WO2025219951A1

WO2025219951A1 - Venglustat for use in methods for reducing disease related biomarker levels in patients with ganglioside storage disorders

Info

Publication number: WO2025219951A1
Application number: PCT/IB2025/054078
Authority: WO
Inventors: Isabela BATSU; Mikhail Nakhimovitch LEVIT; Sergio Pablo SARDI; Riliang ZHENG
Original assignee: Genzyme Corp
Current assignee: Genzyme Corp
Priority date: 2024-04-19
Filing date: 2025-04-17
Publication date: 2025-10-23
Anticipated expiration: 2026-10-19

Abstract

Provided herein is a method in which venglustat is administered to a patient with a ganglioside storage disorder, e.g. a GM2 gangliosidosis such as Sandhoff disease or Tay-Sachs disease, GM1 gangliosidosis, or sialidosis. The method is designed to reduce the levels of disease related biomarkers in the patient.

Description

VENGLUSTAT FOR USE IN METHODS FOR REDUCING DISEASE RELATED BIOMARKER LEVELS

IN PATIENTS WITH GANGLIOSIDE STORAGE DISORDERS

Provided herein is a method in which venglustat is administered to a patient with a ganglioside storage disorder, e.g. a GM2 gangliosidosis such as Sandhoff disease or Tay-

5 Sachs disease, GM1 gangliosidosis, or sialidosis. The method is designed to reduce the levels of disease related biomarkers in the patient. Also provided are pharmaceutical formulations for use in the instant methods.

BACKGROUND

The GM2 gangliosidoses are ultra-rare, progressive autosomal recessive lysosomal storage

10 disorders with no currently approved treatments. The conditions occur due to pathogenic variants in the HEXA and/or HEXB genes leading to a deficiency in the enzyme - hexosaminidase A (Tay-Sachs disease) or a combined deficiency of -hexosaminidase A and B (Sandhoff disease). A related condition occurs in patients having a deficiency in the GM2 activator protein, encoded by the GMA2 gene (the so-called GM2 AB variant). In each case,

15 affected individuals are impaired in lysosomal degradation of the GM2 ganglioside and other glycolipids, causing accumulation of these in the lysosomes of cells and elsewhere in the body. This accumulation causes cytotoxic effects, especially in neurons, which leads to progressive neurological impairment including motor deficits, progressive weakness, hypotonia, decreased responsiveness, vision deterioration, and seizures. Several potential

20 approaches have been investigated for the treatment of GM2 gangliosidoses, including enzyme replacement therapy (“ERT”, in which a functioning version of the deficient enzyme is injected into the patient), substrate reduction therapy (“SRT”, in which agents, typically small molecule drugs, are administered to modulate the glycosphingolipid pathway in a bid to reduce the accumulation of storage lipids), pharmacological chaperone therapy (in which

25 agents are administered to assist in proper protein folding and trafficking to the lysosome), gene therapy (in which functional versions of the deficient genes are introduced into the patient), and stem cell therapy (in which haematopoietic stem cells are transplanted into the patient). Early studies, including with ERT, showed that increasing or restoring P-hexosaminidase activity in a human patient could impact storage lipids in visceral tissue,

30 but that the impact on neuronal tissue was limited (see, e.g., Johnson et al., Birth Defects Grig. Artic. Ser. (1973) 9: 120-124). Direct administration of therapeutics into the brain, e.g. using gene therapy vectors which carry functional copies of the HEXA or HEXB genes, has shown some promising results in animal models (see, e.g., Gray-Edwards et al., Hum. Gene Ther. (2018) 29:312-326) but has yet to be fully assessed in humans (see, e.g., Flotte et al.,

35 Nat Med. (2022) 28(2) : 251-259 for details of initial results on a trial in two human subjects). Studies on the impact of SRT in treating GM2 gangliosidoses have shown some promise in animal models, but those have not translated into any authorised therapies for human use. For example, iminosugar-based inhibitors of the enzyme glucosylceramide synthase (“GCS”) such as miglustat have been studied in mouse models of GM2 gangliosidoses (see, e.g., Platt

40 et al., Phil. Trans. R. Soc. Lond. B (2003) 358:947-954) and in late onset Tay-Sachs disease patients (See, e.g., Shapiro et al., Genet Med. (2009) 11 (6):425— 433), although this has not led to any authorised treatment. Miglustat, also known as NB-DNJ, is authorised for the treatment of Gaucher disease and Pompe disease, two other lysosomal storage diseases leading to toxic substrate accumulation.

Other ultra-rare conditions exist in which a deficiency in a ganglioside-processing enzyme causes accumulation of storage lipids. GM1 gangliosidosis is a progressive, neurosomatic disorder caused by mutations in p-galactosidase, encoded by the GLB1 gene. -galactosidase cleaves P-linked galactose residues from the non-reducing end of glycan moieties found in various glycoconjugates, such as GM1. Reduction in p-galactosidase activity leads to the accumulation of GM1 ganglioside and its asialo derivative GAI, primarily in lysosomes of neuronal tissue. Clinical symptoms include hepatosplenomegaly, cardiomyopathy, skeletal disease, and seizures, especially in early onset forms of the disease, as well as cerebellar dysfunction and progressive dementia in late onset forms. Several therapeutic strategies have been proposed for treating GM1 gangliosidosis, including several similar strategies to those discussed above in connection with GM2 gangliosidoses. Thus, ERT, SRT, pharmaceutical chaperone therapy, gene therapy and stem cell transplantation have all been suggested. While animal models have been investigated, there are no authorised treatments for the condition - several clinical trials of gene therapies are underway (see, e.g., Rha et al., App. Clin. Genet. (2021) 14:209-233). Sialidosis is caused by a deficiency in the enzyme neuraminidase- 1 (typically due to mutations in the NEU1 gene) which leads to progressive accumulation of oligosaccharides and sialylated glycoproteins in cells. Symptoms of sialidosis may include growth problems, visual defects, myoclonus, ataxia, and seizures. There are no authorised treatments for sialidosis.

Venglustat (also known as (.S)-quiniiclidin-3-yl 2-(2-(4-fluorophenyl)thiazol-4-yl)propan-2- ylcarbamate) is a small molecule drug which has been proposed to be useful in the treatment of conditions including lysosomal storage diseases such as Gaucher disease and Fabry disease (see, e.g., international patent application No. PCT/US2012/029417, published as WO 2012/129084, the contents of which is hereby incorporated by reference in its entirety). It has been suggested that venglustat, which is an inhibitor of the enzyme glucosylceramide synthase (GCS), might act in these treatments by reducing levels of the glycosphingolipid GL1. Early phase clinical studies have shown that venglustat exhibits an acceptable safety profde when administered in dosages of up to around 15 mg per day to healthy individuals (see, e.g., Peterschmitt et al., Clin Pharmacol Drug Dev (2021) 10( 1): 86— 98). The impact of venglustat in human patients with ganglioside storage disorders has not been reported.

In the absence of any authorised treatment options, there is an urgent need for agents which can impact the pathophysiology and/or clinical course of GM2 gangliosidoses and other ganglioside storage disorders.

SUMMARY

The present disclosure describes the results of clinical studies which monitored the impact of venglustat on biomarkers of gangliosidoses in human patients. These results have enabled the development of methods which are designed to reduce the levels of specific biomarkers in the plasma and/or cerebrospinal fluid (CSF) of the patient. Those biomarkers include sphingolipid-containing compounds such as GM1, GM2, and GM3 which are postulated to have a cytotoxic effect in vivo. Without wishing to be bound by theory, the present application is thus directed to methods in which administration of venglustat to a human patient reduces the level of these disease-related sphingolipid-containing compounds. The methods may additionally reduce the levels of proteins associated with lysosomal storage diseases and/or neurodegeneration in the CSF, as set out below.

Thus, in a first aspect the disclosure provides a method of reducing the level of a biomarker in a biological fluid of a human subject in need thereof suffering from a ganglioside storage disorder, the method comprising administering to the subject an effective amount of venglustat or a pharmaceutically acceptable salt thereof, wherein the biomarker is selected from the group consisting of GM1, GM2, GM3, GL1, and combinations thereof.

In a further aspect, the disclosure provides a method of administering venglustat, or a pharmaceutically acceptable salt thereof, to a human subject in need thereof suffering from a ganglioside storage disorder, wherein the venglustat or pharmaceutically acceptable salt thereof is administered in an effective amount to reduce the level of a biomarker in a biological fluid of the subject.

In embodiments, the biological fluid is selected from the group consisting of plasma and CSF.

In embodiments, the biomarker is selected from: (a) GM1, optionally in combination with GM3 and/or GL1; (b) GM2, optionally in combination with GM3 and/or GL1; and (c) GM3, optionally in combination with GM2 and/or GL1.

In embodiments, the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of the biomarker by at least about 30%, e.g. by at least about 40%, 50%, 60%, 70% or 80%.

In embodiments, the venglustat or pharmaceutically acceptable salt thereof is administered orally, e.g. in the form of a tablet or capsule.

In embodiments, the venglustat or pharmaceutically acceptable salt thereof is administered once daily.

In embodiments, either the subject is aged 18 or over and the venglustat or pharmaceutically acceptable salt thereof is administered in a dosage (calculated as the free base) of about 15 mg per day; or the subject is aged below 18 and the venglustat or pharmaceutically acceptable salt thereof is administered in a dosage (calculated as the free base) of:

(a) about 15 mg per day to a subject having a body weight of > 50 kg;

(b) about 12 mg per day to a subject having a body weight of 30 kg to < 50 kg;

(c) about 6 mg per day to a subject having a bodyweight of 15 kg to < 30 kg; or

(d) about 4 mg per day to a subject having a bodyweight of 10 kg to < 15 kg. In embodiments, the venglustat is in the form of venglustat free base, or a pharmaceutically acceptable salt of venglustat, optionally venglustat L-malate salt.

In embodiments, the ganglioside storage disorder is selected from a GM2 gangliosidosis (e.g., Sandhoff disease, or Tay-Sachs disease), and GM1 gangliosidosis.

In a further aspect, the disclosure provides a method of reducing the level of a biomarker in a biological fluid of a human subject in need thereof suffering from a GM2 gangliosidosis, the method comprising administering to the subject an effective amount of venglustat or a pharmaceutically acceptable salt thereof, wherein the biomarker is GM2, optionally in combination with GM3 and/or GL1.

In embodiments: (a) the GM2 gangliosidosis is Tay-Sachs disease and the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of GM2 in the CSF by at least about 30%; or (b) the GM2 gangliosidosis is Sandhoff disease and the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of GM2 in the CSF by at least about 40%.

In embodiments, the GM2 gangliosidosis is juvenile GM2 gangliosidosis and the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of (or is effective to reduce the level of) GM2 in the CSF by at least about 30%.

In embodiments, the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of GM2 in the plasma to a level between about 200 and about 750 ng/mL.

In a further aspect, the disclosure provides a method of reducing the level of a biomarker in a biological fluid of a human subject in need thereof suffering from GM1 gangliosidosis, the method comprising administering to the subject an effective amount of venglustat or a pharmaceutically acceptable salt thereof, wherein the biomarker is GM1, optionally in combination with GM3 and/or GL1.

In embodiments, the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of GM1 in the CSF to a level between about 25 and 65 ng/mL, and/or reduces the level of GM1 in the plasma to a level between about 40 and about 115 ng/mL.

In embodiments, the ganglioside storage disorder from which the subject suffers is a GM2 gangliosidosis, and: (i) the method also reduces the level of CD63 in the CSF of the subject; (ii) the venglustat or pharmaceutically acceptable salt thereof is administered in an effective amount to also reduce the level of CD63 in the CSF of the subject; or (iii) the administration of an effective amount of venglustat or a pharmaceutically acceptable salt thereof also reduces the level of CD63 in the CSF of the subject.

In embodiments: (a) the GM2 gangliosidosis is adult Tay-Sachs disease, or adult Sandhoff disease, and the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of (or is effective to reduce the level of) CD63 in the CSF by at least about 30%; or (b) the GM2 gangliosidosis is juvenile GM2 gangliosidosis and the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of (or is effective to reduce the level of) CD63 in the CSF by at least about 35%.

In embodiments, the ganglioside storage disorder from which the subject suffers is juvenile GM2 gangliosidosis, and: (i) the method also reduces the level of NFL in the CSF of the subject; (ii) the venglustat or pharmaceutically acceptable salt thereof is administered in an effective amount to also reduce the level of NFL in the CSF of the subject; or (iii) the administration of an effective amount of venglustat or a pharmaceutically acceptable salt thereof also reduces the level of NFL in the CSF of the subject.

In embodiments, the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of (or is effective to reduce the level of) NFL in the CSF by at least about 50%.

A further aspect of the disclosure provides venglustat, or a pharmaceutically acceptable salt thereof, for use in a method as defined hereinbefore.

A further aspect of the disclosure provides use of venglustat, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for use in a method as defined hereinbefore.

Additional features and advantages of the methods disclosed herein will be apparent from the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows a part of the glycosphingolipid pathway, highlighting the relationship between various ganglioside glycosphingolipids (GM1, GM2, GM3), the enzymes which are responsible for the processing of those glycosphingolipids and the diseases which arise as a result of mutations in the corresponding genes.

Fig. 2 shows the level of GM2 in the primary population (patients with Tay-Sachs disease or Sandhoff disease) who received venglustat or placebo. Results are shown for CSF GM2 levels at week 104 (Fig. 2A) and plasma GM2 levels from weeks 12-104 (Fig. 2B).

Fig. 3 shows the level of GM2 in patients with Tay-Sachs disease and Sandhoff disease who received venglustat or placebo. Results are shown for CSF GM2 levels at week 104 (Fig. 3A) and plasma GM2 levels from weeks 12-104 (Fig. 3B).

Fig. 4 shows the level of GM3 in patients with Tay-Sachs disease and Sandhoff disease who received venglustat or placebo. Results are shown for CSF GM3 levels at week 104 (Fig. 4A) and plasma GM3 levels from weeks 12-104 (Fig. 4B). indicates that the biomarker was not measured. Fig. 5 shows the level of GL1 in patients with Tay-Sachs disease and Sandhoff disease who received venglustat or placebo. Results are shown for CSF GL1 levels at week 104 (Fig. 5A) and plasma GL1 levels from weeks 12-104 (Fig. 5B).

Fig. 6 shows the correlation between baseline hexosaminidase levels and the change in GM2 level in CSF between baseline and 104 weeks in the primary population (patients with Tay- Sachs disease and Sandhoff disease) who received venglustat. The Spearman coefficient of correlation is shown.

Fig. 7 shows the level of GM1 in patients with GM1 gangliosidosis who received venglustat. Results are shown for CSF GM1 levels at week 104 (Fig. 7A) and plasma GM1 levels from weeks 12-104 (Fig. 7B). indicates that the biomarker was not measured.

Fig. 8 shows the level of GM2 in patients with a GM2 gangliosidosis who received venglustat. Results are shown for CSF GM2 levels at week 104 (Fig. 8A) and plasma GM2 levels from weeks 12-104 (Fig. 8B). indicates that the biomarker was not measured.

Fig. 9 shows the level of GL1 in patients with GM1 and GM2 gangliosidoses who received venglustat. Results are shown for CSF GL1 levels at week 104 (Fig. 9A) and plasma GL1 levels from weeks 12-104 (Fig. 9B).

Fig. 10 shows the level of CD63 at baseline in CSF samples from patients in the primary and secondary populations. The box plots show, from left-to-right: patients in the primary population who received placebo (n=15); patients in the primary population who received venglustat (n=30); patients with juvenile GM2 gangliosidosis in the secondary population who received venglustat (n=7); and patients with GM1 gangliosidosis in the secondary population who received venglustat (n=4). The y-axis denotes the CD63 levels in relative units on a linear scale.

Fig. 11 shows the percent change in level of CD63 at week 104 (as compared to baseline) in CSF samples from patients in the primary and secondary populations. The box plots show, from left-to-right: patients in the primary population who received placebo (n=15); patients in the primary population who received venglustat (n=29); patients with juvenile GM2 gangliosidosis in the secondary population who received venglustat (n=7); and patients with GM1 gangliosidosis in the secondary population who received venglustat (n=4).

