Treatment of gm1 gangliosidosis

Abstract

The present invention relates to compounds of Formula (I) for use in the treatment of GM1 gangliosidosis. In particular, the present invention relates to compounds of Formula (I) for use in reducing the accumulation of GM1 ganglioside in the brain and / or spinal cord of a subject, increasing neuronal survival in a subject, and / or preventing neuronal degeneration in a subject.

Description

Technical Field

[0001] This invention relates to compounds of formula (I),

[0002]

[0003] It is used to treat GM1 ganglioside storage syndrome. In particular, the present invention relates to compounds of formula (I) for reducing the accumulation of GM1 gangliosides in the brain and / or spinal cord of a subject, improving neuronal survival in the subject, and / or preventing neuronal degeneration in the subject. Background Technology

[0004] GM1 ganglioside storage disorder is a hereditary lysosomal storage disorder that gradually destroys neurons in the brain and spinal cord. GM1 ganglioside storage disorder is caused by a genetic mutation in the GLB1 gene and is inherited in an autosomal recessive manner. The GLB1 gene provides the instructions for the production of β-galactosidase, an enzyme found in lysosomes. β-galactosidase helps break down several molecules, including GM1 gangliosides. GM1 gangliosides are important for the normal function of neurons in the brain.

[0005] GLB1 gene variants produce a form of β-galactosidase that is less efficient than the normal form of the enzyme in breaking down GM1 gangliosides. Without sufficient functional β-galactosidase, GM1 gangliosides cannot be broken down when no longer needed. This substance accumulates to toxic levels in many tissues and organs, particularly in the brain. Damage caused by GM1 ganglioside accumulation leads to neuronal destruction, resulting in numerous signs and symptoms of GM1 ganglioside storage disease.

[0006] In GM1 ganglioside storage disorders, the severity of symptoms and the timing of their first appearance can vary considerably. Generally, early manifestations of symptoms (such as those occurring in infants or in type I GM1 ganglioside storage disorders) are often part of a more severe and rapidly progressing disease. Signs and symptoms that children with GM1 ganglioside storage disorders may experience include distinctive facial features (coarse facial features), hypotonia (low muscle tone), enlarged liver and spleen (hepatosplenomegaly), excessive startle reflexes, developmental regression, skeletal abnormalities, deformities, organ enlargement, cardiomyopathy, interstitial lung disease, seizures, ataxia, dysarthria, and visual disturbances.

[0007] Currently, there is no treatment for GM1 ganglioside storage disease. Therefore, there is a need for treatment for GM1 ganglioside storage disease.

[0008] WO2015 / 147639 A1 describes a wide range of deoxynojirimycin derivatives, including compounds of the following formula (referred to herein as compounds of formula (I)):

[0009] .

[0010] The compound of formula (I) is a potent dual inhibitor of glucocerebromidase and nonlysosomal glucocerebromidase (GBA2, UniProt code: Q9HCG7). This compound is known by the trade name "nizubaglustat".

[0011] WO2022 / 069709 A1 describes two crystalline forms of the compound of formula (I); "type 3" and "type 2". The X-ray diffraction pattern of the "type 3" crystalline form is visible in WO2022 / 069709 A1. Figure 6 A. The X-ray diffraction pattern of the “Type 2” crystal form can be seen in Figure 2 of WO2022 / 069709 A1. Summary of the Invention

[0012] The inventors have tested compound (I) (i.e., nizubaglustat) in a human induced pluripotent stem cell model of GM1 ganglioside storage disease and found that this compound can reduce GM1 ganglioside accumulation in GLB1-deficient brain organoids in a dose-dependent manner, accompanied by improved neuronal survival. The inventors have also tested compound (nizubaglustat) in an animal (mouse) model of GM1 ganglioside storage disease (strain: B6.129P2(FVB)-Glb1). tm1Adz The compound of formula (I) (i.e., nizubaglustat) was tested in / J) and found to be suitable for the treatment of GM1 ganglioside storage disease and / or symptoms associated with GM1 ganglioside storage disease. The compound also showed no toxicity to human brain cells.

[0013] In a first aspect, the present invention provides a compound of formula (I),

[0014] ,

[0015] It is used to treat GM1 ganglioside storage disorder in subjects.

[0016] In another aspect, the present invention provides a compound of formula (I),

[0017]

[0018] It is used to reduce the accumulation of GM1 gangliosides in the brain and / or spinal cord of a subject, preferably a subject suffering from GM1 ganglioside storage disease.

[0019] In another aspect, the present invention provides a compound of formula (I),

[0020]

[0021] It is used to improve neuronal survival in subjects, preferably those suffering from GM1 ganglioside storage disease.

[0022] In another aspect, the present invention provides a compound of formula (I),

[0023]

[0024] It is used to prevent neuronal degeneration in subjects, preferably those suffering from GM1 ganglioside storage disease.

[0025] In one aspect, the present invention provides a compound of formula (I),

[0026]

[0027] It is used to improve the survival of subjects, preferably those suffering from GM1 ganglioside storage disease.

[0028] The medical applications of this invention can be expressed in any suitable form. Therefore, in another aspect, the invention provides a method for treating GM1 ganglioside storage disease, the method comprising administering to a subject suffering from GM1 ganglioside storage disease an amount of compound (I) effective in treating GM1 ganglioside storage disease in the subject:

[0029] .

[0030] In another aspect, the present invention provides the use of the compound of formula (I) for producing a medicament for treating subjects suffering from GM1 ganglioside storage syndrome.

[0031] .

[0032] In another aspect, the present invention provides the use of the compound of formula (I) for producing a medicament for reducing the accumulation of GM1 gangliosides in the brain and / or spinal cord of a subject, preferably wherein the subject suffers from GM1 ganglioside storage disease.

[0033] .

[0034] In another aspect, the present invention provides the use of the compound of formula (I) for producing a medicament for improving neuronal survival in a subject, preferably wherein the subject suffers from GM1 ganglioside storage disease.

[0035] .

[0036] In another aspect, the present invention provides the use of the compound of formula (I) for producing a medicament for preventing neuronal degeneration in a subject, preferably wherein the subject suffers from GM1 ganglioside storage disease.

[0037] .

[0038] In one aspect, the present invention provides the use of the compound of formula (I) for improving the survival of a subject, preferably wherein the subject suffers from GM1 ganglioside storage disease.

[0039] .

[0040] The present invention also provides a method for improving the survival of a subject, the method comprising administering to a subject suffering from GM1 ganglioside storage disease an amount of compound of formula (I) that effectively improves the survival of the subject:

[0041] .

[0042] For any of the foregoing aspects, the compound of formula (I) is preferably in crystalline form. Preferably, the crystalline form exhibits a reflection at 17.8 ± 0.2° in X-ray powder diffraction patterns, expressed in terms of 2θ values, optionally wherein the reflection at 17.8 ± 0.2° is one of the four strongest reflections in the X-ray powder diffraction pattern. WO2022 / 069709 A1 (which is incorporated herein by reference) describes a suitable crystalline form of the compound of formula (I). Preferably, the compound of formula (I) is in the form of “type 3” as described in WO2022 / 069709 A1.

[0043] Preferably, for any of the foregoing aspects, the compound is applied as a pharmaceutical composition to the target. The pharmaceutical composition may comprise the compound as well as at least one pharmaceutically acceptable carrier, diluent, or excipient.

[0044] The compound of formula (I) can also be called "nizubaglustat".

[0045] Other preferred embodiments of the compounds used according to the invention are presented throughout the specification and particularly in the examples.

[0046] Unless expressly indicated otherwise, each aspect or embodiment defined herein may be combined with any other aspect or embodiment. Specifically, any feature indicated as preferred or advantageous may be combined with one or more other features indicated as preferred or advantageous. Attached Figure Description

[0047] These and other aspects of the invention will now be described with reference to the accompanying drawings, in which:

[0048] Figure 1 The preparation of a GM1 ganglioside storage disease model in human iPSC cells is illustrated. Human iPSC G22 cells were transduced using a 4D-Nucleofector with a plasmid expressing CRISPR / Cas9 and a custom-designed guide RNA targeting exon 2 of the GLB1 locus. Monoclonal antibodies were generated and screened for the presence of a homozygous frameshift in the GLB1 coding sequence that would lead to knockout (determined by the absence of β-galactosidase activity). Clones Ex2-3 were selected, amplified, and used to generate brain organoids containing microglia. After 45 days of maturation, WT and KO brain organoids were exposed to a carrier, 5 nM, or 500 nM of compound (I) (also referred to herein as “nizubaglustat”) for 14, 18, 20, and 24 weeks and subjected to immunostaining. GM1 accumulation was measured using a cholera toxin subunit-B FITC conjugate.

[0049] Figure 2A The sequencing results of the GLB1 locus after homozygous frameshift in the GLB1 coding sequence are shown (top figure). Due to the 1 bp deletion in clone 2.3, the elastin binding protein (EBP) will also be out of frame from amino acid 25, which will predict that the protein will be nonfunctional and / or unstable at the mRNA or protein level (bottom figure).

[0050] Figure 2B The image shows β-galactosidase (β-gal.) staining in the clone-derived Ex 2-3 GLB1 null (GLB1 null) line. Figure 2B In the middle (top left), blue β-gal staining was present in organoid tissue sections, confirming the activity of this enzyme. In contrast, insufficient substrate conversion was associated with less blue staining or the presence of β-gal enzyme. The low levels of blue observed in the Ex 2-3 GLB1 deletion line represent nonspecific background levels. Figure 2B (Top right). Figure 2B The figure below illustrates the same effect, but for exemplary complete organoids (bottom left, G22 control organoid and bottom right, EX 2-3 GLB1 deletion line).

[0051] Figure 2C The study illustrates the neuronal phenotype of the cells (Neun), as well as the presence of microglia (Iba1) and astrocytes (GFAP). DAPI is used to provide information about nucleated cells and helps normalize the quantitative analysis of specific biomarkers.

[0052] Figure 3(Above figure) shows the accumulation of GM1 gangliosides in GLB1-deficient brain organoids and controls with or without nizubaglustat treatment. Figure 3 (The figure below) shows the results of the apoptosis test with and without nizubaglustat.

[0053] Figure 4 (Above) shows representative images of organoids treated with the carrier or nizubaglustat at week 24. Figure 4 (The figure below) shows the results of cell density tests with and without nizubaglustat.

