disease area, or by other diagnostic methods described further herein, e.g. by performing one or more PET-CT scans.
  • an effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • the oligomers of the present invention are administered by slow continuous infusion over a long period, such as more than 24 hours, in order to minimize any unwanted side effects.
  • An effective dose of the inhibitor of the present invention may also be administered using a weekly, biweekly or triweekly dosing period.
  • the dosing period may be restricted to, e.g., 8 weeks, 12 weeks or until clinical progression has been established.
  • treatment according to the present invention may be provided as a daily dosage of the inhibitor of the present invention in an amount of about 0.1-100 mg/kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses every 24, 12,
  • the quantity of the immune cell or the population of immune cells administered to a subject in need thereof is between 10 4 to 10 9 cells per kg.
  • the quantity of cells injected is 10 6 or 10 7 cells per kg.
  • the immune cell or the population of immune cells of the invention can be administrated is 1, 2, 3, 4 or 5 times to the subject in need thereof.
  • compositions for example, the form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the subject, etc.
  • compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular, intracerebral (also known as intraparenchymal) or subcutaneous administration and the like.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze- dried compositions.
  • these may be in organic solvent such as DMSO, ethanol which upon addition, depending on the case, of sterilized water or physiological saline permit the constitution of injectable solutions.
  • compositions include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.
  • the inhibitorcells of the invention are delivered in a manner consistent with conventional methodologies associated with management of the disease or disorder for which treatment is sought.
  • an effective amount of the inhibitor cells of the invention administered to a subject in need of such treatment for a time and under conditions sufficient to prevent or treat the disease or disorder.
  • Nanocapsules can generally entrap compounds in a stable and reproducible way. To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 pm) are generally designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles may be easily made.
  • Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs)). MLVs generally have diameters of from 25 nm to 4 pm.
  • Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core.
  • SUVs small unilamellar vesicles
  • the physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations.
  • the inhibitor or the composition according to the invention may be delivered directly into the brain to directly reach neurons ⁇
  • Methods of delivery of the inhibitor or the composition of the invention to neurons and/or astrocytes includes generally any method suitable for delivery the inhibitor or the composition of the invention to the neurons such that at least a portion of cells of a selected synaptically connected cell population is transduced.
  • the inhibitor or the composition of the invention may be delivered to neuronal cells of the central nervous system.
  • the inhibitor or the composition of the invention is preferentially delivered to cortical neurons but without excluding the possibility of delivering the inhibitor or composition of the invention to neurons in other areas of the central nervous system such as neurons in the spinal cord, brainstem (medulla, pons, and midbrain), cerebellum, diencephalon (thalamus, hypothalamus), telencephalon (hippocampus, corpus striatum, cerebral cortex), or combinations thereof, or preferably any suitable subpopulation thereof.
  • the inhibitor or the composition of the invention may be administered by stereotaxic microinjection.
  • patients have the stereotactic frame base fixed in place (screwed into the skull).
  • the brain with stereotactic frame base (MRI compatible with fiducial markings) is imaged using high resolution MRI.
  • the MRI images are then transferred to a computer which runs stereotactic software.
  • a series of coronal, sagittal and axial images are used to determine the target (site of inhibitor or composition injection) and trajectory.
  • the software directly translates the trajectory into 3 dimensional coordinates appropriate for the stereotactic frame. Burr holes are drilled above the entry site and the stereotactic apparatus positioned with the needle implanted at the given depth.
  • the inhibitor or the composition of the invention is then injected at the target sites.
  • Additional routes of administration may also comprise local application of the inhibitor or the composition of the invention under direct visualization, e. g., superficial cortical application, or other non-stereotactic application.
