Crystalline form of pharmaceutical compound

Abstract

To provide stable and / or non-deliquescent crystalline forms of pharmaceutical compounds, which are substantially non-hygroscopic and / or have relatively high melting points.SOLUTION: The present invention provides a crystalline form that is a crystalline free base, and displays a reflection at 17.8±0.2° stated as a 2θ value in an X-ray powder diffraction pattern, wherein the reflection at 17.8±0.2° is one of the four strongest reflections in the X-ray powder diffraction pattern.SELECTED DRAWING: None

Description

Detailed Description of the Invention

[0001] [Field of the Invention] The present invention relates to a crystalline form of compound (I). [ka] The present invention also relates to a method for making the crystalline form of Compound (I) and to pharmaceutical compositions comprising the crystalline form of Compound (I). Additionally, the present invention relates to methods of using this crystalline form of Compound (I) as a medicine and in the treatment of diseases involving abnormal levels of glucosylceramide and / or elevated levels of glycosphingolipids.

[0002] [Background to the invention] The crystalline state of a compound can be important when the compound is used for pharmaceutical purposes, since the geometry, particle size, polymorphism, solvation, or hydration of the crystalline state of a compound can affect the filtration, flow, tableting, dissolution, and bioavailability of a pharmaceutical agent.

[0003] Deoxynojirimycin derivatives are an important class of molecules in medicinal chemistry and drug discovery. N-(hydroxyethyl)-deoxynojirimycin has been marketed as miglitol as an antidiabetic drug for type 2 diabetes. Miglitol also acts as a broad-spectrum inhibitor of several intestinal glycosidases (maltase, sucrose, and lactase) (Hillebrand et al., Diabetes, 1986, 35, A93-A93) (Scott and Spencer, Drugs, 2000, 59, 521-549).

[0004] N-Butyl-deoxynojirimycin (miglustat, Zavesca®) was developed as an inhibitor of glucosylceramide synthase (also called ceramide glucosyltransferase, EC 2.4.1.80, UniProt code: Q16739) and is used in outpatient clinics to treat patients with lysosomal storage disorders, Gaucher disease (Platt et al., J. Biol. Chem., 1994, 269, 8362-8365) (Cox et al., Lancet, 2000, 355, 1481-1485) and Niemann-Pick disease type C (Pineda et al., Orphanet J. Rare Dis., 2018, 13, 140).

[0005] WO 2015 / 147639 discloses compound (I) [ka] This paper describes novel derivatives of deoxynojirimycin, including: which are effective in the treatment of diseases associated with abnormal levels of cytosolic or lysosomal glucosylceramide and / or elevated levels of glycosphingolipids. Compound (I) is a potent dual inhibitor of glucosylceramide synthase and non-lysosomal glycosylceramidase (GBA2, UniProt code: Q9HCG7).

[0006] The therapeutic compound useful for treating abnormal levels of glucosylceramide and / or elevated levels of glycosphingolipids is often administered in the form of a tablet.When preparing pharmaceutical compositions and formulations for use in such tablets, it is highly desirable to have the crystalline form of the therapeutic compound with low levels of hygroscopicity and / or low levels of deliquescence, thereby allowing the compound to be compressed into desired shape or size.

[0007] Additionally, the relatively high melting points of the therapeutic compounds (usually above about 80° C.) favor resistance to degradation, thereby facilitating storage of the therapeutic compounds and increasing their shelf life, which is desirable for any pharmaceutical agent.

[0008] When considering the handling, manufacturing and storage of pharmaceutical agents, it is also particularly beneficial for the therapeutic compound to be non-hygroscopic or substantially non-hygroscopic. When a pharmaceutical agent exhibits hygroscopic properties, a number of problems can arise, such as: Difficult to reduce a substance to small particles by grinding, or to powder; Undesirable moisture, which interferes with proper reactions and forms undesirable end products resulting in minimum quality and reduced shelf life; Formation of soft tablets during manufacturing or penetration of moisture inside the packaging; Powder adhesion on the conveyor, which may affect the filling process; There is a risk of this occurring.

[0009] It is therefore desirable to have crystalline forms of therapeutic compounds that are non-hygroscopic or substantially non-hygroscopic.

[0010] Crystalline forms of Compound (I) have not been previously reported. Thus, there is a need for stable and / or non-deliquescent crystalline forms of Compound (I), which are preferably substantially non-hygroscopic and / or have a relatively high melting point.

[0011] [Summary of the Invention] In a first aspect, the present invention provides a crystalline form of compound (I). [ka] Each aspect or embodiment defined in this specification may be combined with any other aspect(s) or embodiment(s) unless expressly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature(s) indicated as being preferred or advantageous.

[0012] In a further aspect, the present invention provides pharmaceutical compositions comprising the crystalline forms of Compound (I) described herein.

[0013] In a further aspect, the present invention provides a crystalline form of Compound (I) as described herein, or a pharmaceutical composition as described herein, for use in therapy.

[0014] In a further aspect, the present invention provides a crystalline form of Compound (I) as described herein, or a pharmaceutical composition as described herein, for use as a medicament.

[0015] Another aspect of the present invention relates to a crystalline form of Compound (I) as described herein, or a pharmaceutical composition as described herein, for use in treating a disease involving abnormal levels of glucosylceramide and / or elevated levels of glycosphingolipids.

[0016] In a further aspect, the present invention provides a crystalline form of Compound (I) as described herein, or a pharmaceutical composition as described herein, for use in the treatment of Niemann-Pick disease type C.

[0017] In a further aspect, the present invention provides a method of treating a disease involving abnormal levels of glucosylceramide and / or elevated levels of glycosphingolipids in a human or animal patient, comprising the step of administering to a patient in need thereof a therapeutically effective amount of a crystalline form of Compound (I) described herein, or a pharmaceutical composition described herein.

[0018] In a further aspect, the present invention provides a method of treating Niemann-Pick Type C in a human or animal patient, comprising administering to a patient in need thereof a therapeutically effective amount of a crystalline form of Compound (I) described herein, or a pharmaceutical composition described herein.

[0019] In a further aspect, the present invention provides a method of preparing a crystalline form of Compound (I) described herein, comprising the step of contacting a sample of Compound (I) with a solvent.

[0020] In a further aspect, the present invention provides a crystalline form of Compound (I) obtainable by carrying out the methods described herein.

[0021] In a further aspect, the present invention provides the use of the free base of Compound (I) for preparing a crystalline form of Compound (I).

[0022] In a further aspect, the present invention provides a method of preparing a crystalline form of Compound (I), comprising the step of crystallizing the free base of Compound (I).

[0023] In a further aspect, the present invention provides a crystalline form of Compound (I) obtained by a process for preparing a crystalline form of Compound (I), comprising the step of crystallizing the free base of Compound (I).

[0024] Further preferred embodiments of compounds according to the invention can be found throughout the specification, especially in the Examples.

[0025] The present inventors have surprisingly discovered a stable, non-deliquescent crystalline form of Compound (I), which has additional advantageous properties, such as a substantial lack of hygroscopicity and a relatively high melting point.

[0026] Unexpectedly, the inventors have further discovered crystalline forms of Compound (I) that exhibit good solubility in water. These properties make the crystalline forms of the present invention particularly suitable for use in pharmaceutical compositions.

[0027] Crystallization of therapeutic compounds often involves the use of different salts. Usually, salts undergo crystallization easily, and the resulting material facilitates the subsequent crystallization of the therapeutic compound. For this reason, the use of salts is often the preferred method for crystallizing therapeutic compounds. Therefore, it is surprising that the inventors have discovered a crystalline free base form of compound (I).

[0028] Because neither crystalline free base form of a therapeutic compound requires the presence of a counterion, the concentration of the therapeutic compound in a powder of the free base crystalline form is typically higher than in the corresponding salt form, which is highly beneficial as it reduces the cost of producing the therapeutic compound.

[0029] Without wishing to be bound by theory, it is believed that the crystalline forms of the present invention tend to exhibit the beneficial effects discussed above due to their crystalline structure.

[0030] These and other aspects of the invention will now be described with reference to the accompanying drawings. [Brief description of the drawings]

[0031] [Figure 1] FIG. 1 shows exemplary images obtained using polarized optical microscopy of a crystalline form of Compound (I), designated Form 2, obtained by equilibration with acetonitrile; left: as a dry powder, right: dispersion in paraffin oil. [Diagram 2] FIG. 1 shows the X-ray powder diffraction pattern of Form 2 obtained by equilibration with acetonitrile. [Figure 3A] FIG. 2 shows the TG-FTIR thermogram of Form 2 obtained by equilibration with acetonitrile. [Figure 3B] FIG. 1 shows a differential scanning calorimetry (DSC) thermogram of Form 2 obtained by equilibration with acetonitrile. [Figure 4A]FIG. 2 shows the dynamic vapor sorption (DVS) isotherms of Form 2 obtained by equilibration with acetonitrile: change in water content (red curve) and relative humidity (blue curve) as a function of time. [Figure 4B] FIG. 1 shows the DVS isotherm of Form 2 obtained by equilibration with acetonitrile: change in water content as a function of relative humidity. [Diagram 5] FIG. 1 shows exemplary images obtained using polarized optical microscopy of a crystalline form of Compound (I), designated Form 3, obtained by equilibration with acetonitrile, anisole, or ethyl acetate, respectively. [Figure 6A] FIG. 1 shows the X-ray powder diffraction pattern of Form 3 obtained by equilibration with acetonitrile. [Figure 6B] FIG. 1 shows overlays of X-ray powder diffraction patterns of Form 3 obtained by equilibration with TBME, water, isopropanol, ethyl acetate, acetonitrile or anisole, respectively. [Figure 7] FIG. 1 shows an overlay of X-ray powder diffraction patterns of two crystalline forms of Compound (I) obtained by equilibration with acetonitrile: 1) Form 3 (Example 3), and 2) Form 2 (Example 2). [Figure 8A] FIG. 1 shows the TG-FTIR thermogram of Form 3 obtained by equilibration with ethyl acetate. [Figure 8B] FIG. 1 shows the TG-FTIR thermogram of Form 3 obtained by equilibration with isopropanol. [Figure 8C] FIG. 1 shows a DSC thermogram of Form 3 obtained by equilibration with ethyl acetate. [Figure 9A] FIG. 1 shows DVS isotherms of Form 3 obtained by equilibration with ethyl acetate: change in water content (red curve) and relative humidity (blue curve) as a function of time. [Figure 9B] FIG. 1 shows the DVS isotherm of Form 3 obtained by equilibration with ethyl acetate: change in water content as a function of relative humidity. [Figure 10]FIG. 1 shows an approximation of the midsagittal level obtained following a segmentation protocol based on Paxinos & Franklin "The Mouse Brain Atlas, 2nd Edition," showing stereotaxic coordinates. [Figure 11] Graph showing percentage change in mouse body weight between postnatal day (PND) 11 and 9 weeks. [Figure 12] Graph showing glucosylceramide C16:0 and C18:0 levels after repeated oral administration from PND11 to 70. [Figure 13] FIG. 1 shows the clinical signs scores from PND 56 to 70 in NPC(- / -) vehicle and AZ-3102 treated mice. [Figure 14] FIG. 1 shows tremor scores in NPC(- / -) vehicle and AZ-3102 treated mice from PND 56 to 70. [Figure 15] Figure 1: Region of interest (ROI) definition. The image shows the outline of the ROIs for the cerebellum, hippocampal formation, corpus callosum and striatum (caudate putamen). [Figure 16] FIG. 1 shows brain immunohistochemistry: Calbindin-D28k labeling in NCP(- / -) and NPC(+ / -) vehicle-treated mice compared to NPC(+ / +, wild type mice) and NPC(- / -) treated mice. [Figure 17] FIG. 1 summarizes the mechanism of action of the crystalline form of Compound (I) (AZ-3102).

