EA045552B1 - CRYSTAL FORMS OF PHARMACEUTICAL COMPOUND - Google Patents

Description

Field of technology to which the present invention relates

The present invention relates to the crystalline form of compound (I),

The present invention also relates to a method for producing a crystalline form of the compound (I), and to pharmaceutical compositions containing a crystalline form of the compound (I). In addition, the present invention relates to methods of using this crystalline form of compound (I) as a drug and to methods of using it in the treatment of diseases associated with abnormal levels of glucosylceramide and/or elevated levels of glycosphingolipids.

BACKGROUND OF THE INVENTION

The crystalline form of a compound may be important when the compound is used for pharmaceutical purposes. This is because morphology, particle size, polymorphism, solvation or hydration of the crystalline form of a compound can affect the filtration, flowability, tabletting, solubility and bioavailability of a pharmaceutical agent.

Deoxynojirimycin derivatives represent an important class of compounds in medicinal chemistry and drug development. N-(hydroxyethyl)-deoxynojirimycin is marketed as miglitol and is used as an antidiabetic agent for the treatment of type 2 diabetes. Miglitol also acts as a broad-spectrum inhibitor of several intestinal glycosidases (maltase, sucrase and lactase) (Hillebrand et al, Diabetes , 1986, Vol. 35, A93-A93; Scott and Spencer, Drugs, 2000, Vol. 59, 521-549).

N-butyldeoxynojirimycin (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 clinically to treat patients with the lysosomal storage disease Gaucher disease (Platt et al. , J. Biol. Chem., 1994, Vol. 269, 8362-8365; Cox et al., Lancet, 2000, Vol. 355, 1481-1485) and Niemann-Pick disease type C (Pineda et al. Orphanet J. Rare Dis., 2018, Vol. 13, 140).

WO 2015/147639 A1 describes new deoxynojirimycin derivatives, including compound (I),

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 glucosylceramidase (GBA2, UniProt code: Q9HCG7).

Therapeutic compounds useful for treating abnormal glucosylceramide levels and/or elevated glycosphingolipid levels are often administered in tablet form. In the preparation of pharmaceutical compositions and preparations for use in tablet form, it is highly desirable to have a crystalline form of the therapeutic compound that has a low level of hygroscopicity and/or a low level of absorption of moisture from the air resulting in spreading, allowing the compound to be compressed into the desired shape with the appropriate size.

In addition, the relatively high melting point (typically above about 80° C.) of the therapeutic compound contributes to its resistance to degradation, thereby facilitating storage and increasing the shelf life of the therapeutic compound, which is desirable for any pharmaceutical agent.

It is also particularly advantageous for the therapeutic compound to be non-hygroscopic or substantially non-hygroscopic from the point of view of handling, manufacturing and storage of the pharmaceutical. If a pharmaceutical product exhibits hygroscopic properties, many problems may arise, such as:

the difficulty of crushing the material into small particles or powder when crushing it;

the presence of unwanted moisture, which promotes the occurrence of desired reactions, and leads to the formation of undesirable end products, which leads to poor quality of the product and a shortening of its shelf life;

formation of soft tablets during manufacturing or moisture ingress into the packaging;

- 1 045552 powder sticking to conveyor equipment can affect the filling process.

Thus, it is desirable to have a crystalline form of a therapeutic compound that is non-hygroscopic or substantially non-hygroscopic.

No crystalline forms of compound (I) have been previously reported. Accordingly, there is a need for stable and/or non-deliquescent crystalline forms of compound (I), which are preferably substantially non-hygroscopic and/or which have relatively high melting points.

Brief Disclosure of the Present Invention

In a first aspect, the present invention relates to a crystalline form of compound (I)

Each aspect or embodiment of the present invention, as defined herein, may be combined with any other aspect(s) or embodiment(s) unless expressly indicated otherwise. In particular, any feature indicated as preferred or advantageous may be combined with any other feature or features indicated as preferred or advantageous.

In another aspect, the present invention relates to a pharmaceutical composition containing a crystalline form of compound (I) described herein.

In another aspect, the present invention relates to a crystalline form of compound (I) described herein, or a pharmaceutical composition described herein, for use in therapy.

In yet another aspect, the present invention relates to a crystalline form of compound (I) described herein or a pharmaceutical composition described herein for use as a drug.

In another aspect, the present invention relates to a crystalline form of compound (I) described herein, or a pharmaceutical composition described herein, for use in the treatment of a disease associated with abnormal levels of glucosylceramide and/or elevated levels of glycosphingolipids.

In yet another aspect, the present invention relates to a crystalline form of compound (I) described herein or a pharmaceutical composition described herein for use in the treatment of Niemann-Pick disease type C.

In another aspect, the present invention relates to a method of treating a disease associated with abnormal levels of glucosylceramide and/or elevated levels of glycosphingolipids in humans or animals, wherein the method comprises administering to a patient in need thereof a therapeutically effective amount of a crystalline form of compound (I) described in herein, or a pharmaceutical composition described herein.

In yet another aspect, the present invention relates to a method of treating Niemann-Pick disease type C in humans or animals, wherein the method comprises 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 in this document.

In yet another aspect, the present invention relates to a method for preparing a crystalline form of compound (I) described herein, wherein the method comprises contacting a sample of compound (I) with a solvent.

In yet another aspect, the present invention relates to a crystalline form of compound (I) obtained by carrying out the method described herein.

In yet another aspect, the present invention relates to the use of the free base of compound (I) to obtain a crystalline form of compound (I).

In yet another aspect, the present invention relates to a method for preparing a crystalline form of compound (I), wherein the method involves crystallizing the free base of compound (I).

In yet another aspect, the present invention relates to a crystalline form of compound (I) obtained by a process for preparing a crystalline form of compound (I), wherein the method involves crystallizing the free base of compound (I).

Other preferred embodiments of the invention are described herein, and in particular in the examples.

The inventors of the present invention have unexpectedly obtained a crystalline form of compound (I) which is stable and non-deliquescent. The present invention has provided additional beneficial properties of this compound, such as a significant lack of hygroscopicity and a relatively high melting point of the crystalline form of compound (I).

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The inventors of the present invention unexpectedly obtained a crystalline form of compound (I), which further showed good solubility in water. These properties make the crystalline forms of the invention particularly suitable for use in pharmaceutical compositions.

Crystallization of therapeutic compounds often involves the use of various salts. In general, salts readily crystallize, and the resulting material facilitates subsequent crystallization of therapeutic compounds. For this reason, the use of salt is often the preferred method for crystallizing a therapeutic compound. It is therefore very surprising that the inventors of the present invention were able to obtain the free base crystalline form of compound (I).

Since the free base crystalline form of any therapeutic compound does not require the presence of a counterion, the concentration of the therapeutic compound in the free base crystalline form powder is typically higher than in the corresponding salt form, which is very beneficial because it reduces the cost of drug production.

Without wishing to be bound by any theory, it is believed that the crystalline forms of the invention exhibit the beneficial effects described above due to their crystalline structure.

Brief description of the figures

These and other aspects of the present invention will be further described with reference to the accompanying drawings, which show the following:

In fig. 1 shows illustrative polarized light microscopy images of a crystalline form of compound (I), designated Form 2, prepared by equilibration with acetonitrile; left: in the form of dry powder, right: in paraffin oil.

In fig. 2 shows an X-ray powder diffraction pattern of Form 2 obtained by equilibration with acetonitrile.

In fig. 3A shows a TG-FTIR thermogram of Form 2 obtained by equilibration with acetonitrile.

In fig. 3B shows a differential scanning calorimetry (DSC) thermogram of Form 2 obtained by equilibration with acetonitrile.

In fig. 4A shows the dynamic vapor sorption (DVA) isotherm of Form 2 obtained by equilibration with acetonitrile: change in water content (red curve) and relative humidity (blue curve) as a function of time.

In fig. 4B shows the isotherm of EAF Form 2 obtained by equilibration with acetonitrile: change in water content as a function of relative humidity.

In fig. 5 shows illustrative polarized light microscopy images of a crystalline form of compound (I), designated Form 3, prepared by equilibration with acetonitrile, anisole, or ethyl acetate, respectively.

In fig. 6A shows an X-ray powder diffraction pattern of Form 3 obtained by equilibration with acetonitrile.

In fig. 6B shows an overlay of X-ray powder diffraction patterns of Forms 3 obtained by equilibration with TBME, water, isopropanol, ethyl acetate, acetonitrile, or anisole, respectively.

In fig. Figure 7 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).

In fig. 8A shows a TG-FTIR thermogram of Form 3 obtained by equilibration with ethyl acetate.

In fig. 8B shows a TG-FTIR thermogram of Form 3 obtained by equilibration with isopropanol.

In fig. 8C shows a DSC thermogram of Form 3 obtained by equilibration with ethyl acetate.

In fig. 9A shows the isotherm of Form 3 EAF obtained by equilibration with ethyl acetate: change in water content (red curve) and relative humidity (blue curve) as a function of time.

In fig. 9B shows the isotherm of EAF Form 3 prepared by equilibration with ethyl acetate: change in water content as a function of relative humidity.

In fig. Figure 10 shows approximate mediosagittal levels obtained according to the sectioning protocol according to Paxinos & Franklin The Mouse Brain Atlas, 2nd edition, with stereotactic coordinates indicated.

In fig. 11 shows the percentage change in body weight of mice between postnatal day (PND) 11 and week 9 of life.

In fig. 12 shows the levels of glucosylceramides C16:0 and C18:0 after regular oral administration to group mice aged PND 11-70.

In fig. Figure 13 shows an assessment of clinical signs in NPC(-/-) mice from the group aged PND 56-3 045552

70 treated with vehicle and AZ-3102.

In fig. 14 shows tremor scores in NPC(-/-) mice from the PND 56-70 group treated with vehicle and AZ-3102.

In fig. Figure 15 shows the definition of regions of interest (ROI). The image shows the ROI outlines of the cerebellum, hippocampus, corpus callosum, and striatum (dorsal striatum, caudate-putamen).

In fig. Figure 16 shows brain immunohistochemistry: calbindin-D28k labeling in vehicle-treated NCP(-/-) and NPC(+/-) mice compared with NPC(+/+, wild-type) and NPC(-/-) mice. , receiving AZ-3102.

In fig. 17 shows a generalized mechanism of action of the crystalline form of compound (I) (AZ3102).

Detailed Disclosure of the Present Invention

Unless otherwise specified, scientific and technical terms used in connection with the present invention have the meanings commonly understood by those skilled in the art. The meaning and definitions of the terms used should be clear, but in the event of any hidden ambiguity, the definitions given here take precedence over any dictionary or third-party definition.

It should be understood that expressions presented in the singular are often used for convenience, but all expressions and definitions presented in the singular are intended to cover the plural unless expressly stated otherwise or the context requires. The terms comprising, including and having are intended to be inclusive and imply that there may be additional elements other than those listed. In addition, it should be understood that all references, including journal articles, books, patents, technical literature, etc., mentioned herein are incorporated herein in their entirety and for all purposes by reference.

The term approximately, used here for numeric measures, refers to their value within 10% of the underlying parameter (i.e., plus or minus 10%), and the term approximately, used at the beginning of a line of numeric measures, refers to each value (i.e., i.e. the expressions about 1, 2 and 3 are equivalent to the expressions about 1, about 2 and about 3). For example, a temperature specified as approximately 85°C may include temperatures in the range of 75 to 95°C.

