Technical Field

The invention relates to crystalline forms of obeticholic acid of formula I, with the systematic name (3a,5 ,6a,7a)-6-ethyl-3,7-dihydroxycholan-24-oic acid, a method of their preparation and use for the production of a dosage form. These crystalline forms can be advantageously used to increase purity of obeticholic acid and also for the preparation of its amorphous form.

(I)

Obeticholic acid is a semi-synthetic bile acid analogue with an agonist effect upon the farnesoid X receptor (FXR). It is designed for the treatment of liver diseases, e.g. primary biliary cirrhosis (PBC), non-alcoholic steatohepatitis (NASH) or primary sclerosing cholangitis (PSC).

Background Art

Obeticholic acid was first mentioned in the patent application WO2002072598. It describes its isolation by means of column chromatography, which generally produced amorphous substances; however, the patent application does not disclose any more detailed data about the character of the product.

It was followed by two process patent applications WO2006122977 and US20090062526 dealing with synthesis of obeticholic acid.

The most recently published patent application WO2013192097 describes two solid forms of obeticholic acid: Crystalline Form C and Amorphous Form 1. It describes preparation of Crystalline Form C and then its re-precipitation to Amorphous Form 1. This patent application also mentions other crystalline forms of obeticholic acid, which, however, are not suitable for use in the pharmaceutical industry for various reasons.

Many pharmaceutical solid compounds can exist in various crystalline forms that are considered as polymorphs and hydrates/solvates, having different crystal units and thus different physicochemical properties such as the melting point, solubility, dissolution rate, as well as bioavailability. To distinguish individual solid phases of a compound, several solid- state analytic methods can be used, e.g. X-ray powder diffraction, solid-state NMR and Raman spectroscopy, as well as thermoanalytic methods.

Discovering new solid phases (polymorphs, solvates and hydrates) of an active pharmaceutical ingredient offers an opportunity to select a suitable modification with desired physicochemical properties and processability and improve the characteristics of the chemical product. For this reason, there is an obvious need of novel solid forms (polymorphs, solvates, hydrates) of obeticholic acid. Disclosure of Invention

This object of the present invention is crystalline forms of obeticholic acid. The first of the forms, identified as 1-2, is a monohydrate and is characterized by an easy preparation process, high crystallinity and purification capability. Its significant advantage consists in the possibility of its re-drying to an amorphous form. Thus, there is no need to transform the crystalline form of obeticholic acid to a salt (e.g. sodium or ammonium) and subsequently prepare the amorphous form by precipitation. The other form, identified as 1-3, exhibits high stability; the melting point of Form 1-3 is significantly higher than the melting points of Forms 1-2 or C.

The invention provides Crystalline Form 1-2 of obeticholic acid, exhibiting the following characteristic reflections in the X-ray powder pattern measured by CuKa radiation: 3.1; 6.2; 9.9; 12.4 and 16.7 ± 0.2° 2-theta. In some embodiments, Crystalline Form 1-2 of obeticholic acid is characterized by the following further reflections in the X-ray powder pattern: 4.9; 8.9; 10.9 and 15.8 ± 0.2° 2-theta.

In some embodiments, Crystalline Form 1-2 of obeticholic acid is further characterized by the differential scanning calorimetry curve with the melting point at 80.9°C. In some embodiments, Crystalline Form 1-2 of obeticholic acid is in the form of monohydrate. The invention further provides Crystalline Form 1-3 of obeticholic acid, exhibiting the following characteristic reflections in the X-ray powder pattern measured by CuKa radiation: 8.0; 10.6; 16.0 and 17.6 ± 0.2° 2-theta. In some embodiments, Crystalline Form 1-3 of obeticholic acid is characterized by the following further reflections in the X-ray powder pattern: 10.0; 13.2 and 15.2 ± 0.2° 2-theta.

The invention further provides the use of Crystalline Form 1-2 and/or Crystalline Form 1-3 of obeticholic acid for the preparation of an amorphous form of obeticholic acid.

