RU2806322C2 - Stable polymorphic form of 6-fluoro-9-methyl-9h-beta-carboline and its applications - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: crystalline polymorphic form of 6-fluoro-9-methyl-9H-β -carboline of Formula (I), wherein the crystalline polymorph has an X-ray powder diffraction pattern containing peaks at 2-theta angles of 11.3, 17.1, 17.6, 18.0, 22.5, 23.2 and 29.4 degrees with deviation± 0.2 degrees. The invention also relates to a pharmaceutical composition for the treatment and/or prevention of hearing impairment, dizziness or vestibular disorder, containing a crystalline polymorphic form of 6-fluoro-9-methyl-9H-β -carboline according to the invention together with at least one pharmaceutically acceptable carrier, excipient, and/or diluent. Crystalline polymorphic form of 6-fluoro-9-methyl-9H-β-carboline of Formula (I) is prepared by A1) providing 6-fluoro-9-methyl-9H-β-carboline of Formula (I); B1) dissolution of 6-fluoro-9-methyl-9H-β-carboline in a mixture of a polar solvent and a non-polar solvent; or dissolving 6-fluoro-9-methyl-9H-β-carboline first in a polar solvent and adding a non-polar solvent to the resulting solution 6-fluoro-9-methyl-9H-β-carboline in a polar solvent, where the polar solvent is dichloromethane, acetone, isopropanol or a mixture thereof, or a mixture with water, and the non-polar solvent is methyl tert-butyl ether, n-heptane, cyclohexane or a mixture thereof, and the ratio of polar solvent and non-polar solvent is in the range from 1:2 to 1:10; and concentration of 6-fluoro-9-methyl-9H-β-carboline in a mixture of polar solvent and non-polar solvent is in the range from 50 mm to 200 mm; C1) heating a solution or suspension of 6-fluoro-9-methyl-9H-β-carboline to temperatures ranging from 40°C up to 100°C; D1) stirring the resulting solution for 10 minutes at the same temperature; E1) cooling the resulting solution to a temperature in the range from -10°C to +30°C to obtain the crystalline polymorphic form 6-fluoro-9-methyl-9H-β-carboline; and F) isolating crystalline polymorphic form B 6-fluoro-9-methyl-9H-β -carboline of Formula (I).

(I)

EFFECT: stable crystalline form of 6-fluoro-9-methyl-9H-β-carboline, for the treatment and/or prevention of hearing impairment, dizziness or vestibular disorders.

14 cl, 16 dwg, 6 tbl, 9 ex

Description

The present invention relates to a stable crystalline polymorphic form of 6-fluoro-9-methyl-9H-β-carboline of formula (I), a method for producing said crystalline polymorphic form of 6-fluoro-9-methyl-9H-β-carboline and a pharmaceutical composition, containing the specified crystalline polymorphic form of 6-fluoro-9-methyl-9H-β-carboline. Furthermore, the present invention relates to the use of said stable crystalline polymorphic form and to the use of a pharmaceutical composition containing said stable crystalline polymorphic form in the treatment and/or prevention of hearing impairment, dizziness or vestibular disorders.

BACKGROUND OF THE INVENTION

Polymorphism is the ability of a chemical to exist in more than one distinct crystalline form, having different arrangements of molecules in the crystal lattice. Although polymorphs of the same compounds are chemically identical, each polymorph has its own unique combination of chemical, mechanical, thermal, and physical properties. Differences in the physicochemical properties of different crystalline forms make polymorphism a potentially important issue for pharmaceutical companies (Erdemir et al.; Curr. Opin. Drug Discov. Dev. 2007, 746-755). Difficulties and inconsistencies encountered in product characterization and development can be attributed to polymorphism, and it is generally accepted in the pharmaceutical industry that polymorphism is a critical aspect to consider when developing new drug candidates. Active pharmaceutical ingredients (APIs) are often delivered to patients in a solid state because solid forms provide convenience, physical and chemical stability, ease of product handling, and low manufacturing costs. Since each solid form has unique physicochemical properties, understanding and controlling the properties of APIs in the solid state is extremely important in the drug development process. Unintended formation of an inappropriate polymorph during the crystallization step can result in pharmaceutical dosage forms that are either ineffective at the intended dose for a given application or can potentially become toxic. For these reasons, regulatory agencies require pharmaceutical companies to control the crystallization process so that the desired polymorph is produced continuously and encourage the use of process analytical technologies to develop the crystallization process.

Recrystallization from solution can be thought of as a self-assembly process in which randomly organized molecules dissolved in a supersaturated solvent or mixture of solvents come together to form an ordered three-dimensional molecular array with a periodic repeating pattern. Crystallization is critical to many processes occurring in nature and in the production of a wide range of materials. The quality of a crystalline product is usually determined by four main criteria: size, purity, morphology and crystal structure. Controlling crystal morphology is important in many applications because particle behavior can have a profound impact on post-crystallization processes. For API development, it is critical to obtain a specific polymorph that ensures bioavailability and stability of the drug in the final dosage form.

The German patent application (DE 10 2007 009264 A1) describes 9-alkyl-β-carbolines which, due to their neuroprotective properties, can be used for the treatment and/or prevention of motor system disorders and/or neurological diseases, such as e.g. Alzheimer's disease or Parkinson's disease.

US patent application (US 2004/038970 A1) describes the use of 3-substituted 2,3,4,9-tetrahydro-1H-β carbolines as active agents for the treatment of various clinical pathologies, including tinnitus. However, the substitution pattern of this group of β-carbolines differs significantly in structure from the substitution pattern of 6-fluoro-9-methyl-β-carboline.

The international patent application (WO 2011/079841 A1) describes β-carbolines, preferably 9-alkyl-β-carbolines, a method for their preparation and a pharmaceutical composition containing said β-carbolines. In addition, this PCT application relates to the use of these β-carbolines for the prevention and treatment of hearing loss, tinnitus, sound shocks, dizziness and balance disorders.

The international patent application (WO 2015/044434 A2) describes fluoro-9-methyl-β-carbolines, including 6-fluoro-9-methyl-9H-β-carboline, a method for its preparation and pharmaceutical compositions containing fluoro-9-methyl -β-carbolines. In addition, this PCT application discloses the medical use of fluoro-9-methyl-β-carbolines for the treatment of acute and chronic ear diseases and hearing disorders, dizziness and balance disorders.

On the other hand, the international patent application (WO 2015/044434 A2) describes 6-fluoro-9-methyl-β-carboline and does not describe any crystalline or polymorphic form thereof.

It is an object of the present invention to provide a stable crystalline polymorphic form of 6-fluoro-9-methyl-β-carboline, especially as an active ingredient for the preparation of pharmaceutical compositions, and to provide such stable pharmaceutical compositions containing said stable crystalline polymorphic form of 6-fluoro-9- methyl-β-carboline, as well as its uses and method for obtaining this stable polymorphic form.

This problem of the present invention is solved using the ideas set forth in the independent claims. Other preferred features, aspects and details of the invention will become apparent from the dependent claims, description, drawings and examples of this application.

The essence of the invention

The present invention first provides several specific crystalline polymorphs of 6-fluoro-9-methyl-β-carboline, also referred to herein as 6-FMC, and discloses their technical features. However, it has surprisingly been found that only one of these specific crystalline polymorphic forms is sufficiently stable and therefore suitable for the preparation of pharmaceutical compositions. All other polymorphic forms cannot be used for pharmaceutical compositions due to their instability.

The compound 6-fluoro-9-methyl-β-carboline (6-FMC) exists in at least four polymorphic forms, designated polymorph A, B, C and T. In practice, 6-FMC is used as a suspension to achieve therapeutic concentrations in the target tissue. It is critical to obtain a specific polymorph that ensures bioavailability and stability of the drug in the final dosage form. In this regard, the chemical, mechanical, thermal and physical properties of each polymorph, both in powder form and as part of the composition, were characterized. Noticeable differences between polymorphs were found. The melting points differ and recrystallization produces orthorhombic forms for polymorph A and monoclinic forms for polymorph B. The X-ray powder diffraction spectra are clearly different, as are the TTNMR and infrared spectroscopy spectra. Polymorphs C and T are difficult to isolate in pure form due to their rapid decomposition, so pure spectra of these polymorphs are difficult to obtain. Polymorph stability studies have shown that polymorphs A, C and T are not suitable for pharmaceutical composition, and polymorph A is not stable but can be converted to polymorph B under the conditions described herein. Surprisingly, it has been discovered that polymorph B is the only polymorph that is sufficiently stable and suitable for the preparation of a pharmaceutical composition containing crystalline 6-fluoro-9-methyl-β-carboline particles of polymorph B. Polymorphs A, C and T are not even stable enough under normal storage conditions of pharmaceutical compositions at room temperature, therefore, polymorphic forms A, C and T of 6-fluoro-9-methyl-β-carboline cannot be used as a crystalline active ingredient in pharmaceutical compositions for the treatment of diseases of the inner ear. Such pharmaceutical compositions are preferably liposomal compositions, ointments, suspensions, gels and emulsions, in which the 6-fluoro-9-methyl-β-carboline polymorph Form B is present in crystalline or micronized form, preferably in the form of microparticles or nanoparticles.

