Background Art

Apixaban, a substance having anticoagulant activity, which is indicated for treatment of both venous and arterial thromboembolism, was first described in EP 1 427 415, which describes the basic synthetic approaches to preparation of apixaban and similar molecules, and it also briefly describes pharmaceutical formulations.

Synthesis of apixaban is also described in the later WO03049681.

WO2006078331 describes two different forms of apixaban, apixaban dihydrate H2-2 and an anhydrous form identified as N-1 there. The examples included in this document indicate that the dihydrate is formed by crystallization from a water : polyethylene glycol mixture as needle-like crystals, which, after heating of the crystallizing matter in the presence of a crystallizing solvent and under treatment by an elevated temperature and/or intensive stirring, are transformed to the anhydrous form N-1. Crystals of the anhydrous form can be obtained in the desired particle size by controlling the crystallization conditions.

Document WO2007001385 describes the same transformation of the dihydrate to the anhydrous form. Document US 20070203178 describes dimethyl formamide and formamide solvates of apixaban. Apixaban is very poorly soluble in water; document WO2011106478 indicates a solubility of only 40 nanog/ml and discusses dependence of the apixaban particle size during the preparation of a formulation by wet and dry granulation and their influence on the dissolution rate and bioavailability of the formulation.

Disclosure of Invention

The invention provides three new crystalline forms of apixaban, identified as AP3, AP4, AP5. These forms have been characterized by means of X-ray powder diffraction with the use of CuKot radiation λ=0.1542 nm, differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). Thermodynamical properties (solubility, physical stability) of these new forms have also been studied.

Form AP3 in accordance with this invention is characterized by the reflections in the X-ray powder pattern presented in Table 1. The diffractogram is shown in Fig. 2. Form AP4 in accordance with this invention is characterized by the reflections in the X-ray powder pattern presented in Table 2. The diffractogram is shown in Fig. 5. Form AP5 in accordance with this invention is characterized by the reflections in the X-ray powder pattern presented in Table 3. The diffractogram is shown in Fig. 8. Table 1: Characteristic diffraction peaks corresponding to form AP3 of apixaban.

Relative

Position d-distance intensity

[°2Th.] [nm] [%]

5.85 1.4997 100.0

6.97 1.2673 14.8

11.84 0.7469 12.4

13.47 0.6570 35.5

15.01 0.5896 12.8

16.06 0.5514 32.3

17.45 0.5079 64.5

18.99 0.4669 21.2

20.44 0.4342 10.2 21.52 0.4126 38.7

22.64 0.3924 29.2

24.37 0.3650 16.2

25.92 0.3435 11.3

26.59 0.3349 5.4

27.15 0.3282 4.3

28.73 0.3105 4.2

30.02 0.2974 11.8

Table 2: Characteristic diffraction peaks corresponding to form AP4 of apixaban.

Relative

Position d-distance intensity

[°2Th.] [nm] [%}

5.44 1.6232 65.0

7.10 1.2437 67.9

10.49 0.8426 10.3

10.92 0.8096 17.8

12.75 0.6940 11.7

13.15 0.6726 12.4

15.16 0.5838 20.4

16.00 0.5536 39.9

17.69 0.5008 22.9

19.05 0.4656 29.3

20.28 0.4376 74.9

21.50 0.4130 79.8

22.72 0.3911 100.0

24.56 0.3621 43.5

25.17 0.3536 26.3

26.94 0.3307 16.6

31.09 0.2875 26.5

36.30 0.2473 12.2 Table 3: Characteristic diffraction peaks corresponding to form AP5 of apixaban.

The crystalline forms AP3, AP4 and AP5 of apixaban in accordance with this invention have been further characterized by differential scanning calorimetry DSC and thermogravknetry TGA. The results are summarized in Table 4. Figs. 3, 4, 6, 7, 9 and 10 present the respective DSC and TGA records of these forms. The TGA analysis of forms AP3, AP4 and AP5 indicates that all the forms contain 5 to 7% of water. In addition, the form AP4 contains ca. 13% of a solvent. This means that they are solvates. Table 4: DSC and TGA analysis of the crystalline forms of apixaban

Properties of the newly prepared crystalline forms and their mutual thermodynamic relationships were studied, which were further compared to the described forms H2-2 and Nl. First, solvent and temperature influenced stability tests of the crystalline forms were conducted. The tests were carried out in an HLC shaker. The samples were dosed into HPLC vials and stirred up in ethyl acetate or 2-propanol. The experiments were carried out at 25°C and the stirring speed of 900 rpm for 11 days. The temperature influenced stability tests were performed in a Memmert vacuum drier without the presence of a solvent. The samples were dosed onto Petri dishes and placed in the drier for 24 hours. The test was conducted at 80°C and normal humidity at the atmospheric pressure.

Occurrence of the transformations shown in Fig. 1 has been observed.

Further on, tests of thermodynamic solubility of the known crystalline forms Nl and H2-2 and the new forms AP3, AP4 and AP5 were conducted. An Eppendorf Thermomixer Comfort shaker was used to carry out the experiments. The samples were dosed into Eppendorf tubes and stirred up with water. The experiments were performed at 37°C and the stirring speed of 900 rpm for 24 h. The results are summarized in Table 5. Table 5: Thermodynamic solubility values of crystalline forms of apixaban

The above mentioned thermodynamic solubility results show that the thermodynamic solubility of crystalline form AP3 is more than double as compared to the anhydrous form Nl and nearly 80% higher than that of the dihydrate H2-2. So the assumption can be m ade that the bioavailability of the new crystalline forms AP3 and AP5 will be better, compared to the known forms Nl and H2-2.

