WO2021019214A1

WO2021019214A1 - Treatment of pitt-hopkins syndrome

Info

Publication number: WO2021019214A1
Application number: PCT/GB2020/051764
Authority: WO
Inventors: David Brown
Original assignee: Healx Ltd
Priority date: 2019-07-26
Filing date: 2020-07-23
Publication date: 2021-02-04
Also published as: GB201910755D0

Abstract

The present invention relates to compositions and kits comprising: (i) minocycline, or a pharmaceutically acceptable salt thereof; and (ii) Compound A, or a pharmaceutically acceptable salt thereof, wherein Compound A is selected from the following list: amitriptyline, sulindac, ibudilast, cannabidiol, prochlorperazine, iamotrigine, topiramate, sertraline, amantadine, bumetanide, nicardipine and verapamil. The compositions and kits are useful in the treatment of Pitt-Hopkins syndrome.

Description

TREATMENT OF RPT-HOPKINS SYNDROME

FIELD OF THE INVENTION

This invention relates to a kit, composition and combination therapy comprising minocycline for use in the treatment of Pitt-Hopkins syndrome (PTHS). BACKGROUND OF THE INVENTION

Pitt-Hopkins syndrome is a rare, genetic neurological disorder caused by a molecular variant of TCF4 which is involved in embryologic neuronal differentiation.

PTHS is characterised by distinctive facial features (syndromic facies), developmental delay (psychomotor delay), intellectual disability, early-onset myopia, seizures, constipation and breathing abnormalities (hyperventilation- apneic spells i.e. recurrent episodes where they breathe very fast, often followed by episodes where they struggle to breathe or momentarily stop breathing) and low muscle tone (hypotonia). Further symptoms include repetitive nonfunctional hand movements and behavioural abnormalities such as hyperactivity and anxiety. Many affected individuals meet criteria for autism spectrum disorder.

PTHS is caused by a pathogenic variant of the TCF4 gene found on chromosome 18q21.2. The syndrome was first described in 1978 in two unrelated individuals who shared similar characteristics of dysmorphic facial features, developmental delay, clubbed fingers, and an abnormal breathing pattern. It was presumed to be an autosomal recessive disorder until 2007 when the TCF4 gene, (MIM #602272) was identified, supporting an autosomal dominant inheritance pattern secondary to haploinsuffiency of TCF4. Transcription factor 4, the protein product of TCF4, is a basic helix-loop-helix E-protein believed to be involved in early brain development and neuronal differentiation.

At present there is no effective therapy to treat PTHS. There is a need for therapies for the treatment of PTHS.

Minocycline is a tetracycline antibiotic used in the treatment of bacterial infections and acne vulgaris. Minocycline has the systematic name (4~{S},4~{a}~{S},5~{a}~{R},12~{a}~{R})-4,7-bis(dimethylamino)-

1,10, 11, 12~{a}-tetra hydroxy-3, 12-dioxo-4~{a>, 5, 5~{a},6-tetrahyd ro-4~{H>- tetracene-2-carboxamide. SUMMARY OF THE INVENTION

The present invention is based on in vivo data. The below listed compounds have been identified as being useful in the treatment of Pitt-Hopkins syndrome, when used in combination with minocycline. This is based on the below in vivo data. In a first aspect of the invention, there is provided a composition comprising:

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

(ii) Compound A, or a pharmaceutically acceptable salt thereof, wherein Compound A is selected from the following list: amitriptyline, sulindac, ibudilast, cannabidiol, prochlorperazine, lamotrigine, topiramate, sertraline, amantadine, bumetanide, nicardipine and verapamil.

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

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

(ii) at least one dose of Compound A, or a pharmaceutically acceptable salt thereof, wherein Compound A is selected from the following list: amitriptyline, sulindac, ibudilast, cannabidiol, prochlorperazine, lamotrigine, topiramate, sertraline, amantadine, bumetanide, nicardipine and verapamil.

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

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

(ii) Compound A, or a pharmaceutically acceptable salt thereof, for use in the treatment of Pitt-Hopkins syndrome,

wherein Compound A is selected from the following list: amitriptyline, sulindac, ibudilast, cannabidiol, prochlorperazine, lamotrigine, topiramate, sertraline, amantadine, bumetanide, nicardipine and verapamil.

