Methods for Treating Autism Spectrum Disorders

Abstract

Autism spectrum disorder (ASD) is a neurodevelopmental disorder whose diagnosis is due to impaired social interaction and communication accompanied by repetitive behaviors. To date, no pharmacological treatment has been approved to improve social behavior in patients with ASD. Based on the excitation / inhibition imbalance theory of autism, we hypothesized that bromide ion, which has been used for many years as an antiepileptic drug, may alleviate the core symptoms of ASD. We investigated three genetic mouse models of autism: Oprm1 - / - , Fmr1 - / - and Shank3 Δex13-16- / - The effect of chronic administration of sodium bromide (NaBr) on autism-like symptoms in mice was evaluated. We showed that chronic treatment with NaBr alleviated autism-like behavior in these three models. - / - In mice, these beneficial effects were superior to those of chronic bumetanide treatment. At the transcriptional level, chronic NaBr in Oprm1-deficient mice was associated with increased expression of genes encoding chloride ion transporters, GABAA receptor subunits, oxytocin, and mGlu4 receptors. Finally, we found that Oprm1 - / - We demonstrated a synergistic alleviating effect of chronic NaBr and a positive allosteric modulator (PAM) of the mGlu4 receptor on autistic-like behavior in mice. We demonstrated in heterologous cells that bromide ions act as a PAM of the mGlu4 receptor, providing a molecular mechanism for such synergistic effects. The data reveal the therapeutic potential of bromide ions alone or in combination with a PAM of the mGlu4 receptor for the treatment of ASD.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention is in the field of medicine, particularly neurology.

[0002] 2. Background of the Invention Autism spectrum disorders (ASD) are neurodevelopmental disorders with high heterogeneity and heritability. The diagnosis is reached when social communication and interaction are impaired and the repertoire of behaviors, interests and activities is restricted and repetitive (1). Along with the core symptoms, ASD is often accompanied by neurobehavioral comorbidities, such as high anxiety, cognitive and motor disorders or epilepsy (2-5). Despite the identification of vulnerability genes and environmental risk factors (6-8), the etiology of ASD remains essentially unknown, making the development of pharmacological treatments for these conditions a real challenge.

[0003] Excitation / inhibition (E / I) imbalance appears to be a common mechanistic feature of ASDs (9, 10). The heuristic hypothesis of an exaggerated E / I ratio in ASD was initially proposed by Rubenstein and Merzenich (11) and attracted great interest as a good explanation for the reduced GABA signaling (12, 13) and high incidence of epilepsy (10-30%) (3) in these conditions. Indeed, epilepsy is one of the most frequent comorbid conditions in autism (5, 14), and the incidence of epileptiform EEG or resting-state changes is even higher (15, 16). This suggests common risk factors and / or pathophysiological mechanisms between these conditions (17, 18). However, since its original proposal, the excessive E / I hypothesis in ASD has been challenged by studies of animal models showing reduced arousal, leading to the more general concept of altered E / I homeostasis ( 10 , 19 ).

[0004] The impairment of E / I balance in ASD may result from several neuropathological mechanisms. On the excitatory side, glutamatergic transmission was found to be altered in both patients and animal models, but the directionality differed depending on the genetic mutation / model (9, 20, 21). On the inhibitory side, in line with impaired GABAergic signaling, reduced GABA levels (22) and expression of GABAA and GABAB receptors (23, 24) and genetic polymorphisms of GABAA receptor subunits (25, 26) have been detected in patients with autism. Accordingly, reduced GABAergic neurotransmission has been reported in several ASD models (27–31). Moreover, preclinical studies have shown that low doses of benzodiazepines, which act as positive allosteric modulators (PAMs) of the GABAA receptor (31, 32), or arbaclofen, a GABAB receptor agonist, can improve autistic-like behaviors in animal models (33, 34). Unfortunately, however, clinical trials have failed to demonstrate significant beneficial effects of such compounds in fragile X syndrome (35, 36). Alternatively, GABA neurons remain immature in ASD, resulting in reduced production of chloride ions (Cl - ) from a high to a low intracellular concentration, resulting in a depolarizing Cl-mediated Cl-upole through activated GABAA receptors. - It has also been suggested that the efflux of intracellular Cl is maintained (37). - The concentration is the major Cl - The importer NKCC1 (Na + -K + -2Cl -NKCC1 is under the control of NKCC1 (a symporter of NKCC1) and KCC2, the major chloride exporter. Thus, blocking NKCC1 using bumetanide, a loop diuretic and antiepileptic drug (38, 39), appeared to be a promising therapeutic approach in ASD. Accordingly, bumetanide was found to improve autism-like phenotypes in rodent models of ASD (40) and to alleviate autistic behaviors in small patient cohorts (41, 42). However, clinical benefits, except for the reduction of repetitive behaviors, were not confirmed in large-scale clinical trials (43).

[0005] Bromide ion (Br - Br was the first identified effective treatment for epilepsy (44) and has long been used as an anxiolytic and hypnotic agent (45). With the advent of newer antiepileptic and anxiolytic drugs that are more specific and possibly less toxic, Br - Although the use of Br has gradually decreased, it remains a valuable tool for treating refractory seizures (46, 47). - is Cl - It shares chemical and physical similarities with Cl, and is therefore involved in multiple cellular mechanisms. - These are substitutes for activated GABA. A It involves receptor-mediated anion efflux, and Br - Permeability to Cl - Br - Cl is also involved in a mechanism involving NKCC and KCC cotransporters. - From the perspective of E / I imbalance theory, these characteristics make Br an interesting candidate for ASD treatment. - (WO2018 / 096184).

[0006] Summary of the Invention The present invention is defined by the claims. In particular, the present invention relates to a method of treating autism spectrum disorder (ASD) in a subject in need thereof, comprising administering to the subject a therapeutically effective combination comprising a bromide salt and a positive allosteric modulator of the mGlu4 receptor (PAM).

[0007] Detailed Description of the Invention In this study, we present three genetic mouse models of autism: Oprm1 - / - , Fmr1 - / - and Shank3 Δex13-16- / - In mice, the effects of chronic sodium bromide administration were evaluated by thorough behavioral assessment on the core autism-like symptoms of social deficit and stereotypy, as well as on the frequently comorbid symptom of anxiety. For these three models, alterations in E / I balance and / or changes in the expression of genes involved in this balance have been reported (30, 51-55). The Oprm1 knockout model has the advantage of having limited effects on learning ability (52), allowing a better distinction between autistic features and cognitive impairment. We show that the Br - Treatment ameliorated most of the behavioral deficits observed in these mice and demonstrated increased expression of various genes in the social brain circuit. - / - In mice and in different types of cells, Br - We also found that Br1 increased the expression of the mGlu4 receptor gene and enhanced the effects of the mGlu4 PAM VU0155041 and its agonist glutamate. - This study highlights the therapeutic potential of administration of, and combination of, mGlu4 receptor positive allosteric modulators (PAMs) for the treatment of ASD.

[0008] Methods for Treating Autism Spectrum Disorders In a first aspect, the present invention relates to a method of treating autism spectrum disorder (ASD) in a subject in need thereof, comprising administering to the subject a therapeutically effective combination comprising a bromide salt and a positive allosteric modulator of the mGlu4 receptor (PAM).

[0009] As used herein, the term "subject" or "patient" refers to a mammal, preferably a human. Typically, according to the present invention, a subject refers to any subject suffering from or susceptible to suffering from an autism spectrum disorder (ASD).

[0010] As used herein, the term "autism spectrum disorder" or "ASD" refers to a developmental disorder that affects a subject's communication and behavior. According to the Diagnostic and Statistical Manual of Mental Disorders, autism is characterized by difficulty communicating and interacting with others, restricted interests and activities, and repetitive behaviors (American Psychiatric Association. (2013). Diagnostic and statistical manual of mental disorders (5 th ed.). Arlington, VA: Author).

[0011] As used herein, the term "treatment" or "treating" refers to both prophylactic or preventive treatments and curative or disease-modifying treatments, including treatment of patients at risk of or suspected of having a disease, and treatment of patients who are ill or have been diagnosed with a disease or medical condition. The term also includes suppression of clinical recurrence. Treatment can be administered to a subject who has a medical disorder or is at risk of eventually becoming ill, to prevent, cure, delay the onset, reduce the severity, or ameliorate one or more symptoms of the disorder or recurrent disorder, or to extend the subject's survival beyond that expected in the absence of such treatment. "Therapeutic regimen" refers to a pattern of treatment of a disease, e.g., a dosing pattern used during treatment. Therapeutic regimens can include induction regimens and maintenance regimens. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or a portion of a therapeutic regimen) used for the initial treatment of a disease. The general purpose of an induction regimen is to provide a high level of drug to the patient during the initial period of the treatment regimen. An induction regimen may utilize (partially or entirely) a "loading regimen". This may involve administering a larger amount of drug than a physician would utilize during a maintenance regimen, administering a drug more frequently than a physician would administer during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or a portion of a treatment regimen) used to maintain a patient during disease treatment, e.g., to keep a patient in remission for an extended period of time (months or years). A maintenance regimen may utilize continuous treatment (e.g., administering a drug at regular intervals, such as weekly, monthly, yearly, etc.) or intermittent treatment (e.g., interruption of treatment, intermittent treatment, treatment upon relapse, or treatment upon certain predefined criteria (e.g., pain, disease symptoms, etc.)).

[0012] In particular, the methods of the present invention are particularly useful for alleviating at least one symptom of ASD in a subject in need thereof, wherein said at least one symptom is social behavioral impairment, stereotypic behavior, and / or excessive anxiety.

