Roluperidone for the treatment of lysosomal storage disorders and their symptoms

Abstract

Described herein are methods for treating or preventing a lysosomal storage disorder (LSD), or a symptom of a lysosomal storage disorder, through the administration of Roluperidone to a subject in need thereof. Also provided are methods for reducing lysosomal size and / or cholesterol accumulation in a subject through the administration of Roluperidone to a subject in need thereof.

Description

ROLUPERIDONE FOR THE TREATMENT OF LYSOSOMAL STORAGE DISORDERS AND THEIR SYMPTOMSRELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of, U.S. Provisional Application No. 63 / 592,675 filed on October 24, 2023. The contents of the aforementioned patent application are incorporated herein by reference in their entirety.FIELD OF THE DISCLOSURE

[0002] The present disclosure relates generally to the treatment of lysosomal storage disorders (LSDs), including Niemann-Pick disease.BACKGROUND

[0003] Lysosomal storage disorders (LSDs) are inherited metabolic disorders where a genetic variation disrupts the normal activity of lysosomes in human cells causing abnormal build-up of various toxic materials in the body’s cells due to enzyme deficiencies. It is estimated that LSDs affect about 1 in every 5,000 live births. Although these disorders are considered rare, they are known to affect individuals globally. At present, there are no cures for lysosomal storage disorders and there are not yet specified treatment options for many of the diseases (https: / / rarediseases.org / rare-diseases / lysosomal-storage-disorders / ).

[0004] Niemann-Pick Disease Type Cl is an autosomal recessive, rare, and ultimately fatal lysosomal storage disorder characterized by progressive neurovisceral manifestations due to mutations in the Npcl gene. The defect causes the dramatic decrease of NPC1 protein content and the lysosomal accumulation of cholesterol which induces the degeneration of specific neurons. Like other LSDs, Niemann-Pick Disease Type C does not have a cure. Thus, treatment options are focused on the specific symptoms of each individual. As such, improved methods to treat LSDs and their symptoms, including Niemann-Pick Disease, are needed.SUMMARY

[0005] The present disclosure provides methods of treating or preventing a lysosomal storage disorder (LSD) in a subject in need thereof. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of Roluperidone. In someembodiments, the method comprises administering to the subject a prophylactically effective amount of Roluperidone.

[0006] The present disclosure provides methods of treating or preventing a symptom of a lysosomal storage disorder (LSD) in a subject in need thereof. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of Roluperidone. In some embodiments, the method comprises administering to the subject a prophylactically effective amount of Roluperidone.

[0007] In some embodiments of the preceding methods, the LSD is cholesteryl ester storage disease, Wohnan disease, Hunter syndrome, Hurler’s disease, Scheie syndrome, Hurler- Scheie syndrome, mucopolysaccharidosis (MPS) type III, mucopolysaccharidosis (MPS) type IV (Morquio syndrome), Maroteaux-Lamy syndrome, Sly syndrome, Fabry disease, Gaucher disease, Krabbe disease, metachromatic leukodystrophy, Niemann-Pick disease, Sandhoff disease, Tay-Sachs disease, alpha mannosidosis, beta mannosidosis, Schindler disease, aspartylglucosaminuria, fucosidosis, neuronal ceroid lipofuscinosis, galactosialidosis, infantile sialic acid storage disease, Salla disease, sialuria, mucolipidosis, cystinosis, Danon disease, multiple sulfatase deficiency, proteins GM2-AP deficiency, Farber disease, lysosomal acid lipase deficiency, hyaluronidase deficiency, pycnodysostosis, and / or Pompe disease.

[0008] In some embodiments of the preceding methods, the LSD is Niemann-Pick disease. In some embodiments of the preceding methods, the LSD is Niemann-Pick disease type C. In some embodiments of the preceding claims, the LSD is Niemann-Pick disease type A or B.

[0009] In some embodiments of the preceding methods, the subject has high cholesterol. In some embodiments of the preceding methods, the administration of Roluperidone results in a reduction of cholesterol accumulation in the subject as compared to the cholesterol accumulation in the subject prior to initiating Roluperidone administration. In some embodiments of the preceding methods, the administration of Roluperidone results in a reduction of the lysosomal size of the subject as compared to the lysosomal size of the subject prior to initiating Roluperidone administration. In some embodiments of the preceding methods, the administration of Roluperidone results in a reduction of the lysosomal size of the subject and the reduction of cholesterol accumulation in the subject as compared to the lysosomal size and cholesterol accumulation in the subject prior to initiating Roluperidone administration.

[0010] In some embodiments of the preceding methods, the reduction of cholesterol accumulation in the subject is determined by filipin staining. In some embodiments of the preceding methods, the reduction of cholesterol accumulation in the subject is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% as compared to the cholesterol accumulation in the subject prior to initiating Roluperidone administration. In some embodiments of the preceding methods, the reduction of cholesterol accumulation in the subject occurs within 24 hours, within 48 hours, or within 72 hours of the administration of Roluperidone.

[0011] In some embodiments of the preceding methods, the reduction of lysosomal size of the subject is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% as compared to the lysosomal size of the subject prior to initiating Roluperidone administration. In some embodiments of the preceding methods, the reduction of lysosomal size of the subject occurs within 24 hours, within 48 hours, or within 72 hours of the administration of Roluperidone.

[0012] In some embodiments of the preceding methods, the administration of Roluperidone to the subject does not result in a change in NPC1 expression in the subject as compared to the NPC1 expression in the subject prior to initiating Roluperidone administration. In some embodiments of the preceding methods, the subject is a pediatric subject. In some embodiments of the preceding methods, the subject is an adult.

[0013] In some embodiments of the preceding methods, the method further comprises administering to the subject one or more additional LSD treatment agents. In some embodiments, the one or more additional LSD treatment agents is selected from recombinant enzymes, enzymes plus chaperones, recombinant enzymes plus chaperones, fusion proteins, pharmacological chaperones, miglustat, and insulin growth factor II fusion proteins. In some embodiments of the preceding methods, the treatment of a LSD or a symptom of a LSD further comprises gene therapy. In some embodiments of the preceding methods, the prevention of a LSD or a symptom of a LSD further comprises gene therapy. In some embodiments of the preceding methods, the method further comprises administering to the subject a gene therapy.

[0014] The present disclosure also provides methods of reducing cholesterol, cholesterol levels, cholesterol content, and / or cholesterol accumulation in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically orprophylactically effective amount of Roluperidone. In some embodiments, the subject has cancer, chronic kidney disease, diabetes, HTV7AIDS, Lupus, or Alzheimer’s disease.

[0015] In some embodiments of the preceding methods, the therapeutically or prophylactically effective amount of Roluperidone is administered to the subject once a day. In some embodiments of the preceding methods, the therapeutically or prophylactically effective amount of Roluperidone is administered to the subject twice a day. In some embodiments of the preceding methods, the therapeutically or prophylactically effective amount of Roluperidone is administered orally to the subject. In some embodiments of the preceding methods, the therapeutically or prophylactically effective amount of Roluperidone is between about 1 mg and about 100 mg. In some embodiments of the preceding methods, the therapeutically or prophylactically effective amount of Roluperidone is between 1 mg and 100 mg. In some embodiments, the therapeutically or prophylactically effective amount of Roluperidone is about 16 mg, about 24 mg, about 32 mg, about 40 mg, about 48 mg, about 56 mg, about 64 mg, about 72 mg, about 80 mg, about 88 mg, or about 96 mg. In some embodiments, the therapeutically or prophylactically effective amount of Roluperidone is 16 mg, 24 mg, 32 mg, 40 mg, 48 mg, 56 mg, 64 mg, 72 mg, 80 mg, 88 mg, or 96 mg.

