WO2023126076A1

WO2023126076A1 - Medical use of an amide of y-hydroxybutyric acid in the treatment of fragile x syndrome

Info

Publication number: WO2023126076A1
Application number: PCT/EP2022/050030
Authority: WO
Inventors: Roberto Cacciaglia; Giuseppe Tessitore
Original assignee: Laboratorio Farmaceutico C.T. S.R.L.
Priority date: 2022-01-03
Filing date: 2022-01-03
Publication date: 2023-07-06

Abstract

The present invention relates to an amide of γ-hydroxybutyric acid for medical use in the treatment of Fragile X Syndrome and to a pharmaceutical composition containing this derivative mixed with a physiologically acceptable carrier. The very favourable safety profile of the amide of γ-hydroxybutyric acid makes this compound a suitable candidate for orphan drug designation.

Description

Medical use of an amide of y-hydroxybutyric acid in the treatment of Fragile X Syndrome

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FIELD OF THE INVENTION

The present invention relates to the medical use of an amide of y-hydroxybutyric acid in the treatment of Fragile X Syndrome.

The present invention origins in the pharmaceutical field, especially in the field of orphan drugs.

Specifically the present invention provides for a medicament for the treatment of a rare genetic condition for which no drugs are available in the market.

BACKGROUND ART

Fragile X Syndrome, also referred as FXS, is a rare genetic condition caused by silencing of the Fragile X mental retardation (FMR1 ) gene and the subsequent reduction or loss of FMR protein (FMRP). CGG repeat expansion in the 5’UTR of the FMR1 gene, followed by hypermethylation of the region is the basis of the observed silencing in FXS1.

This event occurs sequentially in successive generations, beginning with small repeat expansion (55-200) causing pre-mutation in one generation with a toxic gain of function in the mRNA, followed by further expansion (>200) to full mutation in subsequent generations with complete silencing of the gene.

FMRP is an RNA binding protein, interacting with over 800mRNAs in the adult neuron. FMRP loss has a cascading effect on several pathways which result in the observed clinical features. Several model organisms have been used to model this disease. Therefore, modelling a complex neurological syndrome like FXS is a continuing process and will require a set of organisms to model all the clinical characteristics, to study various pathobiological aspects and to screen potential drug candidates. Currently, there is no cure or approved drug for the treatment of the underlying causes of FXS. Thus, an orphan drug designation for Fragile X Syndrome is well accepted across regulatory jurisdictions due to the ITS relatively low incidence in the worldwide population and to the low expectations that costs of research and development of a drug can be recovered by its commercialization. Typically, an orphan drug designation according to the Regulation (EC) No. 141/2000 of the European Parliament contemplates a life-threatening or chronically debilitating nature of a disease condition; a medically plausible orphan indication proposition; the prevalence of the medical condition in Ell being not more than 5 in 10,000 individuals; the absence of authorized satisfactory method of diagnosis, the prevention or treatment of the medical condition or the evidence that the new medicine promises significant benefit to those affected by the medical condition compared to existing method(s).

According to the FDA, 21 CFR Part 316 - Orphan Drugs, an orphan drug designation is given in case of a prevalence of a disease condition being less than 200,000 persons. In case the prevalence is more than 200,000, the product may still qualify where there is no reasonable expectation that the costs of research and development associated with the drug for the orphan designated indication can be recovered from sales of the product in the USA.

It is estimated that at present there are at least 200.000 people worldwide affected by Fragile X Syndrome which are not receiving appropriate medical and therapeutical treatment.

At present there is a need to develop reliable in vivo models of FXS for drug discovery.

In addition, recent failures of clinical trials with candidates for treating FXS have highlighted the need for the development and validation of new biomarkers to better measure the clinical outcome of effective treatments.

Therefore, there is an unmet need for new medicaments or drugs which may target the Fragile X Syndrome.

One of the general aim of the present invention resides in providing a medicament, targeting the Fragile X Syndrome.