Accordingly, several stability tests have been carried out on polymorph B of 6-fluoro-9-methyl-β-carboline and the present application has proven that polymorph B is very stable and an ideal form for the desired pharmaceutical compositions. Only under stringent conditions, as described herein, can polymorph B be converted to polymorph A. Conversion of polymorph B to polymorph A can be achieved by supercritical carbon dioxide extraction as well as by vacuum sublimation, while spontaneous conversion of polymorph A to B occurs in the composition immediately after mixing. The reverse process has never been observed. In addition, the third polymorph C and the fourth polymorph T are degraded even during the manufacture of the pharmaceutical composition. Any conversion of polymorph B to polymorph C or to polymorph T cannot be detected even under extreme conditions such as exposure to supercritical carbon dioxide, vacuum sublimation, or extreme heating.

Thus, the present application relates to 6-fluoro-9-methyl-β-carboline polymorph Form B as the only stable polymorph form of 6-fluoro-9-methyl-β-carboline that is suitable for the preparation of the pharmaceutical compositions described herein.

Description of the invention

Accordingly, the present invention relates to a crystalline polymorphic form of 6-fluoro-9-methyl-9H-β-carboline of formula (I)

( I )

wherein the crystalline polymorph of 6-fluoro-9-methyl-9H-β-carboline (herein referred to as polymorph B or polymorph B) has an X-ray powder diffraction pattern containing peaks at 2-theta angles of 11.3, 17.1, 17.6 , 18.0, 22.5, 23.2, and 29.4 degrees with a deviation of ±0.2 degrees.

Preferably, this crystalline polymorph of 6-fluoro-9-methyl-9H-β-carboline has an X-ray powder diffraction pattern containing peaks at 2-theta angle values of 11.3, 14.1, 17.1, 17.6, 18, 0, 19.0, 20.3, 20.6, 22.5, 23.2, 24.3, 25.8, and 29.4 degrees with a deviation of ±0.2 degrees. More precisely, each specified value has a deviation of ±0.2 degrees and can be written as follows: 11.3±0.2 degrees, 14.1±0.2 degrees, 17.1±0.2 degrees, 17.6± 0.2 degrees, 18.0±0.2 degrees, 19.0±0.2 degrees, 20.3±0.2 degrees, 20.6±0.2 degrees, 22.5±0.2 degrees, 23.2±0.2 degrees, 24.3±0.2 degrees, 25.8±0.2 degrees and 29.4±0.2 degrees. Preferably, the deviation is only ±0.15 degrees and more preferably only ±0.1 degrees.

In one embodiment, the crystalline polymorphic form of 6-fluoro-9-methyl-9H-β-carboline (polymorph B) is in a monoclinic form having space group p2 _1/ c, where one molecule is 6-fluoro-9-methyl-9H- β-carboline is located in an asymmetric unit cell having unit cell parameters a=3.85±0.1 Å, b=17.32±0.1 Å, c=13.77±0.1 Å, α=90± 3°, β=91±3° and γ=90±3°.

In one embodiment, the solid-state ¹³ C-NMR spectrum of said crystalline polymorph (polymorph B) of 6-fluoro-9-methyl-9H-β-carboline contains peaks at 28.6, 107.3, 110.2, 111.6, 112.8, 116.6, 121.5, 126.2, 138.0, 155.5, 156.1, 156.7, 157.1, 157.2, 157.7, 158.1 and 158, 4 ppm with a deviation of ±1 ppm, as shown in Fig. 8B. Accordingly, contains peaks at 28.6±1 ppm, 107.3±1 ppm, 110.2±1 ppm, 111.6±1 ppm, 112.8±1 ppm, 116.6±1 ppm, 121.5±1 ppm, 126.2±1 ppm, 138.0±1 ppm, 155.5±1 ppm, 156.1±1 ppm, 156.7±1 ppm, 157.1±1 ppm, 157.2±1 ppm, 157.7±1 ppm, 158.1±1 ppm, and 158.4±1 ppm Preferably the deviation is only ±0.5 ppm. and more preferably only ±0.2 ppm.

In one embodiment, the crystalline polymorphic Form B of 6-fluoro-9-methyl-9H-β-carboline according to the invention has a melting point of 123±1°C.

More preferably, the IR spectrum of said 6-fluoro-9-methyl-9H-β-carboline crystalline polymorph B contains peaks at 803.27, 819.54, 852.30, 864.13, 893.22, 1024.99 , 1126.71, 1148.77, 1183.98, 1272.31, 1283.82, 1333.10, 1362.06, 1426.98, 1448.53, 1481.82, 1554.80 and 1578.94 cm ^{- 1} with a deviation of ±5 cm ^-1 . Preferably the deviation is only ±4 cm ^-1 and more preferably only ±3 cm ^-1 .

Most preferably, the IR spectrum of said crystalline polymorphic form contains peaks at 426.62, 524.31, 558.12, 604.22, 636.09, 704.73, 729.05, 742.59, 803.27, 819 .54, 852.30, 864.13, 893.22, 1024.99, 1126.71, 1148.77, 1183.98, 1272.31, 1283.82, 1333.10, 1362.06, 1426.98 , 1448.53, 1481.82, 1554.80 and 1578.94 cm ^-1 with a deviation of ±5 cm ^-1 .

In the present application, another crystalline polymorph of 6-fluoro-9-methyl-9H-β-carboline (hereinafter polymorph A) has been identified as shown in Fig. 1A. However, this polymorphic form is not stable enough and is described herein as a comparative example, but is not part of the present invention. The crystalline polymorphic form of 6-fluoro-9-methyl-9H-β-carboline (polymorph A) has been fully characterized, while polymorphs C and T are so unstable that even their complete characterization has hardly been possible. This crystalline polymorphic form A is in the orthorhombic form and has space group p2 ₁ 2 ₁ 2 ₁ , one molecule of 6-fluoro-9-methyl-9H-β-carboline is in the unit cell, and the unit cell parameters are a=5.90± 0.1 Å, b=10.35±0.1 Å, c=15.46±0.1 Å, α=90±3°, β=90±3° and γ=90±3°.

The crystal structure of polymorph A (Figure 3A) was characterized in detail using X-ray crystallography. This polymorph crystallizes in the orthorhombic form with space group p2 ₁ 2 ₁ 2 ₁ . The unit cell parameters are as follows: a =5.8986 (1) Å, α=90°; b =10.3506 (3) Å, β=90°; c =15.4572 (5) Å, γ=90°. As shown in Figure 2A, 6-FMC molecules are found in multidimensional layers of pi-stack molecules and orthogonal T-stack molecules. It is also clear to see that there are no hydrates, solvates, or salt-based counterions in the structure.

The composition picture confirmed the identity of the described compound.

The specified polymorphic form A has an X-ray powder diffraction pattern containing peaks at 2-theta angles of 11.9, 13.8, 16.5, 20.0, 23.8, 24.0, 25.6, 26.2, 26, 7 and 28.1 degrees with a deviation of ±0.2 degrees. Particularly characteristic are signals with 2-theta angle values of 11.9, 16.5, 26.7 and 28.1.

Melting points were measured using a Buchi M565 melting point meter with an open capillary. The heating rate was slowed down at 100°C to 1°C per minute to more accurately determine the melting point. The uncorrected melting point was found to be ~125°C ±2°C. The melting point was confirmed using differential scanning calorimetry (DSC). Heating and cooling curves were recorded using differential scanning calorimetry (DSC; Netzsch DSC 204 F1) (Fig. 5A). The melting point was confirmed by heating with DSC. The cooling curve showed a wide range between 65°C and 45°C (Fig. 6A).

The solid-state ¹³ C-NMR spectrum of the indicated crystalline polymorphic form contains peaks at 28.6, 103, 111, 113, 114, 118, 124, 130, 133, 135 and 155-158 (many CF peaks) ppm. with deviation ±1 ppm (Figure 8A). FIG. 8A shows the tNMR spectrum of polymorph A, and FIG. 8B shows the tNMR spectrum of 6-fluoro-9-methyl-9H-β-carboline polymorph B. These spectra show clear differences between the two polymorphs A and B.

Stability of polymorph B of the invention compared to other crystalline forms

The degree of conversion usually depends on the relative stability of the polymorphs, kinetic barriers to phase transformation, and the applied voltage. However, phase transformation is generally not a major concern provided that the transformation occurs in a natural manner, as part of a well-established production process where the critical process parameters of the production process are well understood and controlled.

The most thermodynamically stable polymorphic form of a drug is often selected during development based on minimal potential for conversion to another polymorphic form and based on its higher chemical stability.

In this application, the stability of the 6-fluoro-9-methyl-9H-β-carboline polymorph of the invention (polymorph B) is tested and compared with other 6-fluoro-9-methyl-9H-β-carboline polymorphs (polymorphs A, C and T ) as a comparative example.

An attempt was made to convert the 6-fluoro-9-methyl-9H-β-carboline polymorph of the invention (polymorph B) into another polymorph (such as polymorph A) under different temperature conditions, as described in Example 3.

The crystalline polymorph of 6-fluoro-9-methyl-9H-β-carboline (polymorph B) was dissolved in an organic solvent and the mixture was heated. As described in Example 3, it was not possible to completely convert a crystalline polymorph (polymorph B) to another form (here polymorph A).