Fig. 12 shows the level of NFL at baseline in CSF samples from patients in the primary and secondary populations. The box plots show, from left-to-right: patients in the primary population who received placebo (n=15); patients in the primary population who received venglustat (n=30); patients with juvenile GM2 gangliosidosis in the secondary population who received venglustat (n=7); and patients with GM1 gangliosidosis in the secondary population who received venglustat (n=4).

Fig. 13 shows the percent change in level of NFL at week 104 (as compared to baseline) in CSF samples from patients in the primary and secondary populations. The box plots show, from left-to-right: patients in the primary population who received placebo (n=15); patients in the primary population who received venglustat (n=30); patients with juvenile GM2 gangliosidosis in the secondary population who received venglustat (n=7); and patients with GM1 gangliosidosis in the secondary population who received venglustat (n=4).

DETAILED DESCRIPTION

Although specific embodiments of the present disclosure will now be described with reference to the preparations and schemes, it should be understood that such embodiments are by way of example only and merely illustrative of but a small number of the many possible specific embodiments which can represent applications of the principles of the present disclosure. Various changes and modifications will be obvious to those of skill in the art given the benefit of the present disclosure and are deemed to be within the spirit and scope of the present disclosure as further defined in the appended claims.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, exemplary methods, devices, and materials are now described. All technical and patent publications cited herein are incorporated herein by reference in their entirety.

The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of the art. See, e.g., Michael R. Green and Joseph Sambrook, Molecular Cloning (4^th ed., Cold Spring Harbor Laboratory Press 2012); the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); and MacPherson et al. (1995) PCR 2: A Practical approach.

All numerical designations, e.g., pH, temperature, time, concentration, molecular weight, etc., including ranges, are approximations which are varied (+) or (-) by increments of, e.g., 0. 1 or 1.0, where appropriate. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term “about”, which is used to denote a conventional level of variability. For example, a numerical designation which is “about” a given value may vary by ± 10% of said value; alternatively, the variation may be ± 5%, ± 2%, or ± 1% of the value. It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

As used in the specification and claims, the singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a biomarker” includes a plurality of biomarkers, including mixtures thereof. Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. The term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to”.

As used herein, the term “comprising” or “comprises” is intended to mean that the compositions and methods include the recited elements, but do not exclude others. “Consisting essentially of’ when used to define compositions and methods, shall mean excluding other elements of any essential significance for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. “Consisting of’ shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this disclosure or process steps to produce a composition or achieve an intended result. Embodiments defined by each of these transition terms are within the scope of this disclosure. Use of the term “comprising” herein is intended to encompass both “consisting essentially of’ and “consisting of’.

A “subject”, “individual”, or “patient” is used interchangeably herein, and refers to a human.

The term “healthy individual” as used herein typically denotes an individual who does not suffer from a condition which is amenable to treatment with venglustat. In particular, a healthy individual may be an individual who does not suffer from a lysosomal storage disease selected from a GM2 gangliosidosis (e.g., Sandhoff disease or Tay-Sachs disease), GM1 gangliosidosis, and sialidosis. A healthy individual typically does not have any GBA mutations and typically also lacks pathogenic mutations in other genes encoding enzymes involved in the glycosphingolipid pathway, for example P-hexosaminidase (e.g., Hex A or Hex B), GMl- -galactosidase, GM2 ganglioside activator protein, and neuraminidase.

“Administering” is defined herein as a means of providing an agent (e.g., active ingredient) or a composition containing the agent to a subject in a manner that results in the agent being inside the subject’s body, or prescribing, instructing, managing, or supervising another, including the subject, to so provide said agent or composition. Such an administration can be by any route including, without limitation, oral, dermal, transdermal, transmucosal (e.g., vaginal, rectal, buccal, or sublingual), by injection (e.g., subcutaneous, intravenous, intraperitoneal, intrathecal, intramuscular, intradermal), and by inhalation (e.g., pulmonary, intranasal). Pharmaceutical preparations are, of course, given by forms suitable for each administration route. Administration may also be local or systemic in nature. For example, while oral and injectable routes of administration generally provide systemic exposure, some routes of administration only provide local exposure, such as topical dermal administration and intradermal injection. Intranasal inhalation can provide either local or systemic exposure. The compositions and methods of the present disclosure are typically directed towards enteral, e.g. oral, administration.

As used herein, “co-administration” when referring to a therapeutic use means administration of two or more active ingredients to a patient as part of a regimen for the treatment of a disease or disorder, whether the two or more active agents are given at the same or different times or whether given by the same or different routes of administrations. Concurrent administration of the two or more active ingredients may be at different times on the same day, or on different dates or at different frequencies.

As used herein, the term “simultaneously” when referring to a therapeutic use means administration of two or more active ingredients at or about the same time, and may be by the same route of administration. This may refer to administering the two or more active ingredients in a single dosage form or in multiple separate dosage forms which are administered at or about the same time. For example, this may refer to administering to a patient a single oral tablet or capsule comprising two or more active ingredients, or administering two or more oral tablets or capsules comprising between them two or more active ingredients.

As used herein, the term “separately” when referring to a therapeutic use means administration of two or more active ingredients at or about the same time by different routes of administration, or administration of two or more active ingredients at different times by the same or different routes of administration. For example, the term “separately” includes administering one active ingredient by injection while administering a separate active ingredient orally, when both administrations are taking place at about the same time. In addition, the term “separately” includes administering one active ingredient orally at a particular time of day, e.g. in the morning, while administering a separate active ingredient orally at a different time of day, e.g. one hour later, or three hours later, or in the afternoon or in the evening, or on a different day. Thus, separate administration would also encompass a dosing regimen under which, for example, one drug is taken on days 1, 3, 5, etc., and another drug is taken on days 2, 4, 6, etc.

The phrase “at or about the same time” is understood to generally mean two events taking place with less than 30 minutes between them, e.g. less than 20 minutes, or less than 15 minutes, or less than 10 minutes, or less than 5 minutes. Where an event itself takes place over a period of time, e.g., an intravenous administration of a drug over a period of 60 minutes, “at or about the same time” would include any overlap between such periods of time or the beginning of one such period of time within about 30 minutes of the ending of the previous period of time.

The term “treating” or “treatment” of a subject as used herein typically refers to interventions having a therapeutic or prophylactic effect, e.g., as indicated by some degree of clinical response over a particular time scale (e.g., 104 weeks). The terms “treating” or “treatment” may also, however, include interventions which do not have a measurable or quantifiable clinical response over a particular timescale (e.g., 104 weeks). To the extent that the treatment has a measurable therapeutic or prophylactic effect on a disease, the “treating” or “treatment” of the disease includes: (1) inhibiting the disease, i.e. arresting or reducing the development of the disease or its clinical symptoms; and/or (2) relieving the disease, i.e. causing regression of the disease or its clinical symptoms. “Preventing” or “prevention” of a disease includes causing the clinical symptoms of the disease not to develop in a patient that may be predisposed to the disease but does not yet experience or display symptoms of the disease. A disease which is “amenable to treatment” with or “treatable by” a particular active agent is a disease which can be treated and/or prevented by the active agent in at least some patients who are suffering from the disease or who are predisposed to the disease.

The term “suffering” as it relates to the term “treatment” refers to a patient or individual who has been diagnosed with the disease. The term “suffering” as it relates to the term “prevention” refers to a patient or individual who is predisposed to the disease. A patient may also be referred to being “at risk of suffering” from a disease because of a history of disease in their family lineage or because of the presence of genetic mutations associated with the disease. A patient at risk of a disease has not yet developed all or some of the characteristic pathologies of the disease.

An “effective amount” or “therapeutically effective amount” is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications, or dosages. Such delivery is dependent on a number of variables including, e.g., the time period over which each individual dosage is to be administered, the bioavailability of the therapeutic agent, and the route of administration. It is understood, however, that specific dose levels of the therapeutic agents of the present disclosure for any particular subject depend upon a variety of factors including, for example, the activity of the specific compound employed, the age, body weight, general health, sex, and diet of the subject, the time of administration, the severity of the particular disorder being treated, and the form of administration. Typically, dosage-effect relationships from in vitro and/or in vivo tests initially can provide useful guidance on suitable doses for patient administration. In general, one will desire to administer an amount of the compound that is effective to achieve a plasma, serum, or CSF level commensurate with the concentrations found to be effective in vitro. Determination of these parameters is well within the skill of the art. These considerations, as well as effective formulations and administration procedures are well known in the art and are described in standard textbooks. Consistent with this definition, as used herein, the term “therapeutically effective amount” is an amount sufficient to treat (e.g., improve) one or more symptoms associated with a disease or disorder described herein, ex vivo, in vitro, or in vivo.

As used herein, the term “pharmaceutically acceptable excipient” encompasses any of the standard pharmaceutical excipients, including carriers such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents. Pharmaceutical compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see Remington’s Pharmaceutical Sciences (20th ed., Mack Publishing Co. 2000).

As used herein, the term “pharmaceutically acceptable salt” means a pharmaceutically acceptable acid addition salt or a pharmaceutically acceptable base addition salt of a currently disclosed compound that may be administered without any resultant substantial undesirable biological effect(s) or any resultant deleterious interaction(s) with any other component of a pharmaceutical composition in which it may be contained. Addition salts can be readily prepared using conventional techniques, e.g., by treating a base compound with a defined amount of the chosen mineral or organic acid in an aqueous solvent medium or in a suitable organic solvent such as, for example, methanol or ethanol. Compounds that are positively charged, e.g., containing a quaternary ammonium, may also form salts with the anionic component of various inorganic and/or organic acids. Acids which can be used to prepare pharmaceutically acceptable acid addition salts are those which can form non-toxic acid addition salts, e.g., salts containing pharmacologically acceptable anions, such as chloride, bromide, iodide, nitrate, sulfate or bisulfate, phosphate or acid phosphate, acetate, lactate, citrate or acid citrate, tartrate or bitartrate, succinate, malate, maleate, fumarate, gluconate, saccharate, benzoate, methanesulfonate, and pamoate [i.e., 1,1'- methylene-bis-(2 -hydroxy-3 -naphthoate)] salts. Bases which can be used to prepare the pharmaceutically acceptable base addition salts are those which can form non-toxic base addition salts, e.g., salts containing pharmacologically acceptable cations, such as, alkali metal cations (e.g., potassium and sodium), alkaline earth metal cations (e.g., calcium and magnesium), ammonium or other water-soluble amine addition salts such as N- methylglucamine (meglumine), lower alkanolammonium, and other such bases of organic amines. Addition salts of venglustat are typically acid addition salts. In embodiments, the pharmaceutically acceptable salt of venglustat is venglustat malate, in particular venglustat L- malate.

A mass quantity of venglustat referred to herein corresponds, unless explicitly stated otherwise, to a mass of venglustat calculated as free base. For example, a “15 mg dose of venglustat” refers to an amount of 15 mg of venglustat free base, or to an amount of a salt or prodrug of venglustat which provides an equivalent molar quantity (e.g., 20 mg of venglustat malate salt). Accordingly, references to “venglustat” throughout this specification include the pharmaceutically acceptable salts and prodrugs of venglustat, e.g. as described herein.

The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

The following abbreviations are used herein:

AmAce ammonium acetate

BSA bovine serum albumin

CD63 cluster of differentiation protein 63 antigen

CDI 1 , 1 '-carbonyldiimidazole

CSF cerebrospinal fluid

CV confidence value

DMF dimethylformamide

DNA deoxyribonucleic acid

EDTA ethylenediaminetetraacetic acid

ELISA enzyme-linked immunosorbent assay ERT enzyme replacement therapy

ESI electrospray ionisation

FA formic acid

GAI asialo-monosialotetrahexosyl ganglioside

GA2 gangliotriaosylceramide

GBA glucocerebrosidase

GCS glucosylceramide synthase

GLB1 p-galactosidase gene

GL1 glucosylceramide (also GL-1)

GM1 monosialotetrahexosyl ganglioside

GM2 ganglioside monosialic 2

GM3 monosialodihexosyl ganglioside

Hex p-hexosaminidase

HEXA/B P-hexosaminidase A/B gene

HPC hydroxypropylcellulose

HPLC high performance (or high pressure) liquid chromatography

HSA human serum albumin

IPA isopropyl alcohol

IS internal standard

LLOQ lower limit of quantitation LC/MS/MS liquid chromatography / tandem mass spectrometry MS/MS tandem mass spectrometry NEU1 neuraminidase- 1 gene NFL neurofilament light polypeptide (also Nfl) NPX normalized protein expression PEA proximity extension assay PCR polymerase chain reaction QC/QCs quality control(s) Q.S. a sufficient quantity

RB round bottomed rHA recombinant human albumin SPE solid phase extraction

SRT substrate reduction therapy

TBME methyl tert-butyl ether THF tetrahydrofuran UPLCMS ultra-performance liquid chromatography-mass spectrometry Methods for reducing disease related biomarker levels in patients with ganglioside storage disorders

Venglustat (free base) has a chemical structure according to Formula (I) below, and it may conveniently be provided in the form of a malate addition salt (e.g., prepared as described in the following Examples). Venglustat is an oral GCS inhibitor under development for the treatment of conditions including Fabry disease and Gaucher disease.

The present disclosure and the Examples which follow describe the results of a clinical study in which venglustat was administered to human subjects suffering from GM2 gangliosidoses (Sandhoff disease and Tay-Sachs disease), GM1 gangliosidosis, and sialidosis. Venglustat, in the administered regimen, significantly reduced plasma and CSF levels of biomarkers, including sphingolipids which may be considered toxic and/or part of the underlying pathology of the conditions. Moreover, a correlation could be observed between the activity of the deficient enzyme (hexosaminidase in the case of GM2 gangliosidoses) and the extent of reduction in biomarker levels. The results of the study are supportive of the corrective effect (at least in part) of venglustat on the level of disease-related biomarkers in the plasma and/or CSF of patients suffering from GM2 gangliosidoses and other ganglioside storage disorders.

Accordingly, in one aspect the disclosure provides a method of reducing the level of a biomarker in a biological fluid of a human subject in need thereof suffering from a ganglioside storage disorder, the method comprising administering to the subject an effective amount of venglustat or a pharmaceutically acceptable salt thereof, wherein the biomarker is selected from the group consisting of GM1, GM2, GM3, GL1, and combinations thereof.

In a related aspect, the disclosure provides venglustat, or a pharmaceutically acceptable salt thereof, for use in a method of reducing the level of a biomarker in a biological fluid of a human subject in need thereof suffering from a ganglioside storage disorder, wherein the biomarker is selected from the group consisting of GM1, GM2, GM3, GL1, and combinations thereof. The disclosure also provides venglustat, or a pharmaceutically acceptable salt thereof, for use in a method as defined herein (e.g., as claimed in the appended claims).

In another related aspect, the disclosure provides the use of venglustat, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for use in a method of reducing the level of a biomarker in a biological fluid of a human subject in need thereof suffering from a ganglioside storage disorder, wherein the biomarker is selected from the group consisting of GM1, GM2, GM3, GL1, and combinations thereof. The disclosure also provides the use of venglustat, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for use in a method as defined herein (e.g., as claimed in the appended claims).

A further aspect of the disclosure provides a method of administering venglustat, or a pharmaceutically acceptable salt thereof, to a human subject in need thereof suffering from a ganglioside storage disorder, wherein the venglustat or pharmaceutically acceptable salt thereof is administered in an effective amount to reduce the level of a biomarker in a biological fluid of the subject.

In a further aspect, the disclosure provides a method of treating a human subject in need thereof suffering from a ganglioside storage disorder, the method comprising administering to the subject an effective amount of venglustat or a pharmaceutically acceptable salt thereof, wherein said administration reduces the level of a biomarker in a biological fluid of the subject, wherein the biomarker is selected from the group consisting of GM1, GM2, GM3, GL1, and combinations thereof. The disclosure further provides venglustat, or a pharmaceutically acceptable salt thereof, for use in said method of treating a human subject. The disclosure still further provides the use of venglustat, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for use in said method of treating a human subject.

In embodiments, the biological fluid is selected from the group consisting of blood, a blood fraction, and CSF. In embodiments, the biological fluid comprises (e.g., is) whole blood, or a blood fraction selected from plasma and serum. In embodiments, the biological fluid comprises (e.g., is) whole blood. In other embodiments, the biological fluid comprises (e.g., is) plasma or serum. In embodiments, the biological fluid comprises (e.g., is) plasma. In embodiments, the biological fluid comprises (e.g., is) CSF. In embodiments, the biological fluid is selected from the group consisting of plasma and CSF.

