DISEASE

FIELD OF THE INVENTION

The present invention relates to compounds for use in the treatment and/or prevention of Niemann-Pick C disease.

BACKGROUND OF THE INVENTION

Niemann-Pick C disease, also known as Niemann-Pick disease type C, Niemann-Pick C or NPC, is a fatal autosomal recessive neurodegenerative disease that predominantly affects children. NPC is caused by autosomal recessive mutations in the NPC1 or NPC2 gene, encoding two distinct lysosomal cholesterol-binding proteins, and is linked to dysregulation of intracellular lipid trafficking.

NPC predominantly affects children. Depending on the age of onset, NPC can be categorized as: perinatal (onset at age less than 3 months, including prenatal onset), early- infantile (onset at age from 3 months to less than 2 years), late-infantile (onset at age from 2 years to less than 6 years), juvenile (onset at ages from 6 to 15 years) and adolescent/adult (onset at an age of more than 15 years).

At the cellular level, mutations in NPCl and NPC2 genes profoundly affect the intracellular trafficking of cholesterol and other lipids, and as a consequence lead to lysosomal accumulation of multiple lipid species. Lipids such as glycosphingolipids are generally endocytosed and targeted to the Golgi apparatus, but are mistargeted to lysosomes in NPC [te Vruchte, D. et al, Journal Biological Chemistry , 2004, 279(25), 26167-26175] In patients with NPC, affected cells and tissues are characterized by intralysosomal accumulation of cholesterol, glycosphingolipids, sphingosine, sphingomyelin and lyso(bis)phospatidic acid (LBPA) and present with defects in both lysosomal Ca ²⁺ signaling and endocytosis of lipids and fluid phase material. Thus, enlarged lysosomes are a common cellular phenotype for NPC. This leads to the abnormal accumulation of these lipids within various tissues of the body, including brain, damaging the affected areas.

NPC affects neurologic and psychiatric functions, as well as various internal organs (visceral). Symptoms vary with age of onset and from patient to patient and may include one or more of jaundice at (or shortly after) birth, cholestasis, enlarged spleen and/or liver (hepatosplenomegaly), difficulty with upward and downward eye movements (vertical supranuclear gaze palsy or VSGP), unsteadiness of gait, clumsiness, problems in walking (ataxia), lack of accuracy in voluntary movements (dysmetria), difficulty in posturing of limbs (dystonia), slurred, irregular speech (dysarthria), learning difficulties and progressive intellectual decline (cognitive dysfunction/dementia), psychiatric disorders, sudden loss of muscle tone which may lead to falls (gelastic cataplexy), tremors accompanying movement and, in some cases, seizures, and swallowing problems (dysphagia).

Visceral symptoms are more typically seen in individuals presenting at a younger age. Neurologic and psychiatric symptoms often occur slowly over time, and thus feature more prominently in individuals presenting in the later age groups. Thus, patients with the perinatal and early-infantile forms tend to present with visceral symptoms, cholestasis and development delay, while those with juvenile- and adolescent/adult-onset forms may present with a wide range of neurological manifestations. Hepatosplenomegaly is frequently present in NPC. However, its absence does not exclude the diagnosis, particularly in late-onset cases. Typical neurological manifestations include cerebellar ataxia, dysmetria, dysarthria, and dysphagia. Vertical supranuclear gaze palsy (VSGP), and gelastic cataplexy are characteristic neurological signs. In addition, patients with adult-onset disease often present with neuropsychiatric signs including early cognitive decline and/or psychiatric disturbances. Death from symptoms associated with the disease usually occurs during the second decade of life.

A major influence on the progression and prognosis of NPC is the age of onset of neurological manifestations, which may be broadly grouped as follows: • Pre/perinatal. Neurological involvement is not shown during the neonatal period. Visceral manifestations include neonatal cholestasis. Liver failure occurs in approximately 10 % of patients, with death usually occurring before the age of 6 months. Severe respiratory insufficiency (together with hepatosplenomegaly or more severe liver disease) may present in some infants and may also be fatal.

• Early-infantile (onset at 2-3 months to <2 years of age). Most patients present hepatosplenomegaly. Neurological involvement may initially include delays in motor milestones and hypotonia.

• Late-infantile (onset at 2 to <6 years of age). Most patients present hepatosplenomegaly. Neurological involvement may initially include gait problems, clumsiness, speech delay, cataplexy and VSGP.

• Juvenile (onset at 6-15 years of age). A moderate hepatosplenomegaly is frequent. Neurological involvement may initially include VSGP, school problems, ataxia and sometimes seizures and cataplexy. This is the most common form of the disease in many countries.

• Adolescent/adult (onset at >15 years of age). Absence of hepatosplenomegaly is frequent. Neurological involvement may initially include VSGP, ataxia and dystonia. Patients may also display dementia and psychiatric illnesses.

At present there is only one therapy approved in Europe, the lipid lowering drug miglustat (Zavesca™, Actelion Pharmaceuticals), although a number of experimental therapies including arimoclomol are in clinical trials for this disease.