[0032] [Detailed Description of the Invention] Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. However, the meaning and scope of the above terms should be clear in the event of any potential ambiguity, and the definitions provided herein take precedence over any dictionary definitions or incidental definitions.

[0033] Although singular prefixes such as "a," "an," and "the" are often used for convenience, it should be understood that all instances of the singular are intended to include the plural unless otherwise indicated, either explicitly or by context. 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 set forth in this disclosure, including journal articles, books, patents, technical documents, and the like, are incorporated by reference in their entirety and for all purposes.

[0034] The term "about," as used herein with respect to numerical data, refers to a value within 10% (i.e., plus or minus 10%) of the underlying parameter, and use of the term "about" at the beginning of a series of values ​​modifies each of those values ​​(i.e., "about 1, 2, and 3" refers to about 1, about 2, and about 3). For example, a temperature of "about 85°C" can include temperatures between 75°C and 95°C.

[0035] The term "melting point" in the art. The term "relatively high melting point" as used herein is intended to include crystalline forms that are sufficiently stable to be formulated as pharmaceutical compositions. Preferably, the term "relatively high melting point" describes a melting point greater than about 65°C. Even more preferably, the melting point is greater than about 80°C.

[0036] The term "composition" as used herein is intended to encompass products containing the specified components in the specified amounts, and any product resulting directly or indirectly from the combination of the specified components in the specified amounts. Such terms with respect to pharmaceutical compositions are intended to encompass products including the crystalline form of Compound (I) and, optionally, additional components constituting the carrier, as well as any product resulting directly or indirectly from the combination, complexation or aggregation of any two or more components, or from the dissociation of one or more components, or from other types of reactions or interactions of one or more components. Thus, the pharmaceutical composition of the present invention encompasses any composition comprising the crystalline form of the present invention, and, optionally, a pharma-ceutically acceptable carrier. "Pharmaceutically acceptable" means that the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

[0037] The term "therapeutically effective amount" refers to the amount of a crystalline form or pharmaceutical composition that, when administered to a patient for treating a disease, is sufficient to effect such treatment for the disease. The "therapeutically effective amount" will vary depending on the disease and its severity, as well as the age and weight of the patient being treated. The term "patient" (or "subject") includes, but is not limited to, animals, such as mammals. Preferably, the patient is a human.

[0038] The present invention relates to a crystalline form of compound (I). [ka] The crystalline form of compound (I) may be in any crystalline state. The crystalline form of compound (I) may be a crystalline salt or a crystalline free base (non-ionized form). Preferably, the crystalline form of compound (I) is a crystalline free base. Furthermore, compound (I) may form co-crystals.

[0039] When the crystalline form of Compound (I) is a crystalline salt, the molecular structure of Compound (I) above contains a protonated nitrogen atom.

[0040] The present invention is not limited to a single crystalline form of Compound (I). A solid substance may exist in more than one crystalline form. These alternative crystalline forms are called polymorphs. Each polymorph has 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 the differences in the crystal structure.

[0041] Polymorphic forms may have different mechanical properties, such as flowability and compressibility, which affect the technical properties of the compound. The storage stability and duration of the compound may also depend on the polymorph.

[0042] Polymorphs can be distinguished from one another in various ways. Polymorphs exhibit distinct spectroscopic properties, which can be determined, for example, by infrared spectroscopy, Raman spectroscopy, and 13 It can be determined using C-NMR spectroscopy. Given that each crystalline form refracts X-rays in a different direction, X-ray powder diffraction (XPD) can also be used to characterize polymorphs. Furthermore, thermal methods such as differential scanning calorimetry (DSC) and thermogravimetric analysis (TTA) can provide specific information about a particular polymorph.

[0043] As is well known in the field of powder X-ray diffraction, the relative peak heights in the powder X-ray diffraction spectrum can be used to describe different crystalline forms. Thus, in the "form 3" embodiment, the present invention provides a crystalline form of compound (I) that exhibits a reflection at 17.8±0.2°, specified as 2θ value, in the X-ray powder diffraction pattern, and the reflection at 17.8±0.2° is one of the four strongest reflections in the X-ray powder diffraction pattern. Preferably, the reflection at 17.8±0.2° is one of the three strongest reflections in the X-ray powder diffraction pattern, or the reflection at 17.8±0.2° is one of the two strongest reflections in the X-ray powder diffraction pattern. More preferably, the reflection at 17.8±0.2° is the strongest reflection in the X-ray powder diffraction pattern. Even more preferably, in the "Form 3" embodiment, the crystalline form of Compound (I) exhibits a reflection at 17.8±0.1°, specified as a 2θ value, in an X-ray powder diffraction pattern, wherein the reflection at 17.8±0.1° is one of the four strongest reflections in the X-ray powder diffraction pattern. Preferably, the reflection at 17.8±0.1° is one of the three strongest reflections in the X-ray powder diffraction pattern, or the reflection at 17.8±0.1° is one of the two strongest reflections in the X-ray powder diffraction pattern. More preferably, the reflection at 17.8±0.1° is the strongest reflection in the X-ray powder diffraction pattern.

[0044] The term "strongest reflection" describes the highest peak in an X-ray powder diffraction pattern. The height of a peak in an X-ray powder diffraction pattern is determined based on X-ray intensity (units of counts or counts / second). Thus, the strongest reflection is the reflection in an X-ray powder diffraction pattern that exhibits the highest X-ray intensity. For example, the strongest reflection in the X-ray diffraction pattern shown in Figure 6A is the reflection at 17.8±0.2°, specified as a 2θ value.

[0045] Unless expressly stated to the contrary, all X-ray powder diffraction patterns are determined at about 25° C. using copper K-alpha radiation.

[0046] Preferably, in the "Form 3" embodiment, the crystalline form of Compound (I) further exhibits one or more reflections in an X-ray powder diffraction pattern at one or more of the following angles, specified as 2θ values: 4.1±0.2°, 8.3±0.2°, 12.4±0.2°, 13.6±0.2°, 14.5±0.2°, 14.9±0.2°, 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°. Even more preferably, in the "Form 3" embodiment, the crystalline form of Compound (I) further exhibits one or more reflections in an X-ray powder diffraction pattern at one or more of the following angles, specified as 2θ values: 4.1±0.1°, 8.3±0.1°, 12.4±0.1°, 13.6±0.1°, 14.5±0.1°, 14.9±0.1°, 15.2±0.1°, 17.2±0.1°, 19.3±0.1°, 21.2±0.1°, 22.4±0.1°, 22.9±0.1° and 23.3±0.1°.

[0047] The position of the peaks in the powder X-ray diffraction spectrum is relatively unaffected by experimental details.Thus, the crystalline compound of the present invention can be characterized by a powder X-ray diffraction pattern with a certain peak position.Thus, in the embodiment of "Form 3", the crystalline form of Compound (I) is preferably characterized by reflections at 17.2±0.2°, 17.8±0.2°, 21.2±0.2° and 22.4±0.2°, specified as 2θ values, in the X-ray powder diffraction pattern. More preferably, in the "Form 3" embodiment, the crystalline form of Compound (I) is characterized in an X-ray powder diffraction pattern by reflections 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°, specified as 2θ values. Even more preferably, in the "Form 3" embodiment, the crystalline form of Compound (I) is characterized in an X-ray powder diffraction pattern by reflections at 17.2±0.1°, 17.8±0.1°, 21.2±0.1° and 22.4±0.1°, specified as 2θ values. Most preferably, in the "Form 3" embodiment, the crystalline form of Compound (I) is characterized in an X-ray powder diffraction pattern by reflections 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°, specified as 2θ values.

[0048] The crystalline form of the compound can be characterized by a differential scanning calorimetry (DSC) thermogram. Thus, the crystalline form of Compound (I) is preferably characterized by a DSC thermogram, in the "Form 3" embodiment, showing an endothermic flow onset at about 87°C, and / or a melting point of about 92.4°C, as seen in Figure 8C. Thus, preferably, in the "Form 3" embodiment, the crystalline form of Compound (I) has a melting point of 89°C to 96°C, as determined by DSC. Preferably, in the "Form 3" embodiment, the crystalline form of Compound (I) has a melting point of 90°C to 95°C, as determined by DSC. Even more preferably, in the "Form 3" embodiment, the crystalline form of Compound (I) has a melting point of 91°C to 94°C, as determined by DSC. Most preferably, in the "Form 3" embodiment, the crystalline form of Compound (I) has a melting point of 92°C to 93°C, as determined by DSC.

[0049] The crystalline form of a compound can be characterized by its hygroscopicity. The hygroscopicity of a product represents the increase or decrease in its water content as a function of relative humidity at a certain temperature. A substantially non-hygroscopic product exhibits no or only slight changes in its water content as a result of changes in relative humidity. In a strongly hygroscopic product, the water content can vary widely. Therefore, preferably, the crystalline form of compound (I) is substantially non-hygroscopic.

[0050] The crystalline form of a compound can be characterized by its thermogravimetric analysis trace.Thus, preferably, the crystalline form of compound (I) can be characterized by its thermogravimetric analysis trace.In one embodiment, the crystal of compound (I) is characterized by the thermogravimetric analysis trace shown in Figure 8A or Figure 8B.

[0051] The crystalline form of the compound can also be characterized by its dynamic vapor sorption (DVS) profile. Thus, the crystalline form of compound (I) can preferably be characterized by its DVS profile, as shown in Figures 9A and 9B. Preferably, the crystalline form of compound (I) has a reversible adsorption / desorption profile. Preferably, the DVS profile shows the substantially non-hygroscopic nature of the crystalline form.

[0052] A substantially non-hygroscopic material exhibits a water absorption of less than about 2% at about 95% relative humidity when measured at a temperature of about 25° C. Preferably, the water absorption is less than about 1% at about 95% relative humidity when measured at a temperature of about 25° C. The water absorption value is obtained by measuring the mass increase of the tested crystalline form at about 95% relative humidity and a temperature of about 25° C. compared to the initial mass.

[0053] Thus, the crystalline form of Compound (I) preferably absorbs 0% to 2% water at a relative humidity of about 95% at a temperature of about 25° C. Even more preferably, the crystalline form of Compound (I) absorbs 0% to 1.5% water at a relative humidity of about 95% at a temperature of about 25° C. Most preferably, the crystalline form of Compound (I) absorbs 0% to 1% water at a relative humidity of about 95% at a temperature of about 25° C.

[0054] Moreover, the crystalline forms of the present invention are preferably stable: for example, prolonged incubation (1 week) in ethyl acetate at about 25° C. produced a crystalline form of Compound (I) of good quality, as shown in FIG.