In embodiments, the biomarker is GM1. In embodiments, the biomarker is GM2. In embodiments, the biomarker is GM3. In embodiments, the biomarker is GL1. In embodiments, the biomarker is GM2 and GM3. In embodiments, the biomarker is GM2 and GL1. In embodiments, the biomarker is GM2, GM3 and GL1. In embodiments, the biomarker is GM1 and GM3. In embodiments, the biomarker is GM1 and GL1. In embodiments, the biomarker is GM1, GM3 and GL1. In embodiments, the biomarker does not comprise both GM1 and GM2. In embodiments, the biomarker is selected from: (a) the group consisting of GM1, GM3, GL1, and combinations thereof; and (b) the group consisting of GM2, GM3, GL1, and combinations thereof. In embodiments, the biomarker is selected from: (a) GM1, optionally in combination with GM3 and/or GL1; (b) GM2, optionally in combination with GM3 and/or GL1; and (c) GM3, optionally in combination with GM2 and/or GL1.

In embodiments, the biological fluid is plasma and the biomarker is selected from: (a) GM1, optionally in combination with GM3 and/or GL1; (b) GM2, optionally in combination with GM3 and/or GL1; and (c) GM3, optionally in combination with GM2 and/or GL1. In other embodiments, the biological fluid is CSF and the biomarker is selected from: (a) GM1, optionally in combination with GM3 and/or GL1; (b) GM2, optionally in combination with GM3 and/or GL1; and (c) GM3, optionally in combination with GM2 and/or GL1.

In embodiments, the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of (or is effective to reduce the level of) the biomarker by at least about 10%, e.g. by at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. In embodiments, the level of the biomarker is reduced by at least about 30%, e.g. by at least about 40%, 50%, 60%, 70% or 80%. The reduction in level of the biomarker is typically calculated relative to the baseline level, e.g. to the level which is measured before the initiation of treatment, e.g. shortly before the initiation of treatment (such as, e.g., less than about 1 week before the initiation of treatment). Thus, the reduction can typically be up to, but cannot exceed, 100%. It will be appreciated that methods for the quantification of biomarker levels (e.g. as described herein) typically have a lower limit of quantification and that it may therefore not be possible to demonstrate a reduction of 100% in practice.

In embodiments, the aforementioned reduction in level of the biomarker occurs within a period of about 1 week from initiation of venglustat administration. In embodiments, the reduction in level of the biomarker occurs within a period of about 4 weeks, about 8 weeks, about 12 weeks, about 16 weeks, about 24 weeks, about 30 weeks, about 40 weeks, about 52 weeks, or about 104 weeks. In embodiments, the aforementioned reduction in level of the biomarker in plasma occurs within a period of about 12 weeks from initiation of venglustat administration, or within a period of at least about 12, 16, 24, 30, 40, 52, or 104 weeks. In embodiments, the aforementioned reduction in level of the biomarker in CSF occurs within a period of about 12 weeks from initiation of venglustat administration, or within a period of at least about 12, 16, 24, 30, 40, 52, or 104 weeks. Previous studies of glycosphingolipid biomarkers have shown that the response of CSF biomarkers to a treatment follows a similar profile to the response of plasma biomarkers to that treatment (see, e.g., Schiffmann et al., Brain (2023) 146(2):461-474).

In embodiments, the venglustat or pharmaceutically acceptable salt thereof is administered orally, e.g. in the form of a tablet or capsule. In embodiments, the venglustat or pharmaceutically acceptable salt thereof is administered once daily, e.g. at around the same time every day.

In embodiments, the venglustat or pharmaceutically acceptable salt thereof is administered in a dosage from about 7 mg to about 20 mg per day (calculated as the free base), for example in a dosage of from about 12 mg to about 16 mg per day, e.g., about 15 mg per day (calculated as the free base). In embodiments, the aforementioned dosage, e.g. the dosage of about 15 mg per day, is administered to an adult patient (a patient aged 18 years or over, who may have a bodyweight of > 50 kg). In embodiments, the patient is a juvenile or adolescent patient (a patient aged below 18 years, who may have a bodyweight of < 50 kg) and the venglustat or pharmaceutically acceptable salt thereof is administered in a dosage from about 4 mg to about 15 mg per day (calculated as the free base). In embodiments, the juvenile or adolescent patient has a bodyweight of 10 kg to < 15 kg and the venglustat or pharmaceutically acceptable salt thereof is administered in a dosage of about 4 mg per day (calculated as the free base), e.g. as a once daily dose of a tablet comprising 4 mg of venglustat (calculated as the free base). In embodiments, the juvenile or adolescent patient has a bodyweight of 15 kg to < 30 kg and the venglustat or pharmaceutically acceptable salt thereof is administered in a dosage of about 6 mg per day (calculated as the free base), e.g. as a once daily dose of a tablet comprising 6 mg of venglustat (calculated as the free base). In embodiments, the juvenile or adolescent patient has a bodyweight of 30 kg to < 50 kg and the venglustat or pharmaceutically acceptable salt thereof is administered in a dosage of about 12 mg per day (calculated as the free base), e.g. as a once daily dose of two tablets each comprising 6 mg of venglustat (calculated as the free base). In embodiments, the juvenile or adolescent patient has a bodyweight of > 50 kg and the venglustat or pharmaceutically acceptable salt thereof is administered in a dosage of about 15 mg per day (calculated as the free base), e.g. as a once daily dose of a tablet comprising 15 mg of venglustat (calculated as the free base).

In embodiments, the venglustat is in the form of venglustat free base, a pharmaceutically acceptable salt of venglustat, or a prodrug of venglustat as described herein. In one embodiment, the venglustat is in the form of venglustat malate salt, e.g., venglustat L-malate, optionally in crystalline form.

In embodiments, the venglustat or pharmaceutically acceptable salt thereof is administered in the form of a pharmaceutical composition as described herein.

In embodiments, the venglustat or pharmaceutically acceptable salt thereof is co-administered alongside a therapy for treating the ganglioside storage disorder. In embodiments, the venglustat or pharmaceutically acceptable salt thereof is co-administered alongside enzyme replacement therapy (e.g.. using -hexosaminidase, neuraminidase, or -galactosidase), gene therapy (e.g., to introduce HEXA and/or HEXB, NEU1, or GLB1), substrate reduction therapy with another SRT agent, a pharmacological chaperone therapy, or stem cell therapy. In embodiments, the venglustat or pharmaceutically acceptable salt thereof and the coadministered therapy are administered simultaneously (e.g., in the same oral pharmaceutical dosage form), or separately (e.g., in different pharmaceutical compositions or dosage forms).

In embodiments, the ganglioside storage disorder is a disorder which causes GM1, GM2 and/or GM3 to accumulate in the tissues and/or biological fluids of a human subject. In embodiments, the GM1, GM2 and/or GM3 are the primary storage lipids associated with the disorder. In embodiments, the patient has higher than normal levels of GM1, GM2 and/or GM3 in their CSF and/or blood (e.g., plasma). Normal levels may, for example, be assessed as described herein (see, e.g., Example 4). In embodiments, the ganglioside storage disorder is selected from a GM2 gangliosidosis (e.g., Sandhoff disease, or Tay-Sachs disease), GM1 gangliosidosis, and sialidosis (e.g., sialidosis type I). In embodiments, the ganglioside storage disorder is selected from a GM2 gangliosidosis (e.g., Sandhoff disease, or Tay-Sachs disease), and GM1 gangliosidosis. In embodiments, the ganglioside storage disorder is juvenile GM2 gangliosidosis. GM2 gangliosidoses

The GM2 gangliosidoses are a group of conditions characterised by accumulation of GM2 within the body, especially within the neurons. GM2 gangliosidoses are typically caused by pathogenic mutations in the HEXA and/or HEXB genes, which leads to a deficiency in P- hexosaminidase activity. Symptoms can emerge in infancy (acute infantile GM2 gangliosidosis, in which symptoms typically emerge in the first 6 months of life), in early childhood (subacute juvenile GM2 gangliosidosis, in which symptoms typically emerge between the ages of 2 and 10), or later in adolescence or adulthood (chronic adult GM2 gangliosidoses). Late-onset GM2 gangliosidoses, in particular, tend to go unrecognised or misdiagnosed due to their rarity (see, e.g., Lopshire et al., Mol Genet Metab Rep (2023) 37:el-e8). Juvenile GM2 gangliosidosis typically differs from adult GM2 in the impact of the disease on cognitive function (see, e.g., Maegawa et al., Pediatrics (2006) 118(5) :e 1550- el562).

The present studies indicate that administration of venglustat to patients suffering from GM2 gangliosidoses can lead to a marked reduction in disease biomarkers, especially GM2, in the CSF and plasma. Plasma biomarkers were reduced to levels within the range shown in Table 1 below (indicative of biomarker levels in healthy subjects), while CSF biomarker levels were also reduced.

Thus, in one aspect or embodiment the present disclosure provides a method of reducing the level of a biomarker in a biological fluid of a human subject in need thereof suffering from a GM2 gangliosidosis, the method comprising administering to the subject an effective amount of venglustat or a pharmaceutically acceptable salt thereof, wherein the biomarker is GM2, optionally in combination with GM3 and/or GL1. In a related aspect or embodiment, the disclosure provides venglustat, or a pharmaceutically acceptable salt thereof, for use in a method of reducing the level of a biomarker in a biological fluid of a human subject in need thereof suffering from a GM2 gangliosidosis, wherein the biomarker is GM2, optionally in combination with GM3 and/or GL1. In another related aspect or embodiment, the disclosure provides the use of venglustat, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for use in a method of reducing the level of a biomarker in a biological fluid of a human subject in need thereof suffering from a GM2 gangliosidosis, wherein the biomarker is GM2, optionally in combination with GM3 and/or GL1.

In another aspect, the disclosure provides a method of administering venglustat, or a pharmaceutically acceptable salt thereof, to a human subject in need thereof suffering from a GM2 gangliosidosis, wherein the venglustat or pharmaceutically acceptable salt thereof is administered in an effective amount to reduce the level of a biomarker in a biological fluid of the subject, wherein the biomarker is GM2, optionally in combination with GM3 and/or GL1.

In a further aspect or embodiment the disclosure provides a method of treating a human subject in need thereof suffering from a GM2 gangliosidosis, the method comprising administering to the subject an effective amount of venglustat or a pharmaceutically acceptable salt thereof, wherein said administration reduces the level of a biomarker in a biological fluid of the subject, wherein the biomarker is GM2, optionally in combination with GM3 and/or GL1. In a related aspect or embodiment, the disclosure provides venglustat, or a pharmaceutically acceptable salt thereof, for use in a method of treating a human subject in need thereof suffering from a GM2 gangliosidosis, the method comprising administering to the subject an amount of the venglustat or pharmaceutically acceptable salt thereof effective to reduce the level of a biomarker in a biological fluid of the subject, wherein the biomarker is GM2, optionally in combination with GM3 and/or GL1. In another related aspect or embodiment, the disclosure provides the use of venglustat, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for use in a method of treating a human subject in need thereof suffering from a GM2 gangliosidosis, the method comprising administering to the subject an amount of the venglustat or pharmaceutically acceptable salt thereof effective to reduce the level of a biomarker in a biological fluid of the subject, wherein the biomarker is GM2, optionally in combination with GM3 and/or GL1.

It will be appreciated that embodiments described hereinbefore, e.g. in connection with the method of reducing the level of a biomarker in a human subject in need thereof suffering from a ganglioside storage disorder, may be applied to the methods of reducing the level of a biomarker in a human subject in need thereof suffering from a GM2 gangliosidosis. Further embodiments are set out below.

In embodiments, the GM2 gangliosidosis is selected from Tay-Sachs disease, and Sandhoff disease. In embodiments, the GM2 gangliosidosis is selected from adult (or chronic late- onset) Tay-Sachs disease, and adult (or chronic late-onset) Sandhoff disease. In embodiments, the GM2 gangliosidosis is juvenile GM2 gangliosidosis. In embodiments, the subject has a level of activity of hexosaminidase (e.g., p-hexosaminidase A or p- hexosaminidase B) which is below about 0.6 IU/L at baseline, e.g. below about 0.5, 0.4, 0.3, 0.2, or 0. 1 IU/L. In embodiments, the subject has a level of activity of hexosaminidase (e.g., P-hexosaminidase A or p-hexosaminidase B) which is below about 0.10 IU/L at baseline, e.g. below about 0.08, 0.06, 0.04, 0.02, or 0.01 IU/L. In embodiments, the subject has an undetectable level of activity of hexosaminidase (e.g., P-hexosaminidase A or p- hexosaminidase B) at baseline. In embodiments, the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of (or is effective to reduce the level of) GM2 in the plasma to a level between about 200 and about 750 ng/mL.

In embodiments, the GM2 gangliosidosis is Tay-Sachs disease and the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of (or is effective to reduce the level of) GM2 in the CSF by at least about 30%, e.g. by at least about 40%, 50%, 60%, or 70%. In embodiments, the GM2 gangliosidosis is Tay-Sachs disease and the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of (or is effective to reduce the level of) GM2 in the plasma by at least about 40%, e.g. by at least about 50%, 60%, 70%, or 80%. In embodiments, the GM2 gangliosidosis is Tay- Sachs disease and the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of (or is effective to reduce the level of) GM2 in the plasma to a level of below about 750 ng/mL, below about 600 ng/mL, below about 500 ng/mL, below about 400 ng/mL, below about 300 ng/mL, or below about 250 ng/mL.

In embodiments, the GM2 gangliosidosis is Sandhoff disease and the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of (or is effective to reduce the level of) GM2 in the CSF by at least about 40%, e.g. by at least about 50%, 60%, or 70%. In embodiments, the GM2 gangliosidosis is Sandhoff disease and the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of (or is effective to reduce the level of) GM2 in the plasma by at least about 40%, e.g. by at least about 50%, 60%, 70%, 80%, or 90%. In embodiments, the GM2 gangliosidosis is Sandhoff disease and the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of (or is effective to reduce the level of) GM2 in the plasma to a level of below about 750 ng/mL, below about 600 ng/mL, below about 500 ng/mL, below about 400 ng/mL, below about 300 ng/mL, or below about 200 ng/mL.

In embodiments, the GM2 gangliosidosis is juvenile GM2 gangliosidosis and the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of (or is effective to reduce the level of) GM2 in the CSF by at least about 30%, e.g. by at least about 40%, 50%, 60%, or 70%. In embodiments, the GM2 gangliosidosis is juvenile GM2 gangliosidosis and the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of (or is effective to reduce the level of) GM2 in the plasma by at least about 40%, e.g. by at least about 50%, 60%, 70%, or 80%. In embodiments, the GM2 gangliosidosis is juvenile GM2 gangliosidosis and the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of (or is effective to reduce the level of) GM2 in the plasma to a level of below about 750 ng/mL, below about 600 ng/mL, below about 500 ng/mL, below about 400 ng/mL, below about 300 ng/mL, or below about 250 ng/mL.

GM1 gangliosidosis

GM1 gangliosidosis is typically caused by pathogenic mutations in the GLB1 gene, which leads to a deficiency in p-galactosidase activity and accumulation of GM1 within the body, especially within neuronal tissue. Like the GM2 gangliosidoses, symptoms can emerge in infancy, in early childhood, or later in adolescence or adulthood. The disorder can, however, be considered as a continuum of symptoms which correlate partially with residual enzyme activity (see, e.g., Nicoli et al., Frontiers in Genetics (2021) 12:el-el 1).

The present studies indicate that administration of venglustat to patients suffering from GM1 gangliosidosis can lead to a marked reduction in disease biomarkers, especially GM1, in the CSF and plasma. CSF and plasma biomarkers were reduced in most subjects to levels within the ranges shown in Table 1 below (indicative of biomarker levels in healthy subjects).