In a fourth aspect of the invention, there is provided a kit comprising: (i) at least one dose of minocycline, or a pharmaceutically acceptable salt thereof; and

(ϋ) at least one dose of Compound A, or a pharmaceutically acceptable salt thereof, for simultaneous, separate or sequential use in the treatment of Pitt-Hopkins syndrome, wherein Compound A is selected from the following list: amitriptyline, sulindac, ibudilast, cannabidiol, prochlorperazine, lamotrigine, topiramate, sertraline, amantadine, bumetanide, nicardipine and verapamil. DESCRIPTION OF THE FIGURES

Figure 1 shows the results from minocycline in vivo dose escalation testing.

Figure 2 shows the results from amitriptyline in vivo dose escalation testing.

Figure 3 shows the results from prochlorperazine in vivo dose escalation testing.

Figure 4 shows the results from minocycline and amitriptyline in vivo testing. Figure 5 shows the results from minocycline and prochlorperazine in vivo testing.

Figure 6 shows the results from sulindac in vivo testing.

Figure 7 shows the results from amantadine in vivo testing.

Figure 8 shows the results from verapamil in vivo testing.

Figure 9 shows the results from nicardipine in vivo testing. Figure 10 shows the results from bumetanide in vivo testing.

In the Figures, 'ns' means no significant difference from Wild Type control group. This indicates that the drug-treated Knock Out is behaving like the untreated or treated Wild Type group in the behavioural test, meaning the compound was significantly effective in ameliorating the syndrome's phenotype. WT group and any other group means are statistically different therefore the treatment is not enough to equalize to WT levels

DETAILED DESCRIPTION

There are a number of different manifestations and symptoms in patients with Pitt-Hopkins syndrome. These include: hyperactivity and repetitive behaviour including repetitive nonfunctional hand movements; intellectual impairment, such as difficulties with learning and memory e.g. difficulties with cognitive, executive and language performance, short-term memory, executive function, visual memory, visual-spatial relationships and spatial working memory; stereotypy; social anxiety (i.e. difficulties in social interaction); low muscle tone (hypotonia); constipation, sleep disturbances, seizures; irregular or abnormal breathing patterns and severe nearsightedness (myopia).

In the present invention, and as demonstrated by the below in vivo data, minocycline and Compound A are used to treat one or more of the above symptoms, and are therefore an effective treatment of PTHS. Preferably, minocycline and Compound A are used for the treatment of PTHS, wherein the patient is exhibiting typical symptoms of the syndrome including hyperactivity; social anxiety; intellectual impairment, specifically difficulties with learning and memory; stereotypy; and hypotonia. The term "intellectual impairment" has its normal meaning in the art. It encompasses impairment in learning and memory. Learning impairment may also be called intellectual disability. It encompasses cognitive impairment, delay or limitations in intellectual functions, such as reasoning. Memory impairment 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 (i.e. the temporary storage of information while processing the same or other information) and difficulties with phonological memory (or verbal working memory). This symptom was tested in mice under "fear conditioning" (see in vivo data below).

The term "hyperactivity" has its normal meaning in the art. Hyperactivity may include having very short attention spans, hypersensitivity to visual, auditory, tactile, and olfactory stimuli, distractibility, impulsiveness, restlessness and/or over-activity. This symptom was tested in mice under "open field" (see in vivo data below).

The term "test of daily living" has its normal meaning in the art. It may also mean ability to perform the things normal to a species including any daily activity we perform such as bedding, feeding etc. This symptom was tested in mice under "nesting" (see in vivo data below).

The term "stereotypy" has its normal meaning in the art. It may also be termed as repetitive movements or repetitive behaviour. This symptom was tested in mice under "self-grooming" (see in vivo data below). 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. This symptom was tested in mice under "sociability" (see in vivo data below).

The term "hypotonia" has its normal means in the art. This symptom was test in mice under "test of force". The term "force" has its normal meaning in the art. It may also mean the strength or energy put into an action.

In a preferred embodiment, Compound A is selected from: amitriptyline, prochlorperazine, sulindac, amantadine, verapamil, nicardipine, topiramate, sertraline, cannabidiol and bumetanide.

In a preferred embodiment, Compound A is selected from: amitriptyline, prochlorperazine, sulindac, amantadine, verapamil, nicardipine, and bumetanide.

In a preferred embodiment, Compound A is amitriptyline or prochlorperazine. Most preferably, Compound A is amitriptyline.

Amitriptyline is a tricyclic antidepressant (TCA).

Sulindac is a non-steroidal anti-inflammatory drug (NSAID) used in the treatment of acute and chronic inflammatory conditions, such as arthritis, shoulder bursitis and tendonitis. Ibudilast is an anti-inflammatory and neuroprotective agent. It is commonly used in the treatment of asthma and stroke. Cannabidiol is a phytocannabinoid.