[0054] Figure 5 The modes of action of the crystalline form of compound (I) (labeled “AZ-3102”) are summarized.

[0055] Figure 6 β-gal treated with a carrier or compound of formula (I) at 3 mg / kg / day is shown. - / - The survival of the mice.

[0056] Figure 7 The median body weight and total body weight of mice treated with the carrier and compound of formula (I) (labeled “nizubaglustat”) over time are shown.

[0057] Figure 8 β-galactosidase was shown to be treated with a carrier or with compound (I) (i.e., nizubaglustat) at doses of 0.2, 0.5, 3, and 6 mg / kg / day. - / - The survival of mice. Het: a heterozygous animal used as a healthy control; nizu: nizubaglustat.

[0058] Figure 9 The results of the open field test (OFT) are shown.

[0059] Figure 10 The results of the rotator bar experiment are shown. Data are only for the pilot group and at 16 weeks, where N = 6 / group.

[0060] Figure 11 Mean plasma and brain concentrations of nizubaglustat relative to time (at 24 weeks) are shown. In diseased animals, nizubaglustat exhibited persistent brain concentrations and increased BBB permeability compared to normal (Het) animals.

[0061] Figure 12The levels of GlcCer C16 (plasma) relative to the control were shown after administration of nizubaglustat to healthy animals and GM1 animals.

[0062] Figure 13 The levels of GlcCer C18 (plasma) relative to the control were shown after administration of nizubaglustat to healthy animals and GM1 animals.

[0063] Figure 14 The levels of C16 and C18 (brain) in GlcCer relative to the control were shown after administration of nizubaglustat to healthy animals and GM1 animals.

[0064] Figure 15 The results of the rotarod test (latency) relative to the control were shown in healthy animals and GM1 animals after administration of nizubaglustat.

[0065] Figure 16 The results of the rotarod test (distance traveled) relative to the control were shown after administration of nizubaglustat to healthy animals and GM1 animals.

[0066] Figure 17 Results of the rotarod test (final RPM) relative to the control were shown in healthy and GM1 animals after administration of nizubaglustat. Detailed Implementation

[0067] Unless otherwise defined herein, scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meanings and scopes of terms shall be unambiguous; however, in the event of any potential ambiguity, the definitions provided herein shall prevail over any dictionary or external definition.

[0068] It should be understood that singular prepositions such as "a" or "a kind" are often used for convenience; however, unless explicitly stated otherwise or as the context dictates, all instances of the singular are intended to cover the plural. The terms "comprising," "including," and "having" are intended to be inclusive and mean that additional elements may exist besides those listed. Furthermore, it should be understood that all references (including journal articles, books, patents, technical documents, etc.) mentioned in this disclosure are incorporated herein by reference in their entirety for all purposes.

[0069] The term “approximately” used for numerical data in this article refers to a value within 10% of the base parameter (i.e., plus or minus 10%), and the term “approximately” is used at the beginning of a series of values ​​to modify each value (i.e., “approximately 1, 2, and 3” means approximately 1, approximately 2, and approximately 3). For example, a temperature of “approximately 85°C” can include temperatures from 75°C to 95°C.

[0070] The term "melting point" is well known in the art. The term "relatively high melting point" as used herein is intended to cover crystalline forms that are sufficiently stable to be formulated into pharmaceutical compositions.

[0071] As used herein, the term "composition" is intended to cover products containing specified ingredients in specified amounts, and any products directly or indirectly generated from combinations of specified ingredients in specified amounts. Such a term regarding pharmaceutical compositions is intended to cover products containing a compound of formula (I) and / or a crystalline form of a compound of formula (I) and additional ingredients optionally constituting a carrier, and any products directly or indirectly generated from combinations, complexes, or aggregations of any two or more of the ingredients, or generated from the dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Therefore, pharmaceutical compositions of the present invention cover any composition containing the compounds of the present invention and / or crystalline forms, and optionally pharmaceutically acceptable carriers, diluents, or excipients. "Pharmaceutical acceptable" means that the carrier, diluent, or excipient (if present) must be compatible with the other components of the formulation and harmless to its recipient.

[0072] The terms "therapeuticly effective amount" and "amount with effective delivery" are intended to cover amounts of compounds, crystalline forms, or pharmaceutical compositions sufficient to achieve such treatment of a disease when administered to a patient for treating the disease. Therapeuticly effective amounts will vary depending on the disease and its severity and / or the patient's age and weight. The term "object" (or "patient") includes, but is not limited to, animals, such as mammals. Preferably, the object is a human being.

[0073] This invention provides a compound of formula (I),

[0074]

[0075] It is used to treat GM1 ganglioside storage disorder in subjects.

[0076] This invention provides a compound of formula (I),

[0077]

[0078] It is used to eliminate, alleviate or improve the symptoms of GM1 ganglioside storage syndrome.

[0079] It should be understood that treatment of GM1 ganglioside storage disease can eliminate, reduce or improve one or more symptoms of GM1 ganglioside storage disease without necessarily eliminating, reducing or improving every symptom of GM1 ganglioside storage disease that the patient experienced before treatment.

[0080] "Elimination" of symptoms means that the subject is no longer experiencing the symptoms. "Relief" of symptoms means that the subject experiences a significant improvement in symptoms. Relief is preferably from severe to mild. "Improvement" of symptoms means that the subject experiences a moderate improvement in symptoms.

[0081] Therefore, in some specific implementations, the treatments, uses, etc., provided herein may result in the elimination, reduction, or improvement of one or more symptoms of GM1 ganglioside storage disease. For example, it may reduce one or more symptoms and improve one or more different symptoms.

[0082] As shown in the examples, in a human model of GM1 ganglioside storage disease, treatment with the compound of formula (I) reduced GM1 ganglioside accumulation in brain organoids and improved neuronal survival and health. Therefore, the compound of formula (I) can be used to eliminate, alleviate, or improve various symptoms of GM1 ganglioside storage disease.

[0083] Symptoms of GM1 ganglioside storage disorder may include at least one of the following: distinctive facial features (coarse facial features), hypotonia (low muscle tone), enlarged liver and spleen (hepatosplenomegaly), excessive startle response, developmental regression, skeletal abnormalities, deformities, organ enlargement, cardiomyopathy, interstitial lung disease, seizures, visual impairment, ataxia, and / or dysarthria. The compound of formula (I) can be used to alleviate, eliminate, and / or improve at least one symptom of GM1 ganglioside storage disorder in subjects suffering from GM1 ganglioside storage disorder. Preferably, the compound of formula (I) alleviates, eliminates, and / or improves at least two, at least three, at least four, or at least five symptoms of GM1 ganglioside storage disorder.

[0084] When symptoms of GM1 ganglioside storage disease appear at birth or before 6 months of age, it is called infantile or type I GM1 ganglioside storage disease. If symptoms of GM1 ganglioside storage disease occur in late infancy or early childhood, it is called type II GM1 ganglioside storage disease. When symptoms appear in adolescence or adulthood, the disease is called type III GM1 ganglioside storage disease.

[0085] Compound (I) can be used to treat type I, type II, and / or type III GM1 ganglioside storage disorders. Compound (I) can be used to eliminate, alleviate, or improve the symptoms of type I, type II, and / or type III GM1 ganglioside storage disorders.

[0086] Compound (I) can be used to treat symptoms of diseases exhibiting GM1 ganglioside storage phenotypes. For example, this disease can be Morquio B disease. Morquio B disease is an attenuated phenotype within the β-galactosidase (GLB1) deficiency spectrum. It is characterized by multiple osteodystrophys, ligament laxity, mild coarse facial features, and heart valve defects due to the accumulation of keratin sulfate (primarily in cartilage). Patients with Morquio B disease have normal neurodevelopment. Compound (I) can be used to treat Morquio B disease.

[0087] A method is also provided for eliminating, alleviating, and / or improving one or more of the above-mentioned symptoms by administering a therapeutically effective amount of a compound of formula (I) to a subject in need. The subject may suffer from GM1 ganglioside storage syndrome.

[0088] The medical applications of this invention can be expressed in any suitable form. Therefore, in another aspect, the invention provides a method for treating GM1 ganglioside storage disease, the method comprising administering to a subject suffering from GM1 ganglioside storage disease an amount of compound (I) effective in treating GM1 ganglioside storage disease in the subject:

[0089] .

[0090] In another aspect, the present invention provides the use of the compound of formula (I) for producing a medicament for treating subjects suffering from GM1 ganglioside storage syndrome.

[0091] .

[0092] The present invention also provides compounds of formula (I),

[0093]

[0094] It is used to reduce the accumulation of GM1 gangliosides in the brain and / or spinal cord of a subject, preferably a subject suffering from GM1 ganglioside storage disease.

[0095] A method for reducing GM1 ganglioside accumulation in the brain and / or spinal cord of a subject is also provided, the method comprising administering to the subject a compound of formula (I) that effectively reduces the amount of GM1 ganglioside accumulation in the brain and / or spinal cord of a subject suffering from GM1 ganglioside storage syndrome.

[0096] GM1 (monosialotetrahexosylganglioside) (“prototype” ganglioside) is a member of the ganglioside ganglion family containing one sialic acid residue. GM1 acts as a binding site for both cholera toxin and E. coli heat-labile enterotoxin (Traveler's diarrhea). Suitable methods for determining GM1 ganglioside levels are known to those skilled in the art. Exemplary methods are described in the examples. For example, GM1 ganglioside levels can be measured by conjugating a fluorescein isothiocyanate (FITC) conjugate (C1655, Sigma) to cholera toxin subunit b and using fluorescence microscopy or flow cytometry.

[0097] The statement "reduction of GM1 ganglioside accumulation" encompasses the reduction of GM1 ganglioside levels in the brain and / or spinal cord of the subject after treatment with a compound of formula (I) compared to before treatment. This reduction may occur at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, or at least 6 days after administration of the compound, or at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 13 weeks, at least 14 weeks, at least 15 weeks, at least 16 weeks, at least 17 weeks, at least 18 weeks, at least 19 weeks, at least 20 weeks, at least 21 weeks, at least 22 weeks, at least 23 weeks, at least 24 weeks, at least 25 weeks, at least 26 weeks, at least 27 weeks, at least 28 weeks, at least 29 weeks, or at least 30 weeks after administration of the compound.