[0013] As used herein, the term "social behavior disorder" refers to a condition in which a subject has difficulty with verbal and non-verbal communication. By way of example, symptoms may include abnormal or inappropriate body language, gestures and facial expressions, lack of interest in others or lack of sharing interests or accomplishments, not approaching others or seeking social interaction, coming across as aloof and detaches, preferring to be alone, difficulty understanding the emotions, reactions and non-verbal cues of others, resistance to being touched, difficulty or inability to make friends, delayed learning to speak or not speaking at all, speaking with an irregular tone of voice or a strange rhythm or pitch, repeating words or phrases repeatedly without any intention to communicate, difficulty starting or keeping a conversation going, difficulty communicating needs or desires, inability to understand simple statements or questions and / or taking what is said literally, not noticing humor, sarcasm and sarcasm (American Psychiatric Association. (2013). Diagnostic and statistical manual of mental disorders (5 th ed.). Arlington, VA: Author).

[0014] As used herein, the term "stereotypic behavior" refers to a condition in which a subject suffers from restricted, rigid and / or obsessive behavior, activities and / or interests. By way of example, symptoms may include cognitive impairment, repetitive body movements, constant movement, obsession with novelty, preoccupation with a narrow subject of interest (possibly including numbers or symbols), a strong need for sameness, order and routines (becoming upset when routines or the environment change), clumsiness, irregular postures or odd movements, fascination with spinning objects, moving parts or pieces of toys, and / or over- or under-responsiveness to sensory input (American Psychiatric Association. (2013). Diagnostic and statistical manual of mental disorders (5 th ed.). Arlington, VA: Author).

[0015] As used herein, the term "excessive anxiety" refers to a condition in which a subject suffers from frequent, intense, excessive, persistent worries and fears about life circumstances. By way of example, symptoms may include difficulty concentrating or sleeping, irritability, muscle tension, difficulty controlling emotions or worries, dizziness or palpitations, restlessness, or feeling anxious (US Department of Health and Human Services, National Institutes of Health, National Institute of Mental Health. (2015). NIMH Strategic Plan for Research (NIH Publication No. 02-2650). Retrieved from http: / / www.nimh.nih.gov / about / strategic-planning-reports / index.shtml).

[0016] As used herein, the term "bromide salt" has its ordinary meaning in the art and refers to an inorganic compound consisting of an ionic assembly of bromine in cationic form with one anion. In certain embodiments, the bromide salt is selected from potassium salt, sodium salt, ammonium salt, calcium salt or lithium salt, either alone or as a mixture of two, three, four or five of said salts.

[0017] In a particular embodiment, the bromide salt is sodium bromide (NaBr). In another particular embodiment, the bromide salt is potassium bromide (KBr).

[0018] As used herein, the term "positive allosteric modulator" or "PAM" refers to a class of substances that bind to a receptor and increase agonist affinity (i.e., increase the probability that an agonist will bind to the receptor) and / or increase agonist efficacy (i.e., increase the ability to activate the receptor) (Abdel-Magid AF. Allosteric modulators: an emerging concept in drug discovery. ACS Med Chem Lett. 2015;6(2):104-107. Published 2015 Jan 8. doi:10.1021 / ml5005365).

[0019] As used herein, the term "mGlu4 receptor" or "metabotropic glutamate receptor 4" refers to a protein belonging to group III of the metabotropic glutamate receptor family. Metabotropic glutamate receptors are a family of G protein-coupled receptors that are divided into three groups based on sequence homology, putative signaling mechanisms, and pharmacological properties. Group III receptors have been associated with inhibition of the cyclic AMP cascade. Ligand binding results in conformational changes that trigger signaling through guanine nucleotide-binding proteins (G proteins), modulating the activity of downstream effectors. Signaling leads to inhibition of adenylate cyclase activity (Wu S, Wright RA, Rockey PK, et al. Group III human metabotropic glutamate receptors 4, 7 and 8: molecular cloning, functional expression, and comparison of pharmacological properties in RGT cells. Brain Res Mol Brain Res. 1998;53(1-2):88-97. doi:10.1016 / s0169-328(97)00277-5).

[0020] The MGlu4 receptor is encoded by the GRM4 gene (Gene ID: 2914; Ensembl: ENSG00000124493; OMIM: 604100; UniProt: Q14833).

[0021] By way of example, the PAM of the mGlu4 receptor may be VU0155041 (CAS number: 1093757-42-6), PXT-002331 (CAS number: 2133294-96-7), DT-1687 (CAS number: 1883329-53-0), VU0361737 (CAS number: 1161205-04-4), VU0364770 (CAS number: 61350-00-3), VU0418506, VU001171, VU0652957 (CAS number: 1976050-09-5), PHCCC (CAS number: 179068-02-1) or AB120043 (CAS number: 68-19-9).

[0022] In certain embodiments, the positive allosteric modulator of the mGlu4 receptor has formula (I): [ka] The compound VU0155041 has the following structure:

[0023] As used herein, the term "combination" is intended to refer to all dosage forms that provide a first agent together with a further (second, third...) agent. The agents can be administered simultaneously, separately or sequentially, and in any order. The agents administered in combination have a biological activity in the subject to which they are delivered. Thus, in the context of the present invention, a combination comprises at least two different agents, one agent being a bromide salt and the other agent being a positive allosteric modulator of the mGlu4 receptor. According to the present invention, the combination of the present invention results in a synergistic effect. As used herein, the term "synergistic effect" or grammatical variations thereof means and includes a synergistic effect that occurs in the combination of two or more active compounds, in which the combined activity of two or more active compounds exceeds the sum of the activity of each active compound alone.

[0024] In certain embodiments, the bromide salt is administered chronically. As used herein, the term "chronically" means in a continuous and repetitive manner, but not necessarily at regular intervals.

[0025] In some embodiments, the bromide salt is administered at least once a day. In some embodiments, the bromide salt is administered at least once a week. In some embodiments, the bromide salt is administered at least once every two weeks.

[0026] In some embodiments, the positive allosteric modulator (PAM) of mGlu4 is administered at least once a day. In some embodiments, the positive allosteric modulator (PAM) of mGlu4 is administered at least once a week. In some embodiments, the positive allosteric modulator (PAM) of mGlu4 is administered at least once every two weeks.

[0027] As used herein, the term "therapeutically effective amount" refers to an amount of bromide salt and mGlu4 receptor positive allosteric modulator (PAM) compound sufficient to achieve a therapeutic effect (alleviate at least one symptom of ASD). However, it will be understood that the total daily usage of the compounds and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific level of a therapeutically effective dose for any particular subject will depend on a variety of factors, including the disorder being treated and the severity of the disorder, the activity of the specific compound being utilized, the specific composition being utilized, the age, weight, general health, sex, and diet of the subject, the administration time, route of administration and excretion rate of the specific compound being utilized, the duration of treatment, drugs used in combination or simultaneously with the specific polypeptide being utilized, and similar factors well known in the medical arts. For example, it is well within the skill of the art to start the dosage of the compound at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0028] In certain embodiments, the bromide salt is administered at least 10 mg / kg.

[0029] In certain embodiments, the bromide salt is administered at least 30 mg / kg.

[0030] In certain embodiments, the bromide salt is administered at least 70 mg / kg.

[0031] In some embodiments, the bromide salt is administered at least 125 mg / kg.

[0032] In some embodiments, the bromide salt is administered at least 145 mg / kg.

[0033] In certain embodiments, the bromide salt is administered at least 250 mg / kg.

[0034] In certain embodiments, the bromide salt is administered at least 500 mg / kg.

[0035] In certain embodiments, the allosteric modulator of an mGlu4 receptor is administered at least 1 mg / kg.

[0036] A further object of the present invention relates to a method for enhancing the efficacy of a positive allosteric modulator (PAM) of the mGlu4 receptor administered to a subject suffering from an autism spectrum disorder (ASD), comprising administering to said subject a pharma- ceutical effective amount of a positive allosteric modulator (PAM) of the mGlu4 receptor in combination with a bromide salt.

[0037] In certain embodiments, the present invention relates to a method of enhancing the efficacy of VU0155041 administered to a subject suffering from autism spectrum disorder (ASD), comprising administering to the subject a pharma- tically effective amount of VU0155041 in combination with sodium bromide.

[0038] Parts Kit In a second aspect, the present invention relates to i) a bromide salt and ii) a positive allosteric modulator (PAM) of the mGlu4 receptor as a combined preparation for simultaneous, separate or sequential use in the treatment of autism spectrum disorder (ASD).

[0039] As used herein, the term "concurrent use" refers to the simultaneous use of a bromide salt and a positive allosteric modulator (PAM) of the mGlu4 receptor.

[0040] As used herein, the term "separate use" refers to the non-concurrent use of a bromide salt and a positive allosteric modulator (PAM) of the mGlu4 receptor.

[0041] As used herein, the term "sequential use" refers to the use of a bromide salt and a positive allosteric modulator (PAM) of the mGlu4 receptor that occurs in a certain order.

[0042] In one embodiment, the present invention relates to i) sodium bromide and ii) VU0155041 as a combined preparation for simultaneous, separate or sequential use in the treatment of autism spectrum disorder (ASD).

[0043] therapeutic composition In a third aspect, the present invention relates to a therapeutic composition comprising a bromide salt and a positive allosteric modulator (PAM) of the mGlu4 receptor, for use in treating autism spectrum disorder (ASD) in a subject in need thereof.

[0044] In certain embodiments, the present invention relates to a therapeutic composition comprising sodium bromide and VU0155041, for use in treating autism spectrum disorder (ASD) in a subject in need thereof.

[0045] Typically, the bromide salt and the mGlu4 receptor positive allosteric modulator (PAM) can be combined with a pharma- ceutically acceptable excipient, and optionally a sustained release matrix, such as a biodegradable polymer, to form a therapeutic composition.