[0016] In some embodiments, the therapeutically or prophylactically effective amount of Roluperidone is about 32 mg.

[0017] In some embodiments, the therapeutically or prophylactically effective amount of Roluperidone is about 64 mg.

[0018] In some embodiments, the therapeutically or prophylactically effective amount of Roluperidone is 32 mg. In some embodiments, the therapeutically effective amount of Roluperidone is 64 mg.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 shows the effect of Roluperidone on NPCl protein levels. FIG. 1A shows the densitometric analyses (left) and a representative Western blot (right) of NPC1 protein from healthy and I1061T cells. FIGS. 1B-1D show the densitometric analyses (left) and a representative Western blot (right) of NPC1 protein in I1061T fibroblasts treated with Roluperidone (CA) plus vehicle (Ctr) for 24 hours (FIG. IB), 48 hours (FIG. 1C) and 72 hours (FIG. ID) at the indicated concentrations. 2.5 pM rimcazole (Rim), was used as a positive control of a sigma2 / TMEM97 antagonist. The control of protein loading was done with vinculin. The experiments were carried out four times in duplicate. The statistical analysis was performed with Student’s t-test for FIG. 1A, and one-way ANOVA followed byDunnett's multiple comparisons test for FIGS. 1B-1D where *=p<0.05 versus Ctr; **=p<0.001 versus Ctr; ***=p<0.001 versus healthy.

[0020] FIG. 2 shows the effect of Roluperidone on unesterified cholesterol accumulation. FIG. 2 A shows representative images of filipin staining performed on healthy and II 06 IT fibroblasts treated with vehicle (Ctr), 300 pM cyclodextrin (CD), 2.5 pM rimcazole (Rim), and 3 pM Roluperidone (CA) at 72 hours. FIG. 2B shows the quantification of the mean fluorescence intensity of the filipin stained cells. Data represent means ± standard deviation (SD). Statistical analysis was performed using ANOVA followed by Dunnett's multiple comparisons test where* **p<0.001 versus healthy; °p<0.05 versus I1061T Ctr.

[0021] FIG. 3 shows the effect of Roluperidone on lysosomes. FIG. 3 shows the geometric (geo) mean ratio of Lysotracker™ fluorescence in healthy and I1061T fibroblasts treated with Roluperidone plus vehicle (Ctr) for 24 hours (left), 48 hours (middle), and 72 hours (right) at the indicated concentrations. 2.5 pM rimcazole (Rim) was used as a positive control of a sigma2 / TMEM97 antagonist for the 72-hour treatment. 300 pM cyclodextrin (CD) was used as a positive control of lysosomal reduction. The experiments were performed three times in duplicate for the 24-hour and 48-hour treatments, and two times in duplicate for the 72-hour treatment. The statistical analysis was performed with one-way ANOVA followed by Dunnett's multiple comparisons test where *=p<0.05 versus healthy; **=p<0.01 versus healthy; Ctr ****=p<0.0001 versus healthy; °=p<0.05 versus Ctr; °°=p<0.01 versus Ctr;°°°=p<0.001 versus Ctr.

[0022] FIG. 4 shows the effect of Roluperidone on lysosomes. FIG. 4 shows the geometric (geo) mean ratio of Lysotracker™ fluorescence in healthy and I1061T fibroblasts treated with Roluperidone plus vehicle (Ctr) for 24 hours (left), 48 hours (middle), and 72 hours (right) at the indicated concentrations. 2.5 pM rimcazole (Rim) was used as a positive control of a sigma2 / TMEM97 antagonist for 72-hour treatment. 300 pM cyclodextrin (CD) was used as a positive control of lysosomal reduction. The experiments were performed four times in duplicate. The statistical analysis was performed with one-way ANOVA followed by Dunnett's multiple comparisons test where *=p<0.05 vs healthy (Ctr) and ****=p<0.0001 vs healthy.

[0023] FIG. 5 is a graph showing the effect of Roluperidone on NPC1 localization. The graph illustrates the Pearson’s correlation coefficient between NPC1 and Lamp2 in Healthy and I1061T fibroblasts treated with 2.5pM rimcazole (Rim) or 3 pM Roluperidone (CA) for 72 hours. Pearson’s correlation coefficient was calculated using Image! version 1.52t (NationalInstitutes of Health, Bethesda, MD, USA) software for Windows. The experiments were carried out three times. The statistical analysis was performed with one-way ANOVA followed by the Dunnett's multiple comparisons test, where ****=p<0.0001 vs healthy.DETAILED DESCRIPTION

[0024] Provided herein are, inter alia, methods for treating or preventing a lysosomal storage disorder, or a symptom of a lysosomal storage disorder, in a subject comprising administering a therapeutically or prophylactically effective amount of Roluperidone. Also provided herein are methods for reducing the lysosomal size and cholesterol accumulation in a subject by administering a therapeutically effective amount of Roluperidone. Embodiments herein generally relate to methods of treating lysosomal storage disorders (LSDs) and then- symptoms.

[0025] The methods described herein provide certain advantages over other methods, including, for example, the ability to reduce both lysosomal size and cholesterol accumulation. The methods described herein provide novel treatment options for subjects with LSDs, including, but not limited to Niemann-Pick Disease, or other diseases with similar and / or dissimilar symptoms, including, but not limited to, high cholesterol.Definitions

[0026] The terms used herein have their ordinary meaning and the meaning of such terms is independent at each occurrence thereof. Notwithstanding the foregoing, and except where stated otherwise, the following definitions apply throughout the specification and claims.

[0027] As used here, the terms “patient” or “subject” are used interchangeably.

[0028] As used herein, the term “subject” is interchangeable with the term “subject in need thereof’, both of which refer to a subject having a disease or having an increased risk of developing the disease. A “subject” includes a mammal. The mammal can be, e.g., a human or an appropriate non-human mammal, such as a primate, mouse, rat, dog, cat, cow, horse, goat, camel, sheep, or pig. The subject can also be a bird or fowl. In some embodiments, the subject is a human. In some embodiments, the subject is a human adult, i.e., 18 years of age or older. In some embodiments, the subject is a human child, i.e., 17 years of age or younger. A subject in need thereof can be one who has been previously diagnosed or identified as having a disease or disorder disclosed herein. A subject in need thereof can also be one who is suffering from a disease or disorder disclosed herein. Alternatively, a subject in needthereof can be one who has an increased risk of developing such disease or disorder relative to the population at large (i.e., a subject who is predisposed to developing such disorder relative to the population at large). A subject in need thereof can have a refractory or resistant disease or disorder disclosed herein (i.e., a disease or disorder disclosed herein that does not respond or has not yet responded to treatment). The subject may be resistant at the start of treatment or may become resistant during treatment. In some embodiments, the subject in need thereof received and failed all known effective therapies for a disease or disorder disclosed herein. In some embodiments, the subject in need thereof received at least one prior therapy.

[0029] As used herein, the term “preventing,” “prevent,” or “protecting against” describes reducing or eliminating the onset of the symptoms or complications of such disease, condition, or disorder.

[0030] It is to be appreciated that references to “treating” or “treatment” include the alleviation of established symptoms of a condition. “Treating” or “treatment” of a state, disorder or condition therefore includes: (1) delaying the appearance of clinical symptoms of the state, disorder or condition developing in a human that may be afflicted with or predisposed to the state, disorder, or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder, or condition, (2) inhibiting the state, disorder, or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical or subclinical symptom thereof, or (3) relieving or attenuating the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms.

