LYOPHILISATE OF TREOSULFAN
20220031620 · 2022-02-03
Inventors
Cpc classification
A61K9/19
HUMAN NECESSITIES
C07C309/08
CHEMISTRY; METALLURGY
International classification
Abstract
A lyophilisate of treosulfan is described which possesses favourable characteristics in terms of a short reconstitution time and a high purity and stability and which is particularly useful in the treatment of cancer and for conditioning therapy before transplantation of bone marrow or blood stem cells.
Claims
1. Lyophilisate of treosulfan, wherein the lyophilisate comprises crystalline form B of treosulfan exhibiting an X-ray powder diffraction pattern having characteristic peaks at 20.87 and 23.47±0.20 degrees 2Θ.
2. Lyophilisate according to claim 1, wherein the crystalline form B 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±0.2 degrees 2Θ.
3. Lyophilisate according to claim 1, wherein the crystalline form B exhibits an X-ray powder diffraction pattern essentially as shown in
4. Lyophilisate according to claim 1, wherein the crystalline form B 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-00030 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. Lyophilisate according to claim 1, 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, relative to the combined amount of crystalline form B and crystalline form A.
6. Lyophilisate according to claim 1, 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, relative to the amount of lyophilisate.
7. Lyophilisate according to claim 1 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, relative to the amount of lyophilisate.
8. Lyophilisate according to claim 1, which comprises at least 95% by weight, in particular at least 96% by weight, preferably at least 98% by weight and more preferably at least 99% by weight of treosulfan.
9. Lyophilisate according to claim 1, 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.
10. Lyophilisate according to claim 1, which comprises less than 1% by weight, preferably less than 0.5% by weight and more preferably less than 0.1% by weight of water.
11. Process for preparing the lyophilisate according to claim 1, which process comprises freeze-drying an aqueous solution comprising treosulfan.
12. Process according to claim 11, wherein the aqueous solution comprises water and optionally one or more organic solvents.
13. Process according to claim 12, wherein the organic solvent is acetic acid.
14. Process according to claim 11, which comprises (a) providing the aqueous solution having a first temperature, (b) freezing the aqueous solution, wherein the aqueous solution is cooled from the first temperature to a freezing temperature at a cooling rate of not more than 3 K/min, and (c) drying the frozen solution obtained in step (b) to give the lyophilisate.
15. Process according to claim 14, wherein the cooling rate in step (b) is not more than 2 K/min, preferably not more than 1.5 K/min and more preferably not more than 1.3 K/min, or the cooling rate in step (b) is from 0.05 to 1.5 and preferably from 0.1 to 1.3 K/min.
16. Process according to claim 11, wherein the first temperature is from 15° C. to 95° C., preferably from 20° C. to 50° C. and more preferably from 25° C. to 35° C.
17. Process according to claim 11, wherein the freezing temperature is −40° C. or less, preferably from −60° C. to −40° C. and more preferably from −50° C. to −40° C.
18. Process according to claim 11, wherein the frozen solution is kept at the freezing temperature for at least 1 hour, preferably 1 to 10 hours and more preferably 2 to 8 hours.
19. Process according to claim 14, wherein the drying in step (c) includes a primary drying which is carried out by subjecting the frozen solution to a temperature of −25° C. or higher, preferably a temperature of −15° C. to 0° C., and subjecting the frozen solution to a pressure of 0.03 to 1.0 mbar, preferably 0.1 to 0.6 mbar and more preferably 0.3 to 0.5 mbar, or the drying in step (c) includes a primary drying which is carried out by subjecting the frozen solution to a temperature of 0° C. or higher, preferably a temperature of 0° C. to 60° C., more preferably 20° C. to 60° C., even more preferably 30° C. to 50° C., and subjecting the frozen solution to a pressure of 0.03 to 1.0 mbar, preferably 0.1 to 0.6 mbar and more preferably 0.3 to 0.5 mbar.
20. Process according to claim 19, wherein the primary drying is carried out for at least 5 hour and preferably at least 10 hours.