The term melting point is well known in the art. The term relatively high melting point, as used herein, is intended to describe crystalline forms that are stable enough to be used for the manufacture of 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 above about 80°C.

The term composition as used herein covers a product containing the specified ingredients in the specified amounts, as well as any product that is directly or indirectly the result of a combination of the specified ingredients in the specified amounts. Such term, in relation to a pharmaceutical composition, is intended to include a product containing a crystalline form of compound (I), and, optionally, additional ingredients contained in the carrier, as well as any product that directly or indirectly results from the combination, complexation or aggregation of any two or more ingredients, or dissociation of one or more ingredients, or other types of reactions or interactions of one or more ingredients. Accordingly, the pharmaceutical compositions of the present invention include any composition containing a crystalline form of the invention and, optionally, a pharmaceutically acceptable carrier. By pharmaceutically acceptable is meant that the carrier, diluent or excipient must be compatible with the other ingredients of the composition without causing any harmful effects to the recipient.

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

The present invention relates to the crystalline form of compound (I)

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(i).

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 (in non-ionized form). Preferably, the crystalline form of compound (I) is a crystalline free base. In addition, compound (I) can form cocrystals.

If the crystalline form of compound (I) is a crystalline salt, then the molecular structure of the above compound (I) includes a protonated nitrogen atom.

The present invention is not limited to the single crystalline form of compound (I). Solid materials can exist in more than one crystalline form. These alternative crystal forms are called polymorphs. Each polymorph has a different orientation and/or conformation of molecules in the crystal lattice. Each crystalline state, or polymorph, has a unique set of physicochemical properties due to differences in crystal structure.

Polymorphs may have different mechanical properties, such as powder flow and compressibility, which affect the processing properties of the compound. The stability and shelf life of a compound may also depend on the specific type of polymorph.

Polymorphs can be distinguished from each other in different ways. The polymorphs exhibit distinct spectroscopic properties and can be determined, for example, by infrared spectroscopy, Raman spectroscopy and ¹³ C-NMR spectroscopy. Due to the fact that each crystalline form refracts x-rays differently, powder x-ray diffraction (XRD) can also be used to characterize polymorphs. In addition, thermal techniques such as differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) can also provide unique information about a specific polymorph.

As is well known in the field of powder X-ray diffraction, the relative peak heights of powder X-ray diffraction spectra can be used to describe different crystalline forms. Accordingly, in one aspect regarding Form 3, the present invention relates to a crystalline form of compound (I) characterized by a reflectance in a powder X-ray diffraction pattern indicated as a 2θ value at 17.8±0.2°, wherein the reflectance at 17.8± 0.2° is one of the four strongest reflections in a powder X-ray diffraction pattern. Preferably, the reflection at 17.8±0.2° is one of the three strongest reflections in the powder X-ray diffraction pattern, or the reflection at 17.8±0.2° is one of the two strongest reflections in the powder X-ray 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 aspect regarding Form 3, the crystalline form of compound (I) is characterized by a reflectance in a powder X-ray diffraction pattern indicated as a 2θ value at 17.8±0.1°, wherein the reflectance at 17.8±0.1 ° is one of the four strongest reflections in a powder X-ray 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.

The term strongest reflection describes the highest peak in a powder X-ray diffraction pattern. The peak height in a powder X-ray diffraction pattern is determined by the intensity of the X-ray emission (in units of counts or counts/sec). Thus, the strongest reflection is the reflection that shows the highest x-ray intensity in the powder x-ray diffraction pattern. For example, the strongest reflection in the X-ray powder diffraction pattern shown in FIG. 6A is the reflection, indicated as the value 2θ, at 17.8±0.2°.

Unless otherwise expressly stated, all X-ray powder diffraction patterns were obtained using copper K-alpha radiation at approximately 25°C.

Preferably, in the aspect regarding Form 3, the crystalline form of compound (I) is further characterized by one or more reflections, expressed as a 2θ value, at one or more angles 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° on a powder X-ray diffraction pattern.

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Even more preferably, in the aspect regarding Form 3, the crystalline form of compound (I) is further characterized by one or more reflections, expressed as a 2θ value, at one or more angles of 4.1±0.1°, 8.3±0 ,1°, 12.4+0.1°, 13.6+0.1°, 14.5+0.1°, 14.9+0.1°,

15.2+0.1°, 17.2+0.1°, 19.3+0.1°, 21.2+0.1°, 22.4 ±0.1°, 22.9±0 ,1° and 23.3±0.1° on the powder X-ray diffraction pattern.

The peak positions in a powder X-ray diffraction pattern are relatively insensitive to experimental details. Thus, the crystalline compounds of the invention can be characterized by powder X-ray diffraction patterns with specific peak positions. Accordingly, in the aspect regarding Form 3, the crystalline form of compound (I) is preferably characterized by reflections, expressed as a 2θ value, at 17.2+0.2°, 17.8+0.2°, 21.2+0.2 ° and 22.4±0.2° on the powder X-ray diffraction pattern. More preferably, in the aspect regarding Form 3, the crystalline form of compound (I) is characterized by reflections, expressed as a 2θ value, 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 ° on a powder X-ray diffraction pattern. Even more preferably, in the aspect regarding Form 3, the crystalline form of compound (I) is characterized by reflections, expressed as a 2θ value, at 17.2±0.1°, 17.8+0.1°, 21.2+0 ,1° and 22.4±0.1° on the powder X-ray diffraction pattern. Most preferably, in the aspect regarding Form 3, the crystalline form of compound (I) is characterized by reflections, expressed as a 2θ value, 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 ° on a powder X-ray diffraction pattern.

Crystalline forms of a compound can be characterized using a differential scanning calorimetry (DSC) thermogram. Thus, the crystalline form of compound (I) in the aspect of Form 3 is preferably characterized by a DSC thermogram that shows the onset of endothermic heat flow at approximately 87° C. and/or a melting point of approximately 92.4° C., as shown in FIG. 8C. Accordingly, it is preferable that, in the aspect regarding Form 3, the crystalline form of compound (I) has a melting point of 89 to 96° C. determined by DSC. Preferably, the crystalline form in the form 3 aspect has a melting point of 90 to 95° C., as determined by DSC. Even more preferably, the crystalline form of compound (I) in the form 3 aspect has a melting point of 91 to 94°C as determined by DSC. Most preferably, the crystalline form of compound (I) in the form 3 aspect has a melting point of 92 to 93°C as determined by DSC.

Crystalline forms of a compound can be characterized by their hygroscopicity. The hygroscopicity of a product reflects the increase or decrease in its water content depending on the relative humidity at a certain temperature. Essentially non-hygroscopic products show or are characterized by only minor changes in water content as a result of changes in relative humidity. In highly hygroscopic products, the water content can vary widely. Accordingly, it is preferable that the crystalline form of compound (I) is substantially non-hygroscopic.

Crystalline forms of a compound can be characterized by their thermogravimetric properties. Thus, the crystalline form of compound (I) can preferably be characterized by its thermogravimetric properties. In one embodiment, the crystal of compound (I) is characterized by thermogravimetric properties presented in FIG. 8A or 8B.

Crystalline forms of a compound can also be characterized by a dynamic vapor sorption (DVA) profile. Accordingly, the crystalline form of compound (I) can preferably be characterized by the EAF profile shown in FIG. 9A and 9B. Preferably, the crystalline form of compound (I) has a reversible sorption/desorption profile. Preferably, the chipboard profile shows the substantially non-hygroscopic nature of the crystalline form.

Essentially non-hygroscopic substances have a water absorption of less than about 2% at a relative humidity of about 95%, measured at a temperature of about 25°C. Preferably, water absorption is less than about 1% at a relative humidity of about 95%, measured at a temperature of about 25°C. Water absorption values are obtained by measuring the weight gain of the test crystalline form relative to the initial weight at a relative humidity of approximately 95% and a temperature of approximately 25°C.

Accordingly, the crystalline form of compound (I) preferably absorbs from 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 from 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 from 0% to 1% water at a relative humidity of about 95% at a temperature of about 25°C.

Moreover, the crystalline form of the invention is preferably stable. For example, prolonged incubation in ethyl acetate (1 week) at approximately 25°C resulted in good quality crystalline forms of compound (I) as shown in FIG. 5.

In one aspect regarding Form 2, the present invention provides an additional crystalline form of compound (I). This crystalline form (Form 2) is characterized by a reflection, expressed as a 2θ value, at 16.9±0.2° in the powder X-ray diffraction pattern, where the reflection at 16.9±0.2° is one of the four strongest reflections in the powder X-ray diffraction pattern diffraction pattern. Preferably, the reflection at 16.9±0.2° is one of the three strongest reflections in the powder X-ray diffraction pattern, or the reflection at 16.9±0.2° is one of the two strongest reflections in the powder X-ray 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 in the form 2 aspect is characterized by a reflection, expressed as a 2θ value, at 16.9 ± 0.1° in a powder X-ray 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 powder X-ray diffraction pattern, or the reflection at 16.9±0.1° is one of the two strongest reflections in the powder X-ray diffraction pattern. More preferably, the reflection at 16.9±0.1° is the strongest reflection in the X-ray powder diffraction pattern.

Preferably, the crystalline form of compound (I) in the Form 2 aspect of the present invention is characterized by one or more reflections, expressed as a 2θ value, at one or more angles of 15.2±0.2°, 16.1±0, 2°, 16.5±0.2°, 18.9±0.2°, 23.1±0.2°, 25.5±0.2°, 27.7±0.2° and 28, 5±0.2° on the powder X-ray diffraction pattern. Even more preferably, in the Form 2 aspect of the present invention, the crystalline form of compound (I) is characterized by one or more reflections, expressed as a 2θ value, at one or more angles of 15.2±0.1°, 16.1± 0.1°, 16.5±0.1°, 18.9±0.1°, 23.1±0.1°, 25.5±0.1°, 27.7±0.1° and 28.5±0.1°.

In the Form 2 aspect of the present invention, this crystalline form of compound (I) can also be characterized by reflections, expressed as a 2θ value, at 16.1±0.2°, 16.5±0.2°, 16.9±0 ,2°, 18.9±0.2° and 23.1±0.2° in the powder X-ray diffraction pattern. Preferably, in the Form 2 aspect of the present invention, the crystalline form of compound (I) may also be characterized by reflections, expressed as a 2θ value, at 16.1±0.1°, 16.5±0.1°, 16.9± 0.1°, 18.9±0.1° and 23.1±0.1° in the X-ray powder diffraction pattern.

In the Form 2 aspect of the present invention, the crystalline form of compound (I) can be characterized by a DSC thermogram which shows the onset of endothermic heat flow at approximately 58° C. and a melting point at approximately 70° C., as shown in FIG. 3B. Accordingly, in the Form 2 aspect of the present invention, the crystalline form of compound (I) has a melting point of 67 to 74°C. Preferably, in the Form 2 aspect of the present invention, the crystalline form of compound (I) has a melting point of 68 to 73°C. Even more preferably, in the Form 2 aspect of the present invention, the crystalline form of compound (I) has a melting point of 69 to 72°C. Most preferably, in the Form 2 aspect of the present invention, the crystalline form of compound (I) has a melting point of 69 to 71°C.

The inventors of the present invention have unexpectedly discovered that, in the form 2 aspect, the crystalline form of compound (I) is particularly soluble in water. Accordingly, in the Form 2 aspect of the present invention, it is preferred that the crystalline form of compound (I) has a water solubility of from about 75 mg/ml to about 85 mg/ml, measured at about 25°C. More preferably, in the Form 2 aspect of the present invention, the crystalline form has a water solubility of from about 78 mg/ml to about 82 mg/ml, measured at a temperature of about 25°C. Most preferably, in the Form 2 aspect of the present invention, the crystalline form of compound (I) has a water solubility of approximately 80 mg/ml, measured at approximately 25°C.

