Treatment of GM2 gangliosidosis

Abstract

The present invention relates to a compound of formula (I) for use in treating GM2 gangliosidosis, particularly Sandhoff disease or Tay-Sachs disease. In particular, the present invention relates to a compound of formula (I) for use in increasing the survival time of a subject suffering from GM2 gangliosidosis, particularly Sandhoff disease or Tay-Sachs disease. The compound of formula (I) can also be used as an anti-inflammatory agent. JPEG2025501646000048.jpg36149

Description

[Technical Field]

[0001] The present invention relates to compounds of formula (I) for use in treating GM2 gangliosidoses, in particular Sandhoff disease or Tay-Sachs disease. [ka]

[0002] In particular, the present invention relates to compounds of formula (I) for use in increasing the survival time of a subject suffering from GM2 gangliosidosis, in particular Sandhoff disease or Tay-Sachs disease. [Background technology]

[0003] GM2 gangliosidoses are a group of degenerative / inflammatory disorders affecting the brain caused by deficiencies of various proteins required for lipid breakdown. Examples of GM2 gangliosidoses include Sandhoff disease and Tay-Sachs disease. GM2 gangliosidoses are characterized by rapid neurological deterioration and death, typically before the age of four, due to limited treatment success. GM2 gangliosidoses are the result of inherited defects in genes responsible for encoding the enzymes required to break down GM2 ganglioside (a lipid). This lipid then accumulates in the body, reaching the highest and most abundant levels in the brain. These disorders become apparent at approximately six months of age in severe infantile forms. Juvenile-onset and adult-onset forms of these disorders are associated with a wide range of neurological symptoms, many of which are debilitating and ultimately fatal.

[0004] Normally, GM2 gangliosides are degraded in cellular lysosomes through the coordinated action of the products of three genes: HEXA, HEXB, and GM2A. Deficiency in any one of these genes can result in a lack of HEXA activity toward GM2 gangliosides, resulting in their inability to degrade. The most common form of GM2 gangliosidosis is Tay-Sachs disease, caused by mutations in HEXA. Mutations in HEXB, which encodes the beta subunit of beta-hexosaminidase, cause Sandhoff disease. Approximately 1 in 25 individuals of Ashkenazi Jewish descent are carriers of Tay-Sachs disease, but carrier screening has been successful in reducing the incidence in this population. The prevalence of Sandhoff disease is approximately 1 in 384,000 births, but is higher in certain populations.

[0005] Mouse models of Tay-Sachs and Sandhoff disease have been generated by targeted disruption of the mouse Hexa and Hexb genes. Targeted mutations in the Hexb gene result in a severe neurological phenotype characterized by widespread accumulation of GM2 ganglioside in the brain and spinal cord, leading to spasticity, muscle weakness, rigidity, and ultimately death by 16–17 weeks of age (Gulinello et al., (2008), Behavioral Brain Research:193, 315–319). These mice have been widely used as models for testing potential therapeutic agents targeting GM2 gangliosides.

[0006] In addition to the genetic causes of GM2 gangliosidosis, inflammatory responses (microglial activation, macrophage infiltration, and oxidative damage) are also associated with these lysosomal storage diseases. In particular, inflammation is known to result from excessive GM2 accumulation in the brain, and conversely, chronic brain inflammation is involved in both direct and indirect disease pathogenesis and progression in GM2 gangliosidosis (Jeyakumar et al., (2003), Brain: 126, 974-987). Furthermore, production of the inflammatory cytokines TNFα, IL-1β, and TGFβ1 was shown to be elevated in the brains of Sandhoff disease mice but not in control mice. Subsequent studies confirmed these findings in humans with GM2 gangliosidosis. Finally, macrophage activation and elevated levels of the inflammatory cytokine macrophage inflammatory protein-1α (MIP-1α) have been implicated in the pathogenesis of Sandhoff disease.

[0007] Further evidence for the role of inflammation in GM2 is provided by the use of the anti-inflammatory drug aspirin, which has been shown to significantly slow disease progression in Sandhoff mice (Jeyakumar et al., (2004), Ann. Neurol.:56,642-649). When aspirin was combined with matrix synthesis inhibitory therapy (treatment with compounds that inhibit the synthesis of lipids involved in GM2 gangliosidosis), a synergistic effect (11% improvement in survival time) was found, resulting in a maximum improvement of 73% in survival time.

[0008] Another treatment for GM2 gangliosidosis that has shown promise in recent years is the drug pyrimethamine. Treatment of GM2 gangliosidosis cell lines with pyrimethamine has shown improved enzyme activity and has had some success in early human trials (Ashe et al., (2011), PLOS One:6, e21758). Pyrimethamine, an approved drug for treating malaria, has also been shown to enhance hexosaminidase production (Maegawa et al., (2007), J. Biol. Chem.:282, 9150-9161).

[0009] The primary pharmacological action of therapeutic agents used to treat GM2 gangliosidosis is to inhibit the enzyme glucosylceramide synthase (GCS), which subsequently reduces the biosynthesis of more complex glycosphingolipids (GSLs), thereby inhibiting the formation of glucosylceramide (GlcCer). Substrate synthesis suppression therapy (SRT) is an additional therapeutic principle. SRT is approved for Gaucher disease type 1 and Niemann-Pick disease type C.

[0010] Miglustat (N-butyldeoxynojirimycin), an iminosugar (a synthetic analog of D-glucose) developed as a glucosylceramide synthase inhibitor (also known as ceramide glucosyltransferase, EC 2.4.1.80, UniProt code: Q16739), exhibits extensive distribution and has the ability to reach deep organs such as the brain, bone, and lung. Furthermore, miglustat has been shown to have anti-inflammatory properties. However, clinically, miglustat has not been shown to be therapeutically effective for juvenile GM2 gangliosidosis (Maegawa et al. (2009), Molecular Genetics and Metabolism: 98, 215-224).

[0011] WO 2015 / 147639 describes a wide range of deoxynojirimycin derivatives, including compounds of one formula (herein referred to as compounds of formula (I)). [ka]

[0012] The compounds of formula (I) are potent dual inhibitors of glucosylceramide synthase and non-lysosomal glucosylceramidase (GBA2, UniProt code: Q9HCG7). Summary of the Invention [Problem to be solved by the invention]

[0013] Given the extremely poor survival rates and short life expectancies of patients with GM2 gangliosidoses, such as Sandhoff disease or Tay-Sachs disease, there is a need for novel therapeutic agents that will increase the survival time of patients suffering from these diseases. [Means for solving the problem]

[0014] The present inventors have tested a compound of formula (I) in a mouse model of GM2 gangliosidosis (particularly Sandhoff disease) and found that the compound was highly effective in extending the survival time of affected mice compared to vehicle (placebo)-treated mice. Furthermore, treatment with the compound was shown to significantly improve motor function in treated mice. Thus, in a first aspect, the present invention provides a compound of formula (I) for use in improving motor function in a subject, particularly a subject suffering from GM2 gangliosidosis, particularly Sandhoff disease or Tay-Sachs disease. [ka]

[0015] Preferably, the subject is suffering from Sandhoff disease. Improving motor function improves the quality of life of the subject.

[0016] In a further aspect, the present invention provides a compound of formula (I) for use in treating GM2 gangliosidosis. [ka]

[0017] In a further aspect, the present invention provides a compound of formula (I) for use in treating Sandhoff's disease. [ka]

[0018] In a further aspect, the present invention provides a compound of formula (I) for use in treating Tay-Sachs disease. [ka]

[0019] In a further aspect, the present invention provides a compound of formula (I) for use in eliminating, alleviating or ameliorating the symptoms of GM2 gangliosidosis, particularly Sandhoff disease or Tay-Sachs disease. [ka]

[0020] Preferably, the compounds of formula (I) eliminate, reduce or ameliorate the symptoms of Sandhoff disease.

[0021] In a further aspect, the present invention provides a compound of formula (I) for use in extending the lifespan or life expectancy of a subject suffering from a GM2 gangliosidosis, in particular Sandhoff disease or Tay-Sachs disease, preferably Sandhoff disease. [ka]

[0022] In a further aspect, the present invention provides a compound of formula (I) for use in increasing the survival time of a subject suffering from GM2 gangliosidosis. [ka]

[0023] In a further aspect, the present invention provides a compound of formula (I) for use in increasing the survival time of a subject suffering from Sandhoff disease. [ka]

[0024] In a further aspect, the present invention provides a compound of formula (I) for use in increasing the survival time of a subject suffering from Tay-Sachs disease. [ka]

[0025] The medical use of the present invention may be expressed in any suitable form. Thus, in a further aspect, the present invention provides a method for treating GM2 gangliosidosis, particularly Sandhoff disease or Tay-Sachs disease, comprising administering to a subject suffering from GM2 gangliosidosis, particularly Sandhoff disease or Tay-Sachs disease, a compound of formula (I) in an amount effective to increase the survival time of the subject. [ka]

[0026] Preferably, the method is for treating Sandhoff's disease. Accordingly, the present invention provides a method for treating Sandhoff's disease, comprising administering to a subject suffering from Sandhoff's disease a compound of formula (I) in an amount effective to increase the survival time of said subject.

[0027] In a further aspect, the present invention provides a method of treating Tay-Sachs disease, comprising the step of administering to a subject suffering from Tay-Sachs disease a compound of formula (I) in an amount effective to increase the survival time of said subject. [ka]

[0028] In a further aspect, the present invention provides a method of increasing the survival time of a subject suffering from GM2 gangliosidosis, particularly Sandhoff disease or Tay-Sachs disease, comprising the step of administering to a subject suffering from GM2 gangliosidosis, particularly Sandhoff disease or Tay-Sachs disease, a compound of formula (I) in an amount effective to increase the survival time of said subject. [ka]

[0029] Preferably, the subject is suffering from Sandhoff disease. Accordingly, the present invention provides a method of increasing the survival time of a subject suffering from Sandhoff disease, comprising the step of administering to a subject suffering from Sandhoff disease a compound of formula (I) in an amount effective to increase the survival time of said subject. [ka]

[0030] In a further aspect, the present invention provides a method of increasing survival time of a subject suffering from Tay-Sachs disease, the method comprising the step of administering to a subject suffering from Tay-Sachs disease a compound of formula (I) in an amount effective to increase survival time of said subject. [ka]

[0031] In a further aspect, the present invention provides a method of treating a subject suffering from Sandhoff disease or Tay-Sachs disease and increasing the survival time of the subject, the method comprising the step of administering to a subject suffering from Sandhoff disease or Tay-Sachs disease a compound of formula (I) in an amount effective to increase the survival time of the subject. [ka]

[0032] In a further aspect, the present invention provides the use of a compound of formula (I) for treating a subject suffering from a GM2 gangliosidosis, in particular Sandhoff disease or Tay-Sachs disease. [ka]

[0033] Preferably, the subject is suffering from Sandhoff disease.

[0034] In a further aspect, the present invention provides the use of a compound of formula (I) for increasing the survival time of a subject suffering from a GM2 gangliosidosis, in particular Sandhoff disease or Tay-Sachs disease. [ka]

[0035] Preferably, the subject is suffering from Sandhoff disease.

[0036] In a further aspect, the present invention provides the use of a compound of formula (I) for the manufacture of a medicament for increasing the survival time of a subject suffering from a GM2 gangliosidosis, in particular Sandhoff disease or Tay-Sachs disease. [ka]

[0037] Preferably, the subject is suffering from Sandhoff disease.

[0038] In a further aspect, the present invention provides a compound of formula (I) for use as an anti-inflammatory agent, preferably in a subject suffering from GM2 gangliosidosis. [ka]

[0039] The compounds may exert their anti-inflammatory effects by downregulating inflammatory genes such as ITGAX, TREM2, and CXCL10.

[0040] The present invention also provides the use of a compound of formula (I) in the manufacture of a medicament for increasing the survival time of a subject suffering from GM2 gangliosidosis, in particular Sandhoff disease or Tay-Sachs disease, preferably Sandhoff disease. [ka]

[0041] The present invention also provides the use of a compound of formula (I) in the manufacture of an anti-inflammatory medicament. [ka] In any of the embodiments described herein, the disease is generally GM2 gangliosidosis, but the disease is typically Sandhoff disease or Tay-Sachs disease. In all embodiments, the subject preferably has Sandhoff disease.

[0042] In any of the above embodiments, the compound of formula (I) is preferably crystalline. Preferably, the crystalline form is "Form 3" which exhibits a reflection described as a 2θ value at 17.8±0.2° in an X-ray powder diffraction pattern, and said reflection at 17.8±0.2° is one of the four most intense reflections in said X-ray powder diffraction pattern.

[0043] Preferably, in any of the above aspects, the compound is administered to the subject as a pharmaceutical composition, which may include the compound together with at least one pharmaceutically acceptable carrier, diluent, or excipient.

[0044] Other preferred embodiments of compounds for use according to the present invention appear throughout the specification, particularly in the Examples.

[0045] As explained above, the present inventors have surprisingly discovered that the compound of formula (I) increases the survival time (i.e., life expectancy) of subjects suffering from GM2 gangliosidosis (based on a Sandhoff disease mouse model). Furthermore, the present inventors have discovered that the compound of formula (I) significantly improves the motor skills of subjects, particularly subjects suffering from GM2 gangliosidosis (e.g., Sandhoff disease), and / or positively affects the behavioral assessment of the subjects.

[0046] Unless expressly indicated to the contrary, each aspect or embodiment defined herein may be combined with any other aspect or embodiment(s). In particular, any feature indicated to be preferred or advantageous may be combined with any other feature or features indicated to be preferred or advantageous.