The thermal stability of the polymorphic form of the invention compared to other polymorphic forms is surprising and technically advantageous, particularly for pharmaceutical production and regulation. The search results show that at room temperature and even below room temperature, polymorph A is converted partially or completely to a thermally more stable form, which is polymorph B. This conversion can be accelerated by increasing the temperature. This thermal instability of polymorph A is a disadvantage that makes this polymorph unsuitable for pharmaceutical purposes.

Complete conversion of the polymorphic form of the invention (polymorph B) to polymorph A can only be achieved under harsh conditions, for example, supercritical CO ₂ as described in example 4. The cylinder of the device into which supercritical CO ₂ was introduced was loaded with polymorph B ( Fig.9 A) and heated to 60°C at a pressure of 35 kPa. The time it took for the API to completely dissolve in supercritical CO ₂ was 2.5 hours. The spectrum of the final product is shown in Fig. 9B. It is clear that the conversion of polymorph B to polymorph A occurred. The powder was white (Fig. 9C). However, it is clear that such conditions are not applicable during the preparation of the pharmaceutical compositions of interest. Moreover, the fact that polymorph B can be converted into another polymorphic form only under extreme conditions demonstrates that polymorph B is absolutely stable under the conditions of manufacture, storage and use of the pharmaceutical compositions of interest.

Various dissolution/crystallization conditions were used to attempt to convert polymorph B to polymorph A. As mentioned above, only through complex procedures can polymorph B be completely converted to polymorph A (Example 4). Polymorphic forms of a drug can undergo phase change through a number of manufacturing processes, such as drying, grinding, micronization, wet granulation, spray drying and compression. Exposure to environmental conditions such as humidity and temperature can also cause polymorph transformation. Thus, polymorph A is unstable and therefore not suitable for pharmaceutical processes, whereas polymorph B was stable under all these conditions, including drying, grinding, micronization, wet granulation, spray drying and compression.

Unlike polymorph A, the crystalline (polymorph B) of the invention is very stable, and therefore does not undergo transformation when exposed to a number of manufacturing and formulation processes. This is an unexpected technical advantage.

A third polymorph of 6-fluoro-9-methyl-9H-β-carboline, polymorph C, was prepared after recrystallization of polymorph B in a ternary solvent mixture of heptane, ethanol and water, as described in Example 7 and shown in FIG. 15. The resulting polymorph C was very unstable under standard conditions, and within a couple of weeks at room temperature, 40% to 60% relative humidity, and atmospheric pressure, polymorph C was completely converted to polymorph B.

By dissolving 6-fluoro-9-methyl-β-carboline in acidic water and titrating the solution to pH 12, the fourth polymorph of 6-fluoro-9-methyl-9H-β-carboline, polymorph T, appears to be the less stable polymorph 6 -fluoro-9-methyl-β-carboline. After just a couple of days under standard conditions (room temperature, relative humidity from 40% to 60% and atmospheric pressure), no T polymorph could be detected. This rapid disappearance of the T polymorph has made its complete characterization quite challenging. However, it can be argued that neither of the C and T polymorphs is a hydrate or salt form. Isolation of the indicated polymorph T was unsuccessful. When polymorph A is stored in aqueous media, conversion to polymorph B and polymorph T signals are observed. At least the 2-theta values characteristic of polymorph T can be determined by subtracting the signals belonging to polymorph B from the mixed diffraction pattern. A typical X-ray powder diffraction pattern is shown in FIG. 16. All of these findings clearly highlight that polymorph B is the most stable polymorph of 6-FMC and is, in fact, the only polymorph suitable for preparing the desired pharmaceutical compositions.

Micronization and stability of the polymorph in a poloxamer-based composition:

In a series of experiments, the stability of the polymorph was analyzed. Micronized (≤ 20 μm) polymorphs A and B were added to a phosphate buffer containing a nonionic surfactant as a carrier composition. After 30 h, aliquots were removed, washed, dried, and prepared to determine the identity of the polymorphs using X-ray powder diffractometry (XRPD). XRPD analysis showed that in a sample originally prepared from polymorph A, 60% of the polymorph was converted to polymorph B within those 30 hours (FIG. 10). 48 hours after preparation, XRPD analysis showed 100% conversion of polymorph A to B. These data demonstrate that polymorph B of the invention is a stable crystalline state in the composition. Consistency analysis carried out on a sample originally prepared from polymorph B of the invention at regular intervals over several months showed no conversion of the polymorph (FIG. 12).

In one embodiment, the crystalline polymorphic form of 6-fluoro-9-methyl-9H-β-carboline has a crystalline material particle size of ≤ 200 μm, preferably ≤ 175 μm, more preferably ≤ 150 μm, even more preferably ≤ 125 μm, even more preferably ≤ 100 µm, even more preferably ≤ 75 µm and even more preferably ≤ 50 µm. Most preferably, said crystalline polymorph has a crystal particle size of ≤ 20 μm.

Efficiency in vivo

The present application demonstrates that 6-fluoro-9-methyl-β-carboline (6-FMC) polymorph Form B affects in vivo efficacy as described in Example 9. The results are shown in FIG. 13 and FIG. 14.

The effect of intratympanic administration of 6-FMC polymorph A (0.12 mg), prepared fresh (less than 24 hours before use), on permanent threshold shift was studied in guinea pigs (Fig. 13). Permanent threshold shift (PTS) is defined as the difference between the post-traumatic hearing threshold measured on day 14 and the baseline hearing threshold measured on day 3.

In addition, the effect of intratympanic administration of 6-FMC polymorph B (0.12 mg), prepared at least 48 hours before use, on guinea pig PTS is presented in Fig. 14.

Using a guinea pig model of noise-induced hearing loss (NIHL), the applicant investigated the effectiveness of a single intratympanic application of 6-FMC in the treatment of hearing loss. It was noted that animals treated with 6-FMC polymorph A had a modest improvement in PTS recovery. Compared to vehicle-treated animals, polymorph A treatment demonstrated a 10-18 dB PTS recovery at 16 kHz (FIG. 13). However, clinical use will require a stronger potency.

Surprisingly, during further development it was discovered that there was a clear improvement in efficacy in animals treated with 6-FMC compared to previous experiments (Fig. 14). Surprisingly, a much stronger improvement in PTS was found at all frequencies tested. The level of improvement increased from about 10-18 dB, as seen in FIG. 13, to 23-39 dB, as seen in FIG. 14. This improvement of 30 dB or more observed with polymorphic form B is clinically most significant since hearing is expressed on a logarithmic scale (in decibels). In humans, a hearing loss of 18 dB may hardly be noticed by the subject, whereas a hearing loss of 30 dB makes communication difficult, especially if there is background noise at the same time. Surprisingly, at some frequencies (eg, 4 kHz and 8 kHz, FIG. 14), animals treated with the polymorph B form of 6-FMC exhibited a PTS close to zero.

This strong recovery is important because it elucidates the possibility that treatment with 6-FMC polymorph B can lead to complete hearing recovery after noise-induced hearing loss.

To determine the underlying cause of the increased efficacy data, the applicant conducted a thorough analysis of the experimental procedures and demonstrated that the increased efficacy was dependent on the formulation technology. Initially, the freshly prepared composition containing polymorph A was always used immediately after preparation. However, in experiments that demonstrated increased efficacy, the composition was prepared in advance and stored for at least 48 hours before use. Subsequent analysis of the composition showed that the composition, which was originally prepared with polymorphic form A, underwent complete conversion to polymorphic form B, partially up to 60% within 30 hours (Fig. 10) and completely after 48 hours, as shown in Fig. eleven. Taken together, these data indicate that the identity of the polymorphic form of 6-FMC present in the composition plays a very important role. In addition, Applicant has demonstrated that the Form B polymorph composition results in significantly improved in vivo efficacy data in the guinea pig NIHL model compared to the Form A polymorph composition.

Method for obtaining crystalline polymorphic form

Another aspect of the present invention relates to a method for preparing the crystalline polymorphic form of 6-fluoro-9 H -methyl-β-carboline according to the invention, comprising the following steps:

A1) providing 6-fluoro-9-methyl-9H-β-carboline of formula (I)

(I)

B1) dissolving 6-fluoro-9-methyl-9H-β-carboline in a mixture of a polar solvent and a non-polar solvent; or

dissolving 6-fluoro-9-methyl-9H-β-carboline first in a polar solvent and adding a non-polar solvent to the resulting solution of 6-fluoro-9-methyl-9H-β-carboline in a polar solvent,

where the polar solvent is dichloromethane, acetone, isopropanol or a mixture thereof, or a mixture with water, and the non-polar solvent is methyl tert-butyl ether, n-heptane, cyclohexane or a mixture thereof, and the ratio of the polar solvent and non-polar solvent is range from 1:2 to 1:10;

C1) heating the solution or suspension of 6-fluoro-9-methyl-9H-β-carboline to a temperature in the range of 40°C to 100°C;

D1) stirring the resulting solution for at least 10 minutes at the same temperature;

E1) cooling the resulting solution to a temperature in the range from -10°C to +30°C to obtain a crystalline polymorphic form of 6-fluoro-9-methyl-9H-β-carboline according to claim 1; And

F1) isolation of the crystalline polymorphic form B of 6-fluoro-9-methyl-9H-β-carboline of formula ( I )

(I).

