(19)
(11)EP 2 919 769 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
09.09.2020 Bulletin 2020/37

(21)Application number: 13789831.8

(22)Date of filing:  15.11.2013
(51)International Patent Classification (IPC): 
A61K 31/196(2006.01)
A61K 31/7088(2006.01)
A61P 25/00(2006.01)
A61K 31/00(2006.01)
A61P 25/28(2006.01)
A61K 38/095(2019.01)
(86)International application number:
PCT/EP2013/073942
(87)International publication number:
WO 2014/076235 (22.05.2014 Gazette  2014/21)

(54)

MODULATORS OF INTRACELLULAR CHLORIDE CONCENTRATION FOR TREATING FRAGILE X SYNDROME

MODULATOREN DER INTRAZELLULÄREN CHLORIDKONZENTRATION ZUR BEHANDLUNG DES FRAGILEN X-SYNDROMS

MODULATEURS DE LA CONCENTRATION DE CHLORURE INTRACELLULAIRE POUR LE TRAITEMENT DU SYNDROME X FRAGILE


(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

(30)Priority: 16.11.2012 EP 12193087
16.11.2012 US 201261727235 P

(43)Date of publication of application:
23.09.2015 Bulletin 2015/39

(73)Proprietors:
  • Neurochlore
    13009 Marseille 9 (FR)
  • INSERM - Institut National de la Santé et de la Recherche Médicale
    75654 Paris Cedex 13 (FR)
  • Université d'Aix Marseille
    13284 Marseille Cedex 07 (FR)
  • Centre Hospitalier Universitaire de Brest
    29609 Brest Cedex (FR)

(72)Inventors:
  • BEN-ARI, Yehezkel
    13600 La Ciotat (FR)
  • LEMONNIER, Éric
    29200 Brest (FR)
  • BURNASHEV, Nail
    13009 Marseille (FR)
  • TYZIO, Roman
    13009 Marseille (FR)

(74)Representative: Icosa 
83 avenue Denfert-Rochereau
75014 Paris
75014 Paris (FR)


(56)References cited: : 
WO-A2-2011/011692
US-A1- 2008 139 472
  
  • ERIC LEMONNIER ET AL: "Treating Fragile X syndrome with the diuretic bumetanide: a case report", ACTA PAEDIATRICA, vol. 102, no. 6, 6 May 2013 (2013-05-06), pages e288-e290, XP055090897, ISSN: 0803-5253, DOI: 10.1111/apa.12235
  • SCOTT S. HALL ET AL: "Effects of intranasal oxytocin on social anxiety in males with fragile X syndrome", PSYCHONEUROENDOCRINOLOGY, vol. 37, no. 4, 1 April 2012 (2012-04-01), pages 509-518, XP055056375, ISSN: 0306-4530, DOI: 10.1016/j.psyneuen.2011.07.020
  • MARIAGIULIA TORRIOLI ET AL: "Treatment with valproic acid ameliorates ADHD symptoms in fragile X syndrome boys", AMERICAN JOURNAL OF MEDICAL GENETICS PART A, 1 January 2010 (2010-01-01), pages n/a-n/a, XP055056410, ISSN: 1552-4825, DOI: 10.1002/ajmg.a.33484
  • ERIC LEMONNIER ET AL: "The diuretic bumetanide decreases autistic behaviour in five infants treated during 3 months with no side effects", ACTA PAEDIATRICA, vol. 99, no. 12, 1 July 2010 (2010-07-01), pages 1885-1888, XP055056331, ISSN: 0803-5253, DOI: 10.1111/j.1651-2227.2010.01933.x
  • ADUSEI D C ET AL: "Early developmental alterations in GABAergic protein expression in fragile X knockout mice", NEUROPHARMACOLOGY, PERGAMON PRESS, OXFORD, GB, vol. 59, no. 3, 1 September 2010 (2010-09-01), pages 167-171, XP027110808, ISSN: 0028-3908 [retrieved on 2010-05-12]
  • BEN-ARI Y ET AL: "The GABA excitatory/inhibitory shift in brain maturation and neurological disorders", THE NEUROSCIENTIST, SAGE PUBLICATIONS, US, vol. 18, no. 5, 1 October 2012 (2012-10-01), pages 467-486, XP008160769, ISSN: 1073-8584, DOI: 10.1177/1073858412438697 [retrieved on 2012-04-30]
  
Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


Description

FIELD OF INVENTION



[0001] The present invention relates to the treatment of Fragile X syndrome. More specifically, the present invention relates to a method for treating Fragile X syndrome in a subject in need thereof, wherein said method comprises modulating the intracellular level of chloride, such as, for example, by administering to the subject a modulator of a chloride transporter.

BACKGROUND OF INVENTION



[0002] Fragile X syndrome is the most common inherited form of mental retardation, affecting about 1 in every 4000 boys and about 1 in every 8000 girls.

[0003] Fragile X syndrome (FXS) is a genetic disorder caused by the expansion of a CGG trinucleotide repeat in the 5' untranslated region (5'-UTR) of the Fragile X mental retardation 1 (FMR1) gene, which is located on the X chromosome. The mutation results in a reduced or absent expression of the Fragile X mental retardation protein (FRMP). Even if the exact function of FRMP is not known, experimental evidences have shown that its normal expression is required for normal neural development.

[0004] Depending at least in part on the length of the CGG repeat expansion, FXS patients will present various degrees of symptoms severity. Major symptoms associated with FXS are mental retardation and learning disabilities, in particular delays in learning how to sit, walk and talk. As a consequence, FXS patients usually present nervous or cluttered speech. Moreover, FXS patients may have deficient central executive, working, phonological and/or visual-spatial memories; or difficulty with face encoding.

[0005] Behavioral and emotional problems may also be encountered, such as, for example, hyperactivity, stereotypy, anxiety, seizures, impaired social behavior, cognitive delay, irritability, aggression or self-injurious behavior. Moreover, FXS may also cause ophthalmologic problems including strabismus, and recurrent otitis media and sinusitis during early childhood.

[0006] Due to the high prevalence of Fragile X syndrome, there is a real need for a specific treatment.

[0007] The patent application EP 2 433 635 describes the use of PAK (p21-activated kinases) modulators for treating Fragile X syndrome.

[0008] The patent application WO2012/019990 describes the use of glutamate 5 receptor (mGlu5) antagonists for the treatment of Fragile X syndrome.

[0009] The patent application AU 2011 236093 describes the use of a gamma-aminobutyric acid agonist, in particular of Baclofen, for treating Fragile X syndrome. The drug STX209, a baclofen derivative, is currently under clinical trial for determining its efficacy, safety, and tolerability for the treatment of social withdrawal in adolescents and adults with Fragile X syndrome.

[0010] Other clinical trials are currently in progress, such as, for example, with AFQ056 (a mGluR5 antagonist developed by Novartis), minocycline hydrochloride (an antibiotic which may lower matrix metalloproteinase 9 (MMP9) levels) or Acamprosate (an agonist of a GABA receptor).

[0011] Hall et al. (2012. Psychoneuroendocrinology. 37(4):509-18) teaches about the use of oxytocin, a hormone with prosocial and anxiolytic effects, for treating subjects affected with Fragile X syndrome.

[0012] Torrioli et al. (2010. Am J Med Genet A. 152A(6):1420-7) teaches about the use of valproic acid, for treating subjects affected with Fragile X syndrome.

[0013] However, to the Applicant knowledge, there is currently no drug treatment with prove efficacy for Fragile X syndrome.

[0014] The Applicant surprisingly showed that the intracellular chloride level was significantly increased in a mouse model of FXS as compared to the wild-type situation, leading to an abnormal neuronal activity (see Examples). This was surprising as Adusei et al. (2010. Neuropharmacology. 59(3):167-171) disclose that the chloride transporters NKCC1 and KCC2 are not involved in FXS.

[0015] The present invention thus relates to the use of a modulator of intracellular chloride levels, in particular inhibitor of NKCC, for treating FXS in a subject in need thereof.

SUMMARY



[0016] The invention is defined by the claims. Any subject-matter falling outside the scope of claims is provided for information purposes only. Any references in the description to methods of treatment refer to the compounds, pharmaceutical compositions and medicaments of the present invention for use in a method for treatment of the human or animal body by therapy.

[0017] The present invention thus relates to a composition for treating Fragile X syndrome in a subject in need thereof, wherein said composition comprises an effective amount of a modulator of a chloride transporter as defined in the appended claims.

[0018] In one embodiment, said modulator of chloride transporter is not valproic acid, oxytocin, bendroflumethiazide, benzthiazide, buthiazide, chlorothiazide, cyclothiazide, epithiazide, metalthiazide, hydrochlorothiazide, hydro-flumethiazide, methylclothiazide, polythiazide, trichlormethiazide, chlorthalidone, indapamide, metolazone and quinethazone.

[0019] In one aspect of the disclosure, said modulator is an inhibitor of a transporter involved in the importation of chloride into neurons.

[0020] In another aspect, said inhibitor is an inhibitor of the activity of a transporter involved in the importation of chloride into neurons. In one embodiment, said transporter involved in the importation of chloride into neurons is NKCC1.

[0021] The inhibitor of NKCC is selected from the group comprising bumetanide, furosemide, ethacrynic acid, torsemide, azosemide, muzolimine, piretanide and tripamide; analogs thereof as defined in the appended claims.

[0022] In one embodiment, the effective amount ranges from about 0.01 mg to about 500 mg.

