CRYSTALLINE FORM OF TREOSULFAN

Abstract

A crystalline form of treosulfan and methods to prepare it are described. This crystalline form of treosulfan is useful in pharmaceutical compositions for the treatment of cancer and for conditioning therapy before transplantation of bone marrow or blood stem cells.

Claims

1. Crystalline form B of treosulfan, which exhibits an X-ray powder diffraction pattern having characteristic peaks at 20.87 and 23.47±0.20 degrees 2Θ.

2. Crystalline form B according to claim 1, which exhibits an X-ray powder diffraction pattern having characteristic peaks at 20.87, 23.47, 26.20, 29.65, 30.81, 34.54, 35.30, 36.87 and 46.24±0.20 degrees 2Θ.

3. Crystalline form B according to claim 1, which exhibits an X-ray powder diffraction pattern essentially as shown in FIG. 1.

4. Crystalline form B according to claim 1, which exhibits an X-ray powder diffraction pattern having no peaks in at least one, and preferably in all of the following regions a to f, expressed as degrees 2Θ: TABLE-US-00009 Region Degrees 2Θ a 19.00-19.50 b 20.00-20.65 c 21.50-23.21 d 23.75-24.95 e 27.40-28.35 f 30.00-30.60

5. Treosulfan which comprises at least 96% by weight, in particular at least 97% by weight, preferably at least 98% by weight and more preferably at least 99% by weight of the crystalline form B according to claim 1, relative to the combined amount of the crystalline form B and a crystalline form A.

6. Treosulfan which comprises at least 75% by weight, in particular at least 80% by weight, preferably at least 85% by weight, more preferably at least 90% by weight and even more preferably at least 95% by weight of the crystalline form B according to claim 1, relative to the amount of treosulfan.

7. Treosulfan according to claim 5, which comprises less than 20% by weight, in particular less than 15% by weight, preferably less than 10% by weight and more preferably less than 5% by weight of amorphous phase.

8. Treosulfan according to claim 5, which comprises less than 0.2% by weight, preferably less than 0.1% by weight and more preferably less than 0.05% by weight of methanesulfonic acid.

9. Process for preparing the crystalline form B according to claim 1, which process comprises (A) recrystallizing treosulfan from organic solvent, optionally comprising water, or (B) dissolving treosulfan in organic solvent, optionally comprising water, and allowing evaporation of solvent and optional water to dryness, or (C) dissolving treosulfan in organic solvent, optionally comprising water, and adding anti-solvent.

10. Process according to claim 9, wherein the organic solvent in variant (A), variant (B) and variant (C) is selected from the group of ethers, ketones, esters and alcohols or mixtures thereof and is preferably dioxane, methyl isobutyl ketone, ethyl acetate, tetrahydrofuran and isopropanol or a mixture thereof.

11. Process according to claim 9, wherein in variant (B) a mixture of water and isopropanol is used and water and isopropanol are allowed to evaporate to dryness at room temperature.

12. Process according to claim 11, wherein the mixture comprises about 80% by weight of water and about 20% by weight of isopropanol

13. Process according to claim 9, wherein in variant (C) the anti-solvent is selected from methyl tert-butyl ether, toluene, hexane, pentane, chloroform, and methylene chloride.

14. Pharmaceutical composition comprising the crystalline form B according to claim 1 and optionally at least one pharmaceutically acceptable additive.

15. Pharmaceutical composition according to claim 14, which is in the form of a powder, a tablet, granules or a capsule and in particular in form of a lyophilisate.

16. (canceled)

17. A method of treating cancer comprising administering the crystalline form B of treosulfan according to claim 1 to a patient suffering from cancer and in particular ovarian cancer.

18. A method comprising administering the crystalline form B of treosulfan according to claim 1 to a patient as conditioning therapy before transplantation of bone marrow or of blood stem cells to the patient.

19. The method of claim 17, wherein the crystalline form B of treosulfan is administered in a solution for injection or infusion into the patient.

20. The method of claim 18, wherein the wherein the crystalline form B of treosulfan is administered in a solution for injection or infusion into the patient.

Description

EXAMPLES

Methods

[0059] The following methods have been used for obtaining X-ray powder diffraction (XRPD) patterns, for investigations by means of single-crystal x-ray diffraction (SCXRD) and optical microscopy, for determining the amount of crystalline form B and crystalline form A and the amount of amorphous phase, and for determining the amount of treosulfan, methanesulfonic acid and water.