It should be emphasized that all initial attempts to obtain the crystalline form of 6-FMC resulted in polymorphic form A. Polymorphic form A was obtained by crystallization from non-polar or weakly polar aprotic organic solvents such as toluene, cyclohexane or heptane. Therefore, polymorph Form A was initially considered by the inventors to be a stable polymorph form, since this polymorph Form A was always obtained after recrystallization from n-heptane. The identification of polymorphic form B as a much more stable and much more active form was an unexpected result.

Following the discovery that the B polymorph is pharmacologically highly potent and chemically highly stable, the synthesis procedure described above was developed which selectively leads to the B polymorph.

The isolated crystalline polymorphic form B of 6-fluoro-9-methyl-9H-β-carboline has a powder X-ray diffraction pattern containing peaks at 2-theta angles of 11.3, 17.1, 17.6, 18.0, 22.5, 23.2 and 29.4 degrees with a deviation of ±0.2 degrees, as described above.

Preferably, the crystalline polymorph Form B of 6-fluoro-9-methyl-9H-β-carboline has a powder X-ray diffraction pattern containing peaks at 2-theta angle values of 11.3, 14.1, 17.1, 17.6, 18.0 , 19.0, 20.3, 20.6, 22.5, 23.2, 24.3, 25.8, and 29.4 degrees with a deviation of ±0.2 degrees as described above.

Also preferably, the crystalline polymorphic form of 6-fluoro-9-methyl-9H-β-carboline according to the invention (polymorph B) is in a monoclinic form having space group p2 ₁ c, where one molecule of 6-fluoro-9-methyl-9H- β-carboline is located in an asymmetric unit cell having unit cell parameters a=3.85±0.1 Å, b=17.32±0.1 Å, c=13.77±0.1 Å, α=90± 3°, β=91±3° and γ=90±3° as described above.

More preferably, the solid-state ¹³ C-NMR spectrum of said crystalline polymorph (polymorph B) of 6-fluoro-9-methyl-9H-β-carboline contains peaks at 28.6, 107.3, 110.2, 111.6, 112 .8, 116.6, 121.5, 126.2, 138.0, 155.5, 156.1, 156.7, 157.1, 157.2, 157.7, 158.1 and 158.4 m.d. with a deviation of ±1 ppm, as shown in Fig. 8B.

More preferably, the crystalline polymorph Form B of 6-fluoro-9-methyl-9H-β-carboline has a melting point of 123±1°C as described above.

Even more preferably, the IR spectrum of said 6-fluoro-9-methyl-9H-β-carboline crystalline polymorph B contains peaks at 803.27, 819.54, 852.30, 864.13, 893.22, 1024. 99, 1126.71, 1148.77, 1183.98, 1272.31, 1283.82, 1333.10, 1362.06, 1426.98, 1448.53, 1481.82, 1554.80 and 1578.94 cm ^-1 with a deviation of ±5 cm ^-1 . Most preferably, the IR spectrum of said crystalline polymorph B contains peaks at 426.62, 524.31, 558.12, 604.22, 636.09, 704.73, 729.05, 742.59, 803.27, 819.54, 852.30, 864.13, 893.22, 1024.99, 1126.71, 1148.77, 1183.98, 1272.31, 1283.82, 1333.10, 1362.06, 1426, 98, 1448.53, 1481.82, 1554.80 and 1578.94 cm ^-1 with a deviation of ±5 cm ^-1 as described above.

Preferably, in step B1), the concentration of 6-fluoro-9-methyl-9H-β-carboline in the organic solvent mixture is in the range of 50 mM to 200 mM, preferably 50 mM to 150 mM, more preferably 80 mM to 120 mM .

After performing step C1), the 6-fluoro-9-methyl-9H-β-carboline suspension is also converted into the resulting solution as indicated in step D1).

Preferably, in step C1), the 6-fluoro-9-methyl-9H-β-carboline solution or suspension is heated to a temperature in the range of 40°C to 60°C.

Preferably, in step D1) or after step D1) of the above methods, the following step D2 is performed):

D2) concentrating the mixture of the resulting solution or suspension by evaporating the solvents, preferably under vacuum.

Optionally, in step E1), after cooling the resulting solution, the following step can be performed:

E2) seeding the crystalline polymorphic Form B of 6-fluoro-9-methyl-9H-β-carboline.

Various production methods are described in detail in examples 1 and 2.

In some embodiments, in Step A) of the methods described above, 6-fluoro-9-methyl-9H-β-carboline of formula (I) is a crystalline polymorph of Form A, wherein said polymorph has an X-ray powder diffraction pattern containing peaks at an angle of 2 -theta equal to 11.9, 16.5, 26.7 and 28.1 degrees with a deviation of ±0.2 degrees, or alternatively 11.9, 13.8, 16.5, 20.0, 23.8, 24.0, 25.6, 26.2, 26.7 and 28.1 degrees with a deviation of ±0.2 degrees.

Pharmaceutical composition and medical use

Another aspect of the present invention relates to the medical use of a crystalline polymorph of 6-fluoro-9-methyl-β-carboline (polymorph Form B) of the invention, wherein the crystalline polymorph of 6-fluoro-9-methyl-9H-β-carboline of the invention has a powder X-ray diffraction pattern containing peaks at 2-theta angles of 11.3, 17.1, 17.6, 18.0, 22.5, 23.2 and 29.4 degrees with a deviation of ±0.2 degrees.

Preferably, said crystalline polymorph Form B of 6-fluoro-9-methyl-9H-β-carboline has a powder X-ray diffraction pattern containing peaks at 2-theta angle values of 11.3, 14.1, 17.1, 17.6, 18, 0, 19.0, 20.3, 20.6, 22.5, 23.2, 24.3, 25.8, 28.4 and 29.4 degrees with a deviation of ±0.2 degrees as described above.

Also preferably, said crystalline polymorphic form is in a monoclinic form having space group p2 _1/ c, where one molecule of 6-fluoro-9-methyl-9H-β-carboline is in an asymmetric unit cell having unit cell parameters a=3, 85±0.1 Å, b=17.32±0.1 Å, c=13.77±0.1 Å, α=90±3°, β=91±3° and γ=90±3°, as described above.

More preferably, the solid-state ¹³ C-NMR spectrum of said 6-fluoro-9-methyl-9H-β-carboline crystalline polymorph contains peaks at 28.6, 107.3, 110.2, 111.6, 112.8, 116 .6, 121.5, 126.2, 138.0, 155.5, 156.1, 156.7, 157.1, 157.2, 157.7, 158.1 and 158.4 ppm. with a deviation of ±1 ppm, as shown in Fig. 8B and as above.

Even more preferably, said crystalline polymorph Form B of the pharmaceutical composition has a melting point of 123±1°C.

Even more preferably, the IR spectrum of said crystalline polymorphic form B of the pharmaceutical composition contains peaks at 803.27, 819.54, 852.30, 864.13, 893.22, 1024.99, 1126.71, 1148.77, 1183 .98, 1272.31, 1283.82, 1333.10, 1362.06, 1426.98, 1448.53, 1481.82, 1554.80 and 1578.94 cm ^-1 with a deviation of ±5 cm ^-1 .

In one embodiment, The above crystalline polymorphic form of 6-fluoro-9-methyl-β-carboline (polymorphic form B) according to the invention is useful for the treatment and/or prevention of hearing impairment, dizziness or vestibular disorder.

Preferably, the above crystalline polymorph Form B of 6-fluoro-9-methyl-β-carboline is useful for the treatment and/or prevention of hearing impairment, dizziness or vestibular disorder, wherein the hearing impairment, dizziness or vestibular disorder is selected from the group consisting of Meniere's disease , sudden sensorineural hearing loss, noise-induced hearing loss, age-related hearing loss, autoimmune ear disease, tinnitus, acoustic trauma, blast trauma, labyrinthine deafness, presbycusis, trauma during implantation of an inner ear prosthesis (insertion trauma), dizziness due to diseases of the inner ear and hearing impairment due to antibiotics and cytostatics.

Another aspect of the present invention relates to a pharmaceutical composition containing the above crystalline polymorphic form of 6-fluoro-9-methyl-9H-β-carboline (polymorphic form B) together with at least one pharmaceutically acceptable carrier, excipient, solvent and/or thinner.

In one embodiment, said pharmaceutical composition is useful for the treatment and/or prevention of hearing impairment, dizziness or vestibular disorder.

Preferably, said pharmaceutical composition is useful for the treatment and/or prevention of hearing loss, dizziness or vestibular disorder, wherein the hearing loss, dizziness or vestibular disorder is selected from the group consisting of Meniere's disease, sudden sensorineural hearing loss, noise-induced hearing loss, age-related loss hearing, autoimmune ear disease, tinnitus, acoustic trauma, blast trauma, labyrinthine deafness, presbycusis, trauma during inner ear prosthesis implantation (insertion trauma), vertigo due to inner ear diseases, and hearing loss due to antibiotics and cytostatics.

The above 6-fluoro-9-methyl-β-carboline polymorph Form B or the above pharmaceutical compositions containing 6-fluoro-9-methyl-β-carboline polymorph Form B can be prepared and administered in the form of transdermal administration systems (patches , films), drops, pills, dragees, gels, hydrogels, ointments, syrups, granules, suppositories (ovules), emulsions, dispersions, microcompositions, nanocompositions, liposomes, solutions , juices, suspensions, solutions for infusion or solutions for injections . Preferred are pharmaceutical compositions in the form of liposomes, ointments, suspensions, gels and emulsions. Particularly preferred are preparations in the form of a hydrogel.