[0023] In one embodiment, the composition is administered to the subject in need thereof by subcutaneous, intramuscular, intravenous, intraocular, transdermal, topical, parenteral, intranasal and oral administration, or injection.

[0024] In one embodiment, the subject presents a number of CGG repeats in the 5'-UTR of FMR1 of at least 55, preferably of at least 200. In another embodiment, the subject is diagnosed with Fragile X syndrome. In still another embodiment, the subject is at risk of developing Fragile X syndrome. In one embodiment, the subject is a fetus and the composition is to be administered to his/her mother.

DEFINITIONS



[0025] In the present invention, the following terms have the following meanings:
  • "Fragile X syndrome" refers to a genetic disease associated with and/or caused by to a defect of the expression of the FMR1 gene and/or of the activity of the FMR1-encoded protein, FMRP.
    In one embodiment, Fragile X syndrome refers to a condition wherein the FMR1 gene comprises at least 55 CGG repeats in the 5'-UTR. Preferably, FXS refers to a condition wherein the FMR1 gene comprises at least 200 CGG repeats in the 5'-UTR.
    The expansion repeat in the 5'-UTR of FMR1 causes abnormal expression of the FMR1 gene, and consequently, abnormal function of the FMRP protein. Therefore, the skilled artisan will appreciate that Fragile X syndrome may also refer to a condition caused by and/or associated with one or more of the following: (1) a mutation in FMR1; (2) defective FMR1 expression; (3) increased and/or decreased expression of FMRP; (4) defective FMRP function; (5) increased and/or decreased expression of FMRP's natural binding partners; (6) an increased and/or decreased ability of FMRP to bind to its natural binding partners; (7) decreased or absent arginine methylation of FMRP; (8) increased methylation of FMR1 CpG repeats in the 5' UTR of exon 1; (9) the mislocalization or misexpression of FMRP within the cell (preferably in the neuron) or within the organism.
  • "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented. A subject is successfully "treated" for Fragile X syndrome if, after receiving an effective amount of a modulator according to the present invention, the subject shows observable and/or measurable reduction in or absence of one or more of the following: reduction in one or more of the symptoms associated with the Fragile X Syndrome; and improvement in quality of life issues. The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician.
  • "Effective amount" refers to the level or amount of agent that is aimed at, without causing significant negative or adverse side effects to the target, (1) delaying or preventing the onset of Fragile X syndrome; (2) slowing down or stopping the progression, aggravation, or deterioration of one or more symptoms of Fragile X syndrome; (3) bringing about ameliorations of the symptoms of Fragile X syndrome; (4) reducing the severity or incidence of Fragile X syndrome; or (5) curing Fragile X syndrome. An effective amount may be administered prior to the onset of Fragile X syndrome, for a prophylactic or preventive action. Alternatively or additionally, the effective amount may be administered after initiation of Fragile X syndrome, for a therapeutic action.
  • "Subject" refers to a mammal, preferably a human. In one embodiment of the invention, the subject is a male. In another embodiment of the invention, the subject is a female.
    In one embodiment, the term "healthy subject" refers to a subject not diagnosed with Fragile X syndrome. In another embodiment, a healthy subject does not present symptoms and/or clinical signs of Fragile X syndrome, wherein clinical signs of Fragile X syndromes include mild dysmorphic features, macrocephaly with long face, prominent ears, arched palate, thin face, joint hypermobility, fat feet and macroorchidism. In another embodiment, a healthy subject comprises a FMR1 gene with a number of CGG repeats in the 5'-UTR ranging from 5 to 54. In another embodiment, a healthy subject comprises a FMR1 gene with a number of CGG repeats in the 5'-UTR ranging from 55 to 200.
  • "Modulator" refers to a compound that modulates intracellular chloride level. Preferably, a modulator is a compound whose administration leads to a decrease of intracellular chloride concentration. In one embodiment, said modulator acts on the gene and/or protein expression and/or on the activity of a chloride transporter.
  • "Selective modulator" refers to a selective inhibitor and a selective activator.
  • "Inhibitor" refers to refers to a natural or synthetic compound that has a biological effect to inhibit or significantly reduce or down-regulate the expression of a gene and/or a protein or that has a biological effect to inhibit or significantly reduce the biological activity of a protein. Consequently, "a NKCC inhibitor" refers to a natural or synthetic compound that has a biological effect to inhibit or significantly reduce or down-regulate the expression of the gene encoding for NKCC and/or the expression of the NKCC protein and/or the biological activity of NKCC.
  • "Selective inhibitor" refers to that the affinity of the inhibitor for the chloride transporter for instance NKCC is at least 10-fold, 25-fold, 50-fold, 75-fold, 80-fold, 90-fold, 95 fold, 100-fold, 125-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, preferably 500-fold higher than the affinity for the other chloride transporters in particular KCC2.
  • "About": preceding a figure means plus or less 10% of the value of said figure.
  • "Analog" refers broadly to the modification or substitution of one or more chemical moieties on a parent compound and may include functional derivatives, positional isomers, tautomers, zwitterions, enantiomers, diastereomers, racemates, isosteres or stereochemical mixtures thereof.
  • "Functional derivative" refers to a compound which possesses the capacity to modulate the concentration of chloride into neurons (inhibits the importation or activates the outflow of chloride).

DETAILED DESCRIPTION



[0026] This invention relates to a composition comprising a modulator of intracellular chloride concentration for treating Fragile X syndrome.

[0027] In one embodiment of the invention, the modulator of intracellular chloride concentration is a modulator of a chloride transporter.

[0028] In one embodiment of the invention, the modulator of intracellular chloride concentration is a selective modulator of a chloride transporter.

[0029] In one embodiment, the modulator of a chloride transporter inhibits the importation of chloride into neurons, preferably through the inhibition of transporters involved in the importation of chloride into neurons.

[0030] In another embodiment, the selective modulator of a chloride transporter inhibits the importation of chloride into neurons, preferably through the inhibition of transporters involved in the importation of chloride into neurons.

[0031] In one embodiment of the invention, said modulator is an inhibitor of the chloride transporter involved in the importation of chloride into neurons.

[0032] In another embodiment of the invention, said modulator is a selective inhibitor of the chloride transporter involved in the importation of chloride into neurons.

[0033] In one embodiment of the invention, said modulator is an inhibitor of the protein and/or gene expression of a transporter involved in the importation of chloride into neurons.

[0034] In another embodiment of the invention, said modulator is a selective inhibitor of the protein and/or gene expression of a transporter involved in the importation of chloride into neurons.

[0035] Examples of transporters involved in the importation of chloride into neurons include NKCC (wherein NKCC stands for "Na-K-2Cl co-transporter"), such as, for example, NKCC1. In one embodiment, the modulator of a chloride transporter is thus an inhibitor of NKCC, preferably of NKCC1. In another embodiment, the modulator of a chloride transporter is a selective inhibitor of NKCC, preferably of NKCC1.

[0036] In another embodiment, the inhibitor of a chloride transporter inhibits the activity of the chloride transporter. Examples of such inhibitors include small molecules, antibodies, minibodies, diabodies or fragments thereof binding to the chloride transporter, and antagonists of the chloride transporter.

[0037] Examples of such inhibitors include NKCC inhibitors, such as, for example, NKCC antagonists. In one embodiment, the modulator is an antagonist of NKCC1. In one embodiment, said inhibitor is a selective NKCC inhibitor, preferably a selective NKCC1 inhibitor.

[0038] In one embodiment of the invention, said selective inhibitor interacts directly with the chloride transporter.

[0039] In one embodiment, said selective inhibitor is an antagonist of a chloride transporter importing chloride into neurons.

[0040] In one embodiment of the invention, the inhibitor of a chloride transporter is an inhibitor of NKCC1, such as, for example, a diuretic (such as, for example, a loop diuretic); or a NKKC1 antagonist.

[0041] In one embodiment of the invention, the selective inhibitor decreasing the gene and/or protein expression and/or activity of the chloride co-transporter NKCC1, has a low affinity for KCC2.

[0042] In one embodiment of the invention, the selective inhibitor of the chloride transporter has an affinity for KCC2 inferior than 10-7 M, preferably 10-6 M, more preferably less than 10-5 M.

[0043] In another embodiment of the invention, the selective inhibitor of the chloride transporter has an affinity at least much higher to NKCC1 than to KCC2 (of at least 2 orders of magnitude, preferably of at least 4 orders of magnitude, more preferably of at least 5 orders of magnitude and most preferably of at least 6 orders of magnitude higher binding constant (at least 10-9, preferably more than 10-10).

[0044] In another embodiment of the invention, the selective inhibitor of the chloride transporter does not bind to KCC2 at all.

[0045] In one embodiment of the invention, the selective inhibitor of the chloride transporter refers to a molecule that has an affinity for the NKCC1 at least 10-fold, 25-fold, 50-fold, 75-fold, 80-fold, 90-fold, 95 fold, 100-fold, 125-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, preferably 500-fold higher than its affinity for any one of other isoforms of NKCC transporters comprising NKCC2, KCC transporters comprising KCC1, KCC2, KCC3, KCC4, other transporter chloride including in a nonlimiting list: Cl-HCO3- transporter.