Thus, in one aspect or embodiment the present disclosure provides a method of reducing the level of a biomarker in a biological fluid of a human subject in need thereof suffering from GM1 gangliosidosis, the method comprising administering to the subject an effective amount of venglustat or a pharmaceutically acceptable salt thereof, wherein the biomarker is GM1, optionally in combination with GM3 and/or GL1. In a related aspect or embodiment, the disclosure provides venglustat, or a pharmaceutically acceptable salt thereof, for use in a method of reducing the level of a biomarker in a biological fluid of a human subject in need thereof suffering from GM1 gangliosidosis, wherein the biomarker is GM1, optionally in combination with GM3 and/or GL1. In another related aspect or embodiment, the disclosure provides the use of venglustat, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for use in a method of reducing the level of a biomarker in a biological fluid of a human subject in need thereof suffering from GM1 gangliosidosis, wherein the biomarker is GM1, optionally in combination with GM3 and/or GL1.

In another aspect, the disclosure provides a method of administering venglustat, or a pharmaceutically acceptable salt thereof, to a human subject in need thereof suffering from GM1 gangliosidosis, wherein the venglustat or pharmaceutically acceptable salt thereof is administered in an effective amount to reduce the level of a biomarker in a biological fluid of the subject, wherein the biomarker is GM1, optionally in combination with GM3 and/or GL1.

In a further aspect or embodiment the disclosure provides a method of treating a human subject in need thereof suffering from GM1 gangliosidosis, the method comprising administering to the subject an effective amount of venglustat or a pharmaceutically acceptable salt thereof, wherein said administration reduces the level of a biomarker in a biological fluid of the subject, wherein the biomarker is GM1, optionally in combination with GM3 and/or GL1. In a related aspect or embodiment, the disclosure provides venglustat, or a pharmaceutically acceptable salt thereof, for use in a method of treating a human subject in need thereof suffering from GM1 gangliosidosis, the method comprising administering to the subject an amount of the venglustat or pharmaceutically acceptable salt thereof effective to reduce the level of a biomarker in a biological fluid of the subject, wherein the biomarker is GM1, optionally in combination with GM3 and/or GL1. In another related aspect or embodiment, the disclosure provides the use of venglustat, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for use in a method of treating a human subject in need thereof suffering from GM1 gangliosidosis, the method comprising administering to the subject an amount of the venglustat or pharmaceutically acceptable salt thereof effective to reduce the level of a biomarker in a biological fluid of the subject, wherein the biomarker is GM1, optionally in combination with GM3 and/or GL1.

It will be appreciated that embodiments described hereinbefore, e.g. in connection with the method of reducing the level of a biomarker in a human subject in need thereof suffering from a ganglioside storage disorder, may be applied to the methods of reducing the level of a biomarker in a human subject suffering from GM1 gangliosidosis. Further embodiments are set out below.

In embodiments, the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of (or is effective to reduce the level of) GM1 in the CSF by at least about 30%, e.g. by at least about 40%, 50%, 60%, or 70%. In embodiments, the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of (or is effective to reduce the level of) GM1 in the plasma by at least about 40%, e.g. by at least about 50%, 60%, or 70%.

In embodiments, the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of (or is effective to reduce the level of) GM1 in the CSF to a level of below about 70 ng/mL, below about 65 ng/mL, below about 60 ng/mL, below about 55 ng/mL, below about 50 ng/mL, below about 45 ng/mL, or below about 40 ng/mL. In embodiments, the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of (or is effective to reduce the level of) GM1 in the plasma to a level between about 40 and about 115 ng/mL. In embodiments, the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of (or is effective to reduce the level of) GM1 in the plasma to a level of below about 120 ng/mL, below about 100 ng/mL, below about 90 ng/mL, below about 80 ng/mL, or below about 75 ng/mL.

Sialidosis

Sialidosis is typically caused by pathogenic mutations in the NEU1 gene, which leads to a deficiency in neuraminidase- 1 activity and accumulation of oligosaccharides and sialylated glycoproteins within the body. Sialidosis is a heterogeneous disorder with varying ages of onset and pathologies, and there is currently no approved therapeutic treatment.

The present studies indicate that GM3 is among the oligosaccharides which can accumulate in biological fluids of patients with sialidosis. Administration of venglustat to a patient suffering from sialidosis type I led to a reduction in disease biomarkers, especially GM3, in the CSF and plasma. CSF biomarkers were reduced to levels within the range shown in Table 1 below (indicative of biomarker levels in healthy subjects), while plasma biomarkers reduced to a level below the range shown in Table 1.

Thus, in one aspect or embodiment the present disclosure provides a method of reducing the level of a biomarker in a biological fluid of a human subject in need thereof suffering from sialidosis (e.g., type I sialidosis), the method comprising administering to the subject an effective amount of venglustat or a pharmaceutically acceptable salt thereof, wherein the biomarker is GM3, optionally in combination with GM2 and/or GL1. In a related aspect or embodiment, the disclosure provides venglustat, or a pharmaceutically acceptable salt thereof, for use in a method of reducing the level of a biomarker in a biological fluid of a human subject in need thereof suffering from sialidosis (e.g., type I sialidosis), wherein the biomarker is GM3, optionally in combination with GM2 and/or GL1. In another related aspect or embodiment, the disclosure provides the use of venglustat, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for use in a method of reducing the level of a biomarker in a biological fluid of a human subject in need thereof suffering from sialidosis (e.g., type I sialidosis), wherein the biomarker is GM3, optionally in combination with GM2 and/or GL1.

In another aspect, the disclosure provides a method of administering venglustat, or a pharmaceutically acceptable salt thereof, to a human subject in need thereof suffering from sialidosis, wherein the venglustat or pharmaceutically acceptable salt thereof is administered in an effective amount to reduce the level of a biomarker in a biological fluid of the subject, wherein the biomarker is GM3, optionally in combination with GM2 and/or GL1.

In a further aspect or embodiment the disclosure provides a method of treating a human subject in need thereof suffering from sialidosis (e.g., type I sialidosis), the method comprising administering to the subject an effective amount of venglustat or a pharmaceutically acceptable salt thereof, wherein said administration reduces the level of a biomarker in a biological fluid of the subject, wherein the biomarker is GM3, optionally in combination with GM2 and/or GL1. In a related aspect or embodiment, the disclosure provides venglustat, or a pharmaceutically acceptable salt thereof, for use in a method of treating a human subject in need thereof suffering from sialidosis (e.g., type I sialidosis), the method comprising administering to the subject an amount of the venglustat or pharmaceutically acceptable salt thereof effective to reduce the level of a biomarker in a biological fluid of the subject, wherein the biomarker is GM3, optionally in combination with GM2 and/or GL1. In another related aspect or embodiment, the disclosure provides the use of venglustat, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for use in a method of treating a human subject in need thereof suffering from sialidosis (e.g., type I sialidosis), the method comprising administering to the subject an amount of the venglustat or pharmaceutically acceptable salt thereof effective to reduce the level of a biomarker in a biological fluid of the subject, wherein the biomarker is GM3, optionally in combination with GM2 and/or GL1.

It will be appreciated that embodiments described hereinbefore, e.g. in connection with the method of reducing the level of a biomarker in a human subject in need thereof suffering from a ganglioside storage disorder, may be applied to the methods of reducing the level of a biomarker in a human subject suffering from sialidosis. Further embodiments are set out below.

In embodiments, the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of (or is effective to reduce the level of) GM3 in the CSF by at least about 40%, e.g. by at least about 50%, 60%, 70%, or 80%. In embodiments, the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of (or is effective to reduce the level of) GM3 in the plasma by at least about 40%, e.g. by at least about 50%, 60%, or 70%.

In embodiments, the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of (or is effective to reduce the level of) GM3 in the CSF to a level of below about 30 ng/mL, below about 25 ng/mL, below about 20 ng/mL, or below about 15 ng/mL. In embodiments, the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of (or is effective to reduce the level of) GM3 in the CSF to a level between about 15 and about 30 ng/mL. In embodiments, the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of (or is effective to reduce the level of) GM3 in the plasma to a level of below about 7.0 pg/mL, below about 6.5 pg/mL, below about 6.0 pg/mL, below about 5.5 pg/mL, below about 5.0 pg/mL, below about 4.5 pg/mL, or below about 4.0 pg/mL. In embodiments, the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of (or is effective to reduce the level of) GM3 in the plasma to a level between about 5 and about 20 pg/mL.

Reduction of disease-associated proteins in the CSF

The studies described in the following Examples include an analysis of disease-associated protein levels in the CSF of patients with GM1 gangliosidosis and GM2 gangliosidoses (juvenile and adult). The results of those studies show a reduction in levels of CD63 and/or NFL in certain patient groups receiving venglustat over 104 weeks.

CD63 is a cell-surface glycoprotein which was first detected as a marker of platelet activation. In vivo, CD63 localizes to the membranes of melanosomes and platelet dense bodies. Some cells are enriched in CD63, such as activated basophils and proliferating mast cells, and CD63 is often used in cell biology as a marker for late endocytic compartments. Recently, CD63 has been proposed as a biomarker for multiple lysosomal storage diseases, based on observations in patients with, e.g., Gaucher disease and mucopolysaccharidosis (types I and II). Levels of CD63 in CSF samples taken from those patients were found to be significantly increased as compared to healthy controls, and/or to reduce over time when patients are on treatment (see, e.g., international patent application No. PCT/IB2023/057323, published as WO 2024/018382, the content of which is hereby incorporated by reference in its entirety). Neurofilament light protein (NFL) is the light subunit of neurofilament protein, a major structural component of myelinated axons. CSF measurement of NFL is known to be an indicator of axonal injury in a variety of neurodegenerative diseases, including Parkinson’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis, and multiple sclerosis (see, e.g., Bridel et al., JAMA Neurol. (2019) 76(9): 1035-1048).

Reduction of CD63 in the CSF

Thus, in embodiments the disclosure provides a method of reducing the level of a biomarker, as described herein, wherein the method also reduces the level of CD63 in the CSF of the subject in need thereof suffering from a ganglioside storage disorder, wherein the ganglioside storage disorder is a GM2 gangliosidosis.

In other embodiments, the disclosure provides a method of administering venglustat or a pharmaceutically acceptable salt thereof, as described herein, wherein the venglustat or pharmaceutically acceptable salt thereof is administered in an effective amount to also reduce the level of CD63 in the CSF of the subject in need thereof suffering from a ganglioside storage disorder, wherein the ganglioside storage disorder is a GM2 gangliosidosis.

In other embodiments, the disclosure provides a method of treating a human subject, as described herein, wherein the administration of an effective amount of venglustat or a pharmaceutically acceptable salt thereof also reduces the level of CD63 in the CSF of the subject in need thereof suffering from a ganglioside storage disorder, wherein the ganglioside storage disorder is a GM2 gangliosidosis.

In embodiments, the GM2 gangliosidosis is adult (or chronic late-onset) Tay-Sachs disease, or adult (or chronic late-onset) Sandhoff disease, and the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of (or is effective to reduce the level of) CD63 in the CSF by at least about 10%, e.g. by at least about 15%, 20%, 35%, 30%, 35%, or 40%. In embodiments, the GM2 gangliosidosis is juvenile GM2 gangliosidosis and the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of (or is effective to reduce the level of) CD63 in the CSF by at least about 10%, e.g. by at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%. In embodiments, the aforementioned reduction in level of CD63 in CSF occurs within a period of about 12 weeks from initiation of venglustat administration, or within a period of at least about 12, 16, 24, 30, 40, 52, or 104 weeks from initiation of venglustat administration.

It will be appreciated that the present disclosure also provides venglustat, or a pharmaceutically acceptable salt thereof, for use in a method in accordance with the foregoing embodiments. The present disclosure further provides the use of venglustat, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for use in a method in accordance with the foregoing embodiments.

Reduction of NFL in the CSF

In embodiments the disclosure provides a method of reducing the level of a biomarker, as described herein, wherein the method also reduces the level of NFL in the CSF of the subject in need thereof suffering from a ganglioside storage disorder, wherein the ganglioside storage disorder is juvenile GM2 gangliosidosis.

In other embodiments, the disclosure provides a method of administering venglustat or a pharmaceutically acceptable salt thereof, as described herein, wherein the venglustat or pharmaceutically acceptable salt thereof is administered in an effective amount to also reduce the level of NFL in the CSF of the subject in need thereof suffering from a ganglioside storage disorder, wherein the ganglioside storage disorder is juvenile GM2 gangliosidosis.

In other embodiments, the disclosure provides a method of treating a human subject, as described herein, wherein the administration of an effective amount of venglustat or a pharmaceutically acceptable salt thereof also reduces the level of NFL in the CSF of the subject in need thereof suffering from a ganglioside storage disorder, wherein the ganglioside storage disorder is juvenile GM2 gangliosidosis.

In embodiments, the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of (or is effective to reduce the level of) NFL in the CSF by at least about 20%, e.g. by at least about 30%, 40%, 50%, 60%, or 70%. In embodiments, the aforementioned reduction in level of NFL in CSF occurs within a period of about 12 weeks from initiation of venglustat administration, or within a period of at least about 12, 16, 24, 30, 40, 52, or 104 weeks from initiation of venglustat administration. It will be appreciated that the present disclosure also provides venglustat, or a pharmaceutically acceptable salt thereof, for use in a method in accordance with the foregoing embodiments. The present disclosure further provides the use of venglustat, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for use in a method in accordance with the foregoing embodiments.

Forms of venglustat

The present disclosure contemplates salt forms of venglustat, e.g., venglustat in the form of a pharmaceutically acceptable salt.

Compounds that are basic in nature are generally capable of forming a wide variety of different salts with various inorganic and/or organic acids. Although such salts are generally pharmaceutically acceptable for administration to animals and humans, it is often desirable in practice to initially isolate a compound from the reaction mixture as a pharmaceutically unacceptable salt and then simply convert the latter back to the free base compound by treatment with an alkaline reagent, and subsequently convert the free base to a pharmaceutically acceptable acid addition salt. The acid addition salts of the base compounds can be readily prepared using conventional techniques, e.g. by treating the base compound with a substantially equivalent amount of the chosen mineral or organic acid in an aqueous solvent medium or in a suitable organic solvent such as, for example, methanol or ethanol. Upon careful evaporation of the solvent, the desired solid salt is obtained.

Compounds that are positively charged, e.g., containing a quaternary ammonium, may also form salts with the anionic component of various inorganic and/or organic acids.

Acids which can be used to prepare pharmaceutically acceptable salts of venglustat are those which can form non-toxic acid addition salts, e.g., salts containing pharmacologically acceptable anions, such as chloride, bromide, iodide, nitrate, sulfate or bisulfate, phosphate or acid phosphate, acetate, lactate, citrate or acid citrate, tartrate or bitartrate, succinate, malate, maleate, fumarate, gluconate, saccharate, benzoate, methanesulfonate, and pamoate [i.e., 1,1'- methylene-bis-(2 -hydroxy-3 -naphthoate)] salts .

In one embodiment, the pharmaceutically acceptable salt is a succinate salt. In another embodiment, the pharmaceutically acceptable salt is a 2-hydroxysuccinate salt, e.g., an (S)-2- hydroxysuccinate salt. In another embodiment, the pharmaceutically acceptable salt is a hydrochloride salt (i.e., a salt with HC1). In another embodiment, the pharmaceutically acceptable salt is a malate salt, e.g., an L-malate salt.

The present disclosure also contemplates prodrugs of venglustat. The pharmaceutically acceptable prodrugs disclosed herein are derivatives which can be converted in vivo into venglustat. The prodrugs, which may themselves have some activity, become pharmaceutically active in vivo when they undergo, for example, solvolysis under physiological conditions or enzymatic degradation. Methods for preparing prodrugs of venglustat would be apparent to one of skill in the art based on the present disclosure. In one embodiment, the carbamate moiety of venglustat is modified. For example, the carbamate moiety may be modified by the addition of water and/or one or two aliphatic alcohols. In this case, the carbon-oxygen double bond of the carbamate moiety adopts what could be considered a hemiacetal or acetal functionality. In one embodiment, the carbamate moiety may be modified by the addition of an aliphatic diol such as 1,2-ethanediol.

In one embodiment, the amino group on the quinuclidine moiety is modified. For example, the amino group may be modified to form an acid derivative or a quaternary ammonium salt. The derivative can be formed, for example, by reacting venglustat with an acetylating agent such as an acid chloride, or with an agent such as an alkyl halide.