Another aim of the present invention is to provide a drug for the treatment of Fragile X Syndrome which is effective and safe for the individuals affected by this disease.

A specific aim of the present invention is to provide new uses in the medical field for N-[4-(trifluoromethyl)benzyl]-4-methoxybutyramide, a compound aimed at treating and preventing alcohol misuse, alcoholism and disorders caused with excessive alcohol consumption.

SUMMARY OF THE INVENTION

The present invention origins from the finding that a selected amide of y- hydroxybutyric acid which is used in the treatment of alcohol misuse disorders, unexpectedly, is suitable in the treatment of Fragile X Syndrome.

According to a first aspect, the inventors found that a selected y-hydroxybutyrate analog, which is an amide of y-hydroxybutyric acid, is suitable in the treatment of Fragile X Syndrome and is free of side effects.

The high safety profile of the disclosed amide of y-hydroxybutyric acid is a key factor for developing a drug for treating FXS since other molecules tested in the treatment this syndrome, showed a non-positive safety profile during clinical trials of phase I and II and the relative clinical development was abandoned for this reason.

The selected amide of y-hydroxybutyric acid for the medical uses according to the invention, is N-[4-(trifluoromethyl)benzyl]-4-methoxybutyramide (PubChem SID: 329,974,174) of formula:

a compound provided with a favorable safety profile as evidenced by the clinical trials of phase I and II carried out by the same Applicant for a different indication of use.

The herein disclosed N-[4-(trifluoromethyl)benzyl]-4-methoxybutyramide acts on molecular biomarkers which are specific for the Fragile X Syndrome making this compound a suitable candidate for orphan drug designation for the treatment of FX syndrome.

In addition, the experimental data on safety of N-[4-(trifluoromethyl)benzyl]-4- methoxybutyramide recovered during clinical trials of Phase I and II evidence the safety of this compound and contribute to make it a suitable candidate for the treatment of the Fragile X syndrome.

The inventors, while carrying out certain tests supporting the activity of N-[4- (trifluoromethyl)benzyl]-4-methoxybutyramide in the treatment of alcoholism in accordance with the disclosure of the European Patent EP 0 932 597 B1 , have assessed that the above compound modulates the hippocampal glutamate and GABA transmission, through a double negative/positive allosteric modulation of metabotropic glutamate receptor 5 (mGlu5). Each modulation, either negative or positive, gives different physiologic effects. Negative allosteric modulations (NAM) has shown to reduce drug or alcohol intake, reward, reinforcement, and relapse- related behaviors, while positive allosteric modulations (PAM) improve cognitive function, facilitate the extinction of drug-associated contextual memories, and reverse experimentally-induced deficits in cognitive function.

In addition, the inventors also observed that N-[4-(trifluoromethyl)benzyl]-4- methoxybutyramide, a mGluR5 NAM, is suitable to rescue dendritic spine pathology in mice models of FXS.

In view of the above findings, an amide of y-hydroxybutyric acid which is N-[4- (trifluoromethyl)benzyl]-4-methoxybutyramide or a pharmaceutically acceptable salt thereof, is provided for use in the prevention and/or treatment of Fragile X Syndrome.

In accordance with an another aspect, the present invention provides for a composition comprising N-[4-(trifluoromethyl)benzyl]-4-methoxybutyramide or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, for use in the prevention and/or treatment of Fragile X Syndrome.

Embodiments of the pharmaceutical composition of the invention are defined in the appended claims 2 to 7.

Advantageously, the N-[4-(trifluoromethyl)benzyl]-4-methoxybutyramide is contained in the pharmaceutical composition in a therapeutically effective amount, for example in the range of 10 to 1000 mg per unit of dosage.

In an aspect, the invention concerns with a unit dosage form as defined in claim 8.

DETAILED DESCRIPTION OF THE INVENTION

Certain studies aimed to a better understanding of the underlying molecular mechanisms and pathways involved in FXS have led to the development of specific biomarkers for defining targeted therapeutic strategies intended to reverse the intellectual and behavioral problems of patients with FXS.