  • the inhibitor or the composition of the invention may be delivered intrathecally, in the ventricle or by intravenous injection.
  • the target cells of the inhibitor or the composition of the invention of the present invention are cells of the central nervous systems of a subject afflicted with FCD disease.
  • the subject is a human being, commonly children and adolescents under 18 years particularly.
  • the invention encompasses delivering the inhibitor or the composition of the invention to biological models of the disease.
  • the biological model may be any mammal at any stage of development at the time of delivery, e. g., embryonic, fetal, infantile, juvenile or adult, preferably it is an adult.
  • the target CNS cells may be essentially from any source, especially nonhuman primates and mammals of the orders Rodenta (mice, rats, rabbit, hamsters), Carnivora (cats, dogs), and Arteriodactyla (cows, pigs, sheep, goats, horses) as well as any other non-human system (e. g. zebrafish model system).
  • the method of the invention comprises intracerebral administration through stereotaxic injections.
  • other known delivery methods may also be adapted in accordance with the invention.
  • the inhibitor or the composition of the invention may be injected into the cerebrospinal fluid, e. g., by lumbar puncture.
  • the inhibitor or the composition of the invention may be injected into the spinal cord or into the peripheral ganglia, or the flesh (subcutaneously or intramuscularly) of the body part of interest.
  • the inhibitor or the composition of the invention can be administered via an intravascular approach.
  • the inhibitor or the composition of the invention can be administered intra-arterially (carotid) in situations where the blood-brain barrier is disturbed or not disturbed.
  • the inhibitor or the composition of the invention can be administered during the "opening" of the blood-brain barrier achieved by infusion of hypertonic solutions including mannitol.
  • the inhibitor of the invention may be further identified by screening methods as hereinafter described.
  • Another object of the invention relates to a method for screening an inhibitor of the GluK2-containing kainate receptors.
  • the invention provides a method for screening an inhibitor which is an inhibitor of the GluK2-containing kainate receptors for use in the treatment of FCD.
  • the screening method may measure the binding of a candidate inhibitor of the GluK2-containing kainate receptors, or to cells or membranes bearing the GluK2-containing kainate receptors receptor or a fusion protein thereof by means of a label directly or indirectly associated with the candidate inhibitor.
  • a screening method may involve measuring or, qualitatively or quantitatively, detecting the competition of binding of a candidate inhibitor to the receptor with a labelled competitor (e.g., antagonist).
  • the screening method of the invention comprises the step consisting of: a) providing a plurality of cells expressing the GluK2-containing kainate receptors: b) incubating said cells with a candidate inhibitor ; c) determining whether said candidate inhibitor binds to the GluK2-containing kainate receptors or inhibit the expression of the GluK2 subunit gene of the receptor; and d) selecting the candidate inhibitor that inhibits the GluK2-containing kainate receptors or that prevents the activation of the pathway receptor or that prevents the formation of the heteromeric complex GluK2/GluK5 for example.
  • such screening methods involve providing appropriate cells which express the GluK2-containing kainate receptors.
  • nucleic acids encoding the GluK2 subunit may be employed to co-transfect cells to thereby express the GluK2 receptor.
  • Such a transfection may be achieved by methods well known in the art.
  • cells may be neuronal cells.
  • the candidate inhibitor may be selected from a library of inhibitors previously synthesised, or a library of inhibitors for which the structure is determined in a database, or from a library of inhibitors that have been synthesised de novo or natural inhibitors.
  • the candidate inhibitor may be selected from the group of (a) proteins or peptides, (b) nucleic acids and (c) organic or chemical inhibitors (natural or not).
  • libraries of pre-selected candidate nucleic acids may be obtained by performing the SELEX method as described in documents US 5,475,096 and US 5,270,163.
  • the candidate inhibitor may be selected from the group of antibodies directed against the GluK2 receptor particularly. Combination and kit of part
  • the inhibitor of the invention or the pharmaceutical composition of the invention may comprise a further therapeutic active agent.
  • the present invention also relates to a kit comprising an inhibitor according to the invention and a further therapeutic active agent.
  • anti -F CD or anti-epileptic agents may be added to the pharmaceutical composition or used in combination with the inhibitor of the invention in the case of the treatment of a FCD.