The present disclosure further embraces hydrates, solvates, and polymorphs of venglustat. For example, the venglustat may be in one or more crystalline forms as described in, e.g., international patent application No. PCT/US2014/027081 (published as WO 2014/152215), the entire content of which is incorporated by reference herein. In embodiments, the venglustat is in the form of a malate salt and is in crystalline Form A as described in WO 2014/152215 (see, e.g., claims 1-6). In other embodiments, the venglustat is in the form of a malate salt and is in crystalline Form B as described in WO 2014/152215 (see, e.g., claims 7- 12).

Isotopically-labeled compounds are also within the scope of the present disclosure. As used herein, an “isotopically-labeled compound” refers to a presently disclosed compound including pharmaceutical salts and prodrugs thereof, each as described herein, in which one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds presently disclosed include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, and chlorine, such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F, and ³⁶C1, respectively.

Pharmaceutical compositions

The venglustat, or pharmaceutically acceptable salt thereof, for use in accordance with present disclosure may be formulated as a pharmaceutical composition. The present disclosure thus provides a pharmaceutical composition (e.g., an oral pharmaceutical dosage form) comprising venglustat or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient. The composition may be specifically adapted for use in any of the methods disclosed herein.

The pharmaceutically acceptable excipient can be any such excipient known in the art including those described in, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). Pharmaceutical compositions of the compounds presently disclosed may be prepared by conventional means known in the art including, for example, mixing at least one presently disclosed compound with a pharmaceutically acceptable excipient. In embodiments, the venglustat is in solid crystal form (e.g., crystalline malate salt Form A of venglustat). In other embodiments, the venglustat is in solid amorphous form. In embodiments, the dosage form comprises an amorphous solid dispersion comprising the venglustat with the pharmaceutically acceptable excipient.

In embodiments, the dosage form is a capsule (e.g., a hard capsule) or a tablet (e.g., a chewable tablet, an orally-disintegrating tablet, a dispersible tablet, or a classic tablet or caplet), optionally wherein said dosage form comprises from about 2 to about 30 mg of venglustat (measured as the equivalent amount of free base), e.g., from about 4 mg to about 20 mg, or from about 8 mg to about 12 mg, or about 4 mg, or about 6 mg, or about 8 mg, or about 12 mg, or about 15 mg of venglustat (measured as the equivalent amount of free base).

In embodiments, the dosage form is a classic tablet or caplet (e.g., for swallowing), a chewable tablet, an orally disintegrating tablet, or a dispersible tablet.

In embodiments, the pharmaceutically acceptable excipient comprises one or more of (a) diluent/filler (e.g., cellulose or microcrystalline cellulose, mannitol, or lactose), (b) binder (e.g., povidone, methylcellulose, ethylcellulose, hydroxypropyl cellulose (such as low- substituted hydroxypropyl cellulose), or hydroxypropyl methylcellulose), (c) disintegrant (e.g., crospovidone, sodium starch glycolate, or croscarmellose sodium), (d) lubricant (e.g., magnesium stearate or sodium stearyl fumarate), (e) glidant (e.g., silica or talc), (f) sweetener (e.g., sucralose, acesulfame potassium, aspartame, saccharine, neotame, or advantame), (g) flavor (e.g., apricot flavor), and (h) dye or colorant.

In embodiments, the pharmaceutically acceptable excipient comprises one or more hydrophilic water-soluble or water swellable polymers. In embodiments, the polymer is selected from the group consisting of natural or modified cellulosic polymers, or any mixture thereof.

In embodiments, any one or more pharmaceutically acceptable excipients are present in an amount of 0.01 to 80% by weight, e.g., 0.1 to 60%, or 0.1 to 40%, or 0.1 to 30%, 0.01 to 15%, or 0.01 to 10%, or 0.1 to 20%, or 0.1 to 15% or 0.1 to 10%, or 0.5 to 10%, or 0.5 to 5%, or 1 to 5%, or 2.5 to 5%, or 1 to 3%, or 0. 1 to 1% by weight. In embodiments, the dosage form comprises (a) from 5-95% by weight of diluent(s)/filler(s), e.g., 60-70% or 70-80%, or 65-75%, or 65-70%, or about 68%; (b) from 0.5-5% by weight of lubricant(s), e.g., 1-5%, or

2-4%, or 2-3%, or about 3%; (c) from 2-15% by weight of disintegrant(s), e.g., 4-12%, or 6- 10%, or 7-9%, or about 8%; (d) from 0-12% by weight of binder(s), e.g., 2-10%, or 2-8%, or

3-7%, or 4-6%, or about 5%; (e) from 0-5% by weight of glidant(s), e.g., 0.15-4%, or 1-3%, or 1-2%, or about 1%; and (f) from 0-2% by weight of flavor(s), 0-2% by weight of sweetener(s) and/or 0-2% by weight of color(s), e.g., about 1% each of flavor(s), sweetener(s), and/or color(s). In embodiments, the venglustat is present in an amount of from 3% to 20% by weight (measured as free base).

In embodiments, the oral pharmaceutical composition is a pill, capsule, caplet, tablet, dragee, powder, granule, fdm, lozenge, or liquid. In embodiments, the oral pharmaceutical composition is a capsule or tablet, e.g., a tablet. In an embodiment, the oral pharmaceutical composition is a formulation as described in international patent application No. PCI7IB2021/056673 (published as WO 2022/018695), the entire content of which is incorporated by reference herein.

In embodiments, the formulation is a capsule having the following composition:

In embodiments, the capsule contains 15 mg of venglustat (20. 16 mg of venglustat malate), the fdl mass of the capsule is 165 mg, and the formulation is packaged into a size #3 capsule shell.

In other embodiments, the formulation is a tablet having the following composition:

In embodiments, the tablet contains 15 mg of venglustat (20. 16 mg of venglustat malate), the flavor is apricot flavor, and the weight of the tablet is 150 mg. In other embodiments, the tablet contains 6 mg of venglustat (8.06 mg of venglustat malate), the flavor is apricot flavor, and the weight of the tablet is 60 mg.

In embodiments, the dosage form is a hard-shelled capsule, e.g., wherein said capsule contains a mixture of venglustat (e.g., venglustat malate) and one or more pharmaceutically acceptable excipients. The venglustat and other diluents/carriers may be comprised as granules or pellets, or as a powder, said granules, pellets, or powder being contained within the shell of the capsule. A pharmaceutical composition or dosage form of the present disclosure can include an agent and another carrier, e.g., compound or composition, inert or active, such as a detectable agent, label, adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant, or the like. Carriers also include pharmaceutical excipients and additives, for example, proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars, and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1 to 99.99% by weight or volume. Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid/antibody components, which can also function in a buffering capacity, include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. Carbohydrate excipients are also intended within the scope of this disclosure, examples of which include but are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), and myoinositol.

Carriers which may be used include a buffer or a pH adjusting agent; typically, the buffer is a salt prepared from an organic acid or base. Representative buffers include organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, or phosphate buffers. Additional carriers include polymeric excipients/additives such as polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl-P- cyclodextrin), polyethylene glycols, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates such as “TWEEN 20” and “TWEEN 80”), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).

In solid dosage forms for oral administration (e.g., capsules, tablets, pills, dragees, powders, granules, and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, excipients, or diluents, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, microcrystalline cellulose, calcium phosphate, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, pregelatinized maize starch, polyvinyl pyrrolidone, hydroxypropyl methylcellulose, sucrose, and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, sodium starch glycolate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, sodium lauryl sulphate, acetyl alcohol, and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, silica, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets, and pills, the pharmaceutical compositions can also comprise buffering agents. Solid compositions of a similar type can also be prepared using fdlers in soft and hard-filled gelatin capsules, and excipients such as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

A tablet can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared using binders (for example, gelatin or hydroxypropylmethyl cellulose), lubricants, inert diluents, preservatives, disintegrants (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surfaceactives, and/or dispersing agents. Molded tablets can be made by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent. The tablets and other solid dosage forms, such as dragees, capsules, pills, and granules, can optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the art.

In embodiments, the pharmaceutical compositions are administered orally in a liquid form. Liquid dosage forms for oral administration of an active ingredient include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. Liquid preparations for oral administration may be presented as a dry product for constitution with water or other suitable vehicle before use. In addition to the active ingredient, the liquid dosage forms can contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (e.g., cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, the liquid pharmaceutical compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming, and preservative agents, and the like. Suspensions, in addition to the active ingredient(s) can contain suspending agents such as, but not limited to, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof. Suitable liquid preparations may be prepared by conventional means with a pharmaceutically acceptable additive(s) such as a suspending agent (e.g., sorbitol syrup, methyl cellulose, or hydrogenated edible fats); emulsifying agent (e.g., lecithin or acacia); non-aqueous vehicle (e.g., almond oil, oily esters, or ethyl alcohol); and/or preservative (e.g., methyl or propyl p-hydroxybenzoates, or sorbic acid). The active ingredient(s) can also be administered as a bolus, electuary, or paste.

In some embodiments of the methods described herein, the pharmaceutical composition may take the form of tablets or lozenges formulated for buccal administration in a conventional manner.

Controlled release parenteral compositions can be in form of aqueous suspensions, microspheres, microcapsules, magnetic microspheres, oil solutions, oil suspensions, emulsions, or the active ingredient can be incorporated in biocompatible carrier(s), liposomes, nanoparticles, implants, or infusion devices. Materials for use in the preparation of microspheres and/or microcapsules include, but are not limited to, biodegradable/bioerodible polymers such as polyglactin, poly-(isobutyl cyanoacrylate), poly(2-hydroxyethyl-L- glutamine), and poly(lactic acid). Biocompatible carriers which can be used when formulating a controlled release parenteral formulation include carbohydrates such as dextrans, proteins such as albumin, lipoproteins, or antibodies. Materials for use in implants can be non-biodegradable, e.g., polydimethylsiloxane, or biodegradable such as, e.g., poly(caprolactone), poly(lactic acid), poly(glycolic acid), or poly(ortho esters).

Assays for quantifying glvcosphingolipid levels

The levels of the biomarker (e.g., one or more of the sphingolipids GL1, GM1, GM2 and GM3) in biological fluids may be measured by methods known in the art, e.g. as described in publications which are referenced herein, or by methods as set out in detail herein, e.g. in the examples which follow. In embodiments, the levels of biomarkers are quantified using an LC/MS/MS method, e.g. as described herein. In such methods, the fluid sample (e.g., human plasma, or CSF) may be applied to a column (e.g., HPLC or UPLC column) under conditions which facilitate the separation of glycosphingolipids in the sample. The resulting fractions may then be analysed by mass spectrometry, e.g. by tandem MS/MS, and the peak area(s) of one or more isoforms of each glycosphingolipid of interest calculated. The concentration of each glycosphingolipid of interest in the original sample may then be determined, e.g. by comparison with reference samples having a known concentration of the relevant glycosphingolipid .

In embodiments, the sphingolipid is GL1 and the concentration of total GL1 is determined by summing up the mass spectrometric intensity from the following isomers: GL1 16:0; GL1 18:0; GL1 20:0; GL1 22:0; GL1 23:0; GL1 24: 1; and GL1 24:0. In embodiments, the method employs N-octadecanoyl-D35-psychosine as an internal standard (e.g., when measuring plasma GL1). In embodiments, the method employs 13C6-Glucosylceramide (13C labeled C24:0 GL-1) as an internal standard (e.g., when measuring CSF GL1).

In embodiments, the sphingolipid is GM1 and the concentration of total GM1 is determined by summing up the mass spectrometric intensity from some or all of the following isomers: GM1 Cer34: l; GMl Cer36: l; GMl Cer38: l; GMl Cer40: l; GMl Cer42: l; and GM1 Cer42:2. In embodiments, the method employs N-hexadecanoyl-D9-monosialoganglioside GM1 as an internal standard.

In embodiments, the sphingolipid is GM2 and the concentration of GM2 is determined by summing up the mass spectrometric intensity from the following isomers: GM2 C16:0; GM2 C18:0; GM2 C20:0; GM2 C22:0; GM2 C23:0; GM2 C24:0; and GM2 C24: l. In embodiments, the method employs N-hexadecanoyl-D9-monosialoganglioside GM2 as an internal standard. In embodiments, the sphingolipid is GM3 and the concentration of total GM3 is determined by summing up the mass spectrometric intensity from the following isomers: GM3 C16:0; GM3 C18:0; GM3 C20:0; GM3 C22:0; GM3 C23:0; GM3 C24:0; and GM3 C24: l. In embodiments, the method employs gangliotriaosylceramide (GA2) as an internal standard (e.g., when measuring plasma GM3). In embodiments, the method employs deuterated C16:0 GM3-[palmyl-D3i] as an internal standard (e.g., when measuring CSF GM3).

The level of the disease-associated proteins (CD63 and/or NFL) in CSF may be measured by methods known in the art, e.g. as described in publications which are referenced herein, or by methods as set out in detail herein, e.g. in the examples which follow. In embodiments, the levels of disease-related proteins are quantified using a method selected from an immunoassay (e.g., ELISA, microfluidic ELISA, or bead-based high sensitivity ELISA; capillary Western blot assay; nanoneedle bioarray; Proximity Ligation Assay; or Proximity Extension Assay), an affinity-based assay (e.g., a nucleic acid binding aptamer assay), or a mass spectroscopic assay.

Having been generally described herein, the following non-limiting examples are provided to further illustrate the disclosure.

EXAMPLES

Example 1A: Synthesis of (.S')-aiiinuclidin-3-yl 2-(2-(4-fluorophenyl)thiazol-4-yl)propan-2- ylcarbamate (venglustat)

To a stirred solution of 4-fluorothiobenzamide (8.94 g, 57.6 mmol) in ethanol (70 mL) was added ethyl 4-chloroacetoacetate (7.8 mL, 58 mmol). The reaction was heated at reflux for 4 hours, treated with an addition aliquot of ethyl 4-chloroacetoacetate (1.0 mL, 7.4 mmol), and refluxed for an additional 3.5 hours. The reaction was then concentrated and the residue was partitioned between ethyl acetate (200 mL) and aqueous NaHCCh (200 mL). The organic layer was combined with a backextract of the aqueous layer (ethyl acetate, 1 x 75 mL), dried (Na2SC>4), and concentrated. The resulting amber oil was purified by flash chromatography using a hexane/ethyl acetate gradient to afford ethyl 2-(2-(4-fluorophenyl)thiazol-4-yl)acetate as a low melting, nearly colourless solid (13.58 g, 89%).

To a stirred solution of ethyl 2-(2-(4-fluorophenyl)thiazol-4-yl)acetate (6.28 g, 23.7 mmol) in DMF (50 mL) was added sodium hydride [60% dispersion in mineral oil] (2.84 g, 71.0 mmol). The frothy mixture was stirred for 15 minutes before cooling in an ice bath and adding iodomethane (4.4 mL, 71 mmol). The reaction was stirred overnight, allowing the cooling bath to slowly warm to room temperature. The mixture was then concentrated and the residue partitioned between ethyl acetate (80 mL) and water (200 mL). The organic layer was washed with a second portion of water (1 x 200 mL), dried (Na2SC>4) and concentrated. The resulting amber oil was purified by flash chromatography using a hexane/ethyl acetate gradient to afford ethyl 2-(2-(4-fluorophenyl)thiazol-4-yl)-2-methylpropanoate as a colourless oil (4.57 g, 66%). To a stirred solution of ethyl 2-(2-(4-fluorophenyl)thiazol-4-yl)-2-methylpropanoate (4.56 g, 15.5 mmol) in 1: 1: 1 THF/ethanol/water (45 mL) was added lithium hydroxide monohydrate (2.93 g, 69.8 mmol). The reaction was stirred overnight, concentrated, and redissolved in water (175 mL). The solution was washed with ether (1 x 100 mL), acidified by the addition of 1.0 N HC1 (80 mL) and extracted with ethyl acetate (2 x 70 mL). The combined extracts were dried (Na2SC>4) and concentrated to afford 2-(2-(4-fluorophenyl)thiazol-4-yl)-2- methylpropanoic acid as a white solid (4.04 g, 98%). This material was used in the next step without purification.