Prochlorperazine is a phenothiazine with antiemetix, antipsychotic, antihistaminic and anticholinergic activities used in the treatment of nausea, vomiting and vertigo.

Lamotrigine is a synthetic phenyltriazine which is used as an antiseizure medication. It is commonly used to treat epilepsy and bipolar disorder.

Topiramate is a sulfamate modified fructose diacetonide used in the treatment of epilepsy.

Sertraline is a selective serotonin re-uptake inhibitor (SSRI) used in the treatment of major depressive disorder.

Amantadine is a synthetic tricyclic amine with antiviral, antiparkinsonian, and antihyperalgesic activities.

Bumetanide is a potent sulfa moyla nth ranilic acid derivative belonging to the class of loop diuretics. Nicardipine is a second generation calcium channel blocker used in the treatment of hypertension and stable angina pectoris.

Verapamil is a phenylalkylamine calcium channel blocking agent.

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, fu marie, 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 transderma I 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.

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, 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 Pitt-Hopkins syndrome. An effective dose will be apparent to one skilled in the art, and is dependent on a number of 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 500 mg of minocycline.

In a preferred embodiment, the compositions according to the present invention comprise 1 mg to 500 mg of Compound A.

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 is 10 mg to 500 mg per day of minocycline and 1 mg to 500 mg per day of Compound A.

In an embodiment, the compositions of the invention are administered twice a day. Preferably each dose comprises 5 mg to 250 mg of minocycline and 0.5 mg to 250 mg of Compound A.

In order to treat Pitt-Hopkins syndrome, minocycline and Compound A are used in a chronic dosage regime i.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 a number of 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 i.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 i.e. minocycline and Compound A are taken together or formulated as a single composition. In this embodiment, most preferably, it is formulated as a single composition. Preferably the single daily dose comprises of 10 mg to 500 mg of minocycline and 1 mg to 500 mg of Compound A. It may also be administered sequentially i.e. 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 i.e. minocycline and Compound A 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. Preferably each dose comprises 5 mg to 250 mg of minocycline, with a total daily dosage of minocycline of 10 mg to 500 mg. Preferably each daily dose comprises 0.5 mg to 250 mg of Compound A, with a total daily dosage of 1 mg to 500 mg. Each daily dose may also be administered sequentially i.e. minocycline and Compound A 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 Pitt-Hopkins 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 minocycline, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a patient with Pitt-Hopkins syndrome, wherein the patient has been administered with Compound A 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 Compound A, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a patient with Pitt-Hopkins syndrome, wherein the patient has been administered with minocycline or a pharmaceutically acceptable salt thereof.

STUDY

Pitt-Hopkins syndrome (PTHS) is characterized by cognitive dysfunction, wide mouth and distinctive facial features, and intermittent hyperventilation followed by apnoea (Zweier et a!., 2007). The cognitive dysfunction associated with the loss of one copy of the TCF4 gene, termed haplo-insufficiency, in humans leads to Pitt-Hopkins syndrome, an autism-related disorder associated with pronounced learning deficits.

Pitt-Hopkins syndrome (PTHS) is a genetic neurodevelopmental disorder associated with transcription factor TCF4 mutations/deletions. TCF4 may also be linked to schizophrenia, suggesting that the precise pathogenic mutations are relevant to cellular, synaptic, and behavioural consequences.

The Tcf4 +/- mouse model has been developed to mimic PTHS and therefore assess potential treatments. The mice have deficits in hippocampus-dependent learning and memory, spatial working memory, sociability, stereotypy, hyperactivity and daily living paradigms. The Tcf4 +/- mice also demonstrate hindlimb grip strength deficits. Animals

Mice were purchased from The Jackson Laboratory and maintained on a C57BL/6 background. They were raised on a 12: 12 light:dark cycle with ad libitum access to food and water. Controls consisted of TCF4 +/+. All mice used were heterozygous for the Tcf4 mutation because homozygous mutations result in embryonic /post-partum lethality.

Assay design

Experiments were randomised and blind to the genotype and treatment during all testing and data analysis. Separate investigators prepare and coded dosing solutions, allocate the mice to the study treatment groups, dosed the animals, and collect the behavioural data.

Treatment Groups

In some experiments, there were four treatment groups per compound in the study: Group 1: wild-type littermate mice treated with vehicle (WT + veh); Group 2: Tcf4 +/- KO mice treated with vehicle (Tcf4 +/- + veh), Group 3: WT + drug; and Group 4: Tcf4 +/- + drug.