Miglustat is an N-alkylated iminosugar, an analog of D-glucose that functions as a competitive and reversible inhibitor of the ceramide specific glucosyltransferase (glucosylceramide synthase, GCS), an early step in glycosphingolipid (GSL) biosynthesis. Despite not having a direct effect in cholesterol metabolism, inhibition of GCS by miglustat reduces the accumulation of GSLs in the lysosomes of all cells and delays the progression of neurological symptoms in NPC animal models and patients. Miglustat is also approved for the treatment of Gaucher disease type I and has been studied in other lysosomal storage diseases suggesting that reducing glycolipid accumulation is an accepted therapeutic strategy for lysosomal diseases in which GSLs accumulate (e.g. the mucopolysaccharidoses). It has been reported that miglustat reverses the lipid-trafficking defects in Niemann-Pick C disease patient cells [Lachmann, R.H. et al, Neurobiology Disease , 2004, 16, 654-658; Xu, M. et al, Journal Biological Chemistry , 2012, 287 (47), 39349-39360] In this respect, Lysotracker staining showed that miglustat therapy caused a reduction of the volume of lysosomes in long-lived peripheral blood CD 19 positive B cells isolated from an NPC patient [Lachmann, R.H. et al, Neurobiology Disease , 2004, 16, 654-658] Lysotracker loads into lysosomes dependent on their volume and acidity (emits fluorescence below pH 5.5). In NPC disease lysosomal volume is larger owing to increased lipid storage. Also, increases of GM1 ganglioside have been measured biochemically in the NPC mouse [Lloyd-Evans et al., Nature Medicine , 2008, 14, 1247-1255], have been found in various regions of NPC mouse brain [Sugimoto, Y. et al., PNAS, 2001, 98(22), 12391-12396], as well as in spleen and liver [Fan, M. et al., Journal Lipid Research , 2013, 54 , 2800-2814], and intervention with miglustat in patients was shown to produce striking reductions in GM1 ganglioside plasma content [Fan, M. et al., Journal Lipid Research , 2013, 54 , 2800-2814] and in patient cells [Lachmann et al., Neurobiology of Disease, 2004, 16, 654-658] Treatment with miglustat is frequently associated with gastrointestinal side effects, including diarrhoea, due to inhibition of gut disaccharidase enzymes involved in carbohydrate metabolism. Tremors as well as weight loss are observed frequently, which should be closely monitored especially in pediatric patients [Lyseng-Williamson, Drugs , 2014, 74(1), 61-74] Despite its dose-limiting side effects and compliance issues, miglustat is the only treatment approved to reduce the progression of neurological symptoms in pediatric and adult NPC patients.

Arimoclomol is an experimental therapy that has shown beneficial effects reducing lysosomal volume in NPC patient fibroblasts, and improving neurological impairment in NPC mouse models (Kirkegaard, T. et al., Science Translational Medicine, 2016, 8(355), 355ral l8), and its effects in disease progression are currently being studied in clinical trials in NPC patients. Arimoclomol up-regulates molecular chaperones in cells, including HSP70 [Orphazyme. Technology: about arimoclomol. 2017; Available from: https://www.orphazyme.com/technology/]. In lysosomal storage diseases such as NPC, HSP70 protects lysosomal integrity, improves the activity of several enzymes crucial for lipid metabolism and helps to fold the defective proteins into a functional conformation, allowing the cell to process the accumulated lipids [Kirkegaard, T. et al., Science Translational Medicine, 2016, 8(355), 355ral l8; and Kirkegaard, T. et al Nature, 2010, 463, 549-554] HSP70 levels increased in patients treated with arimoclomol and the accumulation of disease-related lipids, such as unesterified cholesterol and cholesterol metabolite oxy sterol, was reduced [Orphazyme, Company announcement, No. 01/2019, https://www.orphazyme.com/news-feed/2019/l/30/orphazyme-repo rts-positive-results- from-full-data-set-of-phase-iiiii-arimoclomol-trial-in-niema nn-pick-disease-type-c-npc].

However, despite the promising results obtained in clinical trials, there is still a need for new therapies for Niemann-Pick C disease.

SUMMARY OF THE INVENTION

The inventors have surprisingly found new pharmacological strategies for the treatment of Niemann-Pick C disease. The compounds identified by the inventors have shown positive effects in in vitro assays on a Niemann-Pick C disease cell line.

Thus, in one aspect, the present invention relates to a compound selected from the group consisting of amrinone, nicainoprol and piboserod or pharmaceutically acceptable salts thereof, for use in the treatment and/or prevention of Niemann-Pick C disease.

In a second aspect, the present invention relates to a combination comprising two or more compounds selected from the group consisting of amrinone, nicainoprol, piboserod, olanzapine, miglustat, arimoclomol, N-acetylleucine, 2-hydroxypropyl-P-cyclodextrin, 2-hydroxypropyl-Y-cyclodextrin, vorinostat and ursodeoxycholic acid, or pharmaceutically acceptable salts thereof, for use in the treatment and/or prevention of Niemann-Pick C disease, wherein at least one compound is selected from the group consisting of amrinone, nicainoprol and piboserod or a pharmaceutically acceptable salt thereof. In a third aspect, the present invention relates to the use of a compound selected from the group consisting of amrinone, nicainoprol and piboserod or pharmaceutically acceptable salts thereof, for the treatment and/or prevention of Niemann-Pick C disease.

In a fourth aspect, the present invention relates to the use of two or more compounds selected from the group consisting of olanzapine, amrinone, nicainoprol, piboserod, miglustat, arimoclomol, N-acetyl-leucine, 2-hydroxypropyl-P-cyclodextrin, 2- hydroxypropyl-y-cyclodextrin, vorinostat and ursodeoxycholic acid, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment and/or prevention of Niemann-Pick C disease, wherein at least one compound is selected from the group consisting of amrinone, piboserod and nicainoprol or a pharmaceutically acceptable salt thereof.

In a fifth aspect, the present invention relates to a method for the treatment and/or prevention of Niemann-Pick C disease by administration to a patient in need thereof of a compound selected from the group consisting of amrinone, nicainoprol and piboserod or a pharmaceutically acceptable salt thereof.

In a sixth aspect, the present invention relates to a method for the treatment and/or prevention of Niemann-Pick C disease by administration to a patient in need thereof of a combination comprising two or more compounds selected from the group consisting of amrinone, nicainoprol, piboserod, miglustat, arimoclomol, N-acetyl-leucine, 2- hydroxypropyl-P-cyclodextrin, 2-hydroxypropyl-Y-cyclodextrin, vorinostat, olanzapine and ursodeoxycholic acid, or a pharmaceutically acceptable salt thereof, wherein at least one of the compounds is selected from the group consisting of amrinone, nicainoprol and piboserod or a pharmaceutically acceptable salt thereof.

DESCRIPTION OF THE FIGURES

Figures la and lb show the results of the Lysotracker Green screen using a spontaneously immortalized mouse astroglial cell line generated from the NPC mouse model. The results are expressed as arbitrary lysotracker green fluorescence units (RFU (495/515)) per well of 60,000 cells after 6 days of treatment with 50 mM miglustat (NPC + Migl) or amrinone at the indicated concentrations (Figure la), or after 3 days of treatment with 200 mM arimoclomol (NPC + Arim.), or piboserod and nicainoprol at the indicated concentrations (Figure lb). Wild type cells (WT) and untreated NPC cells (NPC UT) are included as controls in each experiment. Data is shown as average ± standard deviation of 6 replicates.