[0046] "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when administered to mammals, particularly humans, as appropriate. Pharmaceutically acceptable carriers or excipients refer to any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid. In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, topical or rectal administration, the active ingredient can be administered alone or in combination with another active ingredient in a unit dosage form, in admixture with a conventional pharmaceutical support, to animals and humans. Suitable unit dosage forms include oral route forms, such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal dosage forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subcutaneous, transdermal, intrathecal and intranasal dosage forms, and rectal dosage forms. Galenic adaptations may be performed for specific delivery in the small intestine or colon. Preferably, the pharmaceutical compositions contain a pharma- ceutically acceptable vehicle for injectable preparations. These may in particular be isotonic and sterile saline solutions (monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc., or mixtures of these salts), or dry compositions, in particular lyophilized compositions, which can be made up into solutions for injection, optionally by adding sterile water or saline. Pharmaceutical forms suitable for injection use include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the preparation must be sterile and fluid to the extent that it is easily syringable. The preparation must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions containing the bromide salts of the present invention and a positive allosteric modulator (PAM) of the mGlu4 receptor (as a free base or a pharmacologically acceptable salt) can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof, and in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The bromide salts and mGlu4 receptor positive allosteric modulators (PAMs) of the present invention can be formulated in neutral or salt form to form compositions. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins), which are formed with inorganic acids such as hydrochloric or phosphoric acid, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating (e.g., lecithin), by the maintenance of the required particle size (in the case of dispersions), and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, such as sugars or sodium chloride. Prolonged absorption of the injectable compositions can be achieved by using in the composition agents that delay absorption, such as aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the required amount of the active polypeptide in an appropriate solvent, with various other ingredients as enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle (containing the basic dispersion medium and the required other ingredients from those enumerated above).In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation methods are vacuum drying and freeze-drying techniques, which produce powders of the active ingredients plus any additional desired ingredients from their previously sterile filtered solutions. Once formulated, the solutions are administered in a manner compatible with the dosage form and in such amounts as are therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as injectable solutions of the types described above, but drug release capsules and the like may also be utilized. For example, when administered parenterally in aqueous solutions, the solutions should be buffered if necessary and the liquid diluent should first be made isotonic with sufficient salt or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, the sterile aqueous media that can be utilized will be known to those of skill in the art in light of the present disclosure. For example, one dose can be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of subcutaneous infusion fluid or injected at the intended site of infusion. There will necessarily be some variation in the dosage depending on the condition of the subject being treated. Multiple administrations are also possible. In addition to the bromide salts and mGlu4 receptor positive allosteric modulators (PAMs) of the present invention formulated for parenteral administration (e.g., intravenous or intramuscular injection), other pharma- ceutically acceptable forms include, for example, tablets and other solid forms for oral administration; liposomal formulations; sustained release capsules; and any other currently used forms.

[0047] In certain embodiments, the present invention relates to a therapeutic composition comprising sodium bromide and VU0155041, wherein the therapeutic composition is for use in treating autism spectrum disorder (ASD) in a subject in need thereof.

[0048] In certain embodiments, the therapeutic composition of the present invention may comprise at least one additional therapeutically active agent. For example, the at least one additional therapeutically active agent may be a diuretic, such as bumetanide (CAS No.: 28395-03-1), anxiolytics, such as clobazam (CAS No.: 22316-47-8), clorazepate (CAS No.: 57109-90-7), nordazepam (CAS No.: 1088-11-5), diazepam (CAS No.: 439-14-5), prazene ... azepam (CAS number: 2955-38-6), alprazolam (CAS number: 28981-97-7), bromazepam (CAS number: 1812-30-2), lorazepam (CAS number: 846-49-1), oxazepam (CAS number: 604-75-1), hydroxyzine (CAS number: 68-88-2), antipsychotics, such as risperidone (CAS number: 106 266-06-2), aripiprazole (CAS number: 129722-12-9), olanzapine (CAS number: 132539-06-1), neuroleptics such as lamotrigine (CAS number: 84057-84-1), carbamazepine (CAS number: 298-46-4), valpromide (CAS number: 2430-27-5), muscle relaxants such as baclofen (C AS number: 1134-47-0), antidepressants such as fluoxetine (CAS number: 54910-89-3), sertraline (CAS number: 79617-96-2), paroxetine (CAS number: 61869-08-7) and / or stimulants such as Ritalin (CAS number: 113-45-1) or caffeine (CAS number: 58-08-2).

[0049] In certain embodiments, the therapeutic compositions of the present invention may include at least one additional compound, such as glutamic acid, vitamin B6, and / or vitamin B12.

[0050] The present invention will be further illustrated by the following figures and examples, which should not be construed as limiting the scope of the invention in any way. [Brief description of the drawings]

[0051] [Figure 1]Chronic sodium bromide dose-dependently alleviated social behavioral impairment in Oprm1- / - mice, with a superior effect to chronic bumetanide. (A) Oprm1+ / + and Oprm1- / - mice were treated once daily for 18 days with either NaBr (0, 125-500 mg / kg: n=14-20 mice per genotype and dose; 10-70 mg / kg: n=8 mice per genotype and dose) or bumetanide (0, 0.5 and 2 mg / kg: n=8-10 mice per genotype and dose). Behavioral testing began on day 8. Social interaction was retested 1 and 2 weeks after chronic treatment was discontinued. (B) In the direct social interaction test (day 9), chronic administration of NaBr (B1) dose-dependently alleviated social impairment in Oprm1-deficient mice at doses >125 mg / kg. There was no detectable effect in Oprm1+ / + mice. Bumetanide (B2) had only a partial effect, increasing the number of nose-touching (low dose) and inhibiting grooming after social contact. At the highest dose, it impaired social interaction in wild-type controls. (C) One week after cessation of treatment, the beneficial effects of bromide administration were maintained at doses above 125 mg / kg. The effect of (one) bumetanide on the duration of nose-touching and grooming after social contact was still detectable. (D) In ​​the three-chamber test, NaBr treatment rescued social preference in Oprm1 mutants from doses of 10 mg / kg onwards, whereas bumetanide increased interest in mice but did not decrease the abnormal interest in objects. Results are shown as scatter plots and mean ± sem. Daggers: effect of genotype; asterisks: effect of treatment; filled stars: genotype x treatment interaction (compared to wild-type vehicle condition); open stars: genotype x treatment x stimulus interaction (mouse vs object); (a) Genotype x treatment interaction (compared to knockout vehicle condition, p<0.001) (two-way ANOVA or three-way ANOVA with stimulus as repeated measure followed by Newman-Keuls post-hoc test). One symbol: p<0.05; two symbols: p<0.01; three symbols: p<0.001.3-Ch: 3-chamber test, M: mouse, MB: marble burying, MS: motor stereotypy, NSF: novelty suppression, SI: social interaction, T: toy, YM: Y-maze. [Diagram 2] Chronic treatment with sodium bromide reduced stereotypic behavior and anxiety in Oprm1- / - mice. For experimental timeline and animal numbers, see Figure 1A. (A) Chronic administration of NaBr (A1) suppressed stereotypic circling episodes in Oprm1- / - mice from doses of 10 mg / kg onwards and less consistently reduced the number of head shakes (doses above 125 mg / kg). In wild-type controls, 500 mg / kg NaBr increased the frequency of grooming episodes and head shakes. Bumetanide (A2) suppressed stereotypic circling and head shakes. (B) In the marble burying test, chronic bromide increased the overall number of buried marbles in both mouse strains, whereas bumetanide had no significant effect. (C) In the Y-maze, NaBr inhibited perseverative same arm returns from a dose of 70 mg / kg. Bumetanide had no significant effect, although a trend towards attenuating perseverance was evident at a dose of 0.5 mg / kg. (D) In ​​the novelty food intake inhibition test, sodium bromide normalized latency to feed in Oprm1-deficient mice to wild-type levels from the lowest dose tested onwards, and increased food intake in all mice. Bumetanide had no significant effect in this test. Results are shown as scatter plots and means ± sem. Daggers: effect of genotype, asterisks: effect of treatment, black stars: genotype × treatment interaction (compared to wild-type vehicle condition) (two-way ANOVA followed by Newman-Keuls post-hoc test). One symbol: p<0.05, two symbols: p<0.01, three symbols: p<0.001. [Diagram 3]Chronic administration of sodium bromide ameliorated social behavioral deficits in Fmr1- / - and Shank3Δex13-16- / - mice. (A) Fmr1- / - or Shank3Δex13-16- / - ("Shank3- / -") and their respective wild-type counterparts were treated with NaBr (0 or 250 mg / kg; n = 8 mice per genotype and treatment) once daily for 18 days. Behavioral testing began on day 8. Social interactions were retested 1 week after cessation of chronic treatment (day 25). (B) In a direct social interaction test, chronic treatment with NaBr normalized interaction parameters to wild-type levels in both Fmr1 and Shank3 mutant strains. (C) One week after cessation of treatment, these beneficial effects were fully maintained in Fmr1- / - mice, but were only detected for some parameters in Shank3Δex13-16- / - mice. (D) In ​​the three-chamber test, chronic administration of NaBr rescued the preference for extended nose contact with the mouse in Shank3Δex13-16- / - mice, resulting in an increased preference ratio. (E) Chronic treatment with sodium bromide suppressed stereotypic circling and head shaking in Fmr1- / - and Shank3Δex13-16- / - mice, and normalized grooming in the latter. (F) NaBr reduced marble burying in Fmr1- / - and Fmr1+ / +, but did not affect the reduction in burying in Shank3Δex13-16- / - mice. (G) In a Y-maze exploration, chronic NaBr suppressed persistent returning to the same arm in both Fmr1 and Shank3 mutant strains. (H) Finally, in a novel environment feeding inhibition test, Fmr1- / - or Shank3Δex13-16- / - mice treated with sodium bromide showed shortened or normalized feeding latencies, respectively, but no change in food intake. Results are shown as scatter plots and means ± sem.Dagger: effect of genotype; black star: genotype x treatment interaction (compared to wild-type vehicle condition); white star: genotype x treatment x stimulus interaction (mouse vs object); (a) genotype x treatment interaction (compared to knockout vehicle condition, p<0.001); (c) genotype x treatment interaction (compared to knockout vehicle condition, p<0.05) (2-way ANOVA or 3-way ANOVA (stimulus on repeated measures) followed by Newman-Keuls post-hoc test). One symbol: p<0.05; two symbols: p<0.01; three symbols: p<0.001. 3-Ch: 3-chamber test; AAR: alternate arm returns; M: mouse; MB: marble burying; MS: motor stereotypy; NSF: novelty feeding suppression; SAR: same arm returns; SPA: spontaneous alternation; SI: social interaction; T: toy; YM: Y-maze. [Figure 4]The beneficial effects of sodium bromide and VU0155041, a positive allosteric modulator of mGlu4 receptors, were synergistic in Oprm1- / - mice. (A) Oprm1+ / + and Oprm1- / - mice were treated once daily for 18 days with either vehicle, NaBr (70 mg / kg), VU0155041 (1 mg / kg), or NaBr and VU0155041 (70 and 1 mg / k, respectively; 8 mice per genotype and dose). Behavioral testing began on day 8. Social interaction was retested 1 and 2 weeks after chronic treatment cessation. (B) In the direct social interaction test, NaBr and VU0155041 treatment showed synergistic effects on the recovery of nose and paw contact duration in Oprm1- / - mice. On the other hand, 1 mg / kg VU0155041 was sufficient to inhibit grooming after social contact. The beneficial effects of NaBr / VU0155041 combination were fully maintained 1 and 2 weeks after treatment cessation. (C) In the three-chamber test, 1 mg / kg VU0155041 increased the duration of nose contact with a mouse in Oprm1- / - mice to that with a toy. 70 mg / kg NaBr and NaBr / VU0155041 combination treatment fully restored the longer nose contact with a mouse. (D) NaBr / VU0155041 combination administration reduced head shaking in Oprm1- / - and Oprm1+ / + mice and (E) normalized marble burying only in Oprm1-deficient mice. (F) VU0155041 treatment was sufficient to suppress persistent returning to the same arm in Oprm1- / - mice exploring a Y-maze, and (G) NaBr administration was sufficient to normalize feeding latencies in a novel environment feeding inhibition test. (H) Only combined NaBr / VU0155041 treatment restored flicking latencies of Oprm1- / - mice to wild-type levels in the tail immersion test at 50°C. Results are shown as scatter plots and mean ± sem.Black star: genotype x NaBr x VU0155041 interaction (compared to wild-type vehicle condition); white star: genotype x stimulus x NaBr x VU0155041 interaction (mouse vs object); (a) genotype x NaBr x VU0155041 interaction (compared to knockout vehicle condition, p<0.001); double dagger: NaBr x VU0155041 interaction; ampersand: genotype x VU0155041 interaction; section: genotype x NaBr interaction (three- or four-way ANOVA followed by Newman-Keuls post-hoc test). One symbol: p<0.05, two symbols: p<0.01, three symbols: p<0.001. 3-Ch: 3-chamber test, M: mouse, MB: marble burying, MS: motor stereotypy, NSF: novelty food inhibition, SI: social interaction, T: toy, TI: tail immersion, YM: Y-maze. [Diagram 5]Bromide ions behave as a PAM for mGlu4 receptors and show synergistic effects with the mGlu4 PAM VU0155041. (A) Signaling cascade of mGlu4 receptors when coupled to the chimeric G protein Gαqi9 (to allow recruitment of the phosphoinositide pathway) and experimental principle of calcium mobilization (panel B) and IP1 accumulation assays (panel C). (B) In calcium mobilization assays, bromide ions behave as a PAM for mGlu4 and showed a broader effect than chloride ions on both pEC50 and Emax. (C) In IP1 accumulation assays, the PAM effect of bromide was confirmed. Supplementation with VU0155041 indeed improved the promotion of mGlu4 signaling as seen by the further increase in ΔpEC50. Results of three independent experiments performed in triplicates are shown as mean ± SEM. Black star: effect of ion concentration on Emax, asterisk: effect of ion concentration on ΔpEC50, compared to physiological conditions (100 mM Cl- + 50 mM gluconate), (a): compared to high chloride conditions (150 mM Cl-, p<0.0001), hashtag: effect of ion concentration and VU0155041 on ΔpEC50, compared to physiological conditions (100 mM Cl- + 50 mM gluconate), (b) and (c): compared to bromide conditions (100 mM Cl- + 50 mM Br-) with or without VU0155041 (p<0.0001) (one-way ANOVA followed by Tukey's post-hoc test). Double symbols: p<0.001, triple symbols: p<0.0001. DAG: diacylglycerol, ER: endoplasmic reticulum, F4: Fluo4 calcium probe, FRET: fluorescence resonance energy transfer, PLC: phospholipase C, IP1 / 2 / 3: inositol mono-, di-, and triphosphates.

