WO2021250402A1

WO2021250402A1 - Composition for treatment of fragile x syndrome

Info

Publication number: WO2021250402A1
Application number: PCT/GB2021/051432
Authority: WO
Inventors: David Brown
Original assignee: Healx Ltd
Priority date: 2020-06-09
Filing date: 2021-06-09
Publication date: 2021-12-16
Also published as: GB202008699D0

Abstract

The present invention relates to compositions and kits comprising: (i) sertraline, or a pharmaceutically acceptable salt thereof; and (ii) metformin, or a pharmaceutically acceptable salt thereof. The compositions and kits are useful in the treatment of Fragile X syndrome.

Description

COMPOSITION FOR TREATMENT OF FRAGILE X SYNDROME

Field of the invention

This invention relates a composition, kit and combination therapy comprising sertraline, or a pharmaceutically acceptable salt thereof, and metformin, or a pharmaceutically acceptable salt thereof, for use in the treatment of Fragile X syndrome.

Background of the invention

Fragile X syndrome, often referred to as Fragile X, is the most common inherited cause of intellectual impairment and the most common monogenic cause of autism. It affects around 1 in 4000 males and 1 in 6000 females worldwide.

There are a wide range of characteristics associated with Fragile X, and typically males are more affected than females. One of the major characteristics associated with Fragile X syndrome is intellectual impairment, such as difficulties with cognitive, executive and language performance. Individuals with Fragile X syndrome typically have social anxiety characterised by social, emotional and communication difficulties related to extreme shyness, poor eye contact and challenges forming peer relationships. Fragile X syndrome is also associated with hyperactivity and disruptive behaviour, such as short attention span, distractibility, impulsiveness, restlessness, over-activity, and sensory problems. Furthermore, individuals with Fragile X syndrome often suffer from seizures.

Fragile X syndrome arises from a mutation in a single gene called Fragile X Mental Retardation Gene 1 (FMR1). The 5' UTR of FMR1 contains a CGG trinucleotide repeat that is polymorphic in the population. Once the repeats exceed 200 in number, methylation of the promoter is triggered, and this in turn causes the lack of expression of the gene and translation of its encoded protein, the Fragile X Mental Retardation Protein (FMRP). FMRP is an RNA-binding protein involved in different steps of mRNA metabolism, such as translational control (in soma and dendritic spines) and RNA transport.

At present, there is no effective therapy to treat Fragile X syndrome. Flowever, there have been considerable efforts to identify pharmacological targets to treat this disorder; in particular, Fragile X syndrome has been a frequent target of repurposing efforts as well as repositioning of drugs in development. Many different standards and methods have been applied to this task. In many cases, repurposing candidates have been identified based primarily on clinical pattern matching, while in others basic disease mechanisms have been studied extensively to identify therapeutic targets, followed by thorough preclinical validation.

Overall, efforts to treat Fragile X syndrome have led to some exciting possibilities, but no definitive successes, despite much effort. This has highlighted the need for new therapies. Sertraline is a selective serotonin re-uptake inhibitor (SSRI) used in the treatment of major depressive disorder, and has the systematic name (lS,4S)-4-(3,4-Dichlorophenyl)-N- methyl-l,2,3,4-tetrahydronaphthalen-l-amine. Metformin is a biguanide hypoglycemic agent used in the treatment of non-insulin-dependent diabetes mellitus, and has the systematic name N,N-Dimethylimidodicarbonimidic diamide.

Summary of the invention

The present invention is based on in vivo data. The combination of sertraline, or a pharmaceutically acceptable salt thereof, with metformin, or a pharmaceutically acceptable salt thereof has been identified as being useful in the treatment of Fragile X syndrome. This can be seen from the in vivo data in the figures.

In a first aspect of the invention, there is provided a composition comprising:

(i) sertraline, or a pharmaceutically acceptable salt thereof; and

(ii) metformin, or a pharmaceutically acceptable salt thereof.

In a second aspect of the invention, a kit comprising:

(i) at least one dose of sertraline, or a pharmaceutically acceptable salt thereof; and

(ii) at least one dose of metformin, or a pharmaceutically acceptable salt thereof.