[0046] Examples of inhibitors of a chloride transporter, preferably of NKCC1, include bumetanide, furosemide, ethacrynic acid, torsemide, azosemide, muzolimine, piretanide, tripamide and analogs, functional derivatives and prodrugs of such compounds; thiazide and thiazide-like diuretics, such as bendroflumethiazide, benzthiazide, chlorothiazide, hydrochlorothiazide, hydro-flumethiazide, methylclothiazide, polythiazide, trichlormethiazide, chlorthalidone, indapamide, metolazone and quinethazone; and analogs, functional derivatives and prodrugs of such compounds.

[0047] Examples of analogs of bumetanide include, but are not limited to: bumetanide aldehyde, bumetanide dibenzylamide, bumetanide diethylamide, bumetanide morpholinoethyl ester, bumetanide 3-(dimethylaminopropyl) ester, bumetanide N,N-diethylglycolamide ester, bumetanide dimethylglycolamide ester, bumetanide pivaxetil ester, bumetanide methoxy(polyethyleneoxy)n-1-ethyl ester, bumetanide benzyltrimethyl-ammonium salt, bumetanide cetyltrimethylammonium salt, pivaloyloxymethyl ester of bumetanide, methyl ester of bumetanide, N,N-dimethylaminoethyl ester of bumetanide, bumetanide [-(C=O)-SH] thioacid, bumetanide S-methyl thioester, bumetanide S-cyanotnethyl thioester, bumetanide S-ethyl thioester, bumetanide S-isoamyl thioester, bumetanide S-octyl thioester, bumetanide S-benzyl thioester, bumetanide S-(morpholinoethyl) thioester, bumetanide S-[3-(dimethylaminopropyl)] thioester, bumetanide S-(N,N-diethylglycolamido) thioester, bumetanide S-(N,N-dimethylglycolamido) thioester, bumetanide S-pivaxetil thioester, bumetanide S-propaxetil thioester, bumetanide S-(methoxypolyethyleneoxy)n-1-ethyl] thioester, bumetanide [-(C=O)-S-] benzyltrimethyl-ammonium thioacid salt and bumetanide [-(C=O)-S] cetyltrimethylammonium thioacid salt; metast-able bumetanide thioacid, bumetanide thioaldehyde, bumetanide O-methyl thioester, bumetanide O-cyanomethyl thioester, bumetanide O-ethyl thioester, bumetanide O-isoamyl thioester, bumetanide O-octyl thioester, bumetanide O-benzyl thioester, bumetanide O-(morpholinoethyl) thioester, bumetanide O-[3-(dimethylaminopropyl)] thioester, bumetanide O-(N,N-diethylglycolamido) thioester, bumetanide O-pivaxetil thioester, bumetanide O-propaxetil thioester, bumetanide O-[methoxy(polyethyleneoxy)n-1 ethyl] thioester, bumetanide [-(C=S)-O-] benzyltrimethyl-ammonium thioacid salt and bumetanide [-(C=S)-O-] cetyltrimethylammonium thioacid salt.

[0048] Preferably, the modulator of the intracellular chloride level is bumetanide and analogs thereof.

[0049] In one embodiment of the invention, the composition comprises an effective amount of a modulator of intracellular chloride concentration.

[0050] According to the invention, the effective amount of a modulator of intracellular chloride concentration is calculated in order to reach a desired intracellular concentration of chloride.

[0051] Indeed, the Applicant surprisingly showed that the intracellular concentration of chloride is more elevated in Fragile X syndrome rodents than in naive ones (see Examples).

[0052] Therefore, according to an embodiment, the effective amount of a modulator of intracellular chloride concentration corresponds to the amount to be administered to a subject in need thereof for reaching the intracellular chloride concentration measured in a healthy subject. Preferably, said healthy subject shares characteristics with the Fragile X syndrome subject to be treated, such as, for example, the same age, sex, diet, weight. More preferably, said healthy subject presents a number of repeats with in the 5'-UTR of the FMR1 gene ranging from 5 to 54.

[0053] In one embodiment of the invention, the effective amount of a modulator ranges from about 0.01 mg to about 500 mg, preferably from about 0.05 mg to about 100 mg, more preferably from about 0.1 mg to about 10 mg and even more preferably from about 0.5 mg to about 1.5 mg.

[0054] According to one embodiment of the invention, the composition of the invention is for treating behavioral and/or cognitive symptoms associated with Fragile X syndrome. In one embodiment, the administration to a subject in need thereof of the composition of the invention results in the alleviation of behavioral symptoms of Fragile X syndrome.

[0055] Examples of behavioral and/or cognitive symptoms of Fragile X syndrome include hyperactivity, stereotypy, anxiety, seizure, impaired social behavior, cognitive delay, hypersensitivity to sensory stimuli, mood disorders, disrupt sleep patterns, irritability, aggression or self-injurious behavior, mental retardation, learning disabilities (such as, for example, delays in learning how to sit, walk and talk), nervous or cluttered speech, difficulty with face encoding and deficient central executive, working, phonological and/or visual-spatial memories.

[0056] In one embodiment of the invention, the behavioral and/or cognitive symptom associated with Fragile X syndrome is not an autistic symptom or a symptom associated with an autism spectrum disorder.

[0057] Methods for assessing the efficacy of the treatment are readily measurable by routing procedures familiar to a physician, such as, for example, use of scales. Examples of scales that may be used for assessing the efficacy of the treatment of Fragile X syndrome include the ABC scale, the GRAM scale and the CGI scale.

[0058] According to one embodiment of the invention, the composition of the invention is for alleviating the occurrence of ocular symptoms and/or otorhinolaryngologic local manifestations associated with Fragile X syndromes. In one embodiment, the administration to a subject in need thereof of the composition of the invention results in the alleviation of ocular symptoms and otorhinolaryngologic local manifestations and/or in the reduction of the occurrence of said symptoms, such as, for example, in the reduction of the occurrence of ophthalmologic problems including strabismus, otitis media and sinusitis.

[0059] According to one embodiment of the invention, the composition of the invention is for treating synaptic defects/symptoms associated with Fragile X syndromes. In one embodiment, the administration to a subject in need thereof of the composition of the invention results in the alleviation of synaptic defects associated with Fragile X syndrome.

[0060] Examples of synaptic defects/symptoms associated with Fragile X syndrome include defective synaptic morphology (such as, for example, an abnormal number, length, and/or width of dendritic spines) and defective synaptic function (such as, for example, enhanced long-term depression (LTD) and/or reduced long-term potentiation (LTP)).

[0061] In one embodiment of the invention, the subject to be treated presents a number of CGG repeats in the 5'-UTR of FMR1 of at least 55, preferably of at least 200.

[0062] In one embodiment of the invention, the subject to be treated is diagnosed with Fragile X syndrome; preferably he/she presents a number of CGG repeats in the 5'-UTR of FMR1 of at least 200. In one embodiment, the diagnosed subject already presents symptoms of Fragile X syndrome. In another embodiment, the diagnosed subject is treated before the onset of Fragile X syndrome symptoms.

[0063] In another embodiment of the invention, the subject to be treated is at risk of developing Fragile X syndrome. In one embodiment of the invention, said risk corresponds to the presence in the kindred of Fragile X syndrome patients. In another embodiment of the invention, said risk correspond to the presence, in the FMR1 gene of at least one of the parents of the subject, of a number of CGG repeats in the 5'-UTR ranging from 55 to 200 or of more than 200.

[0064] In one embodiment of the invention, the subject to be treated is not an autistic patient. Preferably, said subject is not diagnosed with autism or another autism spectrum disorder.

[0065] The Applicant surprisingly showed that delivery was a key event in the development of Fragile X syndrome (see Examples). Therefore, in one embodiment, the composition of the invention is administered to a pregnant woman. According to the invention, the term "pregnant woman" refers to a woman from the conception date to the end of the delivery.

[0066] According to an embodiment, the composition is administered to a pregnant woman when her fetus has been prenatally diagnosed with Fragile X syndrome, preferably when the fetus presents a number of CGG repeats superior to 200. According to an embodiment, the composition is administered to a pregnant woman when her fetus presents a risk of developing Fragile X syndrome. In one embodiment of the invention, said risk corresponds to the presence in the kindred of Fragile X syndrome patients. In another embodiment of the invention, said risk correspond to the presence, in the FMR1 gene of at least one of the parents of the subject, of a number of CGG repeats in the 5'-UTR ranging from 55 to 200 or of more than 200.

[0067] In one embodiment of the invention, the composition is perinatally administered to a child. As used herein, the term "perinatally" refers to a few hours after birth, preferably 10, 8, 6, 5, 4, 3, 2 or 1 hour(s) after birth. Preferably, this embodiment applies when the child has been prenatally diagnosed with Fragile X syndrome, preferably when he/she presents a number of CGG repeats superior to 200. According to an embodiment, the composition may also be perinatally administered when the child presents a risk of developing Fragile X syndrome. In one embodiment of the invention, said risk corresponds to the presence in the kindred of Fragile X syndrome patients. In another embodiment of the invention, said risk correspond to the presence, in the FMR1 gene of at least one of the parents of the subject, of a number of CGG repeats in the 5'-UTR ranging from 55 to 200 or of more than 200.

[0068] In one embodiment of the invention, the subject is a young child. As used herein, the term "young child" refers to a child from 0 to 3 years old.

[0069] In another embodiment of the invention, the subject is a child. In one embodiment, the term "child" may refer to subjects aged from 0 to 12, preferably from 3 to 12. More generally, the term child refers to a subject which is not yet an adolescent.