[0031] The terms "administer", "administering", or "administration" as used in this disclosure refers to either directly administering a disclosed compound or pharmaceutically acceptable salt and / or hydrate of the disclosed compound or composition to a subject, or administering a prodrug derivative or analog of the compound or pharmaceutically acceptable salt and / or hydrate of the compound or composition to the subject, which can form an equivalent amount of active compound within the subject's body.

[0032] The phrase "therapeutically effective amount," as used herein, refers to the amount of a compound disclosed herein, or a composition comprising said compound, administered to a patient already suffering from a disease, condition, or disorder, that is sufficient to cure or at least partially arrest, or relieve to some extent one or more of the symptoms of the disease, disorder, or condition being treated. The effectiveness of such compositions depends uponconditions including, but not limited to, the severity and course of the disease, disorder, or condition, any previous therapy, the patient's health status and response to the drugs, and the judgment of the treating physician. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex, and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed, and like factors well known in the medical arts. Such an amount would be sufficient to elicit the biological or medical response of a tissue system, or patient that is sought by a researcher or clinician. Such a therapeutically effective amount can be determined by one of ordinary skill in the art having regard to their own knowledge, the state of the art, and this disclosure. By way of example only, therapeutically effective amounts may be determined by routine experimentation, including, but not limited to, a dose escalation clinical trial. For any composition described herein, the therapeutically effective amount may be initially determined from cell culture assays.

[0033] The term “prophy lactically effective amount” of a drug (e.g., the cells described herein) is an amount of the drug that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of an injury, disease, pathology or condition, or reducing the likelihood of the onset (or reoccurrence) of an injury, disease, pathology, or condition, or their symptoms. The full prophylactic effect does not necessarily occur by administration of one dose and may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations.

[0034] The term “about” is used herein to mean approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, unless otherwise specified or unless the context clearly dictates otherwise, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20%. In some embodiments, the term “about” refers to a variance of 10%, a variance of 5%, a variance of 3%, or a variance of 1%. For example, “about 64 mg” is equivalent to a range of 57.6 mg to 70.4 (10% variance), or a range of 60.8 mg to 67.2 mg (5% variance), or a range of 63.36 mg to 64.64 mg (1% variance).

[0035] “Comprising” or “comprises” as applied to a particular dosage form, composition, method or process described or claimed herein means that the dosage form, composition or method includes all of the recited elements in a specific description or claim, but does not exclude other elements. “Consists essentially of’ and “consisting essentially of’ means that the described or claimed composition, dosage form, method or process does not exclude other materials or steps that do not materially affect the recited physical, pharmacological, pharmacokinetic properties or therapeutic effects of the composition, dosage form, method, or process. “Consists of’ and “consisting of’ means the exclusion of more than trace elements of other ingredients and substantial method or process steps.

[0036] The term "lysosomal storage disorder" or "LSD" is used in this disclosure to refer to an inherited metabolic disorder where a genetic variation disrupts the normal activity of lysosomes in human cells causing abnormal build-up of various toxic materials in the body’s cells due to enzyme deficiencies. In some embodiments, the LSD is a lipid storage disorder, a mucopolysaccharidosis, a glycoprotein storage disorder, or a mucolipidosis. In some embodiments, the LSD is an inherited metabolic disorder.

[0037] As used herein, “filipin” refers to a polyene macrolide compound that was originally isolated from Streptomyces filipinensis and that exhibits intrinsic fluorescence. Filipin is known in the art as a diagnostic tool for diseases of lipid dysregulation (See, e.g., Dist; et al., The Journal ofPathology, 2003, 200:104-111). Filipin is known in the art to stain unesterified cholesterol. The structure and fluorescent properties of filipin are described in, e.g., in Castanho et al., Eur. J. Biochem., 1992, 207:125-134; Xu et al., J. Biol. Chem., 2010, 285:16844-16853, and WO 2018 / 107059, the relevant disclosures of which are incorporated by reference for the purpose and subject matter referenced herein.

[0038] As used herein, a subject with “high cholesterol”, “hyperlipidemia”, or “hypercholesterolemia” refers to the development of fatty deposits in the blood vessels of the subject. In some embodiments, the accumulation of fatty deposits in the blood vessels limits blood flow. In some embodiments, the accumulation of fatty deposits in the blood vessels increases the subject’s risk of heart attack and / or stroke. In some embodiments, high cholesterol is determined by a blood test to quantify cholesterol levels. In some embodiments, the subject is a human, and the high cholesterol is characterized by total serum cholesterol that is greater than or equal to 200 mg / dl or 5.2 mmol / L. In some embodiments, the subject is a human, and the high cholesterol is characterized by low-density lipoprotein (LDL) greater than or equal 70 mg / dL or greater than or equal to 100 mg / dL. In some embodiments, thesubject is a human, and the high cholesterol is characterized by LDL greater than or equal to 1.8 mmol / L or greater than or equal to 2.6 mmol / L. In some embodiments, the subject is a human, and the high cholesterol is characterized by triglycerides that are greater than 150 mg / dL or 1.7 mmol / L.

[0039] As used herein, an “additional LSD treatment agent”, refers to an agent other than Roluperidone that treats or prevents a LSD or a symptom of a LSD. Non-limiting examples of LSD treatment agents include recombinant enzymes, enzymes plus chaperones, recombinant enzymes plus chaperones, pharmacological chaperones, fusion proteins, miglustat, and insulin growth factor II fusion proteins.

[0040] In some embodiments, the recombinant enzyme is an enzyme replacement therapy. In some embodiments, the recombinant enzyme comprises one or more of FABRAZYME® (agalsidase beta), CEREZYME® (imiglucerase), ELELYSO™ (taliglucerase alfa), MYOZYME® (alglucosidase alfa), LUMIZYME® (alglucosidase alfa), ALDURAZYME® (laronidase), ELAPRASE® (idursulfase), and NAGLAZYME® (galsulfase).

[0041] In some embodiments, the pharmacological chaperones bind to mutated enzymes in the subject and improve the folding and stability of the enzyme. In some embodiments, the pharmacological chaperone can be administered in combination with an enzyme or recombinant enzyme therapy to improve cellular update, improve enzyme / recombinant enzyme stability, and improve cellular localization of the enzyme / recombinant enzyme. See e.g., Parenti G. “Treating lysosomal storage diseases with pharmacological chaperones: from concept to clinics.” EMBO Mol Med. 2009 Aug;l(5):268-79, the relevant disclosures of which are incorporated by reference herein.

[0042] In some embodiments, the fusion protein is an IgG-enzyme fusion protein wherein the IgG domain of an antibody is fused to an enzyme. In some embodiments, the fusion protein includes an IgG domain attached to an enzyme through a flexible linker. In some embodiments, the fusion protein comprises a recombinant enzyme. In some embodiments, the fusion protein includes components that allow the protein to pass the blood-brain barrier. In some embodiments, the fusion protein comprises a human insulin receptor monoclonal antibody (HIRMAb). In some embodiments, the fusion protein comprises one or more post- translational modifications. In some embodiments, the fusion protein acts as an enzyme replacement therapy. In some embodiments, the fusion protein comprises an insulin-like growth factor-II (IGF-II). In some embodiments, the fusion protein comprises the receptor binding motif of IGF-II. In some embodiments, a fusion protein comprising IGF-II enhancesthe ability of the fusion protein to enter cells. See, e.g., Boado et al. “Bi-functional IgG- lysosomal enzyme fusion proteins for brain drug delivery.” Sci Rep 9, 18632 (2019); Pardridge "Blood-brain barrier delivery for lysosomal storage disorders with IgG-lysosomal enzyme fusion proteins." Advanced Drug Delivery Reviews 184 (2022): 114234; and Kan et al. “Insulin-like growth factor II peptide fusion enables uptake and lysosomal delivery of a- N-acetylglucosaminidase to mucopolysaccharidosis type IIIB fibroblasts.” Biochem J. 2014 Mar l;458(2):281-9, the relevant disclosures of which are incorporated by reference herein.