[0098] Compared to the level of GM1 ganglioside accumulation in the brain and / or spinal cord prior to application of compound (I), the level of GM1 ganglioside accumulation in the subject's brain and / or spinal cord can be reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. Compared to the level of GM1 ganglioside accumulation in the brain and / or spinal cord prior to application of compound (I), the level of GM1 ganglioside accumulation in the subject's brain and / or spinal cord can be reduced by at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold. The inventors of this application have unexpectedly discovered that compound (I) (or its crystalline form) reduces the level of GM1 ganglioside by at least 2-fold (after 24 weeks of regular (2 to 3 times per week) application of the compound).

[0099] The present invention also provides compounds of formula (I),

[0100]

[0101] It is used to improve neuronal survival in subjects, preferably those suffering from GM1 ganglioside storage disease.

[0102] A method for improving neuronal survival in a subject is also provided, the method comprising administering to the subject an amount of compound of formula (I) that effectively improves neuronal survival in a subject suffering from GM1 ganglioside storage disease.

[0103] Compound (I) can also be used to improve neuronal health.

[0104] The term "neuronal survival" may mean that the expected lifespan of neurons treated with compound (I) is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, or at least 900% compared to the expected lifespan of neurons without treatment. The neurons with increased survival may be derived from the brain and / or spinal cord of the subject. "Improved survival" means an increase in survival time compared to the absence of treatment with the compound.

[0105] This invention provides a compound of formula (I),

[0106]

[0107] It is used to prevent neuronal degeneration in subjects, preferably those suffering from GM1 ganglioside storage disease.

[0108] A method for preventing neuronal degeneration in a subject is also provided, the method comprising administering to the subject an amount of compound of formula (I) that is effective in preventing neuronal degeneration in a subject suffering from GM1 ganglioside storage disease.

[0109] Compared with the period before application of compound (I), neuronal degeneration was reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and at least 100% after application of compound (I).

[0110] Application of compound (I) can halt neuronal degeneration, provided that the compound is administered to the subject in a therapeutically effective amount. Application of compound (I) can slow neuronal degeneration, provided that the compound is administered to the subject in a therapeutically effective amount.

[0111] This invention provides the use of a compound of formula (I) for producing a medicament for reducing the accumulation of GM1 gangliosides in the brain and / or spinal cord of a subject, preferably wherein the subject suffers from GM1 ganglioside storage disease.

[0112] .

[0113] This invention provides the use of a compound of formula (I) for producing a medicament for improving neuronal survival in a subject, preferably wherein the subject suffers from GM1 ganglioside storage disease.

[0114] .

[0115] This invention provides the use of a compound of formula (I) for producing a medicament for preventing neuronal degeneration in a subject, preferably wherein the subject suffers from GM1 ganglioside storage disease.

[0116] .

[0117] Compound (I) exhibits substantially no toxicity in the subject. Preferably, compound (I) exhibits substantially no neurotoxicity in the subject. The inventors of this application demonstrate that compound (I) neither reduces cell density nor induces apoptosis in cells. This makes compound (I) (or its crystalline form) particularly suitable for treating GM1 ganglioside storage disease.

[0118] The present invention also provides compounds of formula (I),

[0119]

[0120] It is used to improve the survival of subjects, preferably those suffering from GM1 ganglioside storage disease.

[0121] The improvement in the subject's survival extends the subject's lifespan or life expectancy by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or at least 60% compared to the subject's lifespan (or life expectancy) without treatment with compound (I). In other words, the application of compound (I) extends the lifespan of a subject with GM1 ganglioside storage disease compared to the subject's lifespan without treatment with compound (I). Application of compound (I) can prolong the lifespan (and thus delay death) of subjects with GM1 ganglioside storage disease by at least 1 month, at least 3 months, at least 6 months, at least 9 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, at least 10 years, at least 11 years, at least 12 years, at least 13 years, at least 14 years, at least 15 years, at least 16 years, at least 17 years, at least 18 years, at least 19 years, at least 20 years, at least 25 years, at least 30 years, at least 35 years, at least 40 years, at least 45 years, or at least 50 years. Application of compound (I) can prevent death due to the disease in subjects with GM1 ganglioside storage disease. For example, subjects treated with compound (I) can expect to survive at least 5 years longer than those who are not treated with compound (I).

[0122] As illustrated in the examples, the compound of formula (I) (or the crystalline form of compound (I)) can also be used to maintain or improve the healthy weight of subjects suffering from GM1 ganglioside storage disease. Therefore, the present invention provides a compound of formula (I).

[0123]

[0124] It is used to maintain or improve the healthy weight of a subject, preferably the subject suffering from GM1 ganglioside storage disease.

[0125] The term "healthy weight" is intended to encompass the optimal weight for a subject. For human subjects, this is typically indicated by a BMI of 18.5 to 24.9. One of the symptoms of GM1 ganglioside storage syndrome is weight loss. Therefore, the compound of formula (I) (or its crystalline form) is capable of preventing weight loss in subjects suffering from GM1 ganglioside storage syndrome, or improving the subject's weight after application.

[0126] To avoid ambiguity, all embodiments specified herein are not limited to a particular wording to define treatment, and are applied, with necessary modifications, to medical uses or methods of treatment according to the present invention.

[0127] According to all aspects of the invention described herein, preferably, the compound of formula (I) is in crystalline form. The crystalline form of the compound of formula (I) can be any crystalline state. The crystalline form of the compound of formula (I) can be a crystalline salt or a crystalline free base (non-ionized form). Preferably, the crystalline form of the compound of formula (I) is a crystalline free base. Additionally, the compound of formula (I) can form a eutectic.

[0128] Preferably, the crystalline form exhibits reflection at 17.8 ± 0.2° in X-ray powder diffraction pattern, expressed as a 2θ value, wherein the reflection at 17.8 ± 0.2° is one of the four strongest reflections in the X-ray powder diffraction pattern. WO2022 / 069709 A1 (which is incorporated herein by reference) describes a suitable crystalline form of the compound of formula (I). Preferably, the compound of formula (I) is in the form of “type 3” as described in WO2022 / 069709 A1.

[0129] If the crystalline form of the compound of formula (I) is a crystalline salt, then the molecular structure of the compound of formula (I) in this paper contains protonated nitrogen atoms.

[0130] All aspects of the invention described herein are not limited to the single crystalline form of the compound of formula (I). Solid substances can exist in more than one crystalline form. These alternative crystalline forms are called polymorphs. Each polymorph has a different molecular orientation and / or conformation in the crystal lattice. Due to the differences in crystal structure, each crystalline state or "polymorph" exhibits a unique set of physicochemical properties.

[0131] Polymorphic forms can possess different mechanical properties that influence the technical characteristics of the compound, such as flowability and compressibility. The storage stability and duration of the compound can also depend on the polymorph.

[0132] Polymorphs can be distinguished from each other in different ways. Polymorphs clearly exhibit spectral properties, and these properties can be determined using, for example, infrared spectroscopy, Raman spectroscopy, and... 13 C-NMR spectroscopy is used to determine polymorphs. Given that each crystal form refracts X-rays differently, X-ray powder diffraction (XPD) can also be used to characterize polymorphs. Additionally, thermal methods such as differential scanning calorimetry (DSC) can provide unique information about a specific polymorph.

[0133] As is well known in the field of powder X-ray diffraction, the relative peak heights of powder X-ray diffraction patterns can be used to describe different crystal forms. Therefore, in the "Type 3" aspect, the crystal form of the compound of formula (I) shows a reflection at 17.8 ± 0.2° in the X-ray powder diffraction pattern, expressed in 2θ values, optionally wherein the reflection at 17.8 ± 0.2° is one of the four strongest reflections in the X-ray powder diffraction pattern. Preferably, the reflection at 17.8 ± 0.2° is one of the three strongest reflections in the X-ray powder diffraction pattern, or wherein the reflection at 17.8 ± 0.2° is one of the two strongest reflections in the X-ray powder diffraction pattern. More preferably, the reflection at 17.8 ± 0.2° is the strongest reflection in the X-ray powder diffraction pattern. More preferably, in the "type 3" aspect, the crystalline form of the compound of formula (I) exhibits a reflection at 17.8 ± 0.1° in the X-ray powder diffraction pattern, expressed in terms of 2θ values, wherein the reflection at 17.8 ± 0.1° is one of the four strongest reflections in the X-ray powder diffraction pattern. Preferably, the reflection at 17.8 ± 0.1° is one of the three strongest reflections in the X-ray powder diffraction pattern, or wherein the reflection at 17.8 ± 0.1° is one of the two strongest reflections in the X-ray powder diffraction pattern. More preferably, the reflection at 17.8 ± 0.1° is the strongest reflection in the X-ray powder diffraction pattern.

[0134] The term "strongest reflection" describes the highest peak in an X-ray powder diffraction pattern. The height of the peak in a powder X-ray diffraction pattern is determined based on the X-ray intensity (in counts or counts / second). Therefore, the strongest reflection is the reflection that shows the highest X-ray intensity in the X-ray powder diffraction pattern.

[0135] Unless otherwise stated, all X-ray powder diffraction patterns were determined using copper K-α radiation at approximately 25 °C.

[0136] Preferably, in the “type 3” aspect, the crystalline form of the compound of formula (I) also shows, in the X-ray powder diffraction pattern, one or more reflections, expressed in 2θ values, at one or more of the following: 4.1 ± 0.2°, 8.3 ± 0.2°, 12.4 ± 0.2°, 13.6 ± 0.2°, 14.5 ± 0.2°, 14.9 ± 0.2°, 15.2 ± 0.2°, 17.2 ± 0.2°, 19.3 ± 0.2°, 21.2 ± 0.2°, 22.4 ± 0.2°, 22.9 ± 0.2°, and 23.3 ± 0.2°. More preferably, in the "type 3" aspect, the crystalline form of the compound of formula (I) also shows, in the X-ray powder diffraction pattern, one or more reflections, expressed in 2θ values, at one or more of the following: 4.1 ± 0.1°, 8.3 ± 0.1°, 12.4 ± 0.1°, 13.6 ± 0.1°, 14.5 ± 0.1°, 14.9 ± 0.1°, 15.2 ± 0.1°, 17.2 ± 0.1°, 19.3 ± 0.1°, 21.2 ± 0.1°, 22.4 ± 0.1°, 22.9 ± 0.1°, and 23.3 ± 0.1°.