To a stirred and cooled (0 °C) solution of 2-(2-(4-fluorophenyl)thiazol-4-yl)-2- methylpropanoic acid (4.02 g, 15.2 mmol) in THF (100 mL) was added trimethylamine (4.2 mL, 30 mmol) followed by isobutyl chloroformate (3.0 mL, 23 mmol). The reaction was stirred cold for another 1 hour before adding a solution of sodium azide (1.98 g, 30.5 mmol) in water (20 mL). The reaction was stirred overnight, allowing the cooling bath to slowly warm to room temperature. The mixture was then diluted with water (100 mL) and extracted with ethyl acetate (2 x 60 mL). The combined extracts were washed with aqueous NaHCCh (1 x 150 mL) and brine (1 x 100 mL), dried (Na2SC>4) and concentrated. After coevaporating with toluene (2 x 50 mL), the resulting white solid was taken up in toluene (100 mL) and refluxed for 4 hours. (S)-3-quinuclidinol (3.87 g, 30.4 mmol) was then added and reflux was continued overnight. The reaction was concentrated and the residue partitioned between ethyl acetate (100 mL) and aqueous NaHCCh (150 mL). The organic layer was washed with water (1 x 150 mL), dried (Na2SCh) and concentrated. The resulting off-white solid was purified by flash chromatography using a chloroform/methanol/ammonia gradient to afford the title compound as a white solid (4.34 g, 73%). ¹ H NMR (400 MHz, CDCh) 5 7.96-7.88 (m, 2H), 7.16-7.04 (m, 3H), 5.55 (br s, 1H), 4.69-4.62 (m, 1H), 3.24-3.11 (m, 1H), 3.00-2.50 (m, 5H), 2.01-1.26 (m, HH) ppm. ¹³C NMR (400 MHz, CDCh) 5 166.4, 165.1, 163.8 (d, 7=250.3 Hz), 162.9, 155.0, 130.1 (d, 7=3.3 Hz), 128.4 (d, 7= 8.5 Hz), 115.9 (d, 7= 22.3 Hz), 112.5, 71.2, 55.7, 54.2, 47.5, 46.5, 28.0, 25.5, 24.7, 19.6 ppm. Purity: 100 % UPLCMS (210 nm & 254 nm); retention time 0.83 min; (M+l) 390.

Example IB: Preparation of (.S')-Oiiinuclidin-3-yl (2-(2-(4-fluorophenyl)thiazol-4-yl)propan- 2-yl)carbamate (venglustat) in free base form

Step 1: Dimethylation with methyl iodide

Chemical Formula: C₁₃H₁₂FNO₂S Chemical Formula: C₁₅H₁₆FNO₂S

Exact Mass: 265.06 Exact Mass: 293.09

Molecular Weight: 265.30 Molecular Weight: 293.36

A 3N RB flask was equipped with a thermometer, an addition funnel and a nitrogen inlet. The flask was flushed with nitrogen and potassium tert-butoxide (MW 112.21, 75.4 mmol, 8.46 g, 4.0 equiv., white powder) was weighed out and added to the flask via a powder funnel followed by the addition of THF (60 mL). Most of the potassium tert-butoxide dissolved to give a cloudy solution. This mixture was cooled in an ice-water bath to 0-2°C (internal temperature). In a separate flask, the starting ester (MW 265.3, 18.85 mmol, 5.0 g, 1.0 equiv.) was dissolved in THF (18 mL + 2 mL as rinse) and transferred to the addition funnel. This solution was added dropwise to the cooled mixture over a period of 25-30 min, keeping the internal temperature below 5°C during the addition. The reaction mixture was cooled back to 0-2°C. In a separate flask, a solution of methyl iodide (MW 141.94, 47.13 mmol, 6.7 g, 2.5 equiv.) in THF (6 mL) was prepared and transferred to the addition funnel. The flask containing the methyl iodide solution was then rinsed with THF (1.5 mL) which was then transferred to the addition funnel already containing the clear colorless solution of methyl iodide in THF. This solution was added carefully dropwise to the dark brown reaction mixture over a period of 30-40 min, keeping the internal temperature below 10°C at all times during the addition. After the addition was complete, the slightly turbid mixture was stirred for an additional 1 h during which time the internal temperature dropped to 0-5°C. After stirring for an hour at 0-5°C, the reaction mixture was quenched with the slow dropwise addition of 5.0 M aqueous HC1 (8 mL) over a period of 5-7 min. The internal temperature was maintained below 20°C during this addition. After the addition, water (14 mL) was added and the mixture was stirred for 2-3 min. The stirring was stopped and the two layers were allowed to separate. The two layers were then transferred to a 250 mL IN RB flask and the THF was evaporated in vacuo as much as possible to obtain a biphasic layer of THF/product and water. The two layers were allowed to separate. A THF solution of the Step 1 product was used in the next reaction.

Step 2: Hydrolysis of the ethyl ester with LiOH monohydrate reflux, 16 h

Chemical Formula: C₁₅H₁₆FNO₂S Chemical Formula: C₁₃Hi2FNO₂S Exact Mass: 293.09 Exact Mass: 265.06 Molecular Weight: 293.36 Molecular Weight: 265.30

The crude ester in THF was added to the reaction flask. Separately, LiOH.H2O (MW 41.96, 75.0 mmol, 3.15 grams, 2.2 equiv.) was weighed out in a 100 mL beaker to which a stir bar was added. Water (40 mL) was added and the mixture was stirred until all the solid dissolved to give a clear colorless solution. This aqueous solution was then added to the 250 mL RB flask containing the solution of the ester in tetrahydrofuran (THF). A condenser was attached to the neck of the flask and a nitrogen inlet was attached at the top of the condenser. The mixture was heated at reflux for 16 hours. After 16 hours, the heating was stopped and the mixture was cooled to room temperature. The THF was evaporated in vacuo to obtain a brown solution. An aliquot of the brown aqueous solution was analyzed by HPLC and LC/MS for complete hydrolysis of the ethyl ester. Water (15 mL) was added and this aqueous basic solution was extracted with TBME (2 x 40 mL) to remove the t-butyl ester. The aqueous basic layer was cooled in an ice-water bath to 0-10°C and acidified with dropwise addition of concentrated HC1 to pH ~ 1 with stirring. To this gummy solid in the aqueous acidic solution was added TBME (60 mL) and the mixture was shaken and then stirred vigorously to dissolve all the acid into the TBME layer. The two layers were transferred to a separatory funnel and the TBME layer was separated out. The pale yellow aqueous acidic solution was re-extracted with TBME (40 mL) and the TBME layer was separated and combined with the previous TBME layer. The aqueous acidic layer was discarded. The combined TBME layers are dried over anhydrous Na2SC>4, fdtered and evaporated in vacuo to remove TBME and obtain the crude acid as an orange/dark yellow oil that solidified under high vacuum to a dirty yellow colored solid. The crude acid was weighed out and crystallized by heating it in heptane/TBME (3: 1, 5 mL/g of crude) to give the acid as a yellow solid.

Step 3: Formation of hydroxamic acid with NH2OH.HCI

Chemical Formula: C₁₃H₁₂FNO₂S Chemical Formula: C₁3H₁3FN₂O₂S Exact Mass: 265.06 Exact Mass: 280.07 Molecular Weight: 265.30 Molecular Weight: 280.32

The carboxylic acid (MW 265.3, 18.85 mmol, 5.0 g, 1.0 equiv.) was weighed and transferred to a 25 mL IN RB flask under nitrogen. THF (5.0 mL) was added and the acid readily dissolved to give a clear dark yellow to brown solution. The solution was cooled to 0-2°C (bath temperature) in an ice-bath and N, N’ -carbonyldiimidazole (CDI; MW 162.15, 20.74 mmol, 3.36 g, 1.1 equiv.) was added slowly in small portions over a period of 10-15 minutes. The ice-bath was removed and the solution was stirred at room temperature for 1 h. After 1 h of stirring, the solution was again cooled in an ice-water bath to 0-2°C (bath temperature). Hydroxylamine hydrochloride (NH2OH.HCI; MW 69.49, 37.7 mmol, 2.62 g, 2.0 equiv.) was added slowly in small portions as a solid over a period of 3-5 minutes as this addition was exothermic. After the addition was complete, water (1.0 mL) was added to the heterogeneous mixture dropwise over a period of 2 minutes and the reaction mixture was stirred at 0-10°C in the ice-water bath for 5 minutes. The cooling bath was removed and the reaction mixture was stirred under nitrogen at room temperature overnight for 20-22 h. The solution became clear as all of the NH2OH.HCI dissolved. After 20-22 h, an aliquot of the reaction mixture was analyzed by High Pressure Liquid Chromatography (HPLC). The THF was then evaporated in vacuo and the residue was taken up in dichloromethane (120 mL) and water (60 mL). The mixture was transferred to a separatory funnel where it was shaken and the two layers allowed to separate. The water layer was discarded and the dichloromethane layer was washed with IN hydrochloride (HC1; 60 mL). The acid layer was discarded. The dichloromethane layer was dried over anhydrous Na2SC>4, filtered and the solvent evaporated in vacuo to obtain the crude hydroxamic acid as a pale yellow solid that was dried under high vacuum overnight. Step 3 continued: Conversion of hydroxamic acid to cyclic intermediate (not isolated)

Chemical Formula: Ci₃H₁₃FN₂O₂S Chemical Formula: C₁₄H₁₁ FN₂O3S Exact Mass: 280.07 Exact Mass: 306.05 Molecular Weight: 280.32 Molecular Weight: 306.31

The crude hydroxamic acid (MW 280.32, 5.1 g) was transferred to a 250 mL IN RB flask with a nitrogen inlet. A stir bar was added followed by the addition of acetonitrile (50 mL). The solid was insoluble in acetonitrile. The yellow heterogeneous mixture was stirred for 2-3 minutes under nitrogen and CDI (MW 162.15, 20.74 mmol, 3.36 g, 1.1 equiv.) was added in a single portion at room temperature. No exotherm was observed. The solid immediately dissolved and the clear yellow solution was stirred at room temperature for 2-2.5 h. After 2- 2.5 h, an aliquot was analyzed by HPLC and LC/MS which showed conversion of the hydroxamic acid to the desired cyclic intermediate.

The acetonitrile was then evaporated in vacuo to give the crude cyclic intermediate as reddish thick oil. The oil was taken up in toluene (60 mL) and the reddish mixture was heated to reflux for 2 hours during which time, the cyclic intermediate released CO2 and rearranged to the isocyanate (see below).

Chemical Formula: C₁₄H₁₁FN₂O3S Chemical Formula: C13H11FN2OS Exact Mass: 306.05 Exact Mass: 262.06 Molecular Weight: 306.31 Molecular Weight: 262.30

Step 3 continued: Conversion of the isocyanate to the free base ,

Chemical Formula: C₁₃H_{1 1}FN₂OS N₂, 18 h Chemical Formula: C₂₀H₂₄FN₃O₂S

Exact Mass: 262.06 Exact Mass: 389.16

Molecular Weight: 262.30 Molecular Weight: 389.49

The reaction mixture was cooled to 50-60°C and (S)-(+)-quinuclidinol (MW 127. 18, 28.28 mmol, 3.6 g, 1.5 equiv.) was added to the mixture as a solid in a single portion. The mixture was re-heated to reflux for 18 h. After 18 h, an aliquot was analyzed by HPLC and LC/MS which showed complete conversion of the isocyanate to the desired product. The reaction mixture was transferred to a separatory funnel and toluene (25 mL) was added. The mixture was washed with water (2 x 40 mL) and the water layers were separated. The combined water layers were re-extracted with toluene (30 mL) and the water layer was discarded. The combined toluene layers were extracted with IN HC1 (2 x 60 mL) and the toluene layer (containing the O-acyl impurity) was discarded. The combined HC1 layers were transferred to a 500 mL Erlenmeyer flask equipped with a stir bar. This stirring clear yellow/reddish orange solution was basified to pH 10-12 by the dropwise addition of 50% w/w aqueous NaOH. The desired free base precipitated out of solution as a dirty yellow gummy solid which could trap the stir bar. To this mixture was added isopropyl acetate (100 mL) and the mixture was stirred vigorously for 5 minutes when the gummy solid went into isopropyl acetate. The stirring was stopped and the two layers were allowed to separate. The yellow isopropyl acetate layer was separated and the basic aqueous layer was re-extracted with isopropyl acetate (30 mL). The basic aqueous layer was discarded and the combined isopropyl acetate layers were dried over anhydrous Na2SC>4, fdtered into a pre-weighed RB flask and the solvent evaporated in vacuo to obtain the crude free base as beige to tan solid that was dried under high vacuum overnight.

Step 3 continued: Recrystallization of the crude free base

The beige to tan colored crude free base was weighed and re-crystallized from heptane/isopropyl acetate (3: 1, 9.0 mL of solvent/g of crude free base). The appropriate amount of heptane/isopropyl acetate was added to the crude free base along with a stir bar and the mixture was heated to reflux for 10 min (free base was initially partially soluble but dissolved to give a clear reddish orange solution when heated to reflux). The heat source was removed and the mixture was allowed to cool to room temperature with stirring when a white precipitate formed. After stirring at room temperature for 3-4 h, the precipitate was fdtered off under hose vacuum using a Buchner funnel, washed with heptane (20 mL) and dried under hose vacuum on the Buchner funnel overnight. The precipitate was the transferred to a crystallizing dish and dried at 55°C overnight in a vacuum oven. 'H NMR (400 MHz, CDCh) 5 8.04 - 7.83 (m, 2H), 7.20 - 6.99 (m, 3H), 5.53 (s, 1H), 4.73 - 4.55 (m, 1H), 3.18 (dd, J = 14.5, 8.4 Hz, 1H), 3.05 - 2.19 (m, 5H), 2.0 - 1.76 (m, HH) ppm. ¹³ C NMR (100 MHz, CDCh) 5 166.38, 165.02, 162.54, 162.8-155.0 (d, C-F), 130.06, 128.43, 128.34, 116.01, 115.79, 112.46, 71.18, 55.70, 54.13, 47.42, 46.52, 27.94, 25.41, 24.67, 19.58 ppm.

Example 2: Preparation of crystalline forms of (.S')-Oiiinuclidin-3-yl (2-(2-(4- fluorophenyl)thiazol-4-yl)propan-2-yl)carbamate (venglustat) salts

Crystalline salts of (.S)-Quiniiclidin-3-yl (2-(2-(4-fluorophenyl)thiazol-4-yl)propan-2- yl)carbamate may be formed from the free base prepared as described in Example IB.

For example, the free base of (.S)-Quiniiclidin-3-yl (2-(2-(4-fluorophenyl)thiazol-4-yl)propan- 2-yl)carbamate (about 50 mmol) is dissolved IPA (140 ml) at room temperature and fdtered. The fdtrate is added into a 1 L round bottomed flask which is equipped with an overhead stirrer and nitrogen in/outlet. L-malic acid (about 50 mmol) is dissolved in IPA (100 + 30 ml) at room temperature and fdtered. The fdtrate is added into the above 1 L flask. The resulting solution is stirred at room temperature (with or without seeding) under nitrogen for 4 to 24 hours. During this period of time crystals form. The product is collected by fdtration and washed with a small amount of IPA (30 ml). The crystalline solid is dried in a vacuum oven at 55 °C for 72 hours to yield the desired malate salt.

Crystal forms of other salts (e.g., acid addition salts with succinic acid or HC1) may be prepared in an analogous manner.

Example 3 : Assay protocols

Reference standards were obtained from commercial sources (GL1 from Matreya, #1057; C16:0 GM1, Matreya, #1568; C18:0 GM1, Matreya, #1569; C24: l GM1, Matreya, #1570; GM2, Matreya, #SPL1502-EW and #1502; 24: 1 GM2, Matreya, #CUS9753; GM3, Matreya, #SPL1503-EW). Other chemicals, e.g. solvents, were obtained from commercial suppliers as LC/MS or HPLC grade materials.

Measured concentration values obtained using the LC/MS/MS methods described here are influenced by the relative abundance of each individual isomer in the reference material and the instrument and instrument settings. The skilled reader will appreciate that minor adjustments may be made to the methods described herein to obtain equivalent results using different reference materials and/or instruments.

Hexosaminidase activity was measured using a conventional assay (see, e.g., Ben-Yoseph et al., Am J Hum Genet (1985) 37(4): 733-740).