[0055] In the embodiment of "Form 2", the present invention provides a further crystalline form of Compound (I). This crystalline form (Form 2) exhibits a reflection at 16.9±0.2°, specified as a 2θ value, in the X-ray powder diffraction pattern, where the reflection at 16.9±0.2° is one of the four strongest reflections in the X-ray powder diffraction pattern. Preferably, the reflection at 16.9±0.2° is one of the three strongest reflections in the X-ray powder diffraction pattern, or the reflection at 16.9±0.2° is one of the two strongest reflections in the X-ray powder diffraction pattern. More preferably, the reflection at 16.9±0.2° is the strongest reflection in the X-ray powder diffraction pattern. Even more preferably, the crystalline form exhibits a reflection at 16.9±0.1°, specified as a 2θ value, in the X-ray powder diffraction pattern, where the reflection at 16.9±0.1° is one of the four strongest reflections in the X-ray powder diffraction pattern. Preferably, the reflection at 16.9±0.1° is one of the three strongest reflections in the X-ray powder diffraction pattern, or the reflection at 16.9±0.1° is one of the two strongest reflections in the X-ray powder diffraction pattern, more preferably the reflection at 16.9±0.1° is the strongest reflection in the X-ray powder diffraction pattern.

[0056] Preferably, the crystalline form of Compound (I), in the "Form 2" embodiment, exhibits one or more reflections in an X-ray powder diffraction pattern at one or more of the following angles, specified as 2θ values: 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°. Even more preferably, in the "Form 2" embodiment, the crystalline form of Compound (I) exhibits one or more reflections at one or more of the following angles, specified as 2θ values: 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°.

[0057] This crystalline form of Compound (I) may also be characterized in its "Form 2" embodiment by reflections in its X-ray powder diffraction pattern at 16.1±0.2°, 16.5±0.2°, 16.9±0.2°, 18.9±0.2°, and 23.1±0.2°, specified as 2θ values. Preferably, the crystalline form of Compound (I) may also be characterized in its "Form 2" embodiment by reflections in its X-ray powder diffraction pattern at 16.1±0.1°, 16.5±0.1°, 16.9±0.1°, 18.9±0.1°, and 23.1±0.1°, specified as 2θ values.

[0058] The crystalline form of Compound (I), in its "Form 2" embodiment, can be characterized by a DSC thermogram showing an endothermic flow onset at about 58°C and a melting point of about 70°C, as seen in Figure 3B. Thus, the crystalline form of Compound (I), in its "Form 2" embodiment, has a melting point of 67°C to 74°C. Preferably, the crystalline form of Compound (I), in its "Form 2" embodiment, has a melting point of 68°C to 73°C. Even more preferably, the crystalline form of Compound (I), in its "Form 2" embodiment, has a melting point of 69°C to 72°C. Most preferably, the crystalline form of Compound (I), in its "Form 2" embodiment, has a melting point of 69°C to 71°C.

[0059] The inventors of the present application have surprisingly discovered that the crystalline form of Compound (I) in its "Form 2" embodiment is particularly soluble in water. Thus, preferably, the crystalline form of Compound (I) in its "Form 2" embodiment has an aqueous solubility of about 75 mg / mL to about 85 mg / mL when measured at about 25° C. More preferably, the crystalline form in its "Form 2" embodiment has an aqueous solubility of about 78 mg / mL to about 82 mg / mL when measured at about 25° C. Most preferably, the crystalline form of Compound (I) in its "Form 2" embodiment has an aqueous solubility of about 80 mg / mL when measured at about 25° C.

[0060] Methods for measuring solubility, such as shake flask, ultrasonic irradiation, column elution and ultraviolet or visible spectroscopy, are known in the art. Unless expressly stated to the contrary, aqueous solubility is determined using the shake flask method and / or ultrasonic irradiation.

[0061] The present invention also provides pharmaceutical compositions comprising the crystalline forms of Compound (I) described herein.

[0062] Typically, the crystalline forms of Compound (I) are administered to a patient in the form of a pharmaceutical composition or formulation. Such pharmaceutical compositions may be administered to a patient by any acceptable route of administration, including, but not limited to, oral, topical (including transdermal) and parenteral modes.

[0063] The pharmaceutical composition of the present invention is usually prepared by a pharma-ceutically acceptable carrier and one or more optional ingredients. If necessary or desired, the obtained homogeneously blended mixture can then be shaped or filled into tablets, capsules, pills, canisters, cartridges, dispensers, etc., using conventional procedures and equipment.

[0064] When intended for oral administration in solid dosage form (i.e., as capsules, tablets, pills, etc.), the pharmaceutical composition of the present invention usually comprises a crystalline form of Compound (I) as an active ingredient. Preferably, the pharmaceutical composition of the present invention comprises a crystalline form of Compound (I) and does not comprise other ingredients. Preferably, the pharmaceutical composition herein is encapsulated. Preferably, the pharmaceutical composition herein is encapsulated without any other ingredients. The capsule can be a gelatin capsule or a hydroxypropylmethylcellulose (HPMC) capsule. Alternatively, the pharmaceutical composition of the present invention may comprise a crystalline form of Compound (I) as an active ingredient and one or more pharma- ceutically acceptable carriers. Suitable pharma- ceutically acceptable carriers will be known by those skilled in the art and include, for example, fats, water, saline, alcohol (e.g., ethanol), glycerol, polyols, 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 achieving a storage effect, salts for modifying osmotic pressure, coating agents or antioxidants, saccharides such as lactose or glucose; corn, wheat or rice starch; fatty acids such as stearic acid; inorganic salts such as 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, and other commonly used additives such as gelatin, talc, vegetable oils and gum arabic.

[0065] The pharmaceutical composition comprising the crystalline form of Compound (I) can also be administered transdermally or intramucosally 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, azacycloalkan-2-one, and formulated into a patch or similar delivery system.Additional excipients, including gelling agents, emulsifiers, and buffers, can also be used.

[0066] Injections for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Aqueous solvents include, for example, distilled water for injection and / or physiological saline. Examples of non-aqueous solvents include alcohols such as ethanol.

[0067] Preferably, the pharmaceutical composition comprises one or more additional pharmacologic active agents. This combination therapy comprises using the crystalline form of Compound (I) together with one or more additional pharmacologic active agents, either formulated together (e.g., packaged together in a single formulation) or formulated separately (e.g., packaged as individual unit dosage forms).

[0068] In healthy individuals, the core glycosphingolipid glucosylceramide is hydrolyzed in lysosomes by the acid glycosylceramidase (also called glucocerebrosidase or GBA1, EC 3.2.1.45, UniProt code: P04062). In addition, a non-lysosomal glycosylceramidase (GBA2, UniProt code: Q9HCG7) present in the cytoplasm can also process glucosylceramide. As a result, both GBA1 and GBA2 are involved in the psychopathological effects observed in several lysosomal storage disorders.

[0069] In patients with lysosomal storage disorders, defective glycosphingolipid biosynthesis or degradation occurs, resulting in abnormal levels of glucosylceramide and / or other glycosphingolipids.

[0070] Compound (I) has previously been shown to be effective in treating diseases associated with abnormal levels of cytosolic or lysosomal glucosylceramide and / or higher levels of glycosphingolipids (WO 2015 / 147639). Since the bioavailability of the crystalline form of Compound (I) is comparable to that of amorphous Compound (I), the crystalline form of Compound (I) is also effective in treating diseases associated with abnormal levels of cellular glucosylceramide and / or higher levels of glycosphingolipids. In particular, the crystalline form of Compound (I) is effective in treating diseases associated with abnormal levels of cytosolic or lysosomal glucosylceramide and / or higher levels of glycosphingolipids.

[0071] As shown in Example 4, a crystalline form of Compound (I) (Form 3) is effective in treating diseases associated with abnormal levels of cytosolic or lysosomal glucosylceramide and / or elevated levels of glycosphingolipids. Specifically, Form 3 was shown to ameliorate clinical symptoms in Niemann-Pick Type C mice.

[0072] Accordingly, the present invention provides a crystalline form of Compound (I) as described herein, or a pharmaceutical composition as described herein, for use in therapy.

[0073] The invention also provides a crystalline form as described herein, or a pharmaceutical composition as described herein, for use as a medicament.

[0074] Preferably, the present invention provides a crystalline form of compound (I) as described herein or a pharmaceutical composition as described herein for use in treating a disease involving abnormal levels of glucosylceramide and / or higher levels of glycosphingolipids.Preferably, the disease involving abnormal levels of glucosylceramide and / or higher levels of glycosphingolipids is a lysosomal storage disease such as Gaucher disease (types 1, 2 and 3), Fabry disease, GM1 gangliosidosis, GM2 gangliosidosis (such as Tay-Sachs disease, Sandhoff disease and AB variant), sialidosis, Niemann-Pick disease type C and action myoclonus renal failure syndrome, or a symptom of diseases collectively classified as metabolic syndrome, such as obesity, insulin resistance, hyperlipidemia, hypercholesterolemia, polycystic kidney disease, type II diabetes and chronic inflammation, or a neurodegenerative disorder such as Parkinson's disease or dementia with Lewy bodies or atherosclerosis. Even more preferably, the crystalline form of Compound (I), or a pharmaceutical composition comprising the crystalline form of Compound (I), is useful for treating GM1 gangliosidosis and / or GM2 gangliosidosis (such as Tay-Sachs disease, Sandhoff disease and AB variant).

[0075] Even more preferably, the present invention provides a crystalline form of Compound (I) as described herein, or a pharmaceutical composition as described herein, for use in the treatment of Niemann-Pick disease type C.

[0076] The present invention provides a crystalline form of Compound (I) as described herein, or a pharmaceutical composition as described herein, for use in the treatment of Sandhoff's disease.

[0077] The present invention provides a method of treating a disease involving abnormal levels of glucosylceramide and / or elevated levels of glycosphingolipids in a human or animal patient, comprising administering to a patient in need thereof a therapeutically effective amount of a crystalline form of Compound (I) described herein, or a pharmaceutical composition described herein.

[0078] The present invention provides a crystalline form of Compound (I) as described herein, or a pharmaceutical composition as described herein, for use in alleviating symptoms of a disease involving abnormal levels of glucosylceramide and / or elevated levels of glycosphingolipids.

[0079] Even more preferably, the present invention provides a crystalline form of Compound (I) as described herein, or a pharmaceutical composition as described herein, for use in the alleviation of symptoms of Niemann-Pick disease type C.

[0080] The present invention provides a crystalline form of Compound (I) as described herein, or a pharmaceutical composition as described herein, for use in the alleviation of symptoms of Sandhoff Disease.

[0081] The present invention provides a method for alleviating symptoms of a disease involving abnormal levels of glucosylceramide and / or elevated levels of glycosphingolipids in a human or animal patient, comprising the step of administering to a patient in need thereof a therapeutically effective amount of a crystalline form of Compound (I) described herein, or a pharmaceutical composition described herein.