[0137] The positions of peaks in powder X-ray diffraction patterns are relatively insensitive to experimental details. Therefore, the crystalline compounds of the present invention can be characterized by powder X-ray diffraction patterns with certain peak positions. Thus, in the "Type 3" aspect, the crystalline form of the compound (I) in the X-ray powder diffraction pattern is characterized by reflections at 17.2 ± 0.2°, 17.8 ± 0.2°, 21.2 ± 0.2°, and / or 22.4 ± 0.2°, expressed in 2θ values. Any of these reflections may be the strongest reflection (or one of the four strongest reflections) in the X-ray diffraction pattern. The crystalline form of the compound (I) in the X-ray powder diffraction pattern is preferably characterized by reflections at 17.2 ± 0.2°, 17.8 ± 0.2°, 21.2 ± 0.2°, and 22.4 ± 0.2°, expressed in 2θ values. More preferably, in the "type 3" aspect, the crystalline form of the compound of formula (I) is characterized in the X-ray powder diffraction pattern by reflections at the following 2θ values: 4.1 ± 0.2°, 8.3 ± 0.2°, 12.4 ± 0.2°, 13.6 ± 0.2°, 14.5 ± 0.2°, 14.9 ± 0.1°, 15.2 ± 0.2°, 17.2 ± 0.2°, 17.8 ± 0.2°, 19.3 ± 0.2°, 21.2 ± 0.2°, 22.4 ± 0.2°, 22.9 ± 0.2°, and 23.3 ± 0.2°. More preferably, in the “type 3” aspect, the crystalline form of the compound of formula (I) is characterized in the X-ray powder diffraction pattern by reflections at the following 2θ values: 17.2 ± 0.1°, 17.8 ± 0.1°, 21.2 ± 0.1° and 22.4 ± 0.1°. Most preferably, in the “type 3” aspect, the crystalline form of the compound of formula (I) is characterized in the X-ray powder diffraction pattern by reflections, expressed in 2θ values, at the following θ values: 4.1 ± 0.1°, 8.3 ± 0.1°, 12.4 ± 0.1°, 13.6 ± 0.1°, 14.5 ± 0.1°, 14.9 ± 0.1°, 15.2 ± 0.1°, 17.2 ± 0.1°, 17.8 ± 0.1°, 19.3 ± 0.1°, 21.2 ± 0.1°, 22.4 ± 0.1°, 22.9 ± 0.1°, and 23.3 ± 0.1°.

[0138] The crystalline form of the compound can be characterized by a differential scanning calorimetry (DSC) thermogram. Therefore, in the "type 3" aspect, the crystalline form of the compound (I) is preferably characterized by a DSC thermogram showing an initial endothermic heat flux at about 87°C and / or a melting point of about 92.4°C. Therefore, preferably, in the "type 3" aspect, the melting point of the crystalline form of the compound (I) is between 89°C and 96°C, as determined by DSC. Preferably, in the "type 3" aspect, the melting point of the crystalline form is between 90°C and 95°C, as determined by DSC. More preferably, in the "type 3" aspect, the melting point of the crystalline form of the compound (I) is between 91°C and 94°C, as determined by DSC. Most preferably, in the "type 3" aspect, the melting point of the crystalline form of the compound (I) is between 92°C and 93°C, as determined by DSC.

[0139] The relatively high melting point of therapeutic compounds (typically greater than about 80°C) is advantageous for their resistance to decomposition, which in turn helps in the storage of therapeutic compounds and extends their shelf life, which is desirable for any medical agent.

[0140] The crystalline form of a compound can be characterized by its hygroscopicity. The hygroscopicity of a product refers to the increase or decrease in its water content as a function of relative humidity at a given temperature. As a result of changes in relative humidity, products that are substantially non-hygroscopic show no change or only a slight change in their water content. In highly hygroscopic products, the water content can vary widely. Therefore, preferably, the crystalline form of compound (I) is substantially non-hygroscopic. When preparing pharmaceutical compositions and formulations for such tablets, it is particularly desirable to have a crystalline form containing a therapeutic compound with low levels of hygroscopicity and / or low levels of deliquescence, thereby allowing the compound to be compressed into a desired shape or size.

[0141] The essentially non-hygroscopic material exhibits a water absorption rate of less than about 2% when measured at about 95% relative humidity and a temperature of about 25°C. Preferably, the water absorption rate is less than about 1% when measured at about 95% relative humidity and a temperature of about 25°C. The water absorption rate is obtained by measuring the mass increase of the tested crystalline form relative to its initial mass at about 95% relative humidity and a temperature of about 25°C.

[0142] Therefore, the crystalline form of the compound of formula (I) preferably absorbs 0% to 2% water at about 95% relative humidity and at a temperature of about 25°C. More preferably, the crystalline form of the compound of formula (I) absorbs 0% to 1.5% water at about 95% relative humidity and at a temperature of about 25°C. Most preferably, the crystalline form of the compound of formula (I) absorbs 0% to 1% water at about 95% relative humidity and at a temperature of about 25°C.

[0143] In the "Type 2" aspect, the crystalline form of the compound of formula (I) exhibits a reflection at 16.9 ± 0.2° in X-ray powder diffraction pattern, expressed in 2θ values, wherein the reflection at 16.9 ± 0.2° is one of the four strongest reflections in the X-ray powder diffraction pattern. Preferably, the reflection at 16.9 ± 0.2° is one of the three strongest reflections in the X-ray powder diffraction pattern, or wherein the reflection at 16.9 ± 0.2° is one of the two strongest reflections in the X-ray powder diffraction pattern. More preferably, the reflection at 16.9 ± 0.2° is the strongest reflection in the X-ray powder diffraction pattern. Even more preferably, in the "Type 2" aspect, the crystalline form exhibits a reflection at 16.9 ± 0.1° in X-ray powder diffraction pattern, expressed in 2θ values, wherein the reflection at 16.9 ± 0.1° is one of the four strongest reflections in the X-ray powder diffraction pattern. Preferably, the reflection at 16.9 ± 0.1° is one of the three strongest reflections in the X-ray powder diffraction pattern, or the reflection at 16.9 ± 0.1° is one of the two strongest reflections in the X-ray powder diffraction pattern. More preferably, the reflection at 16.9 ± 0.1° is the strongest reflection in the X-ray powder diffraction pattern.

[0144] Preferably, in the “type 2” aspect, the crystalline form of the compound of formula (I) shows one or more reflections in X-ray powder diffraction patterns at one or more of the following values, expressed as 2θ: 15.2 ± 0.2°, 16.1 ± 0.2°, 16.5 ± 0.2°, 18.9 ± 0.2°, 23.1 ± 0.2°, 25.5 ± 0.2°, 27.7 ± 0.2°, and 28.5 ± 0.2°. More preferably, in the "type 2" aspect, the crystalline form of the compound of formula (I) exhibits one or more reflections, expressed in 2θ values, at one or more of the following: 15.2 ± 0.1°, 16.1 ± 0.1°, 16.5 ± 0.1°, 18.9 ± 0.1°, 23.1 ± 0.1°, 25.5 ± 0.1°, 27.7 ± 0.1°, and 28.5 ± 0.1°.

[0145] In the "Type 2" aspect, the crystalline form of the compound of formula (I) in the X-ray powder diffraction pattern is further characterized by reflections at 2θ values ​​of 16.1 ± 0.2°, 16.5 ± 0.2°, 16.9 ± 0.2°, 18.9 ± 0.2°, and 23.1 ± 0.2°. Preferably, in the "Type 2" aspect, the crystalline form of the compound of formula (I) in the X-ray powder diffraction pattern is further characterized by reflections at 2θ values ​​of 16.1 ± 0.1°, 16.5 ± 0.1°, 16.9 ± 0.1°, 18.9 ± 0.1°, and 23.1 ± 0.1°.

[0146] In the "Type 2" aspect, the crystalline form of the compound (I) can be characterized by a DSC thermogram showing the initial endothermic heat flux at about 58°C and the melting point at about 70°C. Therefore, in the "Type 2" aspect, the melting point of the crystalline form of the compound (I) is 67°C to 74°C. Preferably, in the "Type 2" aspect, the melting point of the crystalline form of the compound (I) is 68°C to 73°C. More preferably, in the "Type 2" aspect, the melting point of the crystalline form of the compound (I) is 69°C to 72°C. Most preferably, in the "Type 2" aspect, the melting point of the crystalline form of the compound (I) is 69°C to 71°C.

[0147] Preferably, in the "type 2" aspect, the crystalline form of compound (I) is particularly soluble in water. Therefore, preferably, in the "type 2" aspect, the water solubility of the crystalline form of compound (I), measured at about 25°C, is about 75 mg / mL to about 85 mg / mL. More preferably, in the "type 2" aspect, the water solubility of the crystalline form, measured at about 25°C, is about 78 mg / mL to about 82 mg / mL. Most preferably, in the "type 2" aspect, the water solubility of the crystalline form of compound (I), measured at about 25°C, is about 80 mg / mL.

[0148] Methods for measuring solubility are known in the art; for example, flask shaking, acoustic treatment, column elution, and ultraviolet or visible spectroscopy. Unless explicitly stated otherwise, water solubility is determined using flask shaking and / or acoustic treatment.

[0149] In some embodiments, a therapeutically effective amount (i.e., an amount that effectively delivers the desired effect) of the compound of formula (I) (or its crystalline form) may be from about 0.0001 mg total daily to about 50 mg total daily (mg total / day). In some specific embodiments, the therapeutically effective amount may be at least about 0.001 mg total / day, at least about 0.05 mg total / day, at least about 0.1 mg total / day, at least about 0.25 mg total / day, at least about 0.5 mg total / day, at least about 1 mg total / day, at least about 3 mg total / day, at least about 5 mg total / day, or at least about 8 mg total / day. In other embodiments, the therapeutically active amount may be from about 0.01 to 20 mg total / day, 0.05 to 15 mg total / day, or 0.1 to 12 mg total / day.

[0150] Therefore, the compound of formula (I) (or its crystalline form, preferably "type 3") can be administered to the subject at a dose of about 0.0001 mg total / day to about 50 mg total / day. Preferably, the compound of formula (I) (or its crystalline form, preferably "type 3") is administered to the subject at a dose of about 0.1 mg total / day to about 15 mg total / day.