The invention further provides a method for preparation of the amorphous form of obeticholic acid, comprising drying of Crystalline Form 1-2 in a vacuum drier at the temperature of 40°C or higher, preferably at a temperature in the range of 40 to 80°C. In some embodiments, Crystalline Form 1-2 of obeticholic acid is dried at the temperature of 40°C for at least 20 hours.

The invention further provides a method for preparation of the amorphous form of obeticholic acid, comprising:

a) conversion of Crystalline Form 1-2 and/or Crystalline Form 1-3 of obeticholic acid to its salt;

b) adding an acid;

c) isolation of the obtained amorphous form precipitate by filtration.

In some embodiments, in step a), Crystalline Form 1-2 and/or Crystalline Form 1-3 of obeticholic acid is advantageously converted to the ammonium or sodium salt. In some embodiments, in step b), an organic or inorganic acid is added, preferably hydrochloric or phosphoric acid.

The invention further provides the use of Crystalline Form 1-2 and or Crystalline Form 1-3 of obeticholic acid for the preparation of obeticholic acid with a chemical purity higher than 99.80% in accordance with HPLC.

The invention further provides the use of Crystalline Form 1-2 and/or Crystalline Form 1-3 for the preparation of a pharmaceutical composition.

The invention further provides a pharmaceutical composition containing Crystalline Form 1-2 and/or Crystalline Form 1-3 of obeticholic acid and at least one pharmaceutically acceptable excipient. In some embodiments, at least one pharmaceutically acceptable excipient is selected from the group comprising lactose, microcrystalline cellulose, sodium croscarmellose and magnesium stearate and their combinations. In some embodiments, the pharmaceutical composition has the form of a tablet.

Brief Description of Drawings

Fig. 1: XRPD pattern of Form 1-2 of obeticholic acid

Fig. 2: DSC record of Form 1-2 of obeticholic acid

Fig. 3: TGA record of Form 1-2 of obeticholic acid

Fig. 4: XRPD pattern of Form 1-3 of obeticholic acid

Detailed description of the invention

This invention provides two novel crystalline forms of obeticholic acid.

Form 1-2 exhibits a distinctly crystalline character. The X-ray powder pattern of this salt is shown in Fig. 1. Its characteristic diffractions measured by CuKa radiation are 3.1; 6.2; 9.9; 12.4 and 16.7 ± 0.2 °2-theta. Form 1-2 further exhibits the following characteristic reflections: 4.9; 8.9; 10.9 and 15.8 ± 0.2 °2-theta. Diffraction peaks with a higher relative intensity than 15% are shown in Table 1.

Table 1: Diffraction peaks of Form 1-2 of obeticholic acid

16.37 5.409 20.4

16.71 5.300 39.7

17.88 4.956 18.8

18.55 4.779 13.5

18.97 4.674 16.5

20.43 4.343 9.2

21.08 4.211 9.2

24.52 3.628 4.3

24.96 3.564 4.6

Differential scanning calorimetry (DSC) was applied to measure the melting point of Form 1-2 of obeticholic acid of 80.9 °C (Fig. 2). According to thermogravimetric analysis (TGA), Form 1-2 of obeticholic acid contains 4.3 % of water, which corresponds to one molar equivalent (Fig. 3). Due to its high crystallinity, Form 1-2 is suitable not only for purification of crude obeticholic acid or for preparation of an amorphous form of obeticholic acid, but it is also directly usable in the dosage form.

Form 1-3 also exhibits a strongly crystalline character. The X-ray powder pattern of this salt is shown in Fig. 4. Its characteristic diffractions measured by CuKa radiation are 8.0; 10.6; 16.0; and 17.6 ± 0.2 °2-theta. Form 1-3 further exhibits the following characteristic reflections: 10.0; 13.2 and 15.2 ± 0.2 °2-theta. Diffraction peaks with a higher relative intensity than 15% are shown in Table 2.