List of drawings:

Fig. 1 : Stability of individual crystalline forms

Fig. 2: Diffractogram of apixaban form AP3

Fig. 3: DSC record of apixaban form AP3

Fig. 4: TGA record of apixaban form AP3

Fig. 5: Diffractogram of apixaban form AP4

Fig. 6: DSC record of apixaban form AP4

Fig. 7: TGA record of apixaban form AP4

Fig. 8: Diffractogram of apixaban form AP5

Fig. 9: DSC record of apixaban form AP5

Fig. 10: TGA record of apixaban form AP5

Experimental part:

Analytic methods:

X-ray powder diffraction

The diffractograms were obtained using an X'PERT PRO MPD PANah _j tical powder diffractometer with a graphite monochromator, used radiation CuKa λ=0.1542 nm, excitation voltage: 45 kV, anode current: 40 mA, measured range: 2 to 40° 20, increment: 0.01° 20 with the reflection dwell time of 50 s. The measurement was carried out using a flat powder sample that was placed on a Si plate. For the correction of the primary beam 0.02 rad Soller diaphragms, a 10 mm mask and a 1/4° fixed anti-dispersion diaphragm were used. The irradiated area of the sample was 10 mm, programmable divergence diaphn s were used. For the correction of the secondary beam 0.02 rad Soller diaphragms and 5.0 mm anti- dispersion diaphragm were used. DSC:

DSC measurements were conducted using a Perkin Elmer Pyris 1 DSC device. The samples were weighed and dosed into Al pots and measured in a nitrogen stream (4N ₂ 20 ml/min). The temperature program was set in the range of 50°C to 280°C at the heating rate op 10°C/min. The weight of the samples varied in the range of 3.2 to 3.4 mg.

TGA:

TGA was measured using a Perkin Elmer TGA 6 device. The samples were ^eighed and dosed into ceramic pots and measured in a nitrogen stream (4N ₂ 20 ml/min). The TGA measurements were conducted in the temperature range of 25°C to 300°C at the heating rate of 10°C/min. The weight of the samples varied in the range of 18.5 to 21.2 mg.

Example 1

Preparation of crystalline l-(4-Memoxyphenyl)-7-oxo-6-[4-(2-oxopiperidin-l-yl)phenyl]-4 ,5,6,7-tetrahydrd-lH- pyrazolo[3,4-c]pyridine-3-carboxamide (apixaban), prepared in accordance with the method described in patent WO 2003/026652, in an amount of 200 mg (0.435 mmol) was dissolved apixaban in a hot state. The hot solution was abruptly cooled down to 0 to 5°C and maintained at this temperature for 18 to 24 h. The resulting suspension was filtered and) the product, crystalline form AP3, was dried in vacuo at the room temperature.

Yield: 150 mg (75.0%)

Example 2

Preparation of crystalline form AP3 l-(4-Methoxyphenyl)-7-oxo-6-[4-(2-oxo-l-piperidyl)phenyl]-4, 5-dihydropyrazolo[3,4- c]pyridine-3-carboxamide (apixaban), prepared in accordance with the method described in patent WO 2003/026652, in an amount of 200 mg (0.435 mmol) was dissolved in 0.4 ml of chloroform under reflux. The clear solution was left to spontaneously cool down to the room temperature. During slow evaporation of the solvent the crystalline form AP3 separated.

Yield: 182 mg (92.0%) Example 3

Preparation of crystalline form AP4 l-(4-Methoxyphenyl)-7-oxo-6-[4-(2-oxo-l-piperidyl)phenyl]-4, 5-dihydropyrazolo|3,4- c]pyridine-3-carboxamide (apixaban), prepared in accordance with the method described in patent WO 2003/026652, in an amount of 150 mg (0.326 mmol) was dissolved in 3 ml of chloroform under reflux. The clear solution was abruptly cooled down to 5°C and maintained at this temperature for 1 week. The resulting suspension was filtered and ihe product, crystalline form AP4, was dried in vacuo at the room temperature.

Yield: 99 mg (66.0%)

Example 4

Preparation of crystalline form AP5

l-(4-Methoxyphenyl)-7-oxo-6-[4-(2-oxo-l-piperidyl)phenyl] -4,5-dihydropyrazolo[3,4 c]pyridine-3-carboxamide (apixaban), prepared in accordance with the method described in patent WO 2003/026652, in an amount of 150 mg (0.326 mmol) was dissolved in 3 ml of chloroform under reflux. The clear solution was abruptly cooled down to 0 to 5°C and maintained at this temperature for 24 to 48 h. The resulting suspension was filtered and the product, crystalline form A5, was dried in vacuo at the room temperature.

Yield: 104 mg (69.0%)

Example 5

Solvent controlled change of the crystalline form

The individual crystalline forms H2-2, AP3, AP4 and AP5 were each dosed in an amount of 20 mg into an HPLC vial and suspended in 1 ml of a solvent, ethyl acetate or 2-propanol. The vials were placed in an HLC shaker and shaken at 900 rpm at the room temperature for 11 days. The solid residue was isolated by filtration and further characterized. Example 6

Temperature controlled change of the crystalline form

The individual crystalline forms H2-2, AP3, AP4 and AP5 were each dosed in an amount of 20 mg onto Petri dishes and placed in a Memmert vacuum drier. The samples were heated up to 80°C at the ambient humidity and at the atmospheric pressure for 24 hours.

Example 7

Determination of thermodynamic solubility

The determination was conducted using an Eppendorf Thermomixer Comfort shaker. The individual crystalline forms Nl, H2-2, AP3, AP4 and AP5 were each dosed in an amount of 20 mg into Eppendorf tubes and placed in the shaker. The samples were stirred at 900 rpm at 37°C for 24 h.