[0151] Throughout the treatment period, the dosage of compound (I) (or its crystalline form, preferably "type 3") may remain the same or may be increased or decreased. According to all aspects of the invention, the exact maintenance dose can be routinely determined by a person skilled in the art based on the individual's physical condition, the severity of GM1 ganglioside storage symptoms, and the tolerance of the administered compound. Generally, the exact maintenance dose will be a trade-off between the improvement of GM1 ganglioside storage symptoms and the individual's physiological tolerance to said precise dose (e.g., the observation / possibility of undesirable side effects). This is specific to each individual individual and is routine for a person skilled in the art (e.g., a medical practitioner) to determine the exact maintenance dose for an individual based on the foregoing factors.

[0152] In some specific implementations, the compound of formula (I) (or its crystalline form) may be administered as a single dose once daily. The compound of formula (I) (or its crystalline form) may also be administered in two, three, or more separate doses throughout the day. That is, the daily therapeutically effective dose is divided into two, three, or more separate doses administered to the subject throughout the day. Multiple daily doses do not necessarily need to be the same amount.

[0153] Treatment (regular, e.g., daily administration of the compound of formula (I)) may be administered for a suitable period of time, such as at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks, months, or years. For example, the compound of formula (I) (or its crystalline form) may be administered to the subject for at least 1 month, at least 3 months, at least 6 months, at least 9 months, at least 1 year, at least 2 years, or at least 3 years. If well tolerated, the treatment may be continued indefinitely. Alternatively, the compound of formula (I) (or its crystalline form) may be administered to the subject once.

[0154] The dosage of the compound of formula (I) administered to the subject may be from 3 mg to 9 mg total per day. The dosage of the compound of formula (I) administered to the subject may be about 3 mg total per day, about 6 mg total per day, or about 9 mg total per day. Preferably, the dosage of the compound of formula (I) administered to the subject is 6 mg total per day or 9 mg total per day. More preferably, the dosage of the compound of formula (I) administered to the subject is 9 mg total per day.

[0155] In the context of dosage, the expression "total mg / day" is intended to refer to a daily dose of X mg. For example, a dose of compound (I) of formula (I) of 9 mg total / day means administering 9 mg of compound (I) to a patient within a given day. This can be given in a single dose or multiple doses (e.g., 2, 3, or 4) within a day. The expression "total mg / day" can be expressed equivalently as "mg / day" and has the same meaning. The optimal dosage may depend on the subject's weight. For example, compound (I) can be administered at a dose of 9 mg total / day to a subject weighing 40 kg or more. Compound (I) can be administered at a dose of 6 mg total / day to a subject weighing ≥20 kg and <40 kg. Compound (I) can be administered at a dose of 3 mg total / day to a subject weighing ≥10 kg and <20 kg. Compound (I) is preferably administered once daily.

[0156] Administration can be achieved via oral administration of tablets (e.g., dispersible tablets), pills, capsules, granules, powders, solutions, etc., or via parenteral administration, such as injections (e.g., intra-articular, intravenous, and intramuscular injections), suppositories, ophthalmic solutions, ophthalmic ointments, or agents for external use (e.g., transdermal liquid preparations, ointments, transdermal patches, transmucosal liquid preparations, transmucosal patches, inhalants, etc.).

[0157] Subjects treated with or to be treated with a compound of formula (I) (or its crystalline form) may exhibit one or more symptoms of GM1 ganglioside storage disease. Alternatively, subjects may be treated even if they do not exhibit any symptoms of GM1 ganglioside storage disease. Subjects may be asymptomatic. Subjects treated with a compound of formula (I) (or its crystalline form) may have been diagnosed with GM1 ganglioside storage disease. The diagnosis of GM1 ganglioside storage disease may be based on genetic screening (e.g., to identify GLB1 gene mutations). Alternatively, subjects treated with or to be treated with a compound of formula (I) (or its crystalline form) may not have been diagnosed with GM1 ganglioside storage disease.

[0158] One or more symptoms of GM1 ganglioside storage disorder may include distinctive facial features (coarse facial features), poor muscle tone (hypotonia), enlarged liver and spleen (hepatosplenomegaly), excessive startle response, developmental regression, skeletal abnormalities, deformities, organ enlargement, cardiomyopathy, interstitial lung disease, seizures, ataxia, dysarthria, and visual impairment.

[0159] Subjects treated or to be treated with a compound of formula (I) (or its crystalline form) may have at least one mutation in the GLB1 gene. At least one mutation in the GLB1 gene preferably results in abnormal, insufficient, or reduced activity of β-galactosidase-1.

[0160] Compared to healthy subjects (i.e., those not suffering from GM1 ganglioside storage disease), subjects treated or awaiting treatment with compound (I) (or its crystalline form) may exhibit abnormal or insufficient β-galactosidase-1 activity. The level of β-galactosidase-1 can be measured in samples from subjects using standard biochemical methods.

[0161] According to all aspects of the invention, the object is a mammal, preferably a human. In some specific embodiments, the object is between 28 days and 30 years old. The object can be an infant (i.e., 28 days to 12 months old). The object can be a toddler (i.e., 12 months to 24 months old). The object can be a child (i.e., 24 months to 12 years old). The object can be a teenager (i.e., 12 to 17 years old). The object can be a young adult (i.e., 17 to 30 years old). However, in some embodiments, the invention is also applied to adults older than 30 years of age.

[0162] The compound of formula (I) (or its crystalline form) may be applied to the target as a pharmaceutical composition. The pH of the pharmaceutical composition may be 2.0 to 8.0, 3.0 to 7.0, 4.0 to 7.5, 4.0 to 6.0, or 4.5 to 5.5. Preferably, the pH of the pharmaceutical composition is 4.5 to 5.5. The pharmaceutical composition may also contain an acid and / or a base. For example, the pharmaceutical composition may contain an acid to lower the pH to a desired pH. A base may be used to raise the pH to a desired pH. Those skilled in the art will be readily able to determine suitable acids and bases to control the pH of the pharmaceutical composition. For example, the acid may be citric acid, acetic acid, or hydrochloric acid. For example, the base may be sodium hydroxide or potassium hydroxide.

[0163] The pharmaceutical compositions of the present invention are typically prepared from a pharmaceutically acceptable carrier and one or more optional ingredients. If necessary or desired, the resulting homogeneous blend can then be shaped or loaded into tablets (e.g., dispersible tablets), capsules, pills, cans, cartridges, dispensers, etc., using conventional operations and equipment.

[0164] When intended for oral administration in solid dosage forms (i.e., capsules, tablets (e.g., dispersible tablets), pills, etc.), the pharmaceutical compositions of the present invention typically contain a compound of formula (I) (or its crystalline form) as the active ingredient. The pharmaceutical compositions of the present invention may contain a compound of formula (I) (or its crystalline form) without any other ingredients. The pharmaceutical compositions of the present invention may be contained in capsules. The pharmaceutical compositions of the present invention may be contained in capsules without any other ingredients. The capsules may be gelatin capsules or hydroxypropyl methylcellulose (HPMC) capsules. Alternatively, the pharmaceutical compositions of the present invention may contain a compound of formula (I) (or its crystalline form) as the active ingredient and one or more pharmaceutically acceptable carriers. Suitable pharmaceutically acceptable carriers will be those known to those skilled in the art, such as fats, water, physiological saline, alcohols (e.g., ethanol), glycerol, polyols, aqueous glucose solutions, extenders, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, dispersants, preservatives, sweeteners, colorants, seasoning agents or flavorings, concentrates, diluents, buffers, solvents or solubilizers, chemicals for achieving storage, salts for adjusting osmotic pressure, coating agents or antioxidants, sugars (e.g., lactose or glucose); starch from corn, wheat or rice; fatty acids, such as stearic acid; inorganic salts, such as magnesium aluminate metasilicate or anhydrous calcium phosphate; synthetic polymers, such as polyvinylpyrrolidone or polyalkylene glycols; alcohols, such as stearyl alcohol or benzyl alcohol; synthetic cellulose derivatives, such as methylcellulose, carboxymethylcellulose, ethylcellulose or hydroxypropyl methylcellulose; agents for adjusting the pH of the composition, such as bases and / or acids; and other conventionally used additives, such as gelatin, talc, vegetable oils and gum arabic.

[0165] Oral dosage forms may also be capsule-type delayed-release dosage forms, wherein the compound of formula (I) (or its crystalline form) is encapsulated in a hard or soft soluble container made of a suitable form of gelatin or hydroxypropyl methylcellulose (HPMC) and releases the compound of formula (I) (or its crystalline form) at a time other than immediately after administration; thus, enteric-coated products are delayed-release dosage forms. Capsule-type delayed-release pellets may also be used, wherein the compound of formula (I) (or its crystalline form) is encapsulated in a hard or soft container or "shell". In these cases, the compound of formula (I) (or its crystalline form) itself is in the form of enteric-coated granules, thus delaying the release of the drug until it passes through the intestine. Extended-release capsules and film-coated extended-release capsules are also available.

[0166] Additionally, capsules may be encapsulated in a designated film coating that releases the compound of formula (I) (or its crystalline form) in such a way that at least a reduced dosing frequency is allowed compared to those presented in conventional dosage forms. Some examples include gelatin-coated capsules (solid dosage forms in which the compound of formula (I) (or its crystalline form) is encapsulated in a hard or soft soluble container made of gelatin or HPMC in a suitable form; the capsule is then coated with an additional layer of gelatin or HPMC by a binding process to form a complete seal) and liquid-filled capsules (solid dosage forms in which the compound of formula (I) (or its crystalline form) is encapsulated in a soluble gelatin shell, which is plasticized by the addition of polyols (e.g., sorbitol or glycerol), and therefore has a slightly thicker consistency than hard-shell capsules).

[0167] In some embodiments, the compound of formula (I) (or its crystalline form) may be dissolved or suspended in a liquid carrier or formulated as granules (small particles or grains), pellets (small sterile solid blocks consisting of highly purified compound of formula (I) (or its crystalline form), with or without excipients, which are made by forming granules or by compression and molding), or coated extended-release pellets (such solid dosage forms in which the compound of formula (I) (or its crystalline form) is itself a granule form coated in varying amounts, and which releases the compound of formula (I) (or its crystalline form) in such a way that a reduced dosing frequency is allowed compared to those presented as conventional dosage forms).