21. Process according to claim 19, wherein after the primary drying a secondary drying is carried out by subjecting the product of the primary drying to a temperature of at least 30° C., preferably 30 to 50° C., and subjecting the product of the primary drying to a pressure of 0.03 to 1.0 mbar, preferably 0.1 to 0.6 mbar and more preferably 0.3 to 0.5 mbar.
22. (canceled)
23. A method of treating cancer comprising administering a lyophilisate according to claim 1 to a patient suffering from cancer and in particular ovarian cancer.
24. A method comprising administering a lyophilisate according to claim 1 to a patient in need thereof as a conditioning therapy before transplantation of bone marrow or of blood stem cells into the patient.
Description
EXAMPLES
Methods and Apparatus
[0062] In the following, the methods are given which have been used for obtaining X-ray powder diffraction (XRPD) patterns, for investigations by means of single-crystal X-ray diffraction (SCXRD), 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, acetic acid, methanesulfonic acid and water.
[0063] Moreover, the general procedure for preparing glass vials and for determining the reconstitution behaviour as well the apparatus used for freeze drying are also indicated below.
General Procedure—Preparing Glass Vials
[0064] Glass vials for lyophilization were rinsed before use with purified water and depyrogenized for 2 hours at 300° C. Lyophilization stoppers were autoclaved (121° C., 20 min, 2 bar) and dried for 7 hours at 110° C.
Freeze Dryer
[0065] Freeze drying was carried out in 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.
X-Ray Powder Diffraction (XRPD)
[0066] 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)
[0067] 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
[0068] 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
[0069] 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 hours. 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.
[0070] Crystalline form A and crystalline form B were the only crystalline phases which could be identified.
Determination of Amount of Treosulfan by RP-HPLC
[0071] 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
[0072] 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 Residual Acetic Acid Content by Headspace Gas Chromatography (HS-GC)
[0073] The amount of residual acetic acid was determined by HS-GC after esterification to ethyl acetate.
[0074] For sample preparation, the lyophilisate of one vial was reconstituted with water using 20 ml of water per 1 g of lyophilisate. 500 μl of the reconstituted sample were mixed with 100 μl saturated NaHSO.sub.4-solution and 50 μl of ethanol in a GC-vial. The GC vial was tightly crimped. All samples were prepared in duplicates.
[0075] For preparation of standards, a stock solution of acetic acid of 1 mg/ml was prepared and diluted to 5 individual standards containing 25 μg/ml to 0.5 μg/ml in water. Each stock solution (500 μl) was mixed with 100 μl saturated NaHSO.sub.4 solution and 50 μl of ethanol in a GC-vial.
TABLE-US-00005 c Stock (HOAc) solution Water NaHSO.sub.4 Ethanol Standards: mg/ml μl μl μl μl 0.05% 0.025 125 375 100 50 0.02% 0.01 50 450 100 50 0.01% 0.005 25 475 100 50 0.005% 0.0025 12.5 488 100 50 0.001% 0.0005 2.5 498 100 50
[0076] Standards were prepared in duplicates.
[0077] The GC method for quantification of residual solvents was used to determine the amount of acetic acid in form of its ethyl ester (see Ph.Eur. 2.4.24 Identification and control of residual solvents: System A). The chromatographic conditions used to quantify the amount of ethyl acetate correspond to the USP 467 method for the determination of residual solvents.
[0078] The following gas chromatograph was used:
TABLE-US-00006 Manufacturer Hewlett Packard Type 6890 Headspace 7694, Agilent sampler Technologies Detector FID N2 source Nitrogen generator G1000E, Parker H2 source Hydrogen generator PGH2100
[0079] The gas chromatograph and the head sampler were operated at the following conditions:
TABLE-US-00007 6890N 7694 Headspace sampler GC oven Loop size 1 ml program: Initial 35° C. Oven 85° C. Temperature Initial time 20 min Transfer line 120° C. Rate 25° C./min Loop 100° C. Final 250° C. Equilibration 30 min temperature time Final time 15 min Vial pressure 14 psi Injection Split, 160° C. Pressurization 0.15 min port: Splitless Loop fill 0.5 min Detector FID 270° C. Inject 0.5 min Nitrogen makeup Carrier Gas Nitrogen Flow 3.7 ml/min
Reconstitution Behavior
[0080] The dissolution behavior of the lyophilisates was determined by adding water for injection or 0.45% by weight of aqueous NaCl solution at room temperature to give a final concentration of about 50 mg/ml. The reconstitution process was monitored with regard to dissolution time and behavior.