  • Anti -F CD or anti-epileptic agents include for example aldehydes, aromatic allylic alcohols like Stiripentol, barbiturates like phenobarbital, methylphenobarbital or barb exacl one, benzodiazepines like clobazam, clonazepam, clorazepate, diazepam, midazolam or lorazepam, bromides like potassium bromide, carbamates, felbamate or cenobamate, carboxamides like carbamazepine, oxcarbazepine or eslicarbazepine acetate, fatty acids like the valproates, vigabatrin, progabide, tiagabine or everolimus.
  • aldehydes aromatic allylic alcohols like Stiripentol, barbiturates like phenobarbital, methylphenobarbital or barb exacl one
  • benzodiazepines like clob
  • Another aspect of the present invention relates to i) an inhibitor of the GluK2-containing kainate receptors, and ii) at least one further therapeutic active agent according to the invention, as a combined preparation for simultaneous, separate or sequential use in the treatment of FCD in a subject in need thereof.
  • the term “simultaneous use” denotes the use of an inhibitor of the GluK2-containing kainate receptors and at least one therapeutic active agent occurring at the same time.
  • the term “separate use” denotes the use of an inhibitor of the GluK2- containing kainate receptors and at least one therapeutic active agent not occurring at the same time.
  • sequential use denotes the use of an inhibitor of the GluK2- containing kainate receptors and at least one therapeutic active agent occurring by following an order.
  • FIGURES
  • Figure 1 Examples of neocortical neurons immunostained with an antibody against phospho-S6 Ribosomal protein illustrating typical non-cytomegalic (small arrows) and cytomegalic/dysmorphic neurons (big arrows) at lower (top) and higher (bottom) magnifications in an adult rat with Focal Cortical Dysplasia.
  • Figure 2 Recurrent spontaneous epileptiform discharges recorded ex vivo in an acute neocortical slice with an LFP electrode from an adult FCD rat before (top) and after (bottom) bath application of 5pM UBP310 (under hyperexcitable condition: 5 pM gabazine); note the significant reduction (by around 46 %) in the number of epileptiform discharges (red stars) in the presence of 5pM UBP310.
  • Figure 3 A, Western blotting of GluK2 extracted from a human organotypic cortical slices from a drug-resistant epileptic patient in the presence of an AAV9 control vector or an AAV9 engineered to express a miRNA against grik2; note that GluK2 receptors are highly expressed in human cortical slices (left panel), and that AAV9 with antigrik2 miRNA strongly decrease the GluK2 protein as shown by the bar graph in the right panel.
  • B Field EPSP (fEPSP) evoked by electrical stimulation (triangle) recorded in a human acute slice from a surgically resected FCD in the absence and in the presence of 5 pM UBP310 ; note that fEPSP is strongly reduced in the presence of UBP310.
  • fEPSP Field EPSP
  • FIG. 4 immunostaining of pS6 in cytomegalic neurons in Focal Cortical Dysplasia.
  • the top left photomicrograph shows typical cytomegalic neurons (examples indicated by arrows) immunostained with an antibody against phosphorylated S6 (pS6) protein in Focal Cortical Dysplasia (FCD, ipsilateral side) in the somatosensory cortex of a Swiss mouse; in the lower photomicrographs, DAPI staining is used to identify nuclei (lOx magnification).
  • Figure 5 immunostaining of GluK2 in Wild-Type and Focal Cortical Dysplasia Tissues.
  • the top photomicrographs depict the distribution of GluK2 (using the GluK2 Sysy antibody 180003) within cortical layers IV and V of the somatosensory cortex in both wildtype (WT) and FCD Swiss mice; in the lower photomicrographs, DAPI staining is used to identify nuclei (lOx magnification).
  • the graph illustrates the immunohistochemical (IHC) analysis of GluK2 expression in cortical layers IV and V, showing increased GluK2 expression in the FCD area compared to WT condition (control, CTL).
  • Plasmid electroporations were performed with either wild type (WT) or mutant mTOR expression vectors (pCAG-MTOR WT-IRES-GFP or pCAG-MTOR p.S2215F, respectively, 3.0 pg/pL each), or with non-recombinant vector (pCAG-IRES-GFP; 0.5 pg/pL). All constructs were generated by Genscript (Piscataway, NJ) as described before (Pelorosso et al 2019) and were checked by Sanger sequencing.
  • Electroporations were performed by delivering 40 V voltage pulses using a BTX ECM 830 electroporator (BTX Harvard Apparatus, Holliston, MA) across tweezer-type electrodes (Nepa Gene Co, Chiba, Japan) pinching the head of each embryo through the uterus.
  • BTX ECM 830 electroporator BTX Harvard Apparatus, Holliston, MA
  • tweezer-type electrodes Nepa Gene Co, Chiba, Japan
  • Brains of adult rats were dissected out after transcardial perfusion with AntigenFix (Diapath) and post-fixed overnight at 4°C. Brains were sectioned (100 pm) with a vibratome (Leica VT 1000S, Leica, Nussloch, Germany) and processed for immunohistochemistry as free- floating sections.