[0082] Crystalline forms of Compound (I) (e.g., Form 3) have been shown to be effective in treating a variety of clinical indications, including: 1) disease-related weight loss; 2) Tremor; and / or 3) Ataxic gait can be used to improve

[0083] As shown in Example 4, the crystalline form of compound (I) (e.g., Form 3) effectively penetrates the brain. Therefore, the crystalline form of compound (I) (e.g., Form 3) can be used to treat or alleviate diseases or disease symptoms caused by the brain. For example, the crystalline form of compound (I) (e.g., Form 3) can be used to prevent or reduce cerebellar Purkinje cell loss. The crystalline form of compound (I) (e.g., Form 3) can be used to prevent or reduce neuronal death. The crystalline form of compound (I) (e.g., Form 3) can be used to prevent or reduce brain atrophy.

[0084] It is understood that the increased effectiveness of the crystalline forms of Compound (I) in treating diseases involving abnormal levels of glucosylceramide and / or higher levels of glycosphingolipids is due to the increased potency of the crystalline forms of Compound (I) against glucosylceramide synthase (GCS) and non-lysosomal glucosylcerebrosidase (GBA2).

[0085] The methods described herein may be in vitro methods or in vivo methods.

[0086] Administration may be by either oral administration, such as tablets, pills, capsules, granules, powders, solutions, or parenteral administration, such as injections, such as intra-articular, intravenous and intramuscular injections, suppositories, ophthalmic solutions, eye ointments, or agents for external use, such as transdermal liquid preparations, ointments, transdermal patches, transmucosal liquid preparations, transmucosal patches, inhalants, etc.

[0087] For oral administration, the daily dose is generally about 0.0001-10 mg, preferably 0.001-1 mg or 0.005-5 mg, and more preferably 0.01-0.5 mg per kg of body weight, administered in a single dose or in 2-4 separate doses. For example, for a 70 kg human patient, the optimal daily dose for oral administration is about 0.01-30 mg / day. For intravenous administration, the daily dose is suitably administered once a day or more than twice a day at about 0.00001-10 mg per kg of body weight. Furthermore, transmucosal agents are administered once a day or more than twice a day at a dose of about 0.0001-10 mg per kg of body weight. The dose is appropriately determined in response to each individual case by taking into account symptoms, age, sex, and the like.

[0088] The efficacy of the crystalline forms of Compound (I) in treating diseases involving abnormal levels of glucosylceramide and / or higher levels of glycosphingolipids (e.g., Niemann-Pick disease type C) is so high and the dosages required are relatively low that the crystalline forms of Compound (I) produce fewer side effects than known compounds for treating such diseases, such as Miglitol (dosage of 1200 mg / kg / day).

[0089] The present invention also provides a method for preparing the crystalline forms described herein, comprising contacting a sample of compound (I) with a solvent. Preferably, the solvent is selected from acetonitrile, ethyl acetate, isopropanol, anisole, water and tert-butyl methyl ether (TBME). Even more preferably, the solvent is ethyl acetate, acetonitrile or isopropanol. Preferably, prior to contacting the sample of compound (I) with the solvent, the sample of compound (I) is purified to remove borate esters. Preferably, the sample of compound (I) is purified using chromatography. Even more preferably, the sample of compound (I) is purified using a silica gel chromatography column. Additionally and / or alternatively, the sample of compound (I) is purified by distillation with methanol.

[0090] The crystalline form of Form 3 of Compound (I) can be obtained by any one of the following exemplary methods: 1) A mixture of about 74 mg of compound (I) and 2.0 ml of acetonitrile is stirred at a temperature ranging from 20° C. to 30° C., for example, about 25° C., for 3 days, and then filtered to obtain a crystalline form of compound (I); 2) A mixture of about 74 mg of compound (I) and 2.0 ml of anisole is stirred at a temperature ranging from 20° C. to 30° C., for example, about 25° C., for 3 days, and then filtered to obtain a crystalline form of compound (I); 3) Stirring a mixture of about 82 mg of compound (I) and 1.0 ml of ethyl acetate for 3 days at a temperature ranging from 20° C. to 30° C., for example, about 25° C., and then filtering to obtain a crystalline form of compound (I); 4) Stirring a mixture of about 82 mg of Compound (I) and 1.0 ml of isopropanol for 3 days at a temperature ranging from 20°C to 30°C, for example, about 25°C, and then filtering to obtain a crystalline form of Compound (I); 5) Stirring a mixture of about 45 mg of Compound (I) and 1.0 ml of water for 3 days at a temperature ranging from 20°C to 30°C, for example, about 25°C, and then filtering to obtain a crystalline form of Compound (I); 6) A mixture of about 100 mg of Compound (I) and 3.0 ml of TBME is stirred at a temperature ranging from 20° C. to 30° C., for example, about 25° C., for 3 days, and then filtered to obtain a crystalline form of Compound (I).

[0091] Preferably, prior to mixing of compound (I) with the solvent, the sample of compound (I) is purified to remove borate esters. Preferably, the sample of compound (I) is purified using chromatography. Even more preferably, the sample of compound (I) is purified using a silica gel chromatography column. Additionally and / or alternatively, the sample of compound (I) is purified by distillation with methanol.

[0092] Filtration methods are known to those skilled in the art and include, but are not limited to, filtration through filter paper and sintered glass filtration.

[0093] The method for producing Form 3 of Compound (I) described herein can be carried out on a large scale while maintaining similar ratios of reagents used. For example, the crystalline form of Form 3 of Compound (I) can be obtained by stirring a mixture of Compound (I) and ethyl acetate at a ratio between 0.05-1 g [Compound (I) to ethyl acetate] per mL for 3 days at a temperature ranging from 20° C. to 30° C., e.g., about 25° C., and then filtering to obtain the crystalline form of Compound (I).

[0094] The method for obtaining Form 3 of Compound (I) described herein is highly efficient. The yield of the method described herein is usually higher than 70% (weight ratio of the amount of Form 3 obtained to the amount of Compound (I) initially used). Preferably, the yield of the method described herein is higher than 75%.

[0095] Preferably, the Form 2 crystalline form is formed as an intermediate prior to the formation of the Form 3 crystalline form.

[0096] Preferably, the crystalline form of Form 2, characterized by reflections at 16.1±0.2°, 16.5±0.2°, 16.9±0.2°, 18.9±0.2° and 23.1±0.2°, specified as 2θ values ​​in an X-ray powder diffraction pattern, is formed as an intermediate prior to the formation of the crystalline form of Form 3, characterized by reflections at 17.2±0.2°, 17.8±0.2°, 21.2±0.2° and 22.4±0.2°, specified as 2θ values ​​in an X-ray powder diffraction pattern.

[0097] The present invention also provides crystalline forms of Compound (I) obtainable by carrying out the processes described herein.

[0098] The present invention also provides the use of the free base of Compound (I) to prepare a crystalline form of Compound (I).

[0099] The present invention also provides a method for preparing a crystalline form of Compound (I), comprising the step of crystallizing the free base of Compound (I).

[0100] The present invention also provides a crystalline form of Compound (I) obtained by a process for preparing a crystalline form of Compound (I), comprising the step of crystallizing the free base of Compound (I).

[0101] Among other advantages, the formation of crystalline forms of Compound (I) is believed to be useful for purifying Compound (I). For example, the crystalline forms of Compound (I) obtained by the methods described herein have a purity of greater than 90%, typically greater than 95%.

[0102] The foregoing detailed description has been presented by way of illustration and example and is not intended to limit the scope of the appended claims. Numerous variations in the presently preferred embodiments illustrated herein will be apparent to those of ordinary skill in the art and will remain within the scope of the appended claims and their equivalents.

[0103] The present invention is further disclosed in the following clauses: 1. Crystalline form of compound (I). [ka]

[0104] 2. A crystalline form of clause 1 which is a crystalline free base.

[0105] 3. A crystalline form of clause 1 or clause 2, which exhibits a reflection at 17.8±0.2°, specified as a 2θ value, in an X-ray powder diffraction pattern, wherein the reflection at 17.8±0.2° is one of the four strongest reflections in the X-ray powder diffraction pattern.

[0106] 4. The crystalline form of clause 3, further exhibiting one or more reflections in an X-ray powder diffraction pattern 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°, specified as 2θ values.

[0107] 5. A crystalline form of any one of clauses 1 to 4, characterized in an X-ray powder diffraction pattern by reflections at 17.2±0.2°, 17.8±0.2°, 21.2±0.2° and 22.4±0.2°, specified as 2θ values.

[0108] 6. The crystalline form of any one of clauses 1 to 5, having a melting point of 89°C to 96°C.

[0109] 7. The crystalline form of any one of clauses 1 to 6, having a melting point of 92°C to 93°C.

[0110] 8. The crystalline form of any one of clauses 1 to 7, which is substantially non-hygroscopic.

[0111] 9. A crystalline form of clause 1 or clause 2, which exhibits a reflection at 16.9±0.2°, specified as a 2θ value, in an X-ray powder diffraction pattern, wherein the reflection at 16.9±0.2° is one of the four strongest reflections in the X-ray powder diffraction pattern.

[0112] 10. The crystalline form of clause 9, further exhibiting one or more reflections in an X-ray powder diffraction pattern at one or more of the following angles, specified as 2θ values: 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°.

[0113] 11. A crystalline form of any one of clauses 1, 2, 9 or 10, characterized in an X-ray powder diffraction pattern by reflections at 16.1±0.2°, 16.5±0.2°, 16.9±0.2°, 18.9±0.2° and 23.1±0.2°, specified as 2θ values.

[0114] 12. The crystalline form of any one of clauses 9 to 11, having a melting point of 67°C to 74°C.

[0115] 13. The crystalline form of any one of clauses 9 to 12, having a melting point of 69°C to 71°C.

[0116] 14. The crystalline form of any one of clauses 9 to 13, having an aqueous solubility of 75 mg / mL to 85 mg / mL.

[0117] 15. A pharmaceutical composition comprising the crystalline form of any one of clauses 1 to 14.

[0118] 16. The pharmaceutical composition of clause 15, which is contained in a capsule.

[0119] 17. The pharmaceutical composition of clause 16, which is contained in a capsule without any other ingredients.

[0120] 18. A pharmaceutical composition according to clause 15 or clause 16, comprising at least one pharma- ceutically acceptable carrier.

[0121] 19. A crystalline form of any one of clauses 1 to 14, or a pharmaceutical composition of any one of clauses 15 to 18, for use in therapy.

[0122] 20. A crystalline form of any one of clauses 1 to 14, or a pharmaceutical composition of any one of clauses 15 to 18, for use as a medicament.

[0123] 21. The crystalline form of any one of clauses 1-14 or the pharmaceutical composition of any one of clauses 15-18 for use in treating a disease involving abnormal levels of glucosylceramide and / or elevated levels of glycosphingolipids.

[0124] 22. The crystalline form or pharmaceutical composition for use of clause 21, wherein the disease involving abnormal levels of glucosylceramide and / or higher levels of glycosphingolipids is a lysosomal storage disease such as Gaucher disease, Fabry disease, GM1 gangliosidosis, GM2 gangliosidosis (such as Tay-Sachs disease, Sandhoff disease and AB variants), sialidosis, Niemann-Pick disease type C and action myoclonus renal failure syndrome, or a symptom of diseases collectively classified as metabolic syndromes such as obesity, insulin resistance, hyperlipidemia, hypercholesterolemia, polycystic kidney disease, type II diabetes mellitus and chronic inflammation, or a neurodegenerative disorder such as Parkinson's disease or dementia with Lewy bodies or atherosclerosis.