[0043] As used herein, “cyclodextrin” (CD) is a cyclic oligosaccharide with a hydrophilic outer surface and a somewhat lipophilic central cavity. In aqueous solutions, cyclodextrin can form inclusion complexes with many drugs by taking up a drug molecule, or more frequently some lipophilic moiety of the molecule, into the central cavity. See, e.g., WO 2007 / 012974, the relevant disclosures of which are incorporated by reference for the purpose and subject matter referenced herein. It has been shown that beta-cyclodextrin reduced cholesterol accumulation and neuronal cell loss in a mouse model of Niemann-Pick Disease, Type C. See, e.g., WO 2014 / 022841, the relevant disclosures of which are incorporated by reference for the purpose and subject matter referenced herein.

[0044] As used herein, “rimcazole” (“Rim”), i.e., 9-[3-(3, 5-cis-dimethylpiperazino)propyl] - carbazole, refers to a compound having the following structure:

[0045] Rimcazole is known in the art to be an antagonist of the sigma receptor with an IC50 value of 1480 nM and 386 nM for sigma l and sigma 2 / transmembrane protein 97 (“TMEM97”) receptors, respectively. See, e.g., WO 2001 / 074359 and Job et al. “A behavioral economic analysis of the effects of rimcazole on reinforcing effects of cocaine injection and food presentation in rats” Psychopharmacology (Berl) 2019 Dec; 236(12):3601-3612, the relevant disclosures of which are incorporated by reference herein. A method for preparing Rimcazole is described in W02008 / 040950, the contents of which are incorporated herein in their entirety.

[0046] Roluperidone (also referred to herein as “CA”), i.e., lH-Isoindol-l-one,2-[[l-[2-(4- fluorophenyl)-2-oxoethyl]-4-piperidinyl]methyl]-2,3-dihydro-, hydrochloride, hydrate (1:1:2), refers to a compound having the following structure:■ HCI ■ 2H2O

[0047] Roluperidone, which is also referred to as MIN-101, is a novel cyclic amido derivative, which has antagonistic activities for serotoninergic 5-HT2A, sigma2, alA- adrenergic, and to a lesser extent, alB-adrenergic receptors. Roluperidone is useful in the treatment of symptoms of schizophrenia, including negative symptoms of schizophrenia (U.S. Patent No. 9,732,059). Roluperidone may be synthesized using standard synthetic methods and procedures for the preparation of organic molecules and functional group transformations and manipulations, including the use of protective groups, as can be obtained from the relevant scientific literature or from standard reference textbooks in the field.Although not limited to any one or several sources, recognized reference textbooks of organic synthesis include: Smith, M.B.; March, J. March’s Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th ed.; John Wiley & Sons: New York, 2001; and Greene, T.W.; Wuts, P.G. M. Protective Groups in Organic Synthesis, 3rd; John Wiley & Sons: New York, 1999. A method for preparing Roluperidone is described in U.S. Patent No. 7,166,617, the contents of which are incorporated herein in their entirety.

[0048] Roluperidone is a dihydrate of a hydrochloride salt. The “therapeutically effective amount” of Roluperidone referred to in the methods of the disclosure is based on the corresponding amount of the free base form of Roluperidone that is sufficient to treat or prevent a lysosomal storage disorder, or a symptom of a lysosomal storage disorder, as defined herein. For instance, a therapeutically effective amount of 32.0 mg refers to 32.0 mg of the free base, which is equivalent to 38.4 mg of Roluperidone (the dihydrate hydrochloride salt); and a therapeutically effective amount of 64.0 mg refers to 64.0 mg of the free base, which is equivalent to 76.8 mg of Roluperidone (the dihydrate hydrochloride salt).

[0049] Dosage forms comprising Roluperidone are disclosed in U.S. Patent Nos. 9,458,130, 9,730,920, 10,258,614, 10,799,493, 11,464,744, and 12,048,768, each of which is incorporated herein by reference in its entirety. In some embodiments, the methods of this disclosure are performed using any of the dosage forms disclosed therein.

[0050] As used herein, “Sigma-2,” “o-2”, or “Sigma 2” refers to the Sigma-2 receptor. Sigma receptors are a class of receptors that are expressed in many tissues including the central nervous system. It is known in the art that there are two types of sigma receptors: Sigma- 1 receptors and Sigma-2 receptors. Compounds that exhibit selective binding to Sigma-2 are known to be associated with dystonia and may block calcium channels. See, e.g., WO 1991 / 009594, the relevant disclosures of which are incorporated by reference for the purpose and subject matter referenced herein.Methods

[0051] The disclosure provides methods of treating lysosomal storage disorders, or a symptom of a lysosomal storage disorder (LSD), in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of Roluperidone.

[0052] The disclosure also provides methods of preventing a lysosomal storage disorder, or a symptom of lysosomal storage disorder (LSD), in a subject in need thereof, wherein the method comprises administering to the subject a prophylactically effective amount of Roluperidone.

[0053] In some embodiments, the lysosomal storage disorder is cholesteryl ester storage disease, Wolman disease, Hunter syndrome, Hurler’s disease, Hurler syndrome, Scheie syndrome, Hurler-Scheie syndrome, mucopolysaccharidosis (MPS) type III (SanFilippo syndrome), Morquio syndrome A, Morquio syndrome B, Maroteaux-Lamy syndrome, Sly syndrome, Fabry disease, Gaucher disease, Krabbe disease (globoid cell leukodystrophy), metachromatic leukodystrophy, Niemann-Pick disease, Sandhoff disease, Tay-Sachs disease, alpha mannosidosis, beta mannosidosis, Schindler disease, aspartylglucosaminuria, fucosidosis, neuronal ceroid lipofuscinosis (NCL), galactosialidosis (enzyme protection protein defect), infantile sialic acid storage disease, Salla disease (transmembrane protein defect), sialuria, mucolipidosis, cystinosis, Danon disease, multiple sulfatase deficiency, proteins GM2-AP deficiency (variant AB), Farber disease, lysosomal acid lipase deficiency (early onset or late onset), Morquio (MPS IVA / Type A), Morquio (MPS IVB / Type B), hyaluronidase deficiency, Pycnodysostosis, or Pompe disease.

[0054] In some embodiments, the MPS type III is MPS type IIIA (caused by a deficiency of sulfamidase), MPS type IIIB (caused by a deficiency of a-A-acetylglucosaminidase), MPS type IIIC (caused by a deficiency of heparan acetyl CoA: a-glucosaminide N- acetyltransferase), MPS type IIID (caused by a deficiency of A-acetylglucosamine 6-sulfatase); or MPS type IDE (caused by a deficiency of IV-glucosamine 3-O-sulfatase) (see Fedele, A. O. “Sanfilippo syndrome: causes, consequences, and treatments,” Appl Clin Genet. 2015 Nov 25;8:269-81. doi: 10.2147 / TACG.S57672. PMID: 26648750; PMCID: PMC4664539.).

[0055] In some embodiments, the Gaucher disease is Gaucher disease type I, Gaucher disease type II, or Gaucher disease type III.