Determination of Amount of Water by “Karl Fischer Titration”
[0081] 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 Lyophilisate of Crystalline Form B
[0082] The solution as given in the table below was prepared by weighing 8.0 g of treosulfan in a single use polypropylene (PP) beaker. The required amount of the solvent was added and the treosulfan was dissolved under gentle agitation until a clear solution was obtained. The complete dissolution was checked by visual control. Afterwards the solution was filtered using a 0.2 μm filter. The solution was filled into cleaned and depyrogenized glass vials of a nominal volume of 20 ml.
Composition of Pre-Lyophilization Solution, Target Dose 500 mg Treosulfan Per Vial
[0083]
TABLE-US-00008 Concentration Fill per of treosulfan Solvent vial 50 mg/g 90 wt. % water for 10.0 g injection and 10 wt. % acetic acid
[0084] The filled vials were partially stoppered and the samples were loaded into the freeze dryer and freeze-dried according to the following lyophilization cycle.
Lyophilization Cycle
[0085]
TABLE-US-00009 Shelf Ice condenser Time Step temperature temperature Pressure step # Description [° C.] [° C.] [mbar] [h:min] 1 Loading 25 — 1000 00:01 2 Freezing ramp −45 — 1000 01:00 (1.17 K/min) 3 Freezing −45 — 1000 05:00 4 Vacuum −45 −70 0.05 00:30 adjustment 5 Primary −20 −70 0.05 01:30 drying ramp (0.28 K/min) 6 Primary −20 −70 0.05 109:00 drying 7 Secondary 40 −70 0.05 04:00 drying ramp (0.25 K/min) 8 Secondary 40 −70 0.05 20:00 drying
[0086] All lyophilisates obtained were identified by XRPD analysis as crystalline form B of treosulfan.
[0087] The lyophilisate cakes were well-formed and homogeneous without visible defects. The complete lyophilisate cakes dissolved in 10 ml water for injection at room temperature within less than 30 seconds applying gentle shaking. No pre-heating of the solvent was necessary. The removal of sticky particles adhering to the wall of the vial was also not necessary. The residual amount of water of the lyophilisates was below the limit of quantification of 0.005% by weight.
Properties of Lyophilisates
[0088]
TABLE-US-00010 Amount Reconstitution of water time Below limit of <30 s quantification
Examples 2 and 3—Preparation of Lyophilisate of Form B
[0089] The solutions as given in the table below were prepared by dissolving treosulfan in the respective solvent (30 min, 25° C., ultra-sonic bath). The obtained solutions were filtered and the filtered solutions were filled in cleaned and depyrogenized glass vials (10 vials per formulation) which were stoppered in lyophilization position and sealed in lyophilization bags.
Composition of Pre-Lyophilization Solution, Target Dose 500 mg Treosulfan Per Vial
[0090]
TABLE-US-00011 Concentration Fill amount Example of treosulfan Solvent per vial 2 70 mg/g 98 wt. % water for 7.14 g injection and 2 wt. % acetic acid 3 70 mg/g 94 wt. % water for 7.14 g injection and 6 wt. % acetic acid
[0091] The samples were loaded into the freeze dryer and lyophilized according to the following lyophilization cycle.
Lyophilization Cycle
[0092]
TABLE-US-00012 Ice Shelf condenser Time Step temperature temperature Pressure step # Description [° C.] [° C.] [mbar] [h:min] 1 Loading 25 — 1000 00:01 2 Freezing ramp −45 — 1000 12:00 (0.01 K/min) 3 Freezing −45 — 1000 02:00 4 Annealing ramp −25 — 1000 00:30 (0.67 K/min) 5 Annealing −25 — 1000 05:00 6 Freezing ramp −45 — 1000 00:30 (0.67 K/min) 7 Freezing −45 — 1000 00:30 8 Vacuum −45 −70° C. 0.05 00:30 adjustment 9 Primary −20 −70° C. 0.05 01:30 Drying ramp (0.28 K/min) 10 Primary −20 −70° C. 0.05 14:00 Drying 11 Secondary 40 −70° C. 0.05 04:00 Drying ramp (0.25 K/min) 12 Secondary 40 −70° C. 0.05 20:00 Drying
[0093] All lyophilisates tested were identified by XRPD analysis as crystalline form B of treosulfan.