[0047] These and other aspects of the invention will now be described with reference to the accompanying drawings. [Brief explanation of the drawings]

[0048] [Figure 1] 1 shows an outline of the mode of action of a crystalline compound of formula (I) (AZ-3102). [Figure 2A] 1 shows the X-ray powder diffraction pattern of crystalline "Form 3" of compound of formula (I) obtained by equilibration with acetonitrile. [Figure 2B] 1 shows an overlay of X-ray powder diffraction patterns of crystalline "Form 3" of compound of formula (I) obtained by equilibration with TBME, water, isopropanol, ethyl acetate, acetonitrile, or anisole, respectively. [Figure 3] 1 shows an overlay of X-ray powder diffraction patterns of two crystals of compound of formula (I) obtained by equilibration with acetonitrile: 1) "Form 3", and 2) "Form 2". [Figure 4] 1 shows a schematic description of the testing protocol in a Sandhoff disease mouse model. [Figure 5]1 shows the results of a survival study in a Sandhoff disease mouse model treated with AZ-3102 (Compound (I)) and placebo (vehicle). [Figure 6] 1 shows the results of an open field test (OFT) evaluation in a Sandhoff disease mouse model treated with AZ-3102 (Compound (I)) and placebo (vehicle). [Figure 7] 1 shows the results of the rotarod test in a Sandhoff disease mouse model treated with AZ-3102 (Compound (I)) and placebo (vehicle). [Figure 8] 1 shows the plasma and brain concentration versus time profiles of AZ-3102 after 16 weeks of once-daily dosing. [Figure 9] The mean brain-to-plasma ratio in the cerebellum is shown 2, 4, and 24 hours after constant dosing. [Figure 10] 1 shows the concentration versus time profiles of GlcCer C16:0 and C18:0 in the cerebellum. [Figure 11] 1 shows the survival time of Hexb(- / -) mice at relatively low doses of AZ-3102. [Figure 12] Figure 1 shows gene expression data from RNA-seq in the midbrain. Data are presented as fold expression relative to vehicle-treated Het mice. [Figure 13] 1 shows the effect of AZ-3102 on the production of glial fibrillary acidic protein (GFAP). DETAILED DESCRIPTION OF THE INVENTION

[0049] Unless otherwise defined herein, scientific and technical terms used in the present invention shall have the meanings commonly understood by those skilled in the art. The meaning and scope of these terms should be clear, but in the event of potential ambiguity, the definitions set forth herein shall take precedence over any dictionary or external definitions.

[0050] Although singular prepositions such as "a," "an," and "the" are often used for convenience, it should be understood that all instances of the singular are intended to encompass the plural unless expressly or context dictates otherwise. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements. Furthermore, it should be understood that all references, including journal articles, books, patents, technical literature, and the like, mentioned in this disclosure are incorporated herein by reference in their entirety for all purposes.

[0051] The term "about" as used herein with respect to numerical data refers to a value within a 10% range (i.e., plus or minus 10%) of the underlying parameter, and the use of the term "about" at the beginning of a series of values ​​modifies the respective value (i.e., "about 1, 2, and 3" means about 1, about 2, and about 3). For example, a temperature of "about 85°C" can include temperatures from 75°C to 95°C.

[0052] The term "melting point" is well known in the art. As used herein, the term "relatively high melting point" is intended to encompass crystals that are stable enough to be formulated as a pharmaceutical composition.

[0053] The term "composition" as used herein is intended to encompass a product containing the specified ingredients in the specified amounts, as well as any product obtained directly or indirectly by combining the specified ingredients in the specified amounts. This term, with respect to pharmaceutical compositions, is intended to encompass products comprising a compound of formula (I) and / or a crystal of a compound of formula (I), optionally with additional components constituting a carrier, as well as any product obtained directly or indirectly by the combination, complexation, or aggregation of any two or more of the aforementioned components, or the dissociation of one or more of the aforementioned components, or any other type of reaction or interaction of one or more of the aforementioned components. Thus, pharmaceutical compositions of the present invention encompass any composition comprising a compound and / or crystal of the present invention and, optionally, a pharmaceutically acceptable carrier, diluent, or excipient. "Pharmaceutically acceptable" means that the carrier, diluent, or excipient, if present, must be compatible with the other ingredients of the formulation and not deleterious to the recipient of the formulation.

[0054] The terms "therapeutically effective amount" and "amount effective to achieve an effect" are intended to encompass the amount of a compound, crystal, or pharmaceutical composition that, when administered to a patient to treat a disease, is sufficient to effect this treatment for the disease. The terms "therapeutically effective amount" and "amount effective to achieve an effect" are intended to encompass the amount of a compound, crystal, or pharmaceutical composition that, when administered to a patient to achieve a therapeutic effect (e.g., increase survival time), is sufficient to achieve this therapeutic effect. The therapeutically effective amount varies depending on the disease and its severity, and / or the age and weight of the patient. The term "subject" (or "patient") includes, but is not limited to, animals, such as mammals. Preferably, the subject is a human.

[0055] The present invention provides a compound of formula (I) for use in improving motor function in a subject. [ka]

[0056] The subject may be suffering from a disease involving abnormal glucosylceramide levels and / or elevated glycosphingolipid levels. Preferably, the subject is suffering from GM2 gangliosidosis, particularly Sandhoff disease or Tay-Sachs disease. More preferably, the subject is suffering from Sandhoff disease.

[0057] Also provided is a method for improving motor function in a subject, comprising administering to the subject a compound of formula (I) in an amount effective to improve motor function in the subject. Preferably, the subject is suffering from GM2 gangliosidosis, particularly Sandhoff disease or Tay-Sachs disease. More preferably, the subject is suffering from Sandhoff disease.

[0058] The present invention also provides a compound of formula (I) for use in treating GM2 gangliosidosis, particularly Sandhoff disease or Tay-Sachs disease. Preferably, the compound of formula (I) is used to treat Sandhoff disease. [ka]

[0059] The present invention provides compounds of formula (I) for use in eliminating, alleviating or ameliorating the symptoms of GM2 gangliosidosis, particularly Sandhoff disease or Tay-Sachs disease. [ka]

[0060] Preferably, the compounds of formula (I) are used to eliminate, reduce or ameliorate the symptoms of Sandhoff's disease.

[0061] As shown in the Examples, treating a subject suffering from Sandhoff disease with a compound of Formula (I) eliminated, reduced, or ameliorated certain symptoms of Sandhoff disease, e.g., improved motor function in the subject.

[0062] It will be recognized that treatment of Sandhoff disease may result in the elimination, reduction, or amelioration of one or more symptoms of Sandhoff disease, but may not necessarily eliminate, reduce, or ameliorate all symptoms of Sandhoff disease that the patient experienced prior to treatment.

[0063] Because the clinical symptoms of Sandhoff disease and Tay-Sachs disease are relatively indistinguishable and the biochemical causes of both diseases are similar (Tay-Sachs disease is caused by β-hexosaminidase A deficiency, while Sandhoff disease is associated with β-hexosaminidase A and / or β-hexosaminidase B deficiency), subjects suffering from Tay-Sachs disease are also expected to experience elimination, reduction, or amelioration of certain symptoms associated with Tay-Sachs disease upon administration of a compound of formula (I) (or a crystal thereof).

[0064] "Elimination" of a symptom means that the subject essentially no longer experiences the symptom. "Relief" of a symptom means that the subject experiences a significant improvement in the symptom. The reduction is preferably from severe to mild. "Improvement" of a symptom means that the subject experiences a moderate improvement in the symptom.

[0065] Thus, in certain embodiments, the treatments, uses, etc. provided herein may result in the elimination, reduction, or amelioration of one or more symptoms of Sandhoff disease or Tay-Sachs disease, e.g., one or more symptoms may be alleviated, or one or more different symptoms may be improved.

[0066] Symptoms of Sandhoff disease include at least one of muscle weakness, decreased movement, loss of motor skills, increased reaction to noise, seizures, vision loss, hearing loss, intellectual disability, eye abnormalities, organ enlargement, bone abnormalities, speech disorders, loss of cognitive function, loss of muscle coordination, and / or psychiatric problems. A compound of formula (I) can be used to reduce, eliminate, and / or improve at least one symptom of Sandhoff disease in a subject suffering from Sandhoff disease. Preferably, the compound of formula (I) reduces, eliminates, and / or improves at least two, at least three, at least four, or at least five symptoms of Sandhoff disease. For example, the compound of formula (I) can improve motor function and muscle coordination in a subject.

[0067] Subjects with Tay-Sachs disease also exhibit the above symptoms, and therefore any embodiment described with respect to Sandhoff disease may also be applied to Tay-Sachs disease, or indeed GM2 gangliosidoses in general.

[0068] Thus, viewed alternatively, there is provided a method of eliminating, alleviating, and / or ameliorating 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 thereof, who may be suffering from GM2 gangliosidosis, particularly Sandhoff disease or Tay-Sachs disease.

[0069] Furthermore, the present invention provides compounds of formula (I) for use in extending the lifespan of a subject suffering from GM2 gangliosidosis, particularly Sandhoff disease or Tay-Sachs disease. [ka]

[0070] Preferably, the compound of formula (I) (or a crystal thereof) is used to extend the lifespan of a subject suffering from Sandhoff disease.

[0071] Also provided is a method of extending the lifespan of a subject suffering from GM2 gangliosidosis, particularly Sandhoff disease or Tay-Sachs disease, comprising the step of administering to said subject a compound of formula (I) in an amount effective to extend the lifespan of a subject suffering from GM2 gangliosidosis, particularly Sandhoff disease or Tay-Sachs disease.

[0072] Subjects suffering from GM2 gangliosidosis, particularly Sandhoff disease or Tay-Sachs disease, have a very short life expectancy, especially in the infantile form of the disease. The inventors of the present application surprisingly discovered that a compound of formula (I) (or a crystal thereof) increases life expectancy by at least 22% in a mouse model (comparing treated samples to untreated samples). Thus, a compound of formula (I) (or a crystal thereof, preferably "Form 3") can be used to extend a subject's lifespan (or a subject's 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 lifespan (or life expectancy) that the subject would have in the absence of treatment with a compound of formula (I). In other words, the compound of formula (I) (or a crystal thereof, preferably "Form 3") can be used to extend the lifespan (or life expectancy of a subject) of 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 relative to the lifespan (or life expectancy) that the subject would have in the absence of treatment with the compound of formula (I) (or a crystal thereof, preferably "Form 3").

[0073] The present invention provides a compound of formula (I) for use in increasing the survival time of a subject suffering from GM2 gangliosidosis, in particular Sandhoff disease or Tay-Sachs disease, preferably Sandhoff disease. [ka]

[0074] Preferably, the compound of formula (I) is crystalline (preferably "Form 3" crystalline).

[0075] The compound of formula (I) (or its crystals) may have a lower clearance in the brain than in plasma. Therefore, the compound of formula (I) (or its crystals) is particularly suitable for use in treating GM2 gangliosidosis. For example, the concentration of the compound of formula (I) (or its crystals) in the brain can be maintained for at least 12 hours, at least 24 hours, or at least 48 hours. That is, the concentration of the compound of formula (I) (or its crystals) in the brain does not change (substantially) for at least 12 hours, at least 24 hours, or at least 48 hours after administration (when the brain concentration of the compound of formula (I) or its crystals reaches a steady state or a maximum value based on the administered dose).

[0076] The mortality rate of subjects suffering from Sandhoff disease is very high, especially in the infantile form of the disease. The present inventors unexpectedly discovered that administering the compound of formula (I) (or its crystal, preferably "Form 3") to a subject suffering from Sandhoff disease (HEXB mutation) increases the survival time of the subject, even when the administered dose of the compound is very low. "Increasing survival time" means that the survival time is improved compared to a situation in which the compound is not administered. The subject survives significantly longer than if untreated. Therefore, the compound of formula (I) (or its crystal, preferably "Form 3") can be used to increase the subject's survival time (or the subject's 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 survival time that the subject would have in the absence of treatment with the compound of formula (I). In other words, the compound of formula (I) (or a crystal thereof, preferably "Form 3") can be used to increase the survival time of a subject 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 compared to the survival time that the subject would have in the absence of treatment with the compound of formula (I) (or a crystal thereof).

[0077] Also provided is a method of increasing survival time of a subject suffering from GM2 gangliosidosis, particularly Sandhoff disease or Tay-Sachs disease, comprising the step of administering to said subject a compound of formula (I) (or a crystal thereof, preferably "Form 3") in an amount effective to increase survival time of the subject suffering from GM2 gangliosidosis, particularly Sandhoff disease or Tay-Sachs disease.

[0078] Also provided is a method of treating a subject suffering from GM2 gangliosidosis, particularly Sandhoff disease or Tay-Sachs disease, and increasing the survival time of the subject, comprising the step of administering to a subject suffering from GM2 gangliosidosis, particularly Sandhoff disease or Tay-Sachs disease, a compound of formula (I) (or a crystal thereof, preferably "Form 3"), in an amount effective to increase the survival time of said subject.

[0079] There is also provided the use of a compound of formula (I) (or a crystal thereof, preferably "Form 3") to increase the survival time of a subject suffering from GM2 gangliosidosis, particularly Sandhoff disease or Tay-Sachs disease.

[0080] The present invention provides a compound of formula (I) for use as an anti-inflammatory agent, preferably in a subject suffering from GM2 gangliosidosis. [ka]

[0081] For the avoidance of doubt, all embodiments defined herein are not limited to one particular wording for defining treatment, but also apply mutatis mutandis to the medical use or method of treatment aspects of the invention.

[0082] The methods and uses described herein with respect to Sandhoff disease may also be applied to GM2 gangliosidoses in general, and Tay-Sachs disease in particular.

[0083] According to all aspects of the invention described herein, preferably, the compound of formula (I) is crystalline. The crystalline compound of formula (I) can be in any crystalline state. The crystalline compound of formula (I) can be a salt crystalline or a free base crystalline (non-ionized form). Preferably, the crystalline compound of formula (I) is a free base crystalline. Furthermore, the compound of formula (I) can form co-crystals.