  • Sections were permeabilized in phosphate-buffered saline (PBS) with 0.3% Triton X-100 for 15 min at room temperature, blocked in PBS with 0.3% Triton X-100 and 10% normal goat serum for 1 h at room temperature and incubated in PBS with 0.3% Triton X- 100 and 5% normal goat serum overnight at 4°C with the following primary antibodies: chicken anti-GFP (1/1000; Thermo Fisher Scientific, Waltham, MA; #A10262) and rabbit anti phospho- S6 Ribosomal protein (1 :200, Cell Signalling Technology, #2215).
  • PBS phosphate-buffered saline
  • Sections were washed in PBS and incubated for 2 h at room temperature with the following secondary antibodies: goat anti-chicken Alexa Fluor 488 (1/500; Invitrogen, #A11039), goat anti-rabbit Alexa Fluor 555 (1/500; Invitrogen, #A21428). Sections were counterstained with Hoechst 33342 (1/1000, Thermo Fisher Scientific), washed in PBS and mounted in Fluoromount (Thermo Fisher Scientific). All images were acquired on a LSM800 Zeiss confocal microscope (Zeiss, Jena, Germany) and analyzed with the Fiji software.
  • Acute and cultured slices at 350 pm thickness were prepared from surgical cortical resections from patient diagnosed with drug-resistant epilepsy (AP-HM, Hopital de La Timone, Marseille, France; CHU Pellegrin, Bordeaux, France) as previously described (Le Duigou et al., 2018).
  • AP-HM drug-resistant epilepsy
  • a subset of cultured slices were transduced with AAV9 with antigrik2 miRNA applied directly to slices (4E+10 per slice).
  • rat or human acute slices were transferred in oxygenated normal cerebrospinal fluid (ACSF) at room temperature (>1 hour).
  • the ACSF contained (in mM, Sigma- Aldrich): 126 NaCl, 3.5 KC1, 1.2 NaH2PO4, 26 NaHCO3, 1.3 MgC12, 2.0 CaC12, and 10 glucose (pH 7.4).
  • cortical slices were individually transferred to a recording chamber maintained at 30-32°C and continuously perfused (2-3 ml/min) with oxygenated (95% 02 and 5% CO2) ACSF containing either 5 pM gabazine, or 5 pM gabazine and 50 pM 4-AP in rats and human tissues, respectively.
  • Spontaneous recurrent epileptiform discharges were recorded as local field potentials as previously described (Peret et al., 2014).
  • Immunoblotting with human cortical tissue was detected in fluorescent on Li-COR with Image studio software (Biosciences GmbH) using a rabbit anti-GluK2 (Abeam ab 124702; 1 :2000), a mouse anti-P actin (Sigma A5316; 1 :5000) in Odyssey blocking buffer TBS (Li-COR) and appropriate 800nm fluorophore-conjugated secondary antibodies (IRDye 800; 1 : 15000). The intensity of the signal for GluK2 was normalized to the P actin signal and thereafter to the control condition.
  • FCD type II FCD
  • a rat model induced by in utero electroporation of the recurrent pathogenic variant mTOR p.S2215F in the somatosensory cortex (see methods), was first utilized (Pelorosso et al., 2019).
  • the electroporated somatosensory cortex demonstrated a typical cytoarchitectonic pattern of type II FCD (Pelorosso et al., 2019)(Fig.l); this pattern included neuronal dyslamination and cytomegalic neurons highly-stained with antibody against phospho-S6 Ribosomal protein.
  • GluK2 receptors are highly expressed in cortical slices from a patient with intractable epilepsy; ii) GluK2 protein can be strongly reduced in the presence of AAV9 expression an anti /7 ⁇ 2 miRNA (Fig. 3 A), iii) The amplitude of evoked field EPSP (fEPSP) is strongly reduced in the presence of 5 pM UBP310 (by approximately 50%, Fig. 3B), as well as the frequency of spontaneous recurrent epileptiform discharges (by around 70%, Fig. 3C), in acute cortical slices from a patient with FCD.
  • fEPSP amplitude of evoked field EPSP
  • Brains were freshly frozen in isopentane at -80°C.
  • Fresh frozen sections prepared with a Cryostat at 20 pm were air-dried and fixed by dipping in 4 % paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) for 15 min, before to be stock at -80°C.
  • the sections were incubated with 10 % normal donkey serum for 30 min at room temperature, followed by an incubation with a mixture of the following primary antibodies overnight at 4°C with polyclonal rabbit anti-GluK2 antibody (dilution of 1 :800, SYSY ref.180003) or polyclonal rabbit anti-phospho-S6 ribosomal protein antibody (dilution of 1 : 1000, Cell signaling ref.5364).
  • the sections were then incubated with a mixture of Alexa Fluor 647 labeled species-specific secondary antibodies for 2 h at a dilution of 1 : 1000 (Thermo Fisher Scientific) and mount in Fluoromount + DAPI mounting medium. Images were taken with an epifluorescence microscope Leica DM5000 (Leica Microsystems, Nanterre, France). Results
  • Immunostaining of pS6 in cytomegalic neurons in Focal Cortical Dysplasia is represented in Figure 4.
  • Immunostaining of GluK2 in Wild-Type and Focal Cortical Dysplasia Tissues is represented in Figure 5.
  • the graph illustrates the immunohistochemical (IHC) analysis of GluK2 expression in cortical layers IV and V, showing increased GluK2 expression in the FCD area compared to WT condition (control, CTL).