[0056] In some embodiments, the neuronal ceroid lipofuscinosis (NCL) is Type 1 NCL (i.e., infantile NCL or Santavuori-Haltia disease), Type 2 NCL (i.e., late infantile NCL or Jansky- Bielschowsky disease), Type 3 NCL (i.e., juvenile NCL or Batten disease), Type 4 NCL (i.e., adult NCL or Kufs disease), Type 5 NCL (i.e., Finnish late infantile NCL), Type 6 NCL (i.e., variant late infantile NCL), Type 7 NCL, Type 8 NCL (i.e., Northern epilepsy or progressive epilepsy with mental retardation), Type 8 NCL Turkish late infantile variant, Type 9 NCL, Type 10 NCL, Type 11 NCL, Type 12 NCL, Type 13 NCL, or Type 14 NCL.

[0057] In some embodiments, the mucolipidosis is sialidosis type I (i.e., cherry-red spot myoclonus syndrome), sialidosis type II (i.e., mucolipidosis type I), I-cell disease (i.e., mucolipidosis type II), pseudo-Hurler-poly dystrophy (i.e., mucolipidosis type III), or mucolipidosis type IV.

[0058] In some embodiments, the Niemann-Pick disease is Niemann-Pick disease type A, Niemann-Pick disease type B, or Niemann-Pick disease type C. In some embodiments, the Niemann-Pick disease type C is Niemann-Pick disease type Cl. In some embodiments, the Niemann-Pick disease type C is Niemann-Pick disease type C2.

[0059] In some embodiments, the LSD is characterized by a mutation in the gene encoding NPC intracellular cholesterol transporter 1 (NPC1). NPC1 is a gene encoding a protein that resides in the limiting membrane of endosomes and lysosomes and mediates intracellular cholesterol trafficking through the binding of cholesterol to its N-terminal domain. In some embodiments the mutation in NPC1 results in Niemann-Pick disease type C. In some embodiments, the NPC1 mutation is a T3182— >C transition in exon 21. In some embodiments, this mutation leads to a II 061 T substitution. In some embodiments an II 06 IT substitution affects a putative transmembrane domain protein in NPC1. In some embodiments the II 06 IT substitution leads to hepatosplenomegaly and severe progressive neurological dysfunction in the subject. (See Millat, G. et. al. “Niemann-Pick Cl disease: the I1061T substitution is a frequent mutant allele in patients of Western European descent and correlates with a classic juvenile phenotype” Am J Hum Genet. 1999 Nov;65(5): 1321-9.

[0060] In some embodiments, the administration of Roluperidone does not result in a change in NPC1 expression in the subject.

[0061] In some embodiments, the LSD is characterized by a mutation in the gene encoding NPC intracellular cholesterol transporter 2 (NPC2) gene, the gene encoding sphingomyelin phosphodiesterase 1 (SMPD1), or the gene encoding acid sphingomyelinase (ASM).

[0062] In some embodiments, the ASM mutation is an H421Y mutation, a K576N mutation, an L137P mutation, an fsP189 mutation, an L549P mutation, an S379P mutation, an R441X mutation, an R474W mutation, an F480L mutation, an A196P mutation, a DeltaR608 mutation, an R496L mutation, an fsP330 mutation, or an L302P mutation (https: / / nnpdf.org / diseases / ).

[0063] In some embodiments, symptoms of a lysosomal storage disorder comprise high cholesterol, hepatosplenomegaly (z.e., an enlarged liver and spleen), ataxia, vertical supranuclear gaze palsy, hypotonia, liver disease, respiratory infection, difficulty with speech, difficulty with swallowing and feeding, loss of cognitive skills, seizure, or any combination thereof.

[0064] In some embodiments, the LSD results in an increase in cholesterol levels, cholesterol content, and / or cholesterol accumulation in a subject when compared to a healthy subject. In some embodiments, the increase in cholesterol is associated with an increase in low-density lipoprotein (LDL) particles. In some embodiments the increase in cholesterol results in an increase in lysosomal size as measured by geometric mean ratio fold change when compared to healthy lysosomes. In some embodiments, the cholesterol levels are determined using filipin staining. In some embodiments, the cholesterol levels are determined through analysis of a patient blood sample in a lipid panel.

[0065] In some embodiments, the subject is an adult (i.e., 18 years of age or older).

[0066] In some embodiments, the subject is a pediatric subject (i.e., less than 18 years of age).

[0067] In some embodiments, the subject has a lysosomal storage disorder. In some embodiments, the subject does not have a lysosomal storage disorder, but has one or more diseases with symptoms similar to those of a lysosomal storage disorder. In some embodiments, the symptoms include high cholesterol. In some embodiments, the subject has cancer, chronic kidney disease, diabetes, HTV / AIDS, Lupus, or Alzheimer’s disease.

[0068] In some embodiments, provided herein are methods of reducing cholesterol in a subject in need thereof, wherein the method comprises administering to the subject atherapeutically effective amount of Roluperidone. In some embodiments, provided herein are methods of reducing cholesterol levels in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of Roluperidone. In some embodiments, provided herein are methods of reducing cholesterol content in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of Roluperidone. In some embodiments, provided herein are methods of reducing cholesterol accumulation in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of Roluperidone. As used herein, a reduction in cholesterol, cholesterol levels, cholesterol content, and / or cholesterol accumulation in the subject refers to a reduction as compared to the cholesterol, cholesterol levels, cholesterol content, and / or cholesterol accumulation in the subject prior to initiating Roluperidone administration.

[0069] In some embodiments, provided herein are methods of reducing lysosomal size in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of Roluperidone. As used herein, a reduction in lysosomal size in the subject refers to a reduction as compared to the lysosomal size of the subject prior to initiating Roluperidone administration.

[0070] In some embodiments, provided herein are methods of simultaneously reducing cholesterol accumulation and lysosomal size in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of Roluperidone.

[0071] In some embodiment, the administration of Roluperidone to the subject results in at least one of: (i) a reduction of cholesterol accumulation in the subject, and (ii) a reduction of the lysosomal size of the subject.

[0072] In some embodiments, the administration of Roluperidone to the subject results in a reduction of cholesterol accumulation in the subject. In some embodiments, the administration of Roluperidone to the subject results in a reduction of the lysosomal size of the subject. In some embodiments, the administration of Roluperidone to the subject results in a reduction of the lysosomal size and the cholesterol accumulation of the subject. In some embodiments, the administration of Roluperidone to the subject results in a reduction of the lysosomal size in the brain, spleen, liver, lungs, and / or bone marrow of the subject.

[0073] In some embodiments, the cholesterol accumulation in the subject is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70%after administration of Roluperidone, as compared to the cholesterol accumulation in the subject prior to initiating Roluperidone administration.

[0074] In some embodiments, the cholesterol accumulation is reduced in the subject by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% after administration of Roluperidone to the subject, as compared to the cholesterol accumulation in the subject prior to initiating Roluperidone administration.

[0075] In some embodiments, the cholesterol accumulation is reduced in the subject by 10- 80%, 20-70%, 30-60%, or 40-50% after administration of Roluperidone to the subject, as compared to the cholesterol accumulation in the subject prior to initiating Roluperidone administration.

[0076] In some embodiments, the lysosomal size is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% after administration of Roluperidone to the subject, as compared to the lysosomal size of the subject prior to initiating Roluperidone administration.

[0077] In some embodiments, the lysosomal size is reduced about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% after administration of Roluperidone to the subject, as compared to the lysosomal size of the subject prior to initiating Roluperidone administration.