Methods for measuring solubility are known in the art; for example, shake flask method, sonication method, column elution method, and ultraviolet-visible spectroscopy method. Unless otherwise expressly stated, solubility in water is determined using the shake flask method and/or the sonication method.

The present invention also relates to a pharmaceutical composition containing a crystalline form of compound (I) described herein.

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Typically, the crystalline form of compound (I) is administered to a patient in the form of a pharmaceutical composition or drug. Such pharmaceutical compositions can be administered to a patient by any suitable route of administration, including, but not limited to, oral, topical (including transdermal), and parenteral routes of administration.

The pharmaceutical compositions of the invention are typically prepared using a pharmaceutically acceptable carrier and one or more optional ingredients. If necessary or desired, the resulting uniformly mixed mixture may then be formed or loaded into tablets, capsules, pills, containers, cartridges, dispensers, and the like. using normal procedures and equipment.

The pharmaceutical compositions of the invention intended for oral administration in solid dosage form (ie, capsules, tablets, pills, etc.) generally contain a crystalline form of compound (I) as the active ingredient. Preferably, the pharmaceutical composition of the invention contains a crystalline form of compound (I) and does not contain other active ingredients. Preferably, the pharmaceutical composition of the invention is contained in a capsule. Preferably, the pharmaceutical composition of the invention is contained in a capsule without any other active ingredient. The capsule may be a gelatin capsule or a hydroxypropyl methylcellulose (HPMC) capsule. Alternatively, the pharmaceutical composition of the invention may contain a crystalline form of compound (I) as an active ingredient and one or more pharmaceutically acceptable carriers. Suitable pharmaceutically acceptable carriers are known to those skilled in the art and include, for example, fats, water, saline, alcohol (e.g. ethanol), glycerin, polyols, aqueous glucose, thinning agent, disintegrating agent, binder, lubricant, wetting agent, stabilizer, emulsifier, dispersant, preservative, sweetener, coloring agent, flavoring agent, concentrating agent, diluent, buffering agent, solvent or solubilizing agent, chemical compound for effective storage, salt for altering osmotic pressure, coating agent or antioxidant, saccharides such as lactose or glucose; corn, wheat or rice starch; fatty acids such as stearic acid; inorganic salts such as magnesium metasilicate aluminate or anhydrous calcium phosphate; synthetic polymers such as polyvinylpyrrolidone or polyalkylene glycol; alcohols such as stearyl alcohol or benzyl alcohol; synthetic cellulose derivatives such as methylcellulose, carboxymethylcellulose, ethylcellulose or hydroxypropylmethylcellulose; as well as other commonly used additives such as gelatin, talc, vegetable oil and gum arabic.

The pharmaceutical composition containing crystalline forms of compound (I) can also be administered transdermally or transmucosally using known delivery systems and excipients. For example, the pharmaceutical composition can be mixed with penetration enhancers such as propylene glycol, polyethylene glycol monolaurate, azacycloalkan-2-one, and the like, and it can be included in a patch or similar delivery system. Additional excipients may also be used, including gelling agents, emulsifiers and buffers.

Injectable preparations intended for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Aqueous solvents include, for example, distilled water for injection and/or saline. Examples of non-aqueous solvents include alcohols such as ethanol.

Preferably, the pharmaceutical composition contains one or more additional pharmaceutically active agents. Such combination therapy involves the use of a crystalline form of compound (I) in combination with one or more additional pharmaceutically active agents, either presented together (for example, formulated together in a single preparation) or presented separately (for example, packaged in separate unit dosage forms ).

In healthy humans, a basic glycosphingolipid such as glucosylceramide is hydrolyzed in lysosomes by acid glucosylceramidase (also called glucocerebrosidase or GBA1, EC 3.2.1.45, UniProt code: P04062). In addition, non-lysosomal glucosylceramidase (GBA2, UniProt code: Q9HCG7), located in the cytoplasm, is also capable of processing glucosylceramide. As a result, both GBA1 and GBA2 are involved in the neuropathological effects observed in several lysosomal storage diseases.

Patients with lysosomal storage disease have defects in the biosynthesis or degradation of glycosphingolipids, resulting in abnormal levels of glucosylceramide and/or other glycosphingolipids.

Previously, in publication WO 2015/147639 A1, compound (I) was shown to be effective in the treatment of diseases associated with abnormal levels of cytosolic or lysosomal glucosylceramide and/or elevated levels of glycosphingolipids. Since the bioavailability of crystalline forms of compound (I) is comparable to the bioavailability of amorphous compound (I), then crystalline forms

- 8 045552 forms of compound (I) are also effective in the treatment of diseases associated with abnormal levels of cellular glucosylceramide and/or elevated levels of glycosphingolipids. In particular, crystalline forms of compound (I) are effective in the treatment of diseases associated with abnormal levels of cytosolic or lysosomal glucosylceramide and/or elevated levels of glycosphingolipids.

As shown in Example 4, the crystalline form of compound (I) (Form 3) is effective in the treatment of diseases associated with abnormal levels of cytosolic or lysosomal glucosylceramide and/or elevated levels of glycosphingolipids. In particular, Form 3 has been shown to improve clinical signs in mice with Niemann-Pick disease type C.

Accordingly, the present invention relates to a crystalline form of compound (I) described herein or a pharmaceutical composition described herein for use in therapy.

The present invention also provides a crystalline form as described herein or a pharmaceutical composition as described herein for use as a drug.

Preferably, the present invention relates to a crystalline form of compound (I) described herein, or a pharmaceutical composition described herein, for use in the treatment of a disease associated with abnormal levels of glucosylceramide and/or elevated levels of glycosphingolipids. Preferably, the disease accompanied by abnormal levels of glucosylceramide and/or elevated levels of glycosphingolipids is a lysosomal storage disease such as Gaucher disease (types 1, 2 and 3), Fabry disease, GM1 gangliosidoses, GM2 gangliosidoses (such as Tay-Sachs disease , Sandhoff disease and variant AB), sialidosis, Niemann-Pick disease type C and myoclonus movement syndrome, renal failure, or a symptom of one of the diseases collectively classified as metabolic syndrome, for example obesity, insulin resistance, hyperlipidemia, hypercholesterolemia, polycystic kidney disease, type II diabetes and chronic inflammation, or a neurodegenerative disease such as Parkinson's disease or dementia with Lewy bodies, or atherosclerosis. Even more preferably, a crystalline form of compound (I) or a pharmaceutical composition containing a crystalline form of compound (I) is used for the treatment of GM1 gangliosidoses and/or GM2 gangliosidoses (such as Tay-Sachs disease, Sandhoff disease and variant AB).

Even more preferably, the present invention relates to a crystalline form of compound (I) described herein or a pharmaceutical composition described herein for use in the treatment of Niemann-Pick disease type C.

The present invention relates to a crystalline form of compound (I) described herein or a pharmaceutical composition described herein for use in the treatment of Sandhoff's disease.

The present invention also provides a method for treating a disease associated with abnormal levels of glucosylceramide and/or elevated levels of glycosphingolipids in humans or animals, wherein the method comprises 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.

The present invention relates to a crystalline form of compound (I) described herein, or to a pharmaceutical composition described herein, for use in alleviating the symptoms of a disease associated with abnormal levels of glucosylceramide and/or elevated levels of glycosphingolipids.

The present invention even more preferably relates to a crystalline form of compound (I) described herein or a pharmaceutical composition described herein for use in alleviating the symptoms of Niemann-Pick disease type C.

The present invention relates to a crystalline form of compound (I) described herein or a pharmaceutical composition described herein for use in alleviating the symptoms of Sandhoff's disease.

The present invention also provides a method for alleviating symptoms of a disease associated with abnormal levels of glucosylceramide and/or elevated levels of glycosphingolipids in human or animal patients, wherein the method comprises administering to the patient in need thereof a therapeutically effective amount of a crystalline form of compound (I) described herein, or a pharmaceutical composition described herein.

The crystalline form of compound (I) (eg Form 3) can be used to improve various clinical symptoms, such as:

1) weight loss associated with the disease;

2) tremor; and/or

3) ataxic gait.

As shown in Example 4, the crystalline form of compound (I) (eg, Form 3) effectively

- 9 045552 penetrates the brain. Thus, the crystalline form of compound (I) (eg, Form 3) can be used to treat or alleviate a disease or symptoms of a disease occurring in the brain. For example, a crystalline form of compound (I) (eg, Form 3) can be used to prevent or reduce the loss of Purkinje cells in the cerebellum. The crystalline form of compound (I) (eg, form 3) can be used to prevent or reduce neuronal death. The crystalline form of compound (I) (eg, Form 3) can be used to prevent or reduce cerebral atrophy.

The high efficacy of the crystalline form of compound (I) in the treatment of diseases associated with abnormal levels of glucosylceramide and/or elevated levels of glycosphingolipids is believed to be a result of the high activity of the crystalline form of compound (I) against glucosylceramide synthase (GCS) and non-lysosomal glucosylcerebrosidase (GBA2). .

The methods described herein may be in vitro or in vivo methods.

Administration may be either orally, using tablets, pills, capsules, granules, powders, solutions, etc., or by parenteral administration, such as injections, including intra-articular, intravenous and intramuscular injections, by administration using suppositories, ophthalmic solutions, ophthalmic ointments or topical preparations such as transdermal liquid preparations, ointments, transdermal patches, transmucosal liquid preparations, transmucosal patches, inhalers and the like.

When administered orally, the daily dose is typically about 0.0001 to 10 mg/kg, preferably 0.001 to 1 mg/kg or 0.005 to 5 mg/kg, and more preferably 0.01 to 0.5 mg/kg body weight, and the daily dose can be administered in one dose or in 2-4 separate doses. For example, for a 70 kg person, the optimal daily oral dose is approximately 0.01-30 mg/day. In the case of intravenous administration, the daily dose is suitably administered at a rate of approximately 0.00001 to 10 mg/kg body weight once a day or two or more times a day. In addition, the transmucosal agent is administered at a dose of about 0.0001 to 10 mg/kg body weight once daily or two or more times daily. The dose is selected according to the individual case, taking into account the symptoms, age, sex, etc.

Since the effectiveness of the crystalline form of compound (I) in the treatment of a disease associated with abnormal levels of glucosylceramide and/or elevated levels of glycosphingolipids (for example, such as Niemann-Pick disease type C) is very high and the required dose is relatively low, the crystalline form of the compound (I) causes fewer side effects than known compounds used to treat such diseases, in particular, for example, miglitol (its dose is 1200 mg/kg/day).

The present invention also relates to a method for preparing the crystalline form described herein, wherein the method involves contacting a sample of compound (I) with a solvent. The solvent is preferably 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, before contacting the sample of compound (I) with a solvent, the sample of compound (I) is purified to remove boric acid esters. Preferably, a sample of compound (I) is purified by chromatography. Even more preferably, a sample of compound (I) is purified using a silica gel column chromatography. Additionally and/or alternatively, a sample of compound (I) is purified by distillation with methanol.