[0094] For reconstitution testing, the vials were vented and opened and 10 ml of 0.45% by weight aqueous NaCl solution (room temperature) were added using a 10 ml volumetric pipette. The lyophilisate cakes of both examples 2 and 3 reconstituted within 1 min only. No pre-heating of the solvent was necessary. The removal of sticky particles adhering to the wall of the vial was also not necessary.
[0095] For all lyophilisates, only a very low amount of residual water was determined. Moreover, all samples were free of impurities and showed a similar and high treosulfan content. The acetic acid content was below the detection limit of the HS-GC analysis of 0.003 wt. %.
Properties of Lyophilisates
[0096]
TABLE-US-00013 Amount of Amount Amount of treosulfan of water acetic acid Reconstitution Example [wt. %] [wt. %] [wt. %] time 2 100 <0.1 Below 1 min detection limit 3 100 <0.1 Below 1 min detection limit
Example 4—Preparation of Lyophilisate of Crystalline Form B
[0097] The solution as given in the table below was prepared by weighing 10 g of treosulfan in a 150 ml polypropylene (PP) beaker. The solvent was added and the treosulfan was dissolved under stirring at an ambient temperature of 22° C. The obtained solution was filled into cleaned and depyrogenized glass vials of a nominal volume of 20 ml.
Composition of Pre-Lyophilization Solution, Target Dose about 1000 mg Treosulfan Per Vial
[0098]
TABLE-US-00014 Concentration Fill per of treosulfan Solvent vial 75 mg/g 90 wt. % water for 13.3 g injection and 10 wt. % acetic acid
[0099] The vials were stoppered in lyophilization position and sealed in lyophilization bags. The samples were loaded into the freeze dryer and lyophilized according to the following lyophilization cycle.
Lyophilization Cycle
[0100]
TABLE-US-00015 Ice Shelf condenser Time Step temperature temperature Pressure step # Description [° C.] [° C.] [mbar] [h:min] 1 Loading 45 — atm. 00:20 2 Cooling ramp 15 — atm. 02:10 (0.23 K/min) 3 Isothermal 15 — atm. 06:00 incubation 4 Freezing ramp −45 — 1000 03:20 (0.3 K/min) 5 Freezing −45 — 1000 05:00 6 Annealing ramp −5 — 1000 00:30 (1.33 K/min) 7 Annealing −5 — 1000 05:00 8 Freezing ramp −45 — 1000 01:40 (0.4 K/min) 9 Freezing −45 — 1000 00:30 10 Vacuum −45 −70 0.05 00:30 adjustment 11 Primary −5 −70 0.05 01:30 Drying ramp (0.44 K/min) 12 Primary −5 −70 0.05 70:00 Drying 13 Secondary 40 −70 0.05 04:00 Drying ramp (0.19 K/min) 14 Secondary 40 −70 0.05 20:00 Drying “atm.” means atmospheric pressure
[0101] All lyophilisates tested were identified by XRPD analysis as crystalline form B of treosulfan.
[0102] The obtained lyophilisate cakes were acceptable. For reconstitution testing, the vials were vented and opened and 20 ml of 0.45% by weight aqueous NaCl solution (about 22° C.) were added. The lyophilisate cakes reconstituted within 1.5 min. No pre-heating of the solvent was necessary. The removal of sticky particles adhering to the wall of the vials was also not necessary.
[0103] All samples were free of impurities and had a very high content of treosulfan. The acetic acid content was very low.