[0084] When the crystal of the compound of formula (I) is a salt crystal, the molecular structure of the compound of formula (I) in this case contains a protonated nitrogen atom.

[0085] All aspects of the invention described herein are not limited to a single crystal of the compound of Formula (I). A solid material may exist as more than one crystal. These alternative crystals are called polymorphs. Each polymorph exhibits a different orientation and / or conformation of the molecules in the crystal lattice. Each crystalline state, or "polymorph," exhibits a unique set of physicochemical properties due to differences in the crystal structure.

[0086] Polymorphs may exhibit different mechanical properties, such as flowability and compressibility, which affect the technical properties of the compound. The storage stability and shelf life of the compound may also depend on the polymorph.

[0087] Polymorphs can be distinguished from one another in various ways. Polymorphs exhibit distinct spectroscopic properties, such as infrared spectroscopy, Raman spectroscopy, and 13 Polymorphism can be determined using C-NMR spectroscopy. X-ray powder diffraction (XPD) can also be used to characterize polymorphs, given the fact that each crystal refracts X-rays in a different way. Additionally, thermal methods such as differential scanning calorimetry (DSC) can provide unique information about specific polymorphs.

[0088] As is well known in the field of powder X-ray diffraction, relative peak heights in powder X-ray diffraction spectra can be used to describe various crystals. Thus, in the "Form 3" embodiment, a crystalline compound of Formula (I) exhibits a reflection described as a 2θ value at 17.8±0.2° in an X-ray powder diffraction pattern, wherein the reflection at 17.8±0.2° is one of the four most intense reflections in the X-ray powder diffraction pattern. Preferably, the reflection at 17.8±0.2° is one of the three most intense reflections in the X-ray powder diffraction pattern, or the reflection at 17.8±0.2° is one of the two most intense reflections in the X-ray powder diffraction pattern. More preferably, the reflection at 17.8±0.2° is the most intense reflection in the X-ray powder diffraction pattern. More preferably, in "Form 3" embodiment, the crystalline compound of Formula (I) exhibits a reflection described as a 2θ value at 17.8±0.1° in an X-ray powder diffraction pattern, wherein said reflection at 17.8±0.1° is one of the four most intense reflections in said X-ray powder diffraction pattern. Preferably, said reflection at 17.8±0.1° is one of the three most intense reflections in said X-ray powder diffraction pattern, alternatively, said reflection at 17.8±0.1° is one of the two most intense reflections in said X-ray powder diffraction pattern. More preferably, said reflection at 17.8±0.1° is the most intense reflection in said X-ray powder diffraction pattern.

[0089] The term "most intense reflection" describes the highest peak in an X-ray powder diffraction pattern. Peak heights in an X-ray powder diffraction pattern are determined based on X-ray intensity (in units of counts or counts / second). Thus, the most intense reflection is the reflection that exhibits the highest X-ray intensity in the X-ray powder diffraction pattern. For example, the most intense reflection in the X-ray powder diffraction pattern shown in FIG. 2A is the reflection described as a 2θ value at 17.8±0.2°.

[0090] Unless expressly stated to the contrary, all X-ray powder diffraction patterns are determined at about 25° C. using copper K-α radiation.

[0091] Preferably, in the "Form 3" embodiment, the crystalline compound of Formula (I) further exhibits one or more reflections described as 2θ values ​​at one or more of: 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° in an X-ray powder diffraction pattern. More preferably, in "Form 3" embodiments, the crystalline compound of Formula (I) further exhibits one or more reflections described as 2θ values ​​at one or more of: 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° in an X-ray powder diffraction pattern.

[0092] The peak positions in a powder X-ray diffraction spectrum are relatively insensitive to experimental details. Thus, the crystalline compounds of the present invention can be characterized by a powder X-ray diffraction pattern exhibiting specific peak positions. Thus, in the "Form 3" embodiment, the crystal of the compound of Formula (I) is preferably characterized by reflections described as 2θ values ​​at 17.2±0.2°, 17.8±0.2°, 21.2±0.2°, and 22.4±0.2° in the X-ray powder diffraction pattern. More preferably, in "Form 3" embodiment, a crystalline compound of Formula (I) is characterized by reflections described as 2θ values ​​at 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° in an X-ray powder diffraction pattern. More preferably, in the "Form 3" embodiment, the crystalline compound of Formula (I) is characterized by reflections described as 2θ values ​​at 17.2±0.1°, 17.8±0.1°, 21.2±0.1°, and 22.4±0.1° in an X-ray powder diffraction pattern. Most preferably, in "Form 3" embodiment, a crystalline compound of Formula (I) is characterized by reflections described as 2θ values ​​at 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° in an X-ray powder diffraction pattern.

[0093] Crystals of the compound can be characterized by a differential scanning calorimetry (DSC) thermogram. Thus, in the "Form 3" embodiment, the crystals of the compound of Formula (I) are preferably characterized by a DSC thermogram exhibiting an endothermic heat flow onset at about 87°C and / or a melting point of about 92.4°C. Thus, preferably, in the "Form 3" embodiment, the crystals of the compound of Formula (I) exhibit a melting point determined by DSC of 89°C to 96°C. Preferably, in the "Form 3" embodiment, the crystals exhibit a melting point determined by DSC of 90°C to 95°C. More preferably, in the "Form 3" embodiment, the crystals of the compound of Formula (I) exhibit a melting point determined by DSC of 91°C to 94°C. Most preferably, in the "Form 3" embodiment, the crystals of the compound of Formula (I) exhibit a melting point determined by DSC of 92°C to 93°C.

[0094] A relatively high melting point of a therapeutic compound (usually above about 80° C.) increases resistance to degradation, thereby facilitating the storage and shelf life of the therapeutic compound, which is desirable for any therapeutic agent.

[0095] Crystals of a compound can be characterized by hygroscopicity. The hygroscopicity of a product represents the increase or decrease in water content as a function of relative humidity at a particular temperature. A substantially non-hygroscopic product exhibits no or only a slight change in water content as a result of fluctuations in relative humidity. A strongly hygroscopic product may exhibit a large variation in water content. Therefore, preferably, the crystals of the compound of formula (I) are substantially non-hygroscopic. When preparing pharmaceutical compositions and pharmaceutical formulations for use in such tablets, it is highly desirable to provide crystals of a therapeutic compound that exhibit a low level of hygroscopicity and / or a low level of deliquescence, so that they can be compressed into a desired shape or size.

[0096] A substantially non-hygroscopic material exhibits a water absorption rate of less than about 2% at a relative humidity of about 95% and a temperature at which it is measured of about 25° C. Preferably, the water absorption rate is less than about 1% at a relative humidity of about 95% and a temperature at which it is measured of about 25° C. The water absorption rate value is obtained by measuring the mass increase of a test crystal relative to its initial mass at a relative humidity of about 95% and a temperature of about 25° C.

[0097] Therefore, the crystals of the compound of formula (I) preferably absorb 0% to 2% of water at a relative humidity of about 95% and a temperature of about 25°C. More preferably, the crystals of the compound of formula (I) absorb 0% to 1.5% of water at a relative humidity of about 95% and a temperature of about 25°C. Most preferably, the crystals of the compound of formula (I) absorb 0% to 1% of water at a relative humidity of about 95% and a temperature of about 25°C.

[0098] In a "Form 2" embodiment, the crystal of the compound of Formula (I) exhibits in an X-ray powder diffraction pattern a reflection described as a 2θ value at 16.9±0.2°, wherein said reflection at 16.9±0.2° is one of the four most intense reflections in said X-ray powder diffraction pattern. Preferably, said reflection at 16.9±0.2° is one of the three most intense reflections in said X-ray powder diffraction pattern, or alternatively, said reflection at 16.9±0.2° is one of the two most intense reflections in said X-ray powder diffraction pattern. More preferably, said reflection at 16.9±0.2° is the most intense reflection in said X-ray powder diffraction pattern. Even more preferably, in a "Form 2" embodiment, the crystal exhibits in an X-ray powder diffraction pattern a reflection described as a 2θ value at 16.9±0.1°, wherein said reflection at 16.9±0.1° is one of the four most intense reflections in said X-ray powder diffraction pattern. Preferably, the reflection at 16.9±0.1° is one of the three most intense reflections in the X-ray powder diffraction pattern, alternatively, the reflection at 16.9±0.1° is one of the two most intense reflections in the X-ray powder diffraction pattern, more preferably, the reflection at 16.9±0.1° is the most intense reflection in the X-ray powder diffraction pattern.

[0099] Preferably, in the "Form 2" embodiment, the crystalline compound of Formula (I) exhibits one or more reflections described as 2θ values ​​at one or more of 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° in an X-ray powder diffraction pattern. More preferably, in the "Form 2" embodiment, the crystalline compound of Formula (I) exhibits one or more reflections described as 2θ values ​​at one or more of 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°.

[0100] In the "Form 2" embodiment, the crystalline compound of Formula (I) may be characterized by reflections described as 2θ values ​​at 16.1±0.2°, 16.5±0.2°, 16.9±0.2°, 18.9±0.2°, and 23.1±0.2° in an X-ray powder diffraction pattern. Preferably, in the "Form 2" embodiment, the crystalline compound of Formula (I) may be characterized by reflections described as 2θ values ​​at 16.1±0.1°, 16.5±0.1°, 16.9±0.1°, 18.9±0.1°, and 23.1±0.1° in an X-ray powder diffraction pattern.

[0101] In the "Form 2" embodiment, the crystalline compound of Formula (I) may be characterized by a DSC thermograph exhibiting an endothermic heat flow onset at about 58°C and a melting point of about 70°C. Thus, in the "Form 2" embodiment, the crystalline compound of Formula (I) exhibits a melting point of 67°C to 74°C. Preferably, in the "Form 2" embodiment, the crystalline compound of Formula (I) exhibits a melting point of 68°C to 73°C. More preferably, in the "Form 2" embodiment, the crystalline compound of Formula (I) exhibits a melting point of 69°C to 72°C. Most preferably, in the "Form 2" embodiment, the crystalline compound of Formula (I) exhibits a melting point of 69°C to 71°C.

[0102] Preferably, in the "Form 2" embodiment, the crystals of the compound of Formula (I) are particularly water-soluble. Thus, preferably, in the "Form 2" embodiment, the crystals of the compound of Formula (I) exhibit a water solubility of about 75 mg / mL to about 85 mg / mL when measured at about 25°C. More preferably, in the "Form 2" embodiment, the crystals exhibit a water solubility of about 78 mg / mL to about 82 mg / mL when measured at about 25°C. Most preferably, in the "Form 2" embodiment, the crystals of the compound of Formula (I) exhibit a water solubility of about 80 mg / mL when measured at about 25°C.

[0103] Methods for measuring solubility, such as the shake-flask method, sonication, column elution, and ultraviolet or visible spectroscopy, are known in the art. Unless expressly stated to the contrary, water solubility is determined using the shake-flask method and / or sonication.

[0104] Increasing the survival time of a subject means extending the lifespan or life expectancy of the subject 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 lifespan (or life expectancy) that the subject would have exhibited in the absence of treatment with a compound of Formula (I). In other words, administration of a compound of Formula (I) extends the lifespan of a subject suffering from Sandhoff disease (or other GM2 gangliosidoses) compared to the lifespan that the subject would have exhibited if the subject had not been administered a compound of Formula (I). Administration of a compound of formula (I) can extend the lifespan (and thus delay death) of a subject suffering from Sandhoff 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. Administration of a compound of formula (I) can prevent death from Sandhoff disease (or other GM2 gangliosidoses) in a subject suffering from the disease. For example, a subject treated with a compound of formula (I) can be expected to live at least 5 years longer than they would have if not treated with a compound of formula (I).

[0105] In some embodiments, a therapeutically effective amount (i.e., an amount effective to achieve a desired effect) of the compound of Formula (I) (or a crystal thereof) may range from about 0.0001 milligrams total to about 50 milligrams total of the compound of Formula (I) (or a crystal thereof) per day (mg total / day). In certain embodiments, the therapeutically effective amount may be at least about 0.001 mg total, at least about 0.05 mg total, at least about 0.1 mg total, at least about 0.25 mg total, at least about 0.5 mg total, at least about 1 mg total, at least about 3 mg total, at least about 5 mg total, or at least about 8 mg total. In further embodiments, the therapeutically effective amount may range from about 0.01 to 20 mg total, 0.05 to 15 mg total, or 0.1 to 12 mg total.

[0106] Thus, the compound of formula (I) (or a crystal thereof, preferably "Form 3") may be administered to a subject at a dosage ranging from about 0.0001 mg / day to about 50 mg / day in total. Preferably, the compound of formula (I) (or a crystal thereof, preferably "Form 3") is administered to a subject at a dosage ranging from about 0.1 mg / day to about 15 mg / day in total.

[0107] The dose of the compound of formula (I) (or its crystal, preferably "Form 3") throughout the subject's treatment period may remain the same, or may be increased or decreased. According to all aspects of the present invention, the exact maintenance dose can be routinely determined by those skilled in the art based on the specific subject's physical condition, the severity of the symptoms of Sandhoff disease or other GM2 gangliosidoses, and the tolerability of the administered compound. Generally speaking, the exact maintenance dose is a compromise between the improvement of the symptoms of Sandhoff disease (or other GM2 gangliosidoses) and the specific subject's physiological tolerance of the exact dose (e.g., the observation / possibility of undesirable side effects). The exact maintenance dose is specific to each individual subject, and it is routine for a skilled artisan (e.g., a physician) to determine the exact maintenance dose for a specific subject based on the factors described above.

[0108] In certain embodiments, the compound of formula (I) (or a crystal thereof) can be administered once a day as a single dose. The compound of formula (I) (or a crystal thereof) can be administered as two, three, or more separate doses throughout the day. That is, the therapeutically effective amount per day is divided into two, three, or more doses and administered to the subject separately throughout the day. The multiple doses per day are not necessarily the same amount.