X-Ray Powder Diffraction (XRPD)

[0060] The respective sample was introduced in a standard glass capillary (Ø=0.7 mm) after careful manual grinding with a pestle in a mortar. The X-ray powder diffraction pattern was recorded at room temperature using a Bruker D8 Advance Diffractometer (Cu-Kα1=1.54059 Å, Johansson primary beam monochromator, position sensitive detector) in transmission mode with rotation of the sample. Data were collected in the range of 3 to 50 degrees 2Θ. The tube voltage and current were set to 40 kV and 40 mA, respectively.

Single-Crystal x-Ray Diffraction (SCXRD)

[0061] Single crystal X-ray diffraction data were recorded using a “Rigaku Xcalibur, Sapphire2, large Be window” diffractometer equipped with an X-ray generator containing a molybdenum anode (Mo-Kα=0.71073 Å).

Determination of Amount of Form B and A by XRPD and Rietveld Analysis

[0062] For determining the amount of crystalline form B and A of treosulfan, a respective sample was introduced in a standard glass capillary (Ø=0.7 mm) after careful manual grinding with a pestle in a mortar. The x-ray powder diffraction pattern was recorded at room temperature using a Bruker D8 Advance diffractometer (Cu-Kα1=1.54059 Å, Johansson primary beam monochromator, position sensitive detector) in transmission mode with rotation of the sample. Data were collected in the range of 4 to 50 degrees 2Θ over a period of 4 h. The tube voltage and current were set to 40 kV and 40 mA, respectively. The obtained data were subjected to a quantitative Rietveld analysis by means of the TOPAS software.

Determination of Amount of Amorphous Phase by XRPD and Rietveld Analysis with Internal Standard

[0063] For determining the amount of amorphous phase, a respective sample was mixed with 25% by weight of CaF.sub.2 (Aldrich Chemistry, Lot #MKBP1959V, Calcium Fluoride anhydrous, 99.99%) as internal standard. After careful manual grinding with a pestle in a mortar, the mixture was introduced in a standard glass capillary (Ø=1.0 mm). The x-ray powder diffraction pattern was recorded at room temperature using a Bruker D8 Advance diffractometer (Cu-Kα1=1.54059 Å, Johansson primary beam monochromator, position sensitive detector) in transmission mode with rotation of the sample. Data were collected in the range of 4 to 50 degrees 2Θ over a period of 12 h. The tube voltage and current were set to 30 kV and 30 mA, respectively. The obtained data were subjected to a quantitative Rietveld analysis by means of the TOPAS software.

[0064] Crystalline form A and crystalline form B were the only crystalline phases which could be identified.

Optical Microscopy

[0065] The investigations were performed using a Leica DMRB microscope equipped with a camera system from Kappa, type ZELOS. Samples were examined in purified silicon oil on a purified tray and images were taken with polarized light and lambda slip for higher color contrast.

Determination of Amount of Treosulfan by RP-HPLC

[0066] The amount of treosulfan in a respective sample was determined using reversed-phase high pressure liquid chromatography (RP-HPLC) as indicated in the following:

TABLE-US-00003 HPLC Equipment Agilent Technologies Column Luna C18(2), 5 μm, 250 × 4.6 mm (phenomenex) Mobile phase A 720 ml diluent + 30 ml methanol Isocratic, 25 min Flow rate 0.8 ml/min Column temperature 40° C. Injected volume 20 μl Diluent 697 mg K.sub.2HPO.sub.4/1000 ml, pH 4.5 (H.sub.3PO.sub.4) Detection Refractive index detector Reference solution 50 mg/ml treosulfan in diluent Sample Solution 50 mg/ml treosulfan in solvent for reconstitution

Determination of Amount of Methanesulfonic Acid by HILIC

[0067] The amount of methanesulfonic acid (MSA) was determined using Hydrophilic Interaction Liquid Chromatography (HILIC) as indicated in the following:

TABLE-US-00004 HPLC Equipment Column Nucleodur HILIC (250 × 4.6 mm, 3 μm) Eluent 10 mmol Ammonium formiate in H.sub.2O/acetonitrile (7:93)(Vol/Vol) Flow rate 1.4 ml/min Column temperature 45° C. Injected volume 20 μl Detector 35° C. Run time 1.5 times the retention time of methanesulfonic acid Detection Refractive index detector Reference solution 1 Dissolve methanesulfonic acid in HPLC-grade water to a final concentration of 2.0 mg/ml Reference solution 2 Dilute reference solution 1 with eluent to 0.1 mg/ml. Reference solution 2 is used for quantification of methanesulfonic acid in the test solution. Test solution Dissolve sample to be tested in HPLC-grade water to a final concentration of 20 mg/ml

Determination of Amount of Water by “Karl Fischer Titration”

[0068] About 100 mg of the respective sample was weighed into a glass vial which was sealed with a crimp cap. The sample was transferred into the furnace of a Karl Fischer coulometer type 756, furnace sample processor 774, of Metrohm (Filderstadt, Germany) which was heated to 90° C. The septum of the cap was penetrated by an injection needle, and the generated water vapour was directly transferred into the titration chamber of the Karl Fischer coulometer via dry nitrogen. The measurement was repeated once. Empty glass vials were used for blank correction.