Specifically, the inventors have focused their research on metabotropic glutamate receptors (mGluRs) as suitable biomarkers for screening and testing molecules effective in the treatment of FXS.

Starting from these premises, the inventors observed that:

(i) the compound N-[4-(trifluoromethyl)benzyl]-4-methoxybutyramide acts as a positive modulator of the metabotropic glutamate receptor subtype 5 (mGluR5) and has a high specificity for this receptor resulting in a positive effect on cognitive function and on experimentally-induced deficits of cognitive functions and

(ii) the metabotropic glutamate receptors subtype 5 (mGluR) are an attractive class of CNS receptors suitable as molecular biomarker for FX Syndrome and of valuable pharmacological relevance.

The inventors, matched the above observations (i) and (ii) with (iii) the positive outcome of clinical trials of phases I and II made by the same applicant on (N-[4- (trifluoromethyl)benzyl]-4-methoxybutyramide). The results of in vitro test and of clinical trials showed that this specific molecule target mGluR in human beings and is very safe in human beings and therefore is a suitable candidate for treating the FX Syndrome in substantial absence of severe side effects.

The safety of (N-[4-(trifluoromethyl)benzyl]-4-methoxybutyramide) is a key factor for meeting the aims of this invention because certain compounds which were selected as candidates for treating FX Syndrome, showed a negative profile of safety during pre clinical studies or clinical tests.

Accordingly, a main subject of this invention is the compound 4-methoxy-N-[[4- (trifluoromethyl)phenyl]methyl]-butanamide or a pharmaceutically salts or ester thereof, for use as a medicament in the treatment of Fragile X Syndrome.

In a preliminary stage of their research, the inventors focused their attention on the fact that the absence of the fragile X mental retardation protein, (FMRP) leads to an excessive metabotropic glutamate receptors of Group I, subtype 5 (mGluRs5). These receptors belong to the family C of the G-protein-coupled receptors (GPCRs). They are characterized by a seven transmembrane a-helical domain connected to a large bi-lobed extracellular amino-terminal domain. The orthosteric binding site is contained in the extracellular domain, while the currently known allosteric binding sites reside in the 7^th domain.

The mGluR family includes eight receptor type, subdivided in three groups, (Group I, II and III) based on their structure, preferred signal transduction mechanisms, and pharmacology (Schoepp et al., 1999).

Group I receptors (mGluRI and mGluR5) are coupled to Gaq, leading to the stimulation of phospholipase C and to the increase in intracellular calcium and inositol phosphate levels. mGluR5 have been implicated in a wide range of neurological and/or psychiatric disorders, including addiction.

In the CNS, mGluR5 has been demonstrated to be expressed mainly in the cortex, hippocampus, nucleus accumbens and the caudate-putamen, brain regions known to be involved in emotional and motivational responses, memory and cognitive function whose activities are impaired in individuals affected by FX Syndrome.

In addition, the inventors observed that the absence of the fragile X mental retardation protein, (FMRP) activate long-term depression (LTD) and reduce responsiveness to signals in the hippocampus and other parts of the brain involved in memory and learning. These factors contribute to the neurological and psychiatric symptoms of FXS. The inventors also observed that the reduction of mGluR signaling demonstrate a reversal of the fragile X phenotypes, providing substantial support to the involvement of the mGluR5 pathway in FXS.

Based on these findings the inventors considered the mGluR5 receptor a valuable target for the development of new drugs aimed at treating FXS and the compound 4-methoxy-N-[[4-(trifluoromethyl)phenyl]methyl]-butanamide has been selected as a suitable mGluR5 candidate inhibitor to prove that FXS phenotypes could be corrected by downregulating signaling through group 1 mGluRs.