In a third aspect of the invention, there is provided a composition comprising:

(i) sertraline, or a pharmaceutically acceptable salt thereof; and

(ii) metformin, or a pharmaceutically acceptable salt thereof for use in the treatment of Fragile X syndrome.

In a fourth aspect of the invention, there is provided a kit comprising:

(ii) at least one dose of metformin, or a pharmaceutically acceptable salt thereof, for simultaneous, separate, or sequential use in the treatment of Fragile X syndrome.

In a fifth aspect of the invention, there is provided a method of treating Fragile X syndrome comprising administering to the patient simultaneously, separately, or sequentially: (i) at least one dose of sertraline, or a pharmaceutically acceptable salt thereof; and

In a sixth aspect of the invention, there is provided:

(i) use of sertraline, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a patient with Fragile X syndrome, wherein the patient has been administered metformin, or a pharmaceutically acceptable salt thereof;

(ii) use of metformin, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a patient with Fragile X syndrome, wherein the patient has been administered sertraline, or a pharmaceutically acceptable salt thereof; or

(iii) use of sertraline, or a pharmaceutically acceptable salt thereof, and metformin, or a pharmaceutically acceptable salt thereof, in combination for the manufacture of a medicament for the treatment of a patient with Fragile X syndrome.

Figure 1 shows the results from in vivo open field testing with sertraline and metformin. Figure 2 shows the results from in vivo nesting testing with sertraline and metformin. Figure 3 shows the results from in vivo fear conditioning testing with sertraline and metformin.

Figure 4 shows the results from in vivo sociability testing with sertraline and metformin. Figure 5 shows the results from in vivo hyponeophagia testing with sertraline and metformin.

As Fragile X is a syndrome, there are several different manifestations and symptoms in patients. These include; intellectual impairment, such as difficulties with cognitive, executive and language performance, short-term memory, executive function, visual memory and visual-spatial relationships; autism; social anxiety (/.e. difficulties in social interaction) such as poor eye contact, gaze aversion, prolonged time to commence social interaction, and challenges forming peer relationships; hyperactivity and repetitive behaviour, including very short attention spans, hypersensitivity to visual, auditory, tactile, and olfactory stimuli, distractibility, impulsiveness, restlessness and over-activity; disruptive behaviour, including fluctuating mood, irritability, self-injury and aggression; obsessive compulsive disorder (OCD); ophthalmologic problems, such as strabismus; seizures; difficulties with working memory, which involves the temporary storage of information while processing the same or other information; difficulties with phonological memory (or verbal working memory); and Fragile X-related primary ovarian insufficiency (FXPOI).

In the present invention, and as demonstrated by the below in vivo data, sertraline and metformin in combination are used to treat one or more of the above symptoms and is therefore an effective treatment of Fragile X syndrome. Preferably, sertraline and metformin in combination is used for the treatment of Fragile X syndrome, wherein the patient is exhibiting typical symptoms of the syndrome including social anxiety, hyperactivity, memory loss and/or disruptive behaviour. More preferably, sertraline and metformin in combination is used for the treatment of Fragile X syndrome, wherein the patient is exhibiting hyperactivity, memory loss and/or disruptive behaviour.

The term "hyperactivity" has its normal meaning in the art. Flyperactivity may include having very short attention spans, hypersensitivity to visual, auditory, tactile, and olfactory stimuli, distractibility, impulsiveness, restlessness and/or over-activity.

The term "social anxiety" has its normal meaning in the art. It may also be termed as difficulties in social interaction or low sociability. Social anxiety may include having poor eye contact, gaze aversion, prolonged time to commence social interaction, social avoidance or withdrawal and challenges forming peer relationships.

The term "memory loss" has its normal meaning in the art. It may also be called memory impairment. It refers to an inability to retain information either short-term or long-term. It may include difficulties with cognitive, executive and language performance, executive function, and visual memory. It may also include difficulties with working memory, also called short-term memory (/.e. the temporary storage of information while processing the same or other information) and difficulties with phonological memory (or verbal working memory),

The term "disruptive behaviour" has its normal meaning in the art. It may also include repetitive behaviour. It may also include fluctuating mood, irritability, self-injury, and aggression.