[0109] Treatment (regular, e.g., daily administration of the compound of formula (I)) can be carried out for a suitable period, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks, several months, or several years. For example, the compound of formula (I) (or a crystal thereof) can 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. Treatment can be carried out indefinitely if well tolerated. Alternatively, the compound of formula (I) (or a crystal thereof) can be administered to the subject once.

[0110] Administration may be achieved by oral administration via tablets (e.g., dispersible tablets), pills, capsules, granules, powders, liquids, etc., or parenteral administration via injections, e.g., intra-articular, intravenous, and intramuscular injections, suppositories, ophthalmic solutions, eye ointments, or topical preparations, e.g., transdermal liquid formulations, ointments, transdermal patches, transmucosal liquid formulations, transmucosal patches, inhalers, etc.

[0111] The compound of formula (I) (or a crystal thereof) may be administered to a subject together with an anti-inflammatory agent. In some embodiments, the compound of formula (I) (or a crystal thereof) and the anti-inflammatory agent may be administered simultaneously. In some embodiments, the compound of formula (I) (or a crystal thereof) and the anti-inflammatory agent may be administered separately, for example, sequentially. In some embodiments, the compound of formula (I) (or a crystal thereof) and the anti-inflammatory agent may be co-formulated into a single pharmaceutical composition. Alternatively, the compound of formula (I) (or a crystal thereof) and the anti-inflammatory agent may be formulated into separate pharmaceutical compositions. In either case, the pharmaceutical composition may further comprise one or more pharmaceutically acceptable excipients / solvents.

[0112] The anti-inflammatory agent may be a non-steroidal anti-inflammatory agent, and may be selected from aspirin, ibuprofen, naproxen, diclofenac, celecoxib, mefenamic acid, etoricoxib, and indomethacin.

[0113] Thus, the present invention also provides a therapeutic combination comprising a compound of formula (I) (or a crystal thereof) and an anti-inflammatory agent.

[0114] Alternatively, or in addition, the compound of formula (I) (or a crystal thereof) may itself achieve an anti-inflammatory effect. Preferably, the compound of formula (I) (or a crystal thereof) is an anti-inflammatory agent. Preferably, the compound of formula (I) (or a crystal thereof) increases the expression of anti-inflammatory genes in the brain. Preferably, the compound of formula (I) (or a crystal thereof) decreases the expression of pro-inflammatory genes in the brain. For example, the compound of formula (I) (or a crystal thereof) may decrease the expression of the ITGAX gene in the brain. For example, the compound of formula (I) (or a crystal thereof) may decrease the expression of the TREM2 gene in the brain. For example, the compound of formula (I) (or a crystal thereof) may decrease the expression of the CXCL10 gene in the brain. Preferably, the compound of formula (I) (or a crystal thereof) decreases the expression of pro-inflammatory genes in the intermediate region of the brain. Preferably, the compound of formula (I) (or a crystal thereof) increases the expression of anti-inflammatory genes in the intermediate region of the brain.

[0115] Integrin α-X / β-2 (expressed by the ITGAX gene) is a receptor for fibrinogen. Integrin α-X / β-2 recognizes the sequence GPR in fibrinogen. Integrin α-X / β-2 mediates cell-cell interactions during inflammatory responses. Integrin α-X / β-2 is particularly important in monocyte adhesion and chemotaxis.

[0116] Triggering receptor expressed on myeloid cells 2 (expressed by the TREM2 gene) is a proinflammatory membrane protein likely involved in chronic inflammation; it acts as a receptor for amyloid beta-42, lipoprotein particles, binds to phospholipids such as sphingomyelin, and regulates microglial activation and chemotaxis.

[0117] C-X-C motif chemokine ligand 10 (expressed by the CXCL10 gene) is a proinflammatory molecule involved in the regulation of chemotaxis, differentiation, and activation of peripheral immune cells, cell proliferation, apoptosis, and angiogenesis.

[0118] The compound of formula (I) (or a crystal thereof) may decrease the expression of other genes in the brain, for example, the compound of formula (I) (or a crystal thereof) may decrease the expression of the CYBB gene, CD68 gene, HPGDS gene, C1QB gene, C1QA gene, and / or PLCB2 gene.

[0119] The compound of formula (I) (or a crystal thereof) may increase the expression of other genes in the brain, for example, the compound of formula (I) (or a crystal thereof) may increase the expression of the SLC18A3 gene, the TH gene, the DDC gene, and / or the RET gene.

[0120] A subject who is or should be treated with a compound of formula (I) (or a crystal thereof) may exhibit one or more symptoms of Sandhoff disease or other GM2 gangliosidoses. Alternatively, a subject may be treated even if he or she does not exhibit symptoms of Sandhoff disease or other GM2 gangliosidoses. The subject may be asymptomatic. A subject who is or should be treated with a compound of formula (I) (or a crystal thereof) may have been diagnosed with Sandhoff disease or other GM2 gangliosidoses. The diagnosis of Sandhoff disease or other GM2 gangliosidoses may be based on genetic screening (e.g., to determine HEXB gene mutations). Alternatively, a subject who is or should be treated with a compound of formula (I) (or a crystal thereof) may not have been diagnosed with Sandhoff disease or other GM2 gangliosidoses.

[0121] One or more symptoms of Sandhoff disease may include muscle weakness, decreased movement, loss of motor skills, increased reactivity to noise, seizures, vision loss, hearing loss, intellectual disability, eye abnormalities, organ enlargement, bone abnormalities, speech problems, loss of cognitive function, loss of muscle coordination, and / or psychiatric problems.

[0122] These symptoms are also present in subjects with other GM2 gangliosidoses (e.g., Tay-Sachs disease).

[0123] Treatment with a compound of formula (I) (or a crystal thereof) can increase the survival time of a subject suffering from Sandhoff disease or another GM2 gangliosidoses (e.g., Tay-Sachs disease). In this context, "increasing survival time" means extending the life expectancy of a subject suffering from Sandhoff disease or another GM2 gangliosidoses (e.g., Tay-Sachs disease) who is receiving the treatment, compared to a subject suffering from Sandhoff disease or another GM2 gangliosidoses (e.g., Tay-Sachs disease) who is not receiving the treatment. "Increasing survival time" means improving survival time compared to a situation in which the compound is not administered. The subject lives significantly longer than if they were not treated. Thus, compounds of formula (I) can be used to increase a subject's survival time (or a subject's 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 survival time that the subject would have in the absence of treatment with a compound of formula (I). In other words, the compounds of formula (I) can be used to increase the survival time of a subject 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 compared to the survival time that the subject would have in the absence of treatment with the compounds of formula (I).

[0124] A subject who is or is to be treated with a compound of formula (I) (or a crystal thereof) may have at least one mutation in the HEXB gene, which preferably results in abnormal or reduced activity of the β-hexosaminidase A and / or β-hexosaminidase B enzymes.

[0125] A subject who is or should be treated with a compound of formula (I) (or a crystal thereof) may exhibit at least one mutation in the HEXA gene, which preferably results in an abnormal or reduced activity of the β-hexosaminidase A enzyme.

[0126] A subject being treated or to be treated with a compound of formula (I) (or a crystal thereof) may exhibit abnormal β-hexosaminidase A and / or β-hexosaminidase B activity compared to a healthy subject (i.e., a subject not afflicted with Sandhoff disease or other GM2 gangliosidoses (e.g., Tay-Sachs disease)). The subject may exhibit decreased β-hexosaminidase A and / or β-hexosaminidase B activity compared to a healthy subject (i.e., a subject not afflicted with Sandhoff disease or other GM2 gangliosidoses (e.g., Tay-Sachs disease)). The levels of β-hexosaminidase A and / or β-hexosaminidase enzymes can be measured in a sample from the subject using standard biochemical approaches.

[0127] In addition to increasing the survival time of a subject (or extending the life expectancy of a subject), the compound of formula (I) (or a crystal thereof) may provide at least one additional therapeutic benefit to the subject. For example, the at least one additional therapeutic benefit may be selected from improved motor function, improved behavior, reduced anxiety, improved physical condition, improved balance, reduced seizures, improved speech intelligibility, improved walking, improved swallowing, and improved intelligence.

[0128] According to all aspects of the present invention, the subject is a mammal, preferably a human. In certain embodiments, the subject is between 28 days and 30 years old. The subject may be an infant (i.e., between 28 days and 12 months old). The subject may be a toddler (i.e., between 12 months and 24 months old). The subject may be a child (i.e., between 24 months and 12 years old). The subject may be an adolescent (i.e., between 12 and 17 years old). The subject may be a young adult (i.e., between 17 and 30 years old). However, in some embodiments, the present invention also applies to adults over 30 years old.

[0129] The compound of formula (I) (or a crystal thereof) can be administered to a subject as a pharmaceutical composition. The pharmaceutical composition can have a pH value of 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 pharmaceutical composition has a pH value of 4.5 to 5.5. The pharmaceutical composition can further comprise an acid and / or a base. For example, the pharmaceutical composition can contain an acid to lower the pH to a desired pH value. A base can be used to increase the pH to a desired pH value. Those skilled in the art can easily determine suitable acids and bases for manipulating the pH value of the pharmaceutical composition. For example, the acid can be citric acid, acetic acid, or hydrochloric acid. For example, the base can be sodium hydroxide or potassium hydroxide.

[0130] Typically, the pharmaceutical compositions of the present invention are prepared by combining a pharmaceutically acceptable carrier and one or more optional ingredients, and then, if necessary or desired, the resulting homogeneous blend may be shaped or filled into tablets (e.g., dispersible tablets), capsules, pills, canisters, cartridges, dispensers, and the like using conventional procedures and equipment.

[0131] When intended for oral administration as a solid dosage form (i.e., capsules, tablets (e.g., dispersible tablets), pills, etc.), the pharmaceutical composition of the present invention typically comprises the compound of formula (I) (or a crystal thereof) as an active ingredient. The pharmaceutical composition of the present invention may comprise the compound of formula (I) (or a crystal thereof) without any other ingredients. The pharmaceutical composition of the present invention may be contained in a capsule. The pharmaceutical composition of the present invention may be contained in a capsule without any other ingredients. The capsule may be a gelatin capsule or a hydroxypropylmethylcellulose (HPMC) capsule. Alternatively, the pharmaceutical composition of the present invention may comprise the compound of formula (I) (or a crystal thereof) as an active ingredient and one or more pharmaceutically acceptable carriers. Suitable pharmaceutically acceptable carriers, such as fats, water, saline, alcohols (e.g. ethanol), glycerol, polyols, aqueous glucose solutions, fillers, disintegrants, binders, lubricants, wetting agents, stabilizers, emulsifiers, dispersants, preservatives, sweeteners, colorants, flavorings or fragrances, thickeners, diluents, buffer substances, solvents or solubilizers, chemicals for achieving a storage effect, salts for modifying osmotic pressure, coating agents or antioxidants, sugars, such as lactose or glucose; corn, wheat, or rice starch; fatty acids, such as stearic acid; inorganic salts, For example, magnesium aluminometasilicate 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 hydroxypropylmethylcellulose, 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 will be known to those skilled in the art.

[0132] Additionally, the oral dosage form may be a delayed-release capsule in which the compound of Formula (I) (or its crystals) is encapsulated in a hard or soft soluble container made of a suitable form of gelatin or hydroxypropylmethylcellulose (HPMC), which releases the compound of Formula (I) (or its crystals) at a time other than immediately after administration, and the enteric coating provides a delayed-release dosage form. Delayed-release capsule pellets are also useful, in which the compound of Formula (I) (or its crystals) is encapsulated in a hard or soft container or "shell." In these cases, the compound of Formula (I) (or its crystals) itself is in the form of granules, which are coated with an enteric coating, which delays release of the drug until it enters the intestine. Extended-release capsules and film-coated extended-release capsules are also useful.

[0133] Furthermore, capsules may be coated with a film coating that releases the compound of formula (I) (or its crystals) in a manner that at least reduces the dosing frequency compared to capsules presented as conventional dosage forms. Examples include gelatin-coated capsules (solid dosage forms in which the compound of formula (I) (or its crystals) is encapsulated in a hard or soft, soluble container made of a suitable form of gelatin or HPMC; through a banding process, the capsule is coated with an additional layer of gelatin or HPMC to form a complete seal), and liquid-filled capsules (solid dosage forms in which the compound of formula (I) (or its crystals) is encapsulated in a soluble gelatin shell that has been plasticized by the addition of a polyol such as sorbitol or glycerin and is therefore somewhat more viscous than a hard-shell capsule).

[0134] In some embodiments, the compound of formula (I) (or crystals thereof) may be dissolved or suspended in a liquid medium or may be formulated as granules (small or fine particles), pellets (small sterile solid masses of the highly purified compound of formula (I) (or crystals thereof), with or without excipients, made by forming granules or by compressing and molding), or extended-release coated pellets (solid dosage forms in which the compound of formula (I) (or crystals thereof) itself is in the form of granules to which a coating of varying amounts is applied, thereby releasing the compound of formula (I) (or crystals thereof) in a manner that reduces the dosing frequency compared to dosage forms presented as conventional dosage forms).

[0135] Other forms include pills (small, round, solid dosage forms containing the compound of formula (I) (or its crystals) intended for oral administration), powders (admixtures of dry, micronized compound of formula (I) (or its crystals) with one or more pharmaceutically acceptable excipients, which may be intended for internal or external use), elixirs (clear, pleasant-tasting, sweetened hydroalcoholic liquids containing dissolved compound of formula (I) (or its crystals); intended for oral use), chewing gums (sweetened and flavored insoluble plastic materials of various shapes which, when chewed, release compound of formula (I) (or its crystals) into the oral cavity), syrups (oral solutions containing compound of formula (I) (or its crystals) and high concentrations of sucrose or other sugars; this term also includes sweetened, viscous liquids, including oral suspensions). These include, but are not limited to, any other liquid dosage form prepared in a vehicle), tablets (solid dosage forms containing the compound of formula (I) (or its crystals) with or without suitable diluents), chewable tablets (solid dosage forms containing the compound of formula (I) (or its crystals) with or without suitable diluents that are intended to be chewed, produce a pleasant aftertaste in the mouth, are easy to swallow, and do not leave a bitter or unpleasant aftertaste), coated or delayed-release tablets, dispersible tablets, effervescent tablets, extended-release tablets, film-coated tablets, or extended-release film-coated tablets that are formulated so that the compound of formula (I) (or its crystals) contained therein is available for a long period of time after ingestion. For example, the compound of formula (I) (or its crystals) can be administered to a subject in the form of a dispersible tablet.