Quantitation of GL1 in human plasma by LC/MS/MS

The objective was to validate a method for the determination of GL1 in human K2EDTA plasma using LC/MS/MS. Delipidized human plasma was used for surrogate matrix for calibrators and assay QCs (see, e.g., Zheng et al., Mol Genet Metab Rep (2016) 8:77-79). Authentic human plasma samples were also tested for various assay parameters. Fifty microliters of plasma samples were processed using liquid-liquid extraction, followed by SPE procedure. The extracts were dried down and reconstituted for LC/MS/MS analysis. Seven GL1 isoforms and internal standard (N-octadecanoyl-D35-psychosine)were quantified with chromatographic separation through an Xbridge™ HILIC column and an ESI positive MRM scanning on the tandem mass spectrometer.

The transitions of GL-1 isoforms and internal standard monitored on mass spectrometer are listed in the table below:

LC/MS/MS instrument consisted of either a Waters Acquity UPLC and a Sciex API 4000 triple quadrupole mass spectrometer, or a Shimazu Nexra HPLC and a Sciex API 5000 mass spectrometer. The LC was equipped with a Waters Xbridge™ HILIC column (3.5 pm, 2.1 x 50 mm) operating at 40°C. A 2 minute isocratic elution at 0.5 mL/min was carried out for each sample with a mobile phase composed of 0. 1% formic acid and 2mM ammonium acetate in 5:95 water/methanol. MS detection was performed using with ESI+ ionization. The lonSpray voltage was 5000 V and the temperature was held at 500°C. The entrance potential was 9.00 V. Nitrogen was used as the collision, curtain, nebulizing and auxiliary gas. Spectra were acquired over 2.00 minutes.

The total GL1 peak areas were integrated from summed intensities of 7 GL1 isoforms with Multiquant™ software. The calibration curves were built by linear regression of peak area ratio of the total GL-1 to IS against the nominal concentration of the respective calibration standard.

The method was validated over the concentration range of 0. 1 to 20 pg/mL with linear regression and a weighting factor of 1/x². Acceptable intra- and inter-run accuracy and precision were demonstrated. The method showed no matrix effect in 6 individual human plasma lots. GL1 was stable in plasma up to 26 hours at room temperature, up to 236 days at < -14°C and 1294 days at < -60°C and following 5 freeze/thaw cycles at < -60°C. Interference was not observed in the human plasma under hemolysis and hyperlipidemia conditions. All assay acceptance criteria were met for the assay parameters tested.

Quantitation of GL1 in human CSF by LC/MS/MS

The objective was to validate a method for the quantitation of GL1 in human CSF using LC/MS/MS detection. Fifty microliters of CSF sample with 0.4% Tween 20 was processed with liquid-liquid extraction, followed by LC/MS/MS analysis. Seven GL1 isoforms and internal standard (¹³C6-Glucosylceramide (isotopically labeled C24:0 GL1 with a m/z of 818.70)) were quantified with chromatographic separation through an Atlantis® HILIC Silica Column (3 pm, 2.1x150 mm) and an ESI positive MRM scanning on the tandem mass spectrometer. A gradient elution at 0.6 mL/min was performed for each sample with mobile phase A consisting of 96% acetonitrile, 2% methanol, 1% water, 1% acetic acid, 5 mM ammonium acetate and mobile phase B consisting of 98% methanol, 1% water, 1% acetic acid, 5 mM ammonium acetate. MS detection was performed using a Sciex QTRAP-6500 spectrometer with an lonSpray voltage of 5500 V and an entrance potential of 10.0 V, holding the temperature at 400°C.

The total GL1 peak areas were summed from individually integrated peak areas of 7 GL1 isoforms (the same isoforms as noted above in connection with the plasma GL1 assay). In each case, the daughter ion had a m/z of 264.2. The calibration curves were built by linear regression of peak area ratio of the total GL1 to IS against the nominal concentration of the respective calibration standard.

The method was validated over the concentration range of 2.00 to 200 ng/mL with linear regression and a weighting factor of 1/x. Acceptable intra- and inter-run accuracy and precision were demonstrated. A 5-fold dilution with artificial CSF was acceptable. The method showed no matrix effect in 6 individual human CSF lots containing 0.4% Tween 20. GL1 was stable in human CSF up to 29 hours at room temperature and 5 freeze/thaw cycles, 5.5 months at < -14°C and up to 39 months at < -60°C. Processed sample stability was established for 72 hours at 2°C to 8°C and 10 days at < -14°C. The results met the relative matrix effect acceptance criteria. All assay acceptance criteria were met for the assay parameters tested.

Quantitation of total GM1 in human plasma by LC/MS/MS

The objective was to validate an LC/MS/MS assay for the quantitation of total GM1 in human plasma. Fifty microliters of the human plasma samples were processed using protein precipitation, followed by LC/MS/MS analysis. Six GM1 isoforms and internal standard (N- hexadecanoyl-D9-monosialoganglioside GM1, Matreya, #2057) were detected and the sum of the 6 isoforms was quantified using LC/MS/MS.

HPLC was performed with chromatographic separation through a BEH Amide column (130A, 1.7 pm, 2.1x50 mm) and an ESI positive MRM scan on the tandem mass spectrometer. Mobile phase A was 85% acetonitrile, 10% methanol, 4% water, 1% FA. Mobile phase B was 10% methanol, 89% water, 1% FA. Flow rate was 0.7 mL/min. MS detection was performed using a Sciex QTRAP-6500 spectrometer with an lonSpray voltage of 5400 V and an entrance potential of 10.0 V, holding the temperature at 350°C.

The total GM1 peak areas were integrated from the summed intensities of 6 GM1 isoforms with Multiquant™ software. The calibration curves were built by linear regression of the peak area ratio of the total GM1 to IS against the nominal concentration of the respective calibration standard with 1/x² weighting.

The results demonstrated that the method was precise and accurate from 25.0 to 2000 ng/mL with an LLOQ of 25.0 ng/mL. A 5-fold dilution with delipidized human serum was acceptable. GM1 in human plasma was stable for up to 6 days at ambient temperature, up to 5 freeze/thaw cycles, and up to 12 months at < -14°C and at < -60°C. The results met the relative matrix effect acceptance criteria. All assay acceptance criteria were met for the assay parameters tested.

Quantitation of GM1 in human CSF by LC/MS/MS

The objective was to validate the assay for quantitation of GM1 in human CSF using LC/MS/MS. Fifty microliters of human CSF with 0.3% bovine serum albumin (BSA) was processed using protein precipitation and was analyzed with LC/MS/MS. GM1 and IS (N- hexadecanoyl-D9-monosialoganglioside GM1, Matreya, #2057) were quantified with the chromatographic separation through a Waters BEH amide column (130A, 1.7 pm, 2.1 x 50 mm) and an ESI positive MRM scan on the tandem mass spectrometer, substantially as described above for the GM1 plasma assay. GM1 was quantitated by summing the intensities of GM1 isoforms Cer36: 1 and Cer38: 1 (other isoforms were monitored but not quantitated). The calibration curves were built by linear regression of the peak area ratio of the GM1 to IS against the nominal concentration of the respective calibration standard with 1/x² weighting.

The results demonstrated that the method was precise and accurate from 4 to 160 ng/mL with an LLOQ of 4 ng/mL. A 10-fold dilution with artificial CSF/ 0.3% BSA was acceptable.

GM1 in human CSF / 0.3% BSA was stable for up to 30.1 hours at ambient temperature, up to 5 freeze/thaw cycles, and up to 7 months at < -14°C and up to 12 months < -60°C. All assay acceptance criteria were met for the assay parameters tested.

Quantitation of total GM2 in human plasma by LC/MS/MS

The objective was to validate an LC/MS/MS assay for the quantitation of total GM2 in human plasma by LC/MS/MS. Two major GM2 isoforms (C18:0 and C24: 1) in human plasma are used as GM2 standard in the assay.. Ten microliters of human plasma was processed using protein precipitation and analyzed with LC/MS/MS. Seven GM2 isoforms and internal standard (N-hexadecanoyl-D9-monosialoganglioside GM2, Matreya, #CUS9725) were quantified using LC/MS/MS.

HPLC was performed with chromatographic separation through Waters BEH amide column (130A, 1.7 pm, 2.1x50 mm) using 8.0 min LC gradient program, followed by ESI positive MRM scan from the tandem mass spectrometer. Mobile phase A was 85% acetonitrile, 10% methanol, 4% water, 1% formic acid. Mobile phase B was 10% methanol, 89% water, 1% FA. Flow rate was 0.4 mL/min. MS detection was performed using a Sciex QTRAP-6500 spectrometer with an lonSpray voltage of 5400 V and an entrance potential of 10.0 V, holding the temperature at 350°C.

The results demonstrated that the method was precise and accurate from 50 to 2000 ng/mL with an LLOQ of 50 ng/mL. A 5-fold dilution with delipidized human serum was acceptable. GM2 in human plasma was stable for up to 27.9 hours at ambient temperature, up to 5 freeze/thaw cycles, and up to 6 months at < -14°C and 24 months at < -60°C. All assay acceptance criteria were met for the assay parameters tested.

Quantitation of GM2 in human CSF by LC/MS/MS

The objective was to validate an LC/MS/MS assay for the quantitation of GM2 (Cl 8:0) in human CSF. Fifty microliters of CSF were processed using protein precipitation, followed by LC/MS/MS analysis. The GM2 and internal standard (N-hexadecanoyl-D9- monosialoganglioside GM2, Matreya, #CUS9725, mostly Cl 8:0) were quantified with chromatographic separation through a BEH Amide column and an ESI positive MRM scanning on the tandem mass spectrometer, substantially as described above for the GM2 plasma assay. The temperature in the MS/MS phase was held at 450°C, under which conditions the IS had a Q3 mass of 204.2 Da. C24: 1 GM2 was not quantified in this method. The calibration curves were built by linear regression of the peak area ratio of the GM2 to IS against the nominal concentration of the respective calibration standard with 1/x² weighting.

The method was suitable for determination of GM2 (Cl 8:0) in human CSF with 0.3% BSA over the range of 2.50 ng/mL (LLOQ) to 250 ng/mL. Up to 5-fold dilution with artificial CSF/ 0.3% BSA was acceptable. The GM2 in human CSF was stable for up to 24 hours at ambient temperature, up to 5 freeze/thaw cycles, up to 3 months at < -14°C, and up to 42 months at < -60°C. All assay acceptance criteria were met for the assay parameters tested. Quantitation of total GM3 in human plasma by LC/MS/MS

The objective was to validate the LC/MS/MS assay for the quantitation of total GM3 in human plasma. Twenty microliters of human plasma sample was processed using protein precipitation and analyzed with LC/MS/MS (see, e.g., also Peterschmittet al., Clin Pharm Drug Dev (2021) 10( 1): 86— 98, supplementary materials). Seven GM3 isoforms and internal standard (Gangliotriaosylceramide GA2, Matreya, #1512) were quantified.

HPLC was performed with chromatographic separation through XBridge™ BEC HILIC column (130A, 3.5 pm, 2. 1x50 mm; Waters) using LC gradient program, followed by an ESI positive MRM scan on the tandem mass spectrometer. Mobile phase A was 97% acetonitrile, 2% methanol, 1% acetic acid, 5 mM ammonium acetate. Mobile phase B was 99% methanol, 1% acetic acid, 5 mM ammonium acetate. Flow rate was 1.0 mL/min. MS detection was performed using a Sciex API4000 spectrometer with an lonSpray voltage of 5000 V and an entrance potential of 10.0 V, holding the temperature at 400°C.

The total GM3 peak areas were integrated from summed intensities of the 7 GM3 isoforms with the Multiquant™ software. The calibration curves were built by linear regression of the peak area ratio of the total GM3 to IS against the nominal concentration of the respective calibration standard with 1/x² weighting.

The results demonstrated that the method was precise and accurate from 4.0 to 80 pg/mL with an LLOQ of 4.0 pg/mL. There were no interferences from hemoglobin, bilirubin, or lipids. The GM3 in human plasma was stable for up to 7 days at ambient temperature, up to 5 freeze/thaw cycles, up to 5 days at 2°C-8°C, up to 1 month at < -14°C, and up to 24 months at < -60°C. All assay acceptance criteria were met for the assay parameters tested.

Quantitation of GM3 in human CSF by LC/MS/MS

The objective was to validate an LC/MS/MS assay for the quantitation of the total GM3 in human CSF. Forty microliters of the CSF with 0.3% BSA was processed using protein precipitation and analyzed with LC/MS/MS. Seven GM3 isoforms (the same isoforms which were assessed in the GM3 plasma assay) and internal standard (C16:0 GM3-[palmyl-D31], QI mass = 1184.9 Da, Q3 mass = 265.4 Da) were quantified with chromatographic separation through a BEH Amide column (130A, 1.7 pm, 2. 1x50 mm; Waters) and an ESI positive MRM scan from the tandem mass spectrometer. Mobile phase A was 90% acetonitrile, 5% methanol, 4% water, 1% FA. Mobile phase B was 50% methanol, 49% water, 1% FA. Flow rate was 0.6 mL/min. MS detection was performed using a Sciex Qtrap 6500 spectrometer with an lonSpray voltage of 5500 V and an entrance potential of 10.0 V, holding the temperature at 350°C.

The total GM3 peak areas were integrated from the summed intensities of the 7 GM3 isoforms with the Multiquant™ software. The calibration curves were built by linear regression of the peak area ratio of the total GM3 to IS against the nominal concentration of the respective calibration standard with 1/x² weighting.

The results demonstrated that the method was precise and accurate from 5 to 200 ng/mL with an LLOQ of 5 ng/mL. A 5-fold dilution with artificial CSF/ 0.3% BSA was acceptable. The method showed acceptable extraction recovery and relative matrix effect. GM3 in the human CSF/0.3% BSA was stable for up to 26.1 hours at ambient temperature, up to 5 freeze/thaw cycles, and up to 6 months at < -14°C and 24 months at < -60°C. All assay acceptance criteria were met for the assay parameters tested.

Example 4: Assessment of biomarker levels in healthy individuals

The assays described in Example 3 were employed to test biomarker levels in plasma and CSF samples collected from healthy individuals (sourced from PrecisionMed, Carlsbad, CA for CSF samples; and BioIVT, Westbury, NY for plasma samples). The following table indicates the ranges of values which were measured:

Table 1: Normal ranges for biomarkers in plasma and CSF samples

¹ Plasma GM3 and GL1 were both measured with n = 100 samples, data reported as min to max (i.e., not 95^th percentile)

Example 5: Assessment of biomarker levels in patients with GM2 gangliosidoses and other gangliosidoses

As part of a study on the impact of venglustat in patients with GM2 gangliosidoses and other gangliosidoses, plasma and CSF levels of various biomarkers were measured over a period of 104 weeks. Samples were analysed in accordance with the assays described above. Patient demographics and study details

Patients were recruited in two populations, a primary population of adult late-onset GM2 gangliosidoses (Sandhoff disease and Tay-Sachs disease) and a secondary population including juvenile GM2 gangliosidosis, GM1 gangliosidosis, and sialidosis type I. All patients were at least 2 years of age and > 10 kg in weight at the start of the study. The demographics and baseline characteristics of the two populations are shown in Table 2 below:

Table 2: Demographics and baseline characteristics

Data are presented as mean ± standard deviation, or median [interquartile range ], unless otherwise stated

The study was designed to include 4 main periods:

Period 1 (Day -60 to Day -1): Screening assessments.

Period 2 (Week 0 to Week 104): Primary treatment and analysis period. Participants in the primary population randomly assigned in a 2: 1 ratio to receive venglustat or placebo once daily in a double-blind design. Participants in the secondary population receive venglustat for 104 weeks in an open -label design.

Period 3 (Week 104 to Week 208): Open label period. Participants in both the primary and secondary populations automatically entered into the OLE period following completion of period 2.

Period 4 (Week 208 to Week 214). 6-week post-treatment safety observation period. Screening assessment included medical history and confirmation of diagnosis (where appropriate) by genotyping, e.g. by HEXA / HEXB genotyping for Sandhoff and Tay-Sachs disease in the primary population.

Venglustat was administered at 15 mg dosages once daily to adult patients (age 18 and older). Dosing in patients < 18 years old was in 4 mg, 6 mg, 12 mg or 15 mg dosages once daily (calculated as free base), based on patient bodyweight:

Participants were scheduled for biological sample collection at the following visits: CSF was collected via lumbar puncture in accordance with standard clinical practice. A volume of approximately 7-8 mb was collected, of which the first 1-2 mb was discarded. Plasma was collected in accordance with standard clinical practice.