[0052] Working Example Materials and Methods Animals, breeding methods and housing conditions Oprm1 - / - (B6.129S2-Oprm1 tm1Kff / J)(58), and Shank3 Δex13-16- / -(B6.129-Shank3 tm2Gfng / J , the so-called Shank3B - / - , PDZ domain-deficient) (55) mouse strain was obtained from Jackson Laboratories (Farmington, USA) and bred on a hybrid background (50% 129SVPas-50% C57BL / 6J). Fmr1-KO2 mice (59) were generously provided by R. Willemsen (Erasmus University Medical Center, Rotterdam, The Netherlands) and bred on a C57BL / 6J background. To prevent genetic derivation, equal numbers of male and female mice were born in-house from homozygous parents bred from heterozygous animals. This breeding scheme favored the social impairment in mutant mice by keeping them together during early postnatal development. Unless otherwise stated, animals were group-housed and maintained at controlled temperature (21 ± 1 °C) with a 12-h light / dark cycle (lights on at 7:00 a.m.). Food and water were available ad libitum. Experimental analyses were performed blinded to genotype and experimental conditions. All experimental procedures were performed in accordance with the European Communities Council Directive 2010 / 63 / EU and approved by the Comite d'Ethique en Experimentation animale Val de Loire (C2EA-19).

[0053] Drugs Vehicle (0.9% NaCl; ip, 10 or 20 ml / kg), NaBr (Sigma-Aldrich, Saint-Quentin Fallavier, France) (administered either chronically (10, 30, 70, 125, 250 and 500 mg / kg; ip or os, once daily, in a volume of 20 ml / kg (except in combination with VU0155041: 10 mg / kg)) or acutely (250 mg / kg, 20 ml / kg)), KBr (Sigma-Aldrich, Saint-Quentin Fallavier, France; 145 mg / kg; ip, 20 ml / kg), bumetanide (R&D systems, Minneapolis, USA; 0.5 and 2 mg / kg; ip, 20 ml / kg) or VU0155041 (Cayman Chemicals, Ann Arbor, Mice were treated with 1 mg / kg ip, 10 ml / kg, once daily, from the University of California, San Diego, Santa Cruz, CA, USA. The dose of bumetanide was selected based on previous studies in rodent models of ASD (40, 73). The liminal dose of VU0155041 was set based on our previous studies (51, 60) and pilot experiments (which showed no detectable effects in the social interaction test). When treatment was given chronically, behavioral testing began 8 days after the start of daily dosing. Treatment was maintained for 8–18 consecutive days (see timelines in Figures 1 and 4). This allowed for thorough behavioral phenotyping. Drugs (or vehicle) were administered on the day of testing or 30 min prior to behavioral assays when treatment was given acutely.

[0054] Behavioral experiments When assessing the effects of chronic treatment, experiments were performed consecutively (timeline in Figures 1 and 4) (51, 60). The order of tests was chosen to minimize the occurrence of anxiety in the later assays. Direct social interaction and novelty feeding suppression were performed in four square areas (open field, 50 × 50 cm) separated by opaque gray Plexiglas walls 35 cm high on a white Plexiglas platform (View Point, Lyon, France). Stimulus mice used in the three-chamber test were group-housed male or female wild-type mice aged 8 to 14 weeks and socially naive to the experimental animals.

[0055] social skills Direct social interaction test. On the day of testing, pairs of unfamiliar mice (not cage-mates, age-, sex- and treatment-matched) were introduced into each arena for 10 min (15 lx). Each arena was equipped with a black plastic floor (infrared-transparent). The total time spent in nose contacts (nose-nose, nose-body or nose-anogenital area), the number of these contacts, the time spent in paw contacts and the number of these contacts, grooming episodes (allogrooming) (especially those that occurred immediately (<5 s) after social contact), and the number of pursuit episodes were scored inductively for each animal by a trained experimenter using an ethological keyboard (Labwatcher®, View Point, Lyon, France) on video recordings (infrared-sensitive video camera) (51, 60). The mean duration of nose and paw contacts was calculated from previous data (61-63).