As used herein, a pharmaceutically acceptable salt is a salt with a pharmaceutically acceptable acid or base. Pharmaceutically acceptable acids include both inorganic acids such as hydrochloric, sulphuric, phosphoric, diphosphoric, hydrobromic or nitric acid and organic acids such as citric, fumaric, maleic, malic, ascorbic, succinic, tartaric, benzoic, acetic, methanesulfonic, ethanesulfonic, salicylic, stearic, benzenesulfonic or p- toluenesulfonic acid. Pharmaceutically acceptable bases include alkali metal (e.g. sodium or potassium) and alkali earth metal (e.g. calcium or magnesium) hydroxides and organic bases such as alkyl amines, aryl amines or heterocyclic amines.

The kits or compositions according to the present invention may be administered in a variety of dosage forms. In one embodiment, it may be formulated in a format suitable for oral, rectal, parenteral, intranasal or transdermal administration or administration by inhalation or by suppository.

The kits or compositions according to the present invention may be administered orally, for example as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders, or granules. Preferably, the kits or compositions are formulated such that they are suitable for oral administration, for example tablets and capsules.

The kits or compositions according to the present invention may also be administered parenterally, whether subcutaneously, intravenously, intramuscularly, intrasternally, transdermally, or by infusion techniques. It may also be administered as suppositories.

The kits or compositions according to the present invention may also be administered by inhalation. An advantage of inhaled medications is their direct delivery to the area of rich blood supply in comparison to many medications taken by oral route. Thus, the absorption is very rapid as the alveoli have an enormous surface area and rich blood supply and first pass metabolism is bypassed.

The present invention also provides an inhalation device containing the kits or compositions according to the present invention. Typically said device is a metered dose inhaler (MDI), which contains a pharmaceutically acceptable chemical propellant to push the medication out of the inhaler.

The kits or compositions according to the present invention may also be administered by intranasal administration. The nasal cavity's highly permeable tissue is very receptive to medication and absorbs it quickly and efficiently. Nasal drug delivery is less painful and invasive than injections, generating less anxiety among patients. By this method absorption is very rapid and first pass metabolism is usually bypassed, thus reducing inter- patient variability. Further, the present invention also provides an intranasal device containing the kits or compositions according to the present invention.

The kits or compositions according to the present invention may also be administered by transdermal administration. For topical delivery, transdermal and transmucosal patches, creams, ointments, jellies, solutions, or suspensions may be employed. The present invention therefore also provides a transdermal patch containing the kits or compositions according to the present invention.

The kits or compositions according to the present invention may also be administered by sublingual administration. The present invention therefore also provides a sub-lingual tablet comprising the kits or compositions according to the present invention.

The kits or compositions according to the present invention may also be formulated with an agent which reduces degradation of the substance by processes other than the normal metabolism of the patient, such as anti-bacterial agents, or inhibitors of protease enzymes which might be the present in the patient or in commensural or parasite organisms living on or within the patient, and which are capable of degrading the compound.

The compositions according to the present invention may be formulated in a modified- release dosage form. Modified-release dosage forms include controlled delivery, controlled release, delayed release, extended release, immediate release, long-acting, long-acting release, prolonged release, sustained action, sustained release, timed release, extended release, etc., as used in the art. Methods of modified-release dosage form include diffusion systems, dissolution systems, osmotic systems, ion-exchange resins, floating systems, bio-adhesive systems, matrix systems, and stimuli-inducing release.

Liquid dispersions for oral administration may be syrups, emulsions and suspensions. Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. The suspension or solutions for intramuscular injections may contain, together with the active compound(s), a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.

Solutions for injection or infusion may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions. In an embodiment of the invention, the kits or compositions according to the present invention are administered in an effective amount to treat the symptoms of Fragile X syndrome. An effective dose will be apparent to one skilled in the art and is dependent on several factors such as age, sex, weight, which the medical practitioner will be capable of determining.