Table 2: Diffraction peaks of Form 1-3 of obeticholic acid

Interplanar spacing [A]

Position [°2Th.] [A]=0.1nm Rel. intensity [%]

8.02 11.009 100.0

8.48 10.419 8.7

9.57 9.232 7.5

9.98 8.856 61.3

10.57 8.361 58.8

11.51 7.681 13.8

12.16 7.272 8.2

13.24 6.682 26.3

13.86 6.384 20.6

14.19 6.237 17.3

15.21 5.821 26.7

16.02 5.528 45.6

16.60 5.336 11.1 17.00 5.213 10.4

. 17.61 5.033 32.0

19.21 4.616 13.4

19.90 4.459 9.4

21.17 4.194 10.4

21.78 4.078 3.9

22.44 3.958 5.7

22.74 3.908 5.6

23.45 3.791 3.5

24.25 3.667 6.0

24.61 3.615 7.7

26.70 3.336 2.5

27.37 3.256 1.8

27.98 3.186 1.9

Forms 1-2 and 1-3 can be advantageously used to increase the chemical purity of obeticholic acid. These forms crystallize well and remove impurities from obeticholic acid. Conversion of Form C to Form 1-2 increased the chemical purity from 99.10% (starting Form C) to 99.61% (final Form 1-2). The recrystallization of Form 1-2 alone also increases the chemical purity from the initial 99.61% to the final 99.98% (HPLC). Purification of crude obeticholic acid (HPLC purity 97.59%) can be advantageously achieved by conducting recrystallization through Form 1-2 and in such a case the chemical purity of the final product rises to 99.83% (HPLC) during a single crystallization. Form 1-3 does not exhibit such a high tendency to crystallize as Form 1-2. In spite of this, conversion of Form C to Form 1-3 increased the chemical purity from 99.10% (starting Form C) to 99.48% (final Form 1-3). The recrystallization of Form 1-3 alone increased the chemical purity from the initial 99.48% to the final 99.87% (HPLC). Purification of crude obeticholic acid (HPLC purity 97.59%) can be advantageously achieved by conducting recrystallization through Form 1-3 and in such a case the chemical purity of the final product rose to 98.99% (HPLC) during a single crystallization.

Both the forms, 1-2 and 1-3 can be used to prepare the amorphous form of obeticholic acid with the use of usual procedures. The patent application WO2006122977 discloses re- precipitation via the ammonium salt, the application WO2013192097 discloses re- precipitation via the sodium salt. Both these processes can also be applied without problems to the preparation of the amorphous form from crystalline Forms 1-2 or 1-3 when the target amorphous form is produced in an 87 to 95% yield. Crystalline Form 1-2 can be advantageously used to prepare the amorphous form by mere drying in a vacuum drier, wherein at the temperature of 40°C already the content of the amorphous form starts to increase. Vacuum drying for at least 20 hours at 40°C provides a nearly complete amorphous form. If vacuum drying is carried out at a higher temperature, the drying time is reduced proportionally.

The amorphous form of obeticholic acid can also be prepared directly during the formulation process, e.g. by spray drying or in a fluid bed reactor.

All the prepared forms of obeticholic acid according to this invention (1-2. 1-3 and amorphous forms) can be used for the preparation of pharmaceutical compositions, especially solid dosage forms, e.g. tablets. Such pharmaceutical compositions can contain at least one excipient from the group of fillers (e.g. lactose), binders (e.g. microcrystalline cellulose), disintegrants (e.g. sodium salt of croscarmellose), lubricants (e.g. magnesium stearate), surfactants, etc. These tablets can be coated with common coatings, e.g. polyvinyl alcohol or polyethylene glycol.

The preparation of the novel Crystalline Forms 1-2 and 1-3 comprises the following steps: a) dissolution and/or dispersion of obeticholic acid in a solvent/s;

b) removal of the solvent/s from the mixture from step a).