[0070] In another embodiment of the invention, the subject is an adolescent. In one embodiment, the term "adolescent" may refer to subjects aged from about 12 to 17, but the skilled artisan will appreciate that the length of adolescence may vary from one individual to another.

[0071] In another embodiment, the subject is an adult. In one embodiment, the term "adult" may refer to subjects of more than 17 years old. More generally, the term adult refers to a subject which is no more an adolescent.

[0072] The present invention also relates to a pharmaceutical composition for treating Fragile X syndrome in a subject in need thereof, wherein said pharmaceutical composition comprises an effective amount of a modulator of intracellular chloride concentration and at least one pharmaceutically acceptable excipient.

[0073] As used herein the term "pharmaceutically acceptable excipient" refers to an excipient that does not produce an adverse, allergic or other untoward reaction when administered to an animal, preferably a human. It may include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents. For human administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by FDA Office of Biologics standards.

[0074] Another object of the invention is a medicament for treating Fragile X syndrome comprising an effective amount of a modulator of intracellular chloride concentration.

[0075] The composition, pharmaceutical composition or medicament of the invention may be administered by several routes of administration. Examples of adapted routes of administration include subcutaneous, intramuscular, intravenous, intraocular, transdermal, topical, parenteral, intranasal and oral administration, or injection. The type of form for administration will be matched to the severity of the syndrome as well as to the age, weight, sex of the subject to be treated.

[0076] According to an embodiment, the composition, pharmaceutical composition or medicament of the invention is in a form adapted for injection, preferably selected from the group comprising solutions, such as, for example, isotonic solution, saline solution, sterile aqueous solutions, dispersions, emulsions, suspensions, solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to use, such as, for example, powder, freeze-dried compositions, liposomal forms.

[0077] According to an embodiment, the composition, pharmaceutical composition or medicament of the invention is in a form adapted to oral administration. According to a first embodiment, the form adapted to oral administration is a solid form selected from the group comprising tablets, pills, capsules, soft gelatin capsules, sugarcoated pills, orodispersing tablets, effervescent tablets or other solids. According to a second embodiment, the form adapted to oral administration is a liquid form, such as, for example, a drinkable solution, a buccal spray, liposomal forms.

[0078] According to an embodiment, the composition, pharmaceutical composition or medicament of the invention is in a form adapted for local delivery via the nasal and respiratory routes. Examples of formulations suitable for nasal administration include nasal solutions, sprays, aerosols and inhalants.

[0079] According to an embodiment, the composition, pharmaceutical composition or medicament of the invention is in a form adapted to topical administration. Examples of formulations adapted to topical administration include ointment, paste, eye drops, cream, patch, such as, for example, transdermal patch, gel, liposomal forms.

[0080] According to an embodiment, the composition, pharmaceutical composition or medicament of the invention is in the form of, or comprises, liposomes and/or nanoparticles.

[0081] According to an embodiment, the composition, pharmaceutical composition or medicament of the invention further comprises some excipients, such as, for example, surfactants (e.g., hydroxypropylcellulose); suitable carriers, such as, for example, solvents and dispersion media containing, for example, water, monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils, such as, for example, peanut oil and sesame oil; isotonic agents, such as, for example, sugars or sodium chloride; coating agents, such as, for example, lecithin; agents delaying absorption, such as, for example, aluminum monostearate and gelatin; preservatives, such as, for example, benzalkonium chloride, benzethonium chloride, chlorobutanol, thimerosal; buffers, such as, for example, boric acid, sodium and potassium bicarbonate, sodium and potassium borates, sodium and potassium carbonate, sodium acetate, sodium biphosphate; tonicity agents, such as, for example, dextran 40, dextran 70, dextrose, glycerin, potassium chloride, propylene glycol, sodium chloride; antioxidants and stabilizers, such as, for example, sodium bisulfite, sodium metabisulfite, sodium thiosulfite, thiourea; nonionic wetting or clarifying agents, such as, for example, polysorbate 80, polysorbate 20, poloxamer 282 and tyloxapol; viscosity modifying agents, such as, for example dextran 40, dextran 70, gelatin, glycerin, hydroxyethylcellulose, hydroxymethylpropylcellulose, lanolin, methylcellulose, petrolatum, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, carboxymethylcellulose.

[0082] In one embodiment of the invention, the composition, pharmaceutical composition or medicament of the invention may be used in conjunction with delivery systems that facilitate delivery of the agents to the central nervous system. For example, various blood brain barrier (BBB) permeability enhancers may be used to transiently and reversibly increase the permeability of the blood brain barrier to a treatment agent. Such BBB permeability enhancers include leukotrienes, bradykinin agonists, histamine, tight junction disruptors (e.g., zonulin, zot), hyperosmotic solutions (e.g., mannitol), cytoskeletal contracting agents, and short chain alkylglycerols (e.g., 1-O-pentylglycerol). Oral, sublingual, parenteral, implantation, nasal and inhalational routes can provide delivery of the active agent to the central nervous system. In some embodiments, the compounds of the present invention may be administered to the central nervous system with minimal effects on the peripheral nervous system.

[0083] The blood-brain barrier (BBB) is a physical barrier and system of cellular transport mechanisms between the blood vessels in the central nervous system (CNS) and most areas of the CNS itself. The BBB maintains homeostasis by restricting the entry of potentially harmful chemicals from the blood, and by allowing the entry of essential nutrients. However, the BBB can pose a formidable barrier to delivery of pharmacological agents to the CNS for treatment of disorders or maintaining or enhancing normal and desirable brain functions, such as cognition, learning, and memory.

[0084] According to one embodiment of the invention, the composition, the pharmaceutical composition or the medicament of the invention is administered at least once a day, preferably twice a day, more preferably at least three times a day.

[0085] In one embodiment of the invention, the daily amount of a modulator to be administered to a subject ranges from about 0.01 mg/day to about 500 mg/day, preferably from about 0.05/day mg to about 100 mg/day, more preferably from about 0.1 mg/day to about 10 mg/day and even more preferably from about 0.5 mg/day to about 1.5 mg/day.

[0086] According to one embodiment of the invention, the administration dose of the pharmaceutical composition is determined by the skilled artisan and personally adapted to each subject.

[0087] In one embodiment of the invention, the modulator is administered in a sustained-release form. In one embodiment of the invention, the composition comprises a delivery system that controls the release of the modulator. Examples of suitable carriers for sustained or delayed release include gelatin; gum Arabic; xanthan polymers; thermoplastic resins such as, for example polyvinyl halides, polyvinyl esters, polyvinylidene halides and halogenated polyolefins; elastomers such as, for example, brasiliensis, polydienes, and halogenated natural and synthetic rubbers; and flexible thermoset resins such as polyurethanes, epoxy resins; biodegradable polymers.

[0088] In another embodiment of the invention, the modulator of the present invention is administered in combination with other therapies that could include: speech therapy, behavioral therapy, sensory integration occupational therapy, special education, or individualized educational plans, and, when necessary, treatment of physical abnormalities.

[0089] Another object of the invention is a modulator of a chloride transporter for use in a method for treating Fragile X syndrome in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of a modulator of a chloride transporter. In one embodiment of the invention, said effective amount is calculated in order to reach a desired intracellular concentration of chloride.

[0090] In one embodiment of the invention, the method of treating comprises administering to the subject the composition, the pharmaceutical composition or the medicament of the invention.

[0091] In one embodiment of the invention, the method is for treating behavioral and/or cognitive symptoms of Fragile X syndrome.

[0092] In another embodiment of the invention, the method is for treating ocular symptoms and/or otorhinolaryngologic local symptoms of Fragile X syndrome.

[0093] In another embodiment, the method of the invention is for treating synaptic symptoms/defects of Fragile X syndrome.

[0094] In one embodiment of the invention, the subject in need of a treatment presents a number of CGG repeats in the 5'-UTR of FMR1 of at least 55, preferably of at least 200. In one embodiment of the invention, the subject to be treated is diagnosed with Fragile X syndrome, and already presents or not symptoms of Fragile X syndrome. In another embodiment of the invention, the subject in need of a treatment is at risk of developing Fragile X syndrome.

[0095] The present invention also relates to a method for treating Fragile X syndrome in a fetus, and comprises administering to his/her mother an effective amount of a modulator of a chloride transporter.

[0096] In one embodiment of the invention, the subject is for perinatally treating a child.

[0097] In one embodiment of the invention, the subject is a young child, a child, an adolescent or an adult.

[0098] In one embodiment of the invention, the method of the invention comprises subcutaneously, intramuscularly, intravenously, intraocularly, transdermally, topically, parenterally, intranasally or orally administering the modulator of the invention, or its injection.

[0099] In one embodiment of the invention, the method of the invention comprises administering the modulator of the invention at least once a day, preferably twice a day, more preferably at least three times a day. In another embodiment of the invention, a daily amount of a modulator ranging from about 0.01 mg/day to about 500 mg/day, preferably from about 0.05/day mg to about 100 mg/day, more preferably from about 0.1 mg/day to about 10 mg/day and even more preferably from about 0.5 mg/day to about 1.5 mg/day is administered to the subject.

[0100] The present invention also relates to a method for decreasing the intracellular concentration of chloride in a Fragile X syndrome subject, preferably the neuronal intracellular concentration of chloride.

[0101] Another object of the invention is a method for modulating the intracellular chloride concentration of a subject, wherein the method comprises administering to the subject in need thereof an effective amount of a modulator of a chloride transporter.