In other experiments, there were the following treatments groups: (i) wild-type littermate mice treated with vehicle (WT-veh); (ii) Tcf4 +/- KO mice treated with vehicle (KO-veh); (iii) KO + drug (in various doses), (iv) KO + drug combination

This included:

1. Fear conditioning (a test of learning and memory);

2. Open field (a measure of hyperactivity)

3. Nesting (a test of daily living)

4. Self-grooming (an assessment of stereotypy).

5. Sociability/Partition test (an assessment of social anxiety)

6. Test of Force (a test of hind limb strength).

The term "learning and memory" 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 (i.e. the temporary storage of information while processing the same or other information) and difficulties with phonological memory (or verbal working memory).

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

The term "test of daily living" has its normal meaning in the art. It may also mean ability to perform the things normal to a species including any daily activity we perform such as bedding, feeding etc.

The term 'self-grooming' has its normal meaning in the art. It may also mean selfcleaning and maintaining normal appearance of skin, hair, fur etc. Carried to excess via persistent repetition of an act it may be referred to as 'stereotypy'.

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 "force" has its normal meaning in the art. It may also mean the strength or energy put into an action.

1. Fear Conditioning

Tcf4 +/- mice show reduced freezing in the context of clued fear conditioning test indicating hippocampus-dependent memory and learning deficiency. 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.

2. Open Field

The open-field apparatus was used to test hyperactivity. Tcf4+/- mice show hyperactivity when compared to WT littermates in distance travelled per minute during a 30 minutes trial Open Field. The apparatus was a grey PVC-enclosed arena 50x9x30 cm divided into a lOxlOcm 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 3min. The number of squares entered by the whole body (locomotor activity) were counted. The movement of the mouse around the field was recorded with a video tracking device for 3min (version NT4.0, Viewpoint).

3. 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 tom 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.

4. Self-oroomino Tcf4 +/- mice groom themselves significantly more than WT mice indicating higher levels of stereotype behaviour than control mice. Usually in a sitting position, the mouse will lick its fur, groom with the forepaws, or scratch with any limb. Often the mouse will mix all of these grooming behaviors. Grooming typically follows a sequence of four behaviors:

Elliptical Stroke: Elliptical asymmetric movements of the forepaws over the nose and muzzle, alternating the major and minor paw.

Unilateral Stroke: Alternating strokes of the forepaw across the vibrissae and the eye.

Bilateral Stroke: Large symmetric bilateral strokes of the forepaws that begin behind the ears and pass over the whole face.

Body Licking: Licking of the whole body, typically beginning rostrally and working caudally to the tail.

5. Partition test

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 behaviours 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.

6. Test of Force

Tcf4+/- mice show a significant deficit in hindlimb strength (not forelimb) when compared with WT mice. Neuromuscular function of the hindlimbs was tested with a grip strength meter (San Diego Instruments). Mice were scruffed by the back of the neck, held by the tail, and lifted into an upright position. Then, the mice were lowered toward the apparatus, allowed to grasp the smooth metal grid (hindlimbs only), and pulled backwards in the horizontal plane. The force applied to the grid at the moment the grasp was released was recorded.

Statistical Analysis of Behavioural Data

Data were analysed by two-way analysis of variance (ANOVA) followed by posttest comparisons where appropriate using Tukey's Multiple Comparison Test. Data are represented as the mean and standard error of the mean (SEM).

Conclusion

In the experiments of Figure 1, a dose escalation study was carried out, decreasing the concentration of the minocycline to determine the dose at which it is no longer effective for all or the majority of behavioral tests. At doses of 15 mg/kg or 7 mg/kg, minocycline was no longer effective across all behavioural tests. However, at 30 mg/kg, minocycline was effective for all behavioural tests.

In the experiments of Figure 2, a dose escalation study was carried out, decreasing the concentration of the amitriptyline to determine the dose at which it is no longer effective for all or the majority of behavioral tests. At a dose of 2.5 mg/kg, amitriptyline was no longer effective across all behavioural tests. However, at 5 mg/kg and 10 mg/kg, amitriptyline was effective for almost all behavioural tests.

In the experiments of Figure 3, a dose escalation study was carried out, decreasing the concentration of the prochlorperazine to determine the dose at which it is no longer effective for all or the majority of behavioral tests. At doses of 0.8 mg/kg or 0.4 mg/kg, prochlorperazine was no longer effective across all behavioural tests. However, at 1.5 mg/kg, prochlorperazine was effective for all behavioural tests.