Properties of Lyophilisates
[0104]
TABLE-US-00016 Amount of Amount of treosulfan acetic acid Reconstitution [wt. %] [wt. %] time 100 0.07 1.5 min
Example 5—Preparation of Lyophilisate of Crystalline Form B
[0105] A pre-lyophilization solution was prepared by mixing 52.5 g of treosulfan and 603.75 g of water under stirring at a temperature of 30° C. Stirring was continued for 30 minutes until complete dissolution of the treosulfan. After filtration using a 0.2 μm membrane filter, the filtered solution was filled into cleaned and depyrogenized glass vials.
Composition of Pre-Lyophilization Solution, Target Dose 5000 mg Treosulfan Per Vial
[0106]
TABLE-US-00017 Concentration Fill per of treosulfan Solvent vial 80 mg/g Water for injection 62.5 g
[0107] The vials were stoppered in lyophilization position and sealed in lyophilization bags. The samples were loaded into the freeze dryer and lyophilized according to the following lyophilization cycle.
Lyophilization Cycle
[0108]
TABLE-US-00018 Ice Shelf condenser Time Step temperature temperature Pressure step # Description [° C.] [° C.] [mbar] [h:min] 1 Loading 30 — atm. 00:01 2 Freezing ramp −45 — atm. 01:15 (1.0K/min) 3 Freezing −45 — atm. 03:00 4 Annealing ramp −5 — atm. 00:35 (1.14K/min) 5 Annealing −5 — atm. 02:00 6 Freezing ramp −45 — atm. 00:35 (1.14K/min) 7 Freezing −45 — atm. 01:00 8 Vacuum adjustment −45 −70 0.38 00:30 9 Primary Drying 40 −70 0.38 01:25 ramp (1.0K/min) 10 Primary/Secondary 40 −70 0.38 77:00 Drying “atm.” means atmospheric pressure
[0109] The lyophilisates obtained were identified by XRPD analysis as crystalline form B of treosulfan.
[0110] 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.
[0111] All lyophilisates showed a very high amount of treosulfan and a very low amount of residual water.
Properties of Lyophilisates
[0112]
TABLE-US-00019 Amount of Amount of treosulfan Water Reconstitution [% by weight] [% by weight] time 99.97 0.04 30 5
[0113] A sample of the lyophilisate was stored at 80° C. for 96 h. The stored sample still showed a very high content of treosulfan of >99.4% by weight. The methanesulfonic acid content was lower than 0.05% at the beginning of the testing and 0.05% after the storage proving the lyophilisate to be very stable.
Properties of Lyophilisate after Storage at 80° C. for 96 h
[0114]
TABLE-US-00020 Amount of Amount of treosulfan methanesulfonic Time [% by weight] acid [% by weight] 0 h 100.3 <0.05 96 h >99.4 0.05
Example 6—Preparation of Lyophilisate of Crystalline Form B
[0115] The solution of the composition as given in the table below was prepared by weighing water into a glass beaker and adjusting its temperature to 20° C. using a water bath. The corresponding amount of treosulfan was added and the mixture was stirred until complete dissolution. The obtained solution was filtered and the filtered solution was immediately filled into cleaned and depyrogenized glass vials which were tempered at 20° C.
Composition of Pre-Lyophilization Solution, Target Dose about 1000 mg Treosulfan Per Vial
[0116]
TABLE-US-00021 Concentration of treosulfan Solvent Fill per vial 56 mg/g Water 17.95 g
[0117] The vials were stoppered in lyophilisation position and sealed in lyophilization bags. The samples were loaded into the freeze dryer and lyophilized according to the following lyophilisation cycle.
Lyophilization Cycle
[0118]
TABLE-US-00022 Ice Shelf condenser Time Step temperature temperature Pressure step # Description [° C.] [° C.] [mbar] [h:min] 1 Loading 20 — atm. 00:01 2 Cooling ramp 15 — atm. 00:17 (0.3K/min) 3 Isothermal 15 — atm. 06:00 incubation 4 Freezing ramp −45 — 1000 03:20 (0.3K/min) 5 Freezing −45 — 1000 05:00 6 Annealing ramp −15 — 1000 00:30 (0.1° C./min) 7 Annealing −15 — 1000 05:00 8 Freezing ramp −45 — 1000 01:40 (0.3K/min) 9 Freezing −45 — 1000 00:30 10 Vacuum −45 −70 0.05 00:30 adjustment 11 Primary Drying −5 −70 0.05 01:30 ramp 12 Primary Drying −5 −70 0.05 70:00 13 Secondary 40 −70 0.05 04:00 Drying ramp 14 Secondary 40 −70 0.05 20:00 Drying
[0119] The lyophilisates obtained were identified as form B of treosulfan by XRPD analysis.