Figure 2 shows the results on cellular glycosphingolipid levels as the quantification of total grey area per cell corresponding to CtxB bound to ganglioside GM1, measured by thresholding using Fiji imaging software. WT (wild type cells), NPC (NPC null cells), NPC+Migl (NPC null cells treated with miglustat 50 mM during 6 days), Amrinone 5 mM (NPC null cells treated with amrinone 5 mM during 6 days)

Figure 3 shows the effect of compounds on increasing expression and lysosomal (punctuate) localization of endogenous HSP70. Top row: representative images of untreated Npc 1 ^+/+ (wild-type) and Npc l ⁷ (null) glia alongside the 200 pm arimoclomol treated Npcl ⁷ positive control (ART). Bottom three rows: Npcl ⁷ glia treated for 72 h with the indicated concentrations of piboserod and nicainoprol .

Figure 4 shows the effect of compounds on HSP70 membrane localization within lysosomal structures. Representative images of untreated Npcl ^+/+ (wild-type) and Npcl ^/_ (null) glia alongside Npcl ⁷ glia treated for 72 h with 200 pm arimoclomol treated Npcl ⁷ positive control (ART) and test compounds piboserod and nicainoprol . White arrows indicate “doughnut” like structures representing lysosome limiting membrane localization of HSP70.

DESCRIPTION OF THE INVENTION

In the first aspect, the present invention relates to a compound selected from the group consisting of amrinone, nicainoprol and piboserod or a pharmaceutically acceptable salt thereof, for use in the treatment and/or prevention of Niemann-Pick C disease. The invention also relates to the use of a compound selected from the group consisting of amrinone, nicainoprol and piboserod or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment and/or prevention of Niemann-Pick C disease.

The invention also relates to a method of treating and/or preventing Niemann-Pick C disease in a subject, comprising administering to said subject a therapeutically effective amount of a compound selected from the group consisting of amrinone, nicainoprol and piboserod or a pharmaceutically acceptable salt thereof.

The terms "treating" and “treatment”, as used herein, means reversing, alleviating, inhibiting the progress of Niemann-Pick C disease, or one or more symptoms of such disease or condition, preferably neurological symptoms.

The terms “preventing” and “prevention”, as used herein, means avoiding or inhibiting the onset of one or more symptoms of Niemann-Pick C disease, preferably neurological symptoms.

In a preferred embodiment, the Niemann-Pick C disease is selected from the group consisting of prenatal, perinatal, early-infantile, late-infantile, juvenile, adolescent and adult Niemann-Pick C disease.

Preferably, the compounds disclosed herein, are used for the treatment of Niemann-Pick C disease.

Olanzapine was developed as an antipsychotic agent and is currently used to treat schizophrenia and bipolar disorders. Olanzapine has the chemical structure depicted below. This compound is commercially available or may be synthesized using a suitable preparation method, such as that disclosed in US 5,229,382. Olanzapine can be used as the free base or as a pharmaceutically acceptable acid addition salt thereof, such as the pamoate, benzoate (including monobenzoate), hydrochloride (including mono- and dihydrochloride), sulphate (including hemi- and monosulphate), mesylate (including mono- and dimesylate), besylate (including mono- and dibesylate), lactate (including monolactate), maleate (including mono- and dimaleate), fumarate (including hemifumarate), acetate (including monoacetate), glycolate (including monoglycolate), tartrate (including mono- and ditartrate), malonate (including hemimalonate), succinate (including hemi succinate), adipate (including hemiadipate), tosylate, napsylate, mandelate, gluconate, ascorbate, and citrate salts, preferably it is used as the free base or as the pamoate salt, preferably as the free base.

Amrinone was developed as a pyridine phosphodiesterase 3 inhibitor, which has been used to improve the prognosis in patients with congestive heart failure. Amrinone is a positive inotopic cardiotonic agent that has been shown to increase the contractions initiated in the heart by stimulating calcium ion influx into the cardiac cell. Amrinone has the chemical structure depicted below.

This compound is commercially available or may be synthesized using a suitable preparation method, such as those disclosed in US 4,072,746 and US 4,107,315. Amrinone can be used as the free base or as a pharmaceutically acceptable acid addition salt thereof, such as the lactate salt. It is preferably used as the lactate salt.

Nicainoprol is an antiarrhythmic agent that has the chemical formula depicted below.

This compound is commercially available or may be synthesized using a suitable preparation method, such as that disclosed in US 4,335,123. Nicainoprol can be used as the free base or as a pharmaceutically acceptable acid addition salt thereof. It is preferably used as the free base.

Piboserod is a serotonin-4 receptor (5-HT4) antagonist that was developed for the treatment of congestive heart failure and has the chemical structure depicted below.

This compound is commercially available or may be synthesized using a suitable preparation method, such as that disclosed in WO 93/18036 Al. Piboserod can be used as the free base or as a pharmaceutically acceptable acid addition salt thereof, such as the hydrochloride salt. It is preferably used as the free base.

The term “pharmaceutically acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. Preferably, as used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

Further, the term “pharmaceutically acceptable salt” refers to any salt, which, upon administration to the recipient is capable of providing (directly or indirectly) a compound as described herein. Due to the nature of the chemical groups in the compounds for use according to the invention, the pharmaceutically acceptable salt of compounds provided herein may be acid addition salts, and they can be synthesized from the parent compound, which contains a basic moiety, by conventional chemical methods. Generally, such salts are, for example, prepared by reacting the free base forms of these compounds with a stoichiometric amount of the appropriate acid in water or in an organic solvent or in a mixture of the two. Generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. Examples of the acid addition salts include mineral acid addition salts such as, for example, hydrochloride, hydrobromide, hydroiodide, sulphate, nitrate, phosphate, and organic acid addition salts such as, for example, acetate, benzoate, maleate, fumarate, citrate, lactate, glycolate, gluconate, oxalate, pamoate, succinate, tartrate, malate, mandelate, malonate, adipate, ascorbate, benzenosulphonate (besylate), methanesulphonate (mesylate), p-toluenesulphonate (tosylate), naphthalenosulphonate (napsylate).