[0078] In some embodiments, the lysosomal size is determined using fluorescence microscopy. In some embodiments, the lysosomal size is determined through cytofluorimetric assays using Lysotracker™ (Life Technology #L7528) fluorescence as a tool. LysoTracker™ provides fluorescence detection for live-cell staining of acidic environments and can be used for labeling and tracing acidic organelles such as lysosomes. In some embodiments, stained and unstained cells can be analyzed by Cytofluorimeter CytoFLEX (Beckman Coulter). In some embodiments, the data provided by the Cytofluorimeter CytoFLEX analysis is the ratio of geometric (geo) mean of LysoTracker™ levels in stained divided by unstained samples. In some embodiments, the lysosomal size is determined using electron microscopy.

[0079] In some embodiments, the reduction of cholesterol accumulation in the subject occurs within 24 hours, within 48 hours, or within 72 hours of the administration of Roluperidone.

[0080] In some embodiments, the reduction of lysosomal size occurs to the subject within 24 hours, within 48 hours, or within 72 hours of the administration of Roluperidone.

[0081] The disclosure provides methods of treating Alzheimer’s disease, or a symptom of Alzheimer’s disease, in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of Roluperidone.

[0082] The disclosure also provides methods of preventing Alzheimer’s disease, or a symptom of Alzheimer’s disease, in a subject in need thereof, wherein the method comprises administering to the subject a prophylactically effective amount of Roluperidone.

[0083] In some embodiments, the therapeutically or prophylactically effective amount of Roluperidone is administered to the subject once or twice a day.

[0084] In some embodiments, the therapeutically or prophylactically effective amount of Roluperidone is administered to the subject once a day.

[0085] In some embodiments, the therapeutically or prophylactically effective amount of Roluperidone is administered to the subject twice a day.

[0086] In some embodiments, the therapeutically or prophylactically effective amount of Roluperidone is administered orally to the subject.

[0087] In some embodiments, the therapeutically or prophylactically effective amount of Roluperidone is between about 1 mg to about 100 mg, between about 4 mg to about 96 mg, between about 5 mg to about 90 mg, between about 6 mg to about 85 mg, between about 16 mg to about 80 mg, between about 25 mg to about 75 mg, or between about 30 mg to about 70 mg.

[0088] In some embodiments, the therapeutically or prophylactically effective amount of Roluperidone is between 1 mg to 100 mg, between 4 mg to 96 mg, between 5 mg to 90 mg, between 6 mg to 85 mg, between 16 mg to 80 mg, between 25 mg to 75 mg, or between 30 mg to 70 mg.

[0089] In some embodiments, the therapeutically or prophylactically effective amount of Roluperidone is about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg, about 27 mg, about 28 mg, about 29 mg, about 30 mg, about 31 mg, about 32 mg, about 33 mg, about 34 mg, about 35 mg, about 36 mg, about 37 mg, about 38 mg, about 39 mg, about 40 mg, about 41 mg, about 42 mg, about 43 mg, about 44 mg, about 45 mg, about 46 mg, about 47 mg, about 48 mg, about 49 mg, about 50 mg,about 51 mg, about 52 mg, about 53 mg, about 54 mg, about 55 mg, about 56 mg, about 57 mg, about 58 mg, about 59 mg, about 60 mg, about 61 mg, about 62 mg, about 63 mg, about 64 mg, about 65 mg, about 66 mg, about 67 mg, about 68 mg, about 69 mg, about 70 mg, about 71 mg, about 72 mg, about 73 mg, about 74 mg, about 75 mg, about 76 mg, about 77 mg, about 78 mg, about 79 mg, about 80 mg, about 81 mg, about 82 mg, about 83 mg, about 84 mg, about 85 mg, about 86 mg, about 87 mg, about 88 mg, about 89 mg, about 90 mg, about 91 mg, about 92 mg, about 93 mg, about 94 mg, about 95 mg, about 96 mg, about 97 mg, about 98 mg, about 99 mg, or about 100 mg.

[0090] In some embodiments, the therapeutically or prophylactically effective amount of Roluperidone is 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg, 61 mg, 62 mg, 63 mg, 64 mg, 65 mg, 66 mg, 67 mg, 68 mg, 69 mg, 70 mg, 71 mg, 72 mg, 73 mg, 74 mg, 75 mg, 76 mg, 77 mg, 78 mg, 79 mg, 80 mg, 81 mg, 82 mg, 83 mg, 84 mg, 85 mg, 86 mg, 87 mg, 88 mg, 89 mg, 90 mg, 91 mg, 92 mg, 93 mg, 94 mg, 95 mg, 96 mg, 97 mg, 98 mg, 99 mg, or 100 mg.

[0091] In some embodiments, the therapeutically or prophylactically effective amount of Roluperidone is about 16 mg, about 24 mg, about 32 mg, about 40 mg, about 48 mg, about 56 mg, about 64 mg, about 72 mg, about 80 mg, about 88 mg, or about 96 mg.

[0092] In some embodiments, the therapeutically or prophylactically effective amount of Roluperidone is 16 mg, 24 mg, 32 mg, 40 mg, 48 mg, 56 mg, 64 mg, 72 mg, 80 mg, 88 mg, or 96 mg.

[0093] In some embodiments, the therapeutically or prophylactically effective amount of Roluperidone is 16 mg.

[0094] In some embodiments, the therapeutically or prophylactically effective amount of Roluperidone is about 16 mg.

[0095] In some embodiments, the therapeutically or prophylactically effective amount of Roluperidone is 24 mg.

[0096] In some embodiments, the therapeutically or prophylactically effective amount of Roluperidone is about 24 mg.

[0097] In some embodiments, the therapeutically or prophylactically effective amount of Roluperidone is 32 mg.

[0098] In some embodiments, the therapeutically or prophylactically effective amount of Roluperidone is about 32 mg.

[0099] In some embodiments, the therapeutically or prophylactically effective amount of Roluperidone is 40 mg.

[0100] In some embodiments, the therapeutically or prophylactically effective amount of Roluperidone is about 40 mg.

[0101] In some embodiments, the therapeutically or prophylactically effective amount of Roluperidone is 48 mg.

[0102] In some embodiments, the therapeutically or prophylactically effective amount of Roluperidone is about 48 mg.

[0103] In some embodiments, the therapeutically or prophylactically effective amount of Roluperidone is 56 mg.

[0104] In some embodiments, the therapeutically or prophylactically effective amount of Roluperidone is about 56 mg.

[0105] In some embodiments, the therapeutically or prophylactically effective amount of Roluperidone is 64 mg.

[0106] In some embodiments, the therapeutically or prophylactically effective amount of Roluperidone is about 64 mg.

[0107] In some embodiments, the subject is administered Roluperidone in combination with one or more LSD treatments. In some embodiments, the one or more LSD treatments comprises treatment with recombinant enzymes or enzymes alone or in combination with chaperones, fusion proteins such as enzymes fused to HIRMAb, miglustat, gene therapy, intravenous administration of insulin growth factor II fusion protein, stem cell transplants, enzyme replacement therapy, substrate reduction therapy, pharmacological chaperone therapy, immunosuppressants, nonsteroidal anti-inflammatory drugs (NSAIDs), or any combination thereof. See Beck, M. (2018), Treatment strategies for lysosomal storage disorders. Dev Med Child Neurol, 60: 13-18.

[0108] The details of one or more embodiments of the disclosure are set forth in the accompanying description above. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. Other features, objects, and advantagesof the disclosure will be apparent from the description and from the claims. In the specification and the appended claims, the singular forms include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents and publications cited in this specification are incorporated by reference.