[0102] The present invention also relates to a method for decreasing the driving force of GABA in a Fragile X syndrome subject.

[0103] In one embodiment of the invention, the method comprises administering to the subject an effective amount of a modulator of a chloride transporter.

[0104] In one embodiment of the invention, said effective amount is calculated in order to reach a desired intracellular concentration of chloride.

[0105] In one embodiment of the invention, the method comprises administering to the subject in need thereof the composition, the pharmaceutical composition or the medicament of the invention.

BRIEF DESCRIPTION OF THE FIGURES



[0106] 

Figure 1 is a combination of graphs showing a comparison of developmental profile of driving force for GABA (DFGABA) in hippocampal slices from pre- and postnatal transgenic mouse model for the Fragile X syndrome versus wild type animals: A. Plot of DFGABA measured with single GABAA channels recordings (for details see Tyzio et al., 2006) from CA3 hippocampal pyramidal cells at prenatal (E20) and postnatal (P0, P7, P14 and P28-37) stages. Note that DFGABA is significantly more positive in Fragile X at ages P0, P14 and P28-37. Also, there is no perinatal hyperpolarizing shift of DFGABA in Fragile X model (at P0 p<0.005, at p14 p<0.05, at P28-37 p<0.005); B. I-V relationships of the currents through GABAA channels at P0 in two CA3 pyramidal cells from Fragile X before and after application of bumetanide. Their reversal potential corresponds to DFGABA; note the shift of reversal potential from depolarizing to hyperpolarizing. On the inset - examples of single GABAA channels openings at different holding potentials; C. Action of oxytocin and bumetanide on DFGABA in CA3 pyramidal cells from newborn (P0) Fragile X and wild type. In the wild type pyramidal cells DFGABA is initially hyperpolarizing at P0 as a result of maternal oxytocin action. Bumetanide application increases hyperpolarization. In slices from Fragile X mouse DFGABA is positive at P0 and both oxytocin and bumetanide shift it to hyperpolarization at P0 (p<0.005).

Figure 2 represents in (A) histograms of average values of DFGABA measured in hippocampal CA3 pyramidal neurons at P15. WT mice (6.8 ±1.1 mV, n=15), FXS mice (15.3 ± 1.9 mV, n=24) and FXS mice pretreated with bumetanide (1.0 ±1.1 mV, n=17, ***P<0,001). (B) shows effects of isoguvacine (10 µM) in rats: Representative traces of spontaneous extracellular field potentials recorded in hippocampal slices at P15 in control, FXS mice and FXS mice pretreated with Bumetanide. Corresponding time-courses of spike frequency changes are shown under each trace. (C) shows average histograms of normalized spike frequency in mice shows WT mice (decrease to 67.9 ± 6.1% n=11, ***P<0,001); FXS mice (increase to 165.8 ±13.5%, n=11, ***P<0,001); FXS mice pretreated with bumetanide (decrease to 80.8 ± 8.2%, n=5, **P<0.05). Two-tails t-test for all data sets. Data presented as means ± S.E.M.

Figure 3 represents whole-cell voltage clamp recordings of spontaneous excitatory postsynaptic currents (sEPSCs) at -70 mV from individual hippocampal CA3 pyramidal neurons in acute brain slices from P15 FXS mice and respective control and bumetanide or SSR pretreated animals. (A) shows representative traces of sEPSCs recorded from mice. (B) shows average values ± S.E.M of sEPSCs frequencies in mice. WT mice (n=12) and FXS mice (n=9, ***P<9×10-5), and FXS mice with maternal pretreatment with bumetanide (n=11, ***P<2.2×10-5) and SSR-treated mice (n=13, ***P<4.5×10-5). One-way ANOVA Fisher's LSD post-hoc test. Data presented as means ± S.E.M.

Figure 4 represents isolation-induced ultrasonic vocalizations (USVs) in P8 WT (n=12), FXS (n=11) and FXS mice with maternal bumetanide pretreatment (n=13). One-way ANOVA Kruskal-Wallis test, Dunn's Multiple Comparison post-hoc test *P<0.05, **P<0.01.


EXAMPLES



[0107] The present invention is further illustrated by the following examples.

Materials and methods


Fragile X knock out mice (FXS mice)



[0108] Mice lacking Fmr1 (referred to as Fmr1-/-) were bred in our animal facility. These mice do not express any Fmr1 protein and lack detectable Fmr1 transcripts. For all experiments, Fmr1-/- mice were used with wild-type littermates as a control group. The offspring were used for experiments at E18, P0, P2, P4, P7, P9, P15 and P30. Experiments were carried out on offspring of either sex and in a blind manner at E18 and P0.

Slice preparation



[0109] Experiments were performed on fetuses and neonatal wild type and Fragile X mice from the embryonic day E20 to the postnatal day P38 (term is E21). All of the research was performed in compliance with the national guidelines on human care and use of laboratory animals and approved by INSERM

[0110] Hippocampal slices (300-500 µm thick) were prepared using a Microm tissue slicer (International GmbH, Germany) as described previously (Tyzio et al., 2006, 2008). Slices were kept in oxygenated (95% O2 / 5% CO2) artificial cerebrospinal fluid (ACSF) of the following composition (in mM): NaCl 126, KCl 3.5, CaCl2 2.0, MgCl2 1.3, NaHCO3 25, NaH2PO4 1.2 and glucose 11 (pH 7.4) at room temperature (20-22°C) at least 1 hour before use.

Electrophysiology



[0111] For recordings, slices were placed into a conventional fully submerged chamber superfused with ACSF at a rate of 2-3 mL/min at room temperature. Patch clamp recordings from visually identified CA3 pyramidal cells in cell-attached configuration were performed using EPC-10 amplifier (HEKA Elektronik Dr. Schulze GmbH, Lambrecht/Pfalz, Germany). Patch pipette solution for recordings of single GABAA channels contained (in mM): NaCl 120, KCl 5, TEA-Cl 20, 4-aminopyridine 5, CaCl2 0.1, MgCl2 10, glucose 10, Hepes-NaOH 10 buffered to pH 7.2-7.3 and GABA 2 µM. Analysis of currents through single channels and current-voltage relationships were performed using Clampfit 9.2 (Axon Instruments, Union City, CA). Recordings were digitized (10 kHz) online with Digidata-1200 interface card (Axon Instruments, Union City, CA) and analyzed offline with Axon package, MiniAnalysis (S (Synaptsoft, Decatur, GA), and Origin (Microcal Software, Northampton, MA).

Whole-cell recordings



[0112] Standard whole-cell recordings were performed in coronal neocortical rat or mouse brain slices at room temperature (20-22°C) from the soma of hippocampal CA3 neurons using an EPC-10 (HEKA Elektronik, Germany) amplifier and filtered at 3-10 kHz. The internal solution contained (in mM): 130 K-gluconate, 10 Na-gluconate, 4 NaCl, 4 MgATP, 4 phosphocreatine, 10 HEPES, and 0.3 GTP (pH 7.3 with KOH). Biocytin (final concentration 0.3-0.5%) was added to the pipette solution to label the neurons from which recordings were obtained. Neurons were visualized by using infrared DIC video microscopy. Spontaneous postsynaptic currents were recorded for 15 min at the reversal potentials for GABAergic currents (-70 mV). It should be specifically noted that all recordings were made in normal ACSF (1.3 mM MgCl2). Stored data were analyzed using the Mini Analysis 6.0.3 (Synaptosoft Inc., Chapell Hill, NC) and Origin (MicroCal, Northampton, MA) software. To minimize potential sampling bias, the pups from at least three deliveries for each condition were studied. Data are expressed as mean ±S.E.M.

Extracellular field potentials and multi-unit activity (MUA) recordings



[0113] Recording were made in the hippocampal slices using tungsten wire electrodes (diameter: 50 µm, California Fine Wire, Grover Beach, CA) and a low-noise multichannel DAM-8A amplifiers (WPI, GB; low-pass filter: 0.1 Hz; high-pass filter: 3 KHz; gain: x1000). The signals were digitized using an analogue-to-digital converter (Digidata 1440A, Axon Instruments, USA). pCLAMP 10.1, Clampfit 10.1 (Axon Instruments, USA), MiniAnalysis 6.03 (Synaptosoft, Decatur, CA) and Origin 7.5 (Microcal Software, USA) programs were used for the acquisition and analysis of the synaptic activities. Sampling interval per signal was 100 microseconds (10 kHz).

Vocalization



[0114] To induce ultrasonic vocalizations, mouse pups were isolated individually from their mother on postnatal day 8, and were placed into an isolation box (23×28×18 cm) located inside a sound attenuating isolation cubicle (54×57×41 cm; Coulbourn Instruments, Allentown, PA, USA), and evaluated for ultrasonic vocalizations during a three-minute test. An ultrasound microphone (Avisoft UltraSoundGate Condenser microphone capsule CM16/CMPA, Avisoft Bioacoustics, Berlin, Germany), placed in the roof of the box, was sensitive to frequencies of 10 to 250 kHz. Vocalizations were recorded using the Avisoft Recorder software (version 4.2) with a sampling rate of 250 kHz in 16-bit format. For acoustical analysis, recordings were transferred to SASLab Pro (version 5.2; Avisoft Bioacoustics) and a fast Fourier transformation was conducted (512 FFT-length, 100% frame, Hamming window and 75%-time window overlap). The accuracy of call detection was verified manually by an educated user. A Pearson correlation coefficient was computed to assess the relationship between Automatic and Manual analysis. There was 0.99 correlations between the two variables. At postnatal day 8, waveform patterns of calls were examined in 45 sonograms from 4 groups of mice (WT, FXS, WT pretreated, FXS pretreated). Each call was identified as one of 10 distinct categories, and only the sonograms where mice emitted at least 50 calls were used for this evaluation.