The Form 3 crystalline form of compound (I) can be obtained by any of the following exemplary methods:

1) stirring a mixture of about 74 mg of compound (I) and 2.0 ml of acetonitrile for three days at a temperature of 20 to 30°C, for example at about 25°C, followed by filtration, to obtain a crystalline form of compound (I) ;

2) stirring a mixture of about 74 mg of compound (I) and 2.0 ml of anisole for three days at a temperature in the range of 20 to 30°C, for example at about 25°C, followed by filtration, to obtain a crystalline form of the compound ( I);

3) stirring a mixture of about 82 mg of compound (I) and 1.0 ml of ethyl acetate for three days at a temperature of 20 to 30°C, for example at about 25°C, followed by filtration, 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 three days at a temperature of 20 to 30°C, for example at about 25°C, followed by filtration, 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 three days at a temperature of 20 to 30°C, for example at about 25°C, followed by filtration, to obtain a crystalline form of compound (I) ;

6) stirring a mixture of approximately 100 mg of compound (I) and 3.0 ml of TBME for three

- 10 045552 days at a temperature in the range from 20 to 30°C, for example at approximately 25°C, followed by filtration, to obtain the crystalline form of compound (I).

Preferably, before mixing compound (I) with a solvent, a sample of compound (I) is purified to remove boric acid esters. Preferably, a sample of compound (I) is purified by chromatography. Even more preferably, a sample of compound (I) is purified using a silica gel column chromatography. Additionally and/or alternatively, a sample of compound (I) is purified by distillation with methanol.

Filtration methods are known to those skilled in the art and include, but are not limited to, paper filter filtration and cellular glass filter filtration.

The methods for preparing Form 3 of compound (I) described herein can be carried out on a larger scale by 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) with ethyl acetate in a ratio of 0.05 to 1 g per 1 ml [compound (I): ethyl acetate] for three days at a temperature of 20 to 30 °C, for example at about 25 °C, followed by filtration to obtain the crystalline form of compound (I).

The methods described herein for preparing Form 3 of compound (I) are very effective. The yield of the methods described herein generally exceeds 70% (expressed as the mass ratio of the amount of Form 3 obtained to the amount of compound (I) originally used). Preferably, the yields are greater than 75% for the methods described herein.

Preferably, crystalline form 2 is formed as an intermediate before the formation of crystalline form 3.

Preferably, crystalline form 2, characterized by reflections expressed as a 2θ value at 16.1±0.2°, 16.5±0.2°, 16.9±0.2°, 18.9±0.2 ° and 23.1±0.2° in the X-ray powder diffraction pattern, forms as an intermediate before the formation of crystalline form 3, characterized by reflections, expressed as a 2θ value, at 17.2±0.2°, 17.8±0, 2°, 21.2±0.2° and 22.4±0.2° in the X-ray powder diffraction pattern.

The present invention also relates to a crystalline form of compound (I) obtained by carrying out the method described herein.

The present invention also relates to the use of the free base of compound (I) to obtain a crystalline form of compound (I).

The present invention also relates to a method for obtaining a crystalline form of compound (I), wherein the method involves crystallizing the free base of compound (I).

The present invention also relates to a crystalline form of the compound (I) obtained by a method for producing a crystalline form of the compound (I), wherein the method involves crystallizing the free base of the compound (I).

It is believed that, in addition to other advantages of the present invention, the formation of a crystalline form of compound (I) is useful for the purification of compound (I). For example, the crystalline form of compound (I) obtained by the methods described herein has a purity of greater than 90%, and typically greater than 95%.

The foregoing detailed description has been presented for the purpose of explaining and illustrating the present invention, and is not intended to limit the scope of the appended claims. Many of the preferred embodiments of the present invention illustrated herein will be apparent to those skilled in the art and are within the scope of the appended claims and their equivalents.

The present invention may be further characterized by the definitions presented in the following paragraphs.

1. Crystalline form of compound (I),

2. The crystalline form according to claim 1, wherein the crystalline form is a crystalline free base.

3. The crystalline form according to claim 1 or 2, wherein the crystalline form is characterized by a reflection, expressed as a 2θ value, at 17.8±0.2° in a powder X-ray diffraction pattern, wherein the reflection at 17.8±0.2° is one of the four strongest reflections in the X-ray powder diffraction pattern.

- 11 045552

4. The crystalline form of claim 3, further characterized by one or more reflections, expressed as a 2θ value, at one or more of angles of 4.1±0.2°, 8.3±0.2°, 12.4±0, 2°,

13.6±0.2°, 14.5±0.2°, 14.9±0.2°, 15.2±0.2°, 17.2±0.2°, 19.3±0 ,2°, 21.2±0.2°, 22.4±0.2°, 22.9±0.2° and 23.3±0.2° in the X-ray powder diffraction pattern.

5. The crystalline form according to any one of paragraphs 1 to 4, characterized by reflections, expressed as a 2θ value, at angles of 17.2±0.2°, 17.8±0.2°, 21.2±0.2° and 22 .4±0.2° on the powder X-ray diffraction pattern.

6. Crystalline form according to any of points 1-5, having a melting point from 89°C to 96°C.

7. Crystalline form according to any of paragraphs 1-6, having a melting point from 92°C to 93°C.

8. The crystalline form according to any one of claims 1-7, wherein the crystalline form is substantially non-hygroscopic.

9. The crystalline form according to claim 1 or claim 2, wherein the crystalline form is characterized by a reflection, expressed as a 2θ value, at an angle of 16.9±0.2° in a powder X-ray diffraction pattern, wherein the reflection at 16.9±0.2° is one of the four strongest reflections in a powder X-ray diffraction pattern.

10. The crystalline form of claim 9, further characterized by one or more reflections, expressed as a 2θ value, at one or more of 15.2±0.2°, 16.1±0.2°, 16.5±0, 2°, 18.9±0.2°, 23.1±0.2°, 25.5±0.2°, 27.7±0.2° and 28.5±0.2° on X-ray powder diffraction pattern.

11. The crystalline form according to any one of paragraphs 1, 2, 9 or 10, characterized by reflections, expressed as a 2θ value, at angles of 16.1±0.2°, 16.5±0.2°, 16.9±0, 2°, 18.9±0.2° and 23.1±0.2° in the X-ray powder diffraction pattern.

12. Crystalline form according to any of paragraphs 9-11, having a melting point from 67 to 74°C.

13. Crystalline form according to any of paragraphs 9-12, having a melting point from 69 to 71°C.

14. Crystalline form according to any of paragraphs 9-13, having a solubility in water from 75 mg/ml to 85 mg/ml.

15. A pharmaceutical composition containing a crystalline form according to any one of paragraphs 114.

16. The pharmaceutical composition according to claim 15, where the pharmaceutical composition is contained in a capsule.

17. The pharmaceutical composition according to claim 16, wherein the pharmaceutical composition is contained in a capsule without any other ingredient.

18. The pharmaceutical composition according to claim 15 or 16, containing at least one pharmaceutically acceptable carrier.

19. The crystalline form according to any of paragraphs 1-14 or the pharmaceutical composition according to any of paragraphs 15-18 for use in therapy.

20. The crystalline form according to any of paragraphs 1-14 or the pharmaceutical composition according to any of paragraphs 15-18 for use as a medicinal product.

21. The crystalline form of any one of claims 1 to 14 or the pharmaceutical composition of any of claims 15 to 18 for use in the treatment of a disease associated with abnormal levels of glucosylceramide and/or elevated levels of glycosphingolipids.

22. A crystalline form or pharmaceutical composition for use according to claim 21, wherein the disease associated with abnormal levels of glucosylceramide and/or elevated levels of glycosphingolipids is a lysosomal storage disease such as Gaucher disease, Fabry disease, GM1 gangliosidoses, GM2 gangliosidoses (such such as Tay-Sachs disease, Sandhoff disease, and variant AB), sialidosis, Niemann-Pick disease type C, and myoclonus movement syndrome, renal failure, or a symptom of one of the diseases collectively classified as metabolic syndrome, such as obesity, insulin resistance, hyperlipidemia , hypercholesterolemia, polycystic kidney disease, type II diabetes, and chronic inflammation or neurodegenerative disease such as Parkinson's disease or dementia with Lewy bodies, or atherosclerosis.

23. A crystalline form or pharmaceutical composition for use according to claim 21 or 22, wherein the disease is a GM1 gangliosidosis or a GM2 gangliosidosis (such as Tay-Sachs disease, Sandhoff disease or variant AB).

24. A method of treating a disease associated with abnormal levels of glucosylceramide and/or elevated levels of glycosphingolipids in human or animal patients, wherein the method comprises administering to the patient in need thereof a therapeutically effective amount of a crystalline form of any one of claims 1 to 14, or pharmaceutical composition according to any one of paragraphs-

- 12 045552 tov 15-18.

25. A method for obtaining a crystalline form according to any one of claims 1 to 8, wherein the method involves contacting a sample of compound (I) with a solvent.

26. The method of claim 25, wherein the solvent is selected from acetonitrile, ethyl acetate, isopropanol, anisole, water and tert-butyl methyl ether (TBME).

27. The method of claim 25 or 26, wherein the sample of compound (I) is purified before contacting the sample of compound (I) with a solvent.

28. The method according to claim 27, wherein a sample of compound (I) is purified by chromatography.

29. The method according to claim 28, wherein a sample of compound (I) is purified using silica gel column chromatography.

30. The method according to any one of paragraphs 27-29, in which the sample of compound (I) after purification does not contain borate esters.

31. The method of any one of claims 25-30, wherein the crystalline form of any of claims 9-14 is formed as an intermediate before the crystalline form of any of claims 1-8 is formed.

32. Crystalline form of compound (I) obtained by implementing the method according to any of paragraphs 25-31.

33. Use of the free base of compound (I) to obtain crystalline form.

34. A method for obtaining a crystalline form of a compound (I), wherein the method involves crystallizing the free base of the compound (I).

35. Crystalline form of compound (I), obtained by the method according to paragraph 34.

36. A method for obtaining a crystalline form of compound (I), where the method involves performing the following steps:

i) adding compound (I) to a purification column to obtain a purified sample of compound (I);

ii) adding a solvent to the purified sample of compound (I) to obtain a suspension of compound (I) in the solvent;

iii) stirring a suspension of compound (I) in a solvent to obtain a crystalline form of compound (I); and iv) separating the crystalline form of compound (I), to obtain a pure sample of the crystalline form of compound (I).

37. The method according to paragraph 36, in which the purified sample of compound (I) does not contain borate esters.

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

39. The method according to any of paragraphs 36-38, in which stage ii is carried out at a temperature of 25-35°C.

40. The method according to any one of paragraphs 36-39, in which the mixing in step (iii) is carried out at a temperature of 20-35°C.

41. The method according to any one of paragraphs 36-40, in which the mixing in step (iii) is carried out for at least 1 hour.

42. The method according to paragraph 41, in which the mixing in step (iii) is carried out for at least 16 hours.

Experimental section

Differential scanning calorimetry (DSC) analyzes were performed on a TA Instruments Q2000 instrument (a closed aluminum cuvette or an aluminum cuvette with a pinhole in the lid was used, heating rate 20 deg/min). The maximum of the peak was taken as the melting temperature.

Dynamic vapor sorption (DVA) analyzes were performed on an SPS11-100n Sorptions Prufsystem from ProUmid (formerly Projekt Messtechnik), August-Nagel-Str. 23, 89079 Ulm (Germany). From 5 to 20 mg of sample was placed into an aluminum sample cuvette. A humidity change rate of 5% per hour was used. The measurement program can be described as follows:

The sample was placed on an aluminum or platinum holder on top of the microbalance, and allowed to equilibrate at 50% relative humidity (RH) before running preset humidity programs:

(1) 2 hours at 50% relative humidity;

(2) 50^0% relative humidity (5%/h); 5 hours at 0% relative humidity;

(3) 0^95% relative humidity (5%/h); 5 hours at 95% relative humidity;

(4) 95^0% relative humidity (5%/h); 5 hours at 0% relative humidity;

(5) 0^95% relative humidity (5%/h); 5 hours at 95% relative humidity;

(6) 95^50% relative humidity (5%/h); 2 hours at 50% relative humidity.