[0125] 23. The crystalline form or pharmaceutical composition for use according to clause 21 or clause 22, wherein the disease is GM1 gangliosidosis or GM2 gangliosidosis (such as Tay-Sachs disease, Sandhoff disease or AB variant).

[0126] 24. A method of treating a disease involving abnormal levels of glucosylceramide and / or elevated levels of glycosphingolipids in a human or animal patient, comprising administering to a patient in need thereof a therapeutically effective amount of a crystalline form of any one of clauses 1-14, or a pharmaceutical composition of any one of clauses 15-18.

[0127] 25. A method for preparing the crystalline form of any one of clauses 1-8, comprising contacting a sample of compound (I) with a solvent.

[0128] 26. The process of clause 25, wherein the solvent is selected from acetonitrile, ethyl acetate, isopropanol, anisole, water and tert-butyl methyl ether (TBME).

[0129] 27. The method of clause 25 or clause 26, wherein the sample of compound (I) is purified prior to contacting the sample of compound (I) with the solvent.

[0130] 28. The method of clause 27, wherein the sample of compound (I) is purified using chromatography.

[0131] 29. The method of clause 28, wherein the sample of compound (I) is purified using silica gel column chromatography.

[0132] 30. The method of any one of clauses 27-29, wherein after purification, the sample of compound (I) is free of borate esters.

[0133] 31. The process of any one of clauses 25 to 30, wherein the crystalline form of any one of clauses 9 to 14 is formed as an intermediate prior to the formation of the crystalline form of any one of clauses 1 to 8.

[0134] 32. A crystalline form of compound (I) obtained by carrying out the process according to any one of clauses 25 to 31.

[0135] 33. Use of the free base of Compound (I) to prepare a crystalline form.

[0136] 34. A method for preparing a crystalline form of Compound (I), comprising crystallizing the free base of Compound (I).

[0137] 35. A crystalline form of compound (I) obtained by the process of clause 34.

[0138] 36. Follow these steps: I. loading compound (I) onto a purification column to produce a purified sample of compound (I); ii. adding a solvent to the purified sample of compound (I) to form a suspension of compound (I) in the solvent; iii. stirring the suspension of Compound (I) in the solvent to produce a crystalline form of Compound (I); and iv. isolating the crystalline form of Compound (I) to produce a pure sample of the crystalline form of Compound (I). A method for preparing a crystalline form of Compound (I), comprising:

[0139] 37. The method of claim 36, wherein the purified sample of compound (I) is free of borate esters.

[0140] 38. The process of clause 36 or 37, wherein the solvent is selected from acetonitrile, ethyl acetate, isopropanol, anisole, water and tert-butyl methyl ether (TBME).

[0141] 39. The method of any one of clauses 36 to 38, wherein step ii is carried out at a temperature between 25 and 35°C.

[0142] 40. The method of any one of clauses 36 to 39, wherein the stirring in step (iii) is carried out at a temperature between 20 and 35°C.

[0143] 41. The method of any one of clauses 36 to 40, wherein the stirring in step (iii) is carried out for at least 1 hour.

[0144] 42. The method of clause 41, wherein the stirring in step (iii) is carried out for at least 16 hours.

[0145] Experimental Section Differential scanning calorimetry (DSC) was performed using a TA Instruments Q2000 instrument (sealed aluminum sample pans or aluminum sample pans with pinholes in the lids, heating rate 20 K / min). The melting points are taken as the peak maximum.

[0146] Dynamic vapor sorption (DVS) measurements were performed using an SPS11-100n "Sorptions Pruefsystem" 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 aluminium sample pan. A humidity change rate of 5% per hour was used. The applied measurement program can be described as follows: Samples were placed in an aluminum or platinum holder on top of the 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→95% RH (5% / hour); 5 hours at 95% RH (4) 95→0% RH (5% / hr); 5 hours at 0% RH (5) 0→95% RH (5% / hour); 5 hours at 95% RH (6) 95→50% RH (5% / hour); 2 hours at 50% RH

[0147] Powder X-ray diffraction was carried out using a Stoe Stadi P diffractometer equipped with a Mythen 1K detector operating with Cu-Kα1 radiation. Measurements with this instrument were performed in transmission with a tube voltage of 40 kV and a tube power of 40 mA. A curved Ge monochromator allows the study with Cu-Kα1 radiation. The following parameters were set: step size of 0.02° 2θ, step time of 12 seconds, scan range of 1.5-50.5° 2θ, and 1° 2θ detection step (detector mode with step scan). For 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 preparations and measurements were carried out in ambient air atmosphere (approximately 25°C).

[0148] Approximate solubility was determined by slowly adding solvent to approximately 10 mg of compound followed by brief shaking and / or sonication. If the material does not dissolve upon addition of at least 10 ml total solvent, the solubility is reported as <1 mg / ml. Experiments were performed at approximately 25°C.

[0149] Thermogravimetric measurements (TG-FTIR) were carried out using a Netzsch Thermo-Microbalance TG209 coupled to a Bruker FTIR spectrometer Vector 22 (sample pan with pinhole, N2 atmosphere, heating rate 10°C / min).

[0150] [Example] The present invention is further illustrated by the following non-limiting examples.

[0151] Example 1 - Crystalline Salt Formation In an attempt to obtain a crystalline form of Compound (I), a high throughput salt screening program was conducted on Compound (I) using 16 different salt formers, identified in Table 1, under six different conditions.

[0152] Initial screening experiments were performed by adding a 0.05 M solution of compound (I) in acetone to each well of a quartz 96-well microtiter plate, followed by the addition of a salt former stock solution at a concentration of 0.1 M. The solvent was evaporated from each well under a stream of nitrogen at room temperature. The solid residue in the wells was examined by polarized optical microscopy. [Table 1]

[0153] Although the goal of this initial experiment was to obtain a 1:1 ratio of compound (I) to salt former, optical microscopy studies revealed some of the experimental conditions favoring the formation of crystalline forms. For example, compound (I) mixed with benzenesulfonic acid, hydrochloric acid, DL-mandelic acid, L-tartaric acid, phosphoric acid, and sulfuric acid formed crystalline residues.

[0154] In further screening experiments, six solvent systems were selected: acetone, acetonitrile, ethyl acetate, ethanol, isopropanol-water (3:1) mixture, and acetone / water (9:1) mixture. To equilibrate the slurry, 200 μL of solvent was added to the residue in each well. The so-prepared microtiter plate was agitated at 400 rpm for 1 day at room temperature (approximately 25° C.). The solvent was then evaporated under a stream of nitrogen, and the resulting solid residue was examined by polarized optical microscopy.

[0155] Based on optical microscopy studies, clues regarding potential salts were found for benzenesulfonic acid, gentisic acid, hydrochloric acid, L-lactic acid, D-mandelic acid, phosphoric acid, L-tartaric acid, and toluenesulfonic acid. Some of the most promising clues were selected for follow-up experiments at the 50-200 mg scale (scale-up experiments).

[0156] Scale-up experiments for all test conditions resulted in the formation of amorphous forms of Compound (I) or liquid crystalline salts of Compound (I). Microscopic examination revealed birefringence in many cases, while filtering of the resulting mixtures was not possible, and no solid material could be recovered in any of the experiments.

[0157] In summary, all experiments with salt formers did not produce crystalline forms of Compound (I).

[0158] Example 2 - Form 2 Crystalline Form Because the experiments in Example 1 did not yield useful crystalline material, the free base form of Compound (I) was investigated.

[0159] Compound (I) was purified using a silica gel column to remove the borate ester, followed by equilibration in acetonitrile for a short period (approximately 1-5 minutes). The solubility of purified Compound (I) in acetonitrile was determined to be between 5 mg / mL and 8 mg / mL. The solubility of purified Compound (I) in other solvents was also tested and is presented in Table 2. These values ​​were determined by adding small aliquots of solvent to approximately 10 mg of solid Compound (I) and shaking / sonicating for a short period at room temperature (approximately 25° C.). [Table 2]

[0160] The crystalline form of Form 2 of Compound (I) resulting from short-term (ca. 1-5 min) equilibration in acetonitrile was examined by polarized optical microscopy, powder X-ray diffraction (PXRD) experiments, TG-FTIR, differential scanning calorimetry (DSC) and dynamic vapor sorption (DVS).

[0161] The results of polarized optical microscopy studies are shown in Figure 1, while the PXRD pattern is illustrated in Figure 2. This Form 2 crystalline form of Compound (I) exhibits strongest 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°.

[0162] Thermogravimetric characterization results (TG-FTIR thermogram) of the crystalline form of Compound (I) Form 2 obtained from short equilibration in acetonitrile can be seen in FIG. 3A, while DSC results are shown in FIG. 3B. These results reveal that the sample contains about 0.5% water, which is released upon heating up to about 120° C. At higher temperatures, thermal decomposition is observed. DSC reveals two prominent endothermic events. There is 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.

[0163] Additionally, the behavior of the crystalline free base sample was studied under various water vapor pressures. At high relative humidity, the sample absorbed approximately 18% water. However, most of the absorbed water was released when the relative humidity returned to 50% RH. The results from the DVS measurements are presented in Figures 4A and 4B.

[0164] A summary of the characteristics of the crystalline form of Compound (I) obtained as part of Example 2 is presented in Table 3. [Table 3]

[0165] The resulting Form 2 crystalline form of Compound (I) had low stability and transitioned to a further Form 3 crystalline form of Compound (I) upon exposure to elevated temperatures (above about 30° C.) for about 1-5 minutes. Alternatively, upon equilibration in a solvent (e.g., acetone) at about 25° C. for more than 5 minutes, Form 2 transitioned to Form 3. Such further free base Form 3 of Compound (I) had good stability and a unique set of physicochemical properties.

[0166] Example 3 - Form 3 Independently, stable crystalline forms of Compound (I) were obtained from suspension equilibrium experiments of purified Compound (I) in various individual solvents, e.g., acetonitrile, ethyl acetate, isopropanol, anisole, water or TBME, respectively, at room temperature (about 25° C.).

[0167] In particular, the crystalline form of Form 3 of Compound (I) was obtained by the following experimental method: 1) About 74 mg of compound (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) About 74 mg of Compound (I) was added to 2.0 ml of anisole, and the suspension was stirred at room temperature (about 25° C.) for 3 days, and then filtered; 3) About 82 mg of Compound (I) was added to 1.0 ml of ethyl acetate, and the suspension was stirred at room temperature (about 25° C.) for 3 days, and then filtered; 4) About 82 mg of Compound (I) was added to 1.0 ml of isopropanol, and the suspension was stirred at room temperature (about 25° C.) for 3 days, and then filtered; 5) About 45 mg of Compound (I) was added to 1.0 ml of water, and the suspension was stirred at room temperature (about 25° C.) for 3 days, and then filtered; 6) Approximately 100 mg of Compound (I) was added to 3.0 ml of TBME, and the suspension was stirred at room temperature (about 25° C.) for 3 days, then filtered.