[0120] All lyophilisates showed a very high content of treosulfan and a very low amount of residual water. Furthermore, the amount of methanesulfonic acid was also very low.
Properties of Lyophilisates
[0121]
TABLE-US-00023 Amount of Amount of Amount of treosulfan water methanesulfonic acid [% by weight] [% by weight] [% by weight] 99.95 0.09 <0.05
[0122] Samples of the lyophilisates were stored at 60° C. for 30 days, 70° C. for 18 days and 80° C. for 5 days. Irrespective of the storage conditions, at the end of the tests all samples dissolved completely within 1.5 minutes in 20 ml 0.45% by weight of aqueous NaCl solution. No pre-heating of the solvent was necessary. The removal of sticky particles adhering to the wall of the vials was also not necessary.
[0123] Moreover, all samples still showed a very high content of treosulfan.
Properties of Lyophilisates after Storage at 60 to 80° C.
[0124]
TABLE-US-00024 Amount of t T treosulfan Reconstitution [d] [° C.] [% by weight] time 0 — 99.95 30 60 99.82 1.5 min 18 70 99.94 1.5 min 5 80 99.94 1.5 min
[0125] Samples of the lyophilisates were also stored at 40° C. and 75% relative humidity (r.H.) for 3 months. All stored samples still showed a very high content of treosulfan and a very low amount of methanesulfonic acid indicating their excellent stability.
Properties of Lyophilisates after Storage at 40° C./75% r.H.
[0126]
TABLE-US-00025 Amount of Amount of Time T/r.H. treosulfan methanesulfonic acid [months] [° C./%] [% by weight] [% by weight] 3 40/75 >99.95 <0.05%
Example 7—Preparation of Lyophilisate of Crystalline Form B
[0127] 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
[0128]
TABLE-US-00026 Concentration Fill per of treosulfan Solvent vial 80 mg/g Water for injection 62.5 g
[0129] The vials were stoppered in lyophilization position and sealed in lyophilization bags. The samples were loaded into the freeze dryer and lyophilized according to the following lyophilization cycle.
Lyophilization Cycle
[0130]
TABLE-US-00027 Ice Shelf condenser Time Step temperature temperature Pressure step # Description [° C.] [° C.] [mbar] [h:min] 1 Loading 30 — 1000 00:01 2 Freezing ramp −45 — 1000 01:04 (1.17K/min) 3 Freezing −45 — 1000 06:00 4 Annealing ramp −10 — 1000 00:35 (1K/min) 5 Annealing −10 — 1000 06:00 6 Freezing ramp −45 — 1000 00:35 (1K/min) 7 Freezing −45 — 1000 03:00 8 Vacuum adjustment −45 ≤−70 0.33 00:30 9 Primary Drying 35 ≤−70 0.33 01:25 ramp (0.94K/min) 10 Primary Drying 35 ≤−70 0.33 62:00
[0131] 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.
[0132] 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
[0133]
TABLE-US-00028 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
[0134] 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
[0135]
TABLE-US-00029 Amount of form A Amount of Amount of and B amorphous crystalline [% by weight] [% by weight] phase treosulfan Form A Form B [% by weight] 96.3 0.5 99.5 3.7
[0136] The XRPD pattern of the lyophilisates is shown in
Example 8—Preparation of Crystalline Form B
[0137] 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 wt. % water and 20 wt. % 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.
[0138] The XRPD pattern of the obtained crystals of form B is shown in
[0139] In addition, a suitable single-crystal of form B was selected under the microscope 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 9—Preparation of Crystalline Form A (Reference)
[0140] 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.
[0141] The XRPD pattern of the dried crystals is shown in
[0142] 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 represented above in the section preceding the examples.