[0109] The foregoing description has been presented only for the purposes of illustration and is not intended to limit the disclosure to the precise form disclosed, but by the claims appended hereto.EXAMPLESExample 1:

[0110] Cell culture

[0111] Healthy and I1061T fibroblasts (Coriell Institute Camden USA, cod. GM05659 (Healthy) and GM18453 (1106 IT)) were grown in air containing 5% CO2 in Dulbecco's Modified Eagle Medium (DMEM) medium, containing 10% v / v fetal calf serum, 2 mM L- glutamine, 0.1 mg / mL gentamicin, and 100 U / mL penicillin. Cells were passaged every 3 days.

[0112] Roluperidone effect on NPC1 protein levels

[0113] To evaluate the effect of Roluperidone onNPCl protein levels, I1061T cells were treated for 24 hours (FIG. IB), 48 hours (FIG. 1C), and 72 hours (FIG. ID) with Roluperidone (CA) at three different concentrations (0.3 pM, 1 pM, and 3 pM), a vehicle control (Ctr), or a positive control, 2.5 pM Rimcazole (Rim) (Tocris #1497). 1.5 mg of each of the compounds were weighed, dissolved in the proper solvent to reach a 3 mM concentration, kept at 4°C, and used within 15 days. The dilutions for the treatment were carried out in order to administer a 1 : 1000 volume. Specifically, 2 pL in 2 mL medium was administered. Ctr was made with DMSO.

[0114] Western Blot Analysis

[0115] Cells were washed twice in phosphate buffer saline (PBS), harvested with 1% v / v trypsin, and pelleted by centrifugation. Pellets were re-suspended in PBS containing 0.1 M sucrose, 0.05 M KC1, 0.04 M KH2PO4, 30 mM EDTA, pH 7.4, and sonicated. The protein concentration was determined by the Lowry method.

[0116] Samples were solubilized in 0.25 mM Tris-HCl, (pH 6.8) containing 20% (w / v) SDS, protease inhibitor cocktail (Merck #PIC0002), then boiled for 2 minutes. 10 pg of solubilized proteins were resolved by SDS-PAGE at 100 V for 1 hour and then electrophoretically transferred to nitrocellulose with Turboblot (Biorad) for 10 min at 1.3 A and 25 V. The nitrocellulose was treated with 3% (w / v) bovine serum albumin (BSA) in Tris Buffered Saline (20 mM Trizma base, 137 mM NaCl, 0.1%, v / v, Tween-20, pH 7.6) and then probed overnight with the anti-NPCl antibodies (1 :5000 Novus Biological #400148). The nitrocellulose was successively incubated for 1 hour at room temperature with HRP- conjugated secondary anti-rabbit antibodies (Bio-Rad Laboratories, Milan, Italy). Proteinbound antibodies were visualized by clarity Enhanced Chemiluminescence (ECL) Western blotting (Bio-Rad Laboratories, Milan, Italy, #1705061), and chemiluminescence was registered through the ChemiDoc MP system (Bio-Rad Laboratories, Milan, Italy).Densitometric analysis derived from Western blots was then carried out using ImageJ version 1.52t (National Institutes of Health, Bethesda, MD, USA) software for Windows. Vinculin was used as a housekeeping protein, which served as an internal control for protein loading. Densitometric calculations were obtained as arbitrary units, derived from the ratio between the intensity of protein band and the housekeeping protein.

[0117] Without wishing to be bound by theory, FIG. 1A demonstrates that there was a strong difference between healthy and I1061T fibroblasts in terms ofNPCl protein content demonstrating that the above method is sensitive in detecting it. FIGS. 1B-1D demonstrate that Roluperidone did not have a statistically significant effect on NPC1 protein expression within the time period and conditions described in Example 1 indicating that physiological changes that occur after treatment with Roluperidone may be independent of the function of NPC1.Example 2:

[0118] Filipin staining

[0119] Healthy and H061T fibroblasts were cultured as in Example 1. To evaluate the effect of Roluperidone on cholesterol accumulation, cells were treated with either 3 pM Roluperidone, 300 pM beta-cyclodextrin, or 2.5 pM Rim for 72 hours. Following treatment, the cells were stained with filipin. A 10 mg / mL filipin stock solution (in PBS) was prepared immediately before use. Cells were fixed in paraformaldehyde (4% solution in PBS) for 10 minutes and washed with PBS. Fibroblasts were then stained with 1 mL filipin workingsolution (0.05 mg / mL in PBS) for 1 hour in the dark, at room temperature. Subsequently, cells were washed three times with PBS, and coverslips were mounted with Fluoroshield™ mounting medium and immediately analyzed through confocal microscopy using a UV filter set (340-380 nm excitation). Images were acquired at 40X magnification (FIG. 2A). Filipin quantification was calculated as mean fluorescence intensity per cell area by using Image! software for Windows (FIG. 2B).

[0120] Without wishing to be bound by theory, Example 2 demonstrates that II 06 IT cells had increased cholesterol accumulation compared to healthy cells (FIG. 2B). Treatment with Roluperidone was able to reduce unesterified cholesterol accumulation in 1106 IT cells demonstrating its potential to be used in subjects with LSDs like Niemann-Pick disease. Additionally, since Example 1 indicates that Roluperidone may function independently of NPC1, the results of Example 2 demonstrate that Roluperidone has the potential to be used in the treatment of high cholesterol broadly, such as, for example, in the treatment of aberrant cholesterol accumulation observed in diseases including cancer and Alzheimer’s disease (see Yang et al., T. “Sigma-2 Receptor — A Potential Target for Cancer / Alzheimer’s Disease Treatment via Its Regulation of Cholesterol Homeostasis.” Molecules 2020, 25, 5439, the relevant disclosures of which are incorporated by reference for the purpose and subject matter referenced herein).Example 3:

[0121] LysoTracker™ Analysis

[0122] Healthy and 1106 IT fibroblasts were cultured as in Example 1 and treated as in Example 2. To evaluate the effect of Roluperidone on lysosomal size, cytofluorimetric assays using Lysotracker™ (Life Technology #L7528) fluorescence as a tool was used and performed according to the manufacturer’s instructions. LysoTracker™ provides fluorescence detection for live-cell staining of acidic environments and can be used for labeling and tracing acidic organelles such as lysosomes. Stained and unstained cells were analyzed by Cytofluorimeter CytoFLEX (Beckman Coulter). Data provided are the ratio of geometric (geo) mean of LysoTracker™ levels in stained divided by unstained samples, collected at 24 hours (FIGS. 3-4, left), 48 hours (FIGS. 3-4, middle), and 72 hours (FIGS. 3-4, right). Example 3 was repeated in duplicate, and the results are illustrated in FIGS. 3-4.

[0123] Without wishing to be bound by theory, Example 3 demonstrates that there was a statistically significant increase in the lysosomal size of I1061T fibroblasts when compared tohealthy cells (FIG. 3). Treatment with Roluperidone showed a trend towards reduced fluorescence of the lysosomes in the I1061T fibroblasts after 48 hours, particularly at a concentration of 0.3 pM, representing a decrease in lysosomal size (FIGs. 3-4). In contrast, rimcazole (Rim) showed a statically significant increase of the signal at 72 hours, representing an increase in lysosomal size (FIGs. 3-4). Although Rim was able to reduce unesterified cholesterol accumulation in the 1106 IT fibroblasts, it was unable to decrease the lysosomal size of I1061T fibroblasts. In contrast, treatment with Roluperidone resulted in a reduction in both unesterified cholesterol accumulation and a reduction in lysosomal size further demonstrating its potential for use in patients with LSDs like Niemann-Pick disease.Example 4:

[0124] NPC1 and Lamp2 co-localization

[0125] NPC1 protein localization at the lysosome is important to perform its function as a cholesterol transporter. Although the NPC1 protein is mutated in I1061T fibroblasts, it is still functional. Once produced, it is retained in the endoplasmic reticulum, before being degraded by cell quality control system. The ability to successfully relocate the residual NPC1 protein to lysosomes could be a strategy to rescue, at least in part, NPC1 physiologic function in I106IT fibroblasts. Example 4 evaluates the effect of Roluperidone on the localization ofNPCl protein at the lysosome using co-immunolocalization experiments with Lamp2 as a lysosomal marker.