In a preferred embodiment, the compositions according to the present invention comprise 10 mg to 300 mg of sertraline, preferably 15, 20, 25, 30, or 40 mg to 250, 200, 150, or 100 mg of sertraline. Preferably, the total daily dose of sertraline does not exceed the maximum daily dose recommended by the manufacturer.

In another preferred embodiment, the compositions according to the present invention comprise 100 mg to 2000 mg of metformin, preferably 150, 200, 250, 300, or 350 mg to 1800, 1600, 1400, 1200, or 1000 mg of metformin. Preferably, the total daily dose of metformin does not exceed the maximum daily dose recommended by the manufacturer.

In a further preferred embodiment, the compositions according to the present invention comprise 10 mg to 300 mg of sertraline and 100 mg to 2000 mg of metformin, preferably 15, 20, 25, 30, or 40 mg to 250, 200, 150, or 100 mg of sertraline and 150, 200, 250, 300, or 350 mg to 1800, 1600, 1400, 1200, or 1000 mg of metformin. Preferably, the total daily doses of sertraline and metformin do not exceed the maximum daily doses recommended by the manufacturer.

Any of the above doses may be administered once a day, twice a day, three times a day or four times a day.

In an embodiment, the compositions of the invention are administered at least once a day. Preferably it is administered as a single daily dose. Preferably the single daily dose comprises 10 mg to 300 mg of sertraline and 100 mg to 2000 mg of metformin, preferably 15, 20, 25, 30, or 40 mg to 250, 200, 150, or 100 mg of sertraline and 150, 200, 250, 300, or 350 mg to 1800, 1600, 1400, 1200, or 1000 mg of metformin.

In an embodiment, the compositions of the invention are administered twice a day. Preferably each dose comprises 5 mg to 150 mg of sertraline and 50 mg to 1000 mg of metformin, preferably 7.5, 10, 12.5, 15, or 20 mg to 125, 100, 75, or 50 mg of sertraline and 75, 100, 125, 150, or 175 mg to 900, 800, 700, 600, or 500 mg of metformin Preferably, the dosage regime is such that the total daily dosage of sertraline does not exceed 300 mg, and the daily dose of metformin does not exceed 2000 mg.

In order to treat Fragile X syndrome, sertraline and metformin are used in a chronic dosage regime (/.e. chronic, long-term treatment).

The kit according to the present invention provides for the administration of more than one drug, and they can be administered simultaneous, sequentially, or separately. It is not necessary that they are packed together (but this is one embodiment of the invention). It is also not necessary that they are administered at the same time. As used herein, "separate" administration means that the drugs are administered as part of the same overall dosage regimen (which could comprise several days), but preferably on the same day. As used herein "simultaneously" means that the drugs are to be taken together or formulated as a single composition. As used herein, "sequentially" means that the drugs are administered at about the same time, and preferably within 1 hour of each other. Preferably, the kit is administered simultaneously (/.e. taken together or formulated as a single composition). Most preferably, it is formulated as a single composition.

In an embodiment of the invention, the kit is administered at least once a day. Preferably it is administered as a single daily dose. Preferably the single daily dose is administered simultaneously (/.e. sertraline and metformin are taken together or formulated as a single composition). In this embodiment, most preferably, it is formulated as a single composition. In this embodiment, preferably the kit comprises of 10 mg to 300 mg of sertraline and 100 mg to 2000 mg of metformin, preferably 15, 20, 25, 30, or 40 mg to 250, 200, 150 or 100 mg of sertraline and 150, 200, 250, 300, or 350 mg to 1800, 1600, 1400, 1200, or 1000 mg of metformin. It may also be administered sequentially (/.e. sertraline and metformin are administered at about the same time, and preferably within about 1 hour of each other).