In an embodiment, the compound for use according to the invention is olanzapine or a pharmaceutically acceptable salt thereof, such as the pamoate, benzoate (including monobenzoate), hydrochloride (including mono- and dihydrochloride), sulphate (including hemi- and monosulphate), mesylate (including mono- and dimesylate), besylate (including mono- and dibesylate), lactate (including monolactate), maleate (including mono- and dimaleate), fumarate (including hemifumarate), acetate (including monoacetate), glycolate (including monoglycolate), tartrate (including mono- and ditartrate), malonate (including hemimalonate), succinate (including hemi succinate), adipate (including hemiadipate), tosylate, napsylate, mandelate, gluconate, ascorbate, and citrate salts; preferably olanzapine (free base) or olanzapine pamoate; more preferably olanzapine (free base).

In an embodiment of the various aspects of the present invention, the compound is amrinone or a pharmaceutically acceptable salt thereof, such as the lactate salt; preferably it is amrinone lactate.

In another embodiment of the various aspects of the present invention, the compound is nicainoprol or a pharmaceutically acceptable salt thereof; preferably it is nicainoprol (free base).

In another embodiment of the various aspects of the present invention, the compound is piboserod or a pharmaceutically acceptable salt thereof, such as the hydrochloride salt; preferably it is piboserod (free base).

The compounds for use according to the invention may be administered by any appropriate route (via), such as, oral (e.g., oral, sublingual, etc.), parenteral (e.g., subcutaneous, intramuscular, intravenous, intramuscular, lumbar intrathecal, intracerebroventricular, etc.), vaginal, rectal, nasal, topical, ophthalmic, etc., preferably oral or parenteral, more preferably oral or intravenous. In particular, oral administration is preferred for piboserod or a pharmaceutically acceptable salt thereof and for nicainoprol or a pharmaceutically acceptable salt thereof; whereas intravenous administration is preferred for amrinone or a pharmaceutically acceptable salt thereof.

In particular, the compounds for use according to the invention are administered as a pharmaceutical composition which comprises the corresponding (active) compound and one or more pharmaceutically acceptable excipients.

The term “pharmaceutically acceptable excipient” refers to a vehicle, diluent, adjuvant, support material, lubricant, filler, solvent, colorants, flavour conditioner, antioxidant, binder, adhesive, disintegrant, anti-adherent, glidants and/or agglutinant that is administered with the active ingredient. The pharmaceutically acceptable excipient necessary to manufacture the desired pharmaceutical composition of the invention will depend, among other factors, on the elected administration route. Said pharmaceutical compositions may be manufactured according to conventional methods known by the skilled person in the art.

The pharmaceutical compositions may be in oral form, either solid or liquid. Suitable dose forms for oral administration may be tablets, capsules, syrups or solutions and may contain conventional excipients known in the art such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example lactose, sugar, maize starch, calcium phosphate, sorbitol or glycine; tabletting lubricants, for example magnesium stearate; disintegrants, for example starch, polyvinylpyrrolidone, sodium starch glycollate or microcrystalline cellulose; or pharmaceutically acceptable wetting agents such as sodium lauryl sulfate. Solid oral compositions may be prepared by conventional methods of blending, filling or tabletting. Repeated blending operations may be used to distribute the active agent throughout those compositions employing large quantities of fillers. Such operations are conventional in the art. The tablets may for example be prepared by wet or dry granulation and optionally coated according to methods well known in normal pharmaceutical practice, in particular with an enteric coating.

The pharmaceutical compositions may also be adapted for parenteral administration, such as sterile solutions, suspensions or lyophilized products in the appropriate unit dosage form. Such pharmaceutical excipients can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and similars. Water or saline aqueous solutions and aqueous dextrose and glycerol solutions, particularly for injectable solutions, are preferably used as vehicles.

The compositions will be prepared using standard methods such as those described or referred to in the Spanish and US Pharmacopoeias and similar reference texts. The compounds for use according to the invention may be administered in a “therapeutically effective amount”, i.e. a nontoxic but sufficient amount of the corresponding compound to provide the desired effect. The amount that is “effective” will vary from subject to subject, depending on the age and general condition of the individual, the particular compound administered, and the like. Thus, it is not always possible to specify an exact “therapeutically effective amount”. However, an appropriate amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

The compounds for use according to the invention will typically be administered once or more times a day, for example 1, 2, 3 or 4 times daily, with typical total daily doses depending on the particular compound and severity of the disease, and may be easily determined by the skilled practitioner.

Typical total daily doses of amrinone or a pharmaceutically acceptable salt thereof are in the range of from 0.1 to 300 mg/kg/day (expressed as amrinone free base), preferably from 0.1 to 30 mg/kg/day (expressed as amrinone free base), more preferably from 1 to 30 mg/kg/day (expressed as amrinone free base). These doses are particularly preferred when amrinone or a pharmaceutically acceptable salt thereof is orally administered. Particularly preferred total daily doses of amrinone or a pharmaceutically acceptable salt thereof are in the range of from 0.1 to 100 mg/kg/day (expressed as amrinone free base), preferably from 0.1 to 10 mg/kg/day (expressed as amrinone free base), more preferably from 1 to 10 mg/kg/day (expressed as amrinone free base) when intravenously administered.

Typical total daily doses of nicainoprol or a pharmaceutically acceptable salt thereof are in the range of from 0.01 to 18000 mg/day (expressed as nicainoprol free base), preferably from 0.01 to 1800 mg/day (expressed as nicainoprol free base), more preferably from 0.1 to 1800 mg/day (expressed as nicainoprol free base). Particularly preferred total daily doses of nicainoprol or a pharmaceutically acceptable salt thereof are in the range of from 0.01 to 1800 mg/day (expressed as nicainoprol free base), preferably from 0.01 to 180 mg/day (expressed as nicainoprol free base), more preferably from 0.1 to 180 mg/day (expressed as nicainoprol free base) when intravenously administered. Particularly preferred total daily doses of nicainoprol or a pharmaceutically acceptable salt thereof are in the range of from 0.1 to 18000 mg/day (expressed as nicainoprol free base), preferably from 0.1 to 1800 mg/day (expressed as nicainoprol free base), more preferably from 1 to 1800 mg/day (expressed as nicainoprol free base) when orally administered.