In the experiments of Figure 4, ineffective dosage amounts of minocycline (15 mg/kg) and amitriptyline (2.5 mg/kg) were combined. The combination fully rescued all behavioural tests, showing a synergistic interaction. In the experiments of Figure 5, ineffective amounts of minocycline (15 mg/kg) and prochlorperazine (0.4 mg/kg) were combined. The combination fully rescued the nesting behavioural test, showing a synergistic interaction. In the experiments of Figure 6, treatment with sulindac rescued fear conditioning, nesting and self-grooming behavioural tests.

In the experiments of Figure 7, treatment with amantadine fully or partially rescued all behavioural tests. In the experiments of Figure 8, treatment with verapamil fully rescued all behavioural tests.

In the experiments of Figure 9, treatment with nicardipine fully or partially rescued all behavioural tests.

In the experiments of Figure 10, treatment with bumetanide fully or partially rescued all behavioural tests, except test of force.

Claims

1. A composition comprising:

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

(ii) Compound A, or a pharmaceutically acceptable salt thereof, wherein Compound A is selected from the following list: amitriptyline, sulindac, ibudilast, cannabidiol, prochlorperazine, lamotrigine, topi ra mate, sertraline, amantadine, bumetanide, nicardipine and verapamil.

2. The composition according to claim 1, wherein Compound A is selected from amitriptyline, prochlorperazine, sulindac, amantadine, verapamil, nicardipine, topiramate, sertraline, cannabidiol and bumetanide.

3. The composition according to claim 1 or 2, wherein Compound A is selected from amitriptyline, prochlorperazine, sulindac, amantadine, verapamil, nicardipine, and bumetanide.

4. The composition according to any preceding claim, wherein Compound A is selected from amitriptyline or prochlorperazine, most preferably amitriptyline.

5. The composition according to any preceding claim, comprising 10 mg to 500 mg of minocycline.

6. The composition according to any preceding claim, comprising 1 mg to 500 mg of Compound A.

7. A kit comprising:

8. The kit according to claim 7, further comprising any of the preferred features of claims 2 to 6.

9. A composition comprising:

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

(ii) Compound A, or a pharmaceutically acceptable salt thereof, for use in the treatment of Pitt-Hopkins syndrome, wherein Compound A is selected from the following list: amitriptyline, sulindac, ibudilast, cannabidiol, prochlorperazine, lamotrigine, topi ra mate, sertraline, amantadine, bumetanide, nicardipine and verapamil.

10. The composition according to claim 9, wherein the patient is exhibiting signs of intellectual impairment, hyperactivity, stereotypy, social anxiety and/or hypotonia.

11. The composition according to claim 9 or 10, wherein administration is by a single daily dose.

12. The composition according to claim 11, wherein the single daily dose comprises 10 mg 500 mg of minocycline and/or 1 mg to 500 mg of Compound A.

13. The composition according to claim 9 or 10, wherein administration is a by a dose twice daily.

14. The compound according to claim 13, wherein the twice daily dose comprises 5 mg to 250 mg of minocycline and 0.5 mg to 250 mg of Compound A.

15. A kit comprising:

(ii) at least one dose of Compound A, or a pharmaceutically acceptable salt thereof, for simultaneous, separate or sequential use in the treatment of Pitt-Hopkins syndrome, wherein Compound A is selected from the following list: amitriptyline, sulindac, ibudilast, cannabidiol, prochlorperazine, lamotrigine, topiramate, sertraline, amantadine, bumetanide, nicardipine and verapamil.

16. The kit according to claim 15, having any of the additional features of claims 9 to 14.

17. The composition or kit according to any of claims 9 to 16, further comprising any of the preferred features of claims 2 to 4.

18. A method of treating Pitt-Hopkins syndrome comprising administering the patient simultaneously, separately or sequentially with:

19. Use of minocycline, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a patient with Pitt-Hopkins syndrome, wherein the patient has been administered with Compound A, or a pharmaceutically acceptable salt thereof, selected from the following list: amitriptyline, sulindac, ibudilast, cannabidiol, prochlorperazine, lamotrigine, topiramate, sertraline, amantadine, bumetanide, nicardipine and verapamil.

20. Use of Compound A, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a patient with Pitt-Hopkins syndrome, wherein the patient has been administered with minocycline, or a pharmaceutically acceptable salt thereof, wherein Compound A is selected from the following list: amitriptyline, sulindac, ibudilast, cannabidiol, prochlorperazine, lamotrigine, topiramate, sertraline, amantadine, bumetanide, nicardipine and verapamil.