In an embodiment of the invention, the kit may be administered twice daily. Preferably each daily dose is administered simultaneously (/.e. sertraline and metformin are taken together or formulated as a single composition). In this embodiment, most preferably, it is formulated as a single composition, which is administered twice daily. In this embodiment, preferably the kit comprises of 5 mg to 150 mg of sertraline and 50 mg to 1000 mg of metformin, preferably 7.5, 10, 12.5, 15, or 20 mg to 125, 100, 75, or 50 mg of sertraline and 75, 100, 125, 150, or 175 mg to 900, 800, 700, 600, or 500 mg of metformin. Each daily dose may also be administered sequentially (/.e. for each dose, sertraline and metformin are administered at about the same time, and preferably within about 1 hour of each other).

The present invention also relates to a method of treating Fragile X syndrome comprising administering the patient with a kit or composition as described herein. This embodiment of the invention may have any of the preferred features described above. The method of administration may be according to any of the routes described above.

The present invention also relates to use of sertraline, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a patient with Fragile X syndrome, wherein the patient has been administered metformin, or a pharmaceutically acceptable salt thereof. This embodiment of the invention may have any of the preferred features described above.

The present invention also relates to use of metformin, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a patient with Fragile X syndrome, wherein the patient has been administered sertraline, or a pharmaceutically acceptable salt thereof. This embodiment of the invention may have any of the preferred features described above.

The present invention also relates to use of sertraline, or a pharmaceutically acceptable salt thereof, and metformin, or a pharmaceutically acceptable salt thereof, in combination for the manufacture of a medicament for the treatment of a patient with Fragile X syndrome. This embodiment of the invention may have any of the preferred features described above.

For the avoidance of doubt, the present invention also embraces prodrugs which react in vivo to give a compound of the present invention.

Study

Animals

FMR1 knockout 2 (FMR1 K02) mice were generated by deletion of the promoter and first exon of FMR1. The FMR1 K02 it is both, protein and mRNA null. In this study we used FMR1 K02 and wild-type (WT) littermates generated on a C57BL/6J background and repeatedly backcrossed onto a C57BL/6J background for more than eight generations. Animal housing

The FMR1 K02 mice were housed in 4-5 per cage groups of the same genotype in a temperature- (21 ± 1 °C) and humidity-controlled room with a 12-hr light-dark cycle (lights on 7 a.m.-7 p.m.)· Food and water were available ad libitum. Mice were housed in commercial plastic cages, and experiments were conducted in accordance with the requirements of the UK Animals (Scientific Procedures) Act, 1986. Protocols were reviewed and approved by the IEB, University of Chile Institute review board. All experiments were conducted with the staff blinded to genotype and drug treatment. Separate investigators prepared and coded the dosing solutions, allocated the mice to the study treatment groups, dosed the animals, and collected the behavioral data.

Treatment Groups

There were seven treatment groups in the study with 10 male mice used per treatment group (all at 2 months of age): Group 1: wild-type littermate mice treated with vehicle, "WT-Veh"; Group 2: FMR1 K02 mice treated with vehicle, "KO-Veh"; Group 3: FMR1 K02 mice treated with 50 mg/kg metformin, "KO + met (50 mg/kg)"; Group 4: FMR1 K02 mice treated with 1 mg/kg sertraline, "KO + Ser (1 mg/kg)"; Group 5: FMR1 K02 mice treated with 5 mg/kg sertraline, "KO + Ser (5 mg/kg)"; Group 6: FMR1 K02 mice treated with 1 mg/kg sertraline and 50 mg/kg metformin, "KO + Ser (1 mg/kg)/Met (50 mg/kg)"; Group 7: FMR1 K02 mice treated with 5 mg/kg sertraline and 50 mg/kg metformin, "KO + Ser (5 mg/kg)/Met (50 mg/kg)".

Behavioral Testing

For experiments, all mice were tested once in the same apparatus. Prior to testing, mice were placed in the apparatus for some minutes before the experiment. The apparatus was cleaned with moist and dry tissues before testing each mouse. The aim was to create a low but constant background mouse odor for all experimental subjects. Testers were blind to the genotype and treatment during all testing and data analysis. We assessed weight loss, fur loss, walking, eyes open, eye discharges and general behavior. All signs indicated that all treatments were well tolerated by the FMR1 K02 mice and WT littermates at all times.

Open Field (^'hyperactivity!