The activity of the above compound in the treatment of FXS can be proven by the experiments on well-established animal models as disclosed by Danio rerio (Zebrafish) where true “wild type” animals (wild caught) can be used, incorporating the genetic diversity present in the natural world, with as large a sample size as required to improve the statistics. As an alternative animal model may be used the rat LEH, Fmr1 ^em1/pwc as reported by Hamilton SM, Green JR, Veeraragavan S, et al., “Fmr1 and Nlgn3 knockout rats: Novel tools for investigating autism spectrum disorders,”. Behav Neurosci. Apr 2014; 128(2): 103-109, doi:10.1037/a0035988; Till SM, Asiminas A, Jackson AD, et al. “Conserved hippocampal cellular pathophysiology but distinct behavioural deficits in a new rat model of FXS,”. Hum Mol Genet. 2015; 24(21): 5977-5984, doi:10.1093/hmg/ddv299; Berzhanskaya J, Phillips MA, Gorin A, et al. “Disrupted Cortical State Regulation in a Rat Model of Fragile X Syndrome,”. 2016, doi: 10.1093/cercor/bhv331 ; Asiminas A, Jackson AD, Louros SR, et al. “Sustained correction of associative learning deficits after brief, early treatment in a rat model of Fragile X Syndrome,”. Sci Transl Med. 2019; 11 ( 494): 1-11 , doi: 10.1126/scitranslmed.aao0498.

In another aspect the invention provides a pharmaceutical composition comprising a therapeutically effective amount of 4-methoxy-N-[[4- (trifluoromethyl)phenyl]methyl]-butanamide or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, for use in the prevention and/or treatment of Fragile X Syndrome.

In another aspect a method of treatment of Fragile X Syndrome is provided wherein said method comprises the administration to an individual in need of treatment of an effective amount of 4-methoxy-N-[[4- (trifluoromethyl)phenyl]methyl]-butanamide or a pharmaceutically acceptable salt thereof.

Typically, the individual in need of treatment according the invention is a human being.

The term “effective amount” as used herein includes either a therapeutically and/or prophylactically effective amount of the active ingredient 4-methoxy-N-[[4- (trifluoromethyl)phenyl]methyl]-butanamide or a pharmaceutically acceptable salt thereof.

As used herein, the term “salt” refers to any salt of a compound according to the present invention prepared from an inorganic or organic acid or base and internally formed salts. Typically, such salts have a physiologically acceptable anion or cation.

The term biomarker as used herein means “a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention”.

A biomarkers can be found in blood, plasma, or other tissues and are generally viewed as a molecular signature able to identify individuals who are at high risk for a specific condition. It can also be detected before disease symptoms and therefore used to predict the occurrence of a condition or the nature and seventy of disease outcomes in an individual. They may be used to evaluate the efficacy of response to a pharmacological treatment.

The compound N-[4-(trifluoromethyl)benzyl]-4-methoxybutyramide may be in crystalline form. In certain embodiments, the crystalline forms of the compound are polymorphs.

The compound N-[4-(trifluoromethyl)benzyl]-4-methoxybutyramide may be administered alone or in combination with one or more active ingredients such as additional amides of y-hydroxybutyric acid.

The pharmaceutical compositions of the present invention encompass any compositions made by mixing the compound of the present invention and a pharmaceutically acceptable carrier. Such compositions are suitable for pharmaceutical use in an animal or human.

A pharmaceutical composition may optionally contain other active ingredients.

The term “carrier” refers to a vehicle, excipient, diluents, or adjuvant with which the therapeutic or active ingredient is administered. Any carrier and/or excipient suitable for the form of preparation desired for administration is contemplated for use with the compounds disclosed herein.

The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g. oral or parenteral (including intravenous).

In preparing the compositions for oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavouring agents, preservatives, colouring agents and the like in the case of oral liquid preparations, such as, for example, suspensions, elixirs and solutions; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like in the case of oral solid preparations such as, for example, powders, hard and soft capsules and tablets, with the solid oral preparations being preferred over the liquid preparations.

In certain embodiments, the compounds of the present invention can be combined as the active ingredient in intimate admixture with a suitable pharmaceutical carrier and/or excipient according to conventional pharmaceutical compounding techniques.