Typical total daily doses of piboserod or a pharmaceutically acceptable salt thereof are in the range of from 0.01 to 8000 mg/day (expressed as piboserod free base), preferably from 0.01 to 80 mg/day (expressed as piboserod free base), more preferably from 1 to 80 mg/day (expressed as piboserod free base). Particularly preferred total daily doses of piboserod or a pharmaceutically acceptable salt thereof are in the range of from 0.01 to 600 mg/day (expressed as piboserod free base), preferably from 0.01 to 60 mg/day (expressed as piboserod free base) when intravenously administered. Particularly preferred total daily doses of piboserod or a pharmaceutically acceptable salt thereof are in the range of from 0.1 to 8000 mg/day (expressed as piboserod free base), preferably from 0.1 to 80 mg/day (expressed as piboserod free base), more preferably from 1 to 80 mg/day (expressed as piboserod free base) when orally administered.

For pediatric patients, doses will be adapted to the corresponding mg/kg dose as determined by the skilled practitioner.

The pharmaceutical compositions may be prepared using standard methods such as those described or referred to in the Spanish and US Pharmacopoeias and similar reference texts.

The term “subject” refers to a mammal, e.g., a human.

In a second aspect, the present invention relates to a combination comprising two or more compounds selected from the group consisting of amrinone, nicainoprol, piboserod, olanzapine, miglustat, arimoclomol, N-acetyl-leucine, 2-hydroxypropyl-P-cyclodextrin (HPBCD), 2-hydroxypropyl-Y-cyclodextrin (HPGCD), vorinostat and ursodeoxycholic acid (UDCA or 3a,7P-dihydroxy-5P-cholanic acid), or a pharmaceutically acceptable salt thereof, for use in the treatment and/or prevention of Niemann-Pick C disease, wherein at least one compound is selected from the group consisting of amrinone, nicainoprol and piboserod or a pharmaceutically acceptable salt thereof.

The invention also relates to the use of a combination comprising two or more compounds selected from the group consisting of amrinone, nicainoprol, piboserod, olanzapine, miglustat, arimoclomol, N-acetyl- leucine, 2-hydroxypropyl-P-cyclodextrin, 2- hydroxypropyl-y-cyclodextrin, vorinostat and ursodeoxycholic acid, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment and/or prevention of Niemann-Pick C disease, wherein at least one compound is selected from the group consisting of amrinone, nicainoprol and piboserod or a pharmaceutically acceptable salt thereof.

The invention also relates to a method of treating and/or preventing Niemann-Pick C disease in a subject, comprising administering to said subject a therapeutically effective amount of a combination comprising two or more compounds selected from the group consisting of amrinone, nicainoprol, piboserod, olanzapine, miglustat, arimoclomol, N- acetyl- leucine, 2-hydroxypropyl-P-cyclodextrin, 2-hydroxypropyl-Y-cyclodextrin, vorinostat and ursodeoxycholic acid, or a pharmaceutically acceptable salt thereof, wherein at least one compound is selected from the group consisting of amrinone, nicainoprol and piboserod or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, the Niemann-Pick C disease is selected from the group consisting of prenatal, perinatal, early-infantile, late-infantile, juvenile, adolescent and adult Niemann-Pick C disease.

The term “combination” refers to a product comprising two or more of the defined compounds, either in a single composition or in several compositions (or units), in which case the corresponding compounds are distributed among the several compositions. Preferably, the combination refers to several compositions, in particular comprising one composition (or unit) per compound (compound as defined above) of the combination. The expression “two or more” when characterizing the combination refers to at least two, preferably 2, 3, or 4 compounds, more preferably, 2 or 3 compounds, even more preferably 2 compounds.

When the combination is in the form of a single composition, the compounds present in the combination are always administered simultaneously.

When the combination is in the form of several compositions (or units), each of them having at least one of the compounds of the combination, the compositions or (units) may be administered simultaneously, sequentially or separately.

Simultaneous administration means that the compounds or compositions (or units) are administered at the same time.

Sequential administration means that the compounds or compositions (or units) are administered at different time points in a chronologically staggered manner.

Separate administration means that the compounds or compositions (or units) are administered at different time points independently of each other. Amrinone, nicainoprol, piboserod and their pharmaceutically acceptable salts have been described in detail above.

Miglustat was originally developed as an antiviral and later developed for the treatment of type I Gaucher disease, but has also been approved for treating progressive neurological complication in subjects suffering from Niemann-Pick C disease [Pineda, M. et al., Ophanet Journal Rare Diseases , 2018, 13, 140] Miglustat has the chemical structure depicted below. This compound is commercially available or may be synthesized using a suitable preparation method, such as that disclosed in EP 0367748 A2. Miglustat can be used as the free base or as a pharmaceutically acceptable acid addition salt thereof, such as the hydrochloride salt. It is preferably used as the free base.

Arimoclomol is in development for the treatment of Niemann-Pick C disease and has the chemical structure depicted below.

This compound is commercially available or may be synthesized using a suitable preparation method, such as that disclosed in WO 00/50403 Al. Arimoclomol can be used as the free base or as a pharmaceutically acceptable acid addition salt thereof, such the maleate and citrate salts. It is preferably used as the citrate salt.

N-acetyl-leucine is in development for the treatment of Niemann-Pick C disease and has the chemical structure depicted below. This compound is commercially available. N-acetyl-leucine can be used as the free acid or as a pharmaceutically acceptable basic addition salt thereof, such the sodium and monoethanolamine salts. It is preferably used as the free acid. This compound (free acid or the corresponding salt form) can be used as a mixture of N-acetyl-L-leucine and N- acetyl-D-leucine, or as a single stereoisomer, preferably N-acetyl-L-leucine.

2-Hydroxypropyl-P-cyclodextrin (HPBCD) is a cyclic oligosaccharide containing 7 D- (+)-glucopyranose units wherein the hydrogens of the hydroxyl groups have been totally or partially substituted by 2-hydroxypropyl groups. Totally substituted means that all the hydrogen atoms of the hydroxyl groups present in the glucopyranose units have been replaced by 2-hydroxypropyl groups. Partially substituted means that not all hydrogen atoms of the hydroxyl groups present in the glucopyranose units have been replaced by 2-hydroxypropyl groups. Preferably, more than 60 mol% of the hydrogen atoms of the hydroxyl groups present in the glucopyranose units have been replaced by 2- hydroxypropyl groups, more preferably more than 70 mol%, still more preferably more than 80 mol%, even more preferably more than 90 mol%. HPBCD is in development for the treatment of Niemann-Pick C disease and is commercially available for example as Kleptose HPB (VTS-270) and Trappsol ^® .