[0168] Other forms include pills (small, round solid dosage forms containing a compound of formula (I) (or its crystalline form) intended for oral administration), powders (dry, finely divided close mixtures of a compound of formula (I) (or its crystalline form) and one or more pharmaceutically acceptable additives, intended for internal or external use), elixirs (clear, pleasant-tasting, sweet hydroalcoholic liquids containing dissolved compounds of formula (I) (or its crystalline form); intended for oral use), chewing gum (sweet and flavored insoluble plastic substances of various shapes that release a substance of formula (I) (or its crystalline form) into the oral cavity when chewed), and syrups (oral solutions containing a compound of formula (I) (or its crystalline form) and a high concentration of sucrose or other sugars; the terms mentioned above... It is also used in any other liquid dosage form prepared in a sweet and viscous carrier, including oral suspensions, tablets (containing a compound of formula (I) (or its crystalline form), a solid dosage form with or without a suitable diluent), chewable tablets (containing a compound of formula (I) (or its crystalline form), a solid dosage form with or without a suitable diluent, intended for chewing, producing a pleasant taste residue in the mouth, easy to swallow and without leaving a bitter or unpleasant aftertaste), coated tablets or delayed-release tablets, dispersible tablets, effervescent tablets, extended-release tablets, film-coated tablets or film-coated extended-release tablets, wherein the tablets are formulated such that the contained compound of formula (I) (or its crystalline form) is available for an extended period of time after ingestion. For example, the compound of formula (I) (or its crystalline form) may be administered to the subject in the form of a dispersible tablet.

[0169] In other forms, tablets for solutions, tablets for suspensions, multilayer tablets, and extended-release tablets can be provided, wherein the tablets are formulated in a way that allows for at least a reduced frequency of administration compared to those presented in conventional dosage forms. Orally disintegrating tablets, orally disintegrating delayed-release tablets, soluble tablets, and tablet sugar-coated (osmotic) tablets are also suitable.

[0170] Injectable and infusion formulations used herein include, but are not limited to, liposome injectable formulations which consist of or form liposomes (typically lipid bilayer vesicles composed of phospholipids for encapsulating compound (I) (or its crystalline form)); injections comprising sterile formulations intended for parenteral use; emulsion injections comprising emulsions of sterile, pyrogen-free formulations intended for parenteral administration; or lipid complex injections.

[0171] Other forms include powders for solution injection, which are sterile formulations intended to be reconstituted to form solutions for parenteral use; powders for suspension injection, which are sterile formulations intended to be reconstituted to form suspensions for parenteral use; lyophilized powders for liposome suspension injection, which are sterile lyophilized formulations intended to be reconstituted for parenteral use, said formulations being formulated in a manner that allows liposomes (typically composed of lipid bilayer vesicles consisting of phospholipids for encapsulating compounds of formula (I) (or their crystalline form) within a lipid bilayer or in an aqueous interlayer) to be formed after reconstitution; or lyophilized powders for solution injection, wherein lyophilization (“lyophilization”) is a process involving the removal of water from a frozen product under extremely low pressure.

[0172] As used herein, “solution for injection” means a liquid formulation comprising a compound of formula (I) (or its crystalline form) dissolved in a suitable solvent or a mixture of mutually miscible solvents suitable for injection. “Solution concentrate for injection” means a sterile formulation intended for parenteral use that, upon addition of a suitable solvent, produces a solution that is in all respects suitable for injection.

[0173] Suspension injections comprise a liquid formulation suitable for injection, consisting of solid particles dispersed throughout the liquid phase (wherein these particles are insoluble), and may also consist of an oil phase dispersed throughout the aqueous phase, or vice versa. Liposome suspension injections comprise a liquid formulation suitable for injection, consisting of an oil phase dispersed throughout the aqueous phase in such a manner as to form liposomes (typically lipid bilayer vesicles composed of phospholipids used to encapsulate a compound of formula (I) (or its crystalline form) within a lipid bilayer or in an aqueous interlayer). Acoustically treated suspension injections comprise a liquid formulation suitable for injection, consisting of solid particles dispersed throughout the liquid phase (wherein these particles are insoluble). Additionally, the product is acoustically treated when gas is bubbled through the suspension, and this results in the solid particles forming microspheres.

[0174] Parenteral carrier systems contain one or more pharmaceutically suitable excipients, such as solvents and cosolvents, solubilizers, wetting agents, suspending agents, thickeners, emulsifiers, chelating agents, buffers, pH adjusters, antioxidants, reducing agents, antimicrobial preservatives, compatibilizers, protectants, tonic modifiers, and special additives. Formulations suitable for parenteral administration suitably comprise sterile oily or aqueous formulations of a compound of formula (I) (or its crystalline form), preferably isotonic with the recipient's blood, but not necessarily.

[0175] The inhalation formulations used herein include, but are not limited to, aerosols (products packaged under pressure and containing a compound of formula (I) (or its crystalline form), which are released after activation of a suitable valve system intended for surface application to the skin and local application to the nose (nasal aerosols), mouth (translingual and sublingual aerosols), or lungs (inhaled aerosols). Foam aerosols are formulations containing a compound of formula (I) (or its crystalline form), a surfactant, an aqueous or non-aqueous liquid, and a propellant, such that if the propellant is in an internal (discontinuous) phase (i.e., an oil-in-water type), a stable foam is emitted, and if the propellant is in an external (continuous) phase (i.e., a water-in-oil type), a spray or rapidly breaking foam is emitted. Metered aerosols are pressurized formulations consisting of a metered-dose valve that allows delivery of a uniform amount of spray after each activation. Powder aerosols are products packaged under pressure and containing a compound of formula (I) (or its crystalline form), in powder form, which are released after activation of a suitable valve system. Aerosol spray is an aerosol product that uses compressed gas as a propellant to provide the force required to expel the product as a wet spray and is suitable for solutions of compounds of formula (I) (or their crystalline form) in an aqueous solvent.

[0176] As briefly discussed above, pharmaceutical compositions comprising compound (I) in crystalline form can also be administered transdermally or transmucosally using known delivery systems and excipients. For example, the pharmaceutical composition can be mixed with a penetration enhancer (e.g., propylene glycol, polyethylene glycol monolaurate, azacycloalkane-2-one, etc.) and incorporated into a patch or similar delivery system. Additional excipients, including gelling agents, emulsifiers, and buffers, may also be used. Transdermal dosage forms used herein include, but are not limited to, patches (drug delivery systems that typically comprise an adhesive backing usually applied to an external part of the body, so that the ingredient (compound (I) or its crystalline form) is passively diffused or actively delivered from a portion of the patch, and thus, depending on the patch, the ingredient (compound (I) or its crystalline form) is delivered to an external surface of the body or into the body. Various types of transdermal patches, such as matrices, reservoirs, and others, are known in the art.

[0177] The surface formulations used herein include a variety of formulations known in the art, such as lotions (emulsion liquid formulations, which are typically used for external application to the skin), enhanced lotions (lotion formulations that deliver an enhanced (I) compound (or its crystalline form) in a form where enhancement does not refer to the strength of the (I) compound (or its crystalline form) in the formulation), gels (semi-solid formulations containing a gelling agent to provide hardness to a solution or colloidal dispersion, wherein the gel may contain suspended particles), and ointments (semi-solid formulations that typically contain less than 20% water and volatiles and more than 50% hydrocarbons, waxes, or polyols as a carrier, wherein the formulation is typically used for external application to the skin or mucous membranes). Other embodiments include enhanced ointments (ointment dosage forms with enhanced compound delivery, where enhancement does not refer to the strength of the compound of formula (I) (or its crystalline form) in the dosage form), creams (emulsion semi-solid dosage forms that typically contain more than 20% water and volatiles and / or less than 50% hydrocarbons, waxes, or polyols may also be used as carriers, where such dosage forms are typically intended for external application to the skin or mucous membranes), and enhanced emulsions (cream dosage forms with enhanced compound delivery, where enhancement does not refer to the strength of the compound of formula (I) (or its crystalline form) in the dosage form). As used herein, “emulsion” refers to a dosage form consisting of a two-phase system comprising at least two immiscible liquids, one of which is dispersed as droplets (internal or dispersed phase) within another liquid (external or continuous phase), typically stabilized with one or more emulsifiers, wherein “emulsion” is used as a dosage form term unless a more specific term is applicable (e.g., cream, lotion, ointment). Other embodiments include suspensions (liquid dosage forms containing solid particles dispersed in a liquid carrier), extended-release suspensions, pastes (semi-solid dosage forms containing a large proportion (20% to 50%) of solids finely dispersed in a fat carrier, wherein the dosage form is typically used for external application to the skin or mucous membranes), solutions (clear homogeneous liquid dosage forms containing one or more chemicals dissolved in a solvent or a mixture of miscible solvents), and powders.

[0178] Surface formulation compositions comprise a compound of formula (I) (or its crystalline form) and one or more inactive pharmaceutical ingredients, such as excipients, colorants, pigments, additives, fillers, emollients, surfactants (e.g., anionic, cationic, amphoteric, and nonionic), penetration enhancers (e.g., alcohols, fatty alcohols, fatty acids, fatty acid esters, and polyols), etc. Many FDA-approved surface inactive ingredients are listed in the FDA's Inactive Ingredients Database, which includes inactive ingredients that the manufacturer also clearly intends to include.

[0179] Preferably, the pharmaceutical composition comprises one or more additional pharmaceutically active agents (e.g., anti-inflammatory agents, such as aspirin). This combination therapy involves the use of a crystalline form of such a compound (I) in combination with one or more additional pharmaceutically active agents, either formulated together (e.g., packaged together in a single formulation) or formulated separately (e.g., packaged as a separate unit dosage form).

[0180] The methods and uses described herein may be in vitro methods (or uses) or in vivo methods (or uses).

[0181] The above methods and uses can be combined with methods for diagnosing GM1 ganglioside storage disease, wherein GM1 ganglioside storage disease is first diagnosed in a subject, and then a compound of formula (I) (or its crystalline form) is administered to the subject with GM1 ganglioside storage disease to achieve the desired therapeutic effect. Diagnostic methods may include genetic screening of the GLB1 gene to determine the presence of at least one mutation. Diagnostic methods may include determining the level of β-galactosidase-1 in the subject.

[0182] The foregoing detailed description has been provided by way of explanation and illustration and is not intended to limit the scope of the appended claims. Many variations of the presently preferred embodiments shown herein will be apparent to those skilled in the art and remain within the scope of the appended claims and their equivalents.