[0136] Other forms include dissolving tablets, suspending tablets, multilayer tablets, and extended-release multilayer tablets formulated to at least reduce the frequency of administration compared to tablets offered as conventional dosage forms. Orally disintegrating tablets, delayed-release orally disintegrating tablets, soluble tablets, sugar-coated tablets, osmotic tablets, and the like are also suitable.

[0137] Injectable and infusion dosage forms used herein include, but are not limited to, liposomal injectables, which consist of or form liposomes (lipid bilayer vesicles, usually composed of phospholipids, used to encapsulate the compound of formula (I) (or a crystal thereof)); injectables, which include sterile preparations intended for parenteral use; emulsion injectables, which include emulsions consisting of sterile, pyrogen-free preparations intended to be administered parenterally; or lipid complex injectables.

[0138] Other forms include a powder for injection as a solution, which is a sterile preparation intended to be reconstituted to form a solution for parenteral use; a powder for injection as a suspension, which is a sterile preparation intended to be reconstituted to form a suspension for parenteral use; a lyophilized powder for injection as a suspension liposome, which is a sterile lyophilized preparation intended to be reconstituted for parenteral use, formulated to form liposomes (lipid bilayer vesicles usually composed of phospholipids used to encapsulate the compound of formula (I) (or its crystals) within the lipid bilayer or aqueous space) upon reconstitution; or a lyophilized powder for injection as a solution (wherein lyophilization is a process involving the removal of water from a product in a frozen state at very low pressure).

[0139] As used herein, "solution injectable" refers to a liquid preparation comprising a compound of formula (I) (or a crystal thereof) dissolved in a suitable solvent or a mixture of mutually miscible solvents, which is suitable for injection. "Concentrated solution injectable" refers to a sterile parenteral preparation which, upon addition of a suitable solvent, gives a solution that meets the requirements for an injection in all respects.

[0140] Suspension injectables include liquid formulations suitable for injection that consist of solid particles dispersed throughout a liquid phase in which the particles are insoluble, which may consist of an oil phase dispersed throughout an aqueous phase, or an aqueous phase dispersed throughout an oil phase. Suspension liposome injectables include liquid formulations suitable for injection that consist of an oil phase dispersed throughout an aqueous phase to form liposomes (lipid bilayer vesicles usually composed of phospholipids used to encapsulate the compound of formula (I) (or its crystals) within the lipid bilayer or aqueous space). Suspension sonicated injectables include liquid formulations suitable for injection that consist of solid particles dispersed throughout an aqueous phase in which the particles are insoluble. Furthermore, the suspension is sonicated while gas is being bubbled through the formulation, thereby forming microspheres of the solid particles.

[0141] Parenteral carrier systems comprise one or more pharmaceutically suitable excipients, such as solvents and cosolvents, solubilizing agents, wetting agents, suspending agents, thickening agents, emulsifying agents, chelating agents, buffers, pH adjusting agents, antioxidants, reducing agents, antimicrobial preservatives, bulking agents, protectants, osmolality adjusting agents, and special additives. Formulations suitable for parenteral administration conveniently comprise sterile oily or aqueous preparations of the compound of formula (I) (or crystals thereof), which are preferably isotonic with the blood of the recipient, although this is not required.

[0142] As used herein, inhalation dosage forms include, but are not limited to, aerosols (preparations containing a compound of formula (I) (or its crystals) in a pressurized package that are released upon actuation of an appropriate valve system intended for topical application to the skin, and for topical application to the nose (intranasal aerosols), mouth (lingual and sublingual aerosols), or lungs (inhalation aerosols)). Bubble aerosols are dosage forms that contain a compound of formula (I) (or its crystals), a surfactant, an aqueous or non-aqueous liquid, and a propellant, and that expel stable bubbles when the propellant is present in the internal (discontinuous) phase (i.e., oil-in-water), or sprays or quickly breaking bubbles when the propellant is present in the external (continuous) phase (i.e., water-in-oil). Metered-dose aerosols are pressurized dosage forms consisting of a metered-dose valve that allows the delivery of a uniform amount of spray with each actuation. Powder aerosols are formulations containing the compound of formula (I) (or its crystals) in powder form that are packaged under pressure and released upon actuation of an appropriate valve system. Aerosol sprays are aerosol formulations that utilize compressed gas as a propellant to generate the force necessary to release the formulation as a wet spray, which can be applied to a solution of the compound of formula (I) (or its crystals) in an aqueous solvent.

[0143] As briefly described above, pharmaceutical compositions containing crystals of Compound (I) 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 such as propylene glycol, polyethylene glycol monolaurate, or azacycloalkan-2-one and incorporated into a patch or similar delivery system. Additional excipients, including gelling agents, emulsifiers, and buffers, can also be used. As used herein, transdermal dosage forms include, but are not limited to, patches (drug delivery systems that often include an adhesive backing layer that is typically applied to an external body site, and in which ingredients (including the compound of Formula (I) (or its crystals)) passively diffuse or are actively transported from a portion of the patch, and depending on the patch, ingredients (including the compound of Formula (I) (or its crystals)) are delivered to the external or internal body surface). Various types of transdermal patches, such as matrices, reservoirs, etc., are known in the art.

[0144] Topical dosage forms as used herein include various dosage forms known in the art, such as lotions (emulsive liquid dosage forms generally intended for topical application to the skin), enhanced lotions (lotion dosage forms which enhance the delivery of the compound of formula (I) (or crystals thereof), where enhancement does not refer to the strength of the compound of formula (I) (or crystals thereof) in the dosage form), gels (semi-solid dosage forms which include a gelling agent to provide rigidity to a solution or colloidal dispersion, where the gel may include suspended particles), and ointments (semi-solid dosage forms which typically include less than 20% water and volatiles and more than 50% hydrocarbons, waxes, or polyols as a vehicle, generally intended for topical application to the skin or mucous membranes). Further embodiments include enhanced ointments (ointment dosage forms that enhance compound delivery, where enhancement does not refer to the strength of the compound of Formula (I) (or its crystals) in the dosage form), creams (emulsion semisolid dosage forms that typically contain more than 20% water and volatiles and / or less than 50% hydrocarbons, waxes, or polyols that may also be used as vehicles, and are generally intended for topical application to the skin or mucous membranes), and enhanced creams (cream dosage forms that enhance compound delivery, where enhancement does not refer to the strength of the compound of Formula (I) (or its crystals) in the dosage form). As used herein, an "emulsion" refers to a dosage form consisting of a two-phase system composed of at least two immiscible liquids, one of which is dispersed as an internal or dispersed phase of droplets within the external or continuous phase of the other liquid, generally stabilized by one or more emulsifiers, where "emulsion" is used as the term for the dosage form unless a more specific term (e.g., cream, lotion, ointment) is applicable. Further embodiments include suspensions (liquid dosage forms containing solid particles dispersed in a liquid vehicle), extended release suspensions, pastes (semi-solid dosage forms containing a large proportion of solids, 20-50%, finely dispersed in a fatty vehicle, generally intended for topical application to the skin or mucous membranes), solutions (clear, homogeneous liquid dosage forms containing one or more chemicals dissolved in a solvent or mixture of mutually miscible solvents), and powders.

[0145] Topical dosage form compositions comprise a compound of formula (I) (or a crystal thereof) 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. A variety of FDA-approved topical inactive ingredients can be found in the FDA's "Inactive Ingredients Database," which lists inactive ingredients specifically intended as such by the manufacturer.

[0146] Preferably, the pharmaceutical composition comprises one or more additional pharmaceutically effective agents (e.g., anti-inflammatory agents such as aspirin). This combination therapy encompasses the use of a combination of the crystals of Compound (I) and one or more additional pharmaceutically effective agents, either formulated together (e.g., packaged together in a single formulation) or formulated separately (e.g., packaged as separate unit dosage forms).

[0147] The methods and uses described herein may be in vitro methods (or uses) or in vivo methods (or uses).

[0148] The above-described methods and uses can be combined with a method for diagnosing GM2 gangliosidosis (e.g., Sandhoff disease or Tay-Sachs disease, preferably Sandhoff disease), in which GM2 gangliosidosis (e.g., Sandhoff disease or Tay-Sachs disease, preferably Sandhoff disease) is first diagnosed in a subject, and then compound (I) of formula (I) (or a crystal thereof) is administered to the subject suffering from GM2 gangliosidosis (e.g., Sandhoff disease or Tay-Sachs disease, preferably Sandhoff disease) to achieve a desired therapeutic effect (e.g., increased survival time). The diagnostic method can include genetic screening of the HEXA and / or HEXB genes to determine the presence of at least one mutation. The diagnostic method can include determining the level of β-hexosaminidase A and / or β-hexosaminidase B in the subject. At least one mutation in the HEXA gene can be indicative of Tay-Sachs disease. At least one mutation in the HEXB gene can be indicative of Sandhoff disease.

[0149] The above detailed description has been provided by way of illustration and example, and is not intended to limit the scope of the appended claims. Many variations of the preferred embodiments of the invention illustrated herein will be apparent to those skilled in the art and remain within the scope of the appended claims and their equivalents.

[0150] The invention is further disclosed in the following sections.

[0151] 1. A compound of formula (I) for use in improving motor function in a subject. [ka]

[0152] 2. The compound for use according to clause 1, wherein the subject is suffering from a disease involving abnormal levels of glucosylceramide and / or elevated levels of glycosphingolipids.

[0153] 3. A compound for use according to clause 1 or 2, wherein the subject is suffering from a GM2 gangliosidosis, in particular Sandhoff disease or Tay-Sachs disease, preferably Sandhoff disease.

[0154] 4. A compound of formula (I) for use in treating GM2 gangliosidosis, in particular Sandhoff disease or Tay-Sachs disease, preferably Sandhoff disease. [ka]

[0155] 5. A compound of formula (I) for use in alleviating the symptoms of GM2 gangliosidosis, in particular Sandhoff disease or Tay-Sachs disease, preferably Sandhoff disease. [ka]

[0156] 6. A compound of formula (I) for use in extending the lifespan of a subject suffering from GM2 gangliosidosis, in particular Sandhoff disease or Tay-Sachs disease, preferably Sandhoff disease. [ka]

[0157] 7. A compound of formula (I) for use in increasing the survival time of a subject suffering from GM2 gangliosidosis, in particular Sandhoff disease or Tay-Sachs disease, preferably Sandhoff disease. [ka]

[0158] 8. The compound for use according to any one of clauses 1 to 7, which is a crystal.

[0159] 9. The compound for use according to clause 8, wherein the crystals are crystals of the free base.

[0160] 10. The compound for use according to clause 8 or 9, wherein the crystals exhibit a reflection in an X-ray powder diffraction pattern described as a 2θ value at 17.8±0.2°, said reflection at 17.8±0.2° being one of the four most intense reflections in said X-ray powder diffraction pattern.

[0161] 11. The compound for use according to clause 10, wherein the crystals further exhibit one or more reflections described as 2θ values ​​at one or more of: 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° in an X-ray powder diffraction pattern.

[0162] 12. The compound for use according to clause 8 or 9, wherein the crystals exhibit reflections described as 2θ values ​​at 17.2±0.2°, 17.8±0.2°, 21.2±0.2°, and 22.4±0.2° in an X-ray powder diffraction pattern.

[0163] 13. The compound for use according to any one of clauses 8 to 12, wherein the crystals have a melting point of 89°C to 96°C.

[0164] 14. The compound for use according to any one of clauses 8 to 13, wherein the crystals have a melting point of 91°C to 94°C.

[0165] 15. The compound for use according to any one of clauses 8 to 14, wherein the crystals exhibit a melting point of 92°C to 93°C.

[0166] 16. The compound for use according to any one of clauses 8 to 15, wherein the crystals are substantially non-hygroscopic.

[0167] 17. The compound for use according to clause 8 or 9, wherein the crystals exhibit a reflection in an X-ray powder diffraction pattern described as a 2θ value at 16.9±0.2°, said reflection at 16.9±0.2° being one of the four most intense reflections in said X-ray powder diffraction pattern.

[0168] 18. The compound for use according to clause 17, wherein the crystals further exhibit one or more reflections described as 2θ values ​​at one or more of 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° in an X-ray powder diffraction pattern.

[0169] 19. The compound for use according to any one of clauses 7 to 18, wherein increasing survival time extends the life expectancy of the subject by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40%.

[0170] 20. The compound for use according to any one of clauses 7 to 19, wherein increasing survival time extends the life expectancy of a subject to which the compound is administered compared to a subject to which the compound is not administered.

[0171] 21. The compound for use according to any one of clauses 1-20, wherein the compound is administered to a subject at a dosage ranging from about 0.0001 mg / day to about 50 mg / day.

[0172] 22. The compound for use according to any one of clauses 1-21, wherein the compound is administered to a subject at a dosage ranging from about 0.1 mg / day to about 15 mg / day.

[0173] 23. The compound for use according to any one of clauses 1 to 22, wherein the compound is administered to the subject in combination with an anti-inflammatory agent.

[0174] 24. The compound for use according to clause 23, wherein the compound is administered to the subject simultaneously with or sequentially with said anti-inflammatory agent.