Landscapes

  • Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Epidemiology (AREA)
  • Neurosurgery (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Neurology (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne le traitement de la dysplasie corticale focale. Dans cette étude, les inventeurs ont montré que les récepteurs kaïnate (KAR) contenant GluK2 jouent un rôle clé dans des activités épileptiformes au niveau du néocortex dans un modèle animal de type DCF II (DCFII) démontrant que les KAR peuvent constituer une cible antiépileptique majeure pour traiter l'épilepsie chronique dans la DCF. Ainsi, la présente invention concerne un inhibiteur de récepteurs de kaïnate contenant GluK2 (KAR) destiné à être utilisé dans le traitement de la dysplasie corticale focale (DCF) chez un sujet en ayant besoin.
PCT/EP2024/064839 2023-05-30 2024-05-29 Procédé et composition pharmaceutique destinés à être utilisés dans le traitement de la dysplasie corticale focale Pending WO2024246162A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP23305851 2023-05-30
EP23305851.0 2023-05-30

Publications (1)

Publication Number Publication Date
WO2024246162A1 true WO2024246162A1 (fr) 2024-12-05

Family

ID=86899083

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2024/064839 Pending WO2024246162A1 (fr) 2023-05-30 2024-05-29 Procédé et composition pharmaceutique destinés à être utilisés dans le traitement de la dysplasie corticale focale

Country Status (1)

Country Link
WO (1) WO2024246162A1 (fr)

Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4816397A (en) 1983-03-25 1989-03-28 Celltech, Limited Multichain polypeptides or proteins and processes for their production
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US5270163A (en) 1990-06-11 1993-12-14 University Research Corporation Methods for identifying nucleic acid ligands
US5475096A (en) 1990-06-11 1995-12-12 University Research Corporation Nucleic acid ligands
WO1999032619A1 (fr) 1997-12-23 1999-07-01 The Carnegie Institution Of Washington Inhibition genetique par de l'arn double brin
WO2001036646A1 (fr) 1999-11-19 2001-05-25 Cancer Research Ventures Limited Inhibition d"expression genique a l"aide d"arn bicatenaire
WO2001068836A2 (fr) 2000-03-16 2001-09-20 Genetica, Inc. Procedes et compositions d'interference d'arn
US6573099B2 (en) 1998-03-20 2003-06-03 Benitec Australia, Ltd. Genetic constructs for delaying or repressing the expression of a target gene
US20140068797A1 (en) 2012-05-25 2014-03-06 University Of Vienna Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription
US8697359B1 (en) 2012-12-12 2014-04-15 The Broad Institute, Inc. CRISPR-Cas systems and methods for altering expression of gene products
WO2015036618A1 (fr) * 2013-09-16 2015-03-19 INSERM (Institut National de la Santé et de la Recherche Médicale) Procédé et composition pharmaceutique destinés à être utilisés dans le traitement de l'épilepsie
WO2022008725A1 (fr) * 2019-07-10 2022-01-13 Inserm (Institut National De La Sante Et De La Recherche Medicale) Composés destinés à être utilisés dans le traitement de l'épilepsie

Patent Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4816397A (en) 1983-03-25 1989-03-28 Celltech, Limited Multichain polypeptides or proteins and processes for their production
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US5270163A (en) 1990-06-11 1993-12-14 University Research Corporation Methods for identifying nucleic acid ligands
US5475096A (en) 1990-06-11 1995-12-12 University Research Corporation Nucleic acid ligands
WO1999032619A1 (fr) 1997-12-23 1999-07-01 The Carnegie Institution Of Washington Inhibition genetique par de l'arn double brin
US6506559B1 (en) 1997-12-23 2003-01-14 Carnegie Institute Of Washington Genetic inhibition by double-stranded RNA
US6573099B2 (en) 1998-03-20 2003-06-03 Benitec Australia, Ltd. Genetic constructs for delaying or repressing the expression of a target gene
WO2001036646A1 (fr) 1999-11-19 2001-05-25 Cancer Research Ventures Limited Inhibition d"expression genique a l"aide d"arn bicatenaire
WO2001068836A2 (fr) 2000-03-16 2001-09-20 Genetica, Inc. Procedes et compositions d'interference d'arn
US20140068797A1 (en) 2012-05-25 2014-03-06 University Of Vienna Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription
US8697359B1 (en) 2012-12-12 2014-04-15 The Broad Institute, Inc. CRISPR-Cas systems and methods for altering expression of gene products
WO2015036618A1 (fr) * 2013-09-16 2015-03-19 INSERM (Institut National de la Santé et de la Recherche Médicale) Procédé et composition pharmaceutique destinés à être utilisés dans le traitement de l'épilepsie
WO2022008725A1 (fr) * 2019-07-10 2022-01-13 Inserm (Institut National De La Sante Et De La Recherche Medicale) Composés destinés à être utilisés dans le traitement de l'épilepsie

Non-Patent Citations (25)