The compositions include compositions suitable for parenteral, including subcutaneous, intramuscular, and intravenous, nasal, rectal, topical or oral administration.

Suitable route of administration in any given case will depend in part on the nature and severity of the conditions being treated and on the nature of the active ingredient.

An exemplary route of administration is the oral route. The compositions may be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy. The preferred compositions include compositions suitable for oral administration. The oral compositions may be prepared by any of the methods well-known in the art of pharmacy.

The pharmaceutical compositions in solid form may be in the form of tablets, pills, capsules, powders, suppository and as sustained release formulations.

If desired, tablets may be coated by standard aqueous or non-aqueous techniques. In certain embodiments, such compositions and preparations may contain at least 0,5, or 1 percent of active compound. The percentage of active compound in these compositions may, of course, be varied and may conveniently be from 1 to 60%, 5 to 50%, 10 to 30 percent of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that therapeutically active dosage will be obtained. The active compounds can also be administered intranasally as, for example, liquid drops or spray.

The tablets, pills, capsules, and the like may also contain a binder such as gum tragacanth, acacia, com starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin. When a dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as a fatty oil. Various other materials may be present as coatings or to modify the physical form of the dosage unit. For instance, tablets may be coated with shellac, sugar or both. A syrup or elixir may contain, in addition to the active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and a flavouring agent such as cherry or orange flavour. To prevent breakdown during transit through the upper portion of the gastrointestinal tract, the composition be an enteric coated formulation.

Compositions for topical administration include, but are not limited to, ointments, creams, lotions, solutions, pastes, gels, sticks, liposomes, nanoparticles, patches, bandages and wound dressings. In certain embodiments, the topical formulation comprises a penetration enhancer.

Administration of the compositions is performed under a protocol and at a dosage sufficient to reduce the dependency from drugs or alcohol intake.

In certain embodiments the pharmaceutical composition of the invention contain from 1 % to 50% by weight, preferably from 5 to 30% by weight of N-[4- (trifluoromethyl)benzyl]-4-methoxybutyramide with respect to the total weight of the composition.

In some embodiments, in the pharmaceutical compositions of the present invention the active ingredient N-[4-(trifluoromethyl)benzyl]-4-methoxybutyramide is formulated in dosage units. The dosage unit may contain from 1 to 2000 mg, from 5 to 1000 mg, from 30 to 600 mg, especially from 50 to 500 mg of the compound per dosage unit for daily administration.

For example the unit dosage containing a pharmaceutical composizition according to anyone of the herein disclosed embodiments contains 10, 30, 100, 300, 450 and 600 mg of N-[4-(trifluoromethyl)benzyl]-4-methoxybutyramide or in alternative 100, 300, 450 mg. The mentioned dosage units may be administered from one to three a day, preferably once a day. In certain embodiments, the unit dosage is a tablet or capsule containing the pharmaceutical composition in an amount as herein disclosed or in the alternative the unit dosage is an aqueous solution containing the pharmaceutical composition in an amount as herein disclosed.

With respect to formulations with respect to any variety of routes of administration, methods and formulations for the administration of drugs are disclosed in Remington’s Pharmaceutical Sciences, 17th Edition, Gennaro et al. Eds., Mack Publishing Co., 1985, and Remington’s Pharmaceutical Sciences, Gennaro AR ed. 20th Edition, 2000, Williams & Wilkins PA, USA, and Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins Eds. , 2005; and in Ansel’s Pharmaceutical Dosage Forms and Drug Delivery Systems, 8th Edition, Lippincott Williams & Wilkins Eds., 2005, which are herein incorporated as reference.

When used in combination with one or more other active ingredients, the compound of the present invention and the other active ingredient may be used in lower doses than when each is used singly.

A preferred pharmaceutical composition is a formulation in the form for oral administration a day of the active compound according to the invention in mixture with pharmaceutical acceptable carriers, to be administered typically once a day, or in certain conditions twice or more a day.