The dissolution or dispersion in the solvent or mixture of solvents can be carried out at a temperature in the range from 20°C to the boiling point of the solvents. Subsequently, the mixture is usually cooled down to a temperature of 0°C to 30°C, preferably to the range of 20°C to 25°C, and left to crystallize. The mixture can be seeded. The solid product can be isolated either directly by filtration, or concentration of the mixture, or evaporation of the solvents may follow.

The solvents can be selected from a group of solvents of general formula ROH, RCN, RCOR or RCOOR wherein R can be a CI to C4 carbon chain, preferably ethyl acetate, isopropyl acetate, butyl acetate, acetonitrile or 2-butanone.

The term "room temperature" refers, for the purposes of the text below and above, to the temperature range from 22 to 26°C. Overview of analytic methods

Measurement parameters of XRPD: The diffractograms were measured using an XTERT PRO MPD PANalytical diffractometer, used radiation CuKa (λ= 1.542 A), excitation voltage: 45 kV, anode current: 40 mA, measured range: 2 - 40° 2Θ, increment: 0.02° 2Θ, step dwell time: 200 s, the measurement was carried out on a flat powder sample that was applied on a Si plate. For the setting of the primary optical equipment programmable divergence slits with the irradiated area of the sample of 10 mm, 0.02 rad Soller slits and a ¼° anti-diffusion slit were used. For the setting of the secondary optical equipment an X'Celerator detector with maximum opening of the detection slot, 0.02 rad, Soller slits and a 5.0 mm anti-diffusion slit were used.

The records of differential scanning calorimetr (DSC) were measured using a Discovery DSC device made by TA Instruments. The sample charge in a standard Al pot (40 μϋ) was between 4 and 5 mg and the heating rate was 5°C/min. The temperature program that was used consists of 1 min of stabilization at the temperature of 0°C and then of heating up to 250°C at the heating rate of 5°C/min (amplitude = 0.8°C and period = 60 s). As the carrier gas 5.0 N ₂ was used at the flow of 50 ml/min.

The records of the thermogravimetric analysis (TGA) were measured using a TGA 6 device made by the company Perkin Elmer. The sample charge in a corundum pot was 10-21 mg and the heating rate was 10°C/min. The temperature program that was used consists of 1 minute's stabilization at the temperature of 20°C and then of heating up to 300°C at the heating rate of 10°C/min. As the carrier gas 4.0 N ₂ was used at the flow of 20 ml/min. Chemical purity was measured with the use of liquid chromatography (HPLC):

Device: Waters Acquity UPLC, PDA detection

Sample preparation: Dissolve 25.0 mg of the tested sample in 5.0 ml of 50% acetonitrile Column: - dimension: 1 = 0.10 m, 0 = 2.1 mm

- stationary phase: Kinetex CI 8, 1.7 μηι particles

- column temperature: 50°C

Mobile phase: A: lOmM NH ₄ H ₂ P04 at pH 2.5

B: acetonitrile

Gradient elution: Time Flow

% A % B

(min) (ml/min)

0 0.30 90 10

16 0.30 10 90

20 0.30 10 90

21 0.30 90 10

23 0.30 90 10

Detection: spectrophotometer 195 nm

Injected amount: 10.0 μΐ

Sample temperature: 20°C

Sample concentration: 5.0 mg/ml

Examples

The purpose of the following examples is to elucidate the invention without limiting its scope.

Obeticholic acid was prepared according to the procedure disclosed in the patent application WO2002072598. The chemical purity of crude obeticholic acid prepared this way was 97.59 % (HPLC). The 1H and ^I3 C NMR spectra confirmed the structure of obeticholic acid.