[0175] 25. The compound for use according to clause 23 or 24, wherein the anti-inflammatory agent is aspirin, ibuprofen, naproxen, diclofenac, celecoxib, mefenamic acid, etoricoxib, or indomethacin.

[0176] 26. The compound for use according to any one of clauses 1 to 25, wherein the compound is administered daily to the subject.

[0177] 27. The compound for use according to any one of clauses 1-26, wherein the compound is administered to the subject at least once, at least twice, or at least three times daily.

[0178] 28. The compound for use according to any one of clauses 1 to 27, wherein the subject exhibits one or more symptoms of GM2 gangliosidosis, in particular Sandhoff disease or Tay-Sachs disease, preferably Sandhoff disease.

[0179] 29. The compound for use according to clause 28, wherein one or more symptoms of GM2 gangliosidosis, in particular Sandhoff disease or Tay-Sachs disease, preferably Sandhoff disease, include muscle weakness, decreased movement, loss of motor skills, increased reaction to noise, seizures, vision loss, hearing loss, intellectual disability, eye abnormalities, organ enlargement, bone abnormalities, speech disorders, loss of cognitive function, loss of muscle coordination, and / or psychiatric problems.

[0180] 30. The compound for use according to any one of clauses 1-27, wherein the subject is asymptomatic.

[0181] 31. The compound for use according to any one of clauses 1 to 27, wherein the subject does not exhibit any symptoms of GM2 gangliosidosis, in particular Sandhoff disease or Tay-Sachs disease, preferably Sandhoff disease.

[0182] 32. The compound for use according to any one of clauses 1 to 31, wherein the subject has been diagnosed with GM2 gangliosidosis, in particular Sandhoff disease or Tay-Sachs disease, preferably Sandhoff disease.

[0183] 33. The compound for use according to any one of clauses 28 to 32, wherein the compound increases the survival time of a subject suffering from GM2 gangliosidosis, in particular Sandhoff disease or Tay-Sachs disease, preferably Sandhoff disease, wherein increasing survival time results in a prolongation of the life expectancy of a subject suffering from GM2 gangliosidosis, in particular Sandhoff disease or Tay-Sachs disease, preferably Sandhoff disease, compared to a subject not suffering from GM2 gangliosidosis, in particular Sandhoff disease or Tay-Sachs disease, preferably Sandhoff disease.

[0184] 34. The compound for use according to any one of clauses 1 to 31, wherein the subject has not been diagnosed with GM2 gangliosidosis, in particular Sandhoff disease or Tay-Sachs disease, preferably Sandhoff disease.

[0185] 35. The compound for use according to any one of clauses 1 to 34, wherein the subject has at least one mutation in the HEXB gene.

[0186] 36. A compound for use according to clause 35, wherein the compound increases the survival time of a subject suffering from GM2 gangliosidosis, in particular Sandhoff disease or Tay-Sachs disease, preferably Sandhoff disease, wherein the increased survival time results in an increase in the life expectancy of a subject having at least one mutation in the HEXB gene compared to a subject not having a mutation in the HEXB gene.

[0187] 37. The compound for use according to any one of clauses 1 to 36, wherein the subject exhibits abnormalities in β-hexosaminidase A and / or β-hexosaminidase B activity.

[0188] 38. The compound for use according to any one of clauses 1 to 37, wherein the subject exhibits decreased activity of β-hexosaminidase A and / or β-hexosaminidase B compared to a healthy subject.

[0189] 39. A compound for use according to clause 37 or 38, wherein the compound increases the survival time of a subject suffering from GM2 gangliosidosis, in particular Sandhoff disease or Tay-Sachs disease, preferably Sandhoff disease, wherein increasing survival time results in a prolongation of the life expectancy of a subject exhibiting abnormal or reduced activity of β-hexosaminidase A and / or β-hexosaminidase B compared to a subject exhibiting normal activity of β-hexosaminidase A and / or β-hexosaminidase B.

[0190] 40. The compound for use according to any one of clauses 1 to 39, wherein the compound provides at least one additional therapeutic benefit to a subject selected from improved motor function, improved behavior, reduced anxiety, improved physical condition, improved balance, reduced seizures, improved speech intelligibility, improved gait, improved swallowing, and improved intelligence.

[0191] 41. The compound for use according to any one of clauses 1 to 40, wherein the subject is a human.

[0192] 42. The compound for use according to clause 41, wherein the subject is between 28 days and 30 years old.

[0193] 43. The compound for use according to any one of clauses 1 to 42, wherein the compound is administered to the subject as a pharmaceutical composition exhibiting a pH between 4.0 and 7.5.

[0194] 44. The compound for use according to clause 43, wherein the pharmaceutical composition exhibits a pH of 4.5 to 5.5.

[0195] 45. The compound for use according to clause 43 or 44, wherein the pharmaceutical composition further comprises an acid and / or a base.

[0196] 46. ​​The compound for use according to any one of clauses 43 to 45, wherein the pharmaceutical composition is administered to the subject as a capsule or tablet, for example a dispersible tablet.

[0197] 47. The compound for use according to any one of clauses 1-46, wherein the compound is 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, at least 3 years.

[0198] 48. The compound for use according to any one of clauses 1 to 46, which is administered once to the subject.

[0199] 49. A method for increasing the survival time of a subject suffering from GM2 gangliosidosis, particularly Sandhoff disease or Tay-Sachs disease, preferably Sandhoff disease, comprising the step of administering to a subject suffering from GM2 gangliosidosis, particularly Sandhoff disease or Tay-Sachs disease, preferably Sandhoff disease, a compound of formula (I) in an amount effective to increase the survival time of said subject. [ka]

[0200] 50. A method for treating a subject suffering from GM2 gangliosidosis, particularly Sandhoff disease or Tay-Sachs disease, preferably Sandhoff disease, and increasing the survival time of the subject, comprising the step of administering to a subject suffering from GM2 gangliosidosis, particularly Sandhoff disease or Tay-Sachs disease, preferably Sandhoff disease, a compound of formula (I) in an amount effective to increase the survival time of the subject. [ka]

[0201] 51. Use of a compound of formula (I) for increasing the survival time of a subject suffering from GM2 gangliosidosis, in particular Sandhoff disease or Tay-Sachs disease, preferably Sandhoff disease. [ka] [Example]

[0202] Example 1 - Crystallization of Compound (I) Suspension equilibration experiments of the compound of formula (I) in various solvents, such as acetonitrile, ethyl acetate, isopropanol, anisole, water, or TBME at room temperature (about 25° C.), yielded stable crystals of the compound of formula (I).

[0203] In particular, crystalline Form 3 of the compound of formula (I) was obtained by the following experimental method. 1) About 74 mg of the compound of formula (I) was added to 2.0 ml of acetonitrile, and the suspension was stirred at room temperature (about 25°C) for 3 days and then filtered; 2) adding about 74 mg of the compound of formula (I) to 2.0 ml of anisole, and stirring the suspension at room temperature (about 25°C) for 3 days, followed by filtration; 3) adding about 82 mg of the compound of formula (I) to 1.0 ml of ethyl acetate, stirring the suspension at room temperature (about 25°C) for 3 days, and then filtering; 4) adding about 82 mg of the compound of formula (I) to 1.0 ml of isopropanol, stirring the suspension at room temperature (about 25° C.) for 3 days, and then filtering; 5) Adding about 45 mg of the compound of formula (I) to 1.0 ml of water, stirring the suspension at room temperature (about 25°C) for 3 days, and then filtering; 6) Approximately 100 mg of the compound of formula (I) was added to 3.0 ml of TBME, and the suspension was stirred at room temperature (approximately 25° C.) for 3 days and then filtered.

[0204] The compound of formula (I) was purified using a silica gel column, and then a solvent was added to remove the boric acid ester.

[0205] The resulting crystals of the compound of formula (I) were characterized by powder X-ray diffraction, DSC, and DVS.

[0206] The PXRD pattern of crystals of Form 3 of compound of Formula (I) obtained by suspension equilibration experiments with acetonitrile is shown in Figure 2A. An overlay of the PXRD patterns of crystals of Form 3 of compound of Formula (I) obtained by suspension equilibration experiments with other solvents is shown in Figure 2B. The crystals of compound of Formula (I) show the most intense reflections at 2θ values ​​of 17.2±0.2°, 17.8±0.2°, 21.2±0.2°, and 22.4±0.2°. This is consistent despite the different solvents used to obtain the crystals of compound of Formula (I).

[0207] For comparison, Figure 3 shows an overlay of the PXRD patterns of another crystal (Form 2) and Form 3 of the compound of Formula (I). These two crystals exhibit different PXRD patterns. Form 2 was obtained by short-term (approximately 1-5 minutes) equilibration of the purified compound of Formula (I) (the compound of Formula (I) was purified using a silica gel column to remove borate esters) in acetonitrile. This Form 2 crystal of the compound of Formula (I) exhibits the most intense 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°.

[0208] Powder X-ray diffraction was performed on a Stoe Stadi P diffractometer equipped with a Mythen 1K detector operating at Cu-Kα1 radiation. Measurements on this instrument were performed under a tube voltage of 40 kV and a tube current of 40 mA. A curved Ge monochromator enabled testing at Cu-Kα1 radiation. The following parameters were set: step size 0.02°2θ, step time 12 s, scan range 1.5–50.5°2θ, and detection step (step-scan detection mode) 1°2θ. In a typical sample preparation, approximately 10 mg of sample was placed between two sheets of acetate foil and mounted in a Stoe transmission sample holder. The sample was rotated during the measurement. All sample preparation and measurements were performed under ambient atmosphere (approximately 25°C).

[0209] DSC measurements performed on Form 3 show a sharp endothermic melting peak at about 92° C. with an enthalpy of about 103 J / g. DSC measurements performed on Form 2 show an initial strong endotherm with a peak temperature of about 70° C. and an enthalpy of about 54 J / g, followed by a weaker signal at 81° C. and an enthalpy of about 3 J / g.

[0210] Differential scanning calorimetry (DSC) was performed on a TA Instruments Q2000 instrument (closed aluminum sample pans or aluminum sample pans with a pinhole in the lid, heating rate 20 K / min). The melting point is taken as the maximum peak.

[0211] Furthermore, the behavior of a sample of the crystals of Form 3 of the compound of formula (I), obtained by equilibration with ethyl acetate, was investigated under various water vapor pressures (DVS measurements). At a maximum relative humidity of 95%, the sample absorbed approximately 1.2% water, which was released when the relative humidity returned to 50% RH. The amount of absorbed water was small, and the water sorption was irreversible. Therefore, this crystals of Form 3 of the compound of formula (I) are non-hygroscopic or substantially non-hygroscopic. For Form 2, at high relative humidity, the sample absorbed approximately 18% water, but most of the absorbed water was released when the relative humidity returned to 50% RH. Dynamic vapor sorption (DVS) measurements were performed with the SPS11-100n "Sorptions Profsystem" manufactured by ProUmid (formerly "Projekt Messtechnik"), August-Nagel-Str. 23, 89079 Ulm (Germany). Approximately 5 mg to 20 mg of sample was placed in an aluminum sample pan. A humidity change rate of 5% per hour was used. The applied measurement program can be written as follows: Samples were placed on aluminum or platinum holders on a microbalance and equilibrated at 50% relative humidity (RH) before initiating a predefined humidity program: (1) 50% RH for 2 hours (2) 50 → 0% RH (5% / hour); 5 hours at 0% RH (3) 0 to 95% RH (5% / hour); 95% RH for 5 hours (4) 95 → 0% RH (5% / hour); 5 hours at 0% RH (5) 0 to 95% RH (5% / hour); 95% RH for 5 hours (6) 95 → 50% RH (5% / hour); 2 hours at 50% RH

[0212] Example 2 - Effect of administration of crystals of the compound of formula (I) to mice suffering from Sandhoff disease (Hexb null mutation) In this example, AZ-3102 is the name for Form 3 of the compound of formula (I).

[0213] 1. Introduction Project Background and Overview: GM2 gangliosidoses are a group of degenerative / inflammatory disorders that affect the brain, including Sandhoff disease. These disorders are characterized by rapid neurological deterioration and death, typically before the age of four, due to limited treatment success to date. GM2 gangliosidoses are the result of inherited defects in the gene responsible for encoding the enzyme necessary to break down GM2 ganglioside (a lipid). This lipid then accumulates in the body, reaching the highest levels and greatest abundance in the brain. These disorders become apparent at approximately six months of age in severe infantile forms. Juvenile-onset and adult-onset forms of the disease are associated with a wide range of neurological symptoms, many of which are debilitating and ultimately fatal.

[0214] Normally, GM2 gangliosides are degraded in cellular lysosomes through the coordinated action of the products of three genes: HEXA, HEXB, and GM2A. Defects in any one of these genes can result in a lack of HEXA activity toward GM2 gangliosides, which cannot be degraded. Mutations in HEXB, which encodes the beta subunit of beta-hexosaminidase, cause Sandhoff disease. The incidence of Sandhoff disease is approximately 1 in 384,000 births, but is higher in certain populations.

[0215] A mouse model of Sandhoff disease has been created by targeted disruption of the mouse Hexb gene. Targeted mutations in the Hexb gene result in a severe neurological phenotype characterized by widespread accumulation of GM2 ganglioside in the brain and spinal cord, causing spasticity, muscle weakness, rigidity, and ultimately death by 16–17 weeks of age. These mice have been widely used as a model for testing potential therapies for GM2 gangliosidosis.

[0216] In addition to the genetic causes of GM2 gangliosidosis, inflammatory responses (microglial activation, macrophage infiltration, and oxidative damage) are also associated with these lysosomal storage diseases. In particular, inflammation is known to result from excessive GM2 accumulation in the brain, and conversely, chronic brain inflammation is involved in both direct and indirect disease pathogenesis and progression in GM2 gangliosidosis. This group also demonstrated that production of the inflammatory cytokines TNFα, IL1β, and TGFβ1 was elevated in the brains of Sandhoff disease mice but not in control mice. Subsequent studies confirmed these findings in humans with GM2 gangliosidosis. Finally, macrophage activation and elevated levels of the inflammatory cytokine macrophage inflammatory protein-1α (MIP-1α) are involved in the pathogenesis of Sandhoff disease.