[0126] Healthy and I1061T fibroblasts were cultured as in Example 1. To evaluate the effect of Roluperidone on NPC1 localization, Healthy and I1061T fibroblasts were treated with either vehicle (Ctr), 2.5 pM rimcazole (Rim), or 3 pM Roluperidone for 72 hours. Following treatment, the cells were fixed in 4% paraformaldehyde for 5 minutes before adding methanol (-20°C) for 2 minutes. Permeabilization was then performed using 0.1% Triton for 5 min, then the reaction was blocked with 3% BSA in PBS containing 0.1% Triton. NPC1 antibody (1 TOO, Novus Biological #400148) and LAMP2 (1 :50, Santa Cruz abl 8822) were added and maintained overnight at 4 °C. Staining with 4',6-diamidino-2-phenylindole was used as a control of cell nuclei. After washing with PBS, fixed cells were incubated with Alexa-Fluor 543 anti-mouse and 488 anti-rabbit secondary antibodies (Invitrogen, Carlsbad, CA, USA) for 1 hour at room temperature. Images were captured with a confocal microscope (LCS Leica Microsystems, Wetzlar, Germany) (data not shown).

[0127] Data analysis was performed by calculating Pearson’s correlating coefficient using ImageJ version 1.52t (National Institutes of Health, Bethesda, MD, USA) software forWindows (FIG. 5). The experiments were carried out three times. The statistical analysis was performed with one-way ANOVA followed by Dunnett's multiple comparisons test where ****=p<0.0001 vs Healthy.

[0128] Without wishing to be bound by theory, Example 4 demonstrates that co- immuno localization between NPC1 and Lamp2 (as a lysosomal marker) was seen in healthy fibroblasts, however, Rim and Roluperidone did not have a significant effect on NPC1 localization (as evidenced by enhanced signal compared to control).Equivalents

[0129] The details of one or more embodiments of the disclosure are set forth in the accompanying description above. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. Other features, objects, and advantages of the disclosure will be apparent from the description and from the claims. In the specification and the appended claims, the singular forms include plural referents unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents and publications cited in this specification are incorporated by reference.

[0130] The foregoing description has been presented only for the purposes of illustration and is not intended to limit the disclosure to the precise form disclosed, but by the claims appended hereto.

Claims

CLAIMSWhat is claimed is:

1. A method of treating or preventing a lysosomal storage disorder (LSD) in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically or prophylactically effective amount of Roluperidone.

2. A method of treating or preventing a symptom of a lysosomal storage disorder (LSD) in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically or prophylactically effective amount of Roluperidone.

3. The method of claim 1 or 2, wherein the LSD is cholesteryl ester storage disease, Wolman disease, Hunter syndrome, Hurler’s disease, Scheie syndrome, Hurler-Scheie syndrome, mucopolysaccharidosis (MPS) type in, mucopolysaccharidosis (MPS) type IV (Morquio syndrome), Maroteaux-Lamy syndrome, Sly syndrome, Fabry disease, Gaucher disease, Krabbe disease, metachromatic leukodystrophy, Niemann-Pick disease, Sandhoff disease, Tay-Sachs disease, alpha mannosidosis, beta mannosidosis, Schindler disease, aspartylglucosaminuria, fucosidosis, neuronal ceroid lipofuscinosis, galactosialidosis, infantile sialic acid storage disease, Salla disease, sialuria, mucolipidosis, cystinosis, Danon disease, multiple sulfatase deficiency, proteins GM2-AP deficiency, Farber disease, lysosomal acid lipase deficiency, hyaluronidase deficiency, pycnodysostosis, and / or Pompe disease.

4. The method of any one of claims 1-3, wherein the LSD is Niemann-Pick disease.

5. The method of any one of claims 1-3, wherein the LSD is Niemann-Pick disease type C.

6. The method of any one of claims 1-3, wherein the LSD is Niemann-Pick disease type A or B.

7. The method of any one of claims 1-6, wherein the subject has high cholesterol.

8. The method of any one of claims 1-7, wherein the administration of Roluperidone results in a reduction of cholesterol accumulation in the subject and / or a reduction of the lysosomal size of the subject as compared to the cholesterol accumulation and / or the lysosomal size of the subject prior to initiating Roluperidone administration.

9. The method of claim 8, wherein the reduction of cholesterol accumulation in the subject is determined by filipin staining.

10. The method of claim 8 or 9, wherein the reduction of cholesterol accumulation in the subject is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% as compared to the cholesterol accumulation in the subject prior to initiating the administration of Roluperidone.

11. The method of any one of claims 8-10, wherein the reduction of cholesterol accumulation in the subject occurs within 24 hours, within 48 hours, or within 72 hours of the administration of Roluperidone.

12. The method of claim 8, wherein the reduction of lysosomal size of the subject is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% as compared to the lysosomal size of the subject prior to initiating the administration of Roluperidone.

13. The method of claim 8 or 12, wherein the reduction of lysosomal size of the subject occurs within 24 hours, within 48 hours, or within 72 hours of the administration of Roluperidone.

14. The method of any one of claims 1-13, wherein the administration of Roluperidone to the subject does not result in a change in NPC1 expression in the subject as compared to the NPC1 expression in the subject prior to initiating the administration of Roluperidone.

15. The method of any one of claims 1-14, wherein the subject is a pediatric subject.

16. The method of any one of claims 1-14, wherein the subject is an adult.

17. The method of any one of claims 1-16, wherein the method further comprises administering to the subject one or more additional LSD treatment agents.

18. The method of claim 17, wherein the one or more additional LSD treatment agents is selected from recombinant enzymes, enzymes plus chaperones, recombinant enzymes plus chaperones, fusion proteins, miglustat, and insulin growth factor II fusion protein.

19. The method of any one of claims 1-18, wherein the method further comprises administering to the subject a gene therapy.

20. The method of any one of claims 1-19, wherein the therapeutically or prophylactically effective amount of Roluperidone is administered to the subject once a day.

21. The method of any one of claims 1-19, wherein the therapeutically or prophylactically effective amount of Roluperidone is administered to the subject twice a day.

22. The method of any one of claims 1-21, wherein the therapeutically or prophylactically effective amount of Roluperidone is administered orally to the subject.

23. The method of any one of claims 1-22, wherein the therapeutically or prophylactically effective amount of Roluperidone is between about 1 mg and about 100 mg.

24. The method of any one of claims 1-23, wherein the therapeutically or prophylactically effective amount of Roluperidone is about 16 mg, about 24 mg, about 32 mg, about 40 mg, about 48 mg, about 56 mg, about 64 mg, about 72 mg, about 80 mg, about 88 mg, or about 96 mg.

25. The method of any one of claims 1-24, wherein the therapeutically or prophylactically effective amount of Roluperidone is about 32 mg.

26. The method of any one of claims 1-24, wherein the therapeutically or prophylactically effective amount of Roluperidone is about 64 mg.

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