Example 1—Preparation of Form B Using Water/Isopropanol

[0069] 99.8 mg treosulfan were weighed in a vial (volume 4.0 ml) which was equipped with a PTFE (Polytetrafluoroethylene) sealing and a stirrer. 1.5 ml of a mixture of 80% by weight water and 20% by weight isopropanol preheated to 65° C. were then added. The resulting solution was completely taken up with a syringe (volume 5 ml) and filtered using a 0.2 μm filter into a second vial (volume 4.0 ml). The syringe, second vial and filter had been tempered at 65° C. before use. The solvents were allowed to evaporate from the open vial at room temperature to dryness which resulted in formation of crystals.

[0070] The XRPD pattern of the obtained crystals of form B according to the invention is shown in FIG. 1.

Example 2—Preparation of Form B Using 1,4-Dioxane

[0071] 20.1 mg treosulfan were weighed in a vial (volume 4.0 ml) which was equipped with a PTFE (Polytetrafluoroethylene) sealing and a stirrer. 185 μl of 1,4-dioxane were added at room temperature. After complete dissolution of the solid, the sealing was removed and the solvent was allowed to evaporate to dryness at room temperature.

[0072] The crystals obtained were analyzed by XRPD which showed them to be crystalline form B according to the invention.

Example 3—Preparation of Form B Using Methyl Isobutyl Ketone

[0073] 19.7 mg treosulfan were weighed in a vial (volume 4.0 ml) which was equipped with a PTFE (Polytetrafluoroethylene) sealing and a stirrer. 2.5 ml methyl isobutyl ketone (MIBK) were added at room temperature. After complete dissolution of the solid, the solution was completely taken up with a syringe (volume 5 ml) and filtered using a 0.2 μm filter into a second vial (volume 4.0 ml). The solvent was then allowed to evaporate from the open vial to dryness at room temperature.

[0074] The crystals obtained were analyzed by XRPD which showed them to be crystalline form B according to the invention.

Example 4—Preparation of Form B Using Ethyl Acetate

[0075] 20.0 mg treosulfan were weighed in a vial (volume 4.0 ml) which was equipped with a PTFE (Polytetrafluoroethylene) sealing and a stirrer. 2.0 ml ethyl acetate were added at room temperature. After complete dissolution of the solid, the solution was completely taken up with a syringe (volume 5 ml) and filtered using a 0.2 μm filter into a second vial (volume 4.0 ml). The solvent was then allowed to evaporate from the open vial to dryness at room temperature.

[0076] The crystals obtained were analyzed by XRPD which showed them to be crystalline form B according to the invention.

Example 5—Preparation of Form B Using Tetrahydrofuran

[0077] 50.0 mg treosulfan were weighed in a vial (volume 4.0 ml) which was equipped with a PTFE (Polytetrafluoroethylene) sealing and a stirrer. 0.85 ml tetrahydrofuran (THF) were added at room temperature. After complete dissolution of the solid, the solution was completely taken up with a syringe (volume 5 ml) and filtered using a 0.2 μm filter into a second vial (volume 5.0 ml) which contained 2.85 ml methyl tert-butyl ether (MTBE). The second vial was then carefully shaken which quickly resulted in formation of crystals which were separated by filtration.

[0078] The crystals obtained were analyzed by XRPD which showed them to be crystalline form B according to the invention.

Example 6—Preparation of Form B Using Methyl Ethyl Ketone

[0079] 50.1 mg treosulfan were weighed in a vial (volume 4.0 ml) which was equipped with a PTFE (Polytetrafluoroethylene) sealing and a stirrer. 1.1 ml methyl ethyl ketone (MEK) were added at room temperature. After complete dissolution of the solid, the solution was completely taken up with a syringe (volume 5 ml) and filtered using a 0.2 μm filter into a second vial (volume 5.0 ml) which contained 3.3 ml methyl tert-butyl ether (MTBE) while stirring. This resulted in immediate formation of crystals which were separated by filtration.

[0080] The crystals obtained were analyzed by XRPD which showed them to be crystalline form B according to the invention.

[0081] The crystals were moreover investigated by optical microscopy and a corresponding photomicrograph is shown in FIG. 2.

Example 7—SCXRD Analysis of Form B

[0082] A suitable single-crystal of form B was selected under the microscope from the crystals obtained in accordance with example 3 and was analyzed by means of single-crystal X-ray diffraction (SCXRD). The obtained data are represented above in the section preceding the examples.