[0168] Compound (I) was purified using a silica gel column before the addition of solvent to remove the borate ester.

[0169] The obtained crystalline form of compound (I) was characterized by polarized optical microscopy, powder X-ray diffraction, TG-FTIR, DSC and DVS.

[0170] The results of polarized optical microscopy studies of selected solvents are presented in FIG.

[0171] The PXRD pattern of the crystalline form of Form 3 of Compound (I) obtained from suspension equilibration experiments with acetonitrile is shown in Figure 6A. The overlays of the PXRD patterns of the crystalline form of Form 3 of Compound (I) obtained from suspension equilibration experiments with other solvents are shown in Figure 6B. This crystalline form of Compound (I) exhibits the strongest 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 use of various solvents to obtain the crystalline forms of Compound (I).

[0172] For comparison, Figure 7 shows an overlay of the PXRD patterns of the crystalline forms obtained in Example 2 (Form 2) and this Example (Form 3). The two crystalline forms have different PXRD patterns.

[0173] The results of an exemplary TG-FTIR characterization experiment of the crystalline form of Form 3 of Compound (I) are shown in Figures 8A (ethyl acetate) and 8B (isopropanol).

[0174] Figure 8A reveals that a sample of crystalline form 3 of Compound (I), obtained by equilibration with ethyl acetate, contained about 0.7% ethyl acetate, even though it was dried under vacuum for several days at 40°C. Ethyl acetate is released between about 100°C and 200°C. At higher temperatures, thermal decomposition is observed.

[0175] A sample of Form 3 obtained from a suspension of Compound (I) in isopropanol (and dried in air at room temperature) surprisingly shows that the initial isopropanol content was less than 0.5% (see FIG. 8B).

[0176] An exemplary DSC measurement on a sample of crystalline form Form 3 of Compound (I) obtained by equilibration with ethyl acetate is illustrated in Figure 8C and shows a sharp endothermic melting peak at about 92°C with an enthalpy of about 103 J / g. Thus, the crystalline form of Form 3 of Compound (I) obtained as part of this Example has a higher melting point than the crystalline form from Example 2 (Form 2).

[0177] Furthermore, the behavior of a sample of the crystalline form of Form 3 of Compound (I) obtained by equilibration with ethyl acetate was studied under various water vapor pressures. At the highest relative humidity of 95%, the sample absorbed about 1.2% water, which was released when the relative humidity returned to 50% RH. The results from the DVS measurements are shown in Figures 9A and 9B. The amount of absorbed water is small and the water adsorption is reversible. That is, such a crystalline form of Form 3 of Compound (I) is non-hygroscopic or substantially non-hygroscopic.

[0178] A summary of the crystalline morphological characteristics of Form 3 of Compound (I) obtained as part of Example 3 is presented in Table 4. [Table 4]

[0179] Example 4 -Effect of administration of crystalline forms of Compound (I) to mice suffering from Niemann-Pick disease type C (NPC) In the examples, AZ-3102-00 is the name for Form 3 of Compound (I).

[0180] Purpose of this study The objective of this study was to evaluate treatment of juvenile NPC1 (NPC(- / -)) mice (P11-P70) with a predicted pharmacologically active dose of AZ-3102-00 using oral gavage and to assess PK and histological markers characterizing possible neuropathology.

[0181] Study design Of the 38 mice included in this study, there were 30 NPC(- / -) knockout (KO), 4 NPC(+ / -) heterozygous, and 4 NPC(+ / +) wild-type (WT, Balb / c) mice. NPC1(- / -) mice have premature truncation of the protein, lacking 11 of the 13 transmembrane domains, leaving the first two transmembrane domains intact. NPC1(- / -) mice homozygous for the recessive NIH allele of the Niemann-Pick C1 gene (Npc1m1N) show dual deficiency of sphingomyelinase and glucocerebrosidase activity (JAX#003092). Animals were bred on a BALB / c OlaHsd background.

[0182] Twenty-four NPC1(- / -) mice were treated by oral gavage with AZ-3102-00 from postnatal day 11 (P11) to postnatal day 70 (P70). Age-matched control mice included six NPC(- / -) and four NPC(+ / -) mice treated with vehicle, and four NPC(+ / +) mice received AZ-3102-00. After the last treatment on P70, NPC(- / -) mice (n=4 per time point) were euthanized by IP injection of 600 mg / kg pentobarbital at the following time points: 30 min, 1 h, 2 h, 4 h, 8 h, and 24 h. Control mice were also sacrificed after the last treatment (time not critical). Terminal blood was collected by cardiac puncture into EDTA-coated tubes. Plasma was collected by centrifugation (3000 xg for 10 min at room temperature) and 50 μL aliquots of plasma were transferred to 1.5 mL tubes, frozen on dry ice and stored at -80°C.

[0183] After transcardial perfusion, the brains were removed and bisected. The right hemibrain was post-fixed and embedded in cryomold for further immunohistological analysis. The left hemibrain was frozen on dry ice for further analysis.

[0184] Brains were cryosectioned (12 levels with 5 sections each), and 5 sections per animal were then used for quantitative immunofluorescence labeling of microglia (MAC1) and astrocytes (GFAP) in two brain regions.

[0185] Test system and test system validity Niemann-Pick disease type C (NPC) is an autosomal recessive neurodegenerative disorder associated with mutations in the NPC1 and NPC2 genes and is characterized by the accumulation of unesterified cholesterol and glycosphingolipids (GSLs). Nearly 95% of Niemann-Pick disease type C cases are caused by genetic mutations in the NPC1 gene, referred to as type C1, and 5% are caused by mutations in the NPC2 gene, referred to as type C2. The clinical signs of Niemann-Pick disease types C1 and C2 are similar because both individual genes are involved in the export of lipids, especially cholesterol, from late endosomes or lysosomes. The NPC1 gene codes for a protein located in the inner membrane of the cell and is involved in the trafficking of cholesterol and lipids within the cell. Deficiency of this protein results in abnormal accumulation of lipids and cholesterol in the cell membrane. The NPC2 gene codes for a protein that binds and transports cholesterol. Mice homozygous for the recessive NIH allele of the Niemann-Pick type C1 gene show dual deficiencies of sphingomyelinase and glucocerebrosidase activities. Mutant mice begin to lose weight and exhibit tremors and ataxic gait at about 7 weeks of age. Weight loss continues and the tremors and ataxia become more severe until death at about 12-14 weeks of age. The liver and spleen also become enlarged and Purkinje cells in the cerebellum are severely depleted. Some of these symptoms in mice are similar to those in human Niemann-Pick disease type C patients. [Table 5]

[0186] Compound preparation: At each administration occasion, the dose formulation was divided into aliquots if necessary. [Table 6]

[0187] The required amount of test article was weighed out and dissolved in Elix water (w / w) and the pH was adjusted to acidic pH. No further excipients were added.

[0188] No correction was made for the specific gravity of the test article or the purity / composition of the test article.

[0189] After preparation of each dose, a stock solution was frozen for analysis at the end of the study.

[0190] Stability studies previously performed in connection with the method development and validation study demonstrated that the following bracketed concentrations used in this study, when prepared and stored under the same conditions, are stable in vehicle for at least 24 hours at room temperature, protected from light, for at least 8 days when refrigerated (2-8°C), and for at least 3 weeks in the freezer (≦-15°C) at the bracketed concentrations used in this study (0.01-2 mg / mL):

[0191] animal control Animal Housing Facilities Animals were housed in individual ventilated cages on standard rodent flooring supplied by Rettenmaier. Each cage contained a maximum of five mice. The temperature of the vivarium was maintained at 20-24°C and the relative humidity was maintained at 45-65%. Animals were housed under a constant light cycle (12 h light / dark). Animals had free access to dry, pelleted standard rodent chow (Altromin) and regular tap water. Animals were fed wet food under the guidance of the attending veterinarian.

[0192] Identification Animals were assigned consecutive numbers by classical ear tagging.

[0193] Each cage was identified by a colored card displaying the study number, the animal's sex, the individual registration number (IRN), date of birth, and treatment group assignment. The genotype (transgenic or wild type) of each animal was determined by PCR specific for the transgenic construct. Each mouse was genotyped using DNA isolated from an ear biopsy prior to the start of the study.

[0194] Group assignment Only animals in apparently good health were included in the study. Randomization of group allocation was performed by cage. Animals were assigned to different starting groups (cohorts) including animals from all treatment groups. The number of animals in the starting groups was limited to ensure the same age and homogenous handling.

[0195] Health condition and cage side observations The health status of each individual animal was assessed prior to conduct of the study. During the study, observations were performed daily and any notable cage-side observations were recorded and immediately reported to the study manager and attending veterinarian who would make a decision regarding further action (e.g., euthanasia).

[0196] Body weight and health status were recorded daily for the first week and once a week thereafter.

[0197] Premature Termination and Humane Endpoints No animals had to be prematurely euthanized.

[0198] Materials and Methods: animal [Table A]

[0199] treatment Twenty-four NPC1(- / -) mice (groups D-I) were treated with AZ-3102-00 by oral gavage (10 mL / kg) from postnatal day 11 (P11) to postnatal day 70 (P70). Treatment began at 1.5 mg / kg from P11 to P25, then 3 mg / kg from P26 to P70. Control mice included six NPC1(- / -) and four NPC(+ / +) mice treated with vehicle, and four NPC(+ / +) mice received AZ-3102-00 (groups A-C).

[0200] After the last treatment on P70, NPC(- / -) mice (n=4 per time point; groups D-I) were euthanized by IP injection of 600 mg / kg pentobarbital at the following time points: 30 min, 1 h, 2 h, 4 h, 8 h, and 24 h. Control mice (groups A, B, and C) were also sacrificed after the last treatment (time not critical). [Table 7] Tissue sampling Mice were euthanized by intraperitoneal injection 30 minutes (Group D), 1 hour (+ / - 5 minutes, Group E), 2 hours (+ / - 5 minutes, Group F), 4 hours (+ / - 5 minutes, Group G), 8 hours (+ / - 5 minutes, Group H) and 24 hours (+ / - 5 minutes, Group I) (all times are significant) after the last treatment on P70.

[0201] Six NPC1(- / -) mice in group A, four NPC(+ / -) mice in treated group B, and four NPC(+ / +) mice in group C serving as controls were also euthanized on P70 (approximately 2 hours after the last treatment).

[0202] Mice were terminally euthanized by intraperitoneal injection of pentobarbital (600 mg / kg, dose 10 μL per gram of body weight).

[0203] Blood sampling The thorax was opened and blood was collected by cardiac puncture using a 23-gauge needle. The needle was removed and blood was transferred to a sample tube (MiniCollect® K2EDTA (potassium ethylenediaminetetraacetate)). The tube was inverted completely to promote homogenous distribution of EDTA and prevent clotting. Blood samples were centrifuged at 3000×g for 10 minutes at room temperature (22° C.). A 50 μL aliquot of plasma was transferred to a pre-labeled 1.5 ml LoBind Eppendorf tube, frozen on dry ice, and stored at −80° C.