* Cited by examiner, † Cited by third party
Title
ACKMAN JBANIKSZTEJN LCREPEL VBECQ HPELLEGRINO CCARDOSO CBEN-ARI YREPRESA A: "Abnormal Network Activity in a Targeted Genetic Model of Human Double Cortex", JOURNAL OF NEUROSCIENCE, vol. 29, 2009, pages 313 - 327
BALDASSARI SRIBIERRE TMARSAN EADLE-BIASSETTE HFERRAND-SORBETS SBULTEAU CDORISON NFOHLEN MPOLIVKA MWECKHUYSEN S: "Dissecting the genetic basis of focal cortical dysplasia: a large cohort study", ACTA NEUROPATHOL, vol. 138, 2019, pages 885 - 900, XP036939169, DOI: 10.1007/s00401-019-02061-5
BLÜMCKE I ET AL.: "The clinicopathologic spectrum of focal cortical dysplasias: A consensus classification proposed by an ad hoc Task Force of the ILAE Diagnostic Methods Commission1: The ILAE Classification System of FCD", EPILEPSIA, vol. 52, 2011, pages 158 - 174
CASTRO-TORRES RUBÉN D ET AL: "New Aspects of VEGF, GABA, and Glutamate Signaling in the Neocortex of Human Temporal Lobe Pharmacoresistant Epilepsy Revealed by RT-qPCR Arrays", JOURNAL OF MOLECULAR NEUROSCIENCE, BIRKHAEUSER, CAMBRIDGE, MA, US, vol. 70, no. 6, 6 March 2020 (2020-03-06), pages 916 - 929, XP037149375, ISSN: 0895-8696, [retrieved on 20200306], DOI: 10.1007/S12031-020-01519-6 *
CHALUPNIK PAULINA ET AL: "Kainate Receptor Antagonists: Recent Advances and Therapeutic Perspective", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol. 24, no. 3, 18 January 2023 (2023-01-18), pages 1908, XP093074747, DOI: 10.3390/ijms24031908 *
DICARLO ET AL., NUCLEIC ACIDS RES., vol. 41, pages 4336 - 4343
ENGLOT DJCHANG EF: "Rates and predictors of seizure freedom in resective epilepsy surgery: an update", NEUROSURG REV, vol. 37, 2014, pages 389 - 405
FABRE ET AL., PLOS NEGL. TROP. DIS., vol. 8, 2014, pages 2671
GRATZ ET AL., GENETICS, 2014
GUO ET AL., DEVELOPMENT, vol. 141, pages 707 - 714
HAI ET AL., CELL RES, 2014
HAI ET AL., CELL RES., vol. 24, 2014, pages 122 - 125
HWANG ET AL., PLOS ONE, vol. 8, 2013, pages 68708
LAMBERINK HJ ET AL.: "Seizure outcome and use of antiepileptic drugs after epilepsy surgery according to histopathological diagnosis: a retrospective multi centre cohort study", THE LANCET NEUROLOGY, vol. 19, 2020, pages 748 - 757
LE DUIGOU CSAVARY EMORIN-BRUREAU MGOMEZ-DOMINGUEZ DSOBCZYK ACHALI FMILIOR GKRAUS LMEIER JCKULLMANN DM: "Imaging pathological activities of human brain tissue in organotypic culture", J NEUROSCI METHODS, vol. 298, 2018, pages 33 - 44
MALI ET AL., SCIENCE, vol. 339, 2013, pages 823 - 826
MASHIKO ET AL., DEV. GROWTH DIFFER, vol. 56, 2014, pages 122 - 129
NAJM I ET AL.: "The ILAE consensus classification of focal cortical dysplasia: An update proposed by an ad hoc task force of the ILAE diagnostic methods commission", EPILEPSIA, vol. 63, no. 8, August 2022 (2022-08-01), pages 1899 - 1919
PELOROSSO CWATRIN FCONTI VBUHLER EGELOT AYANG XMEI DMCEVOY-VENNERI JMANENT J-BCETICA V: "Somatic double-hit in MTOR and RPS6 in hemimegalencephaly with intractable epilepsy", HUMAN MOLECULAR GENETICS, vol. 28, 2019, pages 3755 - 3765
PINHEIRO PSLANORE FVERAN JARTINIAN JBLANCHET CCREPEL VPERRAIS DMULLE C: "Selective Block of Postsynaptic Kainate Receptors Reveals Their Function at Hippocampal Mossy Fiber Synapses", CEREBRAL CORTEX, vol. 23, 2013, pages 323 - 331, XP009175513, DOI: 10.1093/cercor/bhs022
RUIZ, A. ET AL., DISTINCT SUBUNITS IN HETEROMERIC KAINATE RECEPTORS MEDIATE IONOTROPIC AND METABOTROPIC FUNCTION AT HIPPOCAMPAL MOSSY FIBER SYNAPSE, vol. 25, 2005, pages 11710 - 11718
SHAKER TAREK ET AL: "Focal Cortical Dysplasia in Childhood Epilepsy", SEMINARS IN PEDIATRIC NEUROLOGY, SAUNDERS, PHILADELPHIA, PA, US, vol. 23, no. 2, 15 June 2016 (2016-06-15), pages 108 - 119, XP029689532, ISSN: 1071-9091, DOI: 10.1016/J.SPEN.2016.06.007 *
SISODIYA SMFAUSER SCROSS JHTHOM M: "Focal cortical dysplasia type II: biological features and clinical perspectives", THE LANCET NEUROLOGY, vol. 8, 2009, pages 830 - 843, XP026448542
YANG ET AL., J. MOL. CELL BIOL., vol. 6, 2014, pages 97 - 99
ZETSCHE ET AL.: "Cpfl is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System", CELL, vol. 163, 2015, pages 836 - 843