X-ray powder diffraction analyzes were performed on a Stoe Stadi P diffractometer

- 13 045552 with a MythenlK detector operating on Cu-Ka1 radiation. Measurements on this device were carried out at a tube voltage of 40 kV and a current of 40 mA. The measurements were carried out using a curved Ge monochromator for Cu-Ka ₁ radiation. The diffractometer was set to a mode with the following parameters: step 0.02° 2θ, step time 12 s, scanning range 1.5-50.5° 2θ, and detector step 1° 2Θ (step-by-step scanning detection mode). For typical sample preparation, approximately 10 mg of sample was placed between two acetate films and the sample was mounted in the sample holder of a Stoe diffractometer. The sample was rotated during the measurement. All sample preparation and measurements were carried out in ambient air (approximately 25 °C).

Approximate solubility was determined by gradually adding solvent to approximately 10 mg of compound, followed by shaking and/or sonication for a short period of time. If a substance did not dissolve when a total of at least 10 mL of solvent was added, the solubility was reported as <1 mg/mL. Experiments were carried out at a temperature of approximately 25°C.

Thermogravimetric measurements, performed by coupled thermogravimetry-infrared spectroscopy (TG-FTIR) analysis, were carried out using a Netzsch ThermoMicrobalance TG 209 coupled to a Bruker FTIR Spectrometer Vector 22 (using pinhole sample cups, N2 atmosphere, heating rate 10° S/min).

Examples.

The following non-limiting examples further illustrate the present invention.

Example 1 Formation of Crystalline Salt

Studies under the program of high-throughput screening of salts of compound (I) were carried out using 16 different salt-forming agents listed in table. 1, while attempting to obtain the crystalline form of compound (I) under six different conditions.

Initial screening experiments were performed by adding a 0.05 M solution of compound (I) in acetone to each well of a 96-microtiter quartz plate, followed by the addition of 0.1 M salt-forming stock solutions. Solvents were evaporated from each well under a stream of nitrogen at room temperature. temperature. Solid residues in the wells were examined using polarized light microscopy.

Table 1

Acids selected for study under the salt screening program

Salt former Molecular mass Stahl class Adipic acid 146.14 1 Benzenesulfonic acid 158.17 2 Benzoic acid 122.12 2 Lemon acid 192.12 1 Fumaric acid 116.07 1 Gentisic acid 154.12 2 Hydrochloric acid 36.46 1 L-lactic acid, 90.08 1 Maleic acid 116.07 1 L-malic acid 134.09 1 DL-mandelic acid 152.15 1 Phosphoric acid 97.99 1 succinic acid 118.09 1 Sulfuric acid 98.08 1 L-tartaric acid 150.09 1 Toluenesulfonic acid 190.22 2

Although the goal of these initial experiments was to obtain a 1:1 ratio of compound (I) to salt former, light microscopy studies revealed some experimental conditions suitable for the formation of the crystalline form. For example, when compound (I) was mixed with benzenesulfonic acid, hydrochloric acid, DL-mandelic acid, L-tartaric acid, phosphoric acid and sulfuric acid, crystalline residues were formed.

In another screening experiment, six solvent systems were selected, namely acetone, acetonitrile, ethyl acetate, ethanol, isopropanol/water (3:1), and acetone/water (9:1). 200 μL of solvent was added to the residue in each well to equilibrate the suspension. Prepared like this

- 14 045552 the microtiter plate was shaken at 400 rpm for one day at room temperature (approximately 25°C). The solvents were then evaporated under a stream of nitrogen, and the resulting solid residues were examined by polarized light microscopy.

Based on light microscopy studies, indications of possible salt formation were found when using 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 samples were selected for subsequent experiments in the 50 to 200 mg scale (upscale experiments).

Scale-up experiments for all conditions tested resulted in the formation of amorphous forms of compound (I) or liquid crystalline salts of compound (I). Although microscopic examination often revealed birefringence, filtration of the resulting mixtures was impossible, and none of the experiments succeeded in isolating solid material.

To summarize, it can be noted that in all experiments with salt formers it was not possible to obtain a crystalline form of compound (I).

Example 2: Crystalline form of Form 2.

Since the experiments described in Example 1 did not result in useful crystalline material, the free base crystalline form of compound (I) was investigated.

Compound (I) was purified using a silica gel column to remove borate esters, followed by brief equilibration with acetonitrile (approximately 1-5 minutes). It was found that the solubility of purified compound (I) in acetonitrile ranged from 5 to 8 mg/ml. The solubility of purified compound (I) in other solvents was also studied, and the results are presented in table. 2. These values were determined by adding small aliquots of solvent to ~10 mg of solid compound (I) and then shaking or sonicating for a short period of time at room temperature (at approximately 25°C).

table 2

Approximate solubility values for purified compound (I)

Solvent Solubility S [mg/ml] Solvent Solubility S [mg/ml] Acetic acid S>120 MEK 40 < S < 50 Acetone 55 < S < 73 Methanol S>160 Acetonitrile 5<S<8 1-propanol 13 < S < 15 DCM S>160 2-propanol 19<S<20 Ethanol 40 < S < 53 THF S>200 Ethyl acetate 12 < S < 13 Water S ~ 80

The crystalline form of form 2 of compound (I), obtained by short (approximately 1-5 min) equilibration with acetonitrile, was studied using polarized light microscopy, powder X-ray diffraction (XRD), TG-FTIR, differential scanning calorimetry experiments (DSC) and dynamic vapor sorption (DSP).

The results of polarized light microscopy are presented in Fig. 1, and the X-ray diffraction pattern obtained using SRD is shown in FIG. 2. This crystal form 2 of compound (I) showed the 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°.

The results of thermoanalytical characterization of crystalline Form 2 of compound (I) obtained by short equilibration with acetonitrile (TG-FTIR thermograms) are presented in FIG. 3A, and the results obtained by DSC are shown in FIG. 3B. The results show that the sample contained approximately 0.5% water, which is released when heated to approximately 120°C. At higher temperatures, thermal decomposition was observed. DSC analysis revealed two significant endothermic events. The first strong endotherm with a peak temperature of approximately 70°C and an enthalpy of approximately 54 J/g was followed by a weaker signal at 81°C and an enthalpy of approximately 3 J/g.

In addition, the behavior of a crystalline free base sample at various water vapor pressures was investigated. 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% relative humidity. The measurement results obtained using the DSP method are presented in Fig. 4A and 4B.

Generalized indicators characterizing the crystalline form of compound (I) obtained in Example 2 are presented in table. 3.

- 15 045552

Table 3

Generalized indicators characterizing crystalline form 2 of compound (I)

Index Meaning/description of the indicator Melting temperature ~ 70°С G playability 18% at 95% relative humidity; hygroscopic form Solubility in water ~ 80 mg/ml

The resulting crystalline form 2 of compound (I) has low stability, and it further transforms into crystalline form 3 of compound (I) when exposed to elevated temperature (more than about 30° C.) for about 1-5 minutes. Alternatively, Form 2 converts to Form 3 when equilibrated with a solvent (eg, acetone) for more than 5 minutes at approximately 25°C. This crystalline form 3 of the free base of compound (I) had better stability and a unique set of physicochemical properties.

Example 3. Form 3.

The stable crystalline form of compound (I) was obtained from independent experiments by equilibrating a suspension of purified compound (I) with various appropriate solvents, for example, acetonitrile, ethyl acetate, isopropanol, anisole, water or TBME, respectively, at room temperature (at approximately 25° WITH).

Specifically, the Form 3 crystalline form of compound (I) was obtained using the following experimental methods:

1) approximately 74 mg of compound (I) was added to 2.0 ml of acetonitrile, and the suspension was stirred for three days at room temperature (approximately 25°C), and then it was filtered;

2) approximately 74 mg of compound (I) was added to 2.0 ml of anisole, and the suspension was stirred for three days at room temperature (approximately 25°C), and then it was filtered;

3) approximately 82 mg of compound (I) was added to 1.0 ml of ethyl acetate, and the suspension was stirred for three days at room temperature (about 25° C.), and then it was filtered;

4) approximately 82 mg of compound (I) was added to 1.0 ml of isopropanol, and the suspension was stirred for three days at room temperature (approximately 25°C), and then it was filtered;

5) approximately 45 mg of compound (I) was added to 1.0 ml of water, and the suspension was stirred for three days at room temperature (approximately 25°C), and then it was filtered;

6) Approximately 100 mg of compound (I) was added to 3.0 ml of TBME, and the suspension was stirred for three days at room temperature (approximately 25° C.) and then filtered.

Before adding the solvent, compound (I) was purified to remove boric acid esters using a silica gel column.

The resulting crystalline form of compound (I) was characterized by polarized light microscopy, powder X-ray diffraction, TG-FTIR, DSC and DSP techniques.

The results of the study using polarized light microscopy for selected solvents are presented in Fig. 5.

The XRD pattern of crystalline form 3 of compound (I) obtained from suspension equilibration experiments with acetonitrile is shown in FIG. 6A. An overlay of PRD diractograms of crystalline forms 3 of compound (I) obtained from suspension equilibration experiments with other solvents is shown in FIG. 6B. This crystalline form of compound (I) shows the strongest reflections at 2Θ values of 17.2±0.2°, 17.8±0.2°, 21.2±0.2° and 22.4±0.2° . These results are consistent with each other, despite the fact that different solvents were used to obtain the crystalline form of compound (I).

For comparison, in FIG. 7 shows an overlay of the PXRD pattern for the crystal form obtained in Example 2 (Form 2) and the crystal form obtained in this Example (Form 3). The two crystal forms have different PDP diractograms.

The results of typical TG-FTIR characterization experiments for crystalline form 3 of compound (I) are shown in FIG. 8A (ethyl acetate) and FIG. 8B (isopropanol).

In fig. 8A shows that a sample of crystalline form 3 of compound (I) prepared by equilibration with ethyl acetate contains approximately 0.7% ethyl acetate despite drying in vacuo at 40° C. for several days. Ethyl acetate is released at temperatures between approximately 100°C and 200°C. At higher temperatures thermal decomposition occurs.

A sample of Form 3, obtained by forming a suspension of compound (I) in isopropanol (IPA) and air drying at room temperature, unexpectedly showed that the initial content

- 16 045552 isopropanol is less than 0.5% (see Fig. 8B).

Typical DSC measurements for a sample of crystalline form 3 of compound (I) obtained by equilibration with ethyl acetate are shown in FIG. 8C; showing a sharp endothermic melting peak at approximately 92°C associated with an enthalpy of approximately 103 J/g. Thus, the crystalline form of Form 3 of compound (I) obtained in this Example has a higher melting point than the crystalline form in Example 2 (Form 2).

In addition, the behavior of a sample of crystalline form 3 of compound (I), obtained by equilibration with ethyl acetate, was studied at various water vapor pressures. At the highest relative humidity of 95%, the sample absorbed approximately 1.2% of water, which was released when the relative humidity returned to 50% relative humidity. The measurement results using the EAF method are shown in Fig. 9A and FIG. 9B. The amount of absorbed water turned out to be insignificant, and water sorption was reversible. Thus, this crystalline form of Form 3 of compound (I) is non-hygroscopic or substantially non-hygroscopic.