Such compositions include, among others, compositions suitable for intravenous, intraperitoneal, intramuscular, subcutaneous, mucocutaneous, rectal, transdermal, topical, buccal, intradermal, intragastric, intradermal, intranasal, intrabuccal, transdermal, intratympanic or sublingual administration. Particularly preferred are administration or injection into the middle ear, as well as local administration through the eardrum.

As the pharmaceutically acceptable carrier, for example, lactose, starch, sorbitol, sucrose, cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, talc, mannitol, ethyl alcohol and the like can be used. Powders, as well as tablets, can consist of 5-95% wt. from such a medium.

Liquid compositions contain solutions, suspensions, sprays and emulsions. For example, water-based injection solutions or based on a mixture of water and propylene glycol for parenteral injection. For the preparation of suppositories, it is preferable to use waxes with a low melting point, fatty acid esters and glycerides.

Pharmaceutical compositions also contain gels and other viscous drug carriers that are biodegradable or non-biodegradable, aqueous or non-aqueous, or microsphere based.

Preferably, the pharmaceutical composition according to the invention is prepared for topical and/or topical administration. A suitable vehicle for otogenic administration, i.e. for administration into the (middle) ear, are organic and inorganic substances which are pharmaceutically acceptable and do not react with the compound of the invention and/or with its additional active agents, for example table salt, alcohols, vegetable oils, benzyl alcohols, alkyl glycols, polyethylene glycols , glycerol triacetate, gelatin, carbohydrates such as lactose or starch, magnesium carbonate (magnesia, chalk), stearate (waxes), talc and petrolatum (petrolatum). The compositions described may be sterilized and/or may contain adjuvants, for example lubricants, preservatives such as thiomersal (i.e. 50% by weight), stabilizers and/or humectants, emulsifiers, salts for regulating osmotic pressure, buffering agents, dyes and/or flavors. These compositions may also contain one or more additional active agents, if desired. Otogenic and/or audiological compositions according to the invention may contain other compounds and/or substances, for example, other biologically active substances such as antibiotics, anti-inflammatory active agents such as steroids, corticoids, analgesics, antipyrines, benzocaines, procaines.

Compositions of the present invention for topical administration may contain other pharmaceutically acceptable compounds and/or substances. In a preferred embodiment of the present invention, a topical excipient is selected that does not enhance the release of crystalline 6-fluoro-9-methyl-β-carboline and possibly additional active agent or active agents into the circulatory system or central nervous system when administered into the ear, middle ear, or ear canal. Possible carrier materials include hydrocarbon acids, anhydrous adsorbents such as hydrophilic petrolatum (Vaseline) and anhydrous lanolin (ie ^Aquaphor® ), and water-in-oil emulsions such as lanolin and cold cream. More preferred are carrier materials that are substantially non-exclusive and which typically contain carrier materials that are water-soluble, as well as oil-in-water emulsion materials (creams or hydrophilic ointments) and water-soluble materials such as -carriers based on polyethylene glycol and aqueous solutions that were gelled with several substances such as methylcellulose, hydroxyethylcellulose and hydroxypropylmethylcellulose.

Description of drawings:

Figure 1 : A) crystals of polymorph A; B) crystals of polymorph B

The crystal structures of polymorphs A and B were obtained by recrystallization in heptane and a mixture of MTBE and acetone or DCM, respectively.

Fig.2:

A) Molecular network of 6-FMC polymorph A in a single crystal (left) and the corresponding chemical structure (right), calculated by diffractometry

B) Molecular network of 6-FMC polymorph B in a single crystal (left) and the corresponding chemical structure (right) calculated by diffractometry

Fig.3:

A) X-ray powder diffraction (XRPD) of polymorph A

B) X-ray powder diffraction (XRPD) of polymorph B

C) Overlay of XRPD signatures calculated using Mercury ^® based on the X-ray structure of polymorph B and the measured structure of polymorph B

Fig.4:

A) Infrared (IR) spectrum of polymorph A

B) Infrared (IR) spectrum of polymorph B

Fig.5:

A) DSC melting curve of polymorph A

B) DSC melting curve of polymorph B

Fig.6:

A) DSC cooling curve of polymorph A

B) DSC cooling curve of polymorph B

Fig.7 :

A) Thermogravimetric analysis of polymorph A

B) Thermogravimetric analysis of polymorph B

Figure 8 : Solid State Nuclear Magnetic Resonance (ssNMR)

A) ¹³ C ttNMR of polymorph A

B) ¹³ C ttNMR of polymorph B

Figure 9 : Supercritical carbon dioxide

A) Spectrum of the polymorph before insertion into the device (polymorph B; ExtrateX supercritical fluid innovation).

B) Spectrum of the polymorph after treating polymorph B with supercritical carbon dioxide (yields polymorph A).

C) Residue after exposure of 6-FMC to supercritical carbon dioxide

The cylinder of the device, into which supercritical CO ₂ was introduced, was loaded with polymorph B (Fig. 9A) and heated to 60°C at a pressure of 35 MPa. The time it took for the API to completely dissolve in supercritical CO ₂ was 2.5 hours. The spectrum of the final product is shown in Fig. 9B. It is clear that there was a transformation from polymorph B to polymorph A. The powder was white (Fig. 9C).

Figure 10: X-ray powder diffraction (XRPD) of the form A polymorph in the poloxamer composition after 30 hours shows 60% conversion of the initially administered polymorph A to the polymorph B of the invention.

Figure 11: After 48 hours, the composition of the initially introduced polymorph A according to the invention undergoes 100% conversion to polymorph B.

Figure 12: After 30 hours, no conversion occurs in the composition of the initially introduced polymorph B according to the invention.

Figure 13: Effect of intratympanic administration of 6-FMC polymorph A (0.12 mg) on PTS in guinea pigs.

Figure 14: Effect of a single intratympanic administration of 6-FMC polymorph B (0.12 mg) on PTS in guinea pigs.

Figure 15: X-ray powder diffraction (XRPD) of polymorph C.

Figure 16: X-ray powder diffraction (XRPD) of B+T polymorphs.

The following examples are included to demonstrate preferred embodiments of the invention. It will be understood by those skilled in the art that the methods described in the examples that follow represent methods discovered by the inventor for the successful practice of the present invention, and thus may be considered to constitute the preferred methods for its practice. However, those skilled in the art should, in light of the present description, understand that many things can be changed in the specific embodiments described and still obtain a like or similar result without departing from the spirit and scope of the invention.

Additional modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art from this description. Accordingly, this description is to be considered illustrative only and is provided for the purpose of disclosing to those skilled in the art the general manner of carrying out the invention. It should be understood that the forms of the invention shown and described herein are to be considered as exemplary embodiments. Elements and materials may be substituted for those illustrated and described herein, the order of parts and processes may be reversed, and certain features of the invention may be used independently, all of which will be apparent to one skilled in the art upon reading this specification. Changes may be made to the elements described herein without departing from the spirit and scope of the invention as described in the following claims.

EXAMPLES

General techniques

1. Solid-state NMR (ssNMR)

ttNMR was determined at CT using a Bruker Advance III HD, 400 MHz ssMAS (in solid sample with magic angle spinning) with a 4 mm rotor and a spin speed of 12.5 kHz and an excitation frequency of 100 MHz. The sample was poured dry into a 4 mm rotor and determined at room temperature.

2.XRPD

XRPD was determined in an open capillary using a STOE Modell Stadi, detector: Mythen Dectris (Cu-K-α-beam monochromator) from 2 to 50 °2-theta

Based on the crystallographic data, the corresponding XRPD values were calculated using the Mercury ^® analysis program and compared with the experimentally determined XRPD values

Sample Preparation: Dry samples (approximately 20 mg) were thoroughly ground in a mortar and filled into a 0.5 mm glass capillary, which was sealed by thoroughly melting.

Data acquisition: XRPD was determined at room temperature using a STOE Modell StudyP, detector: Mythen Dectris 1K using Cu _Kα1 radiation (1.540598 Å) in transmission geometry, a Cu anode long-sharp focus X-ray generator and a curved germanium monochromator . Samples were measured from 2 to 50°2-theta in Debye-Scherrer scanning mode. Peak position accuracy is determined to be ±0.2° 2-theta due to experimental differences such as sample preparation and capillary packing density.

3. IR

The IR spectrum was determined under standard conditions using a Jasco ATR Diamond device (Golden Gate). First, the background was measured without a sample. A visible amount of the sample was then placed on a diamond prism ATR device and screwed together. Samples were measured using 16 scans in the range 4000-400 cm ^-1 . The background was subtracted from the actual scan to obtain the IR spectrum of the sample.

Example 1-1: Preparation of a crystalline polymorph (polymorph A as a reference polymorph) of 6-fluoro-9-methyl-β-carboline (6-FMC)

Method A:

6-fluoro-9-methyl-β-carboline (6-FMC) was suspended in n-heptane (50 ml per 1 g) and the suspension was refluxed. The resulting solution was allowed to cool slowly to RT and left to stand overnight. The precipitate that formed was filtered off, washed with n-heptane, and dried. Polymorph Form A of 6-FMC was obtained as pale yellow-brown needles in 88% yield.