The open-field apparatus was used to test multiple processes including anxiety/hyperactivity and habituation to a novel environment. The apparatus was a grey PVC-enclosed arena 50x9x30 cm divided into a 10x10 cm grid. Mice were brought to the experimental room 5-20min before testing. A mouse was placed into a corner square facing the corner and observed for 3 min. The number of squares entered by the whole body (locomotor activity) and rears (both front paws off the ground, but not as part of grooming) were counted. The latency to the first rear was noted. The movement of the mouse around the field was recorded with a video tracking device for 3 min (version NT4.0, Viewpoint). The latency for the mouse to enter the brightest, the central part of the field, total time spent in this central region, and total activity (as path length in cm) were recorded.

Nesting

The test was performed in individual cages. Normal bedding covered the floor to a depth of 0.5 cm. Each cage was supplied with a "Nestlet," a 5 cm square of pressed cotton batting (Ancare). Mice were placed individually into the nesting cages 1 hr. before the dark phase, and the results were assessed the next morning. Nest building was scored on a 5 point scale.

Score 1: The Nestlet was largely untouched (>90% intact).

Score 2: The Nestlet was partially torn up (50-90% remaining intact).

Score 3: The Nestlet was mostly shredded but often there was no identifiable nest site: < 50% of the Nestlet

Score 4: An identifiable, but flat nest < 90% of the Nestlet was torn up, the material was gathered into a flat nest with walls higher than the mouse height curled up on its side) on less than 50% of its circumference.

Score 5: A (near) perfect nest: >90% of the Nestlet was torn up, the nest was a crater, with walls higher than mouse body height on more than 50% of its circumference.

Fear conditioning

The dependent measure used in contextual fear conditioning was a freezing response following a pairing of an unconditioned stimulus (foot shock), with a conditioned stimulus, a particular context. Freezing is a species-specific response to fear, which has been defined as "absence of movement except for respiration". This may last for seconds to minutes depending on the strength of the aversive stimulus, the number of presentations, and the degree of learning achieved by the subject. Testing involved placing the animal in a novel environment (dark chamber), providing an aversive stimulus (a 1-sec electric shock, 0.2 mA, to the paws), and then removing it. Social Interaction

In the three-chambered sociability task, a subject mouse was evaluated for its exploration of a novel social stimulus (novel mouse). The three-chambered social approach task monitors direct social approach behaviors when a subject mouse is presented with the choice of spending time with either a novel mouse or an empty cup. Sociability is defined as the subject mouse spending more time in the chamber containing the mouse than in the empty chamber. Preference for social novelty is defined as spending more time in the chamber with the novel mouse. The apparatus is a rectangular three-chamber box, where each chamber measures 20 cm (length) x 40.5 cm (width) x 22 cm (height). Dividing walls are made from clear perplex, with small openings (10 cm width x 5 cm height) that allow access into each chamber. The three-chamber task was lit from below (10 lux). The mice were allowed to freely explore the three-chamber apparatus over three 10 min trials. During the trial one wire cup was placed upside down in one of the side chambers and a novel mouse was placed under another wire cup in the other side chamber (novel mouse stimulus), leaving the middle chamber empty. The location of the novel mouse across trials was counterbalanced to minimize any potential confound due to a preference for chamber location. The time spent exploring the novel mice was scored as exploration ratio.

Hyponeophaqia

Mice and rats cannot vomit, due to the tightness of the cardiac sphincter of the stomach, so to overcome the problem of potential food toxicity they have evolved a strategy of first ingesting only very small amounts of novel substances. The amounts ingested then gradually increase until the animal has determined whether the substance is safe and nutritious. Hyponeophagia is used in the behavioural laboratory as a way of measuring anxiety. A highly palatable but novel substance, such as sweet corn, nuts or sweetened condensed milk, is offered to the mice in a novel situation, such as a new cage. The latency to consume a defined amount of the new food is then measured.

The apparatus is a white Perspex base (30 cm²) to which is glued a food well 1.2 cm in diameter, 0.9 cm high, filled with condensed milk. The small arena focuses the attention of the mouse on to the well and the condensed milk, so minimizing the effects of treatments that might affect perception of, or orientation to, the food source. Each white box is occupied by a mouse.