Statistics



[0115] Group measures are expressed as means ±SEM; error bars also indicate SEM. The statistical significance of differences was assessed with the Students t - test. The level of significance was set at P<0.05.

Pharmacology



[0116] All drugs were purchased from Sigma.

Results


Example 1: Developmental excitatory/inhibitory GABA sequence is abolished in hippocampal CA3 pyramidal neurons in FXS mice.



[0117] Delivery is associated with an oxytocin mediated reduction of intracellular chloride ([Cl-]i) leading to an excitatory to inhibitory shift of GABA action (Tyzio et al., 2006). This transient inhibitory shift during delivery exerts a neuro-protective action on central neurons reducing the sequels of anoxic episodes. Here, we analysed this shift in mouse model of fragile X syndrome (FXS).

[0118] To determine the driving force for [Cl-]i (farther driving force of GABA (DFGABA)), we used non-invasive single GABAA channel recordings in embryonic and post-natal hippocampal slices (E20 to P37) from animal models of FXS. In naive age matched and in heterozygote mice, DFGABA followed a sequence described in rats (Tyzio et al., 2006) - but not in mice - with an elevated [Cl-]i and depolarising DFGABA in utero (E20) followed by an abrupt transient oxytocin mediated reduction of [Cl-]i and depolarizing to hyperpolarizing shift of GABA actions restricted to a couple of hours before and after delivery (Figure 1A, B; Table 1).
Table 1: Developmental changes of driving force of GABA in heterozygote (control) mice and FXSs. The data correspond to the graph on Figure 1A.
AgeTypeMean DFGABA, (pA)±SEnCondition
E20 Wild-type 16.5 2.04 6 ACSF
E20 FXS 16.1 1.6 5 ACSF
P0 Wild-type -2.5 0.9 54 ACSF
P0 FXS 5.7 1.3 32 ACSF
P7 Wild-type 12.7 1.2 24 ACSF
P7 FXS 15.7 1.2 24 ACSF
P14 Wild-type 6.8 1.1 15 ACSF
P14 FXS 15.3 2 24 ACSF
P28-37 Wild-type -0.04 1.4 8 ACSF
P28-37 FXS 5.5 8.9 20 ACSF


[0119] Subsequently, GABA again depolarised neurons until P7, followed by a progressive decrement leading to the adult type action where DFGABA is close to the resting membrane potential (Figure 1; Table 1). Therefore, in naive rat and mice neurons, DFGABA is strongly hyperpolarising immediately after birth but depolarising before and during the first post-natal week.

[0120] In contrast, in age matched FXS neurons, DFGABA remained depolarising from E20 to P28-37 with no significant difference between various ages (Figure 1A) indicating that the curve is abolished including the delivery transient reduction. More precisely, in FXS neurons, we observed depolarising DFGABA in embryonic and P7 naive and FXS neurons but significantly more depolarised at other ages (Figure 1A; Table 1). Applications of oxytocin (1µM) shifted DFGABA during the delivery period (P0) in FXS neurons suggesting that oxytocin receptors are operative (Figure 1C, Table 2).
Table 2: Changes of driving force of GABA in CA3 pyramidal cells from hippocampal slices from P0 wild type and FXS mice in presence of bumetanide and oxytocin. The data correspond to the graph on Figure 1C.
AgeTypeMean DFGABA, (pA)±SEnCondition
P0 Wild-type -2.5 0.9 54 ACSF
P0 Wild-type -9.9 2.4 8 Bumetanide
P0 FXS 5.7 1.3 32 ACSF
P0 FXS -6.7 1.4 20 Bumetanide
P0 FXS -8.8 1.6 14 Oxytocin


[0121] The NKCC1 chloride importer antagonist bumetanide that reduces [Cl-]i fully blocked the depolarising DFGABA in FXS neurons at birth confirming the chronic persistent deregulation of chloride transport in autism (Figure 1C, Table 2).

[0122] Present results provide the first demonstration of a permanent [Cl-]i regulation deficiency in FXS lending experimental support to the successful therapeutic actions of bumetanide (compound used in the invention) & oxytocin (reference compound) that reduce [Cl-]i.

Example 2: Maternal pre-treatment with Bumetanide in FXS mice in vitro.



[0123] To test whether depolarizing actions of GABA were associated with neuronal excitation, the effect of the GABAA receptor agonist isoguvacine was tested. In naive neurons the specific GABAAR agonist isoguvacine (2 µM) inhibited or did not affect field potential recordings at P15 (Fig. 2B and C). To test whether maternal treatment with bumetanide before delivery switches the action of GABA from excitatory to inhibitory in offsprings at P15, pregnant females were treated orally 1 day before delivery with bumetanide (2-2.5 mg/kg in drinking water) and juvenile offspring neurons recorded at P15. Maternal pretreatment of FXS with bumetanide restored control DFGABA values in offsprings at P15 (Fig 2A) suppressed the excitatory actions of the GABAAR agonist isoguvacine (Fig 2 B and C) and significantly reduced ongoing activity and frequency of whole-cell recorded glutamatergic sEPSCs (Fig 3). Therefore, excitatory actions of GABA during delivery produce long term deleterious effects on electrophysiological parameters of FXS juvenile neurons. In FXS mice, GABAergic inputs are excitatory and contribute to hyperexcitability of the network. Bumetanide decreases [Cl-]i and consequently reduces contribution of excitatory GABAergic inputs in the overall network activity.

Example 3: Maternal pretreatment with bumetanide restores behavior of FXS mice.



[0124] Isolation and Ultrasonic vocalizations were induced in mice to determine behavioral effect of Bumetanide in a model of FXS. An increased probability of emitting downward and chevron calls in FXS pups than age matched WT (P8). This was rescued by maternal bumetanide pretreatment (Fig. 4, table 3). Therefore, elevated [Cl-]i levels during delivery are involved in the pathogenesis of Fragile X syndrome.
Table 3: analysis of 10 types of calls in the FXS mouse model revealed an increased probability of emitting downward and chevron calls than age matched WT pups (P8). One-way ANOVA Kruskal-Wallis test, with Dunn's Multiple Comparison post-hoc test, and Mann-Whitney test.
P8Isolation-induced USVs mean ± S.E.M
 Chevron callsDownward calls
WT mice 0.6 ± 0.2 (n=12) 10.1 ± 1.8 (n=12)
WT mice +BUM pretreatment 0.5 ± 0.3 (n=9) 13.5 ± 2.8 (n=9)
FXS mice 2.3 ± 0.5 (n=11) 19.72 ± 2.6 (n=11)
FXS mice +BUM pretreatment 0.6 ± 0.1 (n=13) 10.2 ± 1.4 (n=13)
 Statistics
 Chevron callsDownward calls
WT vs. FXS P<0.05 One-way ANOVA Kruskal-Wallis test, Dunn's Multiple Comparison post-hoc test P<0.01 One-way ANOVA Kruskal-Wallis test, Dunn's Multiple Comparison post-hoc test
FXS vs. FXS+BUM pretreatment P<0.05 One-way ANOVA Kruskal-Wallis test, Dunn's Multiple Comparison post-hoc test P<0.05 One-way ANOVA Kruskal-Wallis test, Dunn's Multiple Comparison post-hoc test
WT vs. WT+BUM pretreatment n.s. Mann-Whitney test n.s. Mann-Whitney test

Example 4: Clinical study.



[0125] This example is a case reports presenting the treatment by bumetanide of a 10-years boy with Fragile X syndrome.

[0126] The child was a bilingual boy born in October 2002 from Ukrainian mother and French father. He was the first child and had a younger sister. After a normal maternity, delivery came without difficulties (at birth, weight: 3.335kg, length: 50.5cm and head circumference: 36cm). He was hospitalized at 2 to 8 days post-natal for a spontaneously resorbed hypotonia, his transfontanel cranial ultrasound examination was normal. He was breast-fed during 3 months, sat at 10 months, walked at 19 months, and acquired bladder control at 29 months for daytime and at 36 months for night-time. He had surgical treatment of inguinal hernia (right side) at 10 months and slope right foot treated by physiotherapy. During the spring 2004, the boy presented a malaise when swimming with his father with a sudden generalized tension followed with a brief (a few seconds) spontaneous episode of hypotonia and an empty gaze. Normal EEG was observed during wakefulness.

[0127] The first concerns of the parents came at 2 years because of an onset delay of speech. The child was frequently excited, seemed very anxious, scratched, felt and smelled objects, and refused physical contact and hugs. The absence of interactions with other children was noted at day care center. He was then admitted in a daily child and adolescent psychiatry service in May 2005 (3 half days weekly) until September 2010. Speech rehabilitation was initiated in May 2007. Admitted in kindergarten starting in September 2005, he repeated a year in 2008 and was then accompanied by a special needs assistant before being admitted in specialized institution (French structures named CLIS and IME).