Generalized indicators characterizing crystalline form 3 of compound (I), obtained in Example 3, are presented in table. 4.

Table 4

Generalized indicators characterizing crystalline form 3 of compound (I)

Index Meaning/description of the indicator Melting temperature ~ 92.5°C G playability ~1% at 95% relative humidity; essentially non-hygroscopic form Solubility in water didn't measure

Example 4 Effect of administration of the crystalline form of compound (I) to mice suffering from Niemann-Pick disease type C (NPC).

In this example, AZ-3102-00 is the name of form 3 of compound (I).

Purpose of the study.

The purpose of the study was to evaluate the efficacy of treating young NPC1 mice (NPC(-/-) (ages P11-P70)) with a predicted pharmacologically active dose of AZ-3102-00 using an oral gavage, and to evaluate PK and histological markers for characterization neuropathology that may occur.

Study design.

This study included 38 mice, including: 30 NPC(-/-) knockout (KO) mice, 4 heterozygous NPC(+/-) mice and 4 wild-type NPC(+/+) mice (WT, Balb mice /c). NPC1(-/-) mice have premature truncation of the protein with deletion of 11 of the 13 transmembrane domains, while the first two transmembrane domains remain unchanged. NPC1 (-/-) mice homozygous for the recessive allele of the NIH Niemann Pick type C1 gene (Npc1m1N) are characterized by dual deficiencies in sphingomyelinase and glucocerebrosidase activity (JAX# 003092). The animals were bred from the BALB/c OlaHsd mouse line.

NPC1(-/-) mice were administered AZ-3102-00 via oral gavage from postnatal day 11 (P11) to postnatal day 70 (P70). Age-matched control mice included 6 NPC(-/-) and 4 NPC(+/-) mice treated with vehicle and 4 NPC(+/+) mice treated with AZ3102-00. After the last treatment on day P70, NPC(-/-) mice (n=4 for each time point) were euthanized by intraperitoneal injection of 600 mg/kg pentobarbital at the following time points: 30 min, 1 h, 2 h, 4 h, 8 h and 24 h. After the last treatment, control mice were also killed (not time critical). Terminal blood was collected by cardiac puncture into EDTA-coated tubes. Blood plasma was collected by centrifugation (3000xg for 10 min at room temperature), and 50 μl aliquots of plasma were transferred into 1.5 ml tubes, frozen on dry ice and stored at -80°C.

After transcardial perfusion, the brain was removed and cut in half. The right hemisphere of the brain was postfixed and placed in cryoforms for further immunohistochemical analysis. The left hemisphere of the brain was frozen on dry ice for further analysis.

The brain was subjected to cryotomy (12 levels, 5 sections each). Five sections per animal were then used for quantitative immunofluorescent labeling of microglia (MAC1) and astrocytes (GFAP) in two brain regions.

Test system and justification for the test system.

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 non-esterified cholesterol and glycosphingolipids (GSL). Approximately 95% of cases of the disease

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Niemann-Pick type C is caused by genetic mutations in the NPC1 gene, called Niemann-Pick disease type C1; 5% are caused by mutations in the NPC2 gene, called Niemann-Pick disease type C2. The clinical manifestations of Niemann-Pick disease types C1 and C2 are similar because both respective genes are involved in the release of lipids, especially cholesterol, from late endosomes or lysosomes. The NPC1 gene encodes a protein located in membranes inside cells, and it is involved in the movement of cholesterol and lipids within cells. Deficiency of this protein leads to abnormal accumulation of lipids and cholesterol in cell membranes. The NPC2 gene encodes a protein that binds and transports cholesterol. Mice homozygous for the recessive allele of the NIH gene with Niemann-Pick disease type C1 exhibit dual deficiency in sphingomyelinase and glucocerebrosidase activity. Mutant mice begin to lose weight and exhibit tremors and ataxic gait at approximately 7 weeks of age. Weight loss continues and tremors and ataxia become more severe and continue until death at approximately 12-14 weeks of age. The liver and spleen are also enlarged, and the Purkinje cells in the cerebellum are severely depleted. Some of these signs in mice resemble those of Niemann-Pick disease type C in humans.

Table 5

Test Item Details

Test item Definition/meaning Name of the connection being tested AZ-3102-00 Lot number of the compound being tested CR-20-02149 Product name Not applicable Stability Retest date - April 2021 Compound Not applicable Purity 99.4% Molecular mass 433.5 g/mol expiry date Retest date - April 2021 Storage conditions (for recipe) Storage and stability in media: Store stably under a nitrogen atmosphere at room temperature, protected from light for at least 24 hours; stored in a nitrogen atmosphere in the refrigerator (28°C) for at least 8 days; Stored under nitrogen atmosphere frozen in a freezer (< -15°C) for at least 21 days (3 weeks) Carrier Acidified water Treatment doses 1.5 mg/kg (for Pl 1-P25) and 3 mg/kg (for P26-P70) Route of administration and volume By oral gastric tube, 10 ml/kg Frequency of administration Daily administration for 60 days

Preparing connections.

Dosed preparations were aliquoted where required so that they could be dosed on each administration occasion.

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Table 6

Information about drugs

Dosed drug Form of the drug for administration Procedure Frequency preparation of drugs Installed storage conditions Connection under test Solution Formulations (w/w) were prepared by dissolving the test compound in Ampuwa water under nitrogen atmosphere At least once a week or twice a week if nitrogen atmosphere cannot be used during dissolution 2-8°С in protected from light place

The required amount of test compound was weighed and dissolved in Elix water (w/w) and the pH was adjusted to acidic. No additional excipients were added.

No corrections were made for the specific gravity of the test compound or the purity/composition of the test compound.

After each dose was prepared, the remainder of the solution was frozen for analysis at the end of the study.

Stability analyzes previously performed in connection with method development and validation showed that the test compound was stable in the vehicle when prepared and stored under the same conditions at concentrations adjacent to those used in this study for at least 24 h at room temperature in a place protected from light; for at least 8 days in the refrigerator (2-8°C) and at least 3 weeks in the freezer (<15°C) at concentrations similar to those used in the present study (from 0.01 up to 2 mg/ml).

Animal handling.

Pet accommodation.

Animals were housed in individual ventilated cages on standard rodent bedding supplied by Rettenmaier. Each cage contained no more than five mice. The temperature in the vivarium was maintained in the range of 20 to 24°C, and the relative humidity was maintained in the range of 45% to 65%. Animals were kept under a constant light cycle (12 h light/dark). Animals had unlimited access to dry pelleted standard rodent chow (Altromin) as well as regular tap water. Wet food was provided to the animals under the direction of the treating veterinarian.

Animal identification.

Animals were numbered sequentially using classical designations.

Each cage was identified with a colored card indicating the study number, sex, individual registration numbers (IRN) of the animals, date of birth, and allocation to treatment groups. The genotype (transgenic or wild type) of each animal was determined using GAS specific for the transgenic construct. Each mouse was genotyped using DNA isolated from an ear biopsy prior to the study.

Distribution into groups.

Only animals with satisfactory health were included in the study. Randomization of group assignment was performed for each cell. Animals were assigned to different initial groups (cohorts), including animals in treatment groups. The number of animals in the starting group was limited to ensure the same age of the animals and the same housing conditions.

Animal cage health and monitoring.

Before animals were included in the study, the health status of each individual animal was assessed. During the study, daily observations were made and any observations regarding the behavior of animals in cages were recorded and immediately reported to the study director and the treating veterinarian, who decided on further action (eg, euthanasia).

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The body weight and health status of the animals were recorded daily for the first week and then once a week.

Premature termination of the experiment and humane endpoints

No animals were prematurely euthanized.

Materials and methods

Animals.

Line of mice NPC(-/-) Provider: QPS Austria GmbH, Grambach, Austria Age at the start of the experiment: From the 11th day after birth (Pl 1) to the 70th day after birth (P70) Floor: Mixed Number of animals: 30 NPC(-/-); 4 NPC(+/-); 4 NPC(+/+)

Animal care.

NPC1(-/-) mice (groups D - I) received AZ-3102-00 via oral gavage (10 ml/kg) from postnatal day 11 (P11) to postnatal day 70 (P70). Dosing was started at 1.5 mg/kg from P11 to P25, followed by 3 mg/kg from P26 to P70. Control mice included: 6 NPC1(-/-) and 4 NPC(+/-) mice treated with vehicle, and 4 NPC (+/+) mice treated with AZ3102-00 (groups A-C).

After the last challenge on day P70, NPC(-/-) mice (n=4 for each time point; groups D-I) were euthanized by intraperitoneal injection of 600 mg/kg pentobarbital at the following time points: 30 min, 1 hour, 2 hours, 4 hours. , 8 h and 24 h. Control mice (groups A, B and C) were also sacrificed after the last dosing (not time critical).

Table 7

Description of groups of animals treated

Group Animals Gender in the group Route of administration Injected connection Concentration (a) η A NPC(-/-) Mixed BY Carrier N.A. 6 IN NPC(+/-) Mixed BY Carrier N.A. 4 WITH NPC(+/+) Mixed BY AZ-3102-00 Dose 1 and 3 mg/kg 4 D NPC(-/-) Mixed BY AZ-3102-00 Dose 1 and 3 mg/kg 4 E NPC(-/-) Mixed BY AZ-3102-00 Dose 1 and 3 mg/kg 4 F NPC(-/-) Mixed BY AZ-3102-00 Dose 1 and 3 mg/kg 4 G NPC(-/-) Mixed BY AZ-3102-00 Dose 1 and 3 mg/kg 4 N NPC(-/-) Mixed BY AZ-3102-00 Dose 1 and 3 mg/kg 4 I NPC(-/-) Mixed BY AZ-3102-00 Dose 1 and 3 mg/kg 4 mice received 1 mg/kg during P11-P25 and then 3 mg/kg during P26-P70 P/O - oral administration NA - not applicable

Tissue sampling.

After the last administration on day P70, mice were euthanized by intraperitoneal injection: after 30 min (group D), after 1 h (+/- 5 min, group E), after 2 h (+/- 5 min, group F), after 4 h (+/- 5 min, group G), after 8 h (+/- 5 min, group H) and after 24 h (+/- 5 min, group I) (all times are critical).

NPC1(-/-) mice from group A, 4 NPC(+/-) mice from group B and 4 NPC(+/+) mice from group C

- 20 045552 served as a control and they were also euthanized on day P70 (approximately 2 hours after the last administration).

Before euthanasia, mice were anesthetized with an intraperitoneal injection of pentobarbital (600 mg/kg, dose 10 μl/gram body weight).

Blood sampling.

The thorax of the mice was opened and blood was collected by cardiac puncture with a 23-gauge needle. The needle was removed and the blood was transferred to a sample tube (MiniCollect® K2EDTA (potassium ethylenediaminetetraacetic acid)). The tube was carefully inverted to facilitate uniform distribution of EDTA and prevent clotting. Blood samples were centrifuged at 3000 g for 10 min at room temperature (22°C). Plasma aliquots of 50 μl were transferred to LoBind pre-labeled 1.5-ml Eppendorf tubes frozen on dry ice and stored at -80°C.

Perfusion.