Example 1

Preparation of Form 1-2 of obeticholic acid

Obeticholic acid (1 g, 2.38 mmol, 99.10% HPLC) was dissolved in 3 ml of butyl acetate under moderate reflux. The clear solution was left to slowly cool down to the room temperature and subsequently left to crystallize at the room temperature for approximately 3 hours. The produced crystals were aspirated and dried in a vacuum drier at 25°C for 6 hours. 850 mg of Crystalline Form 1-2 of obeticholic acid was obtained at the chemical purity of 99.61% (HPLC)

Example 2

Preparation of Form 1-3 of obeticholic acid

Obeticholic acid (0.5 g, 1.19 mmol, 97.59% HPLC) was dissolved in 2 ml of acetonitrile under moderate reflux. The clear solution was left to slowly cool down to the room temperature, seeded and subsequently left to crystallize at the room temperature for 24 hours. The produced crystals were aspirated and dried in a vacuum drier at 40°C for 6 hours. 380 mg of Crystalline Form 1-3 of obeticholic acid was obtained at the chemical purity of 98.99% (HPLC).

Preparation of crystals for seeding. An excess of obeticholic acid was stirred in a suspension with acetonitrile at 40°C for 24 hours. After cooling down to the room temperature the suspension was filtered and Crystalline Form 1-3 of obeticholic acid was verified by XRPD in the obtained crystals.

Example 3

Recrystallization of Form 1-2 of obeticholic acid

Obeticholic acid (1.5 g, 3.56 mmol, 99.61% HPLC) was dissolved in 4.5 ml of butyl acetate under moderate reflux. The clear solution was left to slowly cool down to the room temperature and subsequently left to crystallize at the room temperature for approximately 3 hours. The produced crystals were aspirated and dried in a vacuum drier at 25°C for 6 hours. 1.38 g of Crystalline Form 1-2 of obeticholic acid was obtained at the chemical purity of 99.98% (HPLC).

Example 4

Purification of crude obeticholic acid

Obeticholic acid (3.8 g, 9.03 mmol, 97.59% HPLC) was dissolved in 20 ml of butyl acetate under moderate reflux. The clear solution was left to slowly cool down to the room temperature and subsequently left to crystallize at the room temperature for approximately 3 hours. The produced crystals were aspirated and dried in a vacuum drier at 25°C for 25 hours. 2.2 g of Crystalline Form 1-2 of obeticholic acid was obtained at the chemical purity of 99.83% (HPLC). This Form 1-2 was further dried in a vacuum drier at 40°C for 48 hours, providing amorphous obeticholic acid.

Example 5

Preparation of the amorphous form of obeticholic acid from Form 1-2

Obeticholic acid (580 mg) was dissolved in a solution of 8.7 ml of water and 8.7 ml of a 50% solution of sodium hydroxide at 40°C. The obtained solution was slowly added to diluted hydrochloric acid (37%, 9.6 ml) with water (8.7 ml) at 40°C. The produced suspension was stirred at 40°C for 30 minutes and then cooled down to 10°C. The solid fraction was filtered and washed with water. After drying in a vacuum drier (40°C), 526 mg of amorphous obeticholic acid was obtained.

Example 6

Preparation of the amorphous form of obeticholic acid from Form 1-3

Obeticholic acid (500 mg) was dissolved in a solution of 5 ml of water and 0.11 g of 30% ammonia. The obtained solution was maintained at the temperature of 30 - 40°C and 150 mg of phosphoric acid was slowly added. The produced suspension was stirred at 30 - 40°C and then cooled down to 20°C and filtered. The solid fraction was washed with water (3 x 1 ml) and dried in a vacuum drier (40°C). 440 mg of amorphous obeticholic acid was obtained.

Example 7

Pharmaceutical composition of the product - core

The experiments of pharmaceutical compositions were conducted with Crystalline Forms 1-2 and 1-3, as well as with the amorphous form of obeticholic acid.

The following ingredients were charged into a homogenizer: obeticholic acid, lactose, microcrystalline cellulose and sodium croscarmellose. The mixture was homogenized at 20 rpm for 15 min. Finally, magnesium stearate was added and the mixture was homogenized at 20 rpm for another 3 min. The tabletting matter produced in the above mentioned way was compressed in a rotary tabletting machine and used for the production of cores with the approximate weight of 280 mg.