[0128] In February 2008, a pediatric consultation noted a fin and elongated face. At the same time, a diagnosis of pervasive development disorder was made. It was noticed that he used intermittently language with some correct sentences but most often followed by words without links to the context. Another EEG test in March 2008 revealed no abnormality during the day (sleep recording was not possible). A second pediatric consultation revealed hyperactivity, many non-constructive manipulations and behavioral stereotypies (e.g., trolling a telephone for years). He slept readily but had frequent night-time awakenings. Following these repeated sleeping problems, the child received a melatonin treatment (2 mg at night) that ameliorated sleep cycles and reduced night-time awakenings. At this moment, communication became essentially echolalic. Genetic exploration with karyotype and specific research of Fragile X syndrome (FXS) was made in May 2008. A positive identification of FXS mutation was found.

[0129] A completed diagnosis was made at the Centre de Ressources de Bretagne (CRA) in June 2009. Psychomotor examination revealed an axial hypertonicity contrasting with a hypotonia associated with a hypermobility of the hands and feet, difficulty to appreciate height and deepness, absence of lateralization, difficulties in equilibrium and inadequate tonicity. Speech-language pathology was conspicuous that was more pronounced for words of more than syllables, a fast speech flow that handicapped intelligibility. Semantic tests revealed a delay of 3 years with important syntax troubles. The child using essentially isolated words and short verbal expressions, spontaneous sentences were rare and limited to various requests. The Wechsler scale (WPPSI III) was made. Performance IQ was 50 and verbal IQ was 56. During the tests, he explored the environment and objects with an emphasis on smell and hearing sensory modalities. There were clear social interactions deficits, for example the boy was unable to develop relations with his peers and to share pleasure intents, presenting great difficulties with socio-emotive reciprocity.

[0130] Starting from the 5th January 2011, the child received bumetanide twice a day (0.5 mg morning and 0.5 mg evening) during three months. Several biological tests were made at baseline (D0) and then after 7 days (D7), one month (M1), two months (M2) and three months (M3) of treatment. They checked orthostatic hypotension, allergy, cramps, asthenia, diarrhea, myalgia, arthralgia, vertigo and nausea. Blood tests included γ-glutamyltransferase, transaminases, alkaline phosphatases, glycemia, uric acid and creatine, in addition to blood Na+ and K+.

[0131] Five clinical tests were made at baseline (D0) and after three months of treatment (M3) to determine the possible therapeutic efficacy including:
Efficacy of the treatment was measured at baseline (D0) and after three months of treatment (M3) using the GRAM scale (Grille Regulation Adaptation Modulation) for analyzing the dysregulation of the activity in a child; the ABC scale (Aberrant Behavior Checklist) for assessing problem behaviors of children and adults with mental retardation; and the CGI scale (Clinical Global Impression) for assessing the clinical amelioration in a patient following a therapeutic treatment.

[0132] After 90 days of treatment, ABC results reduced from 41 to 27. Moreover, GRAM results were reduced from 56 to 31. Finally, CGI result was 2, revealing a strong amelioration of the clinical state of the patient.

[0133] There was no side effect in the clinical examination made (i.e., orthostatic hypotension, allergy, cramps, asthenia, diarrhea, myalgia, arthralgia, vertigo and nausea). Conventional blood tests were also unaffected (i.e., γ-glutamyltransferase, transaminases, alkaline phosphatases, glycemia, uric acid, creatine).

[0134] Table 4 shows that body weight was not altered and blood Na+ and remained stable. K+ was reduced to a value close to the inferior limit (3.5 mmol/1). Natremia was unaltered by the treatment.
Table 4: Weight and blood ions during the 3 months of treatment.
 Baseline (D0)7 days (D7)1 month (M1)2 months (M2)3 months (M3)
Na+ 141 mmol/l 143 mmol/l 139 mmol/l 140 mmol/l 139 mmol/l
K+ 4.1 mmol/l 3.6 mmol/l 4.3 mmol/l 3.5 mmol/l 3.6 mmol/l
Weight 25 kg 25.2 kg 25.1 kg 25.4 kg 25.7 kg


[0135] Notice that bumetanide treatment continued after 3 months. K+ was reduced to the inferior limit value, thus potassium gluconate syrup was added (i.e., one bulb morning and evening), starting from the 17th May 2011 (4th month of treatment). It led to a recuperation of normal K+ (3.71 mmol/1) one month later (i.e., 11th June 2011).

[0136] Taken together, these results show the efficacy of bumetanide for the treatment of Fragile X syndrome.


Claims

1. A composition for use in treating Fragile X syndrome in a subject in need thereof, wherein said composition comprises a modulator of a chloride transporter, wherein said modulator is an inhibitor of a transporter involved in the importation of chloride into neurons,
wherein said transporter is NKCC1,
wherein the inhibitor of NKCC1 is selected from the group consisting of bumetanide,
an analog of bumetanide, furosemide, ethacrynic acid, torsemide, azosemide, muzolimine, piretanide and tripamide, and
wherein the analog of bumetanide is selected from the group consisting of bumetanide aldehyde, bumetanide dibenzylamide, bumetanide diethylamide, bumetanide morpholinoethyl ester, bumetanide 3-(dimethylaminopropyl) ester, bumetanide N,N-diethylglycolamide ester, bumetanide dimethylglycolamide ester, bumetanide pivaxetil ester, bumetanide methoxy(polyethyleneoxy)n-1-ethyl ester, bumetanide benzyltrimethyl-ammonium salt, bumetanide cetyltrimethylammonium salt, pivaloyloxymethyl ester of bumetanide, methyl ester of bumetanide, N,N-dimethylaminoethyl ester of bumetanide, bumetanide [-(C=O)-SH] thioacid, bumetanide S-methyl thioester, bumetanide S-cyanotnethyl thioester, bumetanide S-ethyl thioester, bumetanide S-isoamyl thioester, bumetanide S-octyl thioester, bumetanide S-benzyl thioester, bumetanide S-(morpholinoethyl) thioester, bumetanide S-[3- (dimethylaminopropyl)] thioester, bumetanide S-(N,N-diethylglycolamido)thioester, bumetanide S-(N,N-dimethylglycolamido)thioester, bumetanide S-pivaxetil thioester, bumetanide S-propaxetil thioester, bumetanide S-(methoxypolyethyleneoxy)n-1-ethyl] thioester, bumetanide [-(C=O)-S-] benzyltrimethyl-ammonium thioacid salt and bumetanide [-(C=O)-S] cetyltrimethylammonium thioacid salt, metastable bumetanide thioacid, bumetanide thioaldehyde, bumetanide O-methyl thioester, bumetanide O-cyanomethyl thioester, bumetanide O-ethyl thioester, bumetanide O-isoamyl thioester, bumetanide O-octyl thioester, bumetanide O-benzyl thioester, bumetanide O-(morpholinoethyl) thioester, bumetanide O-[3-(dimethylaminopropyl)] thioester, bumetanide O-(N,N-diethylglycolamido)thioester, bumetanide O-pivaxetil thioester, bumetanide O-propaxetil thioester, bumetanide O-[methoxy(polyethyleneoxy)n-1 ethyl] thioester, bumetanide [-(C=S)-O-] benzyltrimethyl-ammonium thioacid salt and bumetanide [-(C=S)-O-] cetyltrimethylammonium thioacid salt.
 
2. The composition for use according to claim 1, wherein the inhibitor of NKCC1 is selected from the group consisting of bumetanide, furosemide, ethacrynic acid, torsemide, azosemide, muzolimine, piretanide and tripamide.
 
3. The composition for use according to claim 1 or 2, wherein the inhibitor of NKCC1 is bumetanide.
 
4. The composition for use according to any one of claims 1 to 3, wherein the amount of the composition ranges from about 0.01 mg to about 500 mg.
 
5. The composition for use according to any one of claims 1 to 4, wherein the composition is to be administered to the subject in need thereof by subcutaneous, intramuscular, intravenous, intraocular, transdermal, topical, parenteral, intranasal or oral administration, or injection.
 
6. The composition for use according to any one of claims 1 to 5, wherein the subject in need thereof presents a number of CGG repeats in the 5'-UTR of FMR1 of at least 55, preferably of at least 200.
 
7. The composition for use according to any one of claims 1 to 6, wherein the subject in need thereof is diagnosed with Fragile X syndrome.
 
8. The composition for use according to any one of claims 1 to 6, wherein the subject in need thereof is at risk of developing Fragile X syndrome.
 
9. The composition for use according to any one of claims 1 to 8, wherein the subject in need thereof is a fetus and the composition is to be administered to his/her mother.
 