WO2015044434 describes a method for the synthesis of 6-fluoro-9-methyl-β-carboline (6-FMC), and 6-FMC was obtained as a crude product in the form of mustard yellow crystals. These crystals are in polymorph Form A of the present invention. Elemental analysis of these crystals revealed small amounts of impurities such as water and solvent, and such impurities may cause a decrease in the melting point compared to the highly purified polymorph Form A obtained in this application, which appears as white crystals.

Method B:

1. “Recrystallization of the existing state of the art.” While stirring with a magnetic stirrer, 5 g of a suspension of polymorph B in 200 ml of freshly distilled n-heptane was heated to reflux. Care was taken to ensure that the heating source (oil bath) did not reach the solvent level in the 500 mL round bottom flask. Since the crystals were completely dissolved in 200 ml, only 20 ml more was added instead of the calculated amount of 290 ml. After 5 minutes of reflux, the heating source was removed and stirring was stopped. The solution was isolated from external stirring or cooling to allow slow growth of high purity crystals.

After 12 h of crystallization at room temperature, the crystals were removed by vacuum filtration, suctioning as dry as possible (displacing the loose sediment on the filter using a plastic spatula). The semi-dry product was transferred to a flask and dried for 2 hours at 2.5 Pa at room temperature. The yield on dry matter was 4.52 g (90.4%).

This sample was analyzed by X-ray diffraction and compared with the starting material, which was also re-analyzed to confirm the obtained spectra.

XRPD of the recrystallization product shows that the crystals are mostly "polymorph A" but ∼20% polymorph B is also present; probably because less heptane was used for recrystallization. XRPDs before and after recrystallization are shown as an overlay. To remove the remaining polymorph B, the batch was subjected to a further recrystallization process (see part 3).

2. Recrystallization under harsh conditions (temperature too high, flask too large and heat source 1 cm above solvent level). The same equipment was used as in the first experiment. 3.6 g was refluxed in 120 ml of n-heptane in a 500 ml flask: The solution appeared clear, but above the solvent level, crystals continuously formed during the heating process, which were washed back into the mixture from time to time by dripping boiling heptane.

This mixture (with some pre-formed crystals) cooled and quickly formed crystals in solution (due to the crystals already present). After crystallization was complete, the crystals formed in the flasks appeared as if black dots and a brown smear were present.

3. Second recrystallization: In a 250 ml flask, a suspension of 3.9 g in 225 ml of heptane was refluxed (metal heating block) and the resulting clear solution was refluxed for 10 min, after which the heating source was removed. The reaction mixture was allowed to cool, and as the first crystals appeared, the solution was stirred periodically to avoid the formation of large crystals. After settling for 3 hours, the flocculated crystals were removed by filtration, washed with 15 ml of n-heptane, sucked off to dryness, collected in a smaller flask, and dried overnight in a sublimator at RT and 100 Pa. The resulting crystals turned out to be looser and more voluminous than as a result of the first recrystallization. XRPD showed exclusively the A polymorph.

In a separate experiment, the inventors recorded XRPD of polymorph B as a dry powder and in a capillary impregnated with n-heptane to evaluate the effect of free heptane present during the determination. This is important to ensure that none of the signals detected from the "heptane polymorph" are an artifact due to the inclusion of residual heptane. Additional heptane was confirmed to have no effect on the reported XRPDs.

A) X-ray powder crystallography

X-ray powder diffractometry is currently being considered as a method for determining polymorphism. In addition, demonstration of nonequivalent structure by single crystal X-ray diffraction would confirm the presence of polymorphic structures.

The crystal structure of polymorph A (Figure 1A) was characterized in detail using X-ray crystallography. This polymorph crystallizes in the orthorhombic form with space group p2 ₁ 2 ₁ 2 ₁ . The unit cell parameters are as follows: a =5.8986 (1) Å, α=90°; b =10.3506 (3) Å, β=90°; c =15.4572 (5) Å, γ=90°. As shown in Figure 2A, 6-FMC molecules are found in multidimensional layers of pi-stack molecules and orthogonal T-stack molecules. It is also clear to see that there are no hydrates, solvates, or salt-based counterions in the structure.

Table 1. Bond lengths [Å] and angles [°] for polymorphic form A

B) X-RPD data

X-ray powder diffraction of polymorph A has characteristic signals in degrees 2-theta

Table 2. Values of 2-theta crystalline polymorph A (Figure 3A)

Angle (2θ) Rel. int.
(%) Angle (2θ) Rel. int.
(%) 11.9 69 24.0 49 13.8 eleven 25.6 35 16.5 38 26.2 16 20.0 12 26.7 100 22.4 46 28.1 38 23.8 13 29.9 4.5

C) ttNMR of polymorph A (Fig. 8A)

28.7, 103.6, 111.9, 113.4, 114.6, 114.8, 118.9, 125.4, 131.0, 134.0, 135.8, 155.5, 155, 7, 156.4, 156.6, 157.6, 158.2 and 159.3 ppm.

D) Fourier transform infrared (FT-IR) spectrum of polymorph A (Fig. 4A)

422.64, 446.61, 523.52, 557.49, 604.03, 635.82, 702.67, 743.66, 802.11, 813.34, 848.24, 893.14, 1023, 95, 1069.75, 1152.14, 1183.22, 1275.15, 1363.80, 1427.67, 1450.25, 1480.13, 1560.90 and 1582.19 cm ^-1 .

Example 1-2: Preparation of the crystalline polymorphic form (polymorph B) of 6-fluoro-9-methyl-β-carboline (6-FMC) of the present invention

Method A (isopropanol:n-heptane=1:5):

1 g of 6-fluoro-9-methyl-β-carboline (6-FMC) was suspended in a mixture of isopropanol and n-heptane, and the suspension was refluxed. The resulting solution was cooled and the precipitated solids were filtered, washed with n-heptane and dried. Polymorph Form B of 6-FMC was obtained as a pale yellow-brown powder in 77% yield.

Method B (Dichloromethane: n-heptane=1:4):

2 g of 6-fluoro-9-methyl-β-carboline (6-FMC) was dissolved in 6 ml of DCM and 24 ml of n-heptane was added to the solution. The precipitated solids were filtered, washed with n-heptane and dried. Polymorph Form B of 6-FMC was obtained as a pale yellow-brown powder in 62% yield.

Method C (Dichloromethane: MTBE=1:9.4):

400 mg of 6-fluoro-9-methyl-β-carboline (6-FMC) was dissolved in 1.6 ml of DCM and 15 ml of MTBE was added to the solution. The resulting suspension was concentrated using a rotary evaporator at a temperature of 40°C under a pressure of 65 kPa until a clear solution was obtained. After 2 minutes, evaporation was stopped, the solution was cooled, and the resulting suspension was filtered. The crude crystalline polymorph B of 6-FMC was obtained as a pale brown solid and dried. Yield=75%.

Method D: (Acetone: MTBE=1:2.8):

400 mg of 6-fluoro-9-methyl-β-carboline (6-FMC) was dissolved in 3.6 ml of acetone and 10 ml of MTBE was added to the solution. The resulting solution was concentrated using a rotary evaporator at a temperature of 40°C under a pressure of 45 kPa until a clear solution was obtained. After 7 minutes, the resulting suspension was cooled and filtered. The crude crystalline polymorph B of 6-FMC was obtained as a pale brown solid and dried. Yield=61%.

Method E: (precipitation of free base with water)

200 mg of 6-fluoro-9-methyl-β-carboline (6-FMC) was dissolved in 3.2 ml of DMSO. The clear solution was precipitated in 40 ml of distilled water and the resulting precipitate was centrifuged. The supernatant was removed, and the precipitate was washed 3 times with distilled water to remove residual DMSO. (The process of vortex mixing - centrifugation). After the final washing step, the bright white solids were frozen in liquid nitrogen, lyophilized, and presented as pure polymorph B.

Method F (precipitation of free base with NaOH)

An aqueous suspension of 200 mg of 6-fluoro-9-methyl-β-carboline (6-FMC) was stirred and HCl was added to pH 2. As the salt formed, a clear yellow solution was obtained. To ensure the absence of any particulate matter, it was filtered through a 0.45 μm syringe filter. The solution was stirred again and made alkaline with NaOH to pH 12. During addition, the formation of a thick white precipitate of free base was observed, which was centrifuged. The supernatant was decanted and the solid was washed thoroughly through repeated distilled water, vortex and centrifugation processes (4×). After the last wash, the solid was frozen in liquid nitrogen and lyophilized. Yield: 85% pure polymorph B.

A) X-ray crystallography

The crystal structure of polymorph B (Figure 1B) was also characterized in detail using X-ray crystallography. This polymorph crystallizes in the monoclinic form with space group p2 ₁ c. The unit cell parameters are as follows: a =3.8456 (2) Å, α=90°; b =17.3249 (8) Å, β=91.069 (3)°; c =13.7709 (7) Å, γ=90°. As shown in Figure 2B, the 6-FMC molecules are arranged in ordered layers of zigzag stripes. It is also clear to see that there are no hydrates, solvates, or salt-based counterions in the structure. Analysis showed the absence of any solvent. The composition picture confirmed the identity of the claimed compound.