2-Hydroxypropyl-Y-cyclodextrin (HPBCD) is a cyclic oligosaccharide containing 8 D- (+)-glucopyranose units wherein the hydrogens of the hydroxyl groups have been totally or partially substituted by 2-hydroxypropyl groups. Totally substituted means that all the hydrogen atoms of the hydroxyl groups present in the glucopyranose units have been replaced by 2-hydroxypropyl groups. Partially substituted means that not all hydrogen atoms of the hydroxyl groups present in the glucopyranose units have been replaced by 2-hydroxypropyl groups. Preferably, more than 60 mol% of the hydrogen atoms of the hydroxyl groups present in the glucopyranose units have been replaced by 2- hydroxypropyl groups, more preferably more than 70 mol%, still more preferably more than 80 mol%, even more preferably more than 90 mol%. 2-Hydroxypropyl-y- cyclodextrins (HPGCD) have been disclosed as reducing cholesterol accumulation and restore the functional and molecular abnormalities in NPC patient-derived cells. In addition NPC model mice showed and improved liver status and prolonged survival with HPGCDs [Soga et al., Stem Cells , 2015, 33(4), 1075-1088]

Vorinostat or suberanilohydroxamic acid has been suggested for the treatment of NPC and has the chemical structure depicted below and is commercially available. Vorinostat can be used as the free acid or as a pharmaceutically acceptable basic addition salt thereof, such the potassium salt. It is preferably used as the free acid. Ursodeoxycholic acid, also known as UDCA, ursodiol or 3a,7P-dihydroxy-5P-cholanic acid is in development for the treatment of Niemann-Pick C disease and has the chemical structure depicted below.

This compound is commercially available. UDCA can be used as the free acid or as a pharmaceutically acceptable basic addition salt thereof, such the sodium salt. It is preferably used as the free acid.

The combination for use according to the invention comprises at least one compound which is selected from the group consisting of amrinone, nicainoprol and piboserod or a pharmaceutically acceptable salt thereof. In another embodiment, the combination comprises amrinone or a pharmaceutically acceptable salt thereof, preferably amrinone (free base) or amrinone lactate, more preferably amrinone lactate. Accordingly, the combination comprises amrinone or a pharmaceutically acceptable salt thereof (preferably amrinone free base) and one or more (preferably one) compound selected from the group consisting of olanzapine, nicainoprol, piboserod, miglustat, arimoclomol, N-acetyl-leucine, 2-hydroxypropyl-P-cyclodextrin, 2- hydroxypropyl-y-cyclodextrin, vorinostat and ursodeoxycholic acid or a pharmaceutically acceptable salt thereof.

In another embodiment, the combination comprises nicainoprol or a pharmaceutically acceptable salt thereof, preferably nicainoprol (free base). Accordingly, the combination comprises nicainoprol or a pharmaceutically acceptable salt thereof (preferably nicainoprol free base) and one or more (preferably one) compound selected from the group consisting of olanzapine, amrinone, piboserod, miglustat, arimoclomol, N-acetyl- leucine, 2-hydroxypropyl-P-cyclodextrin, 2-hydroxypropyl-Y-cyclodextrin, vorinostat and ursodeoxycholic acid or a pharmaceutically acceptable salt thereof.

In another embodiment, the combination comprises piboserod or a pharmaceutically acceptable salt thereof, preferably piboserod (free base) or piboserod hydrochloride, more preferably piboserod (free base). Accordingly, the combination comprises piboserod or a pharmaceutically acceptable salt thereof (preferably piboserod free base) and one or more (preferably one) compound selected from the group consisting of olanzapine, amrinone, nicainoprol, miglustat, arimoclomol, N-acetyl-leucine, 2-hydroxypropyl-P-cyclodextrin, 2-hydroxypropyl-Y-cyclodextrin, vorinostat and ursodeoxycholic acid or a pharmaceutically acceptable salt thereof.

In particular, the combination for use according to the invention is administered as a pharmaceutical composition, which comprises the corresponding (active) compounds and a pharmaceutically acceptable excipient, as previously defined. The pharmaceutical composition may comprise one or more active compounds, i.e. all the compounds of the combination may form part of the same pharmaceutical composition or the compounds may be formulated in different pharmaceutical composition (such as a pharmaceutical composition for each compound of the combination).

The combination for use according to the invention will typically be administered once or more times a day, for example 1, 2, 3 or 4 times daily, with typical total daily doses depending on the particular compound and severity of the disease, and may be easily determined by the skilled practitioner.

Examples of preferred total daily doses for amrinone, nicainoprol and piboserod or a pharmaceutically acceptable salt thereof are as previously defined. Typical total daily doses of miglustat or a pharmaceutically acceptable salt thereof are in the range of from 0.1 to 15000 mg/day (expressed as miglustat free base), preferably from 0.1 to 1500 mg/day (expressed as miglustat free base), more preferably from 1 to 600 mg/day (expressed as miglustat free base). These doses are particularly preferred when miglustat or a pharmaceutically acceptable salt thereof is orally administered.

By way of example, typical total daily doses of olanzapine or a pharmaceutically acceptable salt thereof are in the range of from 0.1 to 2500 mg/day (expressed as olanzapine free base), preferably from 0.1 to 20 mg/day (expressed as olanzapine free base), more preferably from 1 to 20 mg/day (expressed as olanzapine free base). These doses are particularly preferred when olanzapine or a pharmaceutically acceptable salt thereof is orally administered.

Typical total daily doses of arimoclomol or a pharmaceutically acceptable salt thereof are in the range of from 0.1 to 10000 mg/day (expressed as arimoclomol free base), preferably from 1 to 1000 mg/day (expressed as arimoclomol free base), more preferably from 150 to 600 mg/day (expressed as arimoclomol free base). These doses are particularly preferred when arimoclomol or a pharmaceutically acceptable salt thereof is orally administered.

Typical total daily doses of N-acetyl-leucine or a pharmaceutically acceptable salt thereof are in the range of from 0.01 to 40 g/day (expressed as N-acetyl-leucine free acid), preferably from 0.01 to 10 g/day (expressed as N-acetyl-leucine free acid), more preferably from 0.1 to 5 g/day (expressed as N-acetyl-leucine free acid). These doses are particularly preferred when N-acetyl-leucine or a pharmaceutically acceptable salt thereof is orally administered.