Example 8—Preparation of Crystalline Form A (Reference)

[0083] About 5 g treosulfan were dissolved in about 80 g of 2-propanol under stirring at 65° C. The resulting solution was then filtered using a 0.2 μm filter and cooled to 15° C. which resulted in the precipitation of crystals. The crystals were collected and dried at about 40° C.

[0084] The XRPD pattern of the dried crystals is shown in FIG. 3 and confirms it to be crystalline form A of treosulfan. The crystalline form A exhibits an XRPD pattern having characteristic peaks at 7.69, 15.43, 18.74, 19.14, 19.77, 20.15, 20.28, 21.24, 21.74, 22.07, 22.96, 23.24, 24.36, 25.29, 28.05, 28.28, 28.97, 30.10 and 40.55±0.20 degrees 2Θ.

[0085] The dried crystals were moreover investigated by optical microscopy and a corresponding photomicrograph is shown in FIG. 4.

[0086] In addition, a suitable single-crystal of form A was selected under the microscope and was analyzed by means of single-crystal X-ray diffraction (SCXRD). The obtained data are presented above in the section preceding the examples.

Example 9—Preparation of Lyophilisate of Crystalline Form B

[0087] The pre-lyophilization solution of the composition as given in the table below was prepared by weighing water into a glass beaker and adjusting its temperature to 30° C. using a water bath. The corresponding amount of treosulfan was added and the mixture was stirred at 30° C. for 30 min. The obtained solution was filtered and the filtered solution was immediately filled into cleaned and depyrogenized glass vials which were tempered at 30° C.

Composition of Pre-Lyophilization Solution, Target Dose about 5000 mg Treosulfan Per Vial

[0088]

TABLE-US-00005 Concentration Fill per of treosulfan Solvent vial 80 mg/g Water for injection 62.5 g

[0089] The vials were stoppered in lyophilisation position and sealed in lyophilization bags. The samples were loaded into a freeze dryer GT 2 (Manufacturer: Hof Sonderanlagenbau (Lohra, Germany)) with 0.4 m.sup.2 shelf area and 8 kg ice condenser capacity including means for differential pressure measurement and lyophilized according to the following lyophilization cycle.

Lyophilization Cycle

[0090]

TABLE-US-00006 Ice Shelf condenser Time Cumulative Step temperature temperature Pressure step time # Description [° C.] [° C.] [mbar] [h:min] [h:min] 1 Loading 30 — 1000 00:01 00:01 2 Freezing ramp −45 — 1000 01:04 01:05 (1.17 K/min) 3 Freezing −45 — 1000 06:00 07:05 4 Annealing ramp −10 — 1000 00:35 07:40 (1 K/min) 5 Annealing −10 — 1000 06:00 13:40 6 Freezing ramp −45 — 1000 00:35 14:15 (1 K/min) 7 Freezing −45 — 1000 03:00 17:15 8 Vacuum −45 ≤−70 0.33 00:30 17:45 adjustment 9 Primary Drying 35 ≤−70 0.33 01:25 19:10 ramp (0.94 K/min) 10 Primary Drying 35 ≤−70 0.33 62:00 81:10

[0091] The obtained lyophilisate cakes were homogenous without any defects. For reconstitution testing, the vials were vented, opened and 100 ml of 0.45% by weight aqueous NaCl solution (room temperature) were added to give a final concentration of treosulfan of 50 mg/ml. The lyophilisate cakes reconstituted within 30 seconds only. No pre-heating of the solvent was necessary. The removal of sticky particles adhering to the wall of the vials was also not necessary.

[0092] All lyophilisates showed a very low amount of residual water and a very low amount of methanesulfonic acid. The latter was even below the limit of detection (LOD) of 0.01% by weight.

Properties of Lyophilisates

[0093]

TABLE-US-00007 Amount of Amount of Amount of methanesulfonic treosulfan water acid Reconstitution [% by weight] [% by weight] [% by weight] time 101.79 0.01 <LOD 30 s

[0094] The lyophilisates obtained were also subjected to XRPD analyses using Rietveld refinement to determine their crystallinity as well as their amount of form A, form B and amorphous phase. Crystalline form A and B were the only crystalline phases which could be detected. The results are given in the following table.

Results of XRPD Analyses

[0095]

TABLE-US-00008 Amount of form A Amount of crystalline and B Amount of treosulfan [% by weight] amorphous phase [% by weight] Form A Form B [% by weight] 96.3 0.5 99.5 3.7

[0096] The XRPD pattern of the lyophilisates is shown in FIG. 5.