It is to be understood that all aspects identified as preferred and advantageous for the derivative of the invention should be deemed as similarly preferred and advantageous also for the pharmaceutical compositions and the relevant uses.

In an additional aspect, the present invention also concerns combination therapies or treatment with a compound N-[4-(trifluoromethyl)benzyl]-4-methoxybutyramide or pharmaceutical composition containing them.

In some embodiments, the compound N-[4-(trifluoromethyl)benzyl]-4- methoxybutyramide and their pharmaceutical compositions and methods of administering them, are useful in treating Fragile X Syndrome when administered in combination with other pharmacological agents or active ingredients.

The following Examples are provided for illustrative and not limiting purposes. EXAMPLES

Example 1

Fragile X Syndrome is caused by mutations in the Fragil Mental Retardation (FMR 1 ) gene, located on the X chromosome. This gene provides the instructions for making a protein called FMRP, which regulates the production of proteins involved in the nerve cell communication.

Reduction or interruption of the production of FMR protein cause therefore cognitive, developmental and behavioural symptoms.

Animal models of FXS may involve techniques for rapid knockdown of gene expression. Among the available in vivo models, we selected two models/techniques to test of (N-[4-(trifluoromethyl)benzyl]-4-methoxybutyramide). First protocol

First technique/protocol to test the in vivo activity on an animal model in the treatment of Fragile X Syndrome as a mGluR5 modulator.

Zebrafish was used as animal model: causing a transient knockdown of FMR 1 gene, through morpholino technique or through chemical induced mutations, there is a reduction of FMRP and increase of mGluR5 (metabotropic glutamate type 5 receptors).

Administration of the compound Fragile X Syndrome as mGluR5 modulator shall restore the dysregulation of excitatory and inhibitory neurotransmission.

Second protocol

The second model used as test for assessing the activity of (N-[4- (trifluoromethyl)benzyl]-4-methoxybutyramide) in Fragile X Syndrome is a specific transgenic rat model (LEH) for autism spectrum disorders (ASD).

In these rats, a zinc-finger nuclease mediated knockout of FMR 1 gene causes loss of FMRP and altered mGluR5 signalling, which can be restored with administration of (N-[4-(trifluoromethyl)benzyl]-4-methoxybutyramide) as mGluR5 modulator.

Example 2

Protocol - clinical trial

Preclinical data suggest that inhibition of the metabotropic glutamate receptor 5 (mGluR5) receptor might hold therapeutic benefits in Fragile X syndrome (FXS). Treatment of Fmr1 knockout mice with mGluR5-negative allosteric modulators (NAMs) has been reported to correct a broad range of phenotypes related to FXS. An early short-term clinical trial with mGluR5 NAMs, including (N-[4- (trifluoromethyl)benzyl]-4-methoxybutyramide) a potent and selective mGluR5 NAM assessing the effects in individuals with FXS.

A suitable protocol with the compound of this application shall include a 12-week, double-blind, parallel-group study of adults and adolescents with FXS as reported by Eriene A Youssef et al. in the publication “Effect of the mGluR5-NAM Basimglurant on Behavior in Adolescents and Adults with Fragile X Syndrome in a Randomized, Double-Blind, Placebo-Controlled Trial:

FragXis Phase 2 Results”, Neuropsychopharmacology (2018) 43, 503-512.

The clinical trial illustrated in the above publication may be used as guideline to put into practice a protocol for assessing the effectiveness of (N-[4- (trifluoromethyl)benzyl]-4-methoxybutyramide) in the treatment of FXS.