[0056] Three-chamber social preference test. The test apparatus consisted of a transparent acrylic box (external walls were light-shielded with black plastic film) with a partition dividing the box into three equal chambers (40 × 20 × 22.5 cm). Two sliding doors (8 × 5 cm) allowed movement between the chambers. A cylindrical wire cage (18 × 9 cm, bars with a diameter of 0.5 cm, spaced 1 cm apart) was used to contain the mouse interactor and the object (soft toy mouse). To minor anxiety, the test was performed under low light conditions (15 lx). Stimulus wild-type mice were habituated to confinement in the wire cage for 2 days prior to testing (20 min / day). On the day of testing, the experimental animals were introduced into the central chamber and allowed to explore the entire apparatus during a 10-min habituation period (the wire cage was empty) after the sliding door was raised. The experimental mice were then returned to the central chamber and confined while the experimenter introduced age- and sex-matched, unfamiliar wild-type animals into a wire cage in one of the end chambers, and a soft toy mouse (8 × 10 cm) into the second wire cage as a control for novelty. The experimental mice were then allowed to explore the apparatus for a 10-min interaction phase. The time spent in each chamber, the time spent contacting the nose with each wire cage (empty: habituation; mouse or toy: interaction), and the number of these nose contacts were scored inductively on the video recordings by a trained experimenter using an ethological keyboard (Labwatcher®, View Point, Lyon, France). The mean duration of nose contacts was calculated from these data (61-63). The relative positions of the stimulus mice (vs. toy) were counterbalanced between groups.

[0057] stereotypic behavior Motor stereotypy. To detect motor stereotypy in mutant versus wild-type animals, mice were individually placed for 10 min in a transparent standard home cage (21 × 11 × 17 cm) containing a 3 cm depth of fresh sawdust (64). Light intensity was set at 30 lux. The number of head-shaking and rearing, burying, grooming, and circling episodes, as well as the total time spent burying, were scored by direct observation by a trained experimenter.

[0058] Y-maze exploration. Spontaneous alternation behavior was used to assess perseverative behavior (65-67). Each Y-maze consisted of three Plexiglas arms (covered with distinct wall patterns) connected together (15 × 15 × 17 cm) (15 lx). The floor was covered with lightly sprayed fresh sawdust to reduce anxiety. Each mouse was placed in the center of the maze and allowed to freely explore the environment for 5 min. The entry patterns into each arm were assessed on video recordings. Spontaneous alternation (SPA), i.e., consecutive entries into each arm forming overlapping triplet sets, alternating arm returns (AAR) and same arm returns (SAR) were scored, and the percentages of SPA, AAR and SAR were calculated as follows: Total / (Number of all arm approaches -2) * 100

[0059] Marble burying. Marble burying was used as a measure of perseverative behavior (68). Mice were individually introduced into transparent cages (21 × 11 × 17 cm) containing 20 marbles (1.5 cm diameter) evenly spaced on a 4 cm deep layer of fresh sawdust. To prevent escape, each cage was covered with a filtering lid. The light intensity in the room was set at 40 lux. After 15 min, the animals were removed from the cages and the number of marbles that were more than half-buried in the sawdust was estimated.

[0060] Anxiety-like behavior Novelty suppression. Novelty suppression (NSF) was measured in 24-h food-deprived mice isolated in standard home cages 30 min before individual testing. Three pellets of regular laboratory chow were placed on a white tissue in the center of each arena, illuminated at 60 lx. Each mouse was placed in a corner of the arena and allowed to explore for up to 15 min. Feeding latency was measured as the time it took to bite the food pellet. Immediately after the feeding event, mice were returned to their home cage (without their cage mates) and allowed to consume the experimental chow for 5 min. Food consumption in the home cage was measured.

[0061] Nociceptive threshold Tail immersion test. The nociceptive threshold was evaluated by sequentially immersing the mouse tail (5 cm from the tip) in water baths at 48°C, 50°C, and 52°C. The latency to withdraw the tail at each temperature was measured, with 10 seconds as the cutoff value.

[0062] Real-time quantitative PCR analysis Brains were removed and placed in a brain matrix (ASI Instruments, Warren, MI, USA). The nucleus accumbens (NAc), caudate putamen (CPu), ventral pallidum / olfactory tubercle (VP / Tu), medial amygdala (MeA) and ventral tegmental area / substantia nigra pars compacta (VTA / SNc) were punched / dissected from 1 mm thick slices (data not shown). Tissues were immediately frozen on dry ice and stored at -80°C until use. Samples were processed individually for each structure, genotype and condition of interest (n=8). RNA was extracted and purified using the Direct-Zol RNA MiniPrep kit (Zymo research, Irvine, USA). cDNA was synthesized using the ProtoScript II Reverse Transcriptase kit (New England BioLabs, Evry-Courcouronnes, France). qRT-PCR was performed in quadruplets using 0.25 μl of cDNA in a final volume of 12 μl in Hard-Shell Thin-Wall 384-Well Skirted PCR plates (Bio-Rad) using the iQ-SYBR Green supermix (Bio-Rad) kit on a CFX384 Touch Real-Time PCR Detection System (Biorad, Marnes-la-Coquette, France). Gene-specific primers were designed using Primer3 software to obtain products of 100–150 bp. Relative expression ratios were normalized to levels of actin and calculated as 2 -ΔΔCt The method was applied to evaluate the difference in expression levels. Gene expression values ​​that differed from the mean by more than two standard deviations were considered outliers and were excluded from further calculations.

[0063] Cell culture and transfection Human embryonic kidney (HEK) 293 cells were transiently transfected by electroporation with rat mGlu4 receptor together with chimeric Gi / Gq proteins to allow phospholipase activation and EAAC1, a glutamate transporter to avoid the influence of extracellular glutamate. Cells were seeded at a density of 100,000 cells / well in PLO-coated black walled clear bottom 96-well plates (Greiner Bio-One) for calcium mobilization and at a density of 50,000 cells / well in PLO-coated black 96-well plates (Greiner Bio-One) for IPOne assays. Cells were cultured in DMEM (Gibco™, Life Technologies) supplemented with 10% fetal bovine serum. To reduce extracellular glutamate concentration, cells were exchanged into GlutaMAX (Gibco™, Life Technologies) 3 hours before the experiment.

[0064] Chloride and bromide buffers for in vitro assays The concentrations of chloride and bromide ions in the buffers used for the in vitro experiments were selected to best match physiological conditions. It has been previously shown that the total amount of halogens (chloride and bromide ions) in cerebrospinal fluid and serum can reach 120-130 mM (69, 70) and that bromide can replace up to 30% of the chloride concentration (71), which gives theoretical chloride and bromide concentrations of 91 mM and 36 mM. We therefore chose a 100 mM chloride dose (NaC6H gluconate) as a physiological control. 11 The buffers were supplemented with O7 to keep osmolality comparable between the buffers (56) and their effects were assessed in vitro with or without the addition of 50 mM chloride or bromide. Chemicals were purchased from Sigma-Aldrich (Merck, L'Isle D'Abeau Chesnes, France). The pH of all buffers was adjusted to 7.4 prior to the experiments.

[0065] For calcium mobilization assays, a buffer containing 100 mM NaCl, 2.6 mM KCl, 1.18 mM MgSO4, 10 mM D-glucose, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1 mM CaCl2, and 0.5% (w / v) bovine serum albumin was used, with no addition of 50 mM NaCl, NaBr, or NaC6H6. 11 O7 was supplemented to give a total anion concentration of 154.6 mM.

[0066] For IP1 accumulation assay, 46 mM NaCl, 4.2 mM KCl, 0.5 mM MgCl2, 10 mM HEPES, 1 mM CaCl2, 50 mM NaC6H 11 A buffer containing 07 and 50 mM LiCl (to avoid degradation of IP1) was used, and 50 mM NaCl, NaBr or NaCH 11 O7 was supplemented to give a total anion concentration of 203.2 mM.

[0067] Calcium mobilization and IP1 accumulation assay Calcium mobilization assay: 24 h after transfection, cells were supplemented with fresh Cl - 1 μM calcium-sensitive fluorescent dye (Fluo-4 AM; Invitrogen, Life Technologies) diluted in buffer (154.6 mM) was loaded for 1 h at 37° C. and 5% CO2. Cells were then washed and maintained in the appropriate buffer supplemented with 4 mM probenecid. Agonists were also diluted in the appropriate buffer. Ca 2+ Emission was measured using a μCell FDSS (Hamamatsu Photonics). After a 20 second baseline recording, fluorescence was recorded for 60 seconds with excitation at 480 nm and emission at 540 nm.

[0068] IP1 accumulation assay: Inositol monophosphate accumulation was measured using the IP-One HTRF (Homogeneous Time Resolved Fluorescence) kit (Cisbio Bioassays, Perkin Elmer, Codolet, France) according to the manufacturer's recommendations (72). Briefly, cells were stimulated to induce IP1 accumulation by treatment with test compounds in appropriate buffers for 30 min at 37°C and 5% CO2 before addition of d2-labeled IP1 and Tb-labeled anti-IP1 antibody. After 1 h of incubation at RT, emissions at 620 nm and 665 nm after excitation at 337 nm were recorded using a Pherastar FS (BMG Labtech).

[0069] Data Analysis and Statistics In vivo experiments Statistical analysis was performed using Statistica 9.0 software (StatSoft, Maisons-Alfort, France). In all comparisons, a value of p<0.05 was considered significant. Statistical significance in behavioral experiments was evaluated using one- or two-way ANOVA (drug, stimuli, and treatment effects) followed by Newman-Keuls post-hoc tests. The significance of quantitative real-time PCR (qRT-PCR) results was evaluated after transformation using two-tailed t-tests as previously described (60). Unsupervised clustering analysis was performed on the transformed qRT-PCR data (Cluster 3.0 and Treeview software) (51, 60) using complete linkage with correlation distances (Pearson correlation) for genotype and treatment. When used for clustering analysis (data not shown), behavioral data were normalized to vehicle-vehicle condition and transformed using the same formula as for qRT-PCR data.