Animals were then transcardially perfused using 0.9% saline. For this purpose, a 23-gauge needle connected to a vial of 0.9% saline was inserted into the left ventricle. The thoracic aorta between the lungs and the liver was clamped with hemostats to block blood flow from the heart to the abdomen while allowing blood to flow to the brain. The right ventricle was opened with scissors. A constant pressure of the perfusion solution was maintained from 100 to 120 mmHg. Art. by connecting the bottle with the solution to a pressure-controlled air compressor. Perfusion was continued until the skull surface became white and only perfusion solution instead of blood emerged from the right ventricle.

Brain sampling.

After perfusion, the skull was opened, the brain was carefully removed and dissected in half on a cooled surface. The left hemisphere was weighed, quickly 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 hours at room temperature.

Histology.

Fabric preparation.

The right hemisphere of the mouse brain was fixed by immersion in a freshly prepared 4% paraformaldehyde solution in PB (pH 7.4) for two hours at room temperature. The hemispheres were then transferred to a 15% sucrose/PBS solution and stored at 4°C until immersed in cryoprotection medium. Tissue blocks were then trimmed as needed, transferred to cryoforms, embedded in OCT medium, frozen in isopentane cooled with dry ice, and stored in an ultra-deep freezer (target temperature -80°C).

Obtaining slices.

Five consecutive cryosections with a thickness of 10 μm in the sagittal direction were obtained using a Leica cryotome. The next 25 slices for each level were discarded. This slice acquisition scheme was repeated for 12 levels and could be modified to obtain slices from the correct levels, for example, if the brain was smaller due to young age or genotype. A total of 12x5 = 60 slices were obtained. Levels for sections were chosen in accordance with the brain atlas (Paxinos and Franklin The Mouse Brain in Stereotaxic Coordinates, 2nd edition, 2001). Slice collection began at ∼0.2 mm lateral to the midline and then continued across the hemisphere to ensure systematic random sampling covering the target regions (see Fig. 10 and Fig. 15). Sections were stored at -20°C. The results included sections from almost the entire brain, and after all sections were collected, the residual tissue block was discarded.

Immunofluorescence of Exp353 (calbindin-B28k).

For each incubation, a homogeneous systematic random set of five sections from one mouse was selected (one section each at levels 2, 4, 6, 8, 10); For information about systematic random sampling, see: http://www.stereology.info/sampling/.

All steps below were performed in Dulbecco's phosphate buffered saline pH 7.2-7.8 (PBS) at room temperature unless otherwise noted:

1) cryosections were air-dried for 45 min and washed in PBS for 10 min;

2) nonspecific binding was blocked with 10% normal donkey serum (Jackson Immuno Research) in 0.1% TritonX-100/PBS for 60 min in a humidified chamber;

3) each section was washed for 3x5 min in PBS;

4) sections were incubated with primary antibodies in 1% normal donkey serum/PBS overnight at 4°C in a humidified chamber; We used a guinea pig polyclonal antibody to Calbindin-D28k (Synaptic Systems, 214005) at a dilution of 1:1000;

5) each section was washed for 3x5 min in PBS;

6) sections were incubated with secondary antibodies in 1% normal donkey serum/PBS for 60 min in a humidified chamber (protected from light); used Cy3-conjugated secondary donkey anti-guinea pig antibody (H+L), (Jackson ImmunoResearch, 706-165-148) diluted

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1:500;

7) each section was washed for 3x5 min in PBS (protected from light);

8) sections were incubated with DAPI working solution for 15 min (protected from light)

9) sections were washed for 2x5 min in PBS (protected from light);

10) sections were washed for 5 min in ddH ₂ O (protected from light);

11) sections were covered with a layer of Mowiol and coverslips (protected from light).

Visualization.

Full scans of stained sections were recorded using a Zeiss AxioScan Z1 automated microscope with high aperture objectives, equipped with a Zeiss Axiocam 506 monochrome camera and a Hitachi 3CCD HV-F202SCL camera, and using Zeiss ZEN 2.3 software.

Quantitative assessment.

Image analysis was performed using Image Pro 10 software (Media Cybernetics). First, target regions (cerebellum and hippocampus or corpus callosum and striatum) were identified in the images by outlining regions of interest (ROIs). Additionally, wrinkles, air bubbles, or any other artifacts that interfered with the measurement were excluded from the ROI. Immunofluorescence within the identified areas was then quantified.

For quantification, background correction was used when necessary and immunoreactive objects were identified using adequate threshold and morphological filtering (by size and shape). Various object characteristics were then quantified, including the percentage of total object area based on ROI size (immunoreactive area is the most comprehensive parameter indicating the presence of differences in immunoreactivity), number of objects normalized to ROI size (object density), average signal intensity of detected objects (average intensity indicates differences in the level of cellular expression of target proteins) and the size of suprathreshold objects. Once the parameters of the target objects are determined in the test run, the quantitative image analysis is launched automatically, so the results are independent of the operator and are fully reproducible.

The raw data were organized using Excel and then analyzed using GraphPad Prism software for statistical analysis and graph preparation. Graphs produced using GraphPad Prism software are part of the study report, and raw data tables are appended to the final report after quality checks have been completed.

Measurement of AZ-3102 and glucosylceramide in mouse plasma and brain tissue.

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

Brain tissues were homogenized in a solution of ultrapure water and methanol (1:1) supplemented with 0.1% formic acid (4 ml per gram of tissue) using a FastPrep 24™ Microtube homogenizer. Tissue homogenates were then mixed with an acetonitrile/ultrapure water/methanol (90:5:5) solution containing 500 nM GlcCer C17:0 and 0.1% formic acid to precipitate the protein. After incubation for 5 min at room temperature, samples were centrifuged (5 min, 13,000 rpm, 20°C), and 50 μl of supernatants were transferred to a siliconized 96-well MTP plate.

AZ-3102 measurements.

First, plasma samples were mixed with an acetonitrile solution containing 50 ng/mL AZ-3101 (internal standard) to precipitate the protein. After incubation for 5 min at room temperature, the samples were centrifuged (5 min, 13,000 rpm, 4°C), and the supernatants were diluted 10-fold with ultrapure water with 0.1% formic acid.

Brain tissues were homogenized in a solution of ultrapure water with methanol (1:1) with the addition of 0.1% formic acid (4 ml per gram of tissue) using a FastPrep 24™ Microtube homogenizer (MP Biomedicals, USA). To precipitate proteins, brain homogenates were mixed with an acetonitrile solution containing 10 ng/ml AZ-3101 (internal standard). After incubation for 5 min at room temperature, the samples were centrifuged (5 min, 13,000 rpm, 4°C), and the supernatants were diluted 10-fold with ultrapure water with 0.1% formic acid.

Diluted plasma and brain tissue supernatants were injected into an Agilent LC liquid chromatography system (Agilent, USA) using an automatic 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 reverse-phase XBridge BEH C8 column (2.1*50 mm, particle size 2.5 μm; Waters, USA) at a temperature of 40° WITH. Mobile phase A consisted of ultrapure water supplemented with 0.1% formic acid. Mobile phase B was acetonitrile supplemented with 0.1% formic acid.

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Registration was carried out in positive ionization mode using an API 5500 triple quadrupole mass spectrometer (AB Sciex, USA) equipped with a Turbo Ion Spray interface.

Data were normalized and quantified using Analyst™ data analysis software (AB Sciex, version 1.6.3). The lower limit of quantitation (LLOQ) for AZ-3102 was 0.2 ng/mL in plasma and 2 ng/g brain tissue samples, respectively.

Glucosylceramide measurement

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

Brain tissues were homogenized in ultrapure water/methanol (1:1) supplemented with 0.1% formic acid (4 ml per gram of tissue) using a FastPrep 24™ Microtube homogenizer. Tissue homogenates were then mixed with an acetonitrile/ultrapure water/methanol (90:5:5) solution containing 500 nM GlcCer C17:0 and 0.1% formic acid to precipitate the protein. After incubation for 5 min at room temperature, samples were centrifuged (5 min, 13,000 rpm, 20°C), and 50 μl of supernatants were transferred to a siliconized 96-well MTP plate.

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 using HPLC-MS/MS in monitoring mode multiple reactions (MRM). Supernatants were analyzed by HPLC-MS/MS using a HALO HILIC column (150*4.6 mm, 2.7 μm) from Advanced Materials Technology to distinguish between galactosyl and glucosyl ceramide isomers. MS/MS data were acquired in positive ionization mode using an API 4000 triple quadrupole (Applied Biosystems, USA), with a Turbo Ion Spray interface. Analysis of GlcCer C 16:0 and GlcCer C18:0 was carried out using a gradient with mobile phase A: 5 mM ammonium acetate in a solution of 94.5% acetonitrile, 2.5% methanol, 2.5% ultrapure water and 0.5% formic acid. acid, and with mobile phase B: ultrapure water and 0.1% formic acid. The LLOQ in brain samples for GlcCer 16:0, GlcCer 18:0, and GlcCer 24:1 was 25.1, 250, and 381.5 pmol per gram of tissue, respectively.

Statistics.

Statistical analysis was performed using GraphPad Prism 9 software. Data were presented as mean ± standard error of the mean (SEM) or mean + SEM.

In vivo data: Differences between groups were tested using two-way ANOVA for repeated measures followed by Bonferroni or Dunnett post hoc analyses.

Histology: The distribution of the data could not be verified due to the low n value and was therefore assumed to be normally distributed. Differences between groups were tested using one-way analysis of variance followed by Dunnett's post hoc analysis. Group A (vehicle-treated NPC −/− mice) was used as the control group for pairwise comparisons.

Results.

Body mass.

In fig. Figure 11 shows the percentage change in body weight between PND 11 and week 9. The graph represents the change in body weight [g] in each group, measured daily during the first week of treatment and then once a week.

The results indicate that the overall health of the animals, as measured by mean percentage body weight gain, was not impaired by AZ-3102, and NPC(-/-) mice of both sexes treated with AZ-3102 gained more weight than mice. NPC(-/-) untreated.

Pharmacokinetics.

Table 8

Correlations of AZ-3102 effects on the brain: plasma values after regular oral administration on days PND 11-70

Parameter Brain: plasma parameters Males Females AUCo-24 0.95 1.37 Stakh 0.26 0.41

Glucosylceramide levels.

In fig. 12 shows the levels of glucosylceramide C16:0 and C18:0 after regular oral administration on days PND 11-70.

Increased levels of glucosylceramide C16:0 and C18:0b were observed in mice treated with AZ-3102

- 23 045552 brain.

Clinical signs.

Definitions of clinical indicators and humane endpoints.

Clinical signs were monitored daily from day P45 until the end of the study (according to the schedule shown in Table 9 below). Parameters of weight loss, general health, clinical data and behavior were recorded and assessed on a point scale. The total score was used for the overall assessment.

Table 9

Table for assessing clinical signs

Experimenter (name/initials):

Line of conduct

Parameter

Experiment#: I Line: NPC1 | Date of?