[0183] Example

[0184] The crystalline form (“Type 3”) of compound (I) has been obtained as described in WO2022 / 069709 A1. Compound (I) (specifically, its “Type 3” crystalline form) is referred to as “nizubaglustat” in the examples.

[0185] Example 1

[0186] 1. Method

[0187] like Figure 1As shown in (Shaker et al., 2021), human iPSC cells G22 (Shaker et al., 2022) were transduced using a 4D-Nucleofector (Lonza) plasmid expressing CRISPR / Cas9 and a custom-designed guide RNA (#62988, Addgene) targeting exon 2 in the GLB1 locus. Single clones were generated and screened for the presence of homozygous frameshifts in the GLB1 coding sequence that would lead to knockout (determined by the absence of β-galactosidase activity). Clones Ex2-3 were selected, amplified, and used to generate brain organoids containing microglia (as described in (Aguado et al., 2023)). After 45 days of maturation, WT and KO brain organoids were exposed to a carrier, 5 nM, or 500 nM of nizubaglustat for 14, 18, 20, and 24 weeks and subjected to immunostaining. GM1 accumulation was measured using the cholera toxin subunit-B FITC conjugate (C1655, Sigma).

[0188] 2. Results and Conclusions

[0189] To evaluate the efficacy of nizubaglustat (a novel and potent dual NLGase (GBA2) and GCS inhibitor currently in clinical development) and to create tools for mechanistic studies, a human brain organoid model of GM1 ganglioside storage disease was developed. Using CRISPR-Cas9, a biallelic one-base-pair (bp) frameshift deletion was generated in exon 2 of the GLB1 gene in healthy hESCs to produce a nonfunctional β-galactosidase ( Figure 2A Due to the deletion of 1 bp in clone 2.3, elastin-binding protein (EBP) will also be deframed from amino acid 25, which will predict that the protein will be nonfunctional and / or unstable at the mRNA or protein level.

[0190] like Figure 2B As shown, staining of the clone-derived Ex 2-3 GLB1 deletion line with β-galactosidase (β-gal.) confirmed the absence of β-gal. enzyme activity. When β-gal. is present, this enzyme converts exogenous substrates (chromogens) to blue. Figure 2B In the middle (top left), blue β-gal staining was present in organoid tissue sections, confirming the activity of this enzyme. In contrast, insufficient substrate conversion was associated with less blue staining or the presence of β-gal enzyme. The low levels of blue observed in the Ex 2-3 GLB1 deletion line represent nonspecific background levels. Figure 2B (Top right). Figure 2BThe figure below illustrates the same effect, but for exemplary complete organoids (bottom left, G22 control organoid and bottom right, EX 2-3 GLB1 deletion line).

[0191] Figure 2C The image shows the neuronal phenotype (Neun) of the cells, as well as the presence of microglia (Iba1) and astrocytes (GFAP). Neun is associated with neuronal differentiation and is a common marker used to indicate the neuronal phenotype present throughout the cell's life. Iba1 (ionized calcium-binding adaptor molecule 1) is a pan-microglia marker, and its expression increases with microglia activation. Glial fibrillary acidic protein (GFAP) is expressed by multiple cell types, including astrocytes. DAPI is used to provide information about nucleated cells and helps normalize the quantitative analysis of specific markers.

[0192] Next, >400 brain organoids were generated from the Ex 2-3 lineage and Gen22 (syngeneic parental control line), and these were cultured for 6 to 24 weeks in the presence of 5 nM, 500 nM nizubaglustat, or a loading control. Analysis of organoid size showed the presence of neurons (NFL and Neun+ve), astrocytes (GFAP+ve), and microglia (IBA1+ve) at later time points in both treated and untreated GLB1-deficient and control organoids.

[0193] like Figure 3 As shown in the figure above, compared with the syngeneic control organoids, GLB1-deficient brain organoids exhibited progressive accumulation of GM1, which became significant at week 18. Importantly, treatment with 5 nM and 500 nM nizubaglustat significantly reduced GM1 accumulation in GLB1-deficient Ex 2-3 brain organoids in a dose-dependent manner at weeks 18, 20, and 24, and was accompanied by improved neuronal survival and health in this human model of GM1 ganglioside storage disease.

[0194] like Figure 3 (See image below) and Figure 4 As shown in the figure below, exposure of control or GLB1-deficient brain organoids to nizubaglustat did not reduce cell density at any time point, nor did it induce apoptosis (in cells cleaved by caspase 3+ve) above baseline levels, indicating that the compound has no significant toxicity to the human brain cell types present in this model.

[0195] Figure 4(Above) shows representative images of organoids treated with the loading agent or nizubaglustat at week 24. Green staining represents GM1 levels, which are quite low in the G22 cell line but significantly increased in the GLB1-deficient (Ex2-3) cell line. Treatment with nizubaglustat significantly reduced GM1 levels in the EX 2-3 cell line.

[0196] in conclusion

[0197] Using CRISPR-Cas9, a 1bp frameshift biallelic deletion of the GLB1 gene in exon 2 was generated in healthy hESCs.

[0198] β-galactosidase staining confirmed the absence of β-galactosidase activity, and the mutation site may disrupt EBP (elastin-binding protein) function.

[0199] GLB1-deficient brain organoids exhibited progressive accumulation of GM1 (as measured by CTXb-streptavidin FITC), which became significant at week 18.

[0200] Treatment with 5 nM and 500 nM nizubaglustat did not decrease cell density at any time point, nor did it induce apoptosis (in caspase-3-positive cells) above baseline levels.

[0201] Treatment with 5 nM or 500 nM nizubaglustat significantly reduced GLB1 deficiency and GM1 accumulation in brain organoids at weeks 18, 20, and 24 in a dose-dependent manner.

[0202] Nizubaglustat, a novel dual inhibitor of nonlysosomal neutral glucosylceramidinase (NLGase, GBA2) and ceramide glucosyltransferase (GCS), exhibits no significant toxicity to the human brain cell types present in this model. It effectively reduces GM1 and has great potential to become an effective treatment for GM1 ganglioside storage disorders.

[0203] 3. References

[0204] 1) Shaker MR, Kahtan A, Prasad R, Lee JH, Pietrogrande G, Leeson HC, et al. Neural Epidermal Growth Factor-Like Like Protein 2 Is Expressed in Human Oligodendroglial Cell Types. Front Cell Dev Biol. 2022;10:803061.

[0205] 2) Shaker MR, Pietrogrande G, Martin S, Lee JH, Sun W and Wolvetang EJ. Rapid and Efficient Generation of Myelinating Human Oligodendrocytes inOrganoids. Front Cell Neurosci. 2021;15:631548.

[0206] 3) Aguado J, Amarilla AA, Taherian Fard A, Albornoz EA, Tyshkovskiy A, Schwabenland M, Pietrogrande G, et al. Senolytic therapy alleviates physiological human brain aging and COVID-19 neuropathology. Nat Aging. 2023;3(12):1561-75.

[0207] Example 2

[0208] background

[0209] In GM1 ganglioside storage disease, GM1 accumulates in lysosomes. This is due to a primary defect in the degradative enzyme β-GAL encoded by the GLB1 gene.

[0210] GM1 production depends on the upstream formation of GlcCer within the Golgi apparatus. AZ-3102 (also known as nizubaglustat) is a potent and selective inhibitor of both GCS and GbA2.

[0211] As demonstrated in a mouse model of GM1 ganglioside storage disease, nizubaglustat has beneficial effects related to nerve... The etiology of ganglioside storage disease is unrelated.

[0212] Nizubaglustat was tested in a mouse model of GM1 ganglioside storage disease to determine its effect on prolonging survival in affected animals. The study was conducted under the guidance of Dr. Jagdeep Walia (MD, MBBS, FRCCP, FCCMG) at Kingston Health Sciences Centre and Queen's University, Kingston, ON, Canada. In short, β-galactosidase-deficient mice (β-gal- / -) were administered either a carrier (acidified water) or nizubaglustat at 3 mg / kg / day. These β-galactosidase-deficient mice (β-gal- / -) were induced by homologous recombination targeting and disrupting the mouse β-gal locus (Hahn, delPilar, Martin et al., 1997). Mice were randomly assigned to treatment groups, with each group containing an equal proportion of males and females (N = 6 mice). All staff members, except the technicians responsible for preparing the formulation, were unaware of the treatment. The drug and / or carrier were administered orally once daily between 8:00 and 12:00 am. Animals were given medication at a rate of 10 mL / kg starting on day 30 after birth until their humane endpoint was reached (N = 6 / dose group). Prophylactic euthanasia was performed on animals to reduce suffering in cases of immobility, tremors or seizures, poor physical condition, inability to perform righting reflexes, 15% weight loss, and near death.

[0213] After observing the last death in one treatment group, the study was unblinded to the PI and sponsor to maintain blinding of ongoing behavioral, pharmacokinetic, and further dose exploration of the group. At the time of this application, the nizubaglustat treatment group presented ( Figure 6 All animals in the treatment group survived and were not prematurely euthanized. In contrast, all animals treated with the loading agent reached their humane endpoint, with a median survival of 147 days (P: 0.0007, log-rank [Mantel-Cox] test). Statistical evaluation was performed using GraphPad Prism (10.2.2). Additionally, body weight over time was provided to assess the general health of the animals. Here, the inventors observed that the growth curves of the loading agent-treated animals were delayed and their performance was poor compared to the treated animals. Figure 7 Although the treated animals were in a plateau phase, they did not deteriorate to the same extent.

[0214] References

[0215] Christopher N. Hahn, Maria del Pilar Martin, Maria Schröder, Marie T.Vanier, Kinuko Suzuki, Yoji Hara, Kunihiko Suzuki, Alessandra d'Azzo, Generalized CNS Disease and Massive GM1-Ganglioside Accumulation in MiceDefective in Lysosomal Acid β-galactosidase, Human Molecular Genetics, Volume 6, Issue 2, February 1, 1997, Pages 205-211

[0216] Example 3

[0217] The beneficial effects of nizubaglustat in a mouse model of GM1 ganglioside storage disease were tested as in Example 2, but at doses of 0.2, 0.5, 3, and 6 mg / kg once daily starting at 4 weeks of age until the mice reached their human endpoint. All treatment groups were blinded to the animal care staff and the principal investigator (PI) of the study. Mice were randomly assigned to treatment groups, with each group containing an equal proportion of males and females (N = 7 to 14 mice / group). Behavioral tests included the rotarod test and the open field test.