[0070] In vitro experiments Data were analyzed using Prism 6 software (GraphPad Software, San Diego, CA, USA). For IP1 accumulation assays, each HTRF ratio was converted to IP1 concentration using a standard curve for each buffer and then normalized to mock-transfected cells to avoid the effect of buffer composition on the HTRF signal. In all experiments, data were fitted using a four-parameter concentration-response curve equation to calculate potency (EC 50 ) to logarithm (logEC 50 ) for clarity, absolute logarithms (pEC 50 ) was used. max pEC represents the maximal response obtained at saturating agonist concentrations. Data shown in the figures represent the mean ± SEM of at least three experiments performed in triplicate. 50 , ΔpEC 50 and E max Statistical differences between were determined using one-way analysis of variance followed by Tukey's post-hoc test.

[0071] result Chronic sodium bromide inhibits Oprm1 - / - More effective than bumetanide in alleviating social behavioral deficits in mice First, the present inventors - / - The effects of NaBr administration over a wide range of doses (10-500 mg / kg) were evaluated and compared with bumetanide administration (0.5 and 2 mg / kg) in mice and their WT counterparts (Figure 1A). Treatment was given chronically to mimic the clinical condition.

[0072] Social interactions were assessed 9 days after chronic NaBr treatment (Figures 1B1-1B2). - / - Mice showed a severe reduction in social interactions. Chronic NaBr treatment significantly reduced the number of nose-touching events (genotype × treatment: F 6,163 = 13.2, p < 0.0001) and mean duration (genotype × treatment: F 6,163= 31.6, p < 0.0001) and number of paw contacts (genotype × treatment: F 6,163 = 14.7, p < 0.0001) and mean duration (genotype × treatment: F 6,163 Chronic NaBr attenuated this defect in mutant mice in a dose-dependent manner, as evidenced by normalization of the AFLP score (F = 14.5, p < 0.0001) from the 125 mg / kg dose. Chronic NaBr also increased the number of chasing episodes in both mouse strains from the 70 mg / kg dose onwards (Treatment: F 6,163 = 5.6, p < 0.0001), and normalized grooming frequency after social contact in the mutants (genotype × treatment: F 6,163 = 32.2, p < 0.0001). In contrast, a single acute injection of NaBr (250 mg / kg) had little effect on social interaction parameters (data not shown). When given chronically, NaBr (70 mg / kg or more) maintained the restoration of nasal contact duration for the highest dose (genotype × treatment: F 6,134 = 53.3, p < 0.0001), and inhibition of grooming after social contact was maintained (genotype × treatment: F 6,134 = 30.4, p < 0.0001 (Figure 1C), produced palliative effects that were detectable even 1 week after treatment was stopped. These effects were largely lost after 2 weeks (data not shown).

[0073] Compared with chronic NaBr, chronic bumetanide at low doses significantly reduced the number of nasal touches (genotype × treatment: F 2,42 = 22.6, p < 0.0001) and pursuit episodes (genotype × treatment: F 2,42 = 12.9, p < 0.0001), but not the duration of nasal contact (genotype × treatment: F 2,42 = 22.6, p < 0.0001), or number of paw contacts (genotype × treatment: F 2,42 = 14.9, p < 0.0001) and duration (genotype × treatment: F 2,42 = 7.3, p < 0.0001). Finally, bumetanide inhibited grooming episodes, especially those that occurred after social contact, from the lowest dose tested onwards (genotype × treatment: F2,42 = 80.7, p < 0.0001). Notably, chronic treatment with bumetanide showed adverse effects on social interaction parameters in WT controls. Depending on the dose, the beneficial effects of bumetanide were not only negative, but also negative, especially on the duration of nose contact (genotype × treatment: F 2,42 = 9.3, p < 0.0001) and post-social grooming episodes (genotype × treatment: F 2,42 = 16.4, p < 0.0001) was still detectable 1 week after cessation of treatment (Figure 1C).

[0074] In the three-chamber test (Fig. 1D), Oprm1 - / - The mice showed severe impairment in social preference as evidenced by comparable numbers of nasal contacts with the mouse and the toy, with the nasal contacts with the toy being even longer than with the mouse. Chronic NaBr completely restored social preference in the mutant mice, which engaged in more frequent (genotype × treatment × stimulus: F 6,160 = 3.5, p < 0.001) and longer (genotype × treatment × stimulation: F 6,160 = 8.9, p < 0.0001), which led to a normalization of the preference ratio at 10 mg / kg NaBr or higher (genotype × treatment: F 6,160 = 11.9, p < 0.0001). In this study, chronic bumetanide at the lowest dose induced more frequent nose contact with the mouse than with the toy (genotype × treatment × stimulus: F 2,42 = 17.7, p < 0.0001). Bumetanide treatment in mutant mice dose-dependently increased the duration of nose contact with the mouse but failed to decrease the duration of nose contact with the toy (genotype × treatment × stimulus: F 2,42 =32.5, p<0.0001). This is because Oprm1 - / - Significant but partial restoration of social preference ratio in mice (genotype × treatment: F 2,42 = 38.4, p < 0.0001).

[0075] In conclusion, chronic, but not acute, NaBr treatment inhibits Oprm1 - / - It dose-dependently restored social behavior in mice, and these beneficial effects were superior to those of chronic bumetanide treatment.

[0076] Sodium bromide is Oprm1 - / - Reduced stereotypic behavior and anxiety in mice Next, we investigated the Oprm1 - / - We assessed the effects of chronic bromide on nonsocial behavior in mice (timeline in Fig. 1A ).

[0077] Regarding stereotypic behavior, Oprm1 - / - Mice spontaneously exhibited stereotypic circling and head shaking (Figures 2A1-A2), which were reduced under NaBr treatment from the lowest dose onwards, whereas the former (genotype × treatment: F 6,161 = 4.6, p < 0.001) compared with the latter (genotype × treatment: F 6,161 = 7.0, p < 0.0001). + / + In control mice, NaBr reduced the number of grooming episodes (genotype × treatment: F 6,161 = 4.5, p < 0.001) and number of head shakes (genotype × treatment: F 6,161 = 7.0, p < 0.0001) in a dose-dependent manner. In both mouse strains, NaBr dose-dependently increased the number of rearing episodes (treatment: F 6,161 = 13.2, p < 0.0001). Under the same conditions, bumetanide inhibited circling and head shaking in mutant mice and reduced the number of rearing episodes in both mouse strains. In the marble burying test (Figure 2B), NaBr treatment did not inhibit the excessive burying in mutant mice (genotype: F 6,163 = 13.6, p < 0.001), and overall increased obscuration (treatment: F 6,163 = 2.4, p < 0.05). Similarly, chronic bumetanide reduced Oprm1 - / - We were unable to suppress excessive burying in mice (genotype: F 2,46= 14.7, p < 0.001). In the Y-maze exploration test (Figure 2C), bromide reduced persistent returning to the same arm in Oprm1-deficient mice to wild-type levels from the 30 mg / kg dose onwards (genotype × treatment: F 6,161 = 6.7, p < 0.0001), whereas bumetanide was unable to suppress them overall, although a decreasing trend was observed at a dose of 0.5 mg / kg (genotype: F 2,46 =19.7, p<0.001).

[0078] The present inventors have - / - Anxiety levels in Oprm1 knockout mice and their WT counterparts were assessed using the novelty food suppression test (Figure 2D). In this test, mutant mice displayed hyperanxiety, increased feeding latency and reduced food intake once returned to their home cage. Chronic bromide normalized feeding latency to wild-type levels in Oprm1 knockout mice from the lowest dose tested onwards (genotype × treatment: F 6,160 = 6.7, p < 0.0001) and increased food intake in both mouse strains (treatment: F 6,160 = 7.0, p < 0.0001). Chronic bumetanide did not significantly improve feeding latency (genotype: F 2,42 = 27.1, p < 0.0001) and food intake (genotype: F 2,42 = 5.6, p < 0.05).

[0079] Taken together, these results suggest that chronic treatment with both bromide and bumetanide inhibits Oprm1 - / - These results indicate that only bromide treatment reduced stereotypic behavior in mice, but had an anxiolytic effect.

[0080] In the next series of experiments, we demonstrated that NaBr administered via the oral route (250 mg / kg, once daily by oral gavage for 4-5 days) significantly inhibited the Oprm1 - / -In mice, it was confirmed that it alleviated autism-like disorders and motor stereotypies, as well as after intraperitoneal injection (data not shown). We also evaluated the behavioral effects of chronic administration of another bromide salt, KBr. In pilot experiments, a dose of KBr equivalent to 250 mg / kg NaBr was toxic, so we reduced the dose to 145 mg / kg KBr, equivalent to 125 mg / kg NaBr. Oprm1 - / - The beneficial effects of NaBr treatment in mice were fully reproduced, if not surpassed, by KBr in tests assessing social, repetitive and anxiety-related behaviors (data not shown). - / - The therapeutic effect of NaBr or KBr in mice was attributed to bromide ions.

[0081] Chronic sodium bromide inhibits Fmr1 - / - and Shank3 Δex13-16- / - Alleviated social behavior disorders, stereotypic behavior and excessive anxiety in mice We then examined the beneficial effects of NaBr on autism-like symptoms in other mouse models of ASD, here in Fmr1-deficient mouse strains and Shank3 Δex13-16 We addressed the question of whether these findings could be generalized to autism-sensitive behaviors in mice (e.g., mice with autism-related phenotype, mice with autism-related phenotype, mice with autism-related phenotype, and mice with autism-related phenotype (knockout mouse strains). To this end, we assessed the effect of chronic administration of NaBr at a dose of 250 mg / kg on autism-sensitive behaviors in these strains (Figure 3A).