[0217] Further evidence for the role of inflammation in GM2 is provided by the use of the anti-inflammatory drug aspirin, which has been shown to significantly slow disease progression in Sandhoff mice. When aspirin was combined with matrix synthesis inhibitory therapy (treatment with compounds that inhibit the synthesis of lipids involved in GM2 gangliosidosis), a synergistic effect (11% improvement in survival time) was found, resulting in a maximum improvement of 73% in survival time.

[0218] Another treatment for GM2 gangliosidosis that has shown promise in recent years is the drug pyrimethamine. Recently, treatment of GM2 gangliosidosis cell lines with pyrimethamine has shown improved enzyme activity and has had some further success in early human trials. Pyrimethamine, an approved drug for treating malaria, has been shown to enhance the production of hexosaminidase.

[0219] Substrate synthesis reduction therapy (SRT), whose primary pharmacological action is to inhibit the formation of glucosylceramide (GlcCer) by inhibiting the enzyme glucosylceramide synthase (GCS) and subsequently reducing the biosynthesis of more complex GSLs, is an additional therapeutic principle. SRT is approved for Gaucher disease type 1 and Niemann-Pick disease.

[0220] Miglustat (N-butyldeoxynojirimycin) is a synthetic analog of the iminosugar D-glucose that primarily inhibits GbA2 (nonlysosomal glucosylceramidase), but at high doses may also inhibit GCS due to its relatively low potency against GCS enzymes. Clinically, miglustat is approved for Gaucher disease type 1 and, in some countries outside the United States, for Niemann-Pick disease type C.

[0221] 2.Purpose The survival and behavioral studies were dose-ranging studies with AZ-3102 and vehicle (control) administered four or more times daily starting on postnatal day 30 to determine efficacy in delaying and / or reducing progressive motor dysfunction in adults and in extending survival time in homozygous mutants.

[0222] 3. Materials and Methods 3.1. Animal Subjects: Hexb- / - mice were obtained from Jackson Laboratories (strain: B6; 129S4-Hexbtm1Rlp / J). The mouse strain was maintained by crossing homozygous - / - males with heterozygous females (resulting in 50% - / - and 50% + / - litters) under specific pathogen-free conditions. All mice were housed in individually ventilated cages (IVCs) with a 12-hour light / dark cycle and free access to food and water. Young adult mice on the same genetic C57BL / 6 background were obtained from Jackson Laboratories. Breeding of Hexb mutants was initially targeted to the needs of "long-term" cohorts, followed by "16-week" biomarker studies.

[0223] 3.2. Breeding: Gender- and age-matched littermates (one male - / - and one female + / -) from synchronized breeding were used. Homozygous mutant Hexb - / - mice, considered in the art to be phenotypically wild-type animals (the mutation is autosomal recessive), and + / - littermates were examined in parallel. Hexb genotype was determined by PCR of DNA isolated from ear clippings obtained at weaning using primers 5'-ATT TTA AAA TTC AGG CCT CGA-3' (common), 5' CAT AGC GTT GGC TAC CCG TGA-3' (mutant), and 5'-CAT TCT GCA GCG GTG CAC GGC-3' (wild-type). Expected breeding time was approximately 19 weeks.

[0224] 3.3. Animal Identification: At weaning, ear clippings were obtained to clearly identify each animal within the same cage, and these samples were used for genotyping (see section 3.2).

[0225] 3.4. Treatment: Within each cohort, mice were randomly assigned to several treatment groups, including a vehicle control. Each cohort was run sequentially. Each cohort had an equal ratio of male and female animals in the long-term cohort, and 8 males and 7 females in the 16-week cohort.

[0226] For all cohorts, drugs and / or vehicle (control) were administered by gavage once daily between 8:00 AM and 12:00 PM from day 30 of age until euthanasia. Within any cohort, animals were prophylactically euthanized to reduce distress due to the occurrence of immobility, tremors or seizures, deterioration of body condition, inability to right, 15% weight loss, and moribundity (see Appendix A for body scoring chart).

[0227] [Table 1]

[0228] Mice were periodically bled before dosing (at 4 weeks of age) and again 4 weeks after dosing (at 8 weeks of age) at 0.5 and 1 hour (see section 3.10).

[0229] Assessments included live animal assessments (for survival benefit and neuromuscular strength) including rotarod and open field. 3.5. Preparation of the formulation:

[0230] [Table 2]

[0231] 1. Introduction and preparation of vehicle controls.

[0232] 1.1. Read the methods before proceeding with the laboratory work.

[0233] 1.2.Read the test plan.

[0234] 1.3. Ensure the work area is clean and select the correct equipment, clean glassware, etc. for testing.

[0235] 1.4. Formulate in ascending order of concentrations.

[0236] 1.5. Starting with the control group - measure the required volume of purified water and stir magnetically. While stirring magnetically, adjust the pH to 3.5-2.5 using 1 M citric acid. Record the volume in the raw data.

[0237] 1.6. Stir for a minimum of 30 minutes. Record the stirring time in the raw data.

[0238] 1.7. Adjust the pH to 4.5-5.5 using 0.1 M NaOH. Record the volume.

[0239] 1.8. Stir using a magnetic stirrer for a minimum of 20 minutes. Record the stirring time in the raw data.

[0240] 1.9. If necessary, take samples at this point.

[0241] 2. Test product formulation 2.1. Weigh out the required amount of test article: For example, if the target concentration for 600 mL is 0.9 mg / mL, weigh out 540 mg.

[0242] 2.2. Add 70% of the final volume of the test article, e.g., 420 mL of purified water, to obtain a thin suspension (which may be difficult to see at these low concentrations).

[0243] 2.3.3 Add up to 3 molar equivalents of citric acid, e.g., 3.74 mL of 1 M aqueous citric acid, and record the pH. The pH should be between 3.5 and 2.5.

[0244] NOTE: The suspension should clarify as an indication of test article dissolution.

[0245] 2.4. The formulation is constantly stirred for 30 minutes or until the material is dissolved. Record the stirring time in the raw data.

[0246] 2.5. Adjust the pH to 4.5-5.5 by adding sodium hydroxide (NaOH 0.1 M). Note: This amount, e.g., 59.8 mL of NaOH, neutralizes 60% of the excess protons from the citric acid, maintaining the solution under acidic conditions.

[0247] 2.6. Make up to final volume with purified water, e.g., 116 mL.

[0248] 2.7. Check and record the final pH. If the pH is not within the range of 4.5-5.5, notify the Study Director. Additional NaOH may be added, but not more than 2.5% of the total volume. Record the volume raw, if necessary.

[0249] 2.8. Stir using a magnetic stirrer for a minimum of 20 minutes. Record the start and end times in the raw data. Continue stirring magnetically.

[0250] 2.9. Continue stirring magnetically and sample at this point if necessary.

[0251] 2.10. Transfer the solution via syringe to the final container while stirring magnetically and flushing with nitrogen.

[0252] 3.6. Cohort: Cohort 1: There were a total of six treatment groups and two vehicle groups using both Hexb+ / - and Hexb- / - animals. AZ-3102 was investigated at doses of 0.5, 1.5, 3.0, and 6.0 mg / kg / day. Dosing occurred daily from day 30 to day 170 unless animals were euthanized earlier per protocol.

[0253] Cohort 2 (16-week biomarker study): There were eight groups using both Hexb+ / - and Hexb- / - animals, which received vehicle control or various doses of AZ-3102 starting at postnatal day 30.

[0254] 3.7 Monitoring - Pharmacodynamics: Mice were examined daily. Behavioral assessments, including subjective and objective measures of general health and body weight (BW), and a neurological assessment score (BNA) were performed twice weekly from postnatal day 56 (=earliest time point of symptom onset in homozygotes).

[0255] Motor function was assessed in cohort 2 using standard methods in mice: the accelerating rotarod test (ROT) for sensorimotor coordination, and the open field locomotor test (OFT) for spontaneous spatial movement investigation.

[0256] Behavioral testing was performed after daily gavage in a separate testing room. Animals were placed in the testing room 30-60 min before the testing procedure to allow for habituation.

[0257] ROT / OFT began at 8 weeks of age, and again at 12 weeks of age, and then every 2 weeks thereafter until a humane endpoint was reached. Each mouse was tested a minimum of four times in the ROT and OFT. The exact number of tests each mouse underwent depended on the number of mice surviving at a particular time point.

[0258] In the ROT, all animals were acclimated to the apparatus for 5 min using a continuous rotation speed of 4–40 revolutions per minute (rpm) the day before the first test. Subsequent experiments did not require prior acclimation. The actual test procedure consisted of a 5-min test with an accelerating rotation speed of 4–40 rpm. Time was measured until the animal fell off the rod. Up to five animals could be tested simultaneously.

[0259] The OFT was conducted in a quiet room under constant lighting conditions using a 40x40cm apparatus. The duration of the test was 5 minutes. All tests were videotaped and then analyzed using tracking software, Smart® version 3.0.05. Measures included mean locomotor activity, walking distance, and time spent in the outer and inner areas of the field.

[0260] 3.8. Sacrifice of mice: At the end of the designated treatment period, mice were humanely euthanized by CO2 asphyxiation followed by transcardial perfusion with Ringer's solution. Blood samples were collected before perfusion. Prior to perfusion, blood samples were collected by cardiac puncture into K2EDTA tubes and stored on wet ice. Whole blood was processed to plasma by centrifugation (3000 g, 10 min at 5°C) within 30 min of collection and stored at -75°C.

[0261] Brain samples were collected, rinsed with saline, and blotted dry. Samples were fixed in 4% PFA for immunohistochemistry, frozen and stored for gene expression and molecular biology analysis, and used frozen for LC-MS / MS analysis.

[0262] 3.9. Blood Sample Collection: Peripheral blood was collected from the animals via the abdominal inferior vena cava at predetermined time points (including baseline).

[0263] Periodic Sampling: All mice underwent periodic sampling by saphenous vein bleeding 0.5 and 1 hour before the first dose and again 4 weeks after dosing (at 8 weeks of age). Plasma was collected and assayed for baseline measurements, AZ-3102, and PD biomarkers.

[0264] A schematic illustration of the test protocol is shown in Figure 4.

[0265] 4.Results Sandhoff disease mouse model (Hexb(- / -)): The Sandhoff disease mouse model targets a mutation in the Hexb gene, which results in a severe neurological phenotype characterized by widespread accumulation of GM2 ganglioside in the brain and spinal cord, causing spasticity, muscle weakness, rigidity, and ultimately death by 16–17 weeks of age (Sango, Yamanaka et al. 1995; Phaneuf, Wakamatsu et al. 1996; Gulinello, Chen et al. 2008). These mice have been widely used as a model for testing potential therapies for GM2 gangliosidosis.

[0266] Hexb(- / -) mice were obtained from Jackson Laboratories (strain: B6; 129S4-Hexbtm1Rlp / J). The mouse strain was maintained by crossing homozygous (- / -) males with heterozygous females (resulting in 50% (- / -) and 50% (+ / -) litters) under specific pathogen-free conditions. All mice were housed in individually ventilated cages (IVCs) with a 12-h light / dark cycle and free access to food and water. Young adult mice on the same genetic C57BL / 6 background were obtained from Jackson Laboratories.

[0267] Within each cohort, mice were randomly assigned to several treatment groups, including a vehicle control (Table 3). Each cohort had an equal ratio of male and female animals (N = 16-18 mice). With the exception of the technician responsible for preparing the formulations, all other staff, including the principal investigator and sponsor, were blinded to the treatments.

[0268] [Table 3]

[0269] All cohorts received drug and / or vehicle via gavage once daily between 8:00 AM and 12:00 PM from day 30 of age until euthanasia. Within any cohort, animals were preemptively euthanized to reduce suffering due to immobility, tremors or seizures, deterioration of body condition, inability to right, 15% weight loss, and moribundity.

[0270] Sandhoff disease mice (Hexb(- / -)) were treated with vehicle or AZ-3102 by daily oral gavage at dose levels of 0 (vehicle, acidified aqueous formulation), 0.5, 1.5, 3, and 6 mg / kg / day. Additionally, animals heterozygous for the Hexb gene (+ / -) were treated with vehicle or 3 mg / kg / day. Treatment continued until the animals reached a humane endpoint. The humane endpoint was blindly determined by animal care staff and consisted primarily of failure to assume a prone position after 10 seconds. Upon reaching this endpoint, further confirmation was given by a senior laboratory technician, after which euthanasia of the animals was initiated. The median survival time of vehicle-treated animals (n = 16–18 per group) was 115 days. Although there was no dose response in AZ-3102-treated animals, the mean median survival time was significantly increased to 26 days, a 22% increase in survival time (p<0.0001, log-rank, Mantle-Cox test) (Figure 5).

[0271] Behavioral assessments were also performed, including the open field test (OFT, ActiMot, TSE systems) and rotarod (RR, IITC Life Sciences). In the OFT, mice were placed in a square box with high walls for 5 min to assess motor skills. Several parameters, including distance traveled (centimeters) and time spent stationary within the box (seconds), were recorded. Motor coordination was assessed using the RR apparatus according to a previously published protocol (Osmon, Vyas et al. 2018). Briefly, mice were placed in a moving cylinder that accelerated from 4 to 40 revolutions per minute (rpm) over a 5-min period. Each mouse underwent three trials on the RR, with a minimum 10-min rest period between each assessment. The highest number achieved for each of the following parameters was recorded: latency to fall, final number of revolutions per minute, and distance traveled.