Ansprüche

1. Zusammensetzung zur Verwendung bei der Behandlung des Fragile-X-Syndroms bei einem davon betroffenen Patienten, wobei die Zusammensetzung einen Modulator eines Chlorid-Transporters umfasst, wobei der Modulator ein Hemmstoff eines Transporters ist, der in den Import von Chlorid in Nervenzellen eingebunden ist,
wobei der Transporter NKCC1 ist,
wobei der Hemmstoff von NKCC1 aus der Gruppe ausgewählt ist, die aus Bumetanid, einem Analog von Bumetanid, Furosemid, Etacrynsäure, Torasemid, Azosemid, Muzolimin, Piretanid und Tripamid besteht, und
wobei das Analog von Bumetanid aus der Gruppe ausgewählt ist, die aus Bumetanidaldehyd, Bumetaniddibenzylamid, Bumetaniddiethylamid, Bumetanidmorpholinoethylester, Bumetanid 3-(Dimethylaminopropyl)-Ester, Bumetanid N,N-Diethylglycolamidester, Bumetaniddimethylglycolamidester, Bumetanidpivaxetilester, Bumetanidmethoxy(polyethylenoxy)n-1-Ethylester, Bumetanidbenzyltrimethyl-Ammonium-Salz, Bumetanidcetyltrimethylammonium-Salz, Bumetanid-Pivaloyloxymethylester, Bumetanid-Methylester, N,N-Bumetanid-Dimethylaminoethylester, Bumetanid [-(C=O)-SH] Thiosäure, Bumetanid-S-Methylthioester, Bumetanid-S-Cyanotnethyl-Thioester, Bumetanid-S-Ethyl-Thioester, Bumetanid-S-Isoamyl-Thioester, Bumetanid-S-Octyl-Thioester, Bumetanid-S-Benzyl-Thioester, Bumetanid-S-(Morpholinoethyl)-Thioester, Bumetanid-S-[3-(Dimethylaminopropyl)]-Thioester, Bumetanid-S(N,N-Diethylglycolamido)-Thioester, Bumetanid-S(N,N-Dimethylglycolamido)-Thioester, Bumetanid-S-Pivaxetil-Thioester, Bumetanid-S-Propaxetil-Thioester, Bumetanid-S-(Methoxypolyethylenenoxy)n-1-Ethyl]-Thioester, Bumetanid [-(C=O)-S-] Benzyltrimethyl-Ammonium Thiosäure-Salz und Bumetanid [-(C=O)-S] Cetyltrimethylammonium Thiosäure-Salz, metastabiler Bumetanid-Thiosäure, Bumetanid Thioaldehyd, Bumetanid O-Methyl Thioester, Bumetanid O-Cyanomethyl Thioester, Bumetanid O-Ethyl Thioester, Bumetanid O-Isoamyl Thioester, Bumetanid O-Octyl Thioester, Bumetanid O-Benzyl Thioester, Bumetanid O-(Morpholinoethyl) Thioester, Bumetanid O-[3-(Dimethylaminopropyl)] Thioester, Bumetanid O-(N,N-Diethylglycolamido)Thioester, Bumetanid O-Pivaxetil Thioester, Bumetanid O-Propaxetil Thioester, Bumetanid O-[Methoxy(polyethylenoxy)n-1-Ethyl]-Thioester, Bumetanid [-(C=S)-O-] Benzyltrimethyl-Ammonium Thiosäure-Salz und Bumetanid [-(C=S)-O-] Cetyltrimethylammonium Thiosäure-Salz besteht.
 
2. Zusammensetzung zur Verwendung nach Anspruch 1, wobei der Hemmstoff von NKCC1 aus der Gruppe ausgewählt ist, die aus Bumetanid, Furosemid, Etacrynsäure, Torsemid, Azosemid, Muzolimin, Piretanid und Tripamid besteht.
 
3. Zusammensetzung zur Verwendung nach Anspruch 1 oder 2, wobei der Hemmstoff von NKCC1 Bumetanid ist.
 
4. Zusammensetzung zur Verwendung nach einem der Ansprüche 1 bis 3, wobei die Menge der Zusammensetzung von etwa 0,01 mg bis etwa 500 mg reicht.
 
5. Zusammensetzung zur Verwendung nach einem der Ansprüche 1 bis 4, wobei die Zusammensetzung dem davon betroffenen Patienten durch subkutane, intramuskuläre, intravenöse, intraokulare, transdermale, topische, parenterale, intranasale oder orale Verabreichung, oder Injektion verabreicht wird.
 
6. Zusammensetzung zur Verwendung nach einem der Ansprüche 1 bis 5, wobei der davon betroffene Patient eine Anzahl von CGG-Repeats in den 5'-UTR von FMR1 von mindestens 55, vorzugsweise von mindestens 200 aufweist.
 
7. Zusammensetzung zur Verwendung nach einem der Ansprüche 1 bis 6, wobei beim davon betroffene Patienten ein Fragile-X-Syndrom diagnostiziert worden ist.
 
8. Zusammensetzung zur Verwendung nach einem der Ansprüche 1 bis 6, wobei der davon betroffene Patient Gefahr läuft, ein Fragile-X-Syndrom zu entwickeln.
 
9. Zusammensetzung zur Verwendung nach einem der Ansprüche 1 bis 8, wobei der davon betroffene Patient ein Fötus ist und die Zusammensetzung seiner/ihrer Mutter zu verabreichen ist.
 


Revendications

1. Une composition pour son utilisation dans le traitement du syndrome de l'X fragile chez un sujet en ayant besoin,
dans laquelle ladite composition comprend un modulateur d'un transporteur du chlorure,
dans laquelle ledit modulateur est un inhibiteur d'un transporteur impliqué dans l'import du chlorure dans les neurones,
dans laquelle ledit transporteur est NKCC1,
dans laquelle l'inhibiteur de NKCC1 est sélectionné parmi le groupe constitué de bumétanide, un analogue du bumétanide, furosémide, acide étacrynique, torsémide, azosémide, muzolimine, pirétanide et tripamine, et
dans laquelle l'analogue du bumétanide est sélectionné parmi le groupe constitué de aldéhyde bumétanide, dibenzylamide bumétanide, diéthylamide bumétanide, ester de morpholinoéthyle bumétanide, ester de 3-(diméthylaminopropyl) bumétanide, ester de N,N-diéthylglycolamide bumétanide, ester de diméthylglycolamide bumétanide, ester de pivaxetil bumétanide, ester de méthoxy(polyéthylèneoxy)n-1-éthyl bumétanide, sel de benzyltriméthylammonuim bumétanide, sel de cétyltriméthylammonium bumétanide, ester de pivaloyloxyméthyle bumétanide, ester de méthyle bumétanide, ester de N,N-diméthylaminoéthyl bumétanide, thioacide [-(C=O)-SH] de bumétanide, thioester de S-méthyl bumétanide, thioester de S-cyanotnéthyl bumétanide, thioester de S-éthyl bumétanide, thioester de S-isoamyl bumétanide, thioester de S-octyl bumétanide, thioester de S-benzyl bumétanide, thioester de S-(morpholinoéthyl) bumétanide, thioester de S-[3-(diméthylaminopropyl)] bumétanide, thioester de S-(N,N-diéthylglycolamido) bumétanide, thioester de S-(N,N-diméthylglycolamido) bumétanide, thioester de S-pivaxetil bumétanide, thioester de S-propaxetil bumétanide, thioester de S-[(méthoxypolyéthylèneoxy)n-1-éthyl] bumétanide, sel de thioacide [-(C=O)-S-] benzyltriméthyl-ammonium bumétanide, sel de thioacide [-(C=O)-S] benzyltriméthyl-ammonium bumétanide et sel de thioacide cétyltriméthylammonium bumétanide, thioacide métastable de bumétanide, thioaldéhyde bumétanide, thioester de O-méthyl bumétanide, thioester de O-cyanométhyl bumétanide, thioester de O-éthyl bumétanide, thioester de O-isoamyl bumétanide, thioester de O-octyl bumétanide, thioester de O-benzyl bumétanide, thioester de O-(morpholinoéthyl) bumétanide, thioester de O-[3-(diméthylaminopropyl)] bumétanide, thioester de O-(N,N-diéthylglycolamido) bumétanide, thioester de O-pivaxetil bumétanide, thioester de O-propaxetil bumétanide, thioester de O-[méthoxy(polyéthylèneoxy)n-1 éthyl] bumétanide, sel de thioacide [-(C=S)-O-] benzyltriméthyl-ammonium bumétanide et sel thioacide [-(C=S)-O-] de cétyltriméthylammonium bumétanide.
 
2. La composition pour son utilisation selon la revendication 1, dans laquelle l'inhibiteur de NKCC1 est sélectionné parmi le groupe constitué de bumétamide, furosémide, acide étacrynique, torsémide, azosémide, muzolimine, pirétanide et tripamide.
 
3. La composition pour son utilisation selon la revendication 1 ou 2, dans laquelle l'inhibiteur de NKCC1 est le bumétanide.
 
4. La composition pour son utilisation selon l'une quelconque des revendications 1 à 3, dans laquelle la quantité de la composition est comprise entre environ 0.01 mg et environ 500 mg.
 
5. La composition pour son utilisation selon l'une quelconque des revendications 1 à 4, dans laquelle la composition est à administrer au sujet en ayant besoin par une administration sous-cutanée, intra-musculaire, intraveineuse, intraoculaire, transdermique, topique, parentérale, intranasale ou orale, ou par injection.
 
6. La composition pour son utilisation selon l'une quelconque des revendications 1 à 5, dans laquelle le sujet en ayant besoin présente un nombre de répétitions CGG dans la région 5'-UTR de FMR1 d'au moins 55, de préférence au moins 200.
 
7. La composition pour son utilisation selon l'une quelconque des revendications 1 à 6, dans laquelle le sujet en ayant besoin est diagnostiqué avec le syndrome de l'X fragile.
 
8. La composition pour son utilisation selon l'une quelconque des revendications 1 à 6, dans laquelle le sujet en ayant besoin présente un risque de développer le syndrome de l'X fragile.
 
9. La composition pour son utilisation selon l'une quelconque des revendications 1 à 8, dans laquelle le sujet en ayant besoin est un fœtus et la composition est à administrer à sa mère.
 




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Cited references

REFERENCES CITED IN THE DESCRIPTION



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Patent documents cited in the description




Non-patent literature cited in the description