Biomarker analysis and results - primary population

Table 3 below shows the changes in CSF biomarker levels in patients with Tay-Sachs disease and Sandhoff disease (median values, with lower and upper quartile range, and number of patients, shown in brackets). Results are shown separately for the placebo group (n = 13 at baseline for Tay-Sachs and n = 5 at baseline for Sandhoff) and the venglustat treated group (n = 29 at baseline for Tay-Sachs and n = 10 at baseline for Sandhoff):

Table 4 below shows the changes in plasma biomarker levels in patients with Tay-Sachs disease and Sandhoff disease (median values, with lower and upper quartile range, and number of patients, shown in brackets):

The extent of CSF GM2 reduction in the whole of the primary population is shown in Figure

2 as a percentage change from baseline. This illustrates significant difference between the patients in the placebo and venglustat treated groups (estimated difference -36.2% with 90% confidence interval [-44.8% to -27.7%], one-sided p-value < 0.0001).

Percentage changes in biomarker levels in the Tay-Sachs and Sandhoff cohorts are shown in Figures 3 to 5. Percent change in CSF levels of GM2, GM3 and GL1 at week 104 are shown in Figs. 3A, 4A and 5A, respectively. Percent change in plasma levels of GM2, GM3 and GL1 at weeks 12-104 in the Tay-Sachs and Sandhoff cohorts are shown in Figs. 3B, 4B and 5B, respectively.

Figure 6 shows the correlation between baseline hexosaminidase level and the percentage change in CSF GM2 in the venglustat treated patients of the primary population.

Biomarker analysis and results - secondary population

Table 5 below shows the changes in CSF biomarker levels in patients with GM1 gangliosidosis (n = 7 at baseline), juvenile GM2 gangliosidosis (n = 7 at baseline), and sialidosis (n = 1). Median values are shown for patients with GM1 and juvenile GM2 gangliosidosis only, with lower and upper quartile range (and number of patients) in brackets.

Table 5: CSF levels of biomarkers in secondary population

GMl (ng/mL) GM2 (ng/mL) GM3 (ng/mL) GL1 (ng/mL)

^GM1 Baseline 57.9 (54.3 to 91.6) (7) - - 7.24 (4.29 to 17.50) (6) gangliosidosis

104 weeks 41.3 (31.2 to 45.9) (4) - - 1.00 (1.00 to 2.23) (5)

Juvenile GM2 Baseline - 188.0 (82.6 to 344.0) (7) - 8.18 (5.33 to 8.74) (7) gangliosidosis

104 weeks - 94.8 (54.2 to 143.0) (7) - 1.00 (1.00 to 1.00) (7)

Sialidosis Baseline - 5.8 (1) 67.0 (1) 22.40 (1)

104 weeks - 5.0 (1) 16.0 (1) 2.28 (1)

” indicates that the biomarker was not measured.

Table 6 below shows the changes in plasma biomarker levels in patients with GM1 gangliosidosis, juvenile GM2 gangliosidosis, and sialidosis. Median values are shown for patients with GM1 and juvenile GM2 gangliosidosis only, with lower and upper quartile range (and number of patients) in brackets.

Table 6: Plasma levels of biomarkers in secondary population

GMl (ng/mL) GM2 (ng/mL) GM3 (gg/iiiL) GL1 (gg/iiiL)

^GM1 Baseline 194 (169 to 374) (7) - - 5.67 (3.94 to 6.72) (7) gangliosidosis

12 weeks 121 (89 to 212) (7) - - 1.19 (0.81 to 1.65) (7)

26 weeks 94 (79 to 205) (7) - - 0.84 (0.81 to 1.46) (7)

52 weeks 93 (83 to 158) (7) - - 1.25 (0.86 to 1.63) (7)

78 weeks 92 (77 to 114) (7) - - 0.94 (0.71 to 1.32) (7)

104 weeks 82 (78 to 107) (6) - - 0.94 (0.70 to 1.14) (6)

Juvenile GM2 Baseline - 782 (523 to 964) (7) - 5.58 (3.25 to 6.93) (7) gangliosidosis

12 weeks - 357 (286 to 479) (7) - 1.09 (0.79 to 1.46) (7)

26 weeks - 314 (261 to 590) (7) - 1.23 (0.84 to 1.54) (7)

52 weeks - 362 (249 to 545) (7) - 1.27 (1.17 to 1.45) (7)

78 weeks - 341 (312 to 471) (7) - 1.06 (0.84 to 1.51) (7)

104 weeks - 330 (210 to 448) (7) - 1.21 (0.79 to 1.33) (7)

Sialidosis Baseline - 376 (1) 13.6 (1) 5.59 (1)

12 weeks -

26 weeks - 170 (1) 2.0 (1) 0.96 (1)

52 weeks - 159 (1) 4.0 (1) 1.12 (1)

78 weeks - 166 (1) 2.0 (1) 0.82 (1)

104 weeks - 233 (1) 4.1 (1) 1.03 (1)

” indicates that the biomarker was not measured.

Percentage changes in biomarker levels in the secondary population cohorts are shown in Figures 7 to 9. Percent change in CSF levels of GM1, GM2, and GL1 at baseline and at week 104 are shown in Figs. 7A, 8A and 9A, respectively. Percent change in plasma levels of GM1, GM2 and GL1 at baseline and at weeks 12-104 are shown in Figs. 7B, 8B and 9B, respectively.

Safety and adverse events

Consistent with earlier studies, venglustat was generally well tolerated in the primary and secondary populations described above. 8 participants from the primary population discontinued the study (2 from the placebo group and 6 from the venglustat treated group). Treatment emergent adverse events were reported with a similar frequency in both placebo and venglustat treatment groups, including injuries, infections, gastrointestinal events, nervous events, musculoskeletal events, respiratory events, psychiatric events, and skin events.

Conclusions

Disease biomarkers were greatly reduced in CSF and in plasma of subjects treated with venglustat. A significant reduction in CSF GM2 level was observed in adult patients with GM2 gangliosidoses as compared to placebo, and marked reductions were observed in GM1 levels in patients with GM1 gangliosidosis, in GM2 levels in patients with juvenile GM2 gangliosidosis, and in GM3 levels in a patient with sialidosis. Example 6: Assessment of disease-associated protein levels in CSF of patients with GM1 and GM2 gangliosidoses

Following the study described in Example 5, CSF samples from the primary population and from the secondary population (GM1 gangliosidosis and juvenile GM2 gangliosidosis only) were tested for levels of CD63 and NFL.

Sample handling and analytical protocols

Samples obtained during the study described in Example 5 were used. The samples had been stored at -70°C (or lower) for varying lengths of time, with baseline samples being collected at least 2 years before analysis, and week 104 samples being collected less than 1 year before analysis. Samples were thawed before testing (some samples had been thawed more than once).

CD63 concentration was measured by Olink® profiling using the Olink Explore HT panel (Olink Proteomics AB, Uppsala, Sweden) according to the manufacturer's instructions, as set out in more detail below. NFL concentration was measured on HD-X™ Automated Immunoassay Analyzer using Simoa® NF-light™ V2 Advantage Kit (Quanterix) according to the manufacturer’s instructions. More generally, methods which could be used to measure the concentration of CD63 or NFL in CSF include immunoassays (e.g., ELISA, microfluidic ELISA, or bead-based high sensitivity ELISA; capillary Western blot assay; nanoneedle bioarray (e.g., from Nanomosaic®), Proximity Ligation Assay (e.g. NULISA®); or PEA), other affinity-based assays (e.g. nucleic acid binding aptamers from Somalogic®), or mass spectroscopy.

For the CD63 Olink assay, 10 pL of CSF samples and Olink controls were placed into a 384- well Sample Source Plate and then diluted 1: 10, 1: 100, 1: 1000 and 1: 100,000 in 384-well Sample Diluent Plates using Mosquito and Dragonfly liquid handlers (both from SPT Labtech). For immunoreaction, these diluted samples were mixed with the Olink® probes (DNA oligo-conjugated antibodies) in 384-well Incubation Plates using the Mosquito handler and incubated at 4 °C for 16-24 hours. The following day, the PCR reagents were added to the immunoreaction mixtures using the Dragonfly handler, and PCR reactions were conducted using ProFlex PCR System (Applied Biosciences). PCR products were pooled using epMotion 5075 liquid handler (Eppendorf) to create libraries, which were then purified using magnetic beads and QC was performed using Bioanalyzer 2100 (Agilent). The libraries were then analyzed by Next Generation Sequencing using NovaSeq 6000 (Illumina). Any samples that failed quality control were excluded from further analysis. The final assay readout is presented in Normalized Protein expression (NPX) values, which is an arbitrary unit on a log2-scale where a higher value corresponds to a higher protein expression. All assay validation data (e.g., detection limits, intra- and inter-assay precision data, etc.), experimental protocols, and data processing are available on the manufacturer's website (www.olink.com). For NFL concentration measurements, CSF samples were diluted 100-fold using Sample dilution buffer (Quanterix) before being analysed using the Simoa assay in accordance with the manufacturer’s protocols.

Results and discussion

The levels of CD63 at baseline in CSF samples from the primary population and secondary population are shown in Figure 10 (CD63 levels are shown on a linear scale and calculated as 2^NPX(BL), j _e 2 to the power of the NPX level at baseline). Figure 11 shows the percent change in the CD63 levels at week 104 in the CSF (as compared to baseline). The percent change is calculated in accordance with the following equation, wherein NPX(104) denotes the NPX value at week 104 and NPX(BL) denotes the NPX value at baseline: 100

The results shown in Figures 10 and 11 suggest that venglustat administration in patients with GM2 gangliosidoses (juvenile and adult) may lead to a reduction in CD63 levels in the CSF.

The levels of NFL at baseline in CSF samples from the primary population and secondary population are shown in Figure 12 (in pg/mL). Figure 13 shows the percent change in the CD63 levels at week 104 in the CSF (as compared to baseline). These results suggest that venglustat administration in patients with juvenile GM2 gangliosidosis may lead to a reduction in NFL levels in the CSF.

It is to be understood that while the disclosure has been described in conjunction with the above embodiments, that the foregoing description and examples are intended to illustrate and not limit the scope of the disclosure. Other aspects, advantages, and modifications within the scope of the disclosure will be apparent to those skilled in the art to which the disclosure pertains.

In addition, where features or aspects are described in terms of Markush groups, those skilled in the art will recognize that such features or aspects are also thereby described in terms of any individual member or subgroup of members of the Markush group.

All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.

Claims

1. A method of reducing the level of a biomarker in a biological fluid of a human subject in need thereof suffering from a ganglioside storage disorder, the method comprising administering to the subject an effective amount of venglustat or a pharmaceutically acceptable salt thereof, wherein the biomarker is selected from the group consisting of GM1, GM2, GM3, GL1, and combinations thereof.

2. A method of administering venglustat, or a pharmaceutically acceptable salt thereof, to a human subject in need thereof suffering from a ganglioside storage disorder, wherein the venglustat or pharmaceutically acceptable salt thereof is administered in an effective amount to reduce the level of a biomarker in a biological fluid of the subject.

3. The method of claim 1 or claim 2, wherein the biological fluid is selected from the group consisting of plasma and CSF.

4. The method of any one of claims 1 to 3, wherein the biomarker is selected from: (a) GM1, optionally in combination with GM3 and/or GL1; (b) GM2, optionally in combination with GM3 and/or GL1; and (c) GM3, optionally in combination with GM2 and/or GL1.

5. The method of any one of claims 1 to 4, wherein the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of the biomarker by at least about 30%, e.g. by at least about 40%, 50%, 60%, 70% or 80%.

6. The method of any one of claims 1 to 5, wherein the venglustat or pharmaceutically acceptable salt thereof is administered orally, e.g. in the form of a tablet or capsule.

7. The method of any one of claims 1 to 6, wherein the venglustat or pharmaceutically acceptable salt thereof is administered once daily.

8. The method of any one of claims 1 to 7, wherein either the subject is aged 18 or over and the venglustat or pharmaceutically acceptable salt thereof is administered in a dosage (calculated as the free base) of about 15 mg per day; or wherein the subject is aged below 18 and the venglustat or pharmaceutically acceptable salt thereof is administered in a dosage (calculated as the free base) of:

(a) about 15 mg per day to a subject having a body weight of > 50 kg;

(b) about 12 mg per day to a subject having a body weight of 30 kg to < 50 kg;

(c) about 6 mg per day to a subject having a bodyweight of 15 kg to < 30 kg; or

(d) about 4 mg per day to a subject having a bodyweight of 10 kg to < 15 kg.

9. The method of any one of claims 1 to 8, wherein the venglustat is in the form of venglustat free base, or a pharmaceutically acceptable salt of venglustat, optionally venglustat L-malate salt.

10. The method of any one of claims 1 to 9, wherein the ganglioside storage disorder is selected from a GM2 gangliosidosis (e.g., Sandhoff disease, or Tay-Sachs disease), and GM1 gangliosidosis.

11. A method of reducing the level of a biomarker in a biological fluid of a human subject in need thereof suffering from a GM2 gangliosidosis, the method comprising administering to the subject an effective amount of venglustat or a pharmaceutically acceptable salt thereof, wherein the biomarker is GM2, optionally in combination with GM3 and/or GL1.

12. The method of claim 11, wherein:

(a) the GM2 gangliosidosis is Tay-Sachs disease and the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of GM2 in the CSF by at least about 30%; or

(b) the GM2 gangliosidosis is Sandhoff disease and the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of GM2 in the CSF by at least about 40%.

13. The method of claim 11, wherein the GM2 gangliosidosis is juvenile GM2 gangliosidosis and the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of (or is effective to reduce the level of) GM2 in the CSF by at least about 30%.

14. The method of any one of claims 11 to 13, wherein the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of GM2 in the plasma to a level between about 200 and about 750 ng/mL.

15. A method of reducing the level of a biomarker in a biological fluid of a human subject in need thereof suffering from GM1 gangliosidosis, the method comprising administering to the subject an effective amount of venglustat or a pharmaceutically acceptable salt thereof, wherein the biomarker is GM1, optionally in combination with GM3 and/or GL1.

16. The method of claim 15, wherein the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of GM1 in the CSF to a level between about 25 and 65 ng/mL, and/or reduces the level of GM1 in the plasma to a level between about 40 and about 115 ng/mL.

17. The method of any one of claims 1 to 16, wherein:

(i) the method also reduces the level of CD63 in the CSF of the subject;

(ii) the venglustat or pharmaceutically acceptable salt thereof is administered in an effective amount to also reduce the level of CD63 in the CSF of the subject; or

(iii) the administration of an effective amount of venglustat or a pharmaceutically acceptable salt thereof also reduces the level of CD63 in the CSF of the subject, wherein the ganglioside storage disorder from which the subject suffers is a GM2 gangliosidosis.

18. The method of claim 17, wherein:

(a) the GM2 gangliosidosis is adult Tay-Sachs disease, or adult Sandhoff disease, and the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of (or is effective to reduce the level of) CD63 in the CSF by at least about 30%; or

(b) the GM2 gangliosidosis is juvenile GM2 gangliosidosis and the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of (or is effective to reduce the level of) CD63 in the CSF by at least about 35%.

19. The method of any one of claims 1 to 18, wherein:

(i) the method also reduces the level of NFL in the CSF of the subject;

(ii) the venglustat or pharmaceutically acceptable salt thereof is administered in an effective amount to also reduce the level of NFL in the CSF of the subject; or

(iii) the administration of an effective amount of venglustat or a pharmaceutically acceptable salt thereof also reduces the level of NFL in the CSF of the subject, wherein the ganglioside storage disorder from which the subject suffers is juvenile GM2 gangliosidosis.

20. The method of claim 19, wherein the administration of venglustat, or the pharmaceutically acceptable salt thereof, reduces the level of (or is effective to reduce the level of) NFL in the CSF by at least about 50%.

21. Venglustat, or a pharmaceutically acceptable salt thereof, for use in a method as claimed in any one of claims 1 to 20.

22. Use of venglustat, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for use in a method as claimed in any one of claims 1 to 20.