[0218] Compared to GM1 mice treated with the loading agent, the nizubaglustat treatment group ( Figure 8 Survival was significantly improved in all animals treated with the drug. Animals treated with the drug reached their humanitarian endpoint with a median survival of 170 days, compared to a median survival of 263 days for animals treated with nizubaglustat (p < 0.0001). Doses as low as 0.2 mg / kg / day also resulted in significantly improved survival (p = 0.0328, log-rank [Mantel-Cox] test). Statistical evaluation was performed using the GraphPad Prism (10.2.2). Doses of 3 and 6 mg / kg / day did not result in statistically significant differences in survival probabilities. Rotball data showed significant, time-related differences in survival rates among mice treated with nizubaglustat compared to controls, indicating improved neuromotor function. Figure 10 ). Figure 9 The results of the open field experiment (OFT) are shown.

[0219] Example 4

[0220] The beneficial effects of nizubaglustat in a mouse model of GM1 ganglioside storage disease were tested as in Examples 2 and 3. Further analysis of the results is described below. Figures 12 to 1 As described in section 8.

[0221] method

[0222] Blood sampling:

[0223] At designated time points, terminal blood samples were drawn from animals via the inferior abdominal vein, including baseline. Plasma was collected at week 24, and baseline measurements, nizubaglustat PK and PD levels, and biomarkers were determined. Samples were collected at the following time points: 5 minutes after administration and at 1, 2, 4, 8, and 24 hours. Blood was collected via cardiac puncture using a 23-gauge needle. The needle was removed, and the blood was transferred to a sample tube (MiniCollect® K2EDTA (potassium ethylenediaminetetraacetate)). The tube was thoroughly inverted to promote uniform distribution of EDTA and prevent clotting. The blood sample was centrifuged at 3000 × g for 10 minutes at room temperature (22°C). 50 µL plasma aliquots were transferred to pre-labeled 1.5 ml LoBind Eppendorf tubes, frozen on dry ice, and stored at -80°C.

[0224] Brain sampling and tissue preparation

[0225] After perfusion with physiological saline, the skull was opened, and the brain was carefully removed and halved on a cooled surface. The left hemisphere was weighed, flash-frozen on dry ice, and stored at -80°C. At room temperature, the mouse right hemisphere was fixed for two hours by immersion in 4% paraformaldehyde (pH 7.4) in freshly prepared PB. Anterior (RB), mid-brain (MB), and caudal (CB) sections were obtained. Subsequently, the hemispheres were transferred to 15% sucrose / PBS and stored at 4°C to ensure cryoprotection. PK and PD analyses were performed on the CB sections.

[0226] Bioanalysis of formulations, nizubaglustat, and biomarkers:

[0227] The concentrations of nizubaglustat in the formulation, plasma, and brain tissue were analyzed using reversed-phase LC-MS / MS. Mouse plasma and brain tissue were used as species matrices. The LLOQ of nizubaglustat was 0.5 ng / mL plasma and 0.5 ng / g brain tissue. The LLOQs of GlcCer C16:0 and C18:0 were 10 pmol and 25 pmol / g tissue, respectively.

[0228] Statistical analysis

[0229] Survival benefits were assessed using Kaplan-Meier survival analysis followed by the Mantle-Cox test. Behavioral tests were performed using ordinary one-way ANOVA and Dunnet's multiple comparison test (GraphPad Prism (version 10.4.1)). Group comparisons were performed using three-way ANOVA Tukey comparison test (GraphPad Prism).

[0230] Rotary bar measurement:

[0231] Motor function was evaluated in mice using an established method: the Accelerated Rotary Bar Performance Test (RR) for sensorimotor coordination. Testing was conducted in a separate testing room after daily tube feeding. Animals were brought to the testing room for 30 to 60 minutes prior to the test to allow for acclimatization. RR assessments were performed at predetermined intervals until a humane endpoint was reached. Each mouse was tested at least three times in the RR. In the RR, all animals were acclimatized to the device for 5 minutes on the first day using a continuous rotation speed of 4 to 40 revolutions per minute (rpm). No prior acclimatization was performed for subsequent experiments. The time until the animal fell off the bar was measured. Each mouse was allowed three trials on the RR, with at least a 10-minute rest between assessments. The highest number achieved for each of the following parameters was recorded: latency to fall, final revolutions per minute, and distance traveled.

[0232] result

[0233] Figure 11 Mean plasma and brain concentrations of nizubaglustat relative to time (at 24 weeks) are shown. In diseased animals, nizubaglustat exhibited persistent brain concentrations and increased BBB permeability compared to normal (Het) animals.

[0234] Figure 12 The levels of GlcCer C16 (plasma) relative to control were shown after administration of (different doses) nizubaglustat in healthy and GM1 animals. Administration of nizubaglustat reduced GlcCer C16 (plasma) levels. The dose-related responses observed with nizubaglustat at 3 and 6 mg / kg / day showed the largest reductions.

[0235] Figure 13The levels of GlcCer C18 (plasma) relative to control were shown after administration of (different doses) nizubaglustat in healthy and GM1 animals. Administration of nizubaglustat reduced GlcCer C18 (plasma) levels. The dose-related responses observed with nizubaglustat at 3 and 6 mg / kg / day showed the largest reductions.

[0236] Figure 14 The levels of GlcCer C16 and C18 (brain) relative to control were shown after administration of (different doses) of nizubaglustat in healthy and GM1 animals. The results indicate that nizubaglustat increases GlcCer C16 and C18 in the brain. After 24 weeks of treatment, all doses were able to produce a significant increase in GlcCer C16 and C18 concentrations in the mouse brain.

[0237] Figure 15 Results of rotarod experiments (latency) relative to control were shown after administration of (different doses) of nizubaglustat in healthy and GM1 animals. Nizubaglustat significantly improved performance on the rotarod. Significant improvements were observed at weeks 12 and 16, but the effect diminished after week 24 (168 days). After week 24, comparisons with untreated animals were not possible because most animals had reached their human endpoints. Each animal was tested three times at each time point; statistical tests: one-way ANOVA and Dunnett's multiple comparison test.

[0238] Figure 16 Results of rotarod tests (distance traveled) relative to control were shown after administration of (different doses) of nizubaglustat to healthy and GM1 animals. Nizubaglustat significantly improved performance on the rotarod. Significant improvements were observed at weeks 12 and 16, but the effect diminished after week 24 (168 days). After week 24, comparisons with untreated animals were not possible because most animals had reached their human endpoints. Each animal was tested three times at each time point; statistical tests were performed using one-way ANOVA and Dunnett's multiple comparison test.

[0239] Figure 17Results of rotarod experiments (final RPM) relative to control were shown in healthy and GM1 animals after administration of (different doses) of nizubaglustat. Nizubaglustat significantly improved performance on the rotarod. Significant improvements were observed at weeks 12 and 16, but the effect diminished after week 24 (168 days). After week 24, comparisons with untreated animals were not possible because most animals had reached their human endpoints. Each animal was tested three times at each time point; statistical tests: one-way ANOVA and Dunnett's multiple comparison test.

Claims

1. Compound of formula (I), It is used to treat GM1 ganglioside storage disorder in subjects.

2. Compound of formula (I), It is used to reduce the accumulation of GM1 gangliosides in the brain and / or spinal cord of a subject, preferably a subject suffering from GM1 ganglioside storage disease.

3. Compound of formula (I), It is used to improve neuronal survival in subjects, preferably those suffering from GM1 ganglioside storage disease.

4. Compound of formula (I), It is used to prevent neuronal degeneration in subjects, preferably those suffering from GM1 ganglioside storage disease.

5. Compound of formula (I), It is used to improve the survival of subjects, preferably those suffering from GM1 ganglioside storage disease.

6. The compound of any one of claims 1 to 5, wherein the compound is in crystalline form.

7. The compound of claim 6, wherein the crystalline form is a crystalline free base.

8. The compound of claim 6 or claim 7, wherein the crystalline form exhibits a reflection at 17.8 ± 0.2° in terms of 2θ value in an X-ray powder diffraction pattern, optionally wherein the reflection at 17.8 ± 0.2° is one of the four strongest reflections in the X-ray powder diffraction pattern.

9. The compound of claim 8, further exhibiting in an X-ray powder diffraction pattern one or more reflections, expressed in 2θ values, at one or more of the following: 4.1 ± 0.2°, 8.3 ± 0.2°, 12.4 ± 0.2°, 13.6 ± 0.2°, 14.5 ± 0.2°, 14.9 ± 0.2°, 15.2 ± 0.2°, 17.2 ± 0.2°, 19.3 ± 0.2°, 21.2 ± 0.2°, 22.4 ± 0.2°, 22.9 ± 0.2°, and 23.3 ± 0.2°.

10. The compound of any one of claims 1 to 9, wherein the compound is administered to the subject at a dose of about 0.1 mg total / day to about 15 mg total / day.

11. The compound of any one of claims 1 to 10, wherein the compound is applied to the object daily.

12. The compound of any one of claims 1 to 11, wherein the object is: (a) Exhibiting one or more symptoms of GM1 ganglioside storage disorder; (b) Does not exhibit any symptoms of GM1 ganglioside storage disorder; (c) Has been diagnosed with GM1 ganglioside storage disorder; or (d) Not diagnosed with GM1 ganglioside storage disease.

13. The compound of any one of claims 1 to 12, wherein the subject is a human being, preferably of age 28 days to 30 years.

14. The compound of any one of claims 1 to 13, wherein the compound is applied to the object as a pharmaceutical composition having a pH of 4.0 to 7.

5.

15. The compound of any one of claims 1 to 14, wherein the compound exhibits substantially no neurotoxicity in the subject.

16. The compound of claim 6 or claim 7, wherein the crystalline form exhibits reflections in X-ray powder diffraction patterns at 17.2 ± 0.2°, 17.8 ± 0.2°, 21.2 ± 0.2° and / or 22.4 ± 0.2°, expressed in 2θ values.

17. The compound of any one of claims 1 to 16, wherein the compound is administered to the subject at a dose of about 3 mg total / day to about 9 mg total / day.

18. The compound of any one of claims 1 to 17, wherein the compound is administered to the subject at a dose of 9 mg total per day.

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