Time:

Clinical data

* Mark in the “Notes” section (deviations from the following clinical examination criteria or symptoms) a: weight loss s: mobility e: msha/skin surface g; stomach i: behavior/activity b: posture d: eyes f: dehydration h: breathing To: temperature t:tremor o: rectal prolapse p: genital prolapse s: did not research specific

Evaluation, result Sum of points Stress level 0 = no stress 0 Stress level 1 = mild stress level that should be monitored closely 1-9 Stress level 2 = medium cipecca, veterinarian should be informed 10-19 Stress level 3 = high stress level, veterinarian and study director should be informed, veterinary assistance or even euthanasia if required 20 or more

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Table 10

Tremor assessment

Tremor assessment Description (Tremor in the animal is observed when the cage is manually tilted and/or when the cage is opened under the work table) Individual behavior rating = 1 “slight incoordination, mild to moderate tremor”: The animal can still walk in a straight line. The animal has mild gait disturbances (wobbly gait and mild tremors). Individual assessment of behavior line = 5 “high level of tremors, uncoordinated movements”: The animal is often unable to walk in a straight line when moving around the cage and has a more pronounced gait disturbance than when scored = 1. Due to severe tremors and loss of coordination, the animal sometimes falls. Individual assessment of the behavior line = 10 “decreased recovery reflex”: The animal exhibits the same symptoms as described for score = 5. Additionally, the animal takes several seconds (approximately at least 2-3 seconds) to assume a sternal recumbent position after being turned onto its back by the examiner.

In fig. 13 shows summary results of overall clinical sign scores for each mouse during PND 56-70 across all score groups and for all groups of NPC(-/-) animals treated with vehicle and AZ-3102. Left: Overall scores for animals and by treatment group, visualized using a gradient, with lower scores in green and higher scores in red. Right side: scores expressed in numerical terms. Cutoff scores greater than 7 are rare in treated animal groups; however, the untreated groups had higher overall scores throughout the study.

The results show that NPC(-/-) mice treated with vehicle had worsening (higher scores) of global clinical signs compared to NPC(-/-) animals treated with AZ3102. This is also shown in Fig. 13: Right side, where scores, eg, greater than 8, were more frequently observed in vehicle-treated NPC(-/-) animals compared to AZ-3102-treated NPC(-/-) animals. By examining these indicators, the inventors found that the occurrence, duration and intensity of tremor were also reduced when animals were treated with AZ-3102 (Fig. 14). High levels of tremor were observed in all vehicle-treated NPC(-/-) animals except one mouse, whereas AZ-3102 essentially eliminated these high levels of tremor in all but one mouse of the 24 animals tested.

In fig. 14 shows tremor scores at postnatal days 56-70 (PND 56-70) in NPC(-/-) mice treated with vehicle and AZ-3102. Pink (=light gray) bars reflect the occurrence and duration of severe tremor (5 points); whereas green (= dark gray) bars reflect the occurrence and duration of mild to moderate tremor. With the exception of one mouse in the untreated group, all other animals showed high levels of tremor (4 out of 5 mice). In contrast, only one mouse in the AZ-3102 group had high levels of tremor (1 of 24). Tremor scores: 0: no tremor; 1: mild incoordination, mild-moderate tremor; 5: high level of tremors, uncoordinated movements; 10: decreased righting reflex (not assessed in any animal).

Histological results.

Defining target areas.

Target regions were marked manually, defining a region of interest (ROI) for subsequent quantitative analysis with fluorescent labeling.

Indicators for quantitative analysis.

The table below shows four standard indicators.

Area size [mm ² ]: this indicator characterizes the average area of the brain area covered by the target area. This indicator is important for checking the correctness of sampling. It is also useful to identify brain atrophy that is part of the phenotype of some animal models.

Immunoreactive area [%]: percentage of the area of interest that is covered by suprathreshold immunoreactive objects (eg: cell somata, neurites, plaques); this is the most comprehensive indicator indicating the presence of overall differences in immunoreactivity.

Object density [number of objects per ^mm2 ]: number of suprathreshold immunoreactive objects normalized to the size of the target area; this measure is particularly useful for detecting changes in neuronal density.

Object brightness [a.u.]: average pixel brightness of suprathreshold immunoreactive objects; this indicator indicates the presence of differences in the level of cellular expression of target proteins.

Object size [ ^μm2 ]: size of suprathreshold immunoreactive objects; this measure is useful for detecting differences in microglial activation or plaque growth.

Kalbindin-B28k.

Calbindin-D28k immunofluorescence was detected using guinea pig polyclonal antibodies, and the signal was quantified in the cerebellum and hippocampus. NCP(-/-) mice showed significantly reduced levels of calbindin-D28k compared to NCP(+/-) and NPC(+/+) mice. Treatment with the test compound significantly increased calbindin-D28k levels in the cerebellum in all groups. All effects are region specific, as no significant differences were found in the hippocampus of mice in the different groups.

In fig. Figure 16 shows brain immunohistochemistry results of calbindin-D28k labeling in vehicle-treated NCP(-/-) and NPC(+/-) mice compared with NPC(+/+, wild-type) and NPC(-/-) mice. ), who received treatment. The graphs represent the average immunofluorescence signal measured within the ROI in 5 mouse brain sections (n=2-4 per group). Data were analyzed using one-way ANOVA and Dunnett's post hoc test. Intervals in the histogram represent group means + SEM. * P value: <0.001.

Using immunohistological methods using calbindin-D28k as a marker for Purkinje cells, we found that treatment with AZ-3102, compared with vehicle-treated NPC(-/-) mice, significantly limited the loss of Purkinje cells in the cerebellum (Fig. 16). ).

Conclusions.

AZ-3102 is a novel oral small molecule compound developed for the treatment of various lysosomal storage disorders. The unique mechanism of action of AZ-3102 lies in its high potency against glucosylceramide synthase (GCS), non-lysosomal glucosylcerebrosidase (GbA2), as well as its brain-penetrating properties. AZ-3102 was studied in a mouse model of Niemann-Pick disease type C [1, 2] in which NPC mice homozygous for the recessive allele of the NIH gene begin to lose body weight and exhibit tremors and ataxic gait at approximately 7 weeks of age [3]. The severity of the disease is associated with significant loss of cerebellar Purkinje cells, and clinical signs worsen until the humane endpoint is reached (usually 12–14 weeks of age) [3]. In this study, we investigated the effectiveness of daily oral administration of AZ-3102 from postnatal day (PND) 11 to 70 in NPC(-/-), NPC(-/+) and WT (NPC(+/)) mice. +); Balb/c) to evaluate the pharmacokinetic (PK) properties of AZ-3102, its ability to modulate glucosylceramide (GlcCer C16:0, GlcCer C18:0) and improve clinical signs of the disease. In addition, the present inventors also examined the effect of treatment on the immunohistochemical marker of Purkinje cells, which correlate with the severity of neuropathology. Following regular daily oral administration of AZ-3102 on days PND 11-70, the animals' general health did not deteriorate as measured by mean percent body weight gain, and NPC(-/-) mice of both sexes treated with AZ-3102 gained more weight than untreated NPC(-/-) animals (Fig. 11). AZ-3102 had a high brain effect: see plasma values (Table 8). Consistent with the targeting of AZ-3102, there was an increase in the number of GlcCer species measured in whole brain homogenates compared with vehicle-treated animals: more than 2-fold for GlcCer 16:0 and approximately 9-fold for GlcCer 18:0 . These two GlcCer species are likely representative of other GlcCer species that will also respond to AZ-3102. To assess whether AZ-3102 reaches sufficient concentrations in the brain to affect more specific features characterizing general health and neuropathology, various clinical features were assessed (Table 9). The scores, summed up over the entire observation period, give a picture of the health status during the period PND 56-70. In fig. Figure 13 provides a graphical representation of the results of assessing the condition of each animal in the observed cohort over time. This figure shows that NPC(-/-) mice treated with vehicle had worsening overall clinical signs (higher scores) compared to NPC(-/-) animals treated with AZ-3102. The quantitative assessment further illustrates (Fig. 13: right side) that higher scores, such as more than 8, were more often observed

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Claims (9)

in a group of NPC(-/-) mice treated with vehicle compared with a group of NPC(-/-) mice treated with AZ-3102. By examining these indicators, the present inventors found that the occurrence, duration, as well as intensity of tremor were also reduced by treatment with AZ-3102 (Fig. 14). High levels of tremor were observed in all vehicle-treated NPC(-/-) animals except one mouse; whereas AZ-3102 essentially eliminated this high level of tremor in all but one mouse of the 24 animals tested. Using immunohistological methods using calbindin-D28k, a marker for Purkinje cells, we found that treatment with AZ-3102, compared with vehicle-treated NPC(-/-) mice, significantly limited the loss of Purkinje cells in the cerebellum (Fig. 16). . This finding is relevant to the clinical picture of Niemann-Pick disease type C in humans, since the death of neurons and, in particular, Purkinje cells, as well as cerebral atrophy are the hallmarks of this disease [4]. Because cerebellar function is important for movement, the improvement in clinical signs and significant reduction in high levels of tremor are likely a result of the survival of Purkinje cells in the cerebellum. In summary, AZ-3102, an orally available highly potent inhibitor (at the nM level) of GCS and GbA2, is able to enter the brain of NPC(-/-) and wild-type animals. Additionally, AZ-3102 inhibited these enzymes in the brain, as is clear from the GlcCer species modulation results, improving clinical signs, significantly reducing high levels of tremor, and limiting the loss of Purkinje cells in the cerebellum, compared with vehicle-treated animals. 1. Loftus, S.K., et al, Murine model of Niemann-Pick C disease: mutation in a cholesterol homeostasis gene. Science, 1997. 277(5323): p. 232-5. 2. Zervas, M., K. Dobrenis, and S.U. 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): p. 49-64. 3. Santiago-Mujica, E., et al., Hepatic and neuronal phenotype of NPC1-/- mice. Heliyon, 2019. 5(3). 4. Vanier, M.T., Niemann-Pick disease type C. Orphanet Journal of Rare Diseases, 2010. 5(1): p. 16. Example 5 Effect of administering the crystalline form of compound (I) to mice suffering from Sandhoff's disease (disruption of the Hexb gene in mice [Hexb(-/-)] through mutations). Mice with Sandhoff's disease were administered a therapeutically effective amount of compound AZ3102. The initial results show that, as with the treatment of Niemann-Pick disease type C, clinical signs in affected animals improved. CLAIM 1. Crystalline form of compound (I), wherein the crystalline form is characterized by a reflection, expressed as a 2θ value, at 17.8±0.2° in the powder X-ray diffraction pattern, with the reflection at 17.8±0.2° being one of the four strongest reflections in the powder X-ray diffraction pattern. 2. The crystalline form of claim 1, further characterized by one or more reflections, expressed as a 2θ value, at one or more angles of 4.1±0.2°, 8.3±0.2°, 12.4±0 ,2°, 13.6±0.2°, 14.5±0.2°, 14.9±0.2°, 15.2±0.2°, 17.2±0.2°, 19 ,3±0.2°, 21.2±0.2°, 22.4±0.2°, 22.9±0.2° and 23.3±0.2° in the X-ray powder diffraction pattern. 3. Crystal form according to claim 1 or 2: (a) having a melting point between 89 and 96°C; and/or (b) the crystalline form is substantially non-hygroscopic. 4. Pharmaceutical composition containing a crystalline form according to any one of claims 1-3. 5. The pharmaceutical composition according to claim 4, wherein the pharmaceutical composition is contained in a capsule. 6. Pharmaceutical composition according to claim 5, where the pharmaceutical composition is contained in a capsule: (a) without any other ingredients; or (b) with at least one pharmaceutically acceptable carrier. 7. Use of a crystalline form according to any of claims 1-3 or a pharmaceutical composition according to any of claims 4-6 in therapy. 8. Use of a crystalline form according to any of claims 1-3 or a pharmaceutical composition according to any of claims 4-6 as a medicine. 9. Use of a crystalline form according to any one of claims 1 to 3 or a pharmaceutical composition according to any of claims 4 to 6 in the treatment of a disease associated with abnormal levels of glucosylceramide and/or elevated levels of glycosphingolipids. -