[0082] Regarding social behavior, during the direct social interaction test (Fig. 3B), Fmr1 - / - and Shank3 Δex13-16- / - In mice, chronic bromide significantly increased the duration of nasal exposure (genotype × treatment-Fmr1:F 1,30 =49.5, p<0.0001; Shank3 Δex13-16 :F 1,28 = 73.0, p < 0.0001) and duration of paw contact (genotype × treatment - Fmr1:F 1,30 =17.7, p<0.0001; Shank3 Δex13-16 :F 1,28= 23.2, p < 0.0001) and the number of chasing episodes (genotype × treatment - Fmr1:F 1,30 =13.7, p<0.0001; Shank3 Δex13-16 :F 1,28 = 11.8, p < 0.0001) and normalized post-social grooming (genotype × treatment-Fmr1:F 1,30 =30.1, p<0.0001; Shank3 Δex13-16 :F 1,28 = 25.0, p < 0.0001). Even one week after cessation of NaBr treatment (Figure 3C), the duration of paw contact (genotype × treatment: F 1,30 = 132.7, p < 0.0001) and grooming frequency after social contact (genotype × treatment: F 1,30 A significant beneficial effect was detected in the 10-mg / kg / day group (=87.0, p<0.0001). Δex13-16 In mice, the effect of the previous bromide treatment on the duration of paw contact was no longer detectable (genotype: F 1,28 = 388.2, p < 0.0001), and grooming after social contact remained efficiently inhibited (genotype × treatment: F 1,28 =55.3, p<0.0001).

[0083] We further assessed social behavior under chronic bromide exposure using the three-chamber test (Figure 3D). In this test, Fmr1 knockout mice made more frequent nose contact with the mouse than with the object, but spent as much time with the live mouse as with the object and made more nose contact with the object, indicating a disrupted social preference. Chronic treatment with NaBr resulted in Fmr1 mutants spending more time exploring the mouse (genotype x treatment x stimulus: F 1,29 = 4.5, p < 0.05), making longer nose contact with the mouse than with the object (genotype × treatment × stimulus: F 1,29 = 18.4, p < 0.001) and normalized the preference ratio (genotype × treatment: F 1,29= 5.2, p < 0.05), but neither stimulus changed the number of nose touches they made (stimulus: F 1,29 = 21.4, p < 0.0001). Similarly, vehicle-treated Shank3 Δex13-16- / - Mice did not spend as much time with the mouse as with the toy in this test, and made more frequent nose contacts with the mouse, but the duration was similar for both stimuli. In contrast, NaBr-treated mutants spent more time with the mouse (genotype × treatment × stimulus: F 1,28 = 4.5, p < 0.05) and made longer nose contact with the conspecific to the toy (genotype × treatment × stimulus: F 1,28 = 26.9, p < 0.0001), this was consistent with no change in the number of nose touches (stimulus: F 1,28 = 37.4, p < 0.0001), and improved preference ratio (genotype × treatment: F 1,28 = 5.9, p < 0.05). Thus, chronic administration of NaBr significantly reduced the oxidative stress in Fmr1-deficient mice and Shank3 mice. Δex13-16 It rescued social behavioral deficits in knockout mice.

[0084] Regarding stereotypic behavior, Fmr1 - / - and Shank3 Δex13-16- / - Mice exhibited more frequent spontaneous grooming (only the latter was significant), circling episodes and head shaking than WT controls (Figure 3E). Chronic treatment with NaBr normalized all these parameters to WT levels (Fmr1-circling, genotype x treatment: F 1,30 = 11.9, p < 0.01; head shake, genotype × treatment: F 1,30 = 68.0, p < 0.001; Shank3 Δex13-16 -Grooming, genotype x treatment: F 1,28 = 10.2, p < 0.01, rotation, genotype × treatment: F 1,28 = 48.4, p < 0.0001; head shake, genotype × treatment: F 1,28 = 5.3, p < 0.05). In the marble burying test (Fig. 3F), chronic NaBr treatment significantly increased Fmr1 + / + and Fmr1 - / -The treatment reduced the number of buried marbles in both groups of mice (treatment effect: F 1,30 =7.3, p<0.05). Shank3 Δex13-16- / - Mice showed severe impairment in marble burying (effect of genotype: F 1,28 =198.7, p<0.0001), and bromide treatment reduced burying in both mutant and WT mice (treatment effect: F 1,28 = 4.8, p < 0.05). In the Y-maze (Figure 3G), Fmr1-deficient mice and Shank3 Δex13-16 Knockout mice showed persistent returning to the same arm more frequently, which was suppressed under chronic bromide (Fmr1-genotype × treatment: F 1,30 = 61.9, p < 0.0001; Shank3 Δex13-16 -Genotype x treatment: F 1,28 = 47.5, p < 0.0001). Thus, chronic treatment with NaBr significantly inhibited Fmr1 - / - and Shank3 Δex13-16- / - In mice, it reduced stereotypic and perseverative behavior.

[0085] Regarding anxiety, the trend for Fmr1-deficient mice to have longer feeding latencies in the novel environment food inhibition test (Figure 3H) did not reach significance, although chronic bromide reduced this latency (genotype × treatment: F 1,30 =6.7, p<0.05). Shank3 Δex13-16- / - Mice were significantly slower to feed in the center of the area, but NaBr administration normalized this latency to WT levels (genotype × treatment: F 1,28 = 35.0, p < 0.0001). Bromide treatment did not affect food intake, but Δex13-16 It was decreased in knockout mice (genotype: F 1,28 = 15.6, p < 0.001). Thus, chronic administration of NaBr significantly increased Fmr1 - / - and Shank3 Δex13-16- / - It has demonstrated anxiolytic properties in mice.

[0086] Chronic sodium bromide inhibits Oprm1 - / -Regulating transcription in the mouse reward circuitry To elucidate the molecular mechanisms involved in the beneficial effects of chronic administration of sodium bromide, we assessed the impact of 2 weeks of NaBr treatment on gene expression across five regions of the brain reward / social circuit: NAc, CPu, VP / Tu, MeA and VTA / SNc in Oprm1-deficient mice. After 1 week under treatment, the mice were subjected to a first session of social interaction and a second session for 45 min before being sacrificed for qRT-PCR experiments (data not shown). We mainly investigated the effects of chloride transporters (Slc12a2 [NKCC1], Slc12a4, 5, 6, 7 [KCC1, 2, 3, 4, respectively], ClCa1), GABA A We focused on genes encoding receptor subunits (Gabra1, 2, 3, 4, 5, Gabrb1, 2) and glutamate receptors (Grm2, 4, 5) and subunits (Grin2a, 2b). In addition, we assessed the expression of marker genes for neuronal expression and plasticity (Fos, Bdnf), social behavior (Oxt), and striatal projection neurons (SPN; Crh, Drd1a, Drd2, Htr6, Pdyn, Penk).

[0087] We performed hierarchical clustering analysis of the qRT-PCR data for each brain region to visualize the effect of NaBr treatment on gene expression (data not shown). - / - Transcriptional profiles in mice were most different under vehicle and NaBr treatment, although mRNA levels were weakly correlated with social interaction parameters (data not shown). These results indicate that bromide induced transcriptional changes by itself, rather than normalizing gene expression in Oprm1 knockouts (as observed for behavioral parameters). This was especially true for CPu (Oprm1 under bromide treatment). - / - Mice showed preferential upregulation of gene expression (clusters a and c)).

[0088] This overall profile was confirmed when focusing on candidate genes (data not shown). We should acknowledge here that the sample number of mice assigned to each experimental condition was small to address the complex effects of genotype and pharmacological treatment, which may have limited the statistical power. For this reason, we focused on gene expression regulation affecting either several brain regions for the same gene or several genes of the same family. Strikingly, chronic administration of NaBr reduced Oprm1 - / - In Oprm1-deficient mice, NaBr increased the expression of all chloride transporters tested. Indeed, the expression of Slc12a2 was decreased in all brain regions except the VP / Tu in mutant mice and was normalized under bromide treatment. Chronic NaBr upregulated the expression of Slc12a5 and Slc12a7 in the NAc, CPu, and MeA of Oprm1-deficient mice, and in the VP / Tu as well. ClCa1 mRNA levels were increased in the NAc, MeA, and VTA / SNc, but decreased in the VP / Tu in mutant mice. They were normalized in the NAc, increased in the VP / Tu, and maintained at high values ​​in the MeA and VTA / SNc by NaBr treatment. In the CPu, bromide increased ClCa1 transcription in both genotypes. As for the GABAergic system, Oprm1 - / - In mice, chronic NaBr inhibited GABA receptor agonism in the NAc and CPu. A Bromide stimulated the expression of Gabra2, which encodes the α2 subunit of the receptor, and this expression remained high in the VP / TU and MeA. Notably, bromide consistently upregulated the expression of Gabra3, Gabra4, Gabra5, Gabrg1, and Gabrb2 in the CPu of mutant mice (data not shown). Bromide treatment downregulated the expression of the early gene Fos in the NAc and VP / Tu and kept it low in the CPu of Oprm1 knockouts. The mRNA levels of Oxt (encoding oxytocin) were upregulated in the Oprm1 - / -The expression of Oxt was decreased in the NAc and VP / Tu of mutant mice, but was normalized in the former and partially normalized in the latter by NaBr. Bromide induced the expression of Oxt in the MeA and VTA / SNc. Finally, chronic NaBr upregulated the expression of Grm4, which encodes the metabotropic glutamate receptor mGlu4, in all brain regions except the VTA / SNc of mutant mice and in the NAc of wild-type controls. Thus, the transcriptional results suggest that bromide treatment increases the Cl - We show that it profoundly affected the expression of transporters, while also regulating the expression of several key players in the GABAergic system, marker genes of neuronal activity and plasticity, as well as genes more specifically involved in the control of social behavior.

[0089] Synergistic effects of chronic bromide and mGlu4 receptor stimulation in Oprm1-deficient mice. Oprm1 under bromide treatment - / - Interested in the increased transcription of Grm4 in mice, whose autism-like symptoms are alleviated by stimulating mGlu4 activity (51), we addressed the question of whether there might be a common mechanism of action between these treatments. - / - and Oprm1 + / + The effects of chronic and borderline doses of NaBr (70 mg / kg) and VU0155041 (1 mg / kg) administered together in mice were examined (Figure 4A).