[0204] Perfusion The animals were then perfused transcardially with 0.9% saline. For this purpose, a 23-gauge needle connected to a bottle containing 0.9% saline was inserted into the left ventricle. The thoracic aorta between the lungs and the liver was clamped with a hemostat to block blood flow from the heart to the abdomen but allow blood flow to the brain. The right ventricle was opened with scissors. The perfusion solution was maintained at a constant pressure of 100-120 mmHg by connecting the solution bottle to a manometer-controlled air compressor. Perfusion was continued until the skull surface turned pale and only the perfusion solution, instead of blood, was discharged from the right ventricle.

[0205] Brain sampling After perfusion, the skull was opened and the brain was carefully removed and divided into two halves on a cold surface. The left hemisphere was weighed, flash frozen on dry ice, and stored at -80°C. The right hemisphere was fixed by immersion in 4% paraformaldehyde in phosphate buffer (pH 7.4) for 2 h at room temperature.

[0206] histology tissue preparation Mouse right hemi-brains were fixed by immersion in freshly prepared 4% paraformaldehyde in PB (pH 7.4) for 2 hours at room temperature. After this, the hemisphere was transferred to 15% sucrose / PBS and stored at 4°C until submerged to ensure cryoprotection. Tissue blocks were then trimmed as required, transferred to cryomolds, embedded in OCT medium, frozen in isopentane cooled on dry ice, and stored in an ultra-low temperature freezer (set at a target temperature of -80°C).

[0207] sectioning Five consecutive cryosections were cut sagittally at 10 μm thickness on a Leica cryotome. The next 25 sections per level were discarded. This collection scheme was repeated for 12 levels and may be modified to collect from the correct level if the brain is smaller, for example due to age or younger genotype. In total, 12×5=60 sections were collected. Sectioning levels were selected according to the brain atlas of Paxinos and Franklin ("The Mouse Brain in Stereotaxic Coordinates", 2nd ed., 2001). Section collection started at a level approximately 0.2 mm lateral to the midline and extended throughout the hemisphere to ensure systematic random sampling through the target region (FIGS. 10 and 15). Sections were stored at −20° C. Nearly the entire brain was sectioned and once all sections had been collected, the remaining tissue block was discarded.

[0208] Immunofluorescence Exp3531 (Calbindin-D28k) For each incubation, a uniform systematic random set of 5 sections per mouse was selected (one section each from levels 2, 4, 6, 8, 10); for information on systematic random sampling please follow the following link: http: / / www.stereology.info / sampling / All steps were carried out in Dulbecco's phosphate-buffered saline pH 7.2-7.8 (PBS) at room temperature unless otherwise noted.

[0209] 1. Cryosections were air dried for 45 minutes and washed in PBS for 10 minutes. 2. Nonspecific binding sites were blocked with 10% normal donkey serum (Jackson Immuno Research) in 0.1% TritonX-100 / PBS for 60 minutes in a humid chamber. 3. Sections were washed in PBS 3 x 5 min each. 4. Sections were incubated with primary antibody in 1% normal donkey serum / PBS overnight at 4° C. in a humid chamber. Guinea pig polyclonal antibody to calbindin-D28k (Synaptic Systems, 214005) 1:1000 5. Sections were washed in PBS for 3 x 5 minutes each. 6. Sections were incubated with secondary antibody in 1% normal donkey serum / PBS for 60 minutes in a humid chamber (protected from light). Donkey anti-guinea pig (H+L), Cy3-conjugated (Jackson ImmunoResearch, 706-165-148), 1:500 7. Sections were washed 3 x 5 minutes each in PBS (protected from light). 8. Sections were incubated with DAPI working solution for 15 minutes (protected from light). 9. Sections were washed 2 x 5 minutes in PBS (protected from light). 10. Sections were washed in ddH2O (protected from light) for 5 minutes. 11. Sections were covered with Mowiol and coverslips (protected from light).

[0210] Imaging Whole-slide scans of stained sections were recorded on a Zeiss automated microscope AxioScan Z1 with high-aperture lenses, equipped with a Zeiss Axiocam506mono and Hitachi 3CCD HV-F202SCL camera and Zeiss ZEN 2.3 software.

[0211] quantitative Image analysis was performed with Image Pro10 (Media Cybernetics). First, the target area (cerebellum and hippocampus or corpus callosum and striatum) was identified by drawing a region of interest (ROI) on the image. Additional ROIs were added to exclude wrinkles, air bubbles, or any other artifacts that would interfere with the measurement. After this, immunofluorescence was quantitatively evaluated within the identified areas.

[0212] For quantification, we used background correction, if necessary, and detected immunoreactive objects by appropriate thresholding and morphological filtering (size, shape). We then quantified various object features: among them, the percentage of cumulative object area based on ROI size (immunoreactive area; this is the most comprehensive parameter that indicates whether there is a difference in immunoreactivity), the number of objects normalized to ROI size (object density), the average signal intensity of the identified objects (average intensity; this indicates whether there is a difference in the cellular expression level of the target protein), and the size of the object above the threshold. Once the parameters of the targeted objects are defined during the test, the quantitative image analysis is performed automatically, and the results are operator-independent and completely reproducible.

[0213] The raw data were structured and sorted in Excel and then transferred to GraphPad Prism for statistical analysis and preparation of graphs. The Prism graphs are part of the study report and a table containing the sorted raw data will be attached to the final report after a quality check has been performed.

[0214] Measurement of AZ-3102 and glucosylceramide in mouse plasma and brain tissue First, plasma samples were protein precipitated with a solution of acetonitrile / ultrapure water / methanol (90:5:5) containing 500 nM of the internal standard glucosylceramide C17:0 (GlcCer C17:0) and 0.1% formic acid. After mixing for 5 min at room temperature, samples were centrifuged for 5 min (13,000 rpm, 20°C) and 50 μL of the supernatant was transferred to a siliconized MTP 96-well plate.

[0215] Brain tissue was homogenized in a solution of ultrapure water:methanol (1:1) containing 0.1% formic acid (4 mL per gram of tissue) using a FastPrep24™ microtube homogenizer. For protein precipitation, the tissue homogenate was then mixed with a solution of acetonitrile / ultrapure water / methanol (90:5:5) containing 500 nM GlcCer C17:0 and 0.1% formic acid. After incubation at room temperature for 5 min, the samples were centrifuged (13000 rpm, 20° C.) for 5 min and 50 μL of the supernatant was transferred to a siliconized MTP 96-well plate.

[0216] Measurement of AZ-3102 For protein precipitation, plasma samples were first mixed with a solution of 50 ng / mL AZ-3101 (internal standard) in acetonitrile. After incubation for 5 min at room temperature, the samples were centrifuged for 5 min (13000 rpm, 4°C) and the supernatant was diluted 10-fold with ultrapure water containing 0.1% formic acid.

[0217] Brain tissue was homogenized in a solution of ultrapure water:methanol (1:1) containing 0.1% formic acid (4 mL per gram of tissue) using a FastPrep 24™ microtube homogenizer (MP Biomedicals, USA). To precipitate proteins, the brain homogenate was mixed with a solution of acetonitrile containing 10 ng / mL AZ-3101 (internal standard). After incubation at room temperature for 5 min, the sample was centrifuged (13000 rpm, 4° C.) for 5 min and the supernatant was diluted 10-fold with ultrapure water containing 0.1% formic acid.

[0218] The diluted plasma and brain tissue supernatants were injected into an Agilent LC system (Agilent, USA) by an automated sample injector (SIL-30, Shimadzu, USA). Analytes were separated by liquid chromatography using a linear gradient of mobile phase B at a flow rate of 0.800 mL / min on a reversed-phase XBridge BEH C8 column (2.1×50 mm, 2.5 μm particle size; Waters, USA) maintained at a temperature of 40° C. Mobile phase A consisted of ultrapure water with 0.1% formic acid. Mobile phase B was acetonitrile with 0.1% formic acid. Acquisition was achieved in positive ionization mode using an API5500 triple quadrupole mass spectrometer (AB Sciex, USA) equipped with a Turbo ion spray interface. Data were calibrated and quantified using the Analyst™ data system (AB Sciex, version 1.6.3). The LLOQ for AZ-3102 was 0.2 ng / mL in plasma samples and 2 ng / g in brain, respectively.

[0219] Measurement of glucosylceramide First, plasma samples were protein precipitated with a solution of acetonitrile / ultrapure water / methanol (90:5:5) containing 500 nM of the internal standard glucosylceramide C17:0 (GlcCer C17:0) and 0.1% formic acid. After mixing for 5 min at room temperature, samples were centrifuged for 5 min (13,000 rpm, 20°C) and 50 μL of the supernatant was transferred to a siliconized MTP 96-well plate.

[0220] Brain tissue was homogenized in a solution of ultrapure water:methanol (1:1) containing 0.1% formic acid (4 mL per gram of tissue) using a FastPrep 24™ microtube homogenizer. For protein precipitation, tissue homogenates were then mixed with a solution of acetonitrile:ultrapure water:methanol (90:5:5) containing 500 nM GlcCer C17:0 and 0.1% formic acid. After incubation at room temperature for 5 min, samples were centrifuged (13000 rpm, 20° C.) for 5 min and 50 μL of supernatant was transferred to a siliconized MTP 96-well plate.

[0221] The concentrations of glucosylceramide C16:0 (GlcCer C16:0), glucosylceramide C18:0 (GlcCer C18:0) and glucosylceramide C24:1 (GlcCer C24:1) in brain samples were quantified by HPLC-MS / MS detection in multiple reaction monitoring mode (MRM). To distinguish between galactosyl-ceramide and glucosyl-ceramide isomers, the supernatant was analyzed by HPLC-MS / MS using a HALO HILIC column (150 × 4.6 mm, 2.7 μm) from Advanced Materials Technology. MS / MS acquisition was achieved in positive ionization mode using an API4000 triple quadrupole equipped with a Turbo ion spray interface (Applied Biosystems, USA). Analysis of GlcCer C16:0 and GlcCer C18:0 was performed using a gradient with mobile phase A: 94.5% acetonitrile, 2.5% methanol, 5 mM ammonium acetate in 2.5% ultrapure water and 0.5% formic acid, and mobile phase B: ultrapure water and 0.1% formic acid. The LLOQs in brain samples for GlcCer 16:0, GlcCer 18:0 and GlcCer 24:1 were 25, 1,250 and 381.5 pmol / g tissue, respectively.

[0222] statistics Statistical analysis was performed with GraphPad Prism 9. Data are expressed as mean ± standard error of the mean (SEM) or mean + standard error of the mean.

[0223] In vivo: Differences between groups were tested with two-way ANOVA for repeated measures followed by Bonferroni or Dunnett post-hoc analysis.

[0224] Histology: Due to the small n, the distribution of the data could not be tested and was therefore assumed to be normal. Differences between groups were tested by one-way ANOVA followed by Dunnett's post-hoc test analysis. Group A (NPC- / -, vehicle-treated) was used as the reference group for pairwise comparisons.

[0225] result body weight Figure 11 shows the percentage change in body weight between PND11 and week 9. The graph represents the evolution of body weight [g] per group, measured daily during the first week of treatment, and weekly thereafter.