The mice are put into new individual temporary holding cages and allowed 20 min in the new cage to adapt. Individual housing prevents social transmission of food preferences. Full cream sweetened condensed milk diluted 50:50 with water is used as the novel food. Mice are food-restricted overnight: remove all food the evening before (also put down clean new bedding as they are coprophagic) then give 1 g/mouse, which is approximately a third of what they would normally eat. The mice are tested the next morning.

The mouse is placed facing away from the well. The latency from being placed in the apparatus to start properly drinking is taken. Sniffs at the milk do not count. Proper drinking is defined as drinking continuously. The test parameter is the latency to drink. The assay is performed between 8 a.m. and 4 p.m.

Statistical Analysis of Behavioral Data

Data were analyzed by one-way ANOVA with a Dunnett's multiple comparison post hoc test in which WT Vehicle group was used as reference and compared with all the other groups.

Interpretation of the statistics:

• NS: The means of WT group vs any other group are not significantly different therefore the treatment equalizes the levels to WT (p > 0.05)

• WT group and any other group means are statistically different therefore the treatment is not enough to equalize to WT levels

• * P < 0.05

• ** P < 0.01

• *** P < 0.001

• **** P < 0.0001

Table 1:

Table 2:

In A, an exploration ratio was used, where the FMR1 mice had a significantly lower exploration ratio than the WT. In B, the time spent with the novel mouse, "NM", vs a familiar mouse, "FM", is represented; in this test the FMR1 KO mice spend the same amount of time with a novel and the familiar mouse while the WT mice spend a significant higher amount of time with the novel mouse vs the familiar one. In B, the time spent with the novel mouse was used to establish the difference in KO FMR1 mice treated with drugs vs the vehicle control group.

Conclusions

From Figure 1 and Table 1, it can be seen that: metformin alone at 50 mg/kg, sertraline alone at 1 mg/kg, and metformin at 50 mg/kg in combination with sertraline at 1 mg/kg did not rescue open field; sertraline alone at 5 mg/kg significantly rescued open field; and, metformin at 50 mg/kg in combination with sertraline at 5 mg/kg fully rescued open field (showing that metformin did not negatively interfere with the effect observed for sertraline alone at 5 mg/kg). From Figure 2 and Table 1, it can be seen that: metformin alone at 50 mg/kg and sertraline alone at 1 mg/kg did not rescue nesting; sertraline alone at 5 mg/kg significantly rescued nesting; and, metformin at 50 mg/kg in combination with sertraline at 1 mg/kg, and metformin at 50 mg/kg in combination with sertraline at 5 mg/kg fully rescued nesting (showing a synergistic effect when compared to the individual drugs or the vehicle for the lower dose of sertraline at 1 mg/kg, and that metformin did not negatively interfere with the effect observed for sertraline alone at 5 mg/kg).

From Figure 3 and Table 1, it can be seen that: metformin alone at 50 mg/kg and sertraline alone at 1 mg/kg did not rescue fear conditioning; sertraline alone at 5 mg/kg significantly rescued fear conditioning; and, metformin at 50 mg/kg in combination with sertraline at 1 mg/kg, and metformin at 50 mg/kg in combination with sertraline at 5 mg/kg fully rescued fear conditioning (showing a synergistic effect when compared to the individual drugs or the vehicle for the lower dose of sertraline at 1 mg/kg, and that metformin did not negatively interfere with the effect observed for sertraline alone at 5 mg/kg).

From Figure 4 and Table 2, it can be seen in test A that: sertraline alone at 1 mg/kg, and sertraline alone at 5 mg/kg did not rescue sociability. From Figure 4 and Table 2, it can be seen in test B that: metformin alone at 50 mg/kg, and metformin at 50 mg/kg in combination with sertraline at 1 mg/kg did not rescue sociability; and, metformin at 50 mg/kg in combination with sertraline at 5 mg/kg fully rescued sociability (showing a synergistic effect for the combination doses when compared to the individual drugs or the vehicle).