[0272] Figure 6 shows the locomotor performance over time for each cohort in the OFT assessment. Both total distance traveled and in-area immobility time improved over the course of the experiment until the animals succumbed to disease pathology. Notably, at 16 weeks, the locomotor performance of the AZ-3102-treated Hexb(- / -) group differed from that of the Hexb(- / -) vehicle-treated group, likely demonstrating a dose-response response in both measures, although not significant at all doses (Figure 6). Similarly, in the RR assessment, at 16 weeks, the effect of AZ-3102 treatment was significant at all dose levels compared to vehicle-treated Hexb(- / -) mice (Figure 7).

[0273] 5. Discussion A mouse model of Sandhoff disease has been created by targeted disruption of the mouse Hexb gene. Targeted mutations in the Hexb gene result in a severe neurological phenotype characterized by widespread accumulation of GM2 ganglioside in the brain and spinal cord, causing spasticity, muscle weakness, rigidity, and ultimately death by 16–17 weeks of age. These mice have been widely used as a model for testing potential therapies for GM2 gangliosidosis.

[0274] In this study, we evaluated AZ-3102, an oral small molecule that exhibits low-nM inhibitory activity against both GCS and GbA2 enzymes, in a mouse model of Sandhoff disease (Hexb(- / -)). Sandhoff disease mice (Hexb(- / -)) were treated with vehicle or AZ-3102 by daily oral gavage at dose levels of 0 (vehicle, acidified aqueous formulation), 0.5, 1.5, 3, and 6 mg / kg / day. In addition, animals heterozygous for the Hexb gene (+ / -) were treated with vehicle or 3 mg / kg / day. Treatment continued until the animals reached a humane endpoint. The median survival time of vehicle-treated animals (n = 16-18 per group) was 115 days. Although there was no dose response in AZ-3102-treated animals, mean median survival time was significantly increased to 26 days, a 22% increase in survival time (p<0.0001, log-rank, Mantle-Cox test). Furthermore, behavioral assessments of both the OFT and RR were significantly improved in AZ-3102-treated animals compared with vehicle-treated Hexb(- / -) animals.

[0275] These data demonstrate that AZ-3102 significantly increases survival time and improves behavioral assessments in an animal model of disease where, without treatment, significant reductions in lifespan are associated with increased neuropathology and impaired motor reflexes. Furthermore, AZ-3102 administered at 3 mg / kg / day to Hexb(+ / -) mice showed no effect on lifespan compared to vehicle-treated Hexb(+ / -) animals, indicating the relatively benign effects of this drug in healthy animals.

[0276] Although no dose-response was observed for survival endpoints, it is noteworthy that an increase in survival time was already present even at 0.5 mg / kg / day. Furthermore, although it is possible to investigate lower doses, behavioral assessments appear to indicate that 0.5 mg / kg / day may be approaching the lower end of efficacy where OFT results at 16 weeks indicate a potential dose-response relationship.

[0277] Further analyses with short-term cohorts will investigate AZ-3102 pharmacokinetics, glycosphingolipid regulation (e.g., GlcCer, GM2), neuropathology, and gene regulation in the presence or absence of AZ-3102 treatment in Hexb(- / -) and Hexb(+ / -) mice.

[0278] Appendix A: Hexb Shivering Mouse Body Scoring Chart: Timeline of untreated Hexb mice: WT and Hexb + / - mice are shivering-free and have a normal lifespan. KO mice begin to show physical symptoms at approximately 10-12 weeks of age, with severe rigidity and / or tremors occurring at approximately 14-16 weeks of age, at which point the KO mice will be euthanized. Most untreated KO mice were used as test controls or breeders.

[0279] The scoring system used to determine whether an animal should be euthanized is shown in Table 4.

[0280] [Table 4]

[0281] Example 3 - Pharmacokinetics (PK) and Pharmacodynamics (PD) Unless otherwise stated, methodology and sampling were the same as in Example 2.

[0282] Plasma and brain PK were determined under steady-state conditions after 16 weeks of administration of AZ-3102-00 (the free base of AZ-3102, referred to throughout this document as AZ-3102 unless otherwise noted). 24 hours after the final dose, animals were euthanized at various time points, and AZ-3102 was measured by LC-MS / MS. Over the dose range of 0.5 to 6 mg / kg / day, plasma concentration profiles showed good separation and dose response. Mean AUC 0~24 The concentrations of C (N = 3 / time point) ranged from 417 to 3607 ng·h / mL, which was slightly underproportional (9-fold) to the predicted exposure ratio of 12-fold (Figure 8 and Table 5). maxThe exposure was also subproportional, ranging from 100 to 674 ng / mL, with an approximately 7-fold difference between the lowest and highest doses. However, the exposure (AUC 0~24 The proportionality of AZ-3102 across doses was well preserved: 6 vs. 3 (1.8-fold); 3 vs. 1.5 (2.0-fold); and 1.5 vs. 0.5 (2.4-fold). Although plasma concentrations decreased over time, brain concentration profiles were maintained with little decrease over 24 hours in most dose groups. Also, decreased clearance in the brain compared with that in plasma was observed in previous studies in rats and dogs (internal data). Importantly, at all dose levels, brain and plasma concentrations over 24 hours were within the IC50 of AZ-3102 for both enzyme targets. 50 At all doses, plasma concentrations were approximately 1 ng / mL after 24 hours, and brain concentrations exceeded 20 ng / g tissue. 50 The in vitro potencies based on were 1.1 ng / mL for GCS and 0.3 ng / mL for GbA2, and therefore concentrations were sufficient to significantly inhibit these enzymes across all doses.

[0283] [Table 5]

[0284] Although plasma concentrations generally decreased over time, brain concentration profiles were relatively maintained over 24 hours in most dose groups, with the exception of NPC heterozygous animals (NPC − / +). This is highlighted in Figure 9, where mean brain-to-plasma ratios are plotted at 2, 4, and 24 hours. In general, at 2 and 4 hours, the ratios of AZ-3102 in Sandhoff disease animals are greater than those in heterozygous animals. Furthermore, at early time points, the ratio appears constant, but at 24 hours, the ratio increases with increasing dose.

[0285] AZ-3102 is a potent inhibitor of both GCS and GbA2. Consistent with this effect, inhibition of GbA2 should result in an increase in GlcCer species in the brain, where GbA2 plays a key role. As a measure of target engagement, GlcCer C16:0 and C18:0 were measured in the cerebellum. As expected, GlcCer C16:0 and C18:0 were increased approximately 10-fold in AZ-3102-treated mice compared to untreated (heterozygous) or Hexb(- / -) untreated mice (Figure 10).

[0286] Example 4 - Further survival studies Subsequent studies investigating lower doses were also conducted (similar to Example 2), using doses of 0.02, 0.06, 0.2, and 0.5 mg / kg / day. The two highest doses were not significantly different from each other in terms of survival time, but each was significantly different from the 0.02 mg / kg / day dose. Median survival times for Hexb(- / -) mice at 0.02, 0.06, 0.2, and 0.5 mg / kg / day were 120, 116, 133, and 138 days. Because the formulation was inspected and accurate dosing confirmed (internal data), it is unclear why survival time was reduced at 0.06 mg / kg / day. Nevertheless, the data confirm the reproducibility of the model at 0.5 mg / kg / day and suggest a dose-response or possibly a threshold effect below 0.02 mg / kg / day (Figure 11, along with data from Figure 5; Example 2).

[0287] With this dosing regimen, a dose of 0.2 mg / kg / day appears to be the minimally effective dose with respect to survival time. However, it is noteworthy that an increase in survival time is already present even at 0.5 mg / kg / day. Furthermore, although lower doses are useful for discerning effects on survival time, a dose of 0.5 mg / kg / day appears to approach the lower limit of efficacy where the OFT results at 16 weeks indicate a potential dose-response relationship.

[0288] Taken together, these data demonstrate that AZ-3102 significantly increases survival time and improves behavioral assessments in an animal model of disease where, without treatment, significant reductions in lifespan are associated with increased neuropathology and impaired motor reflexes. Furthermore, AZ-3102 administered at 3 mg / kg / day to Hexb(+ / -) mice had no effect on lifespan compared to vehicle-treated Hexb(+ / -) mice, indicating the benign effects of this drug in healthy animals.

[0289] Example 5 - Gene Expression Unless otherwise stated, methodology and sampling were the same as in Example 2.

[0290] A heatmap of the top 10 up- and down-regulated genes within the pars intermedius region is shown in Figure 12. While the data are currently being explored for further insight, the most consistent observation is the effect of AZ-3102 on inflammatory gene expression. While not exhaustive, three genes of interest in the pars intermedius are highlighted by way of example: Itgax (gene ID: 16411, encoding integrin α-X, a CD11c surface protein) is associated with microglial activation, a marker of dendritic cells, and associated with neuropathologies, including Alzheimer's disease (Haage, V., Semtner, M., Vidal, RO et al. (2019) Comprehensive gene expression meta-analysis identifies signature genes that distinguish microglia from peripheral monocytes / macrophages in health and glioma. Acta Neuropathol Commun 7, 20). Trem2 (Gene ID: 83433, a proinflammatory membrane protein possibly involved in chronic inflammation (Dhandapani, R., Neri, M., Bernhard, M. et al. (2022) Sustained Trem2 stabilization accelerates microglia heterogeneity and Aβ pathology in a mouse model of Alzheimer's disease. Cell Rep 39, 110883.)) and Cxcl10 (Gene ID: 15945, Chemokine (C-X-C motif) ligand 10, a potential chemoattractant) exhibit chemokine and heparin-binding activity.Normal gene expression in the mouse brain is relatively low compared to other tissues (Yue, F., Cheng, Y., Breschi, A. et al. (2014) A comparative encyclopedia of DNA elements in the mouse genome. Nature 515, 355-364.), which appears to play a role in multiple sclerosis through microglial activation (Tanuma, N., Sakuma, H., Sasaki, A. and Matsumoto, Y. (2006) Chemokine expression by astrocytes plays a role in microglia / macrophage activation and subsequent neurodegeneration in secondary progressive multiple sclerosis. Acta Neuropathol 112, 195-204), and is involved in the neuropathology of GM2 gangliosidosis (Demir, SA, Timur, ZK, Ates, N., Martinez, LA and Seyrantepe, V. (2020) GM2 ganglioside accumulation causes neuroinflammation and behavioral alterations in a mouse model of early onset Tay-Sachs disease.J Neuroinflammation 17,277).

[0291] Example 6 - Immunohistochemistry Unless otherwise stated, methodology and sampling were the same as in Example 2.

[0292] GFAP (glial fibrillary acidic protein), a class III intermediate filament protein, is a cell-specific marker that distinguishes astrocytes from other glial cells during central nervous system development. Intermediate filaments form a meshwork that provides support and strength to cells. Several molecules of glial fibrillary acidic protein bind together to form the type of intermediate filament found in astroglial cells. Astroglial cells support and nurture cells in the brain and spinal cord. When brain or spinal cord cells are damaged through trauma or disease, astroglial cells respond by rapidly producing more glial fibrillary acidic protein. Tables 6 and 7 show a group immunofluorescence summary of Hexb − / − (KO) animals compared to both vehicle-treated Hexb + / − (Het) and AZ-3102-treated Hexb − / − animals, highlighting the significant increase in GFAP staining, particularly in the midbrain. The figures show that the difference between AZ-3102-treated and vehicle-treated Hexb − / − mice in the midsection is even more apparent (FIG. 13), with females showing a statistically significant decrease, while male animals show a trend toward decreased staining.

[0293] [Table 6]

[0294] [Table 7]

Claims

1. A composition for use in increasing the survival time of a subject suffering from GM2 gangliosidosis, the composition comprising a compound of formula (I). 【Chemistry 1】

2. A composition for use as an anti-inflammatory agent, comprising a compound of formula (I). 【Chemistry 2】

3. 3. The composition for use according to claim 1 or 2, which is crystalline.

4. The composition for use according to claim 3, wherein the crystals are crystals of the free base.

5. 4. The composition for use according to claim 3, wherein the crystal exhibits a reflection described as a 2θ value at 17.8±0.2° in an X-ray powder diffraction pattern, and the reflection at 17.8±0.2° is one of the four most intense reflections in the X-ray powder diffraction pattern.

6. 6. The composition for use of claim 5, further exhibiting one or more reflections described as 2θ values ​​at one or more of: 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° in an X-ray powder diffraction pattern.

7. 2. The composition for use of claim 1, wherein increasing survival time extends the subject's life expectancy by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40%.

8. 3. The composition for use according to claim 1 or 2, administered to the subject at a dosage ranging from about 0.1 mg / day to about 15 mg / day in total.

9. The composition for use according to claim 1 , wherein the composition is administered to the subject in combination with an anti-inflammatory agent.

10. 10. The composition for use according to claim 9, wherein the composition is administered to the subject simultaneously with or sequentially with the anti-inflammatory agent.

11. 3. The composition for use according to claim 1 or 2, which is administered to the subject daily.

12. 3. The composition for use according to claim 1 or 2, wherein the subject has at least one mutation in the HEXB gene and / or exhibits reduced activity of β-hexosaminidase A and / or β-hexosaminidase B.

13. 3. The composition for use of claim 1 or 2, wherein the composition provides the subject with at least one additional therapeutic benefit selected from improved motor function, improved behavior, reduced anxiety, improved physical condition, improved balance, reduced seizures, improved speech intelligibility, improved walking, improved swallowing, and improved intelligence.

14. The composition for use according to claim 1 or 2, wherein the subject is a human.

15. The composition for use according to claim 1 or 2, which is administered to the subject in a pharmaceutical composition exhibiting a pH of 4.0 to 7.

5.

16. A composition for use as described in claim 1, wherein the GM2 gangliosidosis is Sandhoff disease or Tay-Sachs disease.

17. A composition for use as described in claim 2 for use in a subject suffering from GM2 gangliosidosis.

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