Table 3. Bond lengths [Å] and angles [°] for polymorphic form B

B) XRPD

The calculated XRPD diffractogram of polymorph B and the determined XRPD diffractogram of polymorph B are shown in Fig. 3B and as an overlay in Fig. 3C. Despite the slight deviation in the absolute values of 2-theta, the diffraction patterns are almost identical relative to each other.

Table 4. Values of 2-theta crystal polymorph B (Fig. 3B)

Angle (2θ) Rel. int. (%) 11.3 31 14.1 8 17.1 20 17.6 47 18.0 62 19.0 21 20.3 8 20.6 14 22.5 100 23.2 94 24.3 12 25.8 13 29.4 38

C) ttNMR of polymorph B (Fig. 8B)

28.6, 107.3, 110.2, 111.6, 112.8, 116.6, 121.5, 126.2, 138.0, 155.5, 156.1, 156.7, 157, 1, 157.2, 157.7, 158.1 and 158.4 ppm.

D) Fourier transform infrared (FT-IR) spectrum of polymorph B (Fig. 4B)

426.62, 524.31, 558.12, 604.22, 636.09, 704.73, 729.05, 742.59, 803.27, 819.54, 852.30, 864.13, 893, 22, 1024.99, 1126.71, 1148.77, 1183.98, 1272.31, 1283.82, 1333.10, 1362.06, 1426.98, 1448.53, 1481.82, 1554.80 and 1578.94 cm ^-1 .

Example 2: DSC and Melting Point Measurement of Crystalline Polymorphs

Powders of 6-FMC polymorphs were heated with stirring, with heating slowing down at temperatures above 100°C (1 degree Celsius per minute). A sample of polymorph A melted at ~125°C. Heating and cooling curves were recorded using differential scanning calorimetry (DSC; Netzsch DSC 204 F1) (Fig. 5A). The melting point was confirmed by heating with DSC. The cooling curve showed a wide range between 65°C and 45°C (Fig. 6A).

The polymorph B sample melted at ~123°C. DSC heating curve analysis demonstrated a transition phase characterized by a slight slowdown in the melting process (Figure 5B). During the cooling process of polymorph B, the curve showed a two-phase transition in the temperature range of 45-75 °C (Fig. 6B). This reflects a decrease in the quality of polymorph B. Diffractometric analysis showed that the conversion of polymorph B to polymorph A correlates with the duration of the melting state until complete conversion. Vacuum sublimation resulted in complete conversion to polymorph A.

Example 3: Polymorph transformation at different temperatures conditions (conversion of polymorph B into polymorph A)

3-1: Polymorph B 6-FMC was dissolved in n-heptane, the suspension was heated (100°C) for 1:45 h with stirring. Then the suspension was cooled and stirred at room temperature for 15 hours. The precipitate was filtered off, washed with n-heptane and air dried. The resulting XRPD spectrum did not reveal any conversion to form A.

3-2: Polymorph B 6-FMC was diluted in toluene, heated to 95°C and cooled to room temperature. The precipitate was filtered off, washed with toluene, and dried in a stream of air. The XRPD spectrum indicated some presence of polymorph A, but this experiment was not reproducible, so it is assumed that there is no conversion of polymorph B to polymorph A under these conditions either.

3-3: A suspension of 6-FMC polymorph B was diluted in n-heptane and heated to 110°C. The reaction mixture was kept for 5 hours 18 minutes in a flask under reflux conditions, followed by gradual cooling to room temperature and stirred for 16 hours. The precipitate was filtered off, washed with heptane and air dried. XRPD proved the immutability of the B polymorph.

3-4: A suspension of 6-FMC polymorph B was diluted in n-heptane and heated to 108°C until the substance was completely dissolved. Polymorph A seed crystals were added to the solution, which was gradually cooled to room temperature. The precipitate was filtered off, washed with n-heptane, and air dried. XRPD proved the immutability of the B polymorph.

Example 4: Polymorph Conversion Using Supercritical CO ₂ (conversion of polymorph B to polymorph A)

Polymorph extraction/conversion with supercritical CO ₂ was performed using 500 mg of crude off-white polymorph B in an ExtrateX rapid expansion supercritical solution (REES) system. The extraction vessel was heated to 60°C at a pressure of 350 bar (35 MPa). The nozzle was a 5 cm long stainless steel capillary with an internal diameter of 0.25 mm at a temperature of 70°C. The expansion vessel was heated to 40°C at 1-5 MPa. Equilibrium time: 2.5 hours; Spray time: 15 min. After this process, polymorph A was obtained as a bright white powder.

The cylinder of the device, into which supercritical CO ₂ was introduced, was loaded with polymorph B (Fig. 9A) and heated to 60°C at a pressure of 35 MPa. The time it took for the API to completely dissolve in supercritical CO ₂ was 2.5 hours. The spectrum of the final product is shown in Fig. 9B. It is clear that there was a transformation from polymorph B to polymorph A. The powder was white (Fig. 9C).

Example 5: Properties of 6-FMC Polymorphs in Powder Form (Melting and Cooling Experiments)

Powders of 6-FMC polymorphs were heated with stirring, with heating slowing down at temperatures above 100°C (1 degree Celsius per minute). A sample of polymorph A melted at ~125°C. Heating and cooling curves were recorded using differential scanning calorimetry (DSC; Netzsch DSC 204 F1) (Fig. 5A). The melting point was confirmed by heating with DSC. The cooling curve showed a wide range between 65°C and 45°C (Fig. 6A).

The polymorph B sample melted at ~123°C. DSC heating curve analysis demonstrated a transition phase characterized by a slight slowdown in the melting process (Figure 5B). During the cooling process of polymorph B, the curve showed a two-phase transition in the temperature range of 45-75 °C (Fig. 6B). This reflects a decrease in the quality of polymorph B. Diffractometric analysis showed that the conversion of polymorph B to polymorph A correlates with the duration of the melting state until complete conversion. Vacuum sublimation at 1-2 kPa resulted in complete conversion to white crystals of polymorph A.

Example 6: Procedure for Extraction of 6-FMC from a Poloxamer-Based Composition (Stability Test for Polymorphs A and B in the Composition)

2 ml of the poloxamer composition with 12 mg/ml 6-FMC was cooled to 4°C, vortexed and transferred to a 2 ml Eppendorf tube. The cooled tube was centrifuged for 2 min using a tabletop centrifuge, and the supernatant polymer solution was pipetted and discarded. The remaining 6-FMC was resuspended and vortexed with 1.5 ml of ice-cold milli-Q water and centrifuged as described above. The supernatant water was again removed using a pipette, and the washing procedure was repeated 3 times (a total of 4 washes). Note: The solution must be kept cool to facilitate the centrifugation process. After the last wash, the remaining white 6-FMC was cooled with liquid nitrogen and freeze-dried overnight at 100 Pa at room temperature.

After 30 hours in the composition, 60% of polymorph form A was converted to polymorph form B (see Fig. 10). Storing the composition for 48 hours or more results in 100% conversion of polymorph A to polymorph B, as shown in FIG. 11.

Example 7: Preparation of the 3rd crystalline polymorph (polymorph C) of 6-fluoro-9-methyl-β-carboline (6-FMC)

818 mg of 6-FMC (polymorph B) was dissolved in 5 ml ethanol and 500 μl water. Then 50 ml of heptane was added, which led to phase separation. Despite phase separation, a rotary evaporator was used at a temperature of 40°C and a pressure of 14 kPa. An oil film separated on the piston wall and suddenly hardened. This crystalline solid was analyzed demonstrating the presence of polymorph B only.

Another 20 ml of heptane was added to the reaction product and heated at reflux (temperature approximately 100°C) for 5 minutes at normal pressure until the solution became clear. The solution was cooled overnight with stirring to room temperature. Darker lumps formed on the piston wall, while a homogeneous crystalline mass formed on the bottom of the piston. The homogeneous crystalline mass consisted of polymorph C.

X-RPD polymorphic form C was determined as shown in Fig. 15, and the characteristic peaks are summarized in Table 5.

Table 5. Values of 2-theta polymorph C

Angle (2θ) Rel. int. (%) Angle (2θ) Rel. int. (%) 4.7 33 23.2 25 9.4 45 23.6 53 11.5 53 23.9 14 14.1 87 24.8 100 17.3 21 25.0 48 18.9 17 26.1 61 19.2 8 28.5 67 22.7 35

Example 8: Preparation of the 4th crystalline polymorph (polymorph T) of 6-fluoro-9-methyl-β-carboline (6-FMC)

250 mg of micronized 6-FMC polymorph B was acidified with HCl followed by sonication at 40°C. The cloudy solution was made alkaline with sodium hydroxide to pH 12, which caused the precipitation of a fine powdery mass. The powder was poured onto weighing paper to air dry. The resulting XRPD revealed a mixture of the new polymorph and polymorph B. The percentage was ~50 to 50%. The new polymorph was clearly different from the A, B, and C polymorphs and was designated the T polymorph.

To test for the presence of water, NMR analysis was performed on a mixture of B/T polymorphs, a mixture of extra-dry B/T polymorphs, and pure polymorph B. None of the samples contained more water than was present in deuterated chloroform. Thus, the analyzes did not reveal any evidence of the presence of hydrates.

The X-RPD of the T polymorph was determined as shown in FIG. 16, and the characteristic peaks are summarized in Table 6.