[0226] The results show that the general health of the animals, as indicated by the mean weight gain, was not disturbed by AZ-3102, and that NPC(- / -) mice of both sexes treated with AZ-3102 gained more weight than NPC(- / -) untreated animals. [Table 8]

[0227] Glucosylceramide levels FIG. 12 shows glucosylceramide C16:0 and C18:0 levels after repeated oral administration from PND11 to 70.

[0228] Mice treated with AZ-3102 show increased levels of glucosylceramides C16:0 and C18:0 in the brain.

[0229] Clinical Signs Clinical scores and humane endpoint definitions Clinical signs were monitored daily (following the template in Table 9 below) starting on P45 until the end of the study. The parameters "weight loss", "general health", "clinical findings" and "cell line specific findings" were recorded and scored according to a score scale. The sum of this score was used for the evaluation. [Table 9] TIFF2024054192000016.tif211149

[0230] Tremor score [Table 10]

[0231] Figure 13 shows the total clinical sign scores from PND56 to 70 across all regions and a summary by treatment group for NPC(- / -) vehicle and AZ-3102 treated mice, respectively. Left: Total scores per animal and treatment group visualized as a gradient, with low scores in green and high scores in red. Right: Summary of scores that reached threshold numbers. Threshold scores greater than 7 rarely occurred for treated groups. However, the untreated group had a higher number of total scores during the course of the study.

[0232] The results show that NPC(- / -) vehicle-treated mice had worse overall clinical signs (higher scores) than NPC(- / -) AZ-3102-treated animals. This is further shown in Figure 13: on the right, for example, scores higher than 8 were observed more frequently in the NPC(- / -) vehicle-treated group than in the NPC(- / -) AZ-3102-treated animals. When examining these scores, we found that the onset, duration and intensity of tremor were also reduced by treatment with AZ-3102 (Figure 14). While high levels of tremor were observed in all cases except one NPC(- / -) vehicle-treated animal, AZ-3102 essentially eliminated these high levels of tremor in all but one of the 24 tested animals.

[0233] Figure 14 shows the tremor scores in NPC(- / -) vehicle and AZ-3102 treated mice at postnatal days 56-70. Pink (=light grey) bars represent the onset and duration of high levels of tremor (score 5), whereas green (=dark grey) bars represent the onset and duration of slight or moderate tremor. Except for one mouse in the untreated group, all other mice showed high levels of tremor (4 out of 5 mice). In contrast, only one mouse in the AZ-3102 treated group had high levels of tremor (1 out of 24 mice). Possible tremor scores: 0: no tremor; 1: slight incoordination, slight to moderate tremor; 5: high level of tremor, uncoordinated movement; 10: reduced righting reflex (no animals reached this score).

[0234] Histological results Defining the target region Target regions were manually outlined by defining regions of interest (ROIs) for subsequent quantitative analysis of fluorescent labels.

[0235] Quantitative analysis readouts The table shown below characterizes the four standard readouts.

[0236] Area size [mm2 ]: These data show the average area per brain section that covers the target region. This information is important to ensure adequate sampling. This information also helps identify brain atrophy, which is part of the phenotype in some animal models.

[0237] Immunoreactive area [%]: the percentage of the ROI that spans immunoreactive objects above the threshold (e.g.: cell bodies, neurites, plaques): this is the most comprehensive parameter that indicates whether there is an overall difference in immunoreactivity.

[0238] Density of the object [mm 2 Number of objects per threshold: the number of immunoreactive objects above threshold, normalized to the size of the target area; this is particularly useful for detecting changes in neuronal density.

[0239] Object intensity [au]: average brightness of pixels of immunoreactive objects above threshold; this indicates whether there are differences in cellular expression levels of the target protein.

[0240] Object size [μm 2 ]: size of immunoreactive object above threshold; this is useful for detecting differences in microglial activation or plaque growth.

[0241] Calbindin-D28k Calbindin-D28k immunofluorescence was detected by guinea pig polyclonal antibody, and the signal was quantified in the cerebellum and hippocampal formation. NCP (- / -) mice show a strong reduction in calbindin-D28k labeling compared to NCP (+ / -) and NPC (+ / +) mice. Treatment with the test article significantly increased calbindin-D28k in the cerebellum in all readouts. All effects are region-specific, since no significant group differences were detected in the hippocampus.

[0242] FIG. 16 shows the results of brain immunohistochemistry: Calbindin-D28k labeling in NCP(- / -) and NPC(+ / -) vehicle-treated mice compared to NPC(+ / +, wild-type mice) and NPC(- / -) treated mice. Graphs represent the mean immunofluorescence signal measured within the ROI in five brain sections per mouse (n=2-4 per group). Data were analyzed by one-way ANOVA and Dunnett's post-hoc test. Bars represent group means+SEM. Bars represent group means+SEM. * Adj. P-value: <0.001.

[0243] Using immunohistological techniques using the calbindin-D28k marker for Purkinje cells, we found that AZ-3102 treatment significantly limited cerebellar Purkinje cell loss compared to vehicle-treated NPC(- / -) mice (Figure 16).

[0244] conclusion AZ-3102 is a novel oral small molecule developed for a variety of lysosomal storage disorders. The unique mechanism of action of AZ-3102 lies in its high potency against glucosylceramide synthase (GCS), non-lysosomal glucosylcerebrosidase (GBA2), and its brain penetrating properties. AZ-3102 has been investigated in a mouse model of Niemann-Pick disease type C [1, 2], where mice homozygous for the recessive NIH allele of the NPC gene begin to lose weight and exhibit tremors and ataxic gait at approximately 7 weeks of age [3]. Disease severity is associated with significant cerebellar Purkinje cell loss, with worsening clinical signs until a humane endpoint is reached (usually at 12–14 weeks of age) [3]. In this study, we investigated daily oral administration of AZ-3102 to NPC(- / -), NPC(- / +) and WT (NPC(+ / +); Balb / c) mice from postnatal day (PND) 11 to 70 to assess its pharmacokinetic (PK) properties, ability to modulate glucosylceramide (GlcCer C16:0, GlcCer C18:0), and ability to ameliorate clinical signs. In addition, we also tested the effect of treatment on immunohistochemical markers for Purkinje cells consistent with neuropathology. After repeated daily oral administration of AZ-3102 from PND 11 to 70, the general health of the animals was not disturbed by AZ-3102, as indicated by the mean weight gain, and AZ-3102-treated NPC(- / -) mice of both sexes gained more weight than NPC(- / -) untreated animals (Figure 11). AZ-3102 demonstrated high brain:plasma exposure (Table 8) and consistent with AZ-3102 target engagement, GlcCer species measured from whole brain homogenates were increased 2-fold for GlcCer 16:0 and nearly 9-fold for GlcCer 18:0 compared to vehicle-treated animals. These two GlcCer species are likely representative of other GlcCer species that may also respond to AZ-3102. To assess whether AZ-3102 reaches sufficient brain concentrations to affect general health and more specific signs of neuropathology, various clinical signs were measured (Table 9). When summed over the observation period, these scores provide a picture of health at PND56-70.Figure 13 is a graphical summary of each animal per cohort observed over time. In this graph, NPC(- / -) vehicle-treated mice had worse overall clinical signs (higher scores) than NPC(- / -) AZ-3102-treated animals. This is further quantified in (Figure 13: right), where, for example, scores higher than 8 were observed more frequently in NPC(- / -) vehicle-treated groups than in NPC(- / -) AZ-3102-treated animals. In examining these scores, we found that the onset, duration and intensity of tremor were also reduced by treatment with AZ-3102 (Figure 14). High levels of tremor were observed in all cases except one NPC(- / -) vehicle-treated animal, while AZ-3102 virtually eliminated these high levels of tremor in all but one of the 24 tested animals. Using immunohistochemical techniques, using the calbindin-D28k marker for Purkinje cells, we found that AZ-3102 treatment significantly limited cerebellar Purkinje cell loss compared to vehicle-treated NP-C(- / -) mice (Figure 16). These findings have relevance to Niemann-Pick disease type C in humans, since neuronal death, particularly Purkinje cell and brain atrophy, are hallmarks of this disease [4]. Since cerebellar function is important for movement, the improvement in clinical signs and the significant reduction in high levels of tremor are likely the result of survival of cerebellar Purkinje cells.

[0245] In summary, AZ-3102, an orally available and highly potent (nM) inhibitor of both GCS and GbA2, was able to stimulate the brains of NPC(- / -) and wild-type animals. Moreover, AZ-3102 inhibited these enzymes in the brain, ameliorating clinical signs, significantly reducing high levels of tremor, and limiting the loss of Purkinje cells in the cerebellum, as evidenced by modulation of GlcCer species, compared to vehicle-treated animals.

[0246] 1. Loftus, SK et al., Murine model of Niemann-Pick C disease: mutation in a cholesterol homeostasis gene. Science, 1997, Vol. 277(No. 5323): pp. 232-5. 2. Zervas, M., K. Dobrenis and SU Walkley, Neurons in Niemann-Pick disease type C accumulate gangliosides as well as unesterified cholesterol and undergo dendritic and axonal alterations. J Neuropathol Exp Neurol, 2001, 60(1): 49-64. 3. Santiago-Mujica, E. et al., Hepatic and neural phenotype of NPC1- / - mice.Heliyon.Heliyon, 2019, Volume 5 (Issue 3). 4. Vanier, M.T., Niemann-Pick disease type C. Orphanet Journal of Rare Diseases, 2010, 5(1):16.

[0247] Example 5 - Effect of administration of a crystalline form of Compound (I) to mice suffering from Sandhoff disease (disruption of the mouse Hexb gene by mutation [Hexb(- / -)]) Mice suffering from Sandhoff disease were given a therapeutically effective dose of AZ-3102, and initial data indicate that it improved the clinical signs of affected animals, similar to the treatment of Niemann-Pick type C.

Claims

1. Crystalline form of compound (I). 【Chemistry 1】

2. 2. The crystalline form of claim 1, which exhibits a reflection at 17.8±0.2°, specified as a 2θ value, in an X-ray powder diffraction pattern, and wherein the reflection at 17.8±0.2° is one of the four strongest reflections in the X-ray powder diffraction pattern.

3. 3. The crystalline form of claim 2, further exhibiting one or more reflections in an X-ray powder diffraction pattern at one or more of the following angles, specified as 2θ values: 4.1±0.2°, 8.3±0.2°, 12.4±0.2°, 13.6±0.2°, 14.5±0.2°, 14.9±0.2°, 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°.

4. A pharmaceutical composition comprising the crystalline form of any one of claims 1 to 3.

5. 5. A pharmaceutical composition according to claim 4 for use in therapy.

6. 5. A pharmaceutical composition according to claim 4 for use in the treatment of diseases involving abnormal levels of glucosylceramide and / or elevated levels of glycosphingolipids.

7. A method for preparing the crystalline form of any one of claims 1 to 3, comprising contacting a sample of compound (I) with a solvent, optionally (a) the solvent is selected from acetonitrile, ethyl acetate, isopropanol, anisole, water and tert-butyl methyl ether (TBME); and / or (b) prior to contacting the sample of compound (I) with the solvent, the sample of compound (I) is purified, optionally, the sample of compound (I) is purified using chromatography; method.

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