Typical total daily doses of 2-hydroxypropyl-P-cyclodextrin or 2-hydroxypropyl-y- cyclodextrin are in the range of from 0.1 to 8000 mg/kg/day, preferably from 0.1 to 3000 mg/kg/day, more preferably from 1 to 1100 mg/kg/day when 2-hydroxypropyl-P- cyclodextrin or 2-hydroxypropyl-y-cyclodextrin is administered intravenous or by intraventricular injection. Typical total monthly doses of 2-hydroxypropyl-P-cyclodextrin or 2-hydroxypropyl-Y-cyclodextrin are in the range of from 0.1 to 5000 mg/month, preferably from 1 to 3600 mg/month, more preferably from 50 to 1800 mg/month when 2-hydroxypropyl-P-cyclodextrin or 2-hydroxypropyl-Y-cyclodextrin is administered by lumbar intrathecal injection.

Typical total daily doses of vorinostat are in the range of from 0.01 to 3000 mg/day, preferably from 0.1 to 400 mg/ day, more preferably from 1 to 400mg/day. These doses are particularly preferred when vorinostat is orally administered.

Typical total daily doses of ursodeoxycholic acid or a pharmaceutically acceptable salt thereof are in the range of from 0.01 to 300 mg/kg/day (expressed as free ursodeoxycholic acid), preferably from 0.1 to 30 mg/kg/day (expressed as free ursodeoxycholic acid), more preferably from 1 to 30 mg/kg/day (expressed as free ursodeoxycholic acid). These doses are particularly preferred when ursodeoxycholic acid or a pharmaceutically acceptable salt thereof is orally administered.

The following examples represent specific embodiments of the present invention. They do not intend to limit in any way the scope of the invention defined in the present description.

Examples

Experimental design

Lysotracker Green test was developed as an initial clinical monitoring assay in NPC patients [Lachmann, R. et ah, Neurobiology Disease , 2004, 16(3) 654-658] and subsequently modified for small molecule screening [Xu, M. et al., ./. Biol. Chem ., 2012, 287(47), 39349-39360] Lysotracker Green loads into lysosomes dependent on their volume, presence of storage molecules and acidity, with larger lysosomal volumes present in NPC disease (with no change in acidic pH [Lloyd-Evans et al, Nature Medicine , 2008, 14, 1247-1255]) owing to the lipid storage and is reduced in cells treated with miglustat where glycolipid biosynthesis is inhibited and lysosomal storage and therefore expansion is reduced. NPC null mouse astroglial cells [Lloyd-Evans, E. et al., Nature Medicine, 2008, 14(11), 1247-1255] were used to do the in vitro tests.

Ganglioside GM1 has been shown to be elevated in animal models of NPC disease as well as in primary human cells and tissues. Cholera Toxin B (CtxB) conjugated to FITC was used to stain fixed cells for ganglioside GM1 [Heyningen, S. Van., Science , 1974, 183(4125), 656-657; O’Hanlon et al, Neurosci Res., 2003, 47(4), 383-390; and Choudhury et al., J Clin Invest., 2002, 109(12), 1541-50] and obtain an accurate representation of the impact of various treatments not only on gangliosides but also as a surrogate marker for changes in the total glycosphingolipid pool. Thus, the compounds were tested for confirmation of effect on NPC glycosphingolipid storage by staining fixed treated cells on day 5 with fluorescein labelled-cholera toxin. If the compounds were efficacious a reduced ganglioside level in the NPC cells should be seen [te Vruchte, D.et al, J. Biol. Chem., 2004, 279(25), 26167-26175] Untreated wild-type and NPC1 null glia cells were used as controls alongside miglustat treated NPC null glia cells as a positive control.

Selected compounds were tested for changes in HSP70 levels by antibody staining since this is a relevant biological marker of arimoclomol and arimoclomol-like compounds.

Materials and methods

Cell lines - Spontaneously immortalized NpcN (hereafter referred to as “NPC null”) and Npcl ^+/+ (referred to as “wild-type”) mouse astrocyte cells were used. Cells were grown as monolayers in T75 tissue culture coated tanks in a Thermo humidified CO2 incubator maintained at 37°C in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% Foetal Bovine Serum (FBS) and 1% L-glutamine. Cells were allowed to grow to 90% confluency before splitting 1 :4-5 in readiness for drug treatments in 6-well plates. [Lloyd-Evans, E. et al, Nature Medicine, 2008, 14(11), 1247-1255; and te Vruchte, Journal Biological Chemistry, 2004, 279(25), 26167-26175] Cell counting - Cell numbers were kept constant based on earlier optimization experiments (60,000 cells per well of a 96 well plate). Trypsinized cells were counted using disposable fast read haemocytometers.

Test compound preparation - Amrinone (Sigma Aldrich), piboserod (Sigma Aldrich), nicainoprol (MedChemExpress), miglustat (MedChemExpress) and arimoclomol (Carbosynth) were solubilized at 10 mM stock concentrations in dimethylsulfoxide (DMSO). All subsequent cellular drug treatments were done in 6 well plates with controls (i.e. DMSO, miglustat or arimoclomol) as well as untreated wild-type and NPC1 null glial cells included in every round (DMSO was added to untreated wild-type and NPC1 null cells to ensure no effect of the solvent). Cells were seeded onto 6 well plates in 1.5 mL volumes of complete DMEM, allowed to begin to attach and then an equal volume of a 2X working concentration of the appropriate test compound in complete DMEM was added to generate the correct final concentration with minimal disruption to the cells.

Qualitative measures of cell death or impaired cell growth - Any cell death/growth inhibition effects were estimated from counting the cells that were seeded into the 6 well plates at the beginning of the treatments compared to the number of cells in the 6 well plates at day 5 of the treatment (for miglustat and amrinone), and at day 3 of the treatment (for arimoclomol, piboserod and nicainoprol).