From Figure 5 and Table 1, it can be seen that: metformin alone at 50 mg/kg, sertraline alone at 1 mg/kg, and sertraline alone at 5 mg/kg did not rescue hyponeophagia; and metformin at 50 mg/kg in combination with sertraline at 1 mg/kg, and metformin at 50 mg/kg in combination with sertraline at 5 mg/kg fully rescued hyponeophagia (showing a synergistic effect for both sets of combination doses when compared to the individual drugs or the vehicle).

Claims

1. A composition comprising:

(iii) sertraline, or a pharmaceutically acceptable salt thereof; and

(iv) metformin, or a pharmaceutically acceptable salt thereof.

2. The composition according to claim 1, comprising 10 mg to 300 mg of sertraline, preferably 15, 20, 25, 30, or 40 mg to 250, 200, 150, or 100 mg of sertraline.

3. The composition according to claim 1 or claim 2, comprising 100 mg to 2000 mg of metformin, preferably 150, 200, 250, 300, or 350 mg to 1800, 1600, 1400, 1200, or 1000 mg of metformin.

4. A kit comprising:

(iii) at least one dose of sertraline, or a pharmaceutically acceptable salt thereof; and

(iv) at least one dose of metformin, or a pharmaceutically acceptable salt thereof.

5. The kit according to claim 4, further comprising any of the preferred features of claim 2 or claim 3.

6. A composition comprising:

(iii) sertraline, or a pharmaceutically acceptable salt thereof; and

(iv) metformin, or a pharmaceutically acceptable salt thereof for use in the treatment of Fragile X syndrome.

7. The composition according to claim 6, wherein the patient is exhibiting signs of hyperactivity, social anxiety, disruptive behaviour, or memory impairment.

8. The composition according to claim 6 or 7, wherein administration of the treatment is by a single daily dose.

9. The composition according to claim 8, wherein the single daily dose comprises 10 mg to 300 mg of sertraline, preferably 15, 20, 25, 30, or 40 mg to 250, 200, 150, or 100 mg of sertraline.

10. The composition according to claim 8 or claim 9, wherein the single daily dose comprises 100 mg to 2000 mg of metformin, preferably 150, 200, 250, 300, or 350 mg to 1800, 1600, 1400, 1200, or 1000 mg of metformin.

11. The composition according to claim 6 or 7, wherein administration of the treatment is by a dose twice daily.

12. The composition according to claim 11, wherein the daily doses comprise 5 mg to 150 mg of sertraline, preferably 7.5, 10, 12.5, 15, or 20 mg to 125, 100, 75, or 50 mg of sertraline.

13. The composition according to claim 11 or claim 12, wherein the daily doses comprise 50 mg to 1000 mg of metformin, preferably 75, 100, 125, 150, or 175 mg to 900, 800, 700, 600, or 500 mg of metformin.

14. A kit comprising:

(iv) at least one dose of metformin, or a pharmaceutically acceptable salt thereof, for simultaneous, separate, or sequential use in the treatment of Fragile X syndrome.

15. The kit according to claim 14, having the additional features of claims 7 to 13.

16. The composition or kit according to any of claims 6 to 15, further comprising any of the preferred features of claim 2 or claim 3.

17. A method of treating Fragile X syndrome comprising administering to the patient simultaneously, separately, or sequentially:

(iii) at least one dose of sertraline, or a pharmaceutically acceptable salt thereof; and (iv) at least one dose of metformin, or a pharmaceutically acceptable salt thereof.

18. The method according to claim 17, having the additional features of claims 7 to 13.

19. Use of sertraline, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a patient with Fragile X syndrome, wherein the patient has been administered metformin, or a pharmaceutically acceptable salt thereof.

20. Use of metformin, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a patient with Fragile X syndrome, wherein the patient has been administered sertraline, or a pharmaceutically acceptable salt thereof.

21. Use of sertraline, or a pharmaceutically acceptable salt thereof, and metformin, or a pharmaceutically acceptable salt thereof, in combination for the manufacture of a medicament for the treatment of a patient with Fragile X syndrome.