[0090] In the direct social interaction test (Figure 4B), 70 mg / kg NaBr significantly inhibited Oprm1 - / - VU0155041 had no detectable effect on behavioral parameters in mice (see Figure 1B). VU0155041 partially rescued the duration of nose contact and inhibited grooming episodes after social contact. When the two treatments were combined, rescued duration of nose contact (genotype × bromide × VU0155041:F 1,56 = 31.1, p < 0.0001) or duration of paw contact (genotype × bromide × VU0155041:F 1,56= 140.3, p < 0.0001), and the normalized number of grooming episodes after social contact (genotype × bromide × VU0155041:F 1,56 Social interaction parameters in mutant mice were normalized to wild-type levels, as evidenced by mean duration of nasal contact (=5.8, p<0.05). Recovery of the mean duration of nasal contact was observed 1 week after cessation of treatment (genotype × bromide × VU0155041:F 1,56 = 40.9, p < 0.0001) and 2 weeks (genotype × bromide × VU0155041:F 1,56 In a three-chamber study, 70 mg / kg NaBr or 1 mg / kg VU0155041 completely preserved Oprm1. - / - In mice, more frequent nose contact with the mouse than with the toy (genotype × NaBr × VU0155041 × stimulus: F 1,55 = 6.7, p < 0.05) (Figure 4C).

[0091] Concerning stereotypic behavior, the combined treatment of NaBr and VU0155041 suppressed Oprm1 - / - and Oprm1 + / + Reduced head shaking in mice (bromide × VU0155041:F 1,56 = 4.1, p < 0.05) (Figure 4D), which normalized marble burying in the mutants (genotype × NaBr × VU0155041:F 1,56 = 9.5, p < 0.001) (Figure 4E). Chronic VU0155041 was sufficient to suppress persistent returning to the same arm during Y-maze exploration in Oprm1-deficient mice (genotype × VU0155041:F 1,56 = 11.3, p < 0.01) (Figure 4F). In the novelty food suppression test, chronic NaBr was similarly sufficient to normalize feeding latency (genotype × VU0155041:F 1,56= 11.3, p < 0.01) (Figure 4G). Finally, bearing in mind that Oprm1-deficient mice have a reduced nociceptive threshold, we tested the effect of bromide and VU0155041 on this parameter. At 50°C, combined treatment with NaBr and VU0155041 normalized flicking latency, whereas each compound alone had no effect (genotype × NaBr × VU0155041:F 1,56 =20.5, p<0.0001) (Figure 4H).

[0092] Taken together, these results suggest that bromide administration and promotion of mGlu4 activity downregulates Oprm1 - / - They have shown that it exerts synergistic beneficial effects on autistic-like behavior in mice.

[0093] Bromide ion acts as a positive allosteric modulator of mGlu4 glutamate receptors Chloride has been shown to promote mGlu4 signaling (56). Here, we assessed whether modulation of mGlu4 activity by bromide, in addition to its ability to induce upregulation of Grm4 expression (data not shown), could result in a synergistic effect of bromide treatment and VU0155041 administration in vivo.

[0094] The present inventors have identified the mGlu4 receptor and i / G q Chimeric G protein Gα qi9 We measured mGlu4 signaling under glutamate stimulation in HEK293T cells transiently expressing mGlu4 (allowing mGlu4 to activate the phosphoinositide pathway). 2+Receptor activation was assessed by measuring release or inositol monophosphate (IP1, FIG. 5A). Experiments were performed in buffers containing either physiological concentrations of chloride ions (100 mM, supplemented with 50 mM gluconate to keep the osmolarity between the media equivalent), 150 mM chloride ions (classical buffer for cell culture studies), or 100 mM chloride ions and 50 mM bromide ions to compare the effect of modulation of chloride and bromide concentrations within the physiological range on mGlu4 activity.

[0095] Physiological concentration of chloride (100 mM Cl - +50 mM gluconate), the addition of 50 nM bromide significantly improved the efficacy of glutamate and reduced the Ca 2+ In the assay, there was a 0.73 ± 0.05 log increase in pEC50 (left panel; ion concentration: F 2,6 = 104.2, p < 0.0001) (Figure 5B). - ), bromide showed higher efficacy (ΔpEC 50 : 0.27 ± 0.04) (right panel; ion concentration: F 2,6 = 66.7, p < 0.0001). Furthermore, bromide enhanced the potency of glutamate, increasing the maximal mGlu4-induced calcium release by 65 ± 9% (E max , Left panel; ion concentration: F 2,21 =56.2, p<0.0001).

[0096] When measuring IP1 production (Figure 5C), bromide inhibited Ca 2+ The efficacy of glutamate (E max , maximum IP1 production) (left panel; ion concentration: F 2,24 = 22.1, p < 0.0001). Despite technical limitations (low amplification at this stage of the signaling cascade), pEC 50 Ca 2+It was consistently elevated in the presence of bromide ions, albeit to a lesser extent than was measured in the assay (left panel; ion concentration: F 2,6 =104.2, p<0.0001; Physiological Cl - Concentration and 50nM Br - ΔpEC between after addition 50 : 0.42 ± 0.03). With the addition of 5 μM VU0155041, bromide significantly increased the potency of glutamate compared to physiological concentrations of chloride (ΔpEC 50 : 0.72 ± 0.03). The combination of bromide and VU0155041 also improved glutamate efficacy by VU0155041 (ΔpEC 50 : 0.39 ± 0.03) or bromide (ΔpEC 50 :0.30±0.03) was more effective than either alone (ion concentration and VU0155041:F 5,12 =49.4, p<0.0001).

[0097] In conclusion, bromide ions behaved as a PAM of mGlu4 receptors in heterologous cells. These PAM effects were superior to those of chloride ions and synergized with those of VU0155041. Collectively, these results provide a molecular mechanism for the synergistic effects of bromide and VU0155041 in Oprm1-deficient mice and suggest that the benefits of bromide treatment in a mouse model of ASD were, at least in part, associated with enhanced mGlu4 activity.

[0098] conclusion In conclusion, this study reports the therapeutic potential of chronic treatment with bromide, alone or in combination with a PAM of the mGlu4 receptor, to alleviate core symptoms of ASD. The beneficial effects of bromide were observed in three mouse models of ASD with different genetic causes, supporting a high translational value. Furthermore, bromide has a long history of use as a medicine, meaning that its pharmacodynamics and toxicity are known. This, together with the long duration of effect and excellent oral bioavailability and brain penetration, are strong advantages for repurposing.

[0099] References Throughout this application various references describe the state of the art to which this invention pertains. The disclosures of these references are incorporated by reference into this disclosure. [Table 1] TIFF2024533116000003.tif247165TIFF2024533116000004.tif239165TIFF2024533116000005.tif242165TIFF20245331160 00006.tif248165TIFF2024533116000007.tif239165TIFF2024533116000008.tif240165TIFF2024533116000009.tif182165

Claims

1. A pharmaceutical composition for use in treating autism spectrum disorder (ASD) in a subject in need thereof, comprising a bromide salt, The pharmaceutical composition is administered to the subject in combination with a positive allosteric modulator (PAM) of the mGlu4 receptor.

2. A pharmaceutical composition for use in treating autism spectrum disorder (ASD) in a subject in need thereof, comprising a positive allosteric modulator (PAM) of the mGlu4 receptor, wherein the pharmaceutical composition comprises: A pharmaceutical composition adapted to be administered to said subject in combination with a bromide salt.

3. 3. The pharmaceutical composition of claim 1 or 2 for alleviating at least one symptom of ASD in a subject in need thereof, wherein the at least one symptom is social behavioral impairment, stereotypic behavior, and / or excessive anxiety.

4. 3. The pharmaceutical composition of claim 1, wherein the bromide salt is sodium bromide.

5. 3. The pharmaceutical composition of claim 1, wherein the bromide salt is potassium bromide.

6. 3. The pharmaceutical composition according to claim 1, wherein the positive allosteric modulator of the mGlu4 receptor is VU0155041.

7. A pharmaceutical composition for use in enhancing the efficacy of a positive allosteric modulator (PAM) of the mGlu4 receptor administered to a subject suffering from autism spectrum disorder (ASD), comprising a bromide salt, comprising: The pharmaceutical composition is administered to the subject in combination with a positive allosteric modulator (PAM) of the mGlu4 receptor.

8. 8. The pharmaceutical composition of claim 7, for use in enhancing the efficacy of VU0155041 administered to a subject suffering from autism spectrum disorder (ASD), wherein the pharmaceutical composition is administered to the subject in combination with VU0155041.

9. 9. The pharmaceutical composition of any one of claims 1, 2, 7, and 8, wherein the bromide salt is administered chronically.

10. 9. The pharmaceutical composition of any one of claims 1, 2, 7, and 8, wherein the bromide salt is administered at a dose of at least 10 mg / kg.

11. A bromide salt and a positive allosteric modulator (PAM) of the mGlu4 receptor as a combined preparation for simultaneous, separate or sequential use in the treatment of autism spectrum disorder (ASD).

12. 12. The combined preparation of claim 11, wherein the bromide salt is sodium bromide and the positive allosteric modulator (PAM) of the mGlu4 receptor is VU0155041.

13. A therapeutic composition comprising a bromide salt and a positive allosteric modulator (PAM) of the mGlu4 receptor, for use in treating autism spectrum disorder (ASD) in a subject in need thereof.

14. 14. The therapeutic composition of claim 13, wherein the bromide salt is sodium bromide and the positive allosteric modulator (PAM) of the mGlu4 receptor is VU0155041.

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