Similar Documents

Publication Publication Date Title
JP6845589B2 (ja) 腱治癒を調節するための物質および方法
US11779600B2 (en) Methods of treating or preventing alzheimer's disease and associated conditions
CN113614222A (zh) 体外人血脑屏障
JP2010500025A (ja) 霊長類ハンチントン遺伝子発現をｉｎｖｉｖｏで抑制する方法および配列
JP6437946B2 (ja) 線維症の検出および処置
KR20070104575A (ko) 표적 유전자의 발현을 억제하는 조성물
JP2018531046A6 (ja) 核酸ベースのｔｉａ−１阻害剤
JP2018531046A (ja) 核酸ベースのｔｉａ−１阻害剤
KR20120055495A (ko) 병소성 전정 질환을 치료하기 위한 세로토닌 5-ｈｔ3 수용체 길항제의 용도
US10016424B2 (en) Method and pharmaceutical composition for use in the treatment of epilepsy
AU2024205206A1 (en) Method for managing pain
Huang et al. MicroRNA-25-3p therapy for intervertebral disc degeneration by targeting the IL-1β/ZIP8/MTF1 signaling pathway with a novel thermo-responsive vector
EP3622958B1 (fr) Utilisation d'un inhibiteur de canal ionique potassique pour le traitement de la dépression et composition pharmaceutique
JP2007514748A (ja) 神経障害を処置するための方法
WO2024246162A1 (fr) Procédé et composition pharmaceutique destinés à être utilisés dans le traitement de la dysplasie corticale focale
Stockwell et al. Therapeutic approaches for targeting the pediatric brain tumor microenvironment
WO2019209704A1 (fr) Compositions de micro-arn 584-5p et méthodes de traitement du cancer
US20170355998A1 (en) Methods and Compositions for Treating Autoimmune Disorders by Targeting Kv1.3 Ion Channels with Functionalized Lipid-Derived Nanovesicles
EP4253540A1 (fr) Acide nucléique antisens et son utilisation
CN117043339A (zh) 用于治疗认知障碍的微小rna 195组合物和方法
WO2025191018A1 (fr) Agent de liaison à mrgpre destiné à être utilisé dans le traitement de troubles inflammatoires et douloureux
Bailey et al. AAV-mediated gene therapy in a SLC13A5 citrate transporter disorder model rescues epileptic and metabolic phenotypes
Bailey et al. AAV-mediated gene therapy for SLC13A5 Citrate Transporter Disorder rescues epileptic and metabolic phenotypes
CN120131598A (zh) 白藜芦醇在制备用于预防或治疗胶质瘤的药物中的应用
WO2025078851A1 (fr) Méthodes de traitement des déficits cognitifs

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24730659

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2024730659

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2024730659

Country of ref document: EP

Effective date: 20260102

ENP Entry into the national phase

Ref document number: 2024730659

Country of ref document: EP

Effective date: 20260102

ENP Entry into the national phase

Ref document number: 2024730659

Country of ref document: EP

Effective date: 20260102

ENP Entry into the national phase

Ref document number: 2024730659

Country of ref document: EP

Effective date: 20260102

ENP Entry into the national phase

Ref document number: 2024730659

Country of ref document: EP

Effective date: 20260102

ENP Entry into the national phase

Ref document number: 2024730659

Country of ref document: EP

Effective date: 20260102