Table 6. Polymorph T 2-theta values obtained by subtracting polymorph B and T from the mixed spectrum

Angle (2θ) Rel. int. (%) Angle (2θ) Rel. int. (%) 9.4 4 22.1 56 15.7 4 22.3 100 15.9 7 13.9 22 17.4 8 24.9 4 19.4 7 26.2 13

Example 9: efficiency in vivo crystalline polymorphic form of 6-FMC according to the invention in the guinea pig model

General Procedures: All experimental validations of in vivo studies were performed in the well-known guinea pig model of NIHL. The guinea pig was chosen because its anatomy is comparable in both structure and size to that of humans. Hearing function was assessed using brain evoked potential audiometry (BERA).

Methods: Adult guinea pigs received intratympanic (it) injection of 6-FMC formulated in a thermosensitive hydrogel or hydrogel alone. All procedures were performed under anesthesia. BERA was used to measure acoustic brainstem evoked potentials (ABR) on days 3 and 14. Auditory stimuli were sinusoidal tones (10 ms duration at 4, 8, 16 kHz) with 5 dB steps from 0 to 90 dB. These measurements were used to calculate PTS. Acoustic trauma was performed on day 0 and consisted of one continuous band (quarter octave centered at 8 kHz) at an SPL of 118 dB for 30 min. Animals were treated 1 hour after the end of the acoustic exposure. The round window was visualized under a surgical microscope through a small hole drilled into the bulla bone of the left ear. 10 μl of gel containing either 6-FMC or vehicle was injected using a Hamilton syringe and a motorized pump onto the round window membrane (RWM) before the hole was sealed with dental cement.

Preparation of the composition: A non-ionic surfactant solution was pre-prepared by dissolving an appropriate amount of non-ionic surfactant in water or PBS buffer and allowing stirring overnight under refrigerated conditions. Once the nonionic surfactant was completely dissolved, the osmolality and pH were adjusted as necessary. The solution was then filtered through a 500 μm sieve to remove undissolved gel particles. This completed the compounding of the carrier. An API (active pharmaceutical ingredient) containing composition was prepared by adding micronized 6-FMC (≤50 µM) at a concentration of 12 mg/ml. For the experiments presented in Fig. 13, the 6-FMC composition was used immediately after preparation. For the experiments in FIG. 14, the 6-FMC formulation was prepared at least 48 hours prior to animal experiments.

Results and discussion

Intratympanic treatment with 6-FMC in polymorph form A or B resulted in a significant reduction in NIHL (Fig. 13 and Fig. 14).

Introduction of polymorph A

For polymorphic form A, a moderate effect could be achieved where PTS was reduced by an average of 7.7 dB (FIG. 13). Overall, noise exposure resulted in a mean PTS of 22.3 dB in vehicle-treated controls and decreased by approximately 7.7 dB to a threshold shift of 14.6 dB in animals treated with polymorph A of 6-FMC.

For polymorph B, this effect was significantly stronger and more significant across all frequencies tested and resulted in a therapeutically beneficial reduction in PTS of at least 23.6 dB to a significant 39.9 dB (Figure 14). Importantly, at some frequencies PTS was reduced to 0, demonstrating that 6-FMC polymorph B treatment has the potential for full recovery from noise-induced hearing loss. Overall, noise exposure resulted in an average PTS of 34.7 dB in vehicle-treated controls and was significantly reduced by an average of 34.1 dB to a 0.6 dB threshold shift in animals treated with polymorph B form of 6-FMC.

Comparison of efficacy data from animal studies using polymorphic forms of 6-FMC A and B demonstrates that polymorphic form B has significantly greater efficacy compared to polymorphic form A. Use of polymorphic form B results in an average reduction in PTS of 34.1 dB compared to an average reduction of 7.7 dB observed in animals fed polymorphic form A. Moreover, animals fed polymorphic form B have been demonstrated to have the potential for full recovery from noise-induced hearing loss, as seen in frequencies of 4 kHz and 8 kHz in Figure 14, where PTS reaches 0. Taken together, the results of in vivo studies using the guinea pig NIHL model suggest that a single intratympanic application of the polymorphic form B of 6-FMC leads to a significant improvement in PTS and has more high efficiency compared to the polymorphic form A.

Claims (26)

1. Crystalline polymorphic form of 6-fluoro-9-methyl-9H-β-carboline of formula (I) ( I ) where the crystalline polymorph has an X-ray powder diffraction pattern containing peaks at 2-theta angles of 11.3, 17.1, 17.6, 18.0, 22.5, 23.2 and 29.4 degrees with a deviation of ±0.2 degrees. 2. The crystalline polymorphic form according to claim 1, wherein the crystalline polymorphic form has a powder X-ray diffraction pattern containing peaks at 2-theta angle values of 11.3, 14.1, 17.1, 17.6, 18.0, 19.0, 20.3, 20.6, 22.5, 23.2, 24.3, 25.8 and 29.4 degrees with a deviation of ±0.2 degrees. 3. The crystalline polymorphic form according to claim 1, wherein the crystalline polymorphic form is in a monoclinic form having a space group p2 _1/ c, where one molecule of 6-fluoro-9-methyl-9H-β-carboline is in an asymmetric unit cell having unit cell parameters a=3.85±0.1 , b=17.32±0.1 , c=13.77±0.1 , α=90±3°, β=91±3° and γ=90±3°. 4. The crystalline polymorphic form according to claim 1, wherein the solid-state ¹³ C-NMR spectrum of said crystalline polymorphic form contains peaks at 28.6, 107.3, 110.2, 111.6, 112.8, 116.6, 121, 5, 126.2, 138.0, 155.5, 156.1, 156.7, 157.1, 157.2, 157.7, 158.1 and 158.4 ppm. with a deviation of ±1 ppm 5. Crystalline polymorphic form according to any one of claims 1 to 4, wherein said crystalline polymorphic form has a melting point of 123°C±1°C. 6. Crystalline polymorphic form according to any one of claims 1 to 4, wherein the particle size of the crystalline polymorphic form is ≤ 200 μm. 7. The crystalline polymorphic form according to claim 6, wherein the particle size of the crystalline polymorphic form is ≤ 20 μm. 8. Pharmaceutical composition for the treatment and/or prevention of hearing impairment, dizziness or vestibular disorder, containing a crystalline polymorphic form of 6-fluoro-9-methyl-9H-β-carboline according to claim 1 together with at least one pharmaceutically acceptable carrier, excipient , and/or diluent. 9. Pharmaceutical composition according to claim 8 in the form of liposomes, ointments, suspensions, gels and emulsions. 10. Crystalline polymorphic form of 6-fluoro-9-methyl-9H-β-carboline according to claim 1, or a pharmaceutical composition according to claim 8, intended for the treatment and/or prevention of hearing impairment, dizziness or vestibular disorder. 11. The crystalline polymorphic form or pharmaceutical composition according to claim 10, wherein hearing loss, dizziness or vestibular disorders are selected from the group consisting of Meniere's disease, sudden sensorineural hearing loss, noise-induced hearing loss, age-related hearing loss, autoimmune ear disease, noise in the ears, acoustic trauma, trauma from an explosion, labyrinthine deafness, presbycusis, trauma during implantation of an inner ear prosthesis (trauma during insertion of the prosthesis), dizziness due to diseases of the inner ear and hearing impairment due to antibiotics and cytostatics. 12. Crystalline polymorphic form of 6-fluoro-9-methyl-9H-β-carboline or pharmaceutical composition according to claim 10, wherein the crystalline polymorphic form or pharmaceutical composition is administered topically and/or locally. 13. A method for obtaining a crystalline polymorphic form of 6-fluoro-9-methyl-9H-β-carboline of formula (I) according to claim 1, including: A1) providing 6-fluoro-9-methyl-9H-β-carboline of formula (I) (I) B1) dissolving 6-fluoro-9-methyl-9H-β-carboline in a mixture of a polar solvent and a non-polar solvent; or dissolving 6-fluoro-9-methyl-9H-β-carboline first in a polar solvent and adding a non-polar solvent to the resulting solution of 6-fluoro-9-methyl-9H-β-carboline in a polar solvent, where the polar solvent is dichloromethane, acetone, isopropanol or a mixture thereof, or a mixture with water, and the non-polar solvent is methyl tert-butyl ether, n-heptane, cyclohexane or a mixture thereof, and the ratio of the polar solvent and non-polar solvent is range from 1:2 to 1:10; and the concentration of 6-fluoro-9-methyl-9H-β-carboline in a mixture of a polar solvent and a non-polar solvent is in the range of 50 mM to 200 mM; C1) heating the solution or suspension of 6-fluoro-9-methyl-9H-β-carboline to a temperature in the range of 40°C to 100°C; D1) stirring the resulting solution for 10 minutes at the same temperature; E1) cooling the resulting solution to a temperature in the range from -10°C to +30°C to obtain a crystalline polymorphic form of 6-fluoro-9-methyl-9H-β-carboline according to claim 1; And F) isolating the crystalline polymorphic form B of 6-fluoro-9-methyl-9H-β-carboline of formula (I) according to claim 1. 14. The method according to claim 13, where after stage E1) and before stage F) the following stage E2 is performed): E2) seeding a crystalline polymorphic form of 6-fluoro-9-methyl-9H-β-carboline.

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