Lysotracker Green phenotypic 96 well screen - Lysotracker ® Green (Molecular Probes) is a widely used fluorescent acidotropic probe for labeling and tracking acidic organelles in live cells. On the final day of the drug screen (day 6 of the treatment for miglustat and amrinone, and at day 3 of the treatment for arimoclomol, piboserod and nicainoprol), cells in 96 well plates were washed once in Dulbecco’s phosphate buffered saline (DPBS) and incubated with 200 nM Lysotracker green (or (N-[2-(dimethylamino)ethyl]-3-{2-[(3,5- dimethyl-lH-pyrrol-2-yl-KN)methylene]-2H-pyrrol-5-yl-

KN}propanamidato)(difluoro)boron), in DPBS for 15 minutes at room temperature prior to removal of the staining reagent, washing with DPBS and reading of the fluorescence emission of the cells on a plate reader (Spectramax Gemini, a monochromator based system). For all readings the gain was set arbitrarily (based on previous knowledge) at 400 and maintained at this level for all the experiments, excitation and emission wavelengths were 485 nm and 515 nm respectively and readings were taken from 5 different points within each well of the 96 well plate (cellbind).

Assay standardization - All data was standardized according to cell number.

Confirmation of effect of test compounds on glvcosphingolipid biology - Fluorescein isothiocyanate (FITC) labelled cholera toxin B subunit (FITC-CtxB, referred to as CtxB; Cholera Toxin B subunit, Sigma Aldrich, ref. C1655) was used to confirm the effect of test compounds on GLS biology owing to its specific binding to ganglioside GM1 on the plasma membrane and membrane structures within cells.

For analysis of total ganglioside GM1 levels, NPC mouse glia were treated for 6 days with the selected test compounds as well as with the positive control miglustat (50 mM) and compared the changes in total FITC-CtxB stained ganglioside GM1 levels (by area measurement on the imaging software Fiji) across the different conditions versus the untreated NPC null and wild-type cells. DPBS washed cells grown in Ibidi 8 well chamberslides were fixed with 3.7% paraformaldehyde for 10 minutes followed by a quench in complete DMEM and two further DPBS washes. These cells were incubated at 4°C with lpg/ml CtxB in DPBS supplemented with 1% bovine serum albumin (BSA, for blocking) and 0.1% saponin (for membrane permeabilization) for 24h followed by 3 x 5 minute washes in DPBS and nuclear counterstaining with 0.5pg/ml Hoechst 33342, cells were then visualized using widefield microscopy.

HSP70 cellular staining - Cells were treated for 3 days with the selected test compounds prior to fixation with paraformaldehyde and staining for HSP-70 with anti-HSP70 antibodies (Mouse anti-human HSP70 antibody; Proteintech). For anti-HSP70 antibody staining, anti-HSP70 antibodies were added at 1:200 and were incubated with fixed cells in DPBS containing 1% BSA and 0.1% saponin 12 h at 4°C followed by 3 x 5min washes in DPBS and then labelling with fluorescent secondary antibodies (Goat Anti-Mouse IgG H&L (DyLight® 488) (ab96871) from Abeam) at 1 : 100 for 30 min at room temperature. Hoechst was added for 10 min after the secondary antibody was removed and the cells were washed 3x5min in PBS. Hoechst (Hoechst 33342; Invitrogen) was added at a 2:5,000 dilution from a 10 mg/ml stock.

Data preparation and analysis - Data was analyzed using Microsoft Excel and statistical analyses were performed using Graphpad Prism 6. Microscopy images were collected on a customized Zeiss Colibri LED system with an Axiocam Mrm CCD camera and Axiovision 4.9.2 software. Post-processing of microscopy images was performed with Adobe Photoshop (any image manipulation was consistent across the dataset) and data analyzed using Fiji 1.0 using thresholding and subsequent area analysis.

Results

Lysotracker Green screen Lysosomal accumulation of glycosphingolipids in NPC null glial cells translates in larger lysosomes and higher Lysotracker green fluorescence than wild type cells as shown in Figures la and lb. Treatment of NPC null glial cells with 50 mM of miglustat during 6 days rescues the wild type phenotype as shown by a significant decrease of lysotracker green fluorescence (Fig la). A similar 6-day treatment with amrinone also significantly reduced lysosome size in NPC null cells, with effects clearly observed at 50 pM but also slightly at 0.5 pM (Fig. la). Treatment of NPC null cells with arimoclomol at 200 pM during 3 days also caused a modest reduction of the size of lysosomes, and similar or higher reductions were observed after treatment with piboserod and nicainoprol at their highest concentrations (Fig. lb). The observed changes in lysotracker fluorescence were not due to a decrease in the number of cells. Changes in cell viability were also determined by seeding the same number of cells in 6 well plates and treating them with the compounds for 6 days (miglustat and amrinone) or 3 days (arimoclomol, piboserod and nicainoprol) at the tested concentrations prior to trypsinization and counting of the 10 pi of cell suspension in fast read haemocytometers. None of the compounds showed any negative impact on cell growth at the test conditions used. Effect of amrinone on cellular glvcosphingolipid levels and storage and on glvcosphingolipid endocvtosis

NPC null cells present higher levels of glycosphingolipids than wild type cells as shown by fluorescent cholera toxin B staining of GM1 gangliosides (Fig 2). The cellular glycosphingolipid storage is reduced after 6 days of treatment with miglustat 50 mM. Treatment with 5 pM of amrinone also causes a significant reduction of glycosphingolipids in NPC null cells (Fig.2). Effect of piboserod and nicainoprol on HSP70

The effect of piboserod and nicainoprol at 50, 100 and 200 pM concentrations, as well as that of the positive control arimoclomol (200 pM) on increasing expression and lysosomal (punctuate) localization of endogenous HSP70 is shown in Figures 3 and 4. The data of Figure 3 clearly show that arimoclomol, the positive control, is capable of increasing the expression of HSP70 in punctate structures corresponding to lysosomes within the Npcl ^~/~ glial cells following a 72 h incubation at 200pM. Piboserod and nicainoprol also increased lysosomal HSP70 expression in the Npcl ^~/~ glia over the tested concentration range (50-200pM). At all concentrations tested there was an increase in expression of punctate (lysosomal) HSP70. It can also be seen that all of the compounds, including the positive control arimoclomol, induced an expression pattern of HSP70 resembling enlarged vesicles with limiting membrane staining of HSP70 but no intravesicular staining (indicated by white arrows in Figure 4), resembling a “doughnut” in appearance. This indicates that HSP70 is potentially bound to the lysosomal membrane where it is believed to stabilize lysosomal function that should result in reduced lipid storage (Kirkegaard et al, Nature , 2010, 463(7280), 549-553).