Example 3

Pharmaceutical composition

(N-[4-(trifluoromethyl)benzyl]-4-methoxybutyramide) (GET73) 50 mg

Cellulose mycrocrystalline (excipient /carrier) 60 mg

Talc (as lubricant) 10 mg

Sodium laurylsulfate (as surfactant) 5 mg

Calcium phosphate (as aggregant-diluent) 200 mg

Magnesium carbonate (as diluent-binder) 100 mg

Example 4

Pharmaceutical composition

(N-[4-(trifluoromethyl)benzyl]-4-methoxybutyramide) (GET73) 150 mg

Maize starch 100 mg Glyceryl behenate (as lubricant) 10 mg

Polysorbate (as surfactant) 10 mg

Magnesium carbonate (as diluent-binder) 150 mg

Lactose (as diluent) 150 mg

Example 5

Pharmaceutical composition with film-coatinq/modified release

(N-[4-(trifluoromethyl)benzyl]-4-methoxybutyramide) (GET 73) 500 mg

Cellulose mycrocrystalline 200 mg

Crospovidone (as anti-aggregant agent) 50 mg

Starch (as diluent-disintegrant) 50 mg

Colloidal silica (as drying agent) 10 mg

Magnesium stearate (as lubricant) 10 mg

Hypromellose (as coating agent) 50 mg

Macrogol (as plasticiser agent) 10 mg

Titanium dioxide (as dye) 10 mg

Example 6

N-[4-(trifluoromethyl)benzyl]-4-methoxybutyramide has been studied in three phase 1 clinical studies and in two phase 2a clinical studies. Overall, 150 subjects have been enrolled into the clinical programme and according to the studies’ methodology, 119 patients have been randomized to receive N-[4- (trifluoromethyl)benzyl]-4-methoxybutyramide. Among them, 74 are healthy volunteers enrolled during phase 1 studies, 45 patients have been exposed to the medication in the phase 2a studies.

Phase 1 73CT-1 -01 (EudraCT number: 2009-015377-12) Haass-Koffler CL, Goodyear K, et al. “A phase I randomized clinical trial testing the safety, tolerability and preliminary pharmacokinetics of the mGluR5 negative allosteric modulator GET 73 following single and repeated doses in healthy volunteers”, Eur J Pharm Sci Nov 2017; 109: 78-85, doi:10.1016/j.ejps.2017.07.031

73 CT-1-01 was a single center, randomized, double blind, placebo-controlled, 2- part Phase 1 study designed to explore the safety and tolerability of N-[4- (trifluoromethyl)benzyl]-4-methoxybutyramide following single ascending dose (Part A) and multiple ascending dose (Part B) oral administration in healthy male subjects aged 18 - 65 inclusive, and with a Body Mass Index (BMI) < 30 kg/m²

Part A: single ascending dose

Forty-eight male subjects were randomized to receive either N-[4- (trifluoromethyl)benzyl]-4-methoxybutyramide (10, 30, 100, 300, 450 and 600 mg) or placebo.

Part B: multiple ascending dose

Thirty-two healthy male subjects were randomized to receive either N-[4- (trifluoromethyl)benzyl]-4-methoxybutyramide (100, 300, 450 mg once a day, and 450 mg b.i.d.) or placebo for 14 days.

Results: In part A of the study eleven Treatment-Emergent Adverse Events (TEAEs) reported and related to N-[4-(trifluoromethyl)benzyl]-4- methoxybutyramide or placebo were classified as mild and one (migraine) as moderate.

In the part B, the most commonly occurring TEAE was headache, which was reported by seven subjects. Only one TEAE was considerate moderate in intensity.

60 subjects were exposed to the medication and in both part A and part B there were no severe TEAEs reported.

Phase 1 73CT-1 ■02 (EudraCT number_2011 002354-31 )

Haass-Koffler CL, Goodyear K, et al. “Administration of the mGluR5 allosteric modulator GET 73 with alcohol: a translational study in rats and humans”, J Psychopharmacol Feb 2018; 33(2): 163-173, doi: 10.1177/0269881117746904. 73CT-1-02 was an open-label, 4-way, randomized, cross-over Phase 1 study designed to determine the effect of alcohol on the bioavailability of N-[4- (trifluoromethyl)benzyl]-4-methoxybutyramide and of its major metabolite when given at 2 dose levels (300 mg single dose and 300 mg t.i.d.) in healthy male subjects aged 18 to 65 inclusive, and with a BMI between 18 and 35 kg/m²

Fourteen subjects have been exposed to the medication. Overall, the incidence of AEs in Study 73CT-1-02 was low; 5 subjects reported a total of 9 AEs. There was no notable dose-related trend in the incidence of AEs and there was no notable difference between dosing in the presence or absence of alcohol. There were no serious or severe AEs. There were no clinically significant findings in any other ECG parameters, clinical laboratory assessments or vital signs.

Phase 1 b/2a Study 73CT-2-03

Haass-Koffler CL, Perciballi R, et al. “An inpatient human laboratory study assessing the safety and tolerability, pharmacokinetics, and biobehavioral effect of GET 73 when co-administered with alcohol in individuals with alcohol use disorder”, Psychopharmacology Nov 2021 ; https://doi.Org/10.1007/s00213-021 -

06008-1 (National Clinical Trial Number: NCT01842503, EudraCT number: 2012-

05515-13).

25 subjects were enrolled and 21 treated with N-[4-(trifluoromethyl)benzyl]-4- methoxybutyramide.

Subjects were treated with 300 mg capsules three times per day for three consecutive days.

10 subjects reported at least one AE with a total number of 20 AEs, possible related or unlikely related to the IND. None of them was assessed as serious, 15 were referred as mild, and 5 as moderate. In a blind analysis among the patients exposed, there was no difference, in terms of number and seventy of the AEs, between the two groups.

Among TEAEs that were considered related to study treatment, headache was the most observed event. Phase Ila

GET73 ¹H-MRS (National Clinical Trial Number: NCT03418623).

Oral, 300 mg oral capsules, administered three times a day. Each subject received the active compound for 3 days and the placebo for 3 days, in a randomized sequence.

In the study, 24 patients have been exposed to the medication.

Among the randomized subjects, 15 reported at least one AE with a total number of 35 AEs, possible related or unlikely related to the IND. None of them was assessed as serious, 27 were referred as mild, 7 as moderate and 1 as severe. Headache, registered in 7 subjects, was the most common side effect.

In conclusion, a total exposure to N-[4-(trifluoromethyl)benzyl]-4- methoxybutyramide is estimated to be 119 subjects.

No serious adverse drug reactions have been identified following analysis of the studies.

No significant new information or new safety concern has been reported on:

- interactions;

- overdose, abuse or misuse;

- positive or negative experiences during pregnancy or lactation;

- experience in special patient groups (i.e. organ impaired);

- effects of long-term treatment;

- patient/consumer and other non-healthcare professional reports.

Claims

1. A compound which is N-[4-(trifluoromethyl)benzyl]-4-methoxybutyramide or a pharmaceutically acceptable salt thereof for use in the prevention and/or treatment of Fragile X Syndrome.

2. A pharmaceutical composition comprising a N-[4-(trifluoromethyl)benzyl]-4- methoxybutyramide or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier for use in the prevention and/or treatment of Fragile X Syndrome.

3. A pharmaceutical composition for use according to claim 2 comprising N-[4- (trifluoromethyl)benzyl]-4-methoxybutyramide as active ingredient in an amount of 5 to 1000 mg.

4. A pharmaceutical composition for use according to claim 3 wherein N-[4- (trifluoromethyl)benzyl]-4-methoxybutyramide is in an amount of 30 to 600 mg, preferably from 50 to 500 mg.

5. A pharmaceutical composition for use according to anyone of claims 2 to 4 wherein the composition is in solid form or in liquid form for oral administration.

6. A pharmaceutical composition for use according to claim 5 wherein the composition is a tablet or an aqueous solution for oral administration.

7. A pharmaceutical composition for use according to anyone of claims 2 to 6 wherein the composition is administered to an individual from one to three per day, preferably once a day.

8. A unit form of dosage comprising a pharmaceutical